Administration of particulate oxygen generators improves skeletal muscle contractile function after ischemia-reperfusion injury in the rat hindlimb.

Extended tourniquet application, often associated with battlefield extremity trauma, can lead to severe ischemia-reperfusion (I/R) injury in skeletal muscle. Particulate oxygen generators (POGs) can be directly injected into tissue to supply oxygen to attenuate the effects of I/R injury in muscle. The goal of this study was to investigate the efficacy of a sodium percarbonate (SPO)-based POG formulation in reducing ischemic damage in a rat hindlimb during tourniquet application. Male Lewis rats were anesthetized and underwent tourniquet application for 3 h at a pressure of 300 mmHg. Shortly after tourniquet inflation, animals received intramuscular injections of either 0.2 mg/mL SPO with catalase (n = 6) or 2.0 mg/mL SPO with catalase (n = 6) directly into the tibialis anterior (TA) muscle. An additional Tourniquet-Only group (n = 12) received no intervention. Functional recovery was monitored by in vivo contractile testing of the hindlimb at 1, 2, and 4 wk after injury. By the 4 wk time point, the Low-Dose POG group continued to show improved functional recovery (85% of baseline) compared with the Tourniquet-Only (48%) and High-Dose POG (56%) groups. In short, the low-dose POG formulation appeared, at least in part, to mitigate the impact of ischemic tissue injury, thus improving contractile function after tourniquet application. Functional improvement correlated with maintenance of larger muscle fiber cross-sectional area and the presence of fewer fibers containing centrally located nuclei. As such, POGs represent a potentially attractive therapeutic solution for addressing I/R injuries associated with extremity trauma.NEW & NOTEWORTHY Skeletal muscle contraction was evaluated in the same animals at multiple time points up to 4 wk after injury, following administration of particulate oxygen generators (POGs) in a clinically relevant rat hindlimb model of tourniquet-induced ischemia. The observed POG-mediated improvement of muscle function over time confirms and extends previous studies to further document the potential clinical applications of POGs. Of particular significance in austere environments, this technology can be applied in the absence of an intact circulation.

Outcomes of Arterial Grafts for the Reconstruction of Military Lower Extremity Arterial Injuries.

INTRODUCTION: Lower extremity (LE) arterial injuries are common in military casualties and limb salvage is a primary goal. Bypass grafts are the most common reconstructions; however, their specific outcomes are largely unreported. We sought to describe the outcomes of LE arterial grafts among combat casualties and their association with limb loss. METHODS: Retrospective cohort study of 2004-2012 Iraq/Afghanistan casualties with LE arterial injury undergoing bypass graft from a database containing follow-up until amputation, death, or military discharge. Primary outcome was composite graft complications (GC-thrombosis, stenosis, pseudoaneurysm, blowout, and/or arteriovenous fistula). RESULTS: Two hundred and twenty-two grafts were included (99 femoral, 73 popliteal, 48 tibial). 56 (26%) had at least one GC; thrombosis was most common in femoral, stenosis most common in popliteal and tibial. GC was not associated with graft level but was associated with synthetic conduit (P = 0.01) and trended towards an association with multiple-level arterial injuries (P = 0.07). Four of eight (50%) synthetic grafts had amputations, all within 72h. Two of the eight synthetic grafts thrombosed, and both limbs were amputated. There were 52 total amputations. Amputation was performed in 13 (23%) of limbs with a GC and 24% of those without (P = 0.93) Overall, 24 (11%) of grafts thrombosed, 16 within 48h and 13 (25%) in limbs undergoing amputation (P = 0.001 for association of thrombosis with amputation). CONCLUSION: GC are common among LE bypass grafts in combat casualties but are not associated with limb loss. Thrombosis is predominantly early and is associated with amputation. Closer attention to ensuring early patency may improve limb salvage.

Utility of the Mangled Extremity Severity Score in Predicting Amputation in Military Lower Extremity Arterial Injury.

BACKGROUND: Effective amputation prediction may help inform appropriate early limb salvage efforts in military lower extremity (LE) arterial injury. The Mangled Extremity Severity Score (MESS) is the most commonly applied system for early amputation prediction but its utility in military trauma is unknown. METHODS: Retrospective cohort study of Iraq and Afghanistan casualties with LE arterial injury who underwent a vascular limb salvage attempt. Retrospectively assessed MESS was statistically explored as an amputation predictor and MESS component surrogates (mechanism, vascular injury characteristics, tourniquet use, and transfusion volume) were used to characterize limb injuries by presenting characteristics and evaluated for amputation prediction. RESULTS: A total of 439 limbs were included with 99 (23%) amputations, 29 (7%) within 48 hr of injury. Median MESS was 5 (interquartile range 4-6) among salvaged limbs and 7 (5-9) among amputations (P < 0.0001). An MESS cutoff of ≥7 had a better receiver operating characteristic sensitivity/specificity profile (area under the curve 0.696 overall, 0.765 amputation within 48 hr) than MESS ≥8 (0.593, 0.621), but amputation rates were only 43% for MESS ≥7 and 50% for ≥8. MESS ≥7 was significantly associated with age, polytrauma, blast or crush mechanism, fracture, tourniquet use, distal (popliteal/tibial) and multiple arterial injuries, and massive transfusion. Amputation was significantly associated with polytrauma, blast or crush mechanism, fracture, and massive transfusion; however, 83 casualties had all 4 characteristics with an amputation rate of only 46%. CONCLUSIONS: In combat casualties with arterial injury, LE amputation after attempted vascular limb salvage is inadequately predicted by existing scoring systems or the presenting characteristics available in this registry. Limb loss is predominantly late and likely because of factors not projectable at initial presentation.

Early Fasciotomy and Limb Salvage and Complications in Military Lower Extremity Vascular Injury.

BACKGROUND: Military guidelines endorse early fasciotomy after revascularization of lower extremity injuries to prevent compartment syndrome, but the real-world impact is unknown. We assessed the association between fasciotomy and amputation and limb complications among lower extremitys with vascular injury. METHODS: A retrospectively collected lower extremity injury database was queried for limbs undergoing attempted salvage with vascular procedure (2004-2012). Limbs were categorized as having undergone fasciotomy or not. Injury and treatment characteristics were collected, as were intervention timing data when available. The primary outcome measure was amputation. Multivariate models examined the impact of fasciotomy on limb outcomes. RESULTS: Inclusion criteria were met by 515 limbs, 335 (65%) with fasciotomy (median 7.7 h postinjury). Of 212 limbs, 174 (84%) with timing data had fasciotomy within 30 min of initial surgery. Compartment syndrome and suspicion of elevated pressure was documented in 127 limbs (25%; 122 had fasciotomy). Tourniquet and shunt use, fracture, multiple arterial and combined arteriovenous injuries, popliteal involvement, and graft reconstruction were more common in fasciotomy limbs. Isolated venous injury and vascular ligation were more common in nonfasciotomy limbs. Fasciotomy timing was not associated with amputation. Controlling for limb injury severity, fasciotomy was not associated with amputation but was associated with limb infection, motor dysfunction, and contracture. Sixty-three percent of fasciotomies were open for >7 d, and 43% had multiple closure procedures. Fasciotomy revision (17%) was not associated with increased amputation or complications. CONCLUSIONS: Fasciotomy after military lower extremity vascular injury is predominantly performed early, frequently without documented compartment pressure elevation. Early fasciotomy is generally performed in severely injured limbs with a subsequent high rate of limb complications.

Can Dynamic Contrast-Enhanced CT Quantify Perfusion in a Stimulated Muscle of Limited Size? A Rat Model.

BACKGROUND: Muscle injury may result in damage to the vasculature, rendering it unable to meet the metabolic demands of muscle regeneration and healing. Therefore, therapies frequently aim to maintain, restore, or improve blood supply to the injured muscle. Although there are several options to assess the vascular outcomes of these therapies, few are capable of spatially assessing perfusion in large volumes of tissue. QUESTIONS/PURPOSES: Can dynamic contrast-enhanced CT (DCE-CT) imaging acquired with a clinical CT scanner be used in a rat model to quantify perfusion in the anterior tibialis muscle at spatially relevant volumes, as assessed by (1) the blood flow rate and tissue blood volume in the muscle after three levels of muscle stimulation (low, medium, and maximum) relative to baseline as determined by the non-stimulated contralateral leg; and (2) how do these measurements compare with those obtained by the more standard approach of microsphere perfusion? METHODS: The right anterior tibialis muscles of adult male Sprague Dawley rats were randomized to low- (n = 10), medium- (n = 6), or maximum- (n = 3) level (duty cycles of 2.5%, 5.0%, and 20%, respectively) nerve electrode coupled muscle stimulation directly followed by DCE-CT imaging. Tissue blood flow and blood volume maps were created using commercial software and volumetrically measured using NIH software. Although differences in blood flow were detectable across the studied levels of muscle stimulation, a review of the evidence suggested the absolute blood flow quantified was underestimated. Therefore, at a later date, a separate set of adult male Sprague Dawley rats were randomized for microsphere perfusion (n = 7) to define blood flow in the animal model with an accepted standard. With this technique, intra-arterial particles sized to freely flow in blood but large enough to lodge in tissue capillaries were injected. Simultaneously, blood sampling at a fixed flow rate was simultaneously performed to provide a fixed blood flow rate sample. The tissues of interest were then explanted and assessed for the total number of particles per tissue volume. Tissue blood flow rate was then calculated based on the particle count ratio within the reference sample. Note that a tissue's blood volume cannot be calculated with this method. Comparison analysis to the non-stimulated baseline leg was performed using two-tailed paired student t-test. An ANOVA was used to compare difference between stimulation groups. RESULTS: DCE-CT measured (mean ± SD) increasing tissue blood flow differences in stimulated anterior tibialis muscle at 2.5% duty cycle (32 ± 5 cc/100 cc/min), 5.0% duty cycle (46 ± 13 cc/100 cc/min), and 20% duty cycle (73 ± 3 cc/100 cc/min) compared with the paired contralateral non-stimulated anterior tibialis muscle (10 ± 2 cc/100 cc/min, mean difference 21 cc/100 cc/min [95% CI 17.08 to 25.69]; 9 ± 1 cc/100 cc/min, mean difference 37 cc/100 cc/min [95% CI 23.06 to 50.11]; and 11 ± 2 cc/100 cc/min, mean difference 62 cc/100 cc/min [95% CI 53.67 to 70.03]; all p < 0.001). Similarly, DCE-CT showed increasing differences in tissue blood volumes within the stimulated anterior tibialis muscle at 2.5% duty cycle (23.2 ± 4.2 cc/100 cc), 5.0% duty cycle (39.2 ± 7.2 cc/100 cc), and 20% duty cycle (52.5 ± 13.1 cc/100 cc) compared with the paired contralateral non-stimulated anterior tibialis muscle (3.4 ± 0.7 cc/100 cc, mean difference 19.8 cc/100 cc [95% CI 16.46 to 23.20]; p < 0.001; 3.5 ± 0.4 cc/100 cc, mean difference 35.7 cc/100 cc [95% CI 28.44 to 43.00]; p < 0.001; and 4.2 ± 1.3 cc/100 cc, mean difference 48.3 cc/100 cc [95% CI 17.86 to 78.77]; p = 0.010). Microsphere perfusion measurements also showed an increasing difference in tissue blood flow in the stimulated anterior tibialis muscle at 2.5% duty cycle (62 ± 43 cc/100 cc/min), 5.0% duty cycle (89 ± 52 cc/100 cc/min), and 20% duty cycle (313 ± 269 cc/100 cc/min) compared with the paired contralateral non-stimulated anterior tibialis muscle (8 ± 4 cc/100 cc/min, mean difference 55 cc/100 cc/min [95% CI 15.49 to 94.24]; p = 0.007; 9 ± 9 cc/100 cc/min, mean difference 79 cc/100 cc/min [95% CI 33.83 to 125.09]; p = 0.003; and 18 ± 18 cc/100 cc/min, mean difference 295 cc/100 cc/min [95% CI 8.45 to 580.87]; p = 0.023). Qualitative comparison between the methods suggests that DCE-CT values underestimate tissue blood flow with a post-hoc ANOVA showing DCE-CT blood flow values within the 2.5% duty cycle group (32 ± 5 cc/100 cc/min) to be less than the microsphere perfusion value (62 ± 43 cc/100 cc/min) with a mean difference of 31 cc/100 cc/min (95% CI 2.46 to 60.23; p = 0.035). CONCLUSIONS: DCE-CT using a clinical scanner is a feasible modality to measure incremental changes of blood flow and tissue blood volume within a spatially challenged small animal model. Care should be taken in studies where true blood flow values are needed, as this particular small-volume muscle model suggests true blood flow is underestimated using the specific adaptions of DCE-CT acquisition and image processing chosen. CLINICAL RELEVANCE: CT perfusion is a clinically available modality allowing for translation of science from bench to bedside. Adapting the modality to fit small animal models that are relevant to muscle healing may hasten time to clinical utility.

Impact of Staged Vascular Management on Limb Outcomes in Wartime Femoropopliteal Arterial Injury.

BACKGROUND: By necessity, wartime arterial injuries undergo staged management. Initial procedures may occur at a forward surgical team (role 2), where temporary shunts can be placed before transfer to a larger field hospital (role 3) for definitive reconstruction. Our objective was to evaluate the impact of staging femoropopliteal injury care on limb outcomes. METHODS: A military vascular injury database was queried for Iraq/Afghanistan casualties with femoropopliteal arterial injuries undergoing attempted reconstruction (2004-2012). Cases were grouped by initial arterial management: shunt placed at role 2 (R2SHUNT), reconstruction at role 2 (R2RECON), and initial management at role 3 (R3MGT). The primary outcome was limb salvage; secondary outcomes were limb-specific complications. Descriptive and intergroup comparative statistics were performed with significance defined at P ≤ 0.05. RESULTS: Of 257 cases, all but 4 had definitive reconstruction before evacuation to Germany (median, 2 days): 46 R2SHUNT, 84 R2RECON, and 127 R3MGT; median Mangled Extremity Severity Score was 6 for all groups. R2SHUNT had median extremity Abbreviated Injury Scale--vascular of 4 (other groups, 3; P < 0.05) and was more likely to have concomitant venous injury and to undergo fasciotomy. Shunts were used for 5 ± 3 hr. About 24% of R2RECON repairs were revised at role 3. Limb salvage rate of 80% was similar between groups, and 62% of amputations performed within 48 hr of injury. Rates of limb and composite graft complications were similar between groups. Thrombosis was more common in R2SHUNT (22%) than R2RECONST (6%) or R3MGT (12%) (P = 0.03). Late (>48 hr) thrombosis rates were similar, whereas 60% of R2SHUNT thromboses occurred on day of injury (P = 0.003 vs. 25% and 0%). CONCLUSIONS: Staged femoropopliteal injury care is associated with similar limb salvage to initial role 3 management. Early thrombosis is likely because of shunt failure but does not lead to limb loss. Current military practice guidelines are appropriate and may inform civilian vascular injury management protocols.

Fatty Acid-Saturated Albumin Reduces High Mortality and Fluid Requirements in a Rat Model of Hemorrhagic Shock Plus Tourniquet and Hypotensive Resuscitation.

Military prehospital care for hemorrhage is often characterized by use of tourniquets (TQ) and permissive hypotensive resuscitation (PHR) with crystalloids or colloids, but these treatments have not been previously combined in an animal model. Although albumin resuscitation solutions have been tested, the potential effects of nonesterified fatty acids (NEFAs) bound to albumin have not been evaluated in vivo, and few studies have investigated concentrated albumin solutions to reduce fluid requirements. We created a militarily relevant rat model of trauma and hemorrhagic shock (T/HS) (27 mL/kg hemorrhage) with TQ and PHR. We investigated the ability of resuscitation with concentrated (250 mg/mL) albumin, followed by Plasmalyte as needed to maintain PHR, to reduce fluid volumes (vs. Plasmalyte alone, N = 17). Albumin was free of nonesterified fatty acids (N = 15) or saturated with oleic acid (OA; N = 13). The model resulted in high (53%) mortality within 3 h of injury. Only OA-saturated albumin was able to significantly reduce mortality (from 47% to 8%) and fluid requirements (from 56 to 6 mL/kg) compared to Plasmalyte alone. Plasma NEFA-binding capacity was saturated earliest in the OA-saturated albumin group. Likewise, OA-saturated albumin tended to increase cell-free hemoglobin in the broncheoalveolar lavage fluid, which was significantly associated with survival. Our findings suggest incorporating TQ and PHR in T/HS models may result in high mortality and fluid requirements and that OA-saturated albumin, but not NEFA-free albumin or Plasmalyte alone, may provide a benefit to early survival and resuscitation volume, though a hemolytic mechanism may have later consequences, so caution is advised.

Noninvasive diagnostics for extremity compartment syndrome following traumatic injury: A state-of-the-art review.

Acute compartment syndrome (ACS) is a serious medical condition that can occur following traumatic injury to an extremity. If left undiagnosed, ACS can eventuate in amputation of the limb or even death. Because of this, fasciotomy to release the pressure within the muscle and restore tissue perfusion is often performed upon suspicion of ACS, as the sequelae to fasciotomy are less severe than those associated with not performing the fasciotomy. Currently, the "gold standard" of diagnosis is based on clinical assessment of such symptoms as pain out of proportion to the injury, obvious high pressure and swelling, pain on passive stretch of the muscles in the affected compartment, and deficits in sensory and/ormotor functions. Diagnosis is often confirmed using invasive measurements of intramuscular pressure (IMP); however, controversy exists as to how direct IMP measurement should be accomplished and threshold pressures for accurate diagnosis. Because of this and the attendant issues with invasive measurements, investigators have been searching over the last 25 years for a noninvasive means to quantitatively measure IMP or perfusion to the limb. The purpose of this review is to summarize the current state of the art of noninvasive devices that could potentially be used to diagnose ACS accurately and objectively. To do this, we divide the discussion into those medical devices that primarily measure mechanical surrogates of IMP (e.g., tissue hardness or myofascial displacement) and those that primarily measure indices of tissue perfusion (e.g., tissue oxygen saturation via near-infraredspectroscopy). While near-infrared spectroscopy-basedtechnologies have shown the most promise, whether such technologies will be of diagnostic benefit await the completion of ongoing clinical trials. LEVEL OF EVIDENCE: Systematic Review, level II.

Predictors and timing of amputations in military lower extremity trauma with arterial injury.

INTRODUCTION: Military lower extremity arterial injuries present threats to life and limb. These injuries are common and limb salvage is a trauma system priority. Understanding the timing and predictors of amputation through the phases of casualty evacuation can help inform future limb salvage efforts. This study characterizes limbs undergoing amputation at different operationally relevant time points. METHODS: A retrospective cohort study of casualties with lower extremity arterial injuries undergoing initial vascular limb salvage in Iraq and Afghanistan was undertaken. Amputations were grouped as having been performed early (in theater at Role 2 or 3) or late (after evacuation to Role 4 or 5). Further distinction was made between late and delayed (after discharge from initial hospitalization) amputations. RESULTS: Four hundred fifty-five casualties met inclusion criteria with 103 amputations (23%). Twenty-one (20%) were performed in theater and 82 (80%) were performed following overseas evacuation. Twenty-one (26% of late amputations) were delayed, a median of 359 days from injury (interquartile range, 176-582). Most amputations were performed in the first 4 days following injury. Amputation incidence was highest in popliteal injuries (28%). Overall, amputation was predicted by higher incidence of blast mechanism and fracture and greater limb and casualty injury severity. Early amputations had higher limb injury severity than late amputations. Delayed amputations had greater incidence of motor and sensory loss and contracture than early amputations. CONCLUSION: Casualty and limb injury severity predict predictors and timing of amputation in military lower extremity arterial injury. Amputation following overseas evacuation was more common than in-theater amputation, and functional loss is associated with delayed amputation. Future limb salvage efforts should focus on postevacuation and rehabilitative care. LEVEL OF EVIDENCE: Epidemiologic study, level III.

Tourniquet use is not associated with limb loss following military lower extremity arterial trauma.

BACKGROUND: The effect of battlefield extremity tourniquet (TK) use on limb salvage and long-term complications following vascular repair is unknown. This study explores the influence of TK use on limb outcomes in military lower extremity arterial injury. METHODS: The study database includes cases of lower extremity vascular injury from 2004 to 2012 with data recorded until discharge from military service. We analyzed all limbs with at least one named arterial injury from the femoral to the tibial level. Tourniquet (TK) and no TK (NTK) groups were identified. Univariate analyses were performed with significance set at p ≤ 0.05. RESULTS: A total of 455 cases were included, with 254 (56%) having a TK for a median of 60 minutes (8-270 minutes). Explosive injuries (53%) and gunshot wounds (26%) predominated. No difference between TK and NTK was present in presence of fracture, level of arterial injury, type of arterial repair, or concomitant venous injury. More nerve injuries were present in the TK group, and Abbreviated Injury Scale extremity and Mangled Extremity Severity Score tended toward greater injury severity. Amputation and mortality rates did not differ between groups, but the incidence of severe edema, wound infection, and foot drop was higher in the TK group. Vascular above-knee amputation, arterial repair complication, and severe edema were higher in the TK group also (p = 0.10). Tourniquet duration of 60 minutes or longer was not associated with increased amputations, but more rhabdomyolysis was present. CONCLUSION: Field TK use is associated with wound infection and neurologic compromise but not limb loss. This may be due to a more severe injury profile among TK limbs. Increased TK times may predispose to systemic, but not limb, complications. LEVEL OF EVIDENCE: Therapeutic/care management, level IV.

Systematic review of prehospital tourniquet use in civilian limb trauma.

BACKGROUND: Military enthusiasm for limb tourniquet use in combat casualty care has resulted in acceptance by the trauma community for use in the prehospital care of civilian limb injuries. To date, there has been no report synthesizing the published data on civilian tourniquet use. The objective of this systematic review was to compile and analyze the content and quality of published data on the civilian use of tourniquets in limb trauma. METHODS: The MEDLINE database was searched for studies on civilian limb tourniquet use in adults published between 2001 and 2017. Search terms were tourniquet, trauma, and injury. Military reports and case series lacking systematic data collection were excluded. Counts and percentages were aggregated and weighted for analysis. RESULTS: Reports were included from six regional trauma centers and one interregional collaboration (total of 572 cases). One national prehospital database report was included but analyzed separately (2,048 cases). All were retrospective cohort studies without prospective data collection. Three reports defined a primary outcome, two had a nontourniquet control group, and no two articles reported the same variables. Limb injury severity and characteristics were inconsistently and incompletely described across reports, as were tourniquet indications and effectiveness. Arterial injury was reported in two studies and was infrequent among cases of tourniquet use. Mortality was low, and limb-specific complications were infrequent but variably reported. CONCLUSION: The rapid increase in the civilian use of tourniquets for limb hemorrhage control has occurred without a large amount or high quality of data. Adoption of a multicenter registry with standardized data collection specific to limb trauma and tourniquet use can serve to improve the trauma community's understanding of the safety and effectiveness of tourniquet use in civilian trauma settings. LEVEL OF EVIDENCE: Systematic review, level IV.

Co-delivery of a laminin-111 supplemented hyaluronic acid based hydrogel with minced muscle graft in the treatment of volumetric muscle loss injury.

Minced muscle autografting mediates de novo myofiber regeneration and promotes partial recovery of neuromuscular strength after volumetric muscle loss injury (VML). A major limitation of this approach is the availability of sufficient donor tissue for the treatment of relatively large VMLs without inducing donor site morbidity. This study evaluated a laminin-111 supplemented hyaluronic acid based hydrogel (HA+LMN) as a putative myoconductive scaffolding to be co-delivered with minced muscle grafts. In a rat tibialis anterior muscle VML model, delivery of a reduced dose of minced muscle graft (50% of VML defect) within HA+LMN resulted in a 42% improvement of peak tetanic torque production over unrepaired VML affected limbs. However, the improvement in strength was not improved compared to a 50% minced graft-only control group. Moreover, histological analysis revealed that the improvement in in vivo functional capacity mediated by minced grafts in HA+LMN was not accompanied by a particularly robust graft mediated regenerative response as determined through donor cell tracking of the GFP+ grafting material. Characterization of the spatial distribution and density of macrophage and satellite cell populations indicated that the combination therapy damps the heightened macrophage response while re-establishing satellite content 14 days after VML to a level consistent with an endogenously healing ischemia-reperfusion induced muscle injury. Moreover, regional analysis revealed that the combination therapy increased satellite cell density mostly in the remaining musculature, as opposed to the defect area. Based on the results, the following salient conclusions were drawn: 1) functional recovery mediated by the combination therapy is likely due to a superposition of de novo muscle fiber regeneration and augmented repair of muscle fibers within the remaining musculature, and 2) The capacity for VML therapies to augment regeneration and repair within the remaining musculature may have significant clinical impact and warrants further exploration.

Fresh whole blood resuscitation does not exacerbate skeletal muscle edema and long-term functional deficit after ischemic injury and hemorrhagic shock.

BACKGROUND: Hemorrhagic shock caused by extremity vascular injuries is common in combat injuries. Fluid resuscitation is the standard treatment for severe hemorrhage (HEM). Tourniquets (TKs) used for HEM control cause ischemia-reperfusion (I/R) injury that induces edema formation in the injured muscle. Resuscitation fluids affect edema formation; however, its effect on long-term functional response remains unknown. The objectives of this study are to (1) compare acute muscle damage; (2) determine long-term functional recovery of ischemic muscle; and (3) compare local and systemic inflammatory response including the expression of junctional proteins following early resuscitation with Hextend and fresh whole blood using a rodent model of combined HEM and TK-induced limb I/R. METHODS: Anesthetized Sprague-Dawley rats underwent 42.5% arterial HEM, followed by 3 hours of TK application. Animals were either not resuscitated or resuscitated with Hextend or fresh whole blood. Two time points were evaluated, 2 and 28 days. Plasma cytokine concentrations were determined at baseline and end resuscitation. At 2 days, edema formation, expression of junctional proteins, and tissue level cytokines concentrations were evaluated. At 28 days, in vivo muscle contractile properties were determined. At both time points, routine histology was performed and graded using a semiquantitative grading system. RESULTS: All animals developed hemorrhagic hypovolemia; the mortality rate was 100% in nonresuscitated rats. Hextend resuscitation exacerbated muscle edema (~11%) and muscle strength deficit (~20%). Fresh whole blood resuscitation presented edema and muscle strength akin to TK only. Fresh whole blood resuscitation upregulated expression of junctional proteins including proangiogenic factors and dampened the inflammatory response. CONCLUSION: Fresh whole blood resuscitation does not exacerbate either TK-induced edema or muscle strength deficit. Fresh whole blood resuscitation may reduce both acute and long-term morbidity associated with extremity trauma. To our knowledge, this is the first study to demonstrate the nature of the resuscitation fluid administered following HEM impacts short- and long-term indices of I/R in skeletal muscle.

A PEGylated platelet free plasma hydrogel based composite scaffold enables stable vascularization and targeted cell delivery for volumetric muscle loss.

Extracellular matrix (ECM) scaffolds are being used for the clinical repair of soft tissue injuries. Although improved functional outcomes have been reported, ECM scaffolds show limited tissue specific remodeling response with concomitant deposition of fibrotic tissue. One plausible explanation is the regression of blood vessels which may be limiting the diffusion of oxygen and nutrients across the scaffold. Herein we develop a composite scaffold as a vasculo-inductive platform by integrating PEGylated platelet free plasma (PFP) hydrogel with a muscle derived ECM scaffold (m-ECM). In vitro, adipose derived stem cells (ASCs) seeded onto the composite scaffold differentiated into two distinct morphologies, a tubular network in the hydrogel, and elongated structures along the m-ECM scaffold. The composite scaffold showed a high expression of ITGA5, ITGB1, and FN and a synergistic up-regulation of ang1 and tie-2 transcripts. The in vitro ability of the composite scaffold to provide extracellular milieu for cell adhesion and molecular cues to support vessel formation was investigated in a rodent volumetric muscle loss (VML) model. The composite scaffold delivered with ASCs supported robust and stable vascularization. Additionally, the composite scaffold supported increased localization of ASCs in the defect demonstrating its ability for localized cell delivery. Interestingly, ASCs were observed homing in the injured muscle and around the perivascular space possibly to stabilize the host vasculature. In conclusion, the composite scaffold delivered with ASCs presents a promising approach for scaffold vascularization. The versatile nature of the composite scaffold also makes it easily adaptable for the repair of soft tissue injuries. STATEMENT OF SIGNIFICANCE: Decellularized extracellular matrix (ECM) scaffolds when used for soft tissue repair is often accompanied by deposition of fibrotic tissue possibly due to limited scaffold vascularization, which limits the diffusion of oxygen and nutrients across the scaffold. Although a variety of scaffold vascularization strategies has been investigated, their limitations preclude rapid clinical translation. In this study we have developed a composite scaffold by integrating bi-functional polyethylene glycol modified platelet free plasma (PEGylated PFP) with adipose derived stem cells (ASCs) along with a muscle derived ECM scaffold (m-ECM). The composite scaffold provides a vasculo-inductive and an effective cell delivery platform for volumetric muscle loss.

A Porcine Urinary Bladder Matrix Does Not Recapitulate the Spatiotemporal Macrophage Response of Muscle Regeneration after Volumetric Muscle Loss Injury.

Volumetric muscle loss (VML) results in irrecoverable loss of muscle tissue making its repair challenging. VML repair with acellular extracellular matrix (ECM) scaffolds devoid of exogenous cells has shown improved muscle function, but limited de novo muscle fiber regeneration. On the other hand, studies using minced autologous and free autologous muscle grafts have reported appreciable muscle regeneration. This raises the fundamental question whether an acellular ECM scaffold can orchestrate the spatiotemporal cellular events necessary for appreciable muscle fiber regeneration. This study compares the macrophage and angiogenic responses including the remodeling outcomes of a commercially available porcine urinary bladder matrix, MatriStem™, and autologous muscle grafts. The early heightened and protracted M1 response of the scaffold indicates that the scaffold does not recapitulate the spatiotemporal macrophage response of the autograft tissue. Additionally, the scaffold only supports limited de novo muscle fiber formation and regressing vessel density. Furthermore, scaffold remodeling is accompanied by increased presence of transforming growth factor and α-smooth muscle actin, which is consistent with remodeling of the scaffold into a fibrotic scar-like tissue. The limited muscle formation and scaffold-mediated fibrosis noted in this study corroborates the findings of recent studies that investigated acellular ECM scaffolds (devoid of myogenic cells) for VML repair. Taken together, acellular ECM scaffolds when used for VML repair will likely remodel into a fibrotic scar-like tissue and support limited de novo muscle fiber regeneration primarily in the proximity of the injured musculature. This is a work of the US Government and is not subject to copyright protection in the USA. Foreign copyrights may apply. Published by S. Karger AG, Basel.

Postischemic conditioning does not reduce muscle injury after tourniquet-induced ischemia-reperfusion injury in rats.

BACKGROUND: The widespread application of tourniquets has reduced battlefield mortality related to extremity exsanguinations. Tourniquet-induced ischemia-reperfusion injury (I/R) can contribute to muscle loss. Postischemic conditioning (PostC) confers protection against I/R in cardiac muscle and skeletal muscle flaps. The objective of this study was to determine the effect of PostC on extremity muscle viability in an established rat hindlimb tourniquet model. METHODS: Rats were randomly assigned to PostC-1, PostC-2, or no conditioning ischemic groups (n = 10 per group). Postischemic conditioning, performed immediately after tourniquet release, consisted of four 15-second cycles (PostC-1) or eight 15-second cycles (PostC-2) of alternating occlusion and perfusion of hindlimbs. Twenty-four hours later, muscles were excised. The primary end points were muscle edema and viability; secondary end points were histologic and markers of oxidative stress. RESULTS: Ischemia-reperfusion injury decreased viability in all tourniquet limbs, but viability was not improved in either PostC group. Likewise, I/R resulted in substantial muscle edema that was not reduced by PostC. The predominant histologic feature was necrosis, but no significant differences were found among groups. Markers of oxidative stress were increased similarly among groups after I/R, although myeloperoxidase activity was significantly increased only in the no conditioning ischemic group. A protective effect from PostC was not observed in our model suggesting that PostC was not effective in reducing I/R skeletal muscle injury or any benefits of PostC were not sustained for 24 hours when tissues were assessed. CONCLUSION: These negative findings are pertinent as the military investigates different strategies to extend the safe time for tourniquet application.

Where's the Leak in Vascular Barriers? A Review.

Edema is typically presented as a secondary effect from injury, illness, disease, or medication, and its impact on patient wellness is nested within the underlying etiology. Therefore, it is often thought of more as an amplifier to current preexisting conditions. Edema, however, can be an independent risk factor for patient deterioration. Improper management of edema is costly not only to the patient, but also to treatment and care facilities, as mismanagement of edema results in increased lengths of hospital stay. Direct tissue trauma, disease, or inappropriate resuscitation and/or ventilation strategies result in edema formation through physical disruption and chemical messenger-based structural modifications of the microvascular barrier. Derangements in microvascular barrier function limit tissue oxygenation, nutrient flow, and cellular waste removal. Recent studies have sought to elucidate cellular signaling and structural alterations that result in vascular hyperpermeability in a variety of critical care conditions to include hemorrhage, burn trauma, and sepsis. These studies and many others have highlighted how multiple mechanisms alter paracellular and/or transcellular pathways promoting hyperpermeability. Roles for endothelial glycocalyx, extracellular matrix and basement membrane, vesiculo-vacuolar organelles, cellular junction and cytoskeletal proteins, and vascular pericytes have been described, demonstrating the complexity of microvascular barrier regulation. Understanding these basic mechanisms inside and out of microvessels aid in developing better treatment strategies to mitigate the harmful effects of excessive edema formation.

Pathophysiological alterations induced by sustained 35-GHz radio-frequency energy heating.

BACKGROUND: Exposure to radio-frequency energy (RFE) of millimeter wavelengths results in a relatively high skin-heating rate, with only a moderate rate of core heating. Yet, prolonged RFE exposure eventuates in severe hypotension and death. In this study, we characterized pathophysiological changes associated with prolonged RFE sufficient to induce hypotension. METHODS: Anesthetized rats were exposed to 35-GHz RFE with a power density of 75 mW/cm2. Cardiovascular and temperature parameters were continuously recorded. Blood factors and histopathology were compared between sham (n=6) and exposed (n=12) animals. RESULTS AND CONCLUSIONS: Using infrared thermography, we confirmed a relatively high temperature (>46 °C) at the skin surface of the irradiated site. Histopathological results included hemorrhage and congestion of blood vessels in the dermis and subcutis of irradiated skin without induction of burn. As in environmental heating, significantly greater levels of serum glucose, creatinine, uric acid, and anion gap were observed in rats exposed to longer-duration RFE (approx. 38-min exposures) than in shorter-duration (approx. 19-min exposures) or sham (time control) animals. However, changes in blood electrolytes or liver enzymes (often seen during heatstroke) were not observed after the RFE exposures. Even without major tissue injury or serum/plasma enzyme and electrolyte changes, rapid cutaneous heating via RFE induced profound hypotension that eventuated in death.

Fresh frozen plasma reduces edema in skeletal muscle following combined limb ischemia-reperfusion injury and hemorrhagic shock in rats.

BACKGROUND: Exsanguination from extremity vascular injuries is the most common potentially survivable injury on the battlefield. Advances in treatment have dramatically improved survival, increasing the need to address associated morbidities including ischemia-reperfusion injury and extremity compartment syndrome. Despite advances, hemorrhagic shock (HS) requiring fluid resuscitation is common. Plasma-based resuscitation for the treatment of HS has been shown to reduce edema and injury in tissues other than muscle. The objective of this study was to determine if fresh frozen plasma (FFP) resuscitation offered protection in a rat model of combined HS and skeletal muscle ischemia-reperfusion injury. METHODS: Anesthetized Sprague-Dawley rats underwent 37.5% arterial hemorrhage, producing HS, followed by 3 hours of tourniquet application. Animals were not resuscitated or resuscitated with either FFP (equal to the shed blood volume) or lactated Ringer's solution (three times shed volume) after 30 minutes of ischemia. They were euthanized 24 hours later, and their muscles were analyzed for edema (wet weight-dry weight). Routine histology was performed on muscle cross-sections stained with hematoxylin and eosin and graded using a semiquantitative grading system. RESULTS: All animals developed HS; the mortality rate was 50% in no resuscitation rats. FFP reduced edema by 13% (p = 0.02) compared with lactated Ringer's solution. Pathology scores were not different between treatment groups. CONCLUSION: FFP resuscitation reduces edema following muscle injury, decreasing the risk of developing extremity compartment syndrome.