STAT6-targeting antisense oligonucleotides against solitary fibrous tumor.

Solitary fibrous tumor (SFT) is a rare, non-hereditary soft tissue sarcoma thought to originate from fibroblastic mesenchymal stem cells. The etiology of SFT is thought to be due to an environmental intrachromosomal gene fusion between NGFI-A-binding protein 2 (NAB2) and signal transducer and activator protein 6 (STAT6) genes on chromosome 12, wherein the activation domain of STAT6 is fused with the DNA-binding domain of NAB2 resulting in the oncogenesis of SFT. All NAB2-STAT6 fusion variations discovered in SFTs contain the C-terminal of STAT6 transcript, and thus can serve as target site for antisense oligonucleotides (ASOs)-based therapies. Indeed, our in vitro studies show the STAT6 3' untranslated region (UTR)-targeting ASO (ASO 993523) was able to reduce expression of NAB2-STAT6 fusion transcripts in multiple SFT cell models with high efficiency (half-maximal inhibitory concentration: 116-300 nM). Encouragingly, in vivo treatment of SFT patient-derived xenograft mouse models with ASO 993523 resulted in acceptable tolerability profiles, reduced expression of NAB2-STAT6 fusion transcripts in xenograft tissues (21.9%), and, importantly, reduced tumor growth (32.4% decrease in tumor volume compared with the untreated control). Taken together, our study established ASO 993523 as a potential agent for the treatment of SFTs.

Reduction of Tumor Growth with RNA-Targeting Treatment of the NAB2-STAT6 Fusion Transcript in Solitary Fibrous Tumor Models.

Solitary fibrous tumor (SFT) is a rare soft-tissue sarcoma. This nonhereditary cancer is the result of an environmental intrachromosomal gene fusion between NAB2 and STAT6 on chromosome 12, which fuses the activation domain of STAT6 with the repression domain of NAB2. Currently there is not an approved chemotherapy regimen for SFTs. The best response on available pharmaceuticals is a partial response or stable disease for several months. The purpose of this study is to investigate the potential of RNA-based therapies for the treatment of SFTs. Specifically, in vitro SFT cell models were engineered to harbor the characteristic NAB2-STAT6 fusion using the CRISPR/SpCas9 system. Cell migration as well as multiple cancer-related signaling pathways were increased in the engineered cells as compared to the fusion-absent parent cells. The SFT cell models were then used for evaluating the targeting efficacies of NAB2-STAT6 fusion-specific antisense oligonucleotides (ASOs) and CRISPR/CasRx systems. Our results showed that fusion specific ASO treatments caused a 58% reduction in expression of fusion transcripts and a 22% reduction in cell proliferation after 72 h in vitro. Similarly, the AAV2-mediated CRISPR/CasRx system led to a 59% reduction in fusion transcript expressions in vitro, and a 55% reduction in xenograft growth after 29 days ex vivo.

Patient preference for the pre-anesthesia evaluation: Telephone versus in-office assessment.

STUDY OBJECTIVE: Pre-anesthesia evaluation (PAE) is designed to reduce patient and family anxiety, identify pre-existing health issues, avoid surgical delays, minimize costs, and tailor an anesthetic plan. If PAE requires a clinic visit, patients must take time off work and may incur travel and childcare costs. A telephone-based Preoperative Assessment Clinic can minimize patient inconvenience, while maintaining high-quality patient care and improving efficiency. We assessed patient satisfaction with a telephone PAE and determined whether patients preferred a telephone PAE or a conventional clinic visit. DESIGN: Prospective, institutional review board-approved study. SETTING: University hospital. PATIENTS: We conducted an IRB-approved telephone survey of 75 adult, post-operative patients. INTERVENTIONS: Telephone survey. MEASUREMENTS: Patients were asked about their preference for a telephone PAE over an in-person evaluation. Survey questions included assessment of patient satisfaction with their anesthesia evaluation, operation, and anesthetic delivered. Delays and day of surgery cancellations were reviewed. MAIN RESULTS: The majority (97%) of patients stated they preferred a telephone PAE. Patient satisfaction was unaffected by driving distance (30±54 mi), ASA physical status or duration of surgery (169±159 min). Even patients who were not satisfied with their anesthetic (N=5) still preferred the telephone-based PAE. No increase in surgical delays or cancellation was noted. CONCLUSION: The majority of patients in this survey preferred a telephone PAE. Given the large catchment area of our hospital of nine counties, telephone-based interviews add to patient convenience and likely increase compliance with the PAE. Even patients who live in close proximity to our hospital (<5 mi) preferred a telephone assessment. A telephone-based PAE provides high patient satisfaction over a traditional office visit while increasing patient convenience. Larger studies are necessary to ensure that telephone PAEs compare well with in-person examinations.

Intraoperative Hemifacial Composite Flap Perfusion Assessment Using Spatial Frequency Domain Imaging: A Pilot Study in Preparation for Facial Transplantation.

BACKGROUND: Vascularized composite allotransplantation represents an important advancement in the field of reconstructive microsurgery and has continued to increase in popularity. The significant clinical morbidity associated with flap failure represents an important barrier to even more widespread use of these techniques. Early identification of vascular compromise has been associated with a higher salvage rate, yet most surgeons rely only on clinical assessment intraoperatively. Spatial frequency domain imaging (SFDI) presents a noncontact, objective measurement of tissue oxygenation over a large field of view. This study aims to evaluate the use of SFDI technology in hemifacial composite flap compromise as could occur during facial transplant. METHODS: Six composite hemifacial flaps were created in three 35-kg Yorkshire pigs and continuously imaged using SFDI before, during, and after 15-minute selective vascular pedicle occlusion. Arterial and venous clamping trials were performed for each flap. Changes in oxyhemoglobin concentration, deoxyhemoglobin concentration, and total hemoglobin were quantified over time. RESULTS: The SFDI successfully measured changes in oxygenation parameters in all 6 composite tissue flaps. Significant changes in oxyhemoglobin, deoxyhemoglobin, and total hemoglobin were seen relative to controls. Early and distinct patterns of alteration were noted in arterial and in venous compromise relative to one another. CONCLUSIONS: The need for noninvasive, reliable assessment of composite tissue graft viability is apparent, given the morbidity associated with flap failure. The results of this study suggest that SFDI technology shows promise in providing intraoperative guidance with regard to pedicle vessel integrity during reconstructive microsurgery.

Near-infrared imaging for the assessment of anastomotic patency, thrombosis, and reperfusion in microsurgery: a pilot study in a porcine model.

BACKGROUND: Advances in microsurgical techniques have increased the use of free tissue transfer. Methods of intraoperative flap perfusion assessment, however, still rely primarily on subjective evaluation of traditional clinical parameters. Anastomotic thrombosis, if not expeditiously identified and revised, can result in flap loss with significant associated morbidity. This study aims to evaluate the use of near-infrared (NIR) fluorescence imaging in the assessment of microsurgical anastomotic patency, thrombosis, and vascular revision. MATERIALS AND METHODS: A model of pedicle thrombosis was created using bilateral abdominal flaps isolated on deep superior epigastric vascular pedicles in four Yorkshire pigs. Following flap elevation, microvascular arterial and venous anastomoses were performed unilaterally, preserving an intact contralateral control flap. Thrombosis was induced at the arterial anastomosis site using ferric chloride, and both flaps imaged using NIR fluorescence angiography. The thrombosed vascular segments were subsequently excised and new anastomoses performed to restore flow. Follow-up imaging of both flaps was then obtained to confirm patency using fluorescence imaging technology. RESULTS: Pedicled abdominal flaps were created and successful anastomotic thrombosis was induced unilaterally in each pig. Fluorescence imaging technology identified large decreases in tissue perfusion of the thrombosed flap within 2 minutes. After successful revision anastomosis, NIR imaging demonstrated dramatic increase in flow to the reconstructed flap, but intensity did not return to pre-thrombosis levels. CONCLUSIONS: Early identification of anastomotic thrombosis is important in successful free tissue transfer. Real-time, intraoperative evaluation of flap perfusion, anastomotic thrombosis, and successful revision can be performed using NIR fluorescence imaging.

Evaluation of wait times for patients seeking cosmetic and reconstructive breast surgery.

BACKGROUND: Patients seeking cosmetic or reconstructive procedures in plastic surgery typically face significant wait times for consultations. Little attention has been given to potential disparities in wait times between elective cosmetic and reconstructive procedures. In this initial pilot study, we audited a broad sample of plastic surgery offices within a single state for wait times in initial consultations for both breast reconstruction and breast augmentation. METHODS: A sample of board-certified plastic surgeons was audited from the American Society of Plastic Surgeons (ASPS) Web site that listed both cosmetic and reconstructive breast surgery. Scripted patient telephone calls were made to 67 plastic surgery clinics within a single state on May 2012. Two calls separated by 7 days were made to each office by the same actor seeking an initial appointment for either breast reconstruction or breast augmentation. Wait times were calculated from the date of the call until the date of appointment offered. RESULTS: There were 72 paired calls completed on 36 plastic surgery clinics. Significant disparities in appointment wait times existed between elective cosmetic versus reconstructive procedures (P = 0.02). Mean wait times for breast reconstruction consultation (26.1 days) were significantly longer than mean consultation wait times for breast augmentation (20.9 days). Interestingly, 17.9% of offices contacted no longer perform certain procedures currently advertised on the ASPS Web site. CONCLUSIONS: Disparities exist in access to care between patients seeking elective breast augmentation and reconstruction after mastectomy. Patients seeking breast augmentation have more rapid access to plastic surgeons. This study did not evaluate possible explanations for the observed differences. Potential causes may include physician preference and compensation benefits for cosmetic procedures.

Risk factors associated with complications in lower-extremity reconstruction with the distally based sural flap: a systematic review and pooled analysis.

The distally based sural fasciocutaneous flap is one of the few options available for local flap reconstruction of soft-tissue defects in the lower one-third of the leg. Few studies have assessed risk factors associated with poor outcomes in this flap. A literature search was performed of MEDLINE, EMBASE, CINAHL and the Cochrane Library for articles evaluating the use of sural artery fasciocutaneous flaps for soft-tissue reconstruction of the leg. Data were pooled and a univariate analysis was performed to identify characteristics associated with increased morbidity. A logistic regression model was created, and odds ratios and p values associated with the development of complications were calculated. Sixty-one papers were identified which included data on 907 patients. The majority of sural flaps were used to cover defects of the heel (28.2%), foot (14.4%) or ankle (25.8%). Trauma was the most common indication, followed by ulcers and open fractures. Flap complications were recorded in 26.4% of cases with a flap loss rate of 3.2%. With multivariate analysis, venous insufficiency and increasing age were independent risk factors for complications. Patients with venous insufficiency had nine times the risk of developing a complication compared to patients without venous insufficiency.

Effects of statins on ischemia-reperfusion complications in breast free flaps.

BACKGROUND: Administration of statins or other cardiovascular medications (CVMs) could potentially protect against the development of ischemia-reperfusion (I/R) injury in free flap reconstruction. The aim of this study was to examine whether the use of statins and other CVMs decreased the rate of I/R injury in autologous free flap breast reconstruction. METHODS: Retrospective chart review was performed on women who had undergone mastectomy and autologous free flap breast reconstruction between 2004 and 2010. Patient characteristics, use of statin and/or CVMs, and I/R-related complications were ascertained. Multivariable logistic regression was used to identify associations between independent risk factors and specific complications. RESULTS: There were 702 free flap breast reconstructions included in this study; 45 performed in patients on statins, 70 in patients on CVMs, and 38 in patients on both. Overall complication rate in patients on statins and patients on CVMs was significantly higher than those not on any medication (46.7% versus 31.5%, P=0.037 and 45.7% versus 31.5%, P=0.017, respectively). When I/R complications were pooled, there were no significant differences between patients not on any medications and those on statins (P=0.26), CVMs (P=0.18), and both (P=0.83.) CONCLUSIONS: Although there may be theoretical pharmacologic benefits of statins and/or CVMs to reduce the incidence of IR injury in autologous free flap breast reconstruction, the results of this study showed no clear advantages when these drugs were used.

Disparity between reported and measured patient weight: can it affect planning in breast reduction surgery?

BACKGROUND: Patient weights are frequently used in surgery for essential calculations including preoperative surgical planning, dosing of medications, and qualification for insurance reimbursement. Often, there is a disparity between patient-reported and actual measured weights. This study examines differences between self-reported and measured weights and implications in breast reduction surgery. METHODS: A review was performed of 197 consecutive women who presented for breast surgery at an academic institution. Self-reported weights were recorded during the initial encounter, and the patients were subsequently weighed on the same day. A paired t-test was used to compare the self-reported and actual measured weights and stratified analysis performed based on body mass index (BMI). The Schnur sliding scale was used to estimate resection weights for reduction mammaplasty. RESULTS: The overall mean difference in reported and measured weight was 3.0 lbs standard deviation (SD) 8.9 (P < 0.0001) with a maximum overestimation of 25 lb and underestimation of 80 lb. Statistically significant differences were found when stratified analysis was performed based on BMI as mean differences in the overweight (BMI 25-30) and obese (BMI > 30) groups were 1.7 lb SD 5.5 (P < 0.026) and 4.9 lbs SD 11.8 (P < 0.0002), respectively. Significant differences in calculated breast reduction resection weights, based on the Schnur sliding scale, were also found when comparing self-reported and measured weights in all groups. CONCLUSIONS: Significant disparities between self-reported and measured weights were identified in patients presenting for breast surgery. These differences can influence important calculations of resection weights for breast reduction surgery. These differences may also affect dosing of medications and preoperative planning.

A novel pilot study using spatial frequency domain imaging to assess oxygenation of perforator flaps during reconstructive breast surgery.

INTRODUCTION: Although various methods exist for monitoring flaps during reconstructive surgery, surgeons primarily rely on assessment of clinical judgment. Early detection of vascular complications improves rate of flap salvage. Spatial frequency domain imaging (SFDI) is a promising new technology that provides oxygenation images over a large field of view. The goal of this clinical pilot study is to use SFDI in perforator flap breast reconstruction. METHODS: Three women undergoing unilateral breast reconstruction after mastectomy were enrolled for our study. The SFDI system was deployed in the operating room, and images acquired over the course of the operation. Time points included images of each hemiabdominal skin flap before elevation, the selected flap after perforator dissection, and after microsurgical transfer. RESULTS: Spatial frequency domain imaging was able to measure tissue oxyhemoglobin concentration (ctO2Hb), tissue deoxyhemoglobin concentration, and tissue oxygen saturation (stO2). Images were created for each metric to monitor flap status and the results quantified throughout the various time points of the procedure. For 2 of 3 patients, the chosen flap had a higher ctO2Hb and stO2. For 1 patient, the chosen flap had lower ctO2Hb and stO2. There were no perfusion deficits observed based on SFDI and clinical follow-up. CONCLUSIONS: The results of our initial human pilot study suggest that SFDI has the potential to provide intraoperative oxygenation images in real-time during surgery. With the use of this technology, surgeons can obtain tissue oxygenation and hemoglobin concentration maps to assist in intraoperative planning; this can potentially prevent complications and improve clinical outcome.

Patient involvement in the decision-making process improves satisfaction and quality of life in postmastectomy breast reconstruction.

BACKGROUND: The patient-physician relationship has evolved from the paternalistic, physician-dominant model to the shared-decision-making and informed-consumerist model. The level of patient involvement in this decision-making process can potentially influence patient satisfaction and quality of life. In this study, patient-physician decision models are evaluated in patients undergoing postmastectomy breast reconstruction. METHODS: All women who underwent breast reconstruction at an academic hospital from 1999-2007 were identified. Patients meeting inclusion criteria were mailed questionnaires at a minimum of 1 y postoperatively with questions about decision making, satisfaction, and quality of life. RESULTS: There were 707 women eligible for our study and 465 completed surveys (68% response rate). Patients were divided into one of three groups: paternalistic (n = 18), informed-consumerist (n = 307), shared (n = 140). There were differences in overall general satisfaction (P = 0.034), specifically comparing the informed group to the paternalistic group (66.7% versus 38.9%, P = 0.020) and the shared to the paternalistic group (69.3% versus 38.9%, P = 0.016). There were no differences in aesthetic satisfaction. There were differences found in the SF-12 physical component summary score across all groups (P = 0.033), and a difference was found between the informed and paternalistic groups (P < 0.05). There were no differences in the mental component score (P = 0.42). CONCLUSIONS: Women undergoing breast reconstruction predominantly used the informed model of decision making. Patients who adopted a more active role, whether using an informed or shared approach, had higher general patient satisfaction and physical component summary scores compared with patients whose decision making was paternalistic.

Fat necrosis in autologous abdomen-based breast reconstruction: a systematic review.

BACKGROUND: Fat necrosis is a common and potentially exasperating complication of autologous breast reconstruction. The authors performed a systematic review of the English literature on autologous breast reconstruction to determine significant patient and surgical factors that are predictors of postoperative fat necrosis. METHODS: A PubMed search using the terms "fat necrosis" and "breast reconstruction" was conducted. Articles were screened using predetermined inclusion and exclusion criteria. Data collected included patient characteristics, reconstructive techniques used, and the specific postoperative morbidity of interest. Patient cohorts were pooled, and the incidence of fat necrosis was calculated in the presence and absence of each risk factor. Chi-square analysis was applied, and p < 0.05 was considered statistically significant. RESULTS: Of 172 articles found, 70 met the inclusion criteria. The mean rate of fat necrosis was 11.3 percent. Deep inferior epigastric artery perforator flaps had the highest rate of fat necrosis (14.4 percent), followed by pedicled transverse rectus abdominis musculocutaneous (12.3 percent), superficial inferior epigastric artery (8.1 percent), and free transverse rectus abdominis musculocutaneous flaps (6.9 percent). Significant predictors of fat necrosis included obesity (p = 0.035), prereconstruction irradiation (p = 0.022), postreconstruction irradiation (p < 0.001), active smoking (p < 0.001), and abdominal scars (p = 0.05). Protective factors included supercharging (p < 0.001) and bilateral reconstruction (p = 0.01). CONCLUSIONS: Although there is little agreement in the literature regarding risk factors for fat necrosis, the authors were able to demonstrate several significant predictors by systematically analyzing 70 articles. Improved knowledge of the risk factors for fat necrosis can help surgeons provide improved preoperative counseling and take measures to minimize the risk of this complication.

Bone flap perfusion assessment using near-infrared fluorescence imaging.

BACKGROUND: Microsurgical vascularized bone flaps are a versatile technique for reconstructing large bone defects. However, the assessment of perfusion is challenging, because clinical examination is difficult intraoperatively and often not possible postoperatively. Therefore, it is important to develop techniques to assess the perfusion of vascularized bone flaps and potentially improve the surgical outcomes. Near-infrared (NIR) fluorescence imaging has previously been shown to provide real-time, intraoperative evaluation of vascular perfusion. The present pilot study investigated the ability of NIR imaging to assess the perfusion of vascularized bone flaps. METHODS: Vascularized bone flaps were created in female Yorkshire pigs using well-established models for porcine forelimb osteomyocutaneous flap allotransplantation (n = 8) and hindlimb fibula flaps (n = 8). Imaging of the bone flaps was performed during harvest using the FLARE intraoperative fluorescence imaging system after systemic injection of indocyanine green. Perfusion was also assessed using the standard of care by clinical observation and Doppler ultrasonography. NIR fluorescence perfusion assessment was confirmed by intermittent clamping of the vascular pedicle. RESULTS: NIR fluorescence imaging could identify bone perfusion at the cut end of the osteotomy site. When the vascular pedicle was clamped or ligated, NIR imaging demonstrated no fluorescence when injected with indocyanine green. With clamp removal, the osteotomy site emitted fluorescence, indicating bone perfusion. The results using fluorescence imaging showed 100% agreement with the clinical observation and Doppler findings. CONCLUSIONS: Vascularized bone transfers have become an important tool in reconstructive surgery; however, no established techniques are available to adequately assess perfusion. The results of our pilot study have indicated that NIR imaging can provide real-time, intraoperative assessment of bone perfusion.

Intercostal neuroma as a source of pain after aesthetic and reconstructive breast implant surgery.

The development of persistent post-operative pain after implant placement for aesthetic or reconstructive breast surgery can lead to significant patient morbidity. Although there are many etiologies for post-operative pain, the diagnosis of an intercostal neuroma is important as this can be treated surgically. We describe three cases of an intercostal neuroma in patients with breast implants. A Tinel's sign can be elicited along the lateral chest wall and a local anesthetic block temporarily alleviates this pain. Surgical management with identification and clipping of the intercostal neuroma and burying into the underlying muscle significantly decreases post-operative pain long term. In patients with persistent pain after breast implant placement, plastic surgeons must be aware of this treatable cause of pain.

Polymer therapy: a novel treatment to improve fat graft viability.

BACKGROUND: Autologous fat grafting is currently undergoing a renaissance. However, fat grafts are limited by unpredictable survival. Poloxamers can act as tissue surfactants. These nonionic surfactants have been shown to stabilize the membranes of damaged cells and to protect against injury and apoptosis in numerous models. This study was designed to investigate the ability of poloxamers to protect harvested adipocytes and to increase fat graft survival. METHODS: Lipoaspirate was obtained from surgical patients. Samples were washed in normal saline, centrifuged at 200 g, treated with various poloxamers or poloxamer components for 30 minutes, centrifuged at 200 g, and implanted into the flanks of nude mice in 1.0-cc, 1.0-g lobules. The grafts were explanted serially for 10 days and at 6 weeks. Endpoints were weight, apoptosis, cell viability, DNA content, and histology. RESULTS: Grafts treated with poloxamers P188, F108, and F127 demonstrated increased graft survival by weight. Fat grafts treated with poloxamers L64 and P188 demonstrated improvement in cell viability, and those treated with poloxamers L64, P188, and F38 demonstrated improved histology. P188-treated grafts demonstrated a 50 percent reduction in apoptosis compared with saline-treated controls (p < 0.05) and an overall 72 percent survival by weight at 6 weeks. P188 demonstrated statistically significant improvement by weight, DNA content, histology, and cell viability (89 percent versus 33 percent). CONCLUSIONS: The authors demonstrate that poloxamers, with membrane-sealing capability, can increase graft survival. Among these poloxamers, P188 demonstrated statistically significant improvement in apoptosis, graft survival by weight, cell viability, DNA content, and histology.

Poloxamer 188 protects against ischemia-reperfusion injury in a murine hind-limb model.

BACKGROUND: Ischemia-reperfusion injury can activate pathways generating reactive oxygen species, which can injure cells by creating holes in the cell membranes. Copolymer surfactants such as poloxamer 188 are capable of sealing defects in cell membranes. The authors postulated that a single-dose administration of poloxamer 188 would decrease skeletal myocyte injury and mortality following ischemia-reperfusion injury. METHODS: Mice underwent normothermic hind-limb ischemia for 2 hours. Animals were treated with 150 microl of poloxamer 188 or dextran at three time points: (1) 10 minutes before ischemia; (2) 10 minutes before reperfusion; and (3) 2 or 4 hours after reperfusion. After 24 hours of reperfusion, tissues were analyzed for myocyte injury (histology) and metabolic dysfunction (muscle adenosine 5'-triphosphate). Additional groups of mice were followed for 7 days to assess mortality. RESULTS: When poloxamer 188 treatment was administered 10 minutes before ischemia, injury was reduced by 84 percent, from 50 percent injury in the dextran group to 8 percent injury in the poloxamer 188 group (p < 0.001). When administered 10 minutes before reperfusion, poloxamer 188 animals demonstrated a 60 percent reduction in injury compared with dextran controls (12 percent versus 29 percent). Treatment at 2 hours, but not at 4 hours, postinjury prevented substantial myocyte injury. Preservation of muscle adenosine 5'-triphosphate paralleled the decrease in myocyte injury in poloxamer 188-treated animals. Poloxamer 188 treatment significantly reduced mortality following injury (10 minutes before, 75 percent versus 25 percent survival, p = 0.0077; 2 hours after, 50 percent versus 8 percent survival, p = 0.032). CONCLUSION: Poloxamer 188 administered to animals decreased myocyte injury, preserved tissue adenosine 5'-triphosphate levels, and improved survival following hind-limb ischemia-reperfusion injury.

A high-throughput model for fat graft assessment.

BACKGROUND AND OBJECTIVES: Current fat-graft animal models require weeks, to months, for results. The purpose of this study was to develop a model for the rapid identification of adipocyte protectants, using apoptosis-specific fluorescence. The goal of our model was to predict long-term fat graft survival within a 10-day period. STUDY DESIGN/MATERIALS AND METHODS: Human fat was obtained from liposuction aspirates, washed with saline, and centrifuged at 200g. The fat was then treated with one of four agents: P188, polyethylene glycol (PEG 8000), lipoic acid (LA), or saline control. Fat lobules were explanted over a 10-day period, and then at six weeks. The explanted fat was measured for apoptosis. Samples were weighed, sent for histology, measured for DNA content, and examined using confocal microscopy. RESULTS: Fat-grafts demonstrated variable apoptosis over the 10-day period. P188 and LA treated samples demonstrated 11-28% less apoptosis during early engraftment than saline treated controls. This early reduction in apoptosis correlated to a approximately 20% reduction in reabsorption by weight six weeks later. P188 and LA samples demonstrated three-times higher DNA content by PICO green analysis when compared to saline controls. PEG 8000 treated samples demonstrated 11% more apoptosis than saline. PEG 8000 treated samples demonstrated an approximately 10% higher level of reabsorption by weight, and two-times higher levels of DNA. Histology of treated samples at six weeks showed architecturally normal fat in P188 and LA treated fat; whereas PEG 8000 had high levels of inflammatory infiltrates, and saline had large amounts of fibrosis. CONCLUSIONS: This model of fat-grafting and early apoptosis can be used to screen agents and grafting methods and predict long-term graft survival. We show that levels of apoptosis within ten days correlate with weight, DNA, and histology, at six weeks. Using this model, long-term adipocyte survival and graft take can be predicted during the first 10 days post-implantation.

Improved survival of murine island skin flaps by prevention of reperfusion injury.

BACKGROUND: Studies have demonstrated that blocking a single specificity of self-reactive immunoglobulin M with a 12-amino acid peptide mimic of the antigen of immunoglobulin M can attenuate murine intestinal and skeletal muscle injury following ischemia and reperfusion. The aim of this study was to ascertain whether peptide (P8) had protective effects in an axial island skin flap model, where tissue loss is attributed to ischemia-reperfusion injury. METHODS: Dorsal lateral thoracic artery island skin flaps (3.5 x 1.5 cm) were elevated in 82 male C57BL/6 mice and rendered ischemic for 10 hours by placing a 7-mm microclamp on the vascular pedicle followed by 7 days of reperfusion. Group I (n = 7), the sham group, had no clamp placed. Group II (n = 21) had clamp placement but no other treatment. Thirty minutes before clamp placement, group III (n = 18) received 0.25 cc of saline intravenously, group IV (n = 18) received 25 microg/0.25 cc P8 peptide, and group V (n = 7) received 25 microg/0.25 cc random 12-mer peptide. Animals in group VI (n = 11) underwent two cycles of 20 minutes of ischemic preconditioning before 10 hours of ischemia. After 1 week of reperfusion, percent necrosis was measured and results were compared using analysis of variance and an unpaired t test. RESULTS: In animals treated with P8 peptide, flap necrosis was 14.61 +/- 2.77 percent. This represents a statistically significant, 56 percent reduction in flap necrosis compared with controls (p < 0.001). CONCLUSION: These data demonstrate that prevention of ischemia-reperfusion injury with P8 peptide produces a significant reduction in necrosis of treated flaps.

Sequential limb ischemia demonstrates remote postconditioning protection of murine skeletal muscle.

BACKGROUND: Ischemic postconditioning, the process of exposing tissues to brief cycles of ischemia-reperfusion after critical ischemia, can mitigate local ischemia-reperfusion injury. Remote protection of skeletal muscle has never been demonstrated in postconditioning models of ischemia-reperfusion injury. METHODS: Mice were subjected to 2 hours of ipsilateral hind limb ischemia followed by reperfusion. Contralateral limb ischemia was subsequently induced for 2 hours after either 0 (n = 6), 20 (n = 6), or 120 (n = 5) minutes of ipsilateral limb reperfusion. These groups were compared with animals subjected to bilateral simultaneous injury (n = 8) and sham animals that did not undergo ischemia (n = 6). The gastrocnemius muscles were harvested for histologic evaluation, and injury was recorded as the percentage of injured fibers. RESULTS: The first limbs undergoing injury in the 20-minute interval group had a 59 percent injury reduction compared with contralateral limbs (16.0 +/- 2.4 percent versus 39.5 +/- 6.5 percent) after 24 hours of reperfusion and 62 percent reduction after 48 hours (24.4 +/- 3.0 percent versus 63.6 +/- 5.5 percent). In animals with no interval or a 120-minute interval between the onset of limb ischemia, there was no significant difference in injury between hind limbs. The injury in these groups was similar to that in hind limbs subjected to simultaneous bilateral ischemia. CONCLUSIONS: A 20-minute reperfusion interval between hind limb ischemia significantly protects against injury in the initially ischemic limb, while similar injury is observed with simultaneous ischemia or an interval of 120 minutes. This study demonstrates remote postconditioning of skeletal muscle and may lead to the development of post hoc therapies.

A novel murine island skin flap for ischemic preconditioning.

BACKGROUND: Ischemia reperfusion injury is a well-known phenomenon affecting skin flap viability. One method to improve flap viability is ischemic preconditioning. Previous murine flap models used random flaps. We developed a single pedicle island skin flap which allows us to create true ischemia by clamping the single pedicle. Our first aim was to describe a novel murine skin flap model with a definable, reproducible injury. Our second aim was to test the usefulness of this model by demonstrating mitigation of injury via ischemic preconditioning. MATERIALS AND METHODS: Dorsal lateral thoracic artery pedicle island skin flaps (3.5 x 1.5 cm) were elevated in 39 male C57/BL6 mice: a Control group (n = 7), 10 h of ischemia (n = 21), and Preconditioning (2 cycles of 20 min ischemia: 20 minutes reperfusion) + 10-h ischemia (n = 11). After flap elevation, a silicon sheet barrier was placed. The axial pedicles were occluded, and the flaps were inset with 6-0 prolene. In all mice, ischemia was followed by 1 wk of reperfusion. At 1 wk, percent necrosis was measured and an analysis of variance was performed. RESULTS: The percent of flap necrosis was 1.1% +/- 1.11% in controls. Animals that were subjected to 10 h of ischemia developed 33.14% +/- 7.23% necrosis. Preconditioned animals that underwent 10 h of ischemia demonstrated a 43% reduction in necrosis (18.82% +/- 5.68%). There was a statistically significant difference among all groups (P < or = 0.001). CONCLUSION: Rat models have been the standard for skin flap experiments. We have developed a novel murine single pedicle island skin flap model with reproducible injury. This model has numerous advantages, including ease of handling, low cost, appropriateness for biomedical studies, and the availability of genetically altered animals. We also confirmed this model's usefulness in a study of mitigation of ischemia reperfusion injury through ischemic preconditioning.