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ANALGESIC EFFICACY FOR TOTAL KNEE ARTHROPLASTY Doctor of Nursing Practice Project: Analgesic Efficacy for Total Knee Arthroplasty Tierra Penick Marian University Leighton School of Nursing Chair: Dr. Bradley Stelflug _______________________ Committee Members: Dr. James Griggs ________________________ Date of Submission: October 31, 2020 1 ANALGESIC EFFICACY FOR TKA 2 Table of Contents Abstract ................................................................................................................................................. 4 Introduction ........................................................................................................................................... 6 Background........................................................................................................................................ 7 Problem Statement .......................................................................................................................... 11 Organizational Gap Analysis of Project Site .............................................................................. 12 Review of the Literature...................................................................................................................... 12 Search Strategy................................................................................................................................ 12 ACB alone versus combined ACB/SNB Therapy ........................................................................... 13 Supplementing FNB with SNB ........................................................................................................ 15 Evidence Based Practice .................................................................................................................. 16 Evidence Based Practice Model .......................................................................................................... 17 Cost-Benefit Analysis .......................................................................................................................... 18 Timeline ............................................................................................................................................... 18 Goals, Objectives, and Expected Outcomes ........................................................................................ 19 Ethical Considerations/Protection of Human Subjects ...................................................................... 19 Project Design...................................................................................................................................... 19 Project Site ....................................................................................................................................... 20 Subject Population........................................................................................................................... 20 Methods ............................................................................................................................................... 21 Procedures ....................................................................................................................................... 21 ANALGESIC EFFICACY FOR TKA 3 Measurement Instruments .............................................................................................................. 22 Data Collection Procedures ............................................................................................................. 22 Data Analysis ................................................................................................................................... 23 Results .................................................................................................................................................. 23 Participants ...................................................................................................................................... 23 Pain Scores ....................................................................................................................................... 24 Opioid Consumption ....................................................................................................................... 25 Length of Hospitalization ................................................................................................................ 25 Discussion ............................................................................................................................................ 26 Conclusion ........................................................................................................................................... 26 References ............................................................................................................................................ 28 Appendix A .......................................................................................................................................... 31 Appendix B .......................................................................................................................................... 32 Appendix C .......................................................................................................................................... 33 Appendix D .......................................................................................................................................... 34 ANALGESIC EFFICACY FOR TKA 4 Abstract Background: The number of annual total knee arthroplasties (TKA) had doubled over the last decade. Although this procedure treated chronic pain and restored mobility from end-stage osteoarthritis and other etiologies, it was associated with acute moderate-to-severe pain in the early postoperative period. Various analgesic techniques such as local infiltration, neuraxial blocks, and peripheral nerve blocks (PNB) were used in conjunction with multimodal anesthesia to prevent postoperative pain following TKA. The large variety of analgesic regimens accompanied with differing institutional and provider preferences challenged the ability to standardize a postoperative analgesic technique for TKA. Local Problem: Anesthesia providers at the project site often utilized the adductor canal block (ACB) alone or combined ACB and popliteal sciatic nerve block (PSNB) techniques for postoperative analgesia following TKA. However, it was unclear which technique was superior. Purpose: To understand the analgesic efficacy between ACB and combined ACB/PSNB following TKA. Methods: A retrospective chart analysis on 100 subjects that received ACB alone or ACB/PSNB for TKA in 2019. Pain was assessed upon post anesthesia recovery unit (PACU) admission, at 12 hours, and at 24 hours. Total opioid consumption was recorded for the first 24 hours. Length of hospitalization (LOH) was recorded per documentation. Results: Pain during PACU stay, pain at 12 hours postoperatively, and opioid consumption at 12 hours postoperatively were all significantly lower in the combined ACB/PSNB group (P = 0.0182, 0.0488, 0.0106 respectively). Pain and opioid consumption at 24 hours and LOH were not significantly different between the two groups. ANALGESIC EFFICACY FOR TKA 5 Conclusion: Combined ACB/PSNB therapy decreased pain and opioid consumption in the first 12 hours postoperatively following TKA. Large randomized controlled trials (RCT) need to be performed to deem the efficacy and incidence of related complications between these two blocks. Keywords: Adductor canal block, sciatic nerve block, femoral nerve block, knee, total knee arthroplasty ANALGESIC EFFICACY FOR TKA 6 Analgesic Efficacy for Total Knee Arthroplasty Introduction Total knee arthroplasty (TKA) was a commonly performed procedure that relieved joint pain and enhanced mobility in the patient with end-stage osteoarthritis and other degenerative etiologies (Li, Ma, & Xiao, 2019). Although this curable procedure often improved quality of life in the long-term, it was knowingly associated with acute moderate-to-severe pain within the first one to three postoperative days (Li, Ma, & Xiao, 2019). As modern medicine shifted focus to multimodal techniques, anesthesia providers sought after approaches to decrease postoperative complications and enhance recovery (Moucha, Weiser, & Levin, 2016). These regimens were often combined with multimodal anesthetics that included neuraxial anesthesia (e.g. spinal blockade), peripheral nerve blocks (PNB), periarticular injections, and non-opioid analgesics (e.g. ketamine or clonidine) (Moucha, Weiser, & Levin, 2016). Multimodal techniques incorporated various pharmacologic and interventional strategies to achieve optimal anesthetic and analgesic outcomes (Gaffney et al., 2017). Ideally, a balanced multimodal approach targeted postoperative pain, prevented significant changes in hemodynamic stability, allowed for early ambulation and mobility, decreased cost, avoided high-dose opioid consumption, and improved overall patient satisfaction (Li, Ma, & Xiao, 2019). As a result, PNBs that spared motor blockade (i.e. allow for ambulation) such as the adductor canal block (ACB) were becoming increasingly popular for lower extremity surgeries such as TKA. However, some studies argued that patients reported posterior and/or lateral pain knee pain when the ACB was used alone for TKA (Nader et al., 2016; Seo et al., 2017). Subsequently, different approaches to block the lower leg and knee, such as the popliteal sciatic nerve block (PSNB), were used in conjunction with the ACB to provide complete coverage (Seo et al., 2017). This project was conducted to determine the ANALGESIC EFFICACY FOR TKA 7 postoperative analgesic effectiveness between two PNB regimens for patients that underwent TKA including ACB alone and combined ACB/PSNB. Background More than 700,000 TKAs were performed annually in the United States, making it one of the most common orthopedic procedures (Terkawi et al., 2017). In fact, the number of annual TKAs had doubled over the past decade (Abdallah et al., 2016). Osteoarthritis (OA), the number one indication for TKA, effected nearly 27 million people in the United States (Gaffney et al., 2017). As a result, it was estimated that there would be a demand for nearly 3.5 million annual TKAs by 2030 (Terkawi et al., 2017). Severe postoperative pain remained a major concern for patients that underwent TKA. In fact, some patients refused or delayed arthroplasty because of reported acute postoperative pain (Gaffney et al., 2017). Although many people had chronic pain relief following TKA, some reported postoperative pain so severe that they would not repeat the surgery again if it were necessary for chronic relief (Gaffney et al., 2017). The goal of TKA was to diminish chronic pain from OA, yet severe acute postoperative pain was associated with chronic post-surgical pain that persisted for longer than six months (Moucha, Weiser, & Levin, 2016; Terkawi et al., 2017). Adequate pain relief was essential for optimal patient satisfaction, rehabilitation following surgery, and physiologic function (Gaffney et al., 2017). There were countless adverse physiologic outcomes associated with uncontrolled pain including cognitive dysfunction, decreased immune function, anxiety, thromboembolism, decreased mobility, insomnia, vasoconstriction leading to end-organ damage, and pneumonia (Gaffney et al., 2017). Subsequently, patients were unable to participate in rehabilitation, and this led to prolonged hospitalization and increased cost of care (Gaffney et al., 2017). ANALGESIC EFFICACY FOR TKA 8 Patients that experienced intense postoperative pain usually required high doses of opioid analgesics for relief (Gaffney et al., 2017). Opioids knowingly provided adequate pain relief as well as enhanced sleep and mood after surgery (Gaffney et al., 2017). Nonetheless, high dose opioids often produced undesirable adverse effects that consequently decreased patient satisfaction (Gaffney et al., 2017). These included pruritis, constipation, urinary retention, respiratory depression, nausea and vomiting, and reduced cognition (Gaffney et al., 2017; Moucha, Weiser, & Levin, 2016). These accompanied with the nations opioid epidemic led providers to favor balanced multimodal analgesia with minimal need for rescue opioids in the postoperative setting (Gaffney et al., 2017). There were more than ten multimodal techniques for postoperative TKA pain at the time of this project (Terkawi et al., 2017). Historically, femoral nerve blockade (FNB) was widely accepted as the gold standard for postoperative analgesia following TKA (Kuang et al., 2017; Zhang, Wang, & Liu, 2019). While FNB improved postoperative pain scores and decreased acute opioid consumption, it was also associated with other, less favorable outcomes such as impaired postoperative mobility (Kuang et al., 2017; Zhang, Wang, & Liu, 2019). While FNB may have spared motor blockade in low concentrations, it often resulted in complete anesthesia, both motor and sensory, to the anterior and medial thigh, knee, lower leg, and foot (NYSORA, 2019). Decreased quadriceps strength delayed postoperative rehabilitation and mobility, prolonged recovery, and lengthened hospitalization (Kuang et al., 2017; Zhang, Wang, & Liu, 2019). In addition, FNB was associated with postoperative falls, increased thromboembolism risk, and inadvertent blood vessel and nerve damage (Li, Ma, & Xiao, 2019). In recent years, the ACB had become a favorable alternative to the FNB. Studies reported ACB to be equal in analgesic effects with minimal effects on quadriceps muscle strength when ANALGESIC EFFICACY FOR TKA 9 compared to FNB (Gao, et al., 2017; Zhang, Wang, & Liu, 2019). The ACB mainly anesthetized the saphenous nerve, the largest sensory branch of the femoral nerve, that supplied sensory innervation to the anteromedial knee, lower leg, and ankle (NYSORA, 2019). It also included articular branches of the obturator nerve and the knee joint (Kuang et al., 2017). These nerves traveled within a triangular-shaped canal of muscles in the distal anteromedial thigh (NYSORA, 2019). Of note, while the ACB was considered a sensory-only block, it had been shown to cause quadriceps motor weakness with large local anesthetic volumes from inadvertent blockade of the nerve that supplied the vastus medialis (NYSORA, 2019). Nonetheless, when compared to the historical FNB, ACB was shown to provide equianalgesic properties with better functional recovery following TKA (Kuang et al., 2017). The utilization of the ACB shifted common multimodal analgesic techniques away from the use of FNB; however, some studies argued that both ACB and FNB resulted in residual posterior knee pain (Abdallah et al., 2016; Nader et al., 2016; Zorrilla-Vaca & Li, 2018). As a result, providers often used supplemental sciatic nerve blocks (SNB) that were shown to significantly reduce posterior knee pain and opioid consumption following TKA (Abdallah et al., 2016; Seo et al., 2017; Terkawi, 2017). There were proximal and distal techniques to blocking the sciatic nerve; however, the distal popliteal SNB approach spared the hamstring motor nerves while anesthetizing the posterolateral aspect of the knee joint (Abdallah, 2014). It was also associated with easier administration and more comfortable positioning than the proximal/infragluteal approach (Abdallah, 2014). Like the FNB, the PSNB may have spared motor blockade in low concentrations, but it often resulted in complete motor and sensory blockade of the lower leg and foot, excluding the anteromedial sensory innervation from the saphenous nerve (NYSROA, 2019). ANALGESIC EFFICACY FOR TKA 10 Although modern techniques improved patient safety, PNBs were associated with unfavorable side effects and adverse events. A major concern for a SNB was the risk for transient dorsiflexion impairment (i.e. foot drop) from blocking the common peroneal nerve, a major branch of the sciatic nerve (Seo et al., 2017). Dorsiflexion impairment was associated with an increased risk for postoperative falls (Gaffney et al, 2017; Seo et al., 2017). When blocking the sciatic nerve, it was common for anesthesia providers to discuss the possibility of foot drop and/or lower extremity weakness with patients in the preoperative setting to avoid postoperative falls. Additionally, some surgeons were concerned that foot drop masked surgical peroneal nerve injury, but surgical nerve palsy usually persisted beyond the 24 hours of foot drop seen with PSNB (Seo et al., 2017). Nonetheless, some surgeons still advised against blocking the sciatic nerve for TKA related to dorsiflexion impairment. The use of ultrasonography (US) allowed experienced providers to visualize real-time spread of local anesthetic, reducing the rate of failed block, intraneural injection, or intravascular injection (NYSORA, 2019). Additionally, electrical peripheral nerve stimulation was often used for motor-blocking techniques such as the PSNB (NYSORA, 2019). Nerve stimulation allowed experienced providers to locate and anesthetize peripheral nerves or plexuses with or without the use of US-guidance (NYSORA, 2019). To promote Enhanced Recovery After Surgery (ERAS) protocols, anesthesia providers at the project site commonly used two different multimodal PNB approaches to target pain following TKA. For the orthopedic surgical patient, ERAS protocols focused on postoperative analgesia, ambulation, and decreased hospitalization for rapid recovery (Oseka & Pecka, 2018). The increased risk for falls and decreased motor strength associated with FNB favored the use of the ACB for postoperative TKA analgesia (Kuang et al., 2017; Zhang, Wang, & Liu, 2019). ANALGESIC EFFICACY FOR TKA 11 Although FNB was still utilized at the project site, ACBs were more commonly used. Additionally, some anesthesia providers supplemented the ACB with a PSNB to target the posterior portion of the knee and knee joint. Problem Statement Inadequate pain relief in the acute postoperative setting remained a major focus for the patient that underwent TKA. Utilizing the most effective multimodal analgesic technique would have diminished postoperative complications related to uncontrolled pain, decreased length of hospitalization (LOH), improved patient satisfaction, decreased opioid consumption, and reduced cost of care. Knowing the efficacy between two common modalities used at the project site, ACB alone and ACB/PSNB combination therapy, would have ensured staff and patients that optimal analgesia was obtained while using minimal resources, expenses, and procedure times. To address the efficacy between postoperative peripheral nerve block analgesia, the following PICOT question was created: For adult patients undergoing TKA, is single-shot ACB and PSNB combination therapy more effective at reducing pain in the first 24 hours postoperatively compared to singleshot ACB alone? To deliver the most effective evidence-based care, this quality improvement project explored the efficacy between these two PNBs. This project retrospectively analyzed pain scores and total opioid consumption within the first 24 hours postoperatively on adult patients that underwent TKA. Once statistical analysis was completed, the DNP student reported findings to the anesthesia department at the project site to deem significance and the need for clinical practice adjustments. ANALGESIC EFFICACY FOR TKA 12 Organizational Gap Analysis of Project Site The discussed clinical gap at the project site was identified by the Section Chief of the Department of Anesthesiology and Medical Director of Surgical Services. Using guidelines set by the Agency for Healthcare Research and Quality (AHRQ, 2016), a gap analysis was performed to guide the discussed DNP project. There was not a standardized protocol for multimodal analgesia or anesthesia for TKA patients at the time of this project. However, most patients underwent general anesthesia with muscle relaxation and received a PNB for postoperative analgesia. This technique was determined by length of surgery (often exceeding two hours), surgeon preference, anesthesia provider preference, and patient factors. While each anesthetic plan was individualized, the decision about PNB lied in the collaborative hands of the surgeon and assigned anesthesia provider. Some surgeons requested that the sciatic nerve was not blocked with fear of dorsiflexion impairment. The anesthesia providers at the project site were unsure whether supplementally blocking the sciatic nerve was necessary for complete postoperative analgesia following TKA. They did not regularly follow uncomplicated post-surgical TKA patients throughout their hospitalization, so it was difficult for them to accurately assess long-term PNB efficacy. Subsequently, in collaboration with project site and Marian University staff, the DNP student proposed this quality improvement project to determine whether combined ACB/PSNB therapy was a more significant postoperative analgesic regimen when compared to ACB alone for the TKA patient. Review of the Literature Search Strategy Review of supportive literature was performed after a specific problem statement was created. The DNP student searched electronic databases including PubMed, Cochrane Library, ANALGESIC EFFICACY FOR TKA 13 and Google Scholar from 2015-2020 without language limitation or region exclusions. Results were filtered to include only clinical trials, systematic reviews, and meta-analyses. A total of 79 articles were included after excluding duplicates. Twenty-one results were found using keywords adductor canal block, sciatic nerve block, and knee. Three of these articles were included based on relevance and abstract review. A second search resulted in 58 results using keywords femoral nerve block, sciatic nerve block, and total knee arthroplasty. Two of these articles were included after excluding studies that did not directly discuss SNB as a complement to FNB in TKA patients. This literature review included a total of 7 peer-reviewed research articles. ACB alone versus combined ACB/SNB Therapy Postoperative analgesic efficacy comparing ACB to ACB/PSNB combination therapy for TKA was published in a peer-reviewed article in 2017 (Seo et al., 2017). This retrospective study performed in 2015-16 evaluated 200 patients that underwent TKA by evaluating pain, opioid consumption and associated complications following each technique (Seo et al., 2017). Patients received continuous ACB catheters in addition to placebo (group A) or local anesthetic (group B) injections for PSNB (Seo et al., 2017). All blocks were performed with US-guidance prior to subarachnoid (i.e. spinal) blockade by the same anesthesiologist, and the TKA was performed by the same surgeon (Seo et al., 2017). Outcomes were measured in intervals over seven postoperative days (POD), and statistical analysis considered significant p-values < 0.05 (Seo et al., 2017). Pain at rest, pain with knee flexion, and PCA requirements were all significantly lower in group B (combined ACB/PSNB therapy) (Seo et al., 2017). The authors stated that posterior knee pain appeared to be the main region of pain relief in those that received PSNB compared to those that received placebo (Seo et al., 2017). Although this study revealed superior analgesic effects using combined ACB/PSNB therapy, 35% (n = 35) that received PSNB ANALGESIC EFFICACY FOR TKA 14 experienced transient foot drop (Seo et al., 2017). This finding warranted the need for further research into the technique for adequately blocking the sciatic nerve using the popliteal approach (Seo et al., 2017). A randomized control trial (RCT) and a retrospective trial were published in 2016 comparing ACB and FNB in the presence of a SNB for patients that underwent TKA (Ardon et al., 2016, Wiesman et al., 2016). The goal of both trials was to determine if it was necessary to block the posterior aspect of the thigh and popliteal fossa using the FNB (Ardon et al., 2016; Wiesman et al., 2016). In other words, investigators wanted to know if dorsal innervation from the FNB was superior to dorsal innervation from combined ACB/SNB (Ardon et al., 2016, Wiesman et al., 2016). Like the study by Seo et al. (2017), these trials evaluated postoperative pain, analgesic consumption, and associated complications for two and three PODs (Ardon et al., 2016; Wiesman et al., 2016). All blocks were performed using US-guidance (motor blocks also utilized nerve stimulators) prior to induction of general anesthesia, although some participants from the retrospective study received spinal anesthesia instead of general (Ardon et al., 2016, Wiesman et al., 2016). All patients and providers were blinded in the RCT (Wiesman et al., 2016). Both trials considered statistically significant p-values of < 0.05 (Ardon et al., 2016; Wiesman et al., 2016). The findings from these two studies reported no significant differences between ACB and FNB in the presence of SNB regarding overall analgesic effect, opioid consumption, or other associated complications (Ardon et al., 2016; Wiesmann et al., 2016). The only significant difference included anterior knee pain during motion on POD 1 (P = 0.002) that was significantly higher for one of the ACB/SNB groups (Ardon et al., 2016). However, median pain scores never exceeded 4/10; therefore, the authors still concluded equianalgesic effects between the groups ANALGESIC EFFICACY FOR TKA 15 (Ardon et al., 2016). Additionally, two patients that received FNB experienced falls (Wiesman et al., 2016). In summary, when evaluating pain, opioid consumption, and other related complications, these two studies suggested ACB to be an equal and maybe even a favorable alternative to FNB in the presence of SNB (Ardon et al., 2016; Wiesman et al., 2016). Another double blinded RCT published in 2016 used a placebo group to determine the analgesic efficacy of ACB following TKA (Nader et al., 2016). Forty patients received preoperative US-guided ACB (group A, n = 20) or saline (Group B, n = 20) in addition to periarticular infiltration and spinal anesthesia (Nader et al., 2016). Pain and opioid consumption were recorded for 36 hours postoperatively, and statistical analysis considered significant pvalues < 0.05 (Nader et al., 2016). Although generalizability from this study was limited by a small sample size, results strongly supported the use of ACB over placebo (Nader et al., 2016). Opioid consumption (P = 0.03) and pain (P = 0.009) were both significantly lower in the first 36 hours in the ACB group (Nader et al., 2016). Time to discharge was also significantly lower in the ACB group (P = 0.007) (Nader et al., 2016). Of note, many of the patients in the ACB group reported posterior knee pain as the primary location of perceived pain (Nader et al., 2016). Supplementing FNB with SNB At the time of this project, there was not a published systematic review or meta-analysis directly evaluating the analgesic efficacy between ACB alone versus ACB/PSNB for TKA. This could have been in part because of the wide array of TKA analgesic modalities or because the ACB was a relatively new block. However, there were published peer-reviewed articles that reported superior analgesia when supplementing FNB with SNB to target residual dorsal knee pain (Abdallah et al., 2016; Zorrilla-Vaca & Li, 2018). These studies supported the need for ANALGESIC EFFICACY FOR TKA 16 complementary posterior knee blockade for adequate postoperative TKA analgesia (Abdallah et al., 2016; Zorrilla-Vaca & Li, 2018). Two meta-analyses aimed to determine if blocking the sciatic nerve resulted in superior postoperative TKA analgesia in the presence FNB (Abdallah et al., 2016; Zorrilla-Vaca & Li, 2018). Both studies reviewed pain, opioid consumption, and recovery outcomes in adult patients that underwent TKA (Abdallah et al., 2016; Zorrilla-Vaca & Li, 2018). Abdallah et al. (2016) reviewed 8 RCTs (n = 379), and Zorrilla-Vaca & Li (2018) reviewed 10 RCTs (n = 514). In the postoperative period, Zorrilla-Vaca and Li (2018) reported reduced pain at rest for 4 hours (P < 0.001) and reduced pain with activity for 12 hours (P = 0.02) when using a single-shot SNB in the presence of FNB. Abdallah et al. (2016) reported decreased pain at rest and activity for 8 hours using supplemental single-shot SNB (P = 0.023 and P < 0.001) and with activity for 36 and 48 hours using supplemental continuous SNB (P = 0.004 and P = 0.031). Both studies reported decreased opioid consumption for 24 hours postoperatively (Abdallah et al., 2016; Zorrilla-Vaca & Li, 2018). Although there was a gap in evidence supporting duration of analgesic efficacy, these meta-analyses concluded that SNB significantly reduced postoperative TKA pain when compared to no SNB in the presence of FNB (Abdallah et al., 2016; Zorrilla-Vaca & Li, 2018). Evidence Based Practice Numerous analgesic regimens have been known to control postoperative TKA pain. The variety of modalities challenged the ability to perform a single RCT using every analgesic technique. Each study in the literature review was limited by differing patient anatomical nerve innervation, approach to blockade, surgical technique, extraneous and confounding variables, and selection of local anesthetic. These variables limited appropriate evaluation of PNB efficacy. Additionally, some available literature included variables that were not included in this project, ANALGESIC EFFICACY FOR TKA 17 including continuous PNB catheters, subarachnoid blockade, patient-controlled analgesics, placebo groups, and control of local anesthetic and adjuncts. However, overall results from the discussed studies supported the need for posterior knee blockade for patients that underwent TKA. Furthermore, these studies supported ACB as a favorable alternative to FNB. This DNP project focused on the two primary outcomes assessed in the literature including pain and opioid consumption. The aim of evaluating length of hospitalization was to guide future studies comparing cost savings between the two blocks. The duration of block efficacy was assessed over the first 24 hours postoperatively, and the popliteal approach to block the sciatic nerve was assessed because it was the most common technique used at the project site. Evidence Based Practice Model The Johns Hopkins Nursing Evidence-Based Practice (JHNEBP) model is a conceptual framework utilized in nursing to ensure current evidence-based practice (EBP) is integrated into the clinical setting (Dang & Dearholt, 2017). See Appendix A for the model diagram. This conceptual model was the framework used for this DNP project as it focuses on the key idea that healthcare professionals are lifelong learners that provide the best quality and evidence-based care (Dang & Dearholt, 2017). This three-step model uses the acronym PET including a problem statement, review of current evidence and literature, and practice translation (Dang & Dearholt, 2017). First, a question was derived from exploring background information gathered by the key project members (Dang & Dearholt, 2017). The PICOT (population, intervention, comparison, outcome, time) format was used because it is a focused question that can be answered with current evidence-based research (JHM, 2018). Following creation of a problem statement, the DNP student performed a comprehensive literature review to collect current, pertinent, and high-quality evidence to support the given problem (Dang & Dearholt, 2017). ANALGESIC EFFICACY FOR TKA 18 Project members then determined how applicable available evidence was to the specific population (Dang & Dearholt, 2017). This is where the discussed quality improvement (QI) project implementation and analysis occurred. Once available current literature and results from the QI project was analyzed, investigators determined that there was a need for clinical practice change at the project site. Cost-Benefit Analysis The costs endured by this DNP project included the time for development, implementation, analysis, and proposed clinical practice change by the project members, university staff, and additional hospital staff that assisted the DNP student in implementation. The benefit of the review of literature and project findings largely outweighed the cost as it helped ensure patients were receiving the best quality and cost-effective PNB for TKA. Timeline Proposed approval of this project was given to the DNP student by university staff on October 11, 2019, and a final PICOT question was approved three days later. Literature review was performed over the next two months and included an annotated bibliography, matrix review, and SWOT analysis. A final project proposal was submitted December 2019. Project site approval and Marian University IRB exemption was given in February 2020. Following approval, the DNP student collaboratively discussed the need for remote Cerner access with the Department of Volunteer Services, and access was granted in April 2020. Data collection and analysis was performed over the summer of 2020. Project findings were presented to Marian University and staff at the project site as a finalized DNP project in October 2020. ANALGESIC EFFICACY FOR TKA 19 Goals, Objectives, and Expected Outcomes The overall aim of this DNP project was to determine which PNB, either ACB alone or ACB/PSNB combination therapy, was more effective at decreasing postoperative pain, opioid consumption, and LOH following TKA. A retrospective chart review was performed to determine pain scores, opioid consumption, and LOH among adult patients that received these multimodal approaches prior to unilateral TKA. The DNP student performed all data extraction and analysis, and she reported the findings to the anesthesia providers at the project site and accompanied staff at Marian University. Ethical Considerations/Protection of Human Subjects The Marian University IRB deemed this DNP project exempt from reviewSee Appendix C. Exempt status was given because this project offered minimal risk to participants, analyzed existing data, and did not have an interventional group. The DNP student also completed online CITI training for research involving human subjects prior to project initiation. All participants included in this project were protected by the Health Insurance Portability and Accountability Act of 1996 (HIPAA). Furthermore, the DNP student and other personnel involved in this project followed the Standards of Care for practice at Marian University. All patient data was immediately de-identified and stored electronically on the DNP students personal laptop. This laptop and Cerner, the EMR used for chart analysis, were password protected to prevent access by unauthorized users. Project Design The comparative groups in this DNP project included ACB alone (group A) and ACB/PSNB combination therapy (group B). Both groups included adult patients that underwent unilateral TKA. All data was retrieved via retrospective chart analysis. ANALGESIC EFFICACY FOR TKA 20 Project Site The project site was a rural, Magnet, Level III Trauma Center. This hospital served approximately nine counties across the Midwest in Indiana and Illinois. Surgical services ranged from dental procedures to open-heart with a total of over 10,000 surgeries performed annually. Orthopedic services, including TKAs, were offered through the joint replacement center and program for sports injuries, joint replacement, and other bone anomalies. The anesthesia group that staffs the project site is a private group that employs Physician Anesthesiologists and Certified Registered Nurse Anesthetists (CRNA) to collaboratively care for surgical patients. The Section Chief of the Department of Anesthesiology and Medical Director of Surgical Services at the project site acted as the liaison for the DNP student throughout this project. Additional project support came from the DNP students program staff. Success of this QI project relied largely on chart reviews performed on patients that underwent TKA in the operating room (OR) at the project site. Although there was a pain assessment protocol for each patient encounter, nursing documentation between nurses was highly variable, especially following PACU discharge. The inconsistency in charting resulted in repeated and missing values, and the DNP student had to adjust outcome intervals accordingly. As a result, pain and opioid consumption were recorded at a time point instead of cumulatively over a time interval following PACU discharge. Although evaluating cumulative data would have likely been more accurate, the inconsistency in charting prevented the ability to do this. Subject Population Subjects included adult patients that underwent primary, unilateral TKA at the project site between January 2019 and April 2019. Inclusion criteria for participants consisted of adult patients over eighteen that underwent unilateral TKA with a general anesthetic and preoperative ANALGESIC EFFICACY FOR TKA 21 ACB or combined ACB/PSNB. Participants were excluded if they were unable to understand the numeric rating scale, classified as American Society of Anesthesiologists (ASA) IV or V, received contralateral or revised TKA within three months of the current surgery, received additional or rescue PNBs, or underwent alternative anesthetic methods (e.g. spinal anesthesia). Additionally, there were two subjects that were excluded because they experienced severe postoperative complications unrelated to the PNB. Methods This project included a retrospective, non-randomized design using a convenience sample to determine the analgesic efficacy of PNB for postoperative TKA analgesia. Procedures All subjects underwent primary, unilateral TKA by different surgeons using a medial parapatellar approach. Following a collaborative decision with the surgeon, anesthesia providers performed all ACBs and PSNBs prior to induction of general anesthesia. ACBs were performed with US-guidance, and PSNBs were performed with US-guidance, peripheral nerve stimulation, or both. All outcome data was recorded separately from the subjects medical record on the DNP students data collection form. This data form was pre-authorized by Marian University staff, and it was coded to exclude all protected health information. Chart reviews were performed through remote access using Cerner, the electronic medical record (EMR) system utilized at the project site. Collection of demographic information included patient age, ASA, and gender. Outcome variables including postoperative pain scores, opioid requirements, and LOH were recorded to determine which block was more effective at decreasing postoperative pain in the first 24 hours following TKA. ANALGESIC EFFICACY FOR TKA 22 Measurement Instruments Pain scores were recorded using an 11-point numeric rating scale (McCaffery & Pasero, 1999). This was the main pain scale utilized at the project site for awake and responsive patients. The numeric rating scale allowed patients to verbally rate pain between 0 and 10, with 0 representing no pain and 10 representing worst pain ever (McCaffery & Pasero, 1999). See Appendix B for scale. Opioid consumption was recorded based on drug, route and dose administered. Total opioid consumption was calculated using an opioid equianalgesic conversion calculator based on American Pain Society guidelines available at https://clincalc.com/Opioids/ (ClinCalc, LLC, 2017). Each opioid was converted to intravenous (IV) morphine milligram equivalents (MME) with a 0% cross-tolerance for acute administration (ClinCalc, LLC, 2017). Outcomes were recorded for 24 hours postoperatively in two separate time intervals (t = 0-12 hours and t = 12-24 hours). Data Collection Procedures The DNP student collected all data via retrospective chart analysis using convenience sampling. Data was recorded and de-identified on an excel data spreadsheet that was preauthorized by university staff. Time intervals were manually calculated and entered in Cerner based on the initial postoperative anesthesia care unit (PACU) time that was documented by the anesthesia provider. LOH was recorded as the length of stay documented in the patients chart. The pain assessment protocol at the project site in the PACU stated that an initial assessment must be recorded upon admission followed by an assessment every fifteen minutes until discharge. If the patient experienced analgesic changes between the fifteen-minute intervals, this pain score was also documented. All pain scores documented during the patients ANALGESIC EFFICACY FOR TKA 23 PACU stay were recorded and presented as an average PACU pain score. Pain scores were also recorded at 12- and 24-hour points (t = 12 hours and t = 24 hours). If there was more than one score documented at a given time point, the average between the scores was recorded. If there was not a pain score documented for a time point, the average between the two most recent pain scores was recorded. One patient was discharged prior to twelve hours post-operatively, and no assumptions were made or included from this patient for data analysis at t = 12 or t = 24 hours. Total opioid consumption was recorded for 24 hours postoperatively in two separate time intervals (t = 0-12 hours and t = 12-24 hours) using the Medication Administration Record (MAR). Opioids included fentanyl, morphine, hydrocodone, hydromorphone, oxycodone, and tramadol. Each administered opioid was recorded based on route and dose, and then they were converted to intravenous MME. Total opioid consumption for each time interval was presented as total MME. Two patients received meperidine (Demerol) as an anti-shivering adjunct. Although meperidine was an opioid analgesic, these were not included in data analysis because they were not administered based on pain reported by the patient. Data Analysis Sigma XL in Microsoft Excel 2016 was used to perform statistical analysis for this project. Comparisons between treatment groups were determined using the Mann-Whitney U test with a statistical significance of P < 0.05. Results Participants Subjects included 100 patients who underwent primary, unilateral TKA. Groups included 50 patients that received ACB (group A) and 50 patients that received combined ACB/PSNB (group B). Group A comprised of 28 female and 22 male subjects, with a mean age of 69.5. Of ANALGESIC EFFICACY FOR TKA 24 the fifty subjects in group A, 18 were classified as ASA 2, and 32 were classified as ASA 3. Group B was comprised of 35 female and 15 male subjects with a mean age of 69.1. Of the fifty subjects in group B, 19 were classified as ASA 2, and 31 were classified as ASA 3. Pain Scores The average PACU pain scores were significantly lower in group B than group A (P = 0.0182). Pain at 12 hours postoperatively was also significantly lower in group B than group A (P = 0.488). There was no significant difference between pain at 24 hours postoperatively (P = 0.2183). PAIN DURING PACU PNB Count Median ACB 50 2.944 Mann-Whitney Statistic P-Value (2-sided adjusted for ties) 2863.50 0.0182 PAIN @ 12 HOURS (t = 12) PNB Count Median ACB 49 2 Mann-Whitney Statistic P-Value (2-sided, adjusted for ties) 2729.00 0.0488 PAIN @ 24 HOURS (t = 24) PNB Count Median ACB 49 3 Mann-Whitney Statistic P-Value (2-sided, adjusted for ties) 2624.50 0.2183 ACB/PSNB 50 1.1 ACB/PSNB 50 1.500 ACB/PSNB 50 2 ANALGESIC EFFICACY FOR TKA 25 Opioid Consumption Opioid consumption over the first 12 hours postoperatively was significantly lower in group B than group A (P = 0.0106). There was not a significant difference between total opioid consumption from 12 to 24 hours postoperatively (P = 0.1784). TOTAL MME 0-12 HOURS PNB Count Median ACB 50 9.500 Mann-Whitney Statistic P-Value (2-sided, adjusted for ties) 2895.00 0.0106 TOTAL MME 12-24 HOURS PNB Count Median ACB 50 8 Mann-Whitney Statistic P-Value (2-sided, adjusted for ties) 2718.00 0.1784 ACB/PSNB 50 8 ACB/PSNB 50 8 Length of Hospitalization There was no significant difference in LOH between the two groups (P = 0.8437). Length of Hospitalization (LOH) PNB Count Median ACB 50 2.400 Mann-Whitney Statistic P-Value (2-sided, adjusted for ties) 2496.00 0.8437 ACB/PSNB 50 2.400 ANALGESIC EFFICACY FOR TKA 26 Discussion This study aimed to determine the efficacy of PSNB in the presence of ACB as a postoperative analgesic technique for TKA. Significant findings included decreased pain and opioid consumption for up to 12 hours postoperatively for combined ACB/PSNB techniques when compared to ACB alone. These findings suggested superior analgesic effects using combined ACB/PSNB for TKA when compared to ACB alone. There was no significance between the two PNBs after twelve hours postoperatively. Additionally, there was no significance in data for LOH between the two PNB techniques. The inability to control for confounding variables such as local anesthetic type, volume, adjuncts (e.g. dexamethasone, epinephrine, etc.), and subsequent duration of action created a major limitation for this retrospective project. Future projects should control for these variables. As mentioned, the retrospective nature of this study presented several limitations. Large sample RCTs need to be performed to accommodate for these. RCTs would allow for control of inconsistent nursing documentation that prevented the DNP student from including all pain scores documented over the 24-hour time frame. Although total opioid consumption consistently reflected the pain scores for each time interval, this limitation likely created subsequent bias. Additionally, RCTs would allow accurate exclusion of participants that have underlying chronic pain syndromes or long-term opioid use that may affect pain and opioid consumption. Lastly, complications such as dorsiflexion impairment in the presence of PSNB and the impact it has on postoperative mobility should also be further evaluated. Conclusion This project suggested that combined ACB/PSNB was a superior analgesic technique for the first 12 postoperative hours following TKA. Postoperative pain following TKA continues to ANALGESIC EFFICACY FOR TKA be a major focus for TKA patients, and inadequate analgesia results in poor postoperative outcomes. Comparative studies including randomized, blinded controlled trials need to be conducted to further determine analgesic efficacy, associated complications, and cost effectiveness between ACB and ACB/PSNB for TKA. 27 ANALGESIC EFFICACY FOR TKA 28 References Abdallah, F. W., Chan V. W., Gandhi R., Koshkin A., Abbas S., & Brull R. (2014, December). The analgesic effects of proximal, distal, or no sciatic nerve block on posterior knee pain after total knee arthroplasty: A double-blind placebo-controlled randomized trial. Anesthesiology, 121(6), 1302-1310. doi: 10.1097/ALN.0000000000000406. PMID: 25099748. Abdallah, F.W., Madjdpour, C. & Brull, R. (2016, February 19). Is sciatic nerve block advantageous when combined with femoral nerve block for postoperative analgesia following total knee arthroplasty? A meta-analysis. Canadian Journal of Anesthesia, 63, 552568. https://doi.org/10.1007/s12630-016-0613-2 Agency for Healthcare Research and Quality (AHRQ). (2016). Gap analysis facilitators guide. Retrieved from https://www.ahrq.gov/patientsafety/capacity/candor/modules/facguide3.html Ardon, A. E., Clendenen, S. R., Porter, S. B., Robards, C. B., & Greengrass, R. A. (2016, June). Opioid consumption in total knee arthroplasty patients: A retrospective comparison of adductor canal and femoral nerve continuous infusions in the presence of a sciatic nerve catheter. Journal of Clinical Anesthesia, 31, 19-26. https://doi.org/10.1016/j.jclinane.2015.12.014 ClinCalc, LLC. (2017). Equivalent opioid calculator. Retrieved from https://clincalc.com/Opioids/ Dang, D., & Dearholt, S. (2017). Johns Hopkins Nursing Evidence-Based Practice: Model and Guidelines (3rd ed.). Indianapolis, IN: Sigma Theta Tau International. ANALGESIC EFFICACY FOR TKA 29 Gaffney, C. J., Pelt, C. E., Gililland, J. M., & Peters, C. L. (2017). Perioperative pain management in hip and knee arthroplasty. The Orthopedic Clinics of North America 48(4), 407-419. doi: 10.1016/j.ocl.2017.05.001 Kuang, M., Ma, J., Fu, L., He, W., Zhao, J., & Ma, X. (2017, October). Is adductor canal block better than femoral nerve block in primary knee arthroplasty? A GRADE analysis of the evidence through a systematic review and meta-analysis. The Journal of Arthroplasty, 32(19), 3238-3248. https://doi.org/10.1016/j.arth.2017.05.015 Li, J. W., Ma, Y. S., & Xiao, L. K. (2019). Postoperative pain management in total knee arthroplasty. Orthopaedic surgery, 11(5), 755761. doi:10.1111/os.12535 McCaffery, M., & Pasero, C. (Eds.) (1999). Pain Clinical Manual (2nd ed.). St. Louis, MO: Mosby. Moucha, C. S. , Weiser, M. C. & Levin, E. J. (2016). Current strategies in anesthesia and analgesia for total knee arthroplasty. Journal of the American Academy of Orthopaedic Surgeons, 24(2), 6073. doi:10.5435/JAAOS-D-14-00259 Nader, A., Kendall, M. C., Manning, D. W., Beal, M., Rahangdale, R., Dekker, R., McCarthy, R. J. (2016). Single-dose adductor canal block with local infiltrative analgesia compared with local infiltrate analgesia after total knee arthroplasty. Regional Anesthesia and Pain Medicine, 41(6), 678684. doi:10.1097/AAP.0000000000000494. New York School of Regional Anesthesia (NYSORA) (2019). Lower extremity blocks. Retrieved from https://www.nysora.com/techniques/lower-extremity/ Oseka, L. & Pecka, S. (2018, February). Anesthetic management in early recovery after surgery protocols for total knee and total hip arthroplasty [PDF file]. American Association of Nurse Anesthetists, 86(1), 32-39. ANALGESIC EFFICACY FOR TKA 30 Seo, J. H., Seo, S. S., Kim, D. H., Park, B. Y., Park, C. H., & Kim, O. G. (2017). Does combination therapy of popliteal sciatic nerve block and adductor canal block effectively control postoperative pain after total knee arthroplasty? Knee Surgery & Related Research, 29(4), 276-281. doi:10.5792/ksrr.17.023 Terkawi, A. S., Mavridis, D., Sessler, D. I., Nunemaker, M. S. , Doais, K. S. , Terkawi, R. S., Nemergut, E. C. (2017). Pain management modalities after total knee arthroplasty: A network meta-analysis of 170 randomized controlled trials. Anesthesiology, 126(5), 923 937. doi:10.1097/ALN.0000000000001607. Wiesmann, T., Piechowiak, K., Duderstadt, S., Haupt, D., Schmitt, J., Eschbach, D., Steinfeldt, T. (2016, January 11). Continuous adductor canal block versus continuous femoral nerve block after total knee arthroplasty for mobilization capability and pain treatment: A randomized and blinded clinical trial. Archives of Orthopaedic Trauma Surgery, 136, 397406. https://doi-org.forward.marian.edu/10.1007/s00402-015-2403-7 Zhang, Z., Wang, Y., & Liu, Y. (2019). Effectiveness of continuous adductor canal block versus continuous femoral nerve block in patients with total knee arthroplasty: A PRISMA guided systematic review and meta-analysis. Medicine, 98(48), e18056. https://doi.org/10.1097/MD.0000000000018056 Zorrilla-Vaca, A., & Li, J. (2018). The role of sciatic nerve block to complement femoral nerve block in total knee arthroplasty: A meta-analysis of randomized controlled trials. Journal of Anesthesia 32, 341350. https://doi-org.forward.marian.edu/10.1007/s00540-0182480-1 ANALGESIC EFFICACY FOR TKA 31 Appendix A ANALGESIC EFFICACY FOR TKA 32 Appendix B ANALGESIC EFFICACY FOR TKA 33 Appendix C Institutional Review Board DATE: 02-24-2020 TO: Tierra Penick FROM: Institutional Review Board RE: IRB #B20.153 TITLE: Peripheral Block Efficacy for Total Knee Arthroplasty SUBMISSION TYPE: New Project ACTION: Determination of Exempt Status DECISION DATE: 02-24-2020 The Institutional Review Board at Marian University has reviewed your protocol and has determined the procedures proposed are appropriate for exemption under the federal regulations. As such, there will be no further review of your protocol and you are cleared to proceed with your project. The protocol will remain on file with the Marian University IRB as a matter of record. Please be mindful of the importance of reporting only deidentified, HIPAA-compliant information about the patient in any exhibit or publication. Although researchers for exempt studies are not required to complete online CITI training for research involving human subjects, the IRB recommends that they do so, particularly as a learning exercise in the case of student researchers. Information on CITI training can be found on the IRBs website: http://www.marian.edu/academics/institutional-review-board. It is the responsibility of the PI (and, if applicable, the faculty supervisor) to inform the IRB if the procedures presented in this protocol are to be modified or if problems related to human research participants arise in connection with this project. Any procedural modifications must be evaluated by the IRB before being implemented, as some modifications may change the review status of this project. Please contact me if you are unsure whether your proposed modification requires review. Proposed modifications should be addressed in writing to the IRB. Please reference the above IRB protocol number in any communication to the IRB regarding this project. Bryan Larsen, Ph.D. Chair, Marian University Institutional Review Board ANALGESIC EFFICACY FOR TKA 34 Appendix D  ...

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Running head: PROVIDER ADHERENCE TO PRACTICE GUIDELINES Marian University Leighton School of Nursing Submitted in partial fulfillment for the degree of Doctor of Nursing Practice Provider Adherence to Practice Guidelines in Overcoming Hypothermia in the Postoperative Period Esmeralda Espinoza Chair: Bradley Stelflug ________________________ (Signature) Committee member: Dana Anders ___________________________ (Signature) Date of Submission: October 5th, 2020 1 PROVIDER ADHERENCE TO PRACTICE GUIDELINES Table of Contents Abstract .................................................................................................................................... 4 Introduction ............................................................................................................................. 6 Background .......................................................................................................................... 6 Problem Statement............................................................................................................... 7 Organizational Gap Analysis of Project Site ................................................................. 9 Review of the Literature ........................................................................................................ 10 Evidence Based Practice: Verification of Chosen Option12 Theoretical Framework/Evidence Based Practice Model ............................................... ....12 Goals & Objectives ............................................................................................................... 13 Project Design ....................................................................................................................... 13 Project Site and Population ............................................................................................. 13 Setting Facilitators and Barriers ..................................................................................... 14 Methods .................................................................................................................................. 14 Measurement Instrument(s) ........................................................................................... 14 Data Collection Procedure ............................................................................................. 14 Conclusion ............................................................................................................................. 16 References .............................................................................................................................. 18 Appendix ..................................................................................................................................... Appendix A-Surgical Care Improvement Project (SCIP) Performance Measures..21 Appendix B-Literature Matrix/SWOT Analysis ........................................................... 22 Appendix C-Conceptual Framework ............................................................................. 23 Appendix D-IRB Approval Letter .................................................................................. 24 2 PROVIDER ADHERENCE TO PRACTICE GUIDELINES Appendix E-Two Sample T-Test 28 Appendix F-Descriptive Statistics28 3 PROVIDER ADHERENCE TO PRACTICE GUIDELINES 4 Abstract Background: The implementation of a standardized warming protocol aims to prevent hypothermia in the postoperative period. Hypothermia in the postoperative period can result in inadvertent adverse outcomes for patients undergoing surgical procedures. In 2011, The Joint Commission (TJC) and Center for Medicare and Medicaid (CMS) issued the Surgical Care Improvement Project (SCIP) Inf-10 guidelines, known as the body temperature management guidelines, to decrease morbidity and mortality in surgical patients undergoing general anesthesia. These guidelines recommend using an active warming system, such as the 3M TM Bair Hugger TM System to warm patients intraoperatively. Review of Literature: Randomized control trials (RCTs) and meta-analysis were included as part of the literature review with the use of Cumulative Index to Nursing and Allied Health Literature (CINAHL), Cochrane Collaboration and Medline databases. Purpose: In this study, the research aims to evaluate the effectiveness of preventing hypothermia in a hospital that adopted the evidence-based body temperature management guidelines. Methods: The author retrospectively analyzed a total of 243 medical records, which included 158 paper medical records and 85 electronic medical records using the hospitals information management system. As part of the study, two groups, each consisting of 50 patient medical records meeting inclusion criteria, were analyzed. A total of 108 paper medical records and 85 electronic medical records were excluded from the study for not meeting criteria. Results: Patients were randomized and divided into two groups. Group A (n=50) consisting of patients who did not receive active warming in 2010, and Group B (n=50) consisting of patients who received the active warming intervention in 2020. Group B revealed a mean body temperature that was 0.1 Celsius higher than Group A. At the end of surgery, Group A PROVIDER ADHERENCE TO PRACTICE GUIDELINES 5 demonstrated nearly a 15% higher incidence of hypothermia when compared to Group B. Descriptive statistics were analyzed using an independent sample t-test, assuming unequal variance for the two groups. There was a statistically significant difference between the two groups (P < 0.028). The studys results support the use of forced air warming (FAW) in the intraoperative period as an effective way of preventing postoperative hypothermia in the Post Anesthesia Care Unit (PACU). Implications/Conclusion: This study assessed the effectiveness of SCIP Inf-10 guidelines in preventing hypothermia in patients undergoing surgical procedures. Prior to 2011, there were no recommendations issued by TJC or CMS to actively warm patients. Active warming, as characterized by the 3M TM Bair Hugger TM System warming system has been shown to be decrease morbidity and mortality, surgical site infections and other complications. Group B, which consisted of patients having undergone surgical procedures in 2020, were actively warmed and experienced a 0% incidence of hypothermia. Group A, which consisted of patients having undergone surgery before the release of SCIP Inf-10 guidelines, experienced nearly a 15% incidence of hypothermia. It is reasonable to conclude that the utilization of body temperature management guidelines is effective in preventing hypothermia in patients undergoing surgical procedures. Keywords: active warming, hypothermia, hypothermia and forced air-warming, hypothermia protocol, intraoperative warming and general anesthesia PROVIDER ADHERENCE TO PRACTICE GUIDELINES 6 Provider Adherence to Practice Guidelines in Overcoming Hypothermia in the Postoperative Period Introduction This project is submitted to the faculty of Marian University Leighton School of Nursing as partial fulfillment of degree requirements for the Doctorate of Nursing Practice (DNP), Nurse Anesthesia track. In accordance with the American Society of Anesthesiologists (ASA), all patients undergoing anesthesia must have five standard physiological parameters monitored including electrocardiogram, circulation, oxygenation, ventilation and body temperature (ASA, 2019). Decades of literature have shown detrimental consequences of unmanaged body temperature regulation well beyond the postoperative period. According to Butterworth, Mackey & Wasnick (2018) cardiac arrhythmias and ischemia, delayed drug metabolism, impaired would healing, increased risk for infection, increased postoperative protein catabolism and stress response, platelet dysfunction as well as increased peripheral vascular resistance, are factors associated with increase morbidity and mortality in hypothermic patients (p. 1214). Background Kurtz et al. (1996) published the first prospective study validating the use of active warming during colorectal surgeries in reducing the incidence of surgical site infections. Redistribution of heat from the body core to the peripheral compartments is attributed as the most common cause of hypothermia (Butterworth, Mackey & Wasnick, 2018). Compared to normothermic patients, those with hypothermia experience frequent complications. Therefore, body temperature regulation is crucial for patients undergoing general anesthesia and necessary for improved patient outcomes. Due to inconsistent compliance with infection prevention measures, TJC and CMS initiated The National Quality Forum Voluntary Consensus for PROVIDER ADHERENCE TO PRACTICE GUIDELINES 7 Hospital Care, also known as Surgical Care Improvement Project (SCIP) guidelines. The SCIP guidelines encompass a set of core measures with recommendations on the administration of antibiotics, monitoring blood glucose levels, perioperative hair removal, urinary catheter management, and perioperative temperature management for patients undergoing surgical procedures (See APPENDIX A). SCIP Inf-10 refers to the temperature body management guidelines for patients undergoing general anesthesia. The recommendations suggest that patients undergoing general anesthesia lasting longer or equal to one hour receive active warming to achieve a temperature of greater than or equal to 36 Celsius within 30 minutes before anesthesia stop time or within 15 minutes of anesthesia end time (SCIP Inf-10, 2020). The body temperature management guidelines were issued to standardize and increase compliance in 2011 (TJC, 2019). The guidelines set forth to create a unified documentation and track the quality of standards to provide quality metrics for standardized guidelines. Problem Statement How will patients having undergone general anesthesia lasting longer than 60 minutes (P), after the introduction and adherence to SCIP Inf-10 guidelines issued in 2011 (I), compare to patients having undergone general anesthesia prior to the institution of these guidelines (C), demonstrate a change in the incidence of hypothermia, identified as less than or equal to 36 Celsius (O) in the postoperative period (T)? Population Fifty medical records were retrospectively analyzed and compared from 2010 to 50 medical records from 2020 to assess the incidence of hypothermia between the two groups. The 2010 group did not receive the active warming intervention and the 2020 group did receive the intervention. Patients over the age of 18 years old having undergone general anesthesia lasting PROVIDER ADHERENCE TO PRACTICE GUIDELINES 8 longer than 60 minutes were included as part of the study. Neuraxial anesthesia, a type of regional anesthesia involving the injection of medication in tissues and nerve roots, were excluded from the study. Cardiac cases are unique, in which purposeful hypothermia is an important factor during surgery (Otto, 2015). Body temperature is therapeutically reduced in cardiac cases and therefore not applicable to the study (Otto, 2015). Cardiac cases are not performed at the research site. All medical records that did not utilize general anesthesia, met the duration of surgical time or did not meet the age requirement of 18 years or older were excluded from inclusion of the study. Setting The research site was a non-for-profit, county-owned hospital located in Northern Indiana. Logansport Memorial Hospital that offers services in womens health, family medicine, general surgery, orthopedics and wound care. The facility performs approximately 4,000 surgical cases a year (LMH, 2019). As previously mentioned, this site does not perform cardiac cases. Intervention Medical records were analyzed for comparison before and after the introduction of the SCIP Inf-10 guidelines at the site of study. The hospital was unable to determine the exact date in which the guidelines were implemented. It is acceptable to assume that the guidelines were not implemented until after its introduction in 2011. According to B. Alexander (personal communication, June 4, 2019), Certified Registered Nurse Anesthetist at LMH confirms the use of SCIP Inf-10 guidelines since 2013. Medical records for patients having surgery before the warming recommendations were analyzed from 2010 and compared to patients who received the warming recommendations in 2020. Comparison PROVIDER ADHERENCE TO PRACTICE GUIDELINES 9 This project aims to determine if the implementation of body management guidelines enhances patient warming and prevents hypothermia. A body temperature less than or equal to 36 Celsius is considered hypothermia. Surgical cases from 2010 and 2020 were randomized to assess body temperatures within 15 minutes of arrival to the PACU. Outcome The desired outcome is to determine if there is a decrease in the incidence of hypothermia for patients having undergone surgery after the implementation of SCIP Inf-10 guidelines. The long-term goal is for anesthesia providers to comply with the use of proper FAW in all patients undergoing general anesthesia, improving patient outcomes. Time After the intervention of FAW during the intraoperative period, the patients goal temperature will be greater than 36 Celsius within 15 minutes of arriving to the PACU after the end of general anesthesia. Organizational Gap Analysis of Project Site Anesthesia providers at the proposed site use FAW intraoperatively to prevent hypothermia in the postoperative period as recommended by the SCIP guidelines. However, the facility has not tracked the incidence of hypothermia since introducing the body management temperature guidelines. Although body temperature is monitored in the PACU and the use of active warming is implemented during surgery, the organization is unaware as to whether adherence has made a positive impact in warming patients. Maintaining body temperature higher than 36 Celsius is a desired quality indicator and necessary for improved patient outcomes. PROVIDER ADHERENCE TO PRACTICE GUIDELINES 10 Review of the Literature A search of available literature was completed with the use of Cumulative Index to Nursing and Allied Health Literature (CINAHL), Cochrane Collaboration and Medline databases. The search terms including active warming, hypothermia, hypothermia and forced air-warming, hypothermia protocol, intraoperative warming and general anesthesia were used. There was a time limit of five years imposed upon articles for review. Articles that did not meet the time requirement along with pediatric related articles were excluded as part of the literature review. RCTs and meta-analysis written in English were selected as pre-search inclusion requirements. Only studies including general anesthesia were selected and rated on evidence classification. All articles including the use of neuraxial anesthesia were excluded from the search. Of articles reviewed, five articles met pre-search inclusion and exclusion criteria to provide evidence for this project (See APPENDIX B). Literature Synthesis Scott and colleagues (2015) determined whether adherence to the SCIP Inf-10 measures of body temperature management is associated with improved outcomes and a reduced incidence of morbidity and mortality. Two groups were designated as the SCIP-compliant and SCIPnoncompliant. The SCIP noncompliant group demonstrated a greater incidence of complications including, congestive heart failure, valvular cardiac disease, peripheral vascular disease, hypertension, pulmonary disease, and renal insufficiency/failure (Scott et al., 2015, p. 118). The primary findings of the study demonstrate a reduced risk of mortality and decreased length of stay with compliance to the SCIP Inf-10 guidelines, which utilizes the use of FAW. This study demonstrates the importance of proper management of body temperature for patients undergoing general anesthesia. PROVIDER ADHERENCE TO PRACTICE GUIDELINES 11 Alderson and colleagues (2014) sought to compare the use forced air warming through a blanket to the use of reflective blankets or clothing to warm patients undergoing anesthesia intraoperatively. Alderson and colleagues concluded the use of FAW to demonstrate and maintain better control of temperature regulation by 0.5 to 1.0 Celsius when compared to other methods of warming. Alparslan et al. (2018) compared FAW systems in prevention of intraoperative hypothermia by comparing tympanic membrane temperatures of patients assigned to FAW with upper body blankets and FAW with underbody blankets. No difference was found between the two groups. Therefore, it was concluded that both methods of FAW possess similar efficacy. The implications for practice suggest that both lower and upper body warming methods are effective in preventing hypothermia in patients undergoing abdominal procedures. As the previous study demonstrated, FAW continues to prove to be an effective method of preventing hypothermia. John et al. (2016) conducted a single-blind randomized study to compare perioperative hypothermia in patients receiving resistive heating or FAW. Core temperatures were monitored from induction of anesthesia through admittance into the PACU. John and colleagues concluded that the use FAW is more effective than resistive heating in preventing postoperative hypothermia. At the end of surgery, the resistive heating group demonstrated nearly a 20% higher incidence of hypothermia when compared to the FAW group (John et al., 2016). These recommendations support the use of FAW to improve patient outcomes in the postoperative period and address the clinical problem of hypothermia seen in the PACU. Nieh & Su (2016) completed a meta-analysis of twenty nice articles to examine proper warming systems for the prevention of hypothermia through the use of meta-analysis procedures, proposed by Higgins. After careful systematic review with meta-analysis, the researchers PROVIDER ADHERENCE TO PRACTICE GUIDELINES 12 summarized that FAW was superior to passive insulation and circulating-water mattresses in preventing the incidence of hypothermia. There is strong evidence that supports the use of FAW as an effective way to prevent hypothermia in those undergoing general anesthesia when compared to other methods of warming. Therefore, it is recommended that FAW be utilized in the intraoperative period to decrease the clinical problem of hypothermia seen in the postoperative period. The proposed site has already implemented the use of FAW. The researcher of the study seeks to assess the effectiveness of this intervention by completion of a retrospective chart review. Evidence Based Practice: Verification of Chosen Option The hospital in which the study was conducted adopted an evidence-based practice guideline for body temperature management as part of a quality improvement initiative. Literature supports the use of FAW as the most effective method in preventing postoperative hypothermia. Theoretical Framework The John Hopkins Nursing Evidenced-Based Practice (JHNEBP) model is a performance improvement theoretical framework that will guide the research project (See APPENDIX C). The JHNEBP model can be used by a single individual or group of researchers to guide a project work. The goal of the model is to discover the latest research findings and quickly incorporate it into patient care using three phases including, identification of the practice question, collection of the evidence and translation of the evidence for use in practice known as the PET process (Schaffer, Sandau & Diedrick, 2013). This project work will assess the intervention of FAW in patients undergoing general anesthesia. PROVIDER ADHERENCE TO PRACTICE GUIDELINES 13 Goals, Objectives and Expected Outcomes The goal of this DNP project is to evaluate whether adherence to the SCIP Inf-10 guidelines reduces the incidence of hypothermia in the postoperative period. The use of FAW is supported by evidence-based research in preventing postoperative hypothermia. The short term objective of the project is to evaluate the incidence of postoperative hypothermia before and after the introduction of SCIP Inf-10 guidelines for body temperature management and provide the facility with the findings so that the organization may assess its performance. It is expected that the incidence of hypothermia will be decreased after the introduction of the guidelines. If there is variation in data and no improvement in the incidence of hypothermia, further education will be necessary to improve this quality indicator. Project Design The DNP project assesses the effectiveness of a performance improvement guideline that has already been implemented at the site. The design of the project is in the form of a retrospective chart review from a single institution to compare the incidence of hypothermia before and after the adoption of the SCIP Inf-10 guidelines. The data for this project will include the collection of qualitative data, specifically the temperature in Celsius for patients having undergone general anesthesia for longer than 60 minutes recovering in the PACU. Project Site and Population Logansport is a rural city located in Northern Indiana with a population of nearly 18,000 residents (U.S.Census, 2020). This study took place at Logansport Memorial Hospital (LMH), a non-for-profit, county-owned regional center that serves the population of Cass County and surrounding north-central Indiana communities (LMH, 2019). LMH provides a full range of surgical, obstetrical, interventional diagnosis, and pain management services. The facility houses PROVIDER ADHERENCE TO PRACTICE GUIDELINES 14 83 in-patient beds and performs approximately 4,000 surgeries a year (LMH, 2019). In 2018, the hospital delivered nearly 500 babies and received 18,000 emergency room visits (LMH, 2019). Setting Facilitators and Barriers The cost effectiveness of evaluating the performance improvement guideline was a facilitator in carrying out the DNP project. No funds were allocated for the completion of the project. However, the disadvantage of both electronic and paper medical records included the chance of human error in documentation. The possibility of misinterpreting and coding information extracted from paper charts were barriers to the study. Incomplete documentation, including missing charts, information that is unrecorded were barriers that may have skewed findings. For the study, it was assumed that no warming device was used if not documented, this was a limitation of the study as providers may have utilized the warming device but omitted documentation. Record keeping is an essential and legal requirement for healthcare professionals. It is possible that patients may have received the active warming intervention but was not reflected accurately in the documentation. Methods After obtaining approval from the Institutional Review Board (IRB) at Marian University (MU), access to anesthesia medical records for patients having undergone surgical procedures during the months of January, February and March of 2010 and 2020 was granted (See APPENDIX D). This institution did not require further approval or authorization to review patients medical charts at the facility. The anesthesia records were analyzed to compare the two groups, the researcher collected the temperature of patients upon arriving to the PACU. The temperatures documented were stored in Excel without any identifiable patient information. PROVIDER ADHERENCE TO PRACTICE GUIDELINES 15 The study is easily replicated by comparing and analyzing temperatures documented in the PACU before and after the adoption of SCIP Inf-10 guidelines at any institution. The minimum, maximum and mean temperatures were calculated for both groups. Further analysis using independent sample t-test, assuming unequal variance for the two groups was completed. Measurement Instrument The effectiveness of the guideline was evaluated using the JHNEBP model. This model provides a three-step process, referred to as the PET (practice question, evidence and translation) process. The researcher evaluated the outcome of this performance improvement guideline by comparing the temperatures of two groups, one group before the introduction of the SCIP-Inf-10 guidelines and second group after the introduction of the guidelines. Temperatures in Celsius were recorded during the postoperative period for comparison using descriptive statistics. The outcome measured the incidence of hypothermia, defined as less than 36 Celsius in the postoperative period. Data Collection The data collector retrospectively analyzed a total of 243 medical records, which included 158 paper medical records and 85 electronic medical records using the hospitals information management system. Charts were chosen randomly and reviewed once by one single data collector. Every patient chart that qualified for inclusion was assigned a numerical identifier for the purpose of data collection. No identifiable patient data was collected. The temperatures recorded in PACU were stored in a Microsoft Excel spreadsheet. Data Analysis Statistical analysis was performed utilizing IBM SPSS Software Version 2020. Descriptive statistics were analyzed using an independent sample t-test, assuming unequal PROVIDER ADHERENCE TO PRACTICE GUIDELINES 16 variance for the two groups (See APPENDIX E). Statistical significance for the test was set at 0.05. There was a statistically significant difference between the two groups (P < 0.028). The minimum, maximum and mean temperatures in Celsius was calculated for each group (See APPENDIX F). Group A (2010 group) mean body temperature was (36.4; n = 50), compared to Group B (2020 group) mean body temperature of (36.5; n = 50) (See APPENDIX F). At the end of surgery, Group A demonstrated nearly a 15% higher incidence of hypothermia when compared to Group B (0% incidence). The final analysis observed that the use of the body temperature management guidelines has not only reduced the incidence of hypothermia in the site of study. Conclusion The Joint Commission (2019) offers guidelines regarding body temperature management to improve patient outcomes. The project intervention analyzed the effectiveness of a quality improvement initiative specifically designed to reduce the incidence of hypothermia in the postoperative period for patients undergoing surgical procedures. The JHNEBP model was a performance improvement theoretical framework that served to guide the research project. This study showed the incidence of hypothermia was reduced by utilizing forced air warming devices. Data was statistically significant, showing no incidence of postoperative with hypothermia with compliance to body temperature management guidelines. As part of quality assurance measures, it is essential for anesthesia providers to comply with the use of active warming systems. Small sample size limits ability to generalize findings outside the setting in which the study was conducted. Future studies in large healthcare systems and multi-center regions would be required to generalize these finding to other practice environments. The results PROVIDER ADHERENCE TO PRACTICE GUIDELINES indicate that the use of intraoperative FAW is effective in preventing hypothermia in the postoperative period. 17 PROVIDER ADHERENCE TO PRACTICE GUIDELINES 18 References Alderson, P., Campbell, G., Smith, A., Warttig, S., Nicholson, A., & Lewis, S. (2014). Thermal insulation for preventing inadvertent perioperative hypothermia. The Cochrane Database of Systematic Reviews, 6(6), 009908. doi:10.1002/14651858.CD009908.pub2 Alparslan, V., Kus, A., Hosten, T., Ertargin, M., Ozdamar, D., Toker, K., & Solak, M. (2018). Comparison of forced-air warming systems in prevention of intraoperative hypothermia. Journal of Clinical Monitoring and Computing, 32(2), 343-349. doi:10.1007/s10877-0170017-z American Psychological Association. (2010). Publication manual of the American Psychological Association (6th ed.). Washington, DC. American Society of Anesthesiologists (ASA). 2019. Standards for basic anesthetic monitoring. Retrieved from https://www.asahq.org/standards-and-guidelines/standards-for-basicanesthetic-monitoring. Body temperature guidelines adhered (B. Alexander, personal communication, June 1, 2019). Bonnel, W., & Smith, K. (2018). Proposal writing for clinical nursing and dnp projects (Second ed.). New York, NY: Springer Publishing Company. Butterworth, J. F., Mackey, D.C., Wasnick, J.D. (2018). Morgan & mikhail's clinical anesthesiology [Sixth edition.]. New York: McGraw-Hill Education. (2018). Retrieved from_http://accessanesthesiology.mhmedical.com.forward.marian.edu/content.aspx?book id=24 44§ionid=193557844 Dearholt, S., Dang, D., (2018). Johns hopkins nursing evidence-based practice: Model and guidelines (3rd ed.). Sigma Theta Tau International Publishing Company. John, M., Crook, D., Dasari, K., Eljelani, F., El-Haboby, A., & Harper, C. (2016). Comparison of PROVIDER ADHERENCE TO PRACTICE GUIDELINES 19 resistive heating and forced-air warming to prevent inadvertent perioperative hypothermia. British Journal of Anaesthesia, 116(2), 249-54. doi:10.1093/bja/aev412 Kurz, A., Sessler, D., & Lenhardt, R. (1996). Perioperative normothermia to reduce the incidence of surgical-wound infection and shorten hospitalization. study of wound infection and temperature group. The New England Journal of Medicine, 334(19), 1209-15. Logansport Memorial Hospital (LMH). (2019). Why LMH? Retrieved from https://www.logansportmemorial.org/Why-LMH/. Moran, K. J., Burson, R., & Conrad, D. (2019). The doctor of nursing practice project. Retrieved from https://ebookcentral.proquest.com Nieh, H., & Su, S. (2016). Meta-analysis: Effectiveness of forced-air warming for prevention of perioperative hypothermia in surgical patients. Journal of Advanc ed Nursing, 72(10), 2294-314. doi:10.1111/jan.13010 Otto, K. A. (2015). Therapeutic hypothermia applicable to cardiac surgery. Anaesthesia and Analgesia, 42(6), 559569. https://doi.org/10.1111/vaa.12299 Schaffer, M., Sandau, K., & Diedrick, L. (2013). Evidence-based practice models for organizational change: Overview and practical applications. Journal of Advanced Nursing, 69(5), 1197-1209. doi:10.1111/j.1365-2648.2012.06122.x SCIP-Inf-10: Surgery patients with perioperative temperature management. (2020). Retrieved from https://www.ihconline.org/media/cms/2z_SCIP-Inf10.pdf Scott, A., Stonemetz, J., Wasey, J., Johnson, D., Rivers, R., Koch, C., & Frank, S. (2015). Compliance with surgical care improvement project for body temperature management (scip inf-10) is associated with improved clinical outcomes. Anesthesiology, 123(1), 11625. doi:10.1097/ALN.0000000000000681 PROVIDER ADHERENCE TO PRACTICE GUIDELINES 20 The Joint Commission [TJC]. Surgical care improvement project core measures set. (2019). Retrieved from https://www.jointcommission.org/-/media/deprecatedunorganized/imported-assets/tjc/system-folders/assetmanager/surgical-careimprovement-projectpdf.pdf?db=web&hash=DF9370CE26B560780613F80261697A03 United States Census Bureau [U.S. Census]. (2019). QuickFacts. Retrieved from https://www.census.gov/quickfacts/logansportcityindiana PROVIDER ADHERENCE TO PRACTICE GUIDELINES Appendix A Adapted from The Joint Commission [TJC]. Surgical care improvement project core measures set. (2019). 21 PROVIDER ADHERENCE TO PRACTICE GUIDELINES 22 Appendix B Adapted from Dearholt, S., Dang, D., (2018). Johns hopkins nursing evidence-based practice: Model and guidelines (3rd ed.). Running head: PROVIDER ADHERENCE TO PRACTICE GUIDELINES 23 Appendix C Citation Variables of Interest (Keywords) Literature Type & Research Tools Research Design & Sample Size Alderson, P., Campbell, G., Smith, A., Warttig, S., Nicholson, A., & Lewis, S. (2014). Thermal insulation for preventing inadvertent perioperative hypothermia. The Cochrane Database of Systematic Reviews, 6(6), 009908. doi:10.1002/146518 58.CD009908.pub2 Warming Level of techniques, evidence=1 warming systems*, thermal insulant*, body temperature regulation, hypothermia , active* warming, thermal or temperature Systemati c Review including RCTs and quasirandomize d controlled N=22 trials Database: COCHRANE Theo retical Foun datio n None # References & SWOT Critique Key Findings #=59 S=systematic review W= Low evidence quality data; some of the references were excluded from the study but still listed O= Further research needed to identify significance in 1 degree Celsius compared to 0.5 degrees Celsius T=Concerned about the potential for skewed results from operating theatre staff changing their behavior when they knew ways of keeping the patient warm had changed 1. There is no clear benefit of extra thermal insulation with compared to standard care 2. Forced air warming (FAW) seems to maintain better than extra insulation, by 0.5 degrees Celsius and 1 degree Celsius 24 PROVIDER ADHERENCE TO PRACTICE GUIDELINES Alparslan, V., Kus, A., Hosten, T., Ertargin, M., Ozdamar, D., Toker, K., & Solak, M. (2018). Comparison of forced-air warming systems in prevention of intraoperative hypothermia. Journa l of Clinical Monitoring and Computing, 32(2), 343-349. doi:10.1007/s10877017-0017-z Intraoperati Level of ve Evidence= hypothermia 1 , forced-air warming systems, underbody and upper body blankets Prospectiv e and randomize d control study N=92 patients None #=25 S= Number of references W=Used two different brands for lower and upper body blankets O= Future studies to compare same manufacture in FAW systems 1. There was no statistical difference between the two groups when intraoperative hemodynamic data was collected in recovery 2. Results showed that use upper and lower body forced-air warming was effective in preventing intraoperative hypothermia in patients undergoing lower abdominal surgery in supine position Equipment; Level of hypothermia Evidence= 1 ; temperature; warming devices RCT singleblinded study None #106 patients undergoing nonemergency surgery were recruited S= Large sample size W= Small pilot study prior to large scale feasibility by recruiting 40 patients undergoing elective surgery O= More research and replication of study plausible T= Hypothermia at end of surgery in both warming groups was common (FAW=36% and resistive 54%) 1. Forced air-warming is more beneficial and effective than resistive heating in preventing postoperative hypothermia 2. Significantly higher rate of hypothermia at end of surgery in resistive heating group compared with forced air-warming (P=0.017) Database: COCHRANE John, M., Crook, D., Dasari K., Elkelani, F., El-Haboby, A., & Harper, C. (2016). Comparison of resistive heating and forced-air warming to prevent inadvertent perioperative hypothermia. British Journal of Anaesthesia, 116(2), 249-54. doi: 10.1093/bja/aev412 25 PROVIDER ADHERENCE TO PRACTICE GUIDELINES Nieh, H., & Su, S. (2016). Metaanalysis: Effectiveness of forced-air warming for prevention of perioperative hypothermia in surgical patients. Journal of Advanced Nursing, 72(10), 2294-314. doi:10.1111/jan.130 10 body Level of temperature; Evidence= forced-air 1 warming; hypothermia ; metaanalysis; nursing; perioperativ e care; systematic review; thermal comfort Systemati c Review incorporat ing metaanalysis N = 29 trials (1875 patients) None #= 55 S= Larger sample size; meta-analysis W=Nonblinded meta-analysis; due to the heterogeneity among trials, the comparison of insulation/warm ing performance between FAW and other warming systems was affected by intervention timing, type of surgery, anesthesia method and sample size O= developed T= Conduct a large-scale and highly randomized control trial to further investigation 1. Forced-air warming was more effective than passive insulation and circulating-water mattresses; 2. There was no statistically significant difference among forcedair warming, resistive heating blankets, radiant warming systems and circulating-water garments 3. Thermal comfort provided by forced-air warming was superior to that of passive insulation, resistive heating blankets and radiant warming systems, but inferior to that of circulating-water mattresses 26 PROVIDER ADHERENCE TO PRACTICE GUIDELINES Scott, A., Stonemetz, J., Wasey, J., Johnson, D., Rivers, R., Koch, C., & Frank, S. (2015). Compliance with surgical care improvement project for body temperature management (scip inf-10) is associated with improved clinical outcomes. Anesthesi ology,123(1), 11625. doi:10.1097/ALN.00 00000000000681 The authors Level of retrospectiv evidence=2 ely analyzed the electronic medical record data from 45,304 inpatients at a single institution to assess whether compliance with SCIP Inf-10 (body temperature managemen t Retrospect None ive study N=45,304 electronic medical records #= 36 S= Large sample size; extensive literature review described with comparison to findings of study W=Participants at low risk for developing cardiovascular complications O= Addressed critiques of SCIP management guidelines T= Anesthesia providers adherence to guidelines; IRB approval from John Hopkins medical center 1. In the SCIP-compliant group, patients demonstrated a decreased incidence of hospital acquired-infections and cardiovascular effects. 2. In the non-SCIP compliant group, patients there was a greater incidence of congestive heart failure, valvular cardiac disease, peripheral vascular disease, hypertension, pulmonary disease, and renal insufficiency/failure 3. The use of active warming is an inexpensive perioperative intervention that can be used to improve patient outcomes and therefore the authors strongly encourage this method to decrease hypothermia in the postoperative period Adapted from Davidson, J. U. (2003). Example knowledgebase development template. In Rankin, S. H., Dumas, M. A., & Reavis, C. (Eds.), Grantsmanship: Developing a program of research, Appendix B (pp. 77-78). Washington, DC: National Organization of Nurse Practitioner Faculties. PROVIDER ADHERENCE TO PRACTICE GUIDELINES Appendix D 27 PROVIDER ADHERENCE TO PRACTICE GUIDELINES Appendix E Appendix F 28  ...

... Diet of the Myotis sodalis (Indiana Bat) at an Urban/ Rural Interface Author(s): Nicole M. Tuttle, David P. Benson, Dale W. Sparks Source: Northeastern Naturalist, 13(3):435-442. Published By: Eagle Hill Institute https://doi.org/10.1656/1092-6194(2006)13[435:DOTMSI]2.0.CO;2 URL: http://www.bioone.org/doi/ full/10.1656/1092-6194%282006%2913%5B435%3ADOTMSI %5D2.0.CO%3B2 BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOnes Terms of Use, available at www.bioone.org/page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. 2006 NORTHEASTERN NATURALIST 13(3):435442 Diet of the Myotis sodalis (Indiana Bat) at an Urban/Rural Interface Nicole M. Tuttle1, David P. Benson1, and Dale W. Sparks2,* Abstract - We conducted a study of the diet of the federally endangered Indiana bat (Myotis sodalis) at an urban/rural interface near Indianapolis International Airport in summer 2004. We used two 1-m2 quadrats covered with window screening to collect guano under a known roost tree. We then examined 20 fecal pellets/week until the bats abandoned the roost (i.e., 13 weeks). The most common orders of insects eaten were: Lepidoptera (35.3% volume, 84.6% frequency), Diptera (27.9%, 73.2%), Coleoptera (16.9%, 62.9%), and Hymenoptera (10.9%, 45.9%). Components of the diet at the ordinal level varied significantly over time. Despite the developed nature of the site, the diet consisted of the same components reported in earlier studies. Introduction A colony of federally-endangered Myotis sodalis Miller and Allen (Indiana bats) near Indianapolis International Airport (the airport, hereafter) has been studied since 1994 (Whitaker et al. 2004). The area surrounding the airport is rapidly developing, and thus offers a unique opportunity to examine responses of bats to urbanization (Duchamp et al. 2004, Sparks et al. 1998, Whitaker et al. 2004). Radio-telemetry studies of Indiana bats foraging near the airport indicate that highly developed areas such as suburbs and strip malls surrounding this site are seldom used by Indiana bats (Sparks et al. 2005a). Instead, this species appears to restrict foraging to less urbanized habitat consisting of a patchwork of forest fragments separated by agricultural fields and grasslands. These areas are associated with the East Fork of White Lick Creek, and are being managed by the Indianapolis Airport Authority in an effort to preserve this colony. This effort is hampered, however, by a lack of knowledge about how bats respond to urbanization (Sparks et al. 1998, 2005a). We are unsure why Indiana bats avoid highly developed areas, but hypothesize these areas provide little suitable food. Objectives of this study were to 1) determine the diet of Indiana bats at the Indianapolis Airport, and 2) compare these results to the diet of the Indiana bat in more rural landscapes, especially nearby portions of rural Indiana (Sparks et al. 2005b). Methods and Materials This study was conducted by obtaining guano from two 1-m2 quadrats covered with window mesh beneath a single roost tree during the 13-week 1 Marian College, 3200 Cold Spring Road, Indianapolis, IN 46222. 2Department of Ecology and Organismal Biology, Indiana State University, Terre Haute, IN 47809. * Corresponding author - dsparks@isugw.indstate.edu. 436 Northeastern Naturalist Vol. 13, No. 3 period beginning on 24 May and ending on 20 August 2004 when the roost was abandoned. We chose to exclusively use guano from this tree for three reasons. First, this tree was used by five radio-tagged Indiana bats during the summers of 2002 and 2003, making it the most consistently used roost tree at the study area. Second, the best data available indicates this tree is only used by Indiana bats, unlike several other roosts we could have chosen. None of the seven other species of bats that have been radio-tracked at the airport have roosted in this tree (Duchamp et al 2004, Everson 2005, Sparks 2003, Whitaker et al. 2004). We specifically avoided obtaining guano from artificial roosts that are used by four species including Indiana bats (Ritzi et al. 2005, Whitaker et al. 2006). Third, little under-story vegetation was present between roosts in this tree used by Indiana bats and the ground, which both maximized the amount of guano collected, and increased the likelihood the guano we collected was fresh. Fecal analysis is known to provide a reasonable estimate of diet in insectivorous bats and does not require sacrificing animals (Whitaker 1988). As such, it is the only practical technique for determining diet of this endangered species. Fecal pellets were stored in plastic bags and frozen until examination. We examined a randomly-selected subsample of 20 pellets each week using established techniques (Murray and Kurta 2002, Whitaker 1988), except for 2 occasions where we analyzed more pellets (22 pellets for the week of 30 May and 38 pellets for the week of 11 June). Each fecal pellet was placed in a Petri dish, covered in 75% ethanol, dissected, and examined under a 1030-power zoom dissecting microscope. Insect remains were identified to the lowest possible taxonomic level (usually family). Each insect type was visually estimated as the percent of the total pellet volume. We present results as both percent volume (average percent of a prey item making up each pellet) and percent frequency (the percent of pellets that contained a food item). Following Whitaker (1988), we used a series of univariate ANOVAs followed by Student-Neuman-Keuls multiple range tests to compare arcsine transformed percent volumes for Lepidoptera, Diptera, Coleoptera, and Hymenoptera across weeks. All statistical analyses were conducted in SPSS 10.0 with an overall rejection level of = 0.05. Results We documented the presence of 9 orders and 26 families of arthropods in the diet (Table 1). Included were 5 pest species: Diabrotica undecimpunctata Barber (spotted cucumber beetle), Cyrtepistomus castaneus (Roelofs) (Asiatic oak weevil), Acrosternum hilare (Say) (green stink bug), mosquitoes (Family: Culicidae), and Mayetolia destructor (Say) (Hessian fly). The overall diet was dominated by moths (37.3% by volume, 84.6% by frequency), flies (27.9%, 73.2%), beetles (16.9%, 62.9%), and wasps/ants (10.9%, 45.9%), and was similar to diets (Fig. 1) reported in earlier studies conducted in more rural areas of Indiana (Belwood 1979, 2006 N.M. Tuttle, D.P. Benson, and D.W. Sparks 437 Table 1. Food eaten by Indiana bat (Myotis sodalis) at a roost in Hendricks County, IN, during summer 2004. Totals for higher-level taxa are found in parentheses. Food eaten Arthopoda (total) Insecta (total) Lepidoptera (total) Diptera (total) Unknown Diptera Nematocera (total) Unknown Nematocera Chironomidae Culicidae Tipulidae Sciaridae Cedciomyiidae (total) Mayetiola destructor Brachycera (total) Muscoidea Assilidae Coleoptera (total) Unknown Coleoptera Cucurlionidae(total) Unknown Cucurlionidae Cyrtepistomus castaneus Chrysomelidae (total) Unknown Chrysomelidae Diabrotica undecimpunctata Chrysochus auratus (Fabricius) Carabidae Scarabaeidae (total) Hymenoptera (total) Unknown Hymenoptera Chalcidoidea Ichneumonidae Braconidae Formicidae Homoptera (total) Cicadellidae Delphacidae Flatidae Trichoptera (total) Hemiptera (total) Unknown Hemiptera Lygaeidae Pentatomidae (total) Acrosternum hilare Neuroptera (total) Chrysopidae Hemerobiidae Unidentified insect (total) Arachnida (total) Araneae Acari % by volume % frequency 100.0 (99.9) (37.3) (27.9) 22.7 (4.4) 0.6 1.8 1.0 1.0 0.1 (< 0.1) < 0.1 (0.9) 0.5 0.4 (16.9) 1.6 (10.6) 2.9 7.7 (1.3) 0.2 1.1 0.5 2.3 0.8 (10.9) 4.5 0.4 5.0 0.1 0.9 (2.0) 1.8 0.2 < 0.1 (1.6) (0.9) 0.3 0.5 (0.1) 0.1 (0.5) < 0.1 0.5 (1.6) (0.1) 0.1 < 0.1 100.0 (100.0) (84.6) (73.2) 57.9 (17.1) 2.9 5.6 4.3 5.7 1.1 (0.4) 0.4 (7.5) 6.8 0.7 (62.9) 16.4 (34.3) 11.1 23.2 (6.8) 2.9 3.9 12.5 5.0 1.4 (45.9) 21.1 0.7 20.7 1.1 4.3 (20.1) 17.9 1.8 0.4 (8.2) (11.0) 3.2 6.4 (1.4) 1.4 (3.1) 0.4 2.9 (5.4) (2.5) 2.1 0.4 Northeastern Naturalist Figure 1. Comparison of diet across the range of Indiana bat (Myotis sodalis) for studies in which diets for more than 20 Indiana bats were included in the analyses. States are abbreviated by postal codes (MI = Michigan, IN = Indiana, MO = Missouri, and KY = Kentucky). 438 Vol. 13, No. 3 2006 N.M. Tuttle, D.P. Benson, and D.W. Sparks 439 Brack 1983, Lee 1993), Kentucky (Kiser and Elliot 1996), and Michigan (Murray and Kurta 2002). Diet at the Indianapolis Airport was least similar to those reported from bats captured just prior to hibernation in Missouri (Brack and LaVal 1985) and at the northern extreme of the range in Michigan (Kurta and Whitaker 1998). We found significant variation over the summer in proportions of lepidopterans (F12,267 = 4.91, P < 0.001), dipterans (F12,267 = 4.90, P < 0.001), coleopterans (F12,267 = 4.91, P < 0.001), and hymenopterans (F12,267 = 4.91, P < 0.001) in the diet (Fig. 2). Lepidopteran consumption peaked in June at 66.5% by volume, but were less than 20% of the diet during 3 other weeks. Diptera were almost absent from the diet in late July and then became increasingly important in August. Coleopterans, particularly Asiatic oak weevils, were the primary food at the beginning of August. Hymenopterans were typically a small proportion of the diet except during the week of 29 June, when they were only slightly less common than dipterans. Discussion Previous studies have recorded significant variation in the diet of Indiana bats across the range of the species, within single colonies through one or more seasons, between reproductive classes, and even between different parts of the night (see review by Murray and Kurta 2002). Similar to previous studies, the diet near Indianapolis shifts several times throughout the maternity season. These fluctuations in diet probably reflect differences in insect availability and changes in diet selection during the course of the maternity season. Barclay and Brigham (1994) suggested members of the genus Myotis Figure 2. Variation in percent-volume of food items consumed by Indiana bats at the Indianapolis International Airport during 2004. Lep = Lepidoptera, Col = Coleoptera, Dip = Diptera, and Hym = Hymenoptera. 440 Northeastern Naturalist Vol. 13, No. 3 have difficulty in distinguishing between insect types because the bats have rapid flight speeds and short echolocation ranges. If so, Myotis spp. should have difficulty in selecting particular prey types. Conversely, several studies (Belwood 1979, Brack and LaVal 1985, Lee 1993) suggest Indiana bats consume some insects disproportionate to their abundance. These observations suggest bats are capable of selecting prey items, but as noted by Whitaker (1995), selection of prey occurs at multiple scales and bats may select prey items in part by modifying where they forage. Despite these differences, it is also important to note that diet of Indiana bats is composed of the same components throughout the range of the bat (Sparks et al. 2005b). In the current as well as all previous studies, diets were dominated by a combination of moths, beetles, flies, caddisflies, and hymenopterans (Belwood 1979; Brack 1983; Brack and LaVal 1985; Kiser and Elliot 1996; Kurta and Whitaker 1998; Lee 1993; Whitaker 1972, 2004). When more than 20 Indiana bats were examined, > 50% of the diet consists of moths, true flies, and beetles (Fig. 1; Sparks et al 2005b), except at the northern extreme of the range (Kurta and Whitaker 1998) where caddisflies are particularly important. Thus, the diet of Indiana bat consists primarily of five orders of insects. Previous authors rarely identified food items below the ordinal scale, but when such identification are made Asiatic oak weevils (Brack 1983, Brack and Whitaker 2004, Kiser and Elliot 1996), spotted cucumber beetles (Brack and Whitaker 2004, Kiser and Elliot 1996), ichneumon wasps (Kiser and Elliot 1996, Kurta and Whitaker 1998, Whitaker 1972), scarab beetles (Kurta and Whitaker 1998, Whitaker 1972), and ants (Kiser and Elliot 1996, Kurta and Whitaker 1998, Whitaker 1972) are all frequently reported. Five of the insects we recorded in the diet of Indiana bats at the airport are pests. The most prevalent of these was the Asiatic oak weevil (7.7% by volume, 23.2% by frequency), which was often consumed in late July. Throughout our study, bats irregularly consumed mosquitoes (1.0% by volume, 4.3% by frequency), spotted cucumber beetles (1.1% by volume, 3.9% by frequency), and green stink bugs (0.1% by volume, 1.4% by frequency). We were surprised to find a wing of a Hessian fly (a pest of wheat) in one sample because wheat is uncommon in the area. Most authors do not identify dipterans below ordinal level, so this agricultural pest may have been overlooked in previous studies. We suspect that in future studies, many other pest species will be detected in the diet of Indiana bats. Indiana bats near Indianapolis Airport ate many of the same foods as do bats in less-developed areas and forage in similar habitats (Sparks et al. 2005b). At present, properties conserved by Indianapolis Airport are providing foods similar to those consumed in more rural areas, but telemetry studies at Indianapolis Airport (Sparks et al. 2005a,b) indicate Indiana bats avoid highly developed lands. Thus, we suspect conservation efforts aimed at these bats must focus on maintaining appropriate foraging habitat. Indiana bats at this site frequently forage on privately-owned parcels 2006 N.M. Tuttle, D.P. Benson, and D.W. Sparks 441 surrounding the conservation lands, and many of these are being developed for commercial or residential use. Thus, we remain concerned that development surrounding this site will eventually negatively impact these bats by removing the remaining viable foraging habitat. As such, examining the types and quantities of insects available within these areas remains an important future project. Acknowledgments We thank the Indianapolis Airport Authority and the Department of Ecology and Organismal Biology at Indiana State University for funding and logistical support. B.L. Everson, B.J. Foster, J.S. Helms, H.L. McDonald, all 3 C.M. Ritzis, J.J. Sheets, and J.K.F. Sparks assisted in radio-telemetry and collection of fecal samples. J.O. Whitaker, Jr. provided assistance throughout the study. Comments by T.C. Carter and 2 anonymous reviewers improved the manuscript. This study was conducted under permits issued by the Indiana State University Animal Care and Use Committee, Indiana Department of Natural Resources, and US Fish and Wildlife Service. Literature Cited Barclay, R.M.R., and R.M. Brigham. 1994. Constraints on optimal foraging: A field test of prey discrimination by echolocating insectivorous bats. Animal Behaviour 48:10131021. Belwood, J.J. 1979. Feeding ecology of an Indiana bat community with emphasis on the endangered Indiana bat, Myotis sodalis. M.Sc. Thesis. University of Florida, Gainesville, FL. Brack, Jr., V. 1983. The non-hibernating ecology of bats in Indiana with emphasis on the endangered Indiana bat, Myotis sodalis. Ph.D. Dissertation, Purdue University, West Lafayette, IN. Brack, Jr., V., and R.K. LaVal. 1985. Food habits of the Indiana bat in Missouri Journal of Mammalogy 66:308315. Brack, Jr., V., and J.O. Whitaker, Jr. 2004. Bats of the Naval Surface Warfare Center at Crane, Indiana. Proceedings of the Indiana Academy of Science 113:6675. Duchamp, J.E., D.W. Sparks, and J.O. Whitaker, Jr. 2004. Foraging-habitat selection by bats at an urban-rural interface: Comparison between a successful and unsuccessful species. Canadian Journal of Zoology 82:11571164. Everson, B.L. 2005. Foraging behavior and seasonal movements of the eastern red bat (Lasiurus borealis) in central Indiana. M.Sc. Thesis. Indiana State University, Terre Haute, IN. Kiser, J.D., and C.L. Elliot. 1996. Foraging habitat, food habits, and roost tree characteristics of the Indiana bat (Myotis sodalis) during autumn in Jackson County, Kentucky. Unpublished Report to Kentucky Department of Fish and Wildlife Resources, Frankfort, KY. Kurta, A., and J.O. Whitaker, Jr. 1998. Diet of the endangered Indiana bat (Myotis sodalis) on the northern edge of its range. American Midland Naturalist 140:280286. Lee, Y.F. 1993. Feeding ecology of the Indiana bat, Myotis sodalis, and resource partitioning with Myotis keenii and Myotis lucifugus. M.Sc. Thesis. University of Tennessee, Knoxville, TN. 442 Northeastern Naturalist Vol. 13, No. 3 Murray, S.W., and Kurta, A. 2002. Spatial and temporal variation in diet. Pp. 182 192, In A. Kurta and J. Kennedy (Eds.). The Indiana Bat: Biology and Management of an Endangered Species. Bat Conservation International, Austin, TX. Ritzi, C.M., B.L. Everson, and J.O. Whitaker, Jr. 2005. Use of bat boxes by a colony of Indiana myotis (Myotis sodalis). Northeastern Naturalist 12:217220. Sparks, D.W. 2003. How does urbanization impact bats? Ph.D. Dissertation. Indiana State University, Terre Haute, IN. Sparks, D.W., J.A. Laborda, and J.O. Whitaker, Jr. 1998. Bats of the Indianapolis International Airport as compared to a more rural community of bats at Prairie Creek. Proceedings of the Indiana Academy of Science 107:171179. Sparks, D.W., C.M. Ritzi, J.E. Duchamp, and J.O. Whitaker, Jr. 2005a. Foraging habitat of the Indiana bat (Myotis sodalis) at an urbanrural interface. Journal of Mammalogy 86:713718 Sparks, D.W., J.O. Whitaker, Jr, and C.M. Ritzi. 2005b. Foraging ecology of the endangered Indiana bat. Pp. 1527, In K.C. Vorees and A. Harrington (Eds.). The Indiana Bats and Coal Mining: A Technical Interactive Forum. Office of Surface Mininng, US Department of the Interior, Alton, IL. Whitaker, Jr., J.O. 1972. Food habits of bats from Indiana. Canadian Journal of Zoology 50:877883. Whitaker, Jr., J.O. 1988. Food habits analysis of insectivorous bats. Pp. 171190, In T.H. Kunz (Ed.). Ecological and Behavioral Methods for the Study of Bats. Smithsonian Institution Press, Washington, DC. Whitaker, Jr., J.O. 1995. Selective versus opportunistic feeding in bats. Bat Research News 35:7577 Whitaker, Jr., J.O. 2004. Prey selection in a temperate-zone, insectivorous bat community. Journal of Mammalogy 85:460469. Whitaker, Jr., J.O., D.W. Sparks, and V. Brack, Jr. 2004. Bats of the Indianapolis International Airport Area, 19912001. Proceedings of the Indiana Academy of Science 113:251261. Whitaker, Jr., J.O., D.W. Sparks, and V. Brack, Jr. 2006. Use of artificial roost structures by bats at the Indianapolis International Airport. Environmental Management 38:2836.  ...

... RESEARCH ARTICLE Epidermal loss of Gq confers a migratory and differentiation defect in keratinocytes Colleen L. Doci1*, Constantinos M. Mikelis2, Juan Luis Callejas-Valera3, Karina K. Hansen4, Alfredo A. Molinolo5, Asuka Inoue6,7, Stefan Offermanns8, J. Silvio Gutkind3 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 College of Arts and Sciences, Marian University Indianapolis, Indianapolis, Indiana, United States of America, 2 Department of Biomedical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, Texas, United States of America, 3 Department of Pharmacology and Moores Cancer Center, University of California San Diego, La Jolla, California, United States of America, 4 Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, United States of America, 5 Department of Pathology, University of California San Diego, La Jolla, California, United States of America, 6 Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, Japan, 7 PRESTO, Japan Science and Technology Agency (JST), Kawaguchi, Saitama, Japan, 8 Department of Pharmacology, Max-Planck Institute for Heart and Lung Research, Bad Nauheim, Germany These authors contributed equally to this work. * cdoci@marian.edu OPEN ACCESS Citation: Doci CL, Mikelis CM, Callejas-Valera JL, Hansen KK, Molinolo AA, Inoue A, et al. (2017) Epidermal loss of Gq confers a migratory and differentiation defect in keratinocytes. PLoS ONE 12(3): e0173692. https://doi.org/10.1371/journal. pone.0173692 Editor: Paul McNeil, Augusta University Medical College of Georgia, UNITED STATES Received: August 13, 2016 Accepted: February 24, 2017 Published: March 16, 2017 Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This research was supported by the Intramural Research Program of the National Institute of Dental and Craniofacial Research, and in part by Marian University Indianapolis. AI received Grant #13408007 from PRESTO, Japan Science and Technology Agency. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Abstract G-protein coupled receptors (GPCRs), which activate heterotrimeric G proteins, are an essential class of transmembrane receptors that are responsible for a myriad of signaling events in normal and pathologic conditions. Two members of the G protein family, Gq and G11, activate one of the main GPCR pathways and function as oncogenes by integrating mitogen-stimulated signaling cascades that are active under malignant conditions. Recently, it has been shown that targeted deletion of G11 and Gq from endothelial cells impairs the Rho-mediated formation of focal adherens junctions, suggesting that G11/q signaling may also play a significant role in cytoskeletal-mediated cellular responses in epithelial cells. Indeed, combined deletion of G11 and Gq confers a significant migratory defect in keratinocytes that delays cutaneous wound healing in an in vivo setting. This delay can be attributed to a defect during the reepithelialization phase due to significantly attenuated migratory capacity of Gq-null keratinocytes under combined G11 deficiency. In fact, cells lacking G11/q demonstrate a severely reduced ability to respond to mitogenic and migratory signals in the microenvironment, leading to inappropriate and premature terminal differentiation. These results suggest that G11/q signaling pathways may be critical for integrating mitogenic signals and cytoskeletal function to achieve normal physiological responses. Emergence of a malignant phenotype may therefore arise from both under- and overexpression of G11/q signaling, implicating its upstream regulation as a potential therapeutic target in a host of pathologic conditions. PLOS ONE | https://doi.org/10.1371/journal.pone.0173692 March 16, 2017 1 / 18 Epidermal loss of Gq confers a migratory and differentiation defect in keratinocytes Competing interests: The authors have declared that no competing interests exist. Introduction The signal transduction mechanisms in the cell that mediate gene expression, cell survival, and growth rely heavily on the function of the largest family of cell-surface molecules, the G-protein coupled receptors (GPCRs) [1]. The GPCR family transduces signals from the cellular microenvironment via receptor coupling and subsequently activating heterotrimeric G proteins. This activation in turn leads to the second-messenger systems that influence transcription, translation, viability, and normal and malignant cell growth. Within the heterotrimeric G proteins, activating mutations in multiple members of the G subunit family have been shown to function as oncogenes. Among the heterotrimeric G proteins, Gq and G11 share around 90% amino acid homology and are the main components of the Gq family of alpha proteins [2]. Gain of function in Gq and G11 leads oncogenic transformation [3, 4] and is particularly prevalent in the development and progression of uveal melanoma, where Gq and G11 share activating mutations and are currently being evaluated for both diagnostic and therapeutic purposes [58]. However, relatively little is known about the pathological consequences of G11 and Gq loss. GNAQ (Gq) and GNA11 (G11) comprise the Gq family of alpha proteins that transduces many of the mitogen-stimulated GPCR signaling cascades. Gq stimulates Phospholipase C (PLC) to achieve downstream activation of Protein Kinase C (PKC) and extracellular signalrelated kinase (ERK) [9]. Combined deletion of G11 and Gq leads to embryonic lethality at day 11 due to myocardial hypertrophy, while global deletion of GNA11 alone causes no apparent behavioral and morphological defects and global deletion of GNAQ alone leads to ataxia and impaired motor control [10, 11]. Thus, due to their compensatory functions, investigation into the role of Gnaq and Gna11 requires conditional deletion of one of these genes in the context of total loss of its compensatory family member. Using this approach, it has been shown that conditional deletion of Gq in the context of Gna11 abolishes the embryonic lethality and hypertrophic response in cardiomyocytes observed previously [12]. This approach has been also used to demonstrate that loss of Gq from endothelial cells under combined G11 deficiency impairs the Rho-mediated formation of focal adherens junctions after histamine stimulation [13]. G11 and Gq have also been found to play a large role in the genetics of the skin [14, 15]. However, it is unclear if cutaneous targeted deletion of G11 and Gq may also have an impact on epidermal cytoskeletal integrity and function. Here, we show that targeted deletion of G11/Gq in the skin does not result in cancer formation, but rather underlies an acute migratory defect in keratinocytes. While not deleterious under physiologic conditions, this defect manifests as a severe attenuation in the rate of cutaneous wound healing. We further demonstrate that this migratory defect may be partly attributed to an inability of G11/q-null keratinocytes to respond to the mitogenic signals that mediate the transition between a differentiated, static cell and a more stem-like phenotype capable of engaging in highly migratory reepithelialization programs. These results suggest that the G11/q signaling pathway may be an important mediator of pathologic stress, where low or inactivated Gq results in a chronic wound phenotype that may contribute to malignant transformation. Therefore, understanding the factors that contribute to Gq expression and regulate its function may have a broad impact in a variety of pathologic disease conditions and open new therapeutic avenues in the future. Materials and methods Mice All animal studies were carried out according to NIH-approved protocols, in compliance with the Guide for the Care and Use of Laboratory Animals. All mice used were in C57BL/6 PLOS ONE | https://doi.org/10.1371/journal.pone.0173692 March 16, 2017 2 / 18 Epidermal loss of Gq confers a migratory and differentiation defect in keratinocytes background, 8 weeks old or older and both males and females were used for experiments. The generation of null alleles of the genes encoding G11 (Gna11) and of the floxed alleles of the genes encoding Gq (Gnaq) have been described previously [16]. For the experiments probing G11 deficiency only, Gna11-/- mice and littermate controls were used. Pups were obtained from a heterozygote mating where Gna11+/- x Gna11+/- animals were crossed, and upon genotyping the Gna11+/+ and Gna11-/- littermates were selected for experiments. Epidermal-specific Gq knockouts were obtained by crossing the aforementioned mice with mice carrying a constitutive Cre-mediated recombination system driven by the K14 promoter (K14-Cre, [17]), where Gna11-/-Gnaqf/f K14-Cre+/- mice were crossed with Gna11-/-Gnaqf/f K14-Cre-/- such that all pups were deficient for G11 and double floxed for Gq. Expression of Gaq was therefore driven by inheritance of the K14-Cre promoter, where 50% of the pups expressed the K14-Cre promoter (Gna11-/-Gnaqf/f K14-Cre+/-, referred to as G11KO/Gq-eKO) and were the experimental group, while the other 50% did not expressing the K14 promoter (Gna11-/-Gnaqf/f K14-Cre-/-, referred to as G11KO/Gq-WT) and were used as controls. Both males and female mice were used for the experiments. Reagents and antibodies Cell culture surfaces were coated with 10 g/ml of poly-L-lysine (Sigma), type I rat tail collagen (BD Biosciences) or fibronectin (Invitrogen). Recombinant human TGF-1 from Peprotech. Deep space black and warp red chromogen kits were from Biocare medical. Antibodies for western blotting and immunohistochemistry were used as described in Table 1. Cell lines, keratinocyte cell culture, and transfections All cells were cultured at 37C in the presence of 5% CO2. HEK293Gq cells, which lack functional Gq and G11, were described previously [18]. Cells were cultured in DMEM (Invitrogen) containing 10% fetal bovine serum (FBS; Sigma-Aldrich) and antibiotic/antimycotic solution (Sigma-Aldrich). Cells were transfected with Turbofect (Fermentas) according to the manufacturers instructions. Fibroblasts and keratinocytes from newborn mouse skin were isolated as previously described [19]. Keratinocytes were cultured in defined keratinocyte serum-free medium (KSFM; Invitrogen) and subdermal fibroblasts were cultured in DMEM 10% FBS. Cells were transfected with 10ng/cm2 of either pCAGGS-Gq or vector control and/ or 200ng/cm2 LifeAct-GFP and assessed 48 hours post transfection. Table 1. Antibodies and experimental conditions. This table details the antibodies, their formulation, experimental application, dilution, and source for all experiments in this manuscript. Antibody Conjugation Primary/Secondary Application Dilution Actin None Primary Western 1:5000 Company Cell Signaling Technology Goat a-rabbit Biotinylated Secondary IHC 1:400 Vector Laboratories Goat a-rabbit HRP Secondary Western 1:40,000 Southern Biotech Goat a-rat Biotinylated Secondary IHC 1:400 Vector Laboratories Gq None Primary Western 1:1000 Santa Cruz Biotechnology K10 None Primary IHC 1:2000 Covance K13 None Primary IHC 1:5000 Abcam K14 None Primary IHC 1:1000 Covance Ki67 None Primary IHC 1:50 Dako Phospho-SMAD2 None Primary Western 1:1000 Cell Signaling Technology SMAD2 None Primary Western 1:1000 Cell Signaling Technology https://doi.org/10.1371/journal.pone.0173692.t001 PLOS ONE | https://doi.org/10.1371/journal.pone.0173692 March 16, 2017 3 / 18 Epidermal loss of Gq confers a migratory and differentiation defect in keratinocytes Immunoblot analysis Cells were lysed in RIPA buffer [50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate] supplemented with protease and phosphatase inhibitors (Thermo Fisher) for 15 minutes on ice, scraped, and then sonicated for 3x10s on 30% power. Lysates were cleared by centrifugation at 10,000xg for 15 minutes at 4C and concentration was determined using BioRad DC protein assay. Ten micrograms total protein was separated by SDS-PAGE and transferred to PVDF membrane overnight at 4C. Membranes were blocked for 1 hour at room temperature in 5% milk in TBST and then probed with primary antibodies overnight at 4C. Membranes were washed four times in TBST, probed with HRP-conjugated secondary antibodies for 1h at RT in 5% milk, washed four times in TBST, and detected using chemiluminescent substrate (Millipore) with the FluorChem E system image analyzer (Cell Biosciences, Santa Clara, CA) Histopathologic and immunohistologic analysis Formalin-fixed paraffin-embedded (FFPE) tissue slides were deparaffinized in 100% xylene, hydrated through a series of graded alcohols (100%, 95%, 80%, and 70%), and incubated in sodium citrate buffer (pH6.0) to unmask the antigen. Endogenous peroxidase activity was blocked by incubate with 3% H2O2, blocked in PBS with 3% BSA, and then incubated with primary antibody in blocking buffer overnight at 4C. The slides were washed in PBS three times, incubated with a biotin-conjugated secondary antibody at room temperature for 30 minutes followed by the avidin-biotin complex (Vector Stain Elite Standard, ABC kit, Vector Laboratories) for 30 minutes at room temperature. For single stained slides, the slides were washed and developed in 3,30 -diaminobenzidine (Sigma FASTDAB tablet, Sigma Chemical) under microscopic observation. The reaction was stopped in tap water and the tissues were counterstained with hematoxylin, dehydrated, and mounted. For double stained images, slides were developed with Deep Space Black chromogen kit according to manufacturers instructions (Biocare Medical). The reaction was stopped in tap water, washed with TBS, blocked in TBS with 3% BSA, and then incubated with primary antibody in blocking buffer overnight at 4C. The slides were washed in TBS three times, incubated with alkaline phosphatase-conjugated secondary antibody at room temperature for 30 minutes followed by the AP complex (Vector Stain Elite AP, ABC kit, Vector Laboratories) for 30 minutes at room temperature. Slides were washed and developed using Warp Red chromogen kit according to manufacturers instructions (Biocare Medical) under microscopic observation. Images were taken using Scanscope (Aperio). Quantification of slides stained for Ki67 was performed using Aperio Scanscope software and the Nuclear v9 algorithm, while quantification of K10 and K13 was performed using IHC Membrane v9 algorithm. H-scores were determined as the product of the staining intensity (0, absent; 1, weak staining; 2, moderate staining; and 3, strong staining) multiplied by the percentage of positive cells quantified. Proliferation assay (EdU incorporation assay) To visualize individual cells synthesizing DNA we used the ClickIt kit (Invitrogen) according to manufacturers instructions. This kit allows for robust statistical analysis in small populations of cells while utilizing the highly-specific labeling methodology of BrdU or [3H]-thymidine incorporation. Briefly, subconfluent cells were grown in 96-well plates in quadruplicate, starved, and transferred to complete media for 16 h. Then EdU (10 M) was incorporated for 4 h and cells were fixed in a 4% paraformaldehide/PBS solution (Electron Microscopy Sciences, PA USA). Samples were counterstained with Hoescht 33342 and visualized under Axio PLOS ONE | https://doi.org/10.1371/journal.pone.0173692 March 16, 2017 4 / 18 Epidermal loss of Gq confers a migratory and differentiation defect in keratinocytes Imager Z1 microscope equipped with ApoTome system controlled by AxioVision software (Carl Zeiss). Colony formation assay For the colony formation analysis, cells were plated in 6-well plates at 100 live cells/well (counted with trypan blue) and cultured until visible colonies around 12 mm in diameter appeared in control wells. The cells were then stained with crystal violet fixing solution (0.05% crystal violet in PBS with 4% paraformaldehyde) for 30 min RT, washed with water, and the resulting colonies were quantified by size and number per plate with ImageJ. Scratch wound closure Scratch assays were performed with keratinocytes or fibroblasts grown to confluence on fibronectin-coated wells and starved overnight. Scratches were made with a plastic pipette tip across the diameter of each well. Phase contrast images of the wound were obtained every 6 h for fibroblasts and every 24 h for the keratinocytes, Quantitative analysis of uncovered wound area was performed using the Axiovision Rel. 4.7 (Carl Zeiss, Thornwood, NY). Calculations were based on three replicates per experiment from three independent experiments. Data mining and GEO analysis Data regarding the expression levels of migratory genes was downloaded from the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/, accessed June 30, 2015) for GSE8531. Significant genes, defined as those with a p-value less than 0.05 and logFC value >2 or <-2, were inputted into the ENRICHR algorithm (http://amp.pharm.mssm.edu/Enrichr/, accessed June 30, 2015). The GO Molecular Function ontology was downloaded and represented according to the internally calculated p-value. Migration assay Cells were serum starved for six hours and placed in the top well of a Boyden chamber while serum-free media or serum-free media supplemented with 5% FBS, 1 ng/ml TGF-1, or 10 ng/ ml TGF-1 was placed in the bottom well. The two chambers were separated by an 8 m pore polyvinylpropylene-free membrane coated with 10 g/ml collagen. Cells were incubated at 37C in a humidified chamber overnight. The next day, the apparatus was disassembled and the membrane was fixed for 30 minutes in methanol at room temperature. The membrane was then stained with hematoxylin for 20 minutes at RT, washed three times with distilled water, mounted face down on glass slides, and nonmigrated cells were scraped off with cotton swabs. Membranes were scanned and quantified using ImageJ. Calculations were based on 6 imaging fields each from three independent experiments. Statistical significance was determined using Students t-test. Immunofluorescence Cells were transfected with LifeAct-GFP and either empty vector or Gq and grown on cover slips coated with collagen I. Cells were starved for 6 hours and then treated for 3 hours with either 0, 1, or 10 ng/ml TGF-1. Cells were fixed with 4% paraformaldehyde, washed in PBS, and mounted on uncharged slides. The images were taken using an Axio Imager Z1 microscope equipped with an ApoTome system. PLOS ONE | https://doi.org/10.1371/journal.pone.0173692 March 16, 2017 5 / 18 Epidermal loss of Gq confers a migratory and differentiation defect in keratinocytes Wound healing assay and experimental mice This study was approved by the Animal Care and User Committee, according to National Institutes of Health (NIH) animal study protocols approved by the Animal Care and Use Committee protocol 12667, National Institute of Dental and Craniofacial Research, in compliance with the Guide for the Care and Use of Laboratory Animals. Animals were housed on 12-h light/dark cycles and received food, standard rodent chow, and water ad libitum in compliance with Association for Assessment and Accreditation of Laboratory Animal Care International guidelines. The animals were observed daily by the investigators and animal care staff. Any animals displaying signs of discomfort, wasting, ruffled hair coat, hunching, or other signs indicative of distress were treated appropriately to alleviate discomfort or euthanized if recommended by animal care staff or the facility veterinary. Mice were anesthetized and dorsal surfaces were shaved and cleaned with betadine topical solution. Fifteen millimeters full-thickness incisional skin wounds were made in the mid-dorsal area. The wound sites were monitored and measured daily utilizing wound closure as endpoint as previously [20]. At indicated time points, the wound field was excised and fixed in aqueous buffered zinc formalin for 24 h, transferred to 70% ethanol, paraffin embedded and sectioned. Wound size was determined as the factor of wound width and length, normalized to the wound area at Day 0. Wound half-life was determined from the nonlinear regression of wound closure over time per animal. Statistical analysis All analyses were performed with a minimum of three technical replicates per experiment from three independent experiments and the means obtained were used for ANOVA or independent t-tests. Additional technical and biological replicates were used as indicated. Statistical analyses, variation estimation and validation of test assumptions were carried out using the Prism 6 statistical analysis program (GraphPad). Asterisks denote statistical significance (nonsignificant or NS, P > 0.05;  P < 0.05;  P < 0.01; and  P < 0.001). All data are reported as mean standard error of the mean (s.e.m.). Results Epidermal integrity is maintained under homeostasis despite targeted deletion of G proteins We have recently shown that activating mutations in Gq function as drivers of oncogenesis in uveal melanoma and are also found in a variety of other malignancies [21, 22], while deletion of Gnaq in the context of Gna11 deficiency appears to have significant defects in developmental and Rho-mediated endothelial cell functions [11, 13]. Deletion of Gnaq alone results in ataxia, impaired motor control, and skin pigmentation [10, 11, 14, 23]. However, a specific functional role for Gq proteins in the skin has not been identified partially due to the compensatory roles that Gq and G11 play in vivo. To determine if Gq proteins might have a specific effect on epidermal function, we used a mouse model harboring global deletion of G11 (G11KO) and targeted deletion of Gq in the skin (Gq-eKO) driven by the K14 promoter (Fig 1A). G11KO/Gq-eKO animals displayed no gross abnormalities (Fig 1B) despite complete deletion of Gq proteins from the skin (Fig 1C). Histologically, both littermate controls bearing global G11 deletion but expressing WT Gq and G11KO/Gq-eKO animals demonstrated no differences in epidermal thickness (Fig 1D). There was a slight decrease in Ki67 proliferating cells that corresponded with a slight increase in K10+ differentiated cells in G11KO/ Gq-eKO animals, suggesting that there might be a stronger tendency towards terminal differentiation within these cells (Fig 1D). However, these animals were largely phenotypically PLOS ONE | https://doi.org/10.1371/journal.pone.0173692 March 16, 2017 6 / 18 Epidermal loss of Gq confers a migratory and differentiation defect in keratinocytes Fig 1. Mice with epidermal loss of Gq do not demonstrate phenotypic defects under normal conditions. A) Schematic representation of the animal model used to derive a delete Gnaq exon 6 (Ex6) from the basal epidermal compartment concomitant with global deletion of Gna11. B) Representative pictures of G11KO/Gq-WT control and G11KO/Gq-eKO mice. C) Western blot demonstrating Gq deletion in the epidermal keratinocytes but not fibroblasts after expression of K14-Cre. D) Normal skin samples from G11KO/Gq-WT control and G11KO/Gq-eKO animals (N = 4 each) were examined for histology (H&E), proliferation (Ki67), and differentiation (K10). https://doi.org/10.1371/journal.pone.0173692.g001 PLOS ONE | https://doi.org/10.1371/journal.pone.0173692 March 16, 2017 7 / 18 Epidermal loss of Gq confers a migratory and differentiation defect in keratinocytes normal under homeostatic conditions and did not present the endogenous tumorigenic susceptibility of the Gs model. Loss of Gq confers a strong migratory defect in keratinocytes To determine if any defect might be present within G11/q-null keratinocytes, we isolated cells from mice and analyzed them according to the key hallmarks of carcinomas, namely increased proliferation, increased colony formation, and increased migration. Similar to what we observed in histologic sections, G11/q-null keratinocytes did not demonstrate any differences in proliferation (Fig 2A). Similarly, no statistical differences could be detected in the size or number of colonies (Fig 2B). However, a remarkable phenotype was observed upon induction of migration, where G11/q-null keratinocytes had a profound defect in their ability to migrate after scratch wounding (Fig 2C). Over a 96-hour time course, control keratinocytes migrated to close the gap formed after a scratch wound assay, while G11/q-null keratinocytes were able to close less than half of the gap in the same time frame (Fig 2C). This was due specifically to loss of G11/q and not due to loss of G11 alone, as G11-null fibroblasts isolated from the same mice showed no such defect and were identical to fibroblasts from control mice (Fig 2C). Together, this suggested that rather than promoting a carcinogenic phenotype, deletion of Gq from the epidermis contributed to a migratory defect. To determine if a similar phenotype had been observed in other studies, we searched for gene expression data related to alteration of a migration phenotype in keratinocytes. By annotating the GEO dataset from a study that used TGF ligands to assess migration-specific genes [24], we found that among the ten most statistically significantly altered ontologies amongst regulated genes, nine were related to G protein signaling events and specifically to events associated with Gq activity, including phospholipase C activity (S1 Fig). TGF- has been largely characterized in cell migration due to its pleiotropic effects in a wide variety of cell types and in particular its contribution to proliferation and migration in cutaneous injury [2527]. Taking advantage of the CRISPR targeted deletion system, we tested the possibility that loss of Gq might be necessary for integrating migration stimulatory signals. First, we compared parental HEK293 cells with G11/Gq CRISPR-deleted HEK293 migration and found that consistent with our previous observations, the G11/q-null cells migrated significantly less under serum-starved and FBS-stimulated conditions (Fig 3A). To further investigate whether this was a migration-specific defect due to Gq expression and not due to competing mitogen signaling or clonality issues from the CRISPR cell cloning, we reintroduced Gq with low levels of transfection plasmid or used empty vector as a control and then assessed their TGF--dependent migration. G11/q-null cells showed a modest, dosedependent response to TGF-, indicating that they are capable of responding to TGF- signaling (Fig 3B). However, reintroduction of Gq resulted in a far more robust response to TGF- and these cells migrated significantly more than empty vector controls (Fig 3B). To confirm that this was not due to a defect in TGF- signaling, we stimulated the cells for 30 minutes and then performed western blots for downstream targets of TGF-, namely SMAD2. Both the Gq-expressing and vector control cells responded to TGF- signaling to approximately the same degree, as indicated by accumulation of p-SMAD2 (Fig 3C). This suggests that TGF- does not signal directly through Gq, but rather that Gq participates at some level in orchestrating the signaling cascade that is initiated upon TGF- in order to facilitate migration. Migration requires reorganization of the actin cytoskeleton, so we imaged vector control and Gq-restored cells after treatment with TGF- via fluorescently-labelled actin. Here, vector control cells acquired a ruffled cytoskeletal appearance at high concentrations of TGF-, but Gq-restored cells were able to achieve a more efficient reorganization of their cytoskeleton at PLOS ONE | https://doi.org/10.1371/journal.pone.0173692 March 16, 2017 8 / 18 Epidermal loss of Gq confers a migratory and differentiation defect in keratinocytes Fig 2. G11/q-null keratinocytes have normal proliferative and stem-like phenotypes but demonstrate a significant migratory defect. Keratinocytes from G11KO/Gq-WT control and G11KO/Gq-eKO animals were assayed for proliferation (A), colony formation (B) and scratch wound closure (C). To confirm the migratory defect, keratinocyte and fibroblast wound closure from G11KO/Gq-WT control and G11KO/Gq-eKO were quantified. Statistical significance was determined using Students t-test, *p<0.05, **p<0.01, ***p<0.001. https://doi.org/10.1371/journal.pone.0173692.g002 PLOS ONE | https://doi.org/10.1371/journal.pone.0173692 March 16, 2017 9 / 18 Epidermal loss of Gq confers a migratory and differentiation defect in keratinocytes Fig 3. Gq enhances TGF- stimulated migration and cytoskeletal remodeling. A) Parental and G11/q-null HEK293 cells were assessed for migration towards serum free media (SFM) or 5% fetal bovine serum (FBS). B) G11/q-null or G11/q-null with transfected, reconstituted expression of Gq were allowed to migrate towards 0, 1, or 10 ng/ml TGF-1. C) Western blot demonstrating efficacy of TGF- signaling in G11/q-null and reconstituted cells after 30 minutes treatment with 0, 1, or 10 ng/ml TGF-1. D) G11/q-null and reconstituted cells co-expressing LifeActGFP were treated with 0, 1, or 10 ng/ml TGF-1 for 3 hours and assessed for cytoskeletal changes. Statistical significance was determined by Students t-test, *p<0.05, **p<0.01, ***p<0.001. https://doi.org/10.1371/journal.pone.0173692.g003 PLOS ONE | https://doi.org/10.1371/journal.pone.0173692 March 16, 2017 10 / 18 Epidermal loss of Gq confers a migratory and differentiation defect in keratinocytes low doses of TGF- (Fig 3D). Together, these results demonstrate that loss of Gq confers a strong migratory defect in epithelial cells that is specific to migration-related signaling events and independent of other growth related signaling cues. Gq-induced migratory defects in keratinocytes leads to significant delay in cutaneous wound healing The ability of keratinocytes to migrate into the wound bed is an essential step in cutaneous wound healing and essential for reepithelialization. As such, we reasoned that the pronounced migratory defect in G11/q-null animals might attenuate wound healing and lead to a delay in wound closure. G11KO/Gq-WT control and G11KO/Gq-eKO littermates were subjected to 15 mm dorsal cutaneous wounds and observed continuously during the wound healing process (Fig 4A). Initial wound sizes were nearly identical between G11KO/Gq-WT control and G11KO/Gq-eKO animals (Fig 4B). Despite this, G11KO/Gq-eKO mice healed significantly later than their control littermates (Fig 4C). This delay in wound healing was directly attributed to loss of both epidermal Gq proteins, Gq and G11 as G11-null control animals alone healed similarly to wild-type animals (S2 Fig). Analysis of the rate of wound healing between G11KO/Gq-WT control and G11KO/Gq-eKO animals demonstrated a significant shift at all time points, leading to a highly significant difference in the wound healing curves (Fig 4D). Nonlinear regression of these curves revealed that the G11KO/Gq-eKO animals had a wound half-life nearly twice that of G11KO/Gq-WT control mice (Fig 4E). Together, this data indicates that the migratory defect incurred upon loss of Gq has significant impact on the rate of cutaneous wound healing and loss of epidermal Gq may lead to the development of chronic wounds. Gq-mediated wound healing defects correlate with insensitivity to the regulation of differentiation signals Defects in the keratinocyte migration impair subsequent reepithelialization phase of wound healing and can be seen by a failure to form an epithelial tongue adjacent to the wound bed. This was confirmed by H&E analysis, where the length of the migrating epithelial tongue in G11KO/Gq-eKO cells was significantly smaller than their G11KO/Gq-WT control counterparts (Fig 5, top row). While migrating cells closest to the wound bed do not demonstrate significant levels of proliferation, the trailing end of the epithelial tongue engages in robust proliferation to facilitate reepithelialization [28]. Compared to normal adjacent epithelium, WT cells demonstrate the spectrum of proliferation associated with this phase of wound healing, where the proximal segment is less proliferative than normal keratinocytes but a sharp doubling in proliferation can be seen at the supporting edge (Fig 5, second row). In contrast, KO cells demonstrate a relatively stable level of proliferation throughout the epithelial tongue that is not overtly different from the adjacent normal cells (Fig 5, second row). In the same manner, G11KO/Gq-eKO animals demonstrate a profound inability to downregulate markers of differentiation at the leading edge of the epithelial tongue. While control littermates show a steady and significant decrease in the early differentiation marker K10 as the epithelial tongue approaches the wound bed, a nearly uniform expression of K10 is observed in all segments of the epithelial tongue of G11KO/Gq-eKO animals (Fig 5, third row). This correlated to a nearly 13-fold reduction in slope for the K10 H-score across the epithelial tongue (Fig 5, third row). Defects in reepithelialization are associated with chronic wounds and malignant transformation in which reciprocal expression of K10 and K13 are often observed [29, 30]. Again, we observed that G11KO/Gq-WT control animals had a steady, nearly linear increase in K13 H-score across the epithelial tongue that was abrogated in the G11KO/Gq-eKO PLOS ONE | https://doi.org/10.1371/journal.pone.0173692 March 16, 2017 11 / 18 Epidermal loss of Gq confers a migratory and differentiation defect in keratinocytes Fig 4. G11KO/Gq-eKO mice have delayed wound healing. G11KO/Gq-WT control and G11KO/Gq-eKO mice received 15 mm incisional wound and closure was monitored over 18 days. A) Gross examination of the wounds in G11KO/Gq-WT control (N = 15) and G11KO/Gq-eKO (N = 10) mice. B) Initial wound size. Statistical significance determined by Students t-test. C) Kaplan-Meier survival curve of wound closure. Statistical significance was determined by log-rank test. D) Wound closure over time. Statistical significance was determined by two-way ANOVA. E) Wound half-life for G11KO/Gq-WT control and G11KO/Gq-eKO animals, as determined from the rate of wound closure over time. Statistical significance was determined by Students t-test, ***p<0.001. https://doi.org/10.1371/journal.pone.0173692.g004 PLOS ONE | https://doi.org/10.1371/journal.pone.0173692 March 16, 2017 12 / 18 Epidermal loss of Gq confers a migratory and differentiation defect in keratinocytes Fig 5. Loss of epidermal Gq correlates with defects in reepithelialization of cutaneous wounds. Top panel: the epithelial tongue as evidenced by H&E staining (yellow dashed line) is diagrammed and the length quantified. Representative stains and quantification of Ki67 (second panel), K10 (third panel), and K13 (bottom panel) are shown. All quantifications are based on an N = 4 mice per group and proliferation is shown relative to normal adjacent epithelium. Slope was determined by linear regression and statistical significance was determined by Students t-test, p<0.05, **p<0.01, ***p<0.001. https://doi.org/10.1371/journal.pone.0173692.g005 PLOS ONE | https://doi.org/10.1371/journal.pone.0173692 March 16, 2017 13 / 18 Epidermal loss of Gq confers a migratory and differentiation defect in keratinocytes animals. While a slight increase was detected, it was still nearly 4 times less than that of the control animals. Together, this data indicates that the migratory defect that results from epidermal loss of Gq manifests in a failure of the epithelial tongue to migrate during wound healing. Further, it suggests that the deficiency in reepithelialization is due to an inability to engage the signaling pathways that originate within the wound bed. Discussion Migration is a complex and highly orchestrated process that is essential for maintaining many cell functions under normal and pathologic conditions. Here, we demonstrate a novel role for Gq in facilitating acquisition of a stress-induced migratory phenotype in epidermal cells in the context of G11 loss. Under normal conditions, loss of G11/q signaling does not demonstrate any overt effects but upon wounding, G11/q-null cells exhibit a remarkable inability to initiate the standard migratory response both in vitro and in vivo. This migratory defect correlates strongly with an inability to engage in the normal differentiation and proliferative programs that are initiated upon wounding and consequently lead to significantly delayed wound closure. These results have strong implications for our current understanding for the management of chronic wounds and aggressive malignancies whose pathologic hallmarks include the acquisition of a migratory phenotype. Migration of keratinocytes, either collectively or as single cells, depends on the integration of internal and external cues to achieve directed movement [31]. These extracellular cues involve growth factors including members of the EGF, FGF, insulin, and VEGF families [32]. G proteins have been shown to be critical in the transactivation of many of these growth factor receptors and in some cases this transactivation has been shown to have pathologic consequences including tumor development and metastasis [3335]. Despite the significant contribution of other G family members with migration, remarkably little is known regarding the specific role of Gq in epidermal motility. LPA has been shown to activate G11/q signaling pathways and drive cell proliferation, differentiation, and migration in keratinocytes, similar to the effect observed upon TGF- stimulation [36]. Conversely, transactivation of the insulin-like growth factor I receptor (IGF1R) by UTP-stimulated P2Y purinergic receptors inhibits PI3K activation in a Gq-dependent manner, blocking subsequent keratinocyte migration [37, 38]. However, in other contexts, Gq is critical for activation of PI3K, MAPK, and other motilityassociated pathways [39], suggesting that the role of Gq in migration may be to integrate multiple and sometimes conflicting extracellular signals to achieve appropriate cellular responses. Wound healing is a clear example of a process that involves such an incorporation of multiple complex signaling networks. In order to restore barrier function, keratinocytes must coordinate migration, proliferation, and differentiation within a dynamic extracellular space. After acute injury, keratinocytes at the margin of wounds begin to migrate towards the wound bed. Proximity to the proinflammatory cytokines secreted at the wound bed leads to a number of phenotypical changes, including downregulation of proliferative and differentiation markers such as Ki67 and K10 [28]. At the same time, epidermal cells trailing the migrating keratinocytes engage in a hyperproliferative program to support the wound healing process [28]. Indeed, WT keratinocytes demonstrate a burst of proliferation at the trailing edge of the epithelial tongue that is nearly twice that of normal epithelium while simultaneously downregulating proliferation at the leading edge. Conversely, G11/q-null keratinocytes show a remarkably dampened response to stimuli, with an only slightly increased level of proliferation in the distal tongue section and a relatively unchanged level of proliferation at the leading edge. A similar trend is observed with respect to differentiation, as G11/q-null cells show little to no downregulation of K10, while WT cells actively engage in dedifferentiation at the wound bed. PLOS ONE | https://doi.org/10.1371/journal.pone.0173692 March 16, 2017 14 / 18 Epidermal loss of Gq confers a migratory and differentiation defect in keratinocytes Failure to enact the reepithelialization phase of wound healing due to diminished keratinocyte migration is clinically associated with chronic wounds. Cutaneous squamous cell carcinomas (SCCs) in particular have been shown to arise from precancerous lesions associated with chronic wounds and impaired wound healing paradigms [40]. Activation of the epithelial stem cell compartment is a critical element of wound healing, and epidermal loss of another G family member, Gs, was associated with subsequent formation of basal cell carcinomas [41]. Therefore, chronic wounds demonstrating loss of Gq function may be more prone to development of precancerous and cancerous lesions in the skin. In addition to activation of the stem cell compartment, chronic wounds and tumors share many other similar mechanistic and molecular manifestations, including irreversible loss of keratinocyte differentiation, uncontrolled proliferation, and enhanced migration [42]. Impaired signaling, proliferation, and migration of fibroblasts in chronic wounds have been observed and are in part attributable to decreased sensitivity to TGF-1 [43]. Given the decreased responsiveness of G11/q-null cells to TGF-1, this suggests that loss of Gq-mediated signaling may contribute to the development of chronic wounds that could progress to SCCs. K13, a common marker of malignant progression, typically replaces expression of K10 during tumor progression and is also seen at higher levels at the leading edge of the epithelial tongue in wound healing [29, 30]. Only cells expressing Gq were able to differentially regulate expression of K13 (Fig 5), indicating that Gq is essential for transmitting the multilayered cues within the wound microenvironment. Taken together, it is tempting then to speculate then that under conditions of chronic inflammation and pathologic injury, loss of Gq may contribute to chronic wound formation and subsequent malignant transformation in the epithelial compartment. Gq and G11 are integral members of the G family of proteins and play multifaceted roles in the regulation of gene expression, viability, and growth. Here, we propose that Gq proteins may have pleiotropic effects in the epithelial compartment under pathologic conditions to coordinate reepithelialization. Loss of epithelial G11/q during the wound healing process manifests in a severe migratory phenotype punctuated by an inability of G11/q-null cells to differentially regulate proliferation and differentiation proximal to the wound bed. As such, Gq proteins are essential for the integration of the complex signaling networks in pathologic conditions and may provide a unique opportunity for clinical intervention. Supporting information S1 Fig. GPCR and Gaq-coupled GPCR ontologies are highly associated with keratinocyte migration. Expression levels of statistically significant differentially regulated genes involved in migration were downloaded and analyzed for ontologic similarities using the ENRICHR program. The GO Molecular Function ontology is represented according to the internally calculated p-value. (TIF) S2 Fig. Wound healing in G11KO mice is not significantly different from wild-type mice. Wild-type C57B/L6 mice and G11KO/Gq-WT mice were given 15 mm incisional wounds and closure was monitored over 18 days. A) Initial wound size. Statistical significance determined by Students t-test. B) Kaplan-Meier survival curve of wound closure. Statistical significance was determined by log-rank test. C) Wound closure over time. Statistical significance was determined by two-way ANOVA. D) Wound half-life for WT and G11KO as determined from the rate of wound closure over time. Statistical significance was determined by Students t-test, ns = not significant. (TIF) PLOS ONE | https://doi.org/10.1371/journal.pone.0173692 March 16, 2017 15 / 18 Epidermal loss of Gq confers a migratory and differentiation defect in keratinocytes Acknowledgments We thank Dr. Roberto Weigert (NIDCR, National Institutes of Health) for providing the LifeAct plasmid. This research was supported by the Intramural Research Program of the National Institute of Dental and Craniofacial Research, and in part by Marian University Indianapolis. A.I. was funded by JST, PRESTO Grant #13408007. Author Contributions Conceptualization: CLD CMM JSG. Formal analysis: CLD CMM. Funding acquisition: CLD JSG. Investigation: CLD CMM JLCV KKH AAM. Methodology: CLD CMM AAM AI SO. Project administration: CLD CMM JSG. 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... The Journal of Undergraduate Neuroscience Education (JUNE), Spring 2020, 18(2):E4-E5 EDITORIAL Faculty for Undergraduate Neuroscience (FUN) Statement on Diversity, Equity, and Inclusion Ronald J. Bayline1, Mary E. Morrison2, Kurt R. Illig3, Veronica G. Martinez-Acosta4, Lora A. Becker5, Carlita B. Favero6, Hewlet G. McFarlane7, Leah A. Chase8, Susan M.L. Banks9, Gerald D. Griffin10, Siobhan Robinson11, Jacqueline K. Rose12, Michelle T. Tong13, Alo C. Basu14, Jason P. Chan15 1 FUN Past President, Washington & Jefferson College, Washington, PA 15301; 2FUN President, Lycoming College, Williamsport, PA 17701; 3FUN President-elect, University of St. Thomas, St. Paul MN 55105; 4FUN Treasurer, University of The Incarnate Word, San Antonio, TX 78209; 5FUN Treasurer-elect, University of Evansville, Evansville IN 47722; 6 FUN Secretary, Ursinus College, Collegeville, PA 19426; 7FUN Presidents Advisory Council, Kenyon College, Gambier OH 43022; 8FUN Presidents Advisory Council, Hope College, Holland MI 49422; 9FUN Councilor, Florida Southern College in Lakeland, FL 33801; 10FUN Councilor, Hope College, Holland MI 49422; 11FUN Councilor, Hamilton College, Clinton NY 13323; 12FUN Councilor, Western Washington University, Bellingham, WA 98225; 13FUN Councilor, University of St. Thomas, St. Paul, MN 55105; 14FUN Councilor and Summer Virtual Meeting Committee Co-chair, College of the Holy Cross, Worcester, MA 01610; 15Summer Virtual Meeting Committee Co-chair, Marian University, Indianapolis, IN 46222. We join our voices with countless others across the country in an unequivocal condemnation of police brutality, institutionalized racism, and all forms of anti-black violence. As a scientific community, we acknowledge that our own past is filled with examples where we have brought violence against Black, Indigenous, and People of Color (BIPOC), from histories as overt as the Tuskegee Syphilis trials (www.history.com, 2019a) or the unethical use of Puerto Rican women as subjects in birth control trials (www.history.com, 2019b) to the more pernicious acts of everyday racism that occur in our colleges and universities. Unfortunately, we are still not far from this history. This past week, George Floyd was killed in Minneapolis, and as COVID-19 ravages the globe and affects all of us in deeply painful ways, our BIPOC communities have disproportionately carried the burden (www.embracerace.org). However, insofar as we have been complicit in racism, we also hold the tools for a robust participation in the work of anti-racism. What we do with students in our classrooms and in our labs is not separate from the work of racial justice. We all have our part to play. Indeed, we as scientists must marshal all our skills (yes, even pipetting!) toward creating a brighter future and more equitable world. As scholar-teachers, we must create neuroscience as an inclusive space for our students, especially for our students of color. FUN commits to taking concrete steps to further educate, equip, and heal our community. We make 4 commitments to action that will be implemented in our organizational structure and future programming: programming, committee membership and leadership opportunities accessible and inclusive, and we commit to taking this step together as a unified community that recognizes the reality and pain of racial inequality. 1. Reflection toward action We commit to intentional reflection on the ways in which we participate in systems of oppression. We believe that only through critical reflection can we begin to identify and then dismantle these systems. We commit to making FUN REFERENCES 2. Science as healing We recognize the need to create a safe space for BIPOC neuroscientists to share stories, struggles, and strategies for healing. We commit to creating this space. 3. Research as resistance We commit to highlighting neuroscience research, especially by undergraduate students, that inform the biological, biopsychology, and behavioral ramifications of chronic oppression. 4. Pedagogy of the oppressed We commit to supporting faculty and programs in their antiracism work, through workshops and sessions on inclusive excellence in STEM which recognize the full humanity and full potential of BIPOC students and perspectives. This means a focus on inclusion as well as diversity, and a rejection of deficit-informed methods. These commitments are just the beginning and we will use our summer virtual meeting to take the first steps. As a diverse and vibrant community, FUN has a responsibility to our members to take action. As a leader in undergraduate neuroscience, we have a duty to the broader scientific community to advance the ideals of diversity, equity, inclusion, and justice. Embracerace.org (2020) Accessed June 16, 2020. https://www.embracerace.org/resources/disproportionate-racialimpacts-of-covid. History.com (2019a) Accessed June 16, 2020. JUNE is a publication of Faculty for Undergraduate Neuroscience (FUN) www.funjournal.org Bayline et al. https://www.history.com/news/the-infamous-40-year-tuskegeestudy. History.com (2019b) Accessed June 16, 2020. https://www.history.com/news/birth-control-pill-history-puertorico-enovid. FUN: Diversity, Equity, and Inclusivity E5 Received June, 14, 2020; revised June, 16, 2020; accepted June, 16, 2020. Address correspondence to: Dr. Mary E. Morrison, Department of Biology, Lycoming College, Williamsport, PA 17701. Email: morrison@lycoming.edu Copyright 2020 Faculty for Undergraduate Neuroscience www.funjournal.org  ...

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... HHS Public Access Author manuscript Author Manuscript Sci Transl Med. Author manuscript; available in PMC 2019 June 28. Published in final edited form as: Sci Transl Med. 2018 July 25; 10(451): . doi:10.1126/scitranslmed.aap8798. Transcriptional signature primes human oral mucosa for rapid wound healing Ramiro Iglesias-Bartolome1,2,^, Akihiko Uchiyama1, Alfredo A. Molinolo2,3, Loreto Abusleme4, Stephen R. Brooks5, Juan Luis Callejas-Valera2,3, Dean Edwards2, Colleen Doci2,+, Marie-Liesse Asselin-Labat6, Mark W. Onaitis6, Niki Moutsopoulos4, J. Silvio Gutkind2,3,*, and Maria I. Morasso1,* Author Manuscript 1.Laboratory of Skin Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, MD, USA 2.Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, Bethesda, MD, USA 3.Department of Pharmacology and Moores Cancer Center, University of California San Diego, La Jolla, CA, USA 4.Oral Immunity and Inflammation Unit, National Institute of Dental and Craniofacial Research, Bethesda, MD, USA 5.Biodata Mining and Discovery Section, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, MD, USA Author Manuscript 6.Moores Cancer Center, University California San Diego, La Jolla, CA, USA Abstract Author Manuscript Oral mucosal wound healing has long been regarded as an ideal system of wound resolution. However, the intrinsic characteristics that mediate optimal healing at mucosal surfaces are poorly understood, particularly in humans. Here we present a unique comparative analysis between human oral and cutaneous wound healing using paired and sequential biopsies during the repair process. Utilizing molecular profiling, we determine that wound-activated transcriptional networks are present at basal state in the oral mucosa, priming the epithelium for wound repair. We show that oral mucosa wound-related networks control epithelial cell differentiation and regulate inflammatory responses, highlighting fundamental global mechanisms of repair and inflammatory responses in humans. The paired comparative analysis allowed for the identification of differentially expressed SOX2 and PITX1 transcriptional regulators in oral versus skin keratinocytes that confer a unique identity to oral keratinocytes. We show that SOX2 and PITX1 transcriptional function has the potential of reprogramming skin keratinocytes to acquire increased * Correspondence should be addressed to: Morasso@nih.gov (M.I.M.); sgutkind@ucsd.edu (J.S.G.). ^Current address: Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA. +Current address: College of Arts and Sciences, Marian University, Indianapolis, USA Accession numbers Raw and analyzed RNA-Seq data have been deposited in the Gene Expression Omnibus (GEO) site (GSE97615, GSE97616, GSE97617). Iglesias-Bartolome et al. Page 2 Author Manuscript cell migration capability and improve wound resolution in vivo. Our data establishes a novel understanding of the biology of healing in human mucosal and cutaneous environments, and provides insights into therapeutic targeting of chronic and non-healing wounds. INTRODUCTION Author Manuscript Improving wound healing resolution is becoming a major medical and social priority due to the drastic increase in traumatic injury, chronic wounds and scarring (1). While several studies characterize in detail the mechanisms and pathways altered in these deficient wounds, a different approach that defines factors involved in accelerated wound healing would allow for the identification of novel therapeutic targets to improve tissue repair. In this regard, oral wound healing, along with embryonic wound healing, have long been considered models of optimal wound resolution characterized by rapid and scarless wound healing (14). Dissecting the different molecular events that drive wound healing resolution in oral mucosa compared with those of the skin will help us define why these specific lesions heal more efficiently and translate those findings to treat deficient healing processes. Author Manuscript Author Manuscript Cutaneous wound healing is well-documented, with the overall classic interpretation for the repair pathway having four systematic phases: hemostasis, inflammation, proliferation and remodeling (5). The molecular circuitries that drive these different phases of cutaneous repair have been characterized, while the unique environment of the oral cavity represents a different wound-healing paradigm that is still poorly understood. Oral wounds heal at an accelerated rate compared to cutaneous wounds, and in vitro or animal model studies have attributed this to a variety of mechanisms including differential inflammatory response, distinct modulation of stem cell, proliferative and differentiation programs, and more efficient epithelial remodeling (1, 3, 6). Despite this progress, the lack of detailed clinical studies in humans comparing oral and cutaneous wound healing in vivo has limited the advancement of our knowledge on the mechanisms mediating accelerated wound healing. To close this gap, we characterized the molecular and histological aspects of wound healing in paired samples of oral mucosa and the skin in healthy human subjects. Wounds were introduced in the oral buccal mucosa and the skin at the same time and sequentially biopsied for comparison at progressive time points. Our results emphasize how biologically the oral buccal mucosa constitutes a unique environment that is characterized by a radically different wound-healing program. We show that the oral cavity is primed for wound repair and that oral mucosa accelerated wound healing might be attributed to the ability of oral keratinocytes to limit epithelial cell differentiation and pro-inflammatory responses. We also present evidence that transcriptional networks established by functionally active transcription factors such as SOX2 and PITX1 mediate this phenotype and can be exploited to reprogram cutaneous keratinocytes to present oral keratinocyte features, including accelerated wound closure. Sci Transl Med. Author manuscript; available in PMC 2019 June 28. Iglesias-Bartolome et al. Page 3 Author Manuscript RESULTS Wound-activated transcriptional networks present at basal state prime the oral mucosa for wound repair. Author Manuscript Utilizing paired and sequential biopsy samples during the healing process, we contrast oral mucosal with cutaneous healing to determine the differential regulation of these processes in the human setting (Fig. 1; Clinicaltrials.gov #NCT01078467, see Methods section for details on clinical study). Clinical screening was performed (Day 0) followed by baseline biopsies to create paired identical wounds in the oral buccal mucosa and skin (Day 1). Importantly, day 1 biopsies allowed for evaluation of homeostatic transcriptional profiles in human mucosal versus cutaneous surfaces. Follow-up biopsies of the wound area(s) were collected at day 3 (48 hours after first biopsy) and day 6 (120 hours after first biopsy) of healing in two different groups. This approach allowed for the evaluation of distinct phases during the physiologic process of human wound healing (Fig. 1). Author Manuscript Analysis of the healing time course revealed that oral wounds resolve significantly faster than skin wounds (Fig. 1C). This was observed following the first wound-inducing biopsy (3 mm wound, Fig. 1C, upper panel) and after the secondary biopsy of the wound area (5 mm wound, Fig. 1C, lower panel) (typical examples are shown in fig. S1, B and C). Oral wounds shrink within minutes of completing the biopsy, probably due to muscle contraction. This is nearly absent in the 5 mm biopsy, and yet both wounds showed similarly high healing rates (Fig. 1D). This suggested that the initial contraction of small wounds may not explain the accelerated healing rate of oral wounds. Histological analysis of the wound sections shows re-epithelialization of the oral mucosa wound at earlier time points when compared to the skin (Fig. 1E). Remarkably, as early as day 3, oral wounds are almost completely covered by squamous epithelium, even in the absence of stromal healing. Author Manuscript To molecularly characterize the human repair process, oral and skin biopsies were subjected to RNA-sequencing (RNA-Seq) (Fig. 2A). Unsupervised clustering analysis of the gene expression data provided three major observations: First, we found that the oral and skin samples cluster separately indicating distinct transcriptional identities, consistent with unique tissue microenvironments. Interestingly, even though there is a significant amount of differentially regulated genes between oral mucosa and skin during wound healing, most of these differences are already evident at starting basal conditions (day 1) (fig. S1). Secondly, we observe that within the skin samples there is clear separation between baseline biopsies and wound biopsies at days 3 and 6. In fact, an overall upregulation of gene activity in skin is evident during the healing process, indicating non-resolution of the skin wounds at the time points evaluated (Fig. 2A, skin). Finally, and in contrast to the skin, the oral day 3 biopsies separate from day 1 and 6, indicating wound healing activity at day 3 that resolves by day 6, signifying healing and return of gene expression to basal conditions by day 6 after wounding (Fig. 2A, oral). Consistent with these observations, ANOVA analysis of the differential gene expression during wound healing in oral and skin revealed few significant gene expression changes during oral healing at day 3 (410) and none at day 6, while a large number of genes are differentially regulated during skin wound healing at both time points (Skin Day 3 / Day 1 Sci Transl Med. Author manuscript; available in PMC 2019 June 28. Iglesias-Bartolome et al. Page 4 Author Manuscript (1473); Skin Day 6 / Day 1 (1836) (Fig. 2B). These results demonstrate a significantly enhanced transcriptional activity during skin wound healing but minimal differential transcriptional regulation in oral wounds, raising the possibility that the transcriptional regulatory networks responsible for the accelerated healing in oral mucosa are already present in the unwounded state. Author Manuscript Author Manuscript To explore the intrinsic differences between oral mucosa and skin at baseline, we determined the significant differentially expressed genes between unwounded (day 1) oral mucosa and unwounded skin (table S1). Transcripts upregulated in the oral environment were consistent with increased keratinocyte activation and with heightened anti-microbial defenses (Day 1, Fig. 2C). Among the upregulated transcripts in the oral samples, we found genes described in wound- activated keratinocytes (7), including keratins 6 (K6) and 16 (K16), small proline rich (SPRR) and S100 proteins, defensins, serpins and annexins, among others (Fig. 2C). Strikingly, Ingenuity Pathway Analysis (IPA) showed that the top processes represented in the transcriptome of the oral mucosa were related to inflammatory skin disorders such as psoriasis, dermatitis and skin hyperplasia (Fig. 2D), conditions in which transcriptional networks resemble those of the wound-activated skin (811). This indicated that gene networks related to increased proliferation, migration and wound resolution were potentially already present in the oral mucosa at basal state. Since psoriasis has been shown to present a particular expression signature of genes, inflammatory cytokines and proteins related to wound healing, we utilized the gene list from the Psoriasis gene signature in IPA (Fig 2D) to perform an unsupervised clustering of our samples (Fig. 2E). Results showed that skin wound samples (days 3 and 6) cluster more closely with oral samples at all days, away from unwounded skin (Fig. 2E). These results provide supporting evidence that wound-activated networks are present in the normal oral epithelium. Interestingly, psoriasis patients have accelerated wound healing with reduced scarring (11, 12) suggesting that the presence of gene networks related to wound healing at basal state might be key for the accelerated wound healing observed in psoriatic skin and oral mucosa. Analysis of the transcriptome of the oral mucosa also revealed that gene networks related to cell movement and migration are highly activated in the unwounded oral mucosa (fig. S2A), showing upregulation at the basal state of numerous genes linked to epithelial and immune cell migration (fig. S2B). Taken together, these results indicate that wound-activated transcriptional networks are present at basal state in the oral mucosa, priming the epithelium for wound repair. Oral mucosa shows reduced differentiation and more actively sustained inflammatory response during wound healing. Author Manuscript Consistent with the possibility that the transcriptional regulatory networks responsible for the accelerated healing in oral mucosa are already present in the unwounded state, we identified 250 genes (table S2) that are present at high levels in the unwounded oral mucosa but are only upregulated in the skin during wound healing (Fig. 3A, black line in CIRCOS plot). While there is ample evidence of the importance of the extracellular matrix and underlying stroma during re-epithelialization (1), the top most significant gene ontologies (GOs) represented in this gene list where processes related to keratinization, epidermal cell Sci Transl Med. Author manuscript; available in PMC 2019 June 28. Iglesias-Bartolome et al. Page 5 Author Manuscript differentiation and responses to biotic stimulus and bacterium (Fig. 3A and table S3). Terms related to epithelial and immune cell migration where also represented (table S3). These results highlight specific gene networks both, intrinsic to keratinocyte biology and related to immune responses, as critical elements mediating the priming of the oral mucosa to wound repair. Indeed, distinct keratin and structural protein expression profiles between oral mucosa and skin are indicative of inherent differences in the epithelial compartments of these tissues (Fig. 3B). Correspondingly, we found that characteristic signature keratins for each epithelia (13) are present: K4 and K13 in the oral mucosa and K1 and K10 in the cornified skin (Fig. 3C). We also examined reciprocal staining for each keratin (K4 and K10) in skin and oral mucosa (fig. S3A), and confirmed the tissue-specific expression for each keratin. Author Manuscript Importantly, stress and wound-activated keratins, including the keratin 6 family (K6A, K6B and K6C) and K16, are uniquely active in unwounded oral epithelium and maintained highly upregulated during wound healing, while they are only expressed in the skin during the wound healing process (Fig. 3B and 3D). These keratins are essential for the in vivo migratory and structural capacity of keratinocytes in murine oral epithelia and during wound healing (14, 15). An added powerful result from analyzing these human datasets is that it has allowed the identification of distinct expression profiles for genes clustered in the epidermal differentiation complex (EDC) (16). The majority of the EDC genes (S100s, SPRRs and cell envelope precursors) that are upregulated in skin during wounding (Fig. 3B), are already expressed at heightened levels at baseline day 1 in the oral mucosa. Author Manuscript Differentiation markers such as involucrin (IVL) usually present in both epithelia, were downregulated in the oral mucosa during healing, while were present in the migratory tongue of the wounded skin (Fig. 3B and fig. S3B). Interestingly, even though the oral mucosa has a more extensive total area of proliferating cells during wound healing (Fig. 3E), there is no significant difference in the number of proliferating cells when this is corrected for surface area involved (Fig. 3E). In aggregate, these results indicate that oral wounds do not engage differentiation pathways during wound healing, maintaining a larger pool of regenerative epithelial keratinocytes that aid in accelerated wound closure, while the activated keratin expression corroborates that the oral mucosa is primed for wound repair. Author Manuscript The differential expression of structural keratins observed between oral and cutaneous samples, reflects the unique characteristics of these distinct epithelia and highlight the fact that the oral buccal mucosa is non-cornified and therefore more exposed to environmental signals (17). As such, the oral epithelium is increasingly exposed to the commensal microbiota that inhabit barrier surfaces. In fact, the oral environment is home to some of the most rich and diverse microbial communities harbored on human body surfaces (18) and is an environment of constant mechanical stimulation during mastication, shown to induce heightened immune responsiveness (19). In accordance to this concept, our data demonstrate that oral epithelia have increased anti-microbial defenses and immune responses at steady state, but minimally up-regulate inflammatory pathways during the healing process (Fig. 4A and figs. S4AB). In contrast, in the cutaneous microenvironment, inflammatory responses are less active at steady state but become upregulated throughout the healing process and do Sci Transl Med. Author manuscript; available in PMC 2019 June 28. Iglesias-Bartolome et al. Page 6 Author Manuscript not resolve by day 6, suggesting of a chronic inflammatory response when compared to the oral mucosa (Fig. 4AC and fig. S4A). Chronic inflammation is a hallmark of non-healing wounds and over-activation of immune processes during healing has been shown to have detrimental effects on wound resolution, delaying closure and increasing fibrosis and scarring (20). Importantly, several immune mediators including proinflammatory cytokines, chemokines and cyclooxygenases showed higher expression in the skin at basal state and are upregulated in skin continuously through day 6 (Fig. 4B). Additionally, markers of fibrosis, including TGF- (21) are upregulated in skin wounds compared to oral wounds (fig. S4C). Analysis of the gene expression changes in skin wound healing from day 3 to day 6 revealed that although gene networks related to keratinocyte differentiation (peptide cross-linking, keratinization) where upregulated by day 6, additional networks related to immune response are still active at this time (fig. S4D). This further corroborates the non-resolution of the inflammatory response in the skin wounds. Author Manuscript Author Manuscript Histological and quantitative evaluation of the cellular infiltrate during healing reflected an acute response in the oral environment with complete resolution by day 6, consistent with transcriptional responses that revert to baseline by day 6 in the oral environment but continue to amplify in the skin (Fig. 4D). Associated with this rapid and controlled inflammatory response in the oral environment, we observe constitutive activation of several pro-resolution molecules (annexin, SLPI, lipoxygenase, IL1RN (22)) in the oral mucosa (Fig. 4C). In particular, we confirmed that Annexin-A1 and SLPI, two proteins that have been shown to ameliorate wound healing by moderating chronic inflammation (2325), are detected at significantly higher protein levels in the oral mucosa and oral wounds compared to skin (Fig. 4E). Altogether, these results show that oral mucosa is primed for wound repair by a series of wound-signature networks that help control epithelial cell differentiation and regulate pro-inflammatory responses. Transcriptional networks in oral mucosa contribute to rapid wound resolution. Rapid oral versus skin wound healing has previously been identified in mouse and pig models (3, 6, 26) and replicated in vitro in oral keratinocyte models (4), suggesting a conserved mechanism in oral keratinocytes that allows for faster wound resolution. Author Manuscript To identify conserved factors/pathways in oral keratinocytes which may be physiologically relevant for improved healing, we searched databases for common overexpressed transcripts that are present in our human oral mucosa dataset (Fig. 2F), and are overexpressed in mouse oral mucosa (26) in vivo, and in human (27) and mouse oral keratinocytes (3) (fig. S5). This analysis identified eight genes that are consistently upregulated in oral mucosa and oral keratinocytes (Fig. 5A), four of which encode for transcriptional regulators (PAX9, PITX1, PITX2, SOX2), one that encodes the LIM-domain only 7 emerin-binding factor (LMO7) (28) and three that encode factors involved in various oral mucosa biological processes (ALDH3A1, ATP1B1 and IGFBP2) (4, 29, 30). Transcription factors have the potential to reprogram cells to specific developmental states (31). Within the group of oral upregulated transcription factors we found the paired-like homeodomain PITX factors (PITX1 and PITX2), the paired-box homeodomain factor PAX9 and the HMG-domain SOX2, which is part of the SOX family of transcriptional drivers of Sci Transl Med. Author manuscript; available in PMC 2019 June 28. Iglesias-Bartolome et al. Page 7 Author Manuscript somatic cell reprogramming (32) (33, 34). We confirmed the differential expression of the PAX9, PITX1, PITX2 and SOX2 transcriptional regulators in primary human oral (NOK) and skin (NHEK) keratinocytes in vitro and in human biopsies, at baseline and during healing (Fig. 5B and C). We determined that PITX1 and SOX2 are expressed in NOK cells and unwounded oral mucosa while their expression is almost undetectable in NHEK cells and skin. Interestingly, the expression of PITX1 and SOX2 increased upon wounding in the epithelial layer of the oral mucosa. PAX9 shows increased expression in epithelia and dermis after wounding in both oral mucosa and skin, and is also expressed at lower levels in NHEK cells. Author Manuscript To functionally validate the role of these factors in controlling a transcription-regulated wound healing program, we analyzed the gene expression changes in NOK cells treated with siRNAs for PAX9, PITX1, PITX2 and SOX2 (Fig. 6 and fig. S6A). We found that knockdown of these factors results in alterations of gene networks related to cell movement and migration, particularly after PITX1 and SOX2 knockdown (Fig. 6B and fig. S6AC). Indeed, siRNA for PITX1 and SOX2 significantly reduced the migration capacity of NOK cells (Fig. 6C). Both factors achieve this effect by acting on different processes, since SOX2 affects gene networks related to the immune and defense response while PITX1 expression results in the alteration of epidermal developmental and differentiation and keratinization pathways (fig. S6B and C). Author Manuscript We then evaluated the functional effects of overexpressing PITX1 and SOX2 in NHEK cells (Fig. 7 and fig. S7), which do not express these factors under normal conditions (Fig. 5B and C). Transcriptomic analysis of NHEK cells transduced with PITX1 and SOX2 revealed a similar pattern of GO biological process as oral keratinocytes, with SOX2 affecting pathways related to the immune response, including cytokines and antibacterial peptides, and PITX1 expression resulting in the alteration of developmental and differentiation pathways (Fig. 7B). Interestingly, both PITX1 and SOX2 expression in skin keratinocytes resulted in reduced expression of the skin differentiation marker KRT1 and overexpression of the oral epithelial marker cornulin (CRNN) (Fig. 7C and fig. S7B). PITX1 also resulted in increased expression of several markers enriched in the oral epithelium, including keratins (KRT13, KRT78), SPRRs (SPRR2A, SPRR2E, SPRR3), LCE3D, IGFBP2 and ALDH3A1 (Fig. 7C). Remarkably, transduction with PITX1 and SOX2 significantly increased the migratory capacity of NHEK cells (Fig. 7D and fig. S7C). These results show that the differential expression of transcriptional regulators in oral versus skin keratinocytes confers a unique identity to oral keratinocytes and determines the potential of reprogramming skin keratinocytes to acquire increased cell migration capability and improve wound resolution. Author Manuscript To investigate the effect of overexpressing SOX2 in epidermal keratinocyte on cutaneous wound healing in vivo, we generated epidermal-specific SOX2 overexpressing mice (35). Mice expressing a tamoxifen-inducible Cre driven by the keratin 14 promotor (K14CreERTM) were crossed with LSL-SOX2 mice to generate K14CreERTM/LSL-SOX2 mice. Next, we compared the kinetics of cutaneous wound healing in K14CreERTM/LSLSOX2 mice activated or not by tamoxifen to express SOX2 in basal keratinocytes (Fig. 8A). Immunohistochemical analysis showed that tamoxifen treatment induced SOX2 expression in epidermal keratinocytes in K14CreERTM/LSL-SOX2 mice (Fig. 8B). Interestingly, short Sci Transl Med. Author manuscript; available in PMC 2019 June 28. Iglesias-Bartolome et al. Page 8 Author Manuscript term SOX2 expression in the skin resulted in an amplification of the K5 stem cell compartment (Fig. 8B). Wound healing was significantly promoted in SOX2 overexpressing skin from 3 to 9 days after wounding compared to mice treated with vehicle (Fig. 8CD). SOX2 overexpression in epithelial keratinocytes lead to skin acanthosis when compared to control skin (Fig. 8E). Histological analysis in SOX2 overexpressing mice Day 5 after wounding showed a larger migratory tongue than control mice (Fig. 8E). Furthermore, SOX2 overexpression significantly increased PCNA positive proliferating epithelial cells (Fig. 8F). These findings demonstrated that genetic or pharmacological approaches to increase the expression or activity of the SOX2 transcription factor in the skin is an efficacious approach to positively regulate cutaneous wound healing. Author Manuscript DISCUSSION Wound healing is a major medical and social priority and broadening our understanding of the mechanisms involved in wound repair is needed to improve wound care. While oral wounds, in distinction to cutaneous wounds, are able to heal quicker with far less complications, the lack of detailed comparative analysis in humans prevented the advancement of our knowledge in this area. Buccal mucosa, gingiva and palate show similar accelerated wound healing when compared to cutaneous wounds (3) (21), despite all having different keratinization and terminal differentiation profiles. This indicates that the oral cavity in general is endowed with mechanisms to increase wound resolution that go beyond local differences in epithelial structure. Author Manuscript In this study, by analyzing paired samples of human oral buccal mucosa and skin at basal conditions and during wound healing, we identify the physiological and molecular determinants for this repair paradigm. Our findings could have widespread implications for wound healing study and interventions. Pathways and/or molecules characterized in this study may facilitate rapid, scar- less healing that can be considered for application to nonoral mucosal sites to promote healing. Author Manuscript To identify and explain the mechanisms that define accelerated wound healing, we analyzed the gene expression signature changes during oral mucosal and skin wound resolution in healthy human subjects. Even before wounding, homeostatic oral mucosa is equipped with transcriptional networks that prime the epithelium for wound repair. In addition, our data indicates that the most significant processes driving the repair in acute wounds of healthy individuals are keratinocyte-driven. These networks are determined in part by the differential expression of a set of transcriptional regulators in oral vs skin keratinocytes, indicating that pathways established during development are responsible for the differential wound resolution capacity of these cells. This priming allows the oral mucosa to rapidly control and limit inflammatory responses, leading to fast inflammatory resolution. In addition, oral keratinocyte activation and reduced differentiation allows for a rapid re-epithelialization of the wound area. Indeed, it has been recently shown that re-epithelialization in mice is dependent on tissue-scale coordination of Sci Transl Med. Author manuscript; available in PMC 2019 June 28. Iglesias-Bartolome et al. Page 9 Author Manuscript proliferation, differentiation and migration (36), with acquisition of stem cell properties in de-differentiated epidermal cells (37). In the long term of human wound repair, the reduced expression of cytokines and fibrosis mediators such as annexin 1 and SLPI potentially contributes to the scarless wound healing observed in the oral mucosa. Author Manuscript Author Manuscript At the core of oral wound healing are transcriptional regulators that hold the key to the activation of the molecular events responsible for accelerated wound resolution. In this study we identify eight factors that define oral keratinocytes and demonstrate that two of them, the transcription factors SOX2 and PITX1, regulate networks involved in wound closure. The SOX and PITX family of transcription factors have important roles in development, ranging from regulation of cell fate to axis and pattern formation (32,34). Our results support that these transcription factors are responsible in establishing an oral mucosa-specific network that primes this epithelia for rapid and efficient wound healing. Furthermore, we show that SOX2 and PITX1 can be exploited to reprogram skin keratinocytes to present oral keratinocyte features, including accelerated wound resolution both in vitro and in vivo. SOX2 in particular is a key factor in adult stem cell maintenance for a myriad of epithelial tissues (38) and has been shown to induce amplification of stem cell in the skin (39). Confirming these results, we show that SOX2 induces an expansion of the K5+ basal/stem cell compartment of the skin in mice, indicating that the wound regenerative capacity of SOX2 might be linked to its stem cell regulatory functions. On the other hand, our data suggests that PITX1 regulates a different set of processes related to the expression of structural proteins, including keratins, LCEs and SPRRs. It is worth noting that PITX2, a member of the PITX family, is an essential component of the genetic network activated by tissue damage during heart repair (40). More studies are needed to identify the specific pathways activated by these transcriptional regulators and their potential for wound repair and tissue regeneration. Overall, we present a unique combination of human clinical data and histological and gene expression analysis that provides a comprehensive comparative analysis of the molecular and cellular mechanisms underlying the different wound healing processes in oral and skin epithelia. Our data indicates that the unique environment of the oral cavity represents a radically different wound-healing program geared towards rapid wound-resolution at every step (fig. S9). Ultimately, this human transcriptomic dataset highlights fundamental global mechanisms of inflammation and repair in humans that will serve as an invaluable resource, providing insights into therapeutic targeting of chronic and non-healing wounds. Supplementary Material Author Manuscript Refer to Web version on PubMed Central for supplementary material. Acknowledgments This work was supported by the Intramural Research Programs of the National Institute of Arthritis and Musculoskeletal and Skin Diseases (M.I.M ZIA AR041124) and the National Institute of Dental and Craniofacial Research (J.S.G. Z01DE00558) of the National Institutes of Health. We thank members of the Laboratory of Skin Biology (LSB) and of the Laboratory of Cancer Biology and Genetics (LCBG) for helpful suggestions and discussions. We also thank Gustavo Gutierrez-Cruz and Stefania DellOrso of the NIAMS Genome Analysis Core Facility and the NIAMS Light Imaging Core Facility. This work utilized the computational resources of the NIH Sci Transl Med. Author manuscript; available in PMC 2019 June 28. Iglesias-Bartolome et al. Page 10 Author Manuscript HPC Biowulf Cluster. We would like to express our gratitude to the participants of the clinical trial #NCT01078467 and research teams whose contributions made this work possible. 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Author Manuscript (A) Schematic of spatiotemporal human biopsy sample collection; Clinicaltrials.gov #NCT01078467. Baseline biopsies were performed to create paired identical wounds in the oral mucosa and skin (day 1). Follow-up biopsies of the wound area(s) were collected at day 3 and day 6 of healing in two different groups. (B) Table showing the time-course of the clinical study and biopsy sampling, and representative H&E pictures of longitudinal sections of biopsies taken at day 1. Thirty healthy subjects were randomized in three groups (with 10 subjects on each group). SM: smooth muscle. See Methods section for more information. (C) Representative pictures and healing time course of oral wounds and skin wounds in group 1 (after 3 mm primary biopsy), and group 2 (after 5 mm secondary biopsy). Markings in millimeters. Error bars represent SDs. Number of samples for each group as follows: 3 mm wounds, day 1 n=29, day 3 n=30, day 6 n=20, day 9 n=9, day 13 n=9, day 15 n=9; 5mm wounds, day 1 n=11, day 3 n=20, day 9 n=21, day 13 n=21, day 15 n=21. (D) Graphs show Sci Transl Med. Author manuscript; available in PMC 2019 June 28. Iglesias-Bartolome et al. Page 13 Author Manuscript healing rate of 3 mm wounds in group 1 and 5 mm wounds in group 2 as well as the contraction percentage immediately after 3 mm biopsy in group 1 and 5 mm biopsy in group 2. n.s.: not significant, *p<0.05. Error bars represent SDs. (E) Representative H&E pictures of oral and skin wounds at day 3 and 6. At day 3 there is almost complete epithelization of the wound, with migratory tongue growing on top of blood clot. In the skin, migratory epithelial tongue is growing under the scab that covers the wound. At day 6, oral wounds are completely resolved, while skin wounds are still undergoing re-epithelialization. Magnification of dotted box is shown on the right. In magnification, epithelium is marked with a dotted line. Author Manuscript Author Manuscript Author Manuscript Sci Transl Med. Author manuscript; available in PMC 2019 June 28. Iglesias-Bartolome et al. Page 14 Author Manuscript Author Manuscript Author Manuscript Figure 2. Wound-activated transcriptional networks are present in the unwounded oral mucosa. Author Manuscript (A) Schematic representation of biopsy site in the mucosa of the cheek and posterior axillary region of the arm (left) and unsupervised clustering analysis of RNA-Seq gene expression data of the 24 paired samples at Day 1, 3 and 6. O: oral, S: skin. Numbers indicate matching subject. Paired oral and skin samples were chosen randomly from four subjects for each day (24 total samples from 12 individual subjects) and were a mix of males and females. (B) CIRCOS plot of the differential gene expression during wound healing (ANOVA). No significant differences were found on Oral Day 6 versus Day 1 (Day 6/1). Ribbon connectors indicate the same genes present in different datasets. Number of genes with differential expression in each comparison: Oral Day 3 versus Day 1 (Day 3/1): 410 genes, Skin Day 3 versus Day 1 (Day 3/1): 1473 genes, Skin Day 6 versus Day 1 (Day 6/1): 1836 genes. See fig. S1a for explanation on CIRCOS plot. (C) Volcano plot indicating differential gene expression between unwounded (Day 1) oral mucosa and skin. Magnification on right panel, highlights the some of the most significantly upregulated genes in the oral mucosa over the skin. (D) IPA analysis results showing Diseases and Functions terms found in upregulated Sci Transl Med. Author manuscript; available in PMC 2019 June 28. Iglesias-Bartolome et al. Page 15 Author Manuscript genes in the oral mucosa over the skin. (E) Unsupervised hierarchical clustering using a psoriasis gene signature with the gene expression of oral mucosa and skin data at baseline (Day 1) and during wound healing (Day 3 and 6). Author Manuscript Author Manuscript Author Manuscript Sci Transl Med. Author manuscript; available in PMC 2019 June 28. Iglesias-Bartolome et al. Page 16 Author Manuscript Author Manuscript Author Manuscript Figure 3. Oral keratinocytes show reduced differentiation during wound healing. Author Manuscript (A) CIRCOS plot summarizing cross-reference of transcripts upregulated during skin wound healing (Skin D3/D1, Skin D6/D1, D=day) with those upregulated in the oral mucosa with respect to the skin at basal conditions (Oral D1/Skin D1). Black line indicates genes upregulated in the unwounded oral mucosa that are upregulated during skin wound healing (250 unique genes; see Table S2). Right panel, graph indicates GO biological process terms enriched in Oral D1/Skin D1 dataset. Ribbon connectors indicate that the same genes are present in different datasets. Oral D1/Skin D1: 760 genes, Skin D3/D1: 971 genes, Skin D3/D1: 1089 genes. (B) Relative mRNA expression levels of keratinization and epidermal cell differentiation markers throughout the wound healing process. (C) Representative pictures of unwounded (D1) and wounded (D3 and D6) oral mucosa and skin stained to show expression of the basal marker keratin 5 (K5, red), and differentiation markers keratin 4 (K4) for the oral mucosa and keratin 10 (K10) for the skin (green).(D) Representative Sci Transl Med. Author manuscript; available in PMC 2019 June 28. Iglesias-Bartolome et al. Page 17 Author Manuscript pictures of unwounded (D1) and wounded (D3) oral mucosa and skin stained to show expression of the basal marker cytokeratin 5 (K5, red), and activated epithelium keratin 6 (K6, green). (E) Representative pictures of unwounded (D1) and wounded (D3 and D6) oral mucosa and skin stained to show expression of the basal marker keratin 5 (K5, green), and proliferation marker PCNA (red). Quantification of number of cells positive for the proliferation marker PCNA per area (mm2). **p<0.01 oral vs skin day 6, no asterisk means not statistically different between oral and skin. Error bars represent SDs of three independent samples. Magnification of the dotted box is shown on the right of each picture. For D1 magnification shows the basal, unwounded expression of corresponding marker. For D3 and D6 magnification shows the migratory tongue or wound area, A, or an adjacent epithelial area to the wound, B. Author Manuscript Author Manuscript Author Manuscript Sci Transl Med. Author manuscript; available in PMC 2019 June 28. Iglesias-Bartolome et al. Page 18 Author Manuscript Author Manuscript Author Manuscript Figure 4. Inflammatory pathways are more active and sustained in skin wounds that in oral wounds. Author Manuscript (A) IPA analysis results showing diseases and functions terms found in differentially regulated genes during the wound healing process included terms related to inflammatory processes. (B) CIRCOS plot showing the genes exclusively upregulated during skin wound healing (black line) and GO biological process terms enriched in this dataset. Ribbon connectors indicate that the same genes are present in different datasets. Oral Day 3/Day 1: 276 genes, Skin Day 3/Day 1: 971 genes. D=day. (C) Relative mRNA expression levels of interleukins, chemokines and other inflammatory regulators during wound healing. (D) Representative pictures of recruitment of immune cells during the wound healing process in the oral mucosa and the skin. Bottom panels show quantification of recruitment of specific immune cell types during the wound healing process in the oral mucosa and the skin. * p<0.05, ** p<0.01, no asterisk means not statistically different, comparisons between day 3 and day 6 vs day 1 oral or skin respectively. Error bars represent SDs of three independent Sci Transl Med. Author manuscript; available in PMC 2019 June 28. Iglesias-Bartolome et al. Page 19 Author Manuscript samples. (E) Representative images of unwounded (D1) and wounded (D3) oral mucosa and skin stained to detect expression of the basal marker keratin 5 (K5, red), and the immunemodulators SLPI (green) and AnnexinA1 (ANXA1, magenta). Magnification of dotted box is shown on the right of each picture. Author Manuscript Author Manuscript Author Manuscript Sci Transl Med. Author manuscript; available in PMC 2019 June 28. Iglesias-Bartolome et al. Page 20 Author Manuscript Author Manuscript Author Manuscript Figure 5. Transcriptional networks in oral keratinocytes contributing to rapid wound resolution. Author Manuscript (A) Relative mRNA expression levels in the wound healing dataset of genes consistently upregulated in oral mucosa and oral keratinocytes, presented by fold change (FC) of the unwounded oral mucosa with respect to the unwounded skin (O1/S1). (B) Protein levels of PAX9, PITX2, PITX1 and SOX2 in primary cultures of human oral (NOK) and skin (NHEK) keratinocytes. (C) Representative pictures of unwounded (D1) and wounded (D3 and D6) oral mucosa and skin stained to show expression of the basal marker keratin 5 (K5, red), and the indicated transcription factor (green). Magnification of the dotted box is shown on the right of each picture. For D1 magnification shows the basal, unwounded expression of corresponding marker. For D3 and D6 magnification shows the migratory tongue or wound area, A, or an adjacent epithelial area to the wound, B. Sci Transl Med. Author manuscript; available in PMC 2019 June 28. Iglesias-Bartolome et al. Page 21 Author Manuscript Author Manuscript Author Manuscript Figure 6. Knockdown of oral signature genes in primary oral keratinocytes. Author Manuscript (A) Protein level of SOX2, PITX1 and total protein (RPS14) after transfection with respective siRNAs in NOK cells. (B) IPA analysis of RNA-seq data from NOK cells treated with siRNAs for SOX2 and PITX1: Diseases and functions terms related to migration and cell movement found in differentially regulated genes. *p0.05 (C) Relative cell migration length of NOK cells transduced with siRNA for SOX2, PITX1 and siControl. Values were determined by 3 microscopic fields in n=3 per group. *p < 0.05 siSOX2 and siPITX1 versus control. Error bars represent SDs of three independent experiments. Sci Transl Med. Author manuscript; available in PMC 2019 June 28. Iglesias-Bartolome et al. Page 22 Author Manuscript Author Manuscript Author Manuscript Figure 7. Overexpression of SOX2 and PITX1 in primary skin keratinocytes. Author Manuscript (A) Representative pictures of NHEK cells transduced with indicated adenoviruses and stained to show overexpression of corresponding proteins. No expression of PITX1 or SOX2 was observed in non-transduced cells (not shown). Bar= 20m. (B) GO biological process terms enriched in datasets of genes differentially regulated by PITX1 and SOX2 overexpression in NHEK cells. (C) Fold change of the expression levels of genes related to differentiation and response to biotic stimulus in NHEK cells transduced with PITX1 and SOX2, presented as log2 fold change (log FC) over GFP expression. (D) Migrating NHEK cells transduced with SOX2, PITX1 and GFP (control) by adenoviral delivery. Images were taken at 0 and 24hs after removal of silicone insert. Values were determined by counting the number of migrating cells at 24hs in 6 microscopic fields in n=3 per group. **p < 0.01, *p < 0.05 adSOX2 and adPITX1 versus control. Error bars represent SDs of three independent experiments. Sci Transl Med. Author manuscript; available in PMC 2019 June 28. Iglesias-Bartolome et al. Page 23 Author Manuscript Author Manuscript Author Manuscript Figure 8. Conditional overexpression of SOX2 contributed to the promotion of cutaneous wound healing. Author Manuscript (A) Schematic representation of the experimental design used for K14CreERTM/LSL-SOX2 mice. Mice are treated with vehicle or tamoxifen five consecutive days by topical application on dorsal skin (Day 1-Day 5). The wounds were created as a 6 mm full-thickness excisional dorsal skin wound by biopsy punch (Day 6). (B) Representative images of unwounded skin stained to show expression of SOX2 (red), and the basal marker keratin 5 (K5, green) in K14CreERTM/LSL-SOX2 mice treated with ethanol as vehicle or tamoxifen. Bar = 50 um. (C) Photographs of the wound areas after topical treatment with vehicle or tamoxifen in K14CreERTM/SOX2 mice at 1, 3, 5, 7, and 9 days after wounding. Bar=1mm. (D) Percent wound area at each time point relative to the original wound area in K14CreERTM/LSLSOX2 mice treated with vehicle or tamoxifen. Quantification of the wound areas in n = 7 wounds per groups was performed using Image J software. *p < 0.05, **p < 0.01 tamoxifen versus vehicle at each day. Error bars represent SDs of seven wounds. (E) Representative Sci Transl Med. Author manuscript; available in PMC 2019 June 28. Iglesias-Bartolome et al. Page 24 Author Manuscript H&E-stained section Day 1(un-wounded) and Day5 (during wound healing, wound edges including epithelial tongue). Skin sections were from K14CreERTM/LSL-SOX2 mice after treatment with tamoxifen or vehicle. Author Manuscript Author Manuscript Author Manuscript Sci Transl Med. Author manuscript; available in PMC 2019 June 28.  ...