Yorkshire Cross Pigs Research Articles

Poster 105: Nanowarming by Inductive Heating of Vitrified Large Articular Cartilage Specimens, In Vivo Testing with Hanford Mini Pigs

Poster 105: Nanowarming by Inductive Heating of Vitrified Large Articular Cartilage Specimens, In Vivo Testing with Hanford Mini Pigs

Fully Guided Third Molar Tooth Bud Ablation in Pigs

The purpose of this study was to selectively ablate the entire volume of targeted third molar (3M) tooth buds in a pig model. This study demonstrates the potential for a less-invasive alternative to contemporary surgical techniques for removal of 3Ms. The investigator developed a mandibular split-mouth animal model study design. The model used pigs because the animals' 3M tooth buds are dimensionally similar to those of humans. The study sample consisted of 5 female Yorkshire-cross pigs at 20weeks of age. The investigator delivered microwave energy thermal doses to thermocoagulate tooth bud tissues inside the bony crypts of targeted 3M tooth buds. Based on the bony crypt dimensions obtained from computed tomography scans of each subject, the microwave thermal dose was predetermined. The mandibles were dissected to visually compare thermocoagulated right-side 3M tooth bud tissues to left-side untreated controls. All 5 study animals were successfully treated. All 5 fully guided third molar tooth bud ablation (3TBA) procedures resulted in thermocoagulation of the entire volume of targeted 3M tooth bud tissues, with no visual evidence of damage to structures beyond the bony crypt. The animal model developed for this study enabled the demonstration of a fully guided 3TBA protocol. The animal model and 3TBA procedure employed in this study appear to be appropriate for use in future long-term animal studies designed to demonstrate the efficacy of 3TBA for inducing molar agenesis.

Chilled Irrigation for Control of Temperature Elevation During Ureteroscopic Laser Lithotripsy: In Vivo Porcine Model.

Introduction: Multiple studies have shown significant heating of fluid within the urinary collecting system with high-power laser settings. Elevated fluid temperatures may cause thermal injury and tissue damage unless appropriately mitigated. A previous in vitro study demonstrated that chilled (CH) (4°C) irrigation slowed temperature rise, decreased plateau temperature, and lowered thermal dose during laser activation with high-power settings. We sought to evaluate the thermal effects of CH, room temperature (RT), and warmed (WM) irrigation during ureteroscopy with laser activation in an in vivo porcine model. Materials and Methods: Seven female Yorkshire cross pigs (45-55 kg) were anesthetized and positioned supine. Retrograde ureteroscopy was performed with a thermocouple affixed 5 mm from the distal end of the ureteroscope. In two pigs, a holmium:YAG laser was activated for 60 seconds at irrigation rates of 8, 12, and 15 mL/min with CH, RT, or WM irrigation. In five pigs, core body temperature was recorded for 1 hour with or without continuous CH irrigation at 15 mL/min. Results: At irrigation rates ≥12 mL/min, temperature curves appeared uniformly offset, WM > RT > CH irrigation. The threshold of thermal tissue injury was reached during laser activation for all irrigation temperatures at 8 mL/min. The threshold was not reached with CH irrigation at 12 or 15 mL/min, or with RT irrigation at 15 mL/min. The threshold was exceeded at all irrigation rates with WM irrigation. There was no significant change in core body temperature after delivering CH irrigation at 15 mL/min compared with no irrigation for 60 minutes. Conclusion: Irrigation with CH saline solution during ureteroscopic laser lithotripsy slows temperature rise, lowers peak temperature, and lengthens the time to thermal injury compared with irrigation with RT or WM saline solutions. Core body temperature was not significantly impacted by CH irrigation.

Hemodynamic Effects of Cardiovascular Medications in a Normovolemic and Hemorrhaged Yorkshire-cross Swine Model.

The Yorkshire-cross swine model is a valuable translational model commonly used to study cardiovascular physiology and response to insult. Although the effects of vasoactive medications have been well described in healthy swine, the effects of these medications during hemorrhagic shock are less studied. In this study, we sought to expand the utility of the swine model by characterizing the hemodynamic changes that occurred after the administration of commonly available vasoactive medications during euvolemic and hypovolemic states. To this end, we anesthetized and established femoral arterial, central venous, and pulmonary arterial access in 15 juvenile Yorkshire-cross pigs. The pigs then received a series of rapidly metabolized but highly vasoactive medications in a standard dosing sequence. After completion of this sequence, each pig underwent a 30-mL/kg hemorrhage over 10 min, and the standard dosing sequence was repeated. We then used standard sta- tistical techniques to compare the effects of these vasoactive medications on a variety of hemodynamic parameters between the euvolemic and hemorrhagic states. All subjects completed the study protocol. The responses in the hemorrhagic state were often attenuated or even opposite of those in the euvolemic state. For example, phenylephrine decreased the mean arterial blood pressure during the euvolemic state but increased it in the hemorrhagic state. These results clarify previously poorly defined responses to commonly used vasoactive agents during the hemorrhagic state in swine. Our findings also demonstrate the need to consider the complex and dynamic physiologic state of hemorrhage when anticipating the effects of vasoactive drugs and planning study protocols.

An immunologically active, adipose-derived extracellular matrix biomaterial for soft tissue reconstruction: concept to clinical trial

Soft tissue reconstruction remains an intractable clinical challenge as current surgical options and synthetic implants may produce inadequate outcomes. Soft tissue deficits may be surgically reconstructed using autologous adipose tissue, but these procedures can lead to donor site morbidity, require multiple procedures, and have highly variable outcomes. To address this clinical need, we developed an “off-the-shelf” adipose extracellular matrix (ECM) biomaterial from allograft human tissue (Acellular Adipose Tissue, AAT). We applied physical and chemical processing methods to remove lipids and create an injectable matrix that mimicked the properties of lipoaspirate. Biological activity was assessed using cell migration and adipogenesis assays. Characterization of regenerative immune properties in a murine muscle injury model revealed that allograft and xenograft AAT induced pro-regenerative CD4+ T cells and macrophages with xenograft AAT additionally attracting eosinophils secreting interleukin 4 (Il4). In immunocompromised mice, AAT injections retained similar volumes as human fat grafts but lacked cysts and calcifications seen in the fat grafts. The combination of AAT with human adipose-derived stem cells (ASCs) resulted in lower implant volumes. However, tissue remodeling and adipogenesis increased significantly in combination with ASCs. Larger injected volumes of porcine-derived AAT demonstrated biocompatibility and greater retention when applied allogeneicly in Yorkshire cross pigs. AAT was implanted in healthy volunteers in abdominal tissue that was later removed by elective procedures. AAT implants were well tolerated in all human subjects. Implants removed between 1 and 18 weeks demonstrated increasing cellular infiltration and immune populations, suggesting continued tissue remodeling and the potential for long-term tissue replacement.

A pig model of tunneled dialysis catheter (TDC) infection and dysfunction: Opportunities for therapeutic innovation.

Although tunneled dialysis catheters (TDC) are far from ideal, they still represent the main form of vascular access for most patients beginning dialysis. Catheters are easy to place and allow patients instant access to dialysis, but regardless of these benefits, catheters are associated with a high incidence of significant complications like bloodstream infections, central venous stenosis, thrombosis, and dysfunction. In the present study, we aim to describe and characterize a swine model of catheter dysfunction and bloodstream infection, that recreates the clinical scenario, to help to serve as a platform to develop therapeutic innovations for this important clinical problem. Six Yorkshire cross pigs were used in this study. Non-coated commercial catheters were implanted in the external jugular recreating the main features of common clinical practice. Catheters were aseptically accessed twice a week for a mock dialysis procedure (flushing in and out) to assess for and identify catheter dysfunction. Animals were monitored daily for infections; once detected, blood samples were collected for bacterial culture and antibiograms. Study animals were euthanized when nonresponsive to treatment. Tissue samples were collected in a standardized fashion for macroscopic inspection and histological analysis. The data analysis revealed an early onset of infection with a median time to infection of 9 days, 40% of the isolates were polymicrobial, and the average time to euthanasia was 20.16 ± 7.3 days. Median time to catheter dysfunction onset was 6 days post-implantation. Postmortem dissection revealed external fibrin sheath and internal thrombosis as the main causes of catheter dysfunction. There was also evidence of central venous stenosis with positive cells for αSMA, CD68, Ki67, Smoothelin, and Vimentin within the venous neointima. The described model represents a reliable and reproducible large animal model of catheter dysfunction and bloodstream infection, which recreates all the main complications of TDC's and so could be used as a validated large animal model to develop new therapies for TDC related infection, thrombosis/dysfunction and central venous stenosis.

Blunt Pulmonary Injury Induces Profound Pulmonary Hypertension in a Porcine Polytrauma Model

Traumatic blunt pulmonary injuries, while typically appearing mild on initial exam, notoriously progress into cases of pulmonary hypertension (PHT) and pulmonary edema often worsened with the fluid resuscitation necessitated by such injuries. Increased hypoxemia in acute lung injury is believed to cause pulmonary vasoconstriction which further increases pulmonary arterial pressure, and worsens hypoxemia in a vicious cycle of persistent PHT. The relationship between pulmonary hemodynamics and pulmonary edema, however is not well understood. We hypothesize that the initial increase in pulmonary vasoconstriction occurs to isolate damaged lung tissue and prevent pulmonary infiltration due to traumatic disruption of the alveolar air-liquid interface. In this study, we tested this hypothesis by examining the development of extravascular lung water in relation to pulmonary vasoconstriction in a porcine polytrauma model of acute lung injury. Pulmonary function and hemodynamics were examined before and after localized blunt right chest injury trauma in anesthetized, mechanically ventilated Yorkshire cross pigs (26+/- 1 kg, n=9). Acute grade II-III lung injury was verified at the end of the experiment. Cardiac output was measured via thermodilution with a SwanGanz catheter and extravascular lung water index (ELWI) and pulmonary capillary permeability were assessed in real time using the Pulse Ion Contour Cardiac Output (PiCCO) method (Getinge). Pulmonary arterial pressure increased within 60 minutes of initial lung injury (19 +/- 1 mm Hg at baseline vs 23 +/- 2 mm Hg) despite a drop in mean arterial pressure (60 +/- 1 mm Hg at baseline to 48 +/- 4 mm Hg after trauma). The ratio of pulmonary vascular resistance to systemic vascular resistance (PVR/SVR), indicated a selective pulmonary vasoconstriction(0.23 +/- 0.01 to 0.41 +/- 0.05, p = 0.054). ELWI was negatively correlated with the PVR/SVR ratio (r=0.44, p=0.010). Central venous pressure was lowest (6 +/- 0.6 mm Hg) right after trauma and increased (8 +/- 0.4 mm Hg) 2 hours after trauma along with the increase in PVR/SVR post trauma. Results suggests that traumatic pulmonary edema was limited via autoregulatory vasoconstriction. While effective in preventing pulmonary fluid extravasation, pulmonary hypertension resulted in a marked increase in heart afterload and accumulation of fluid in the right heart. The magnitude of this effect was such that even a moderate degree of fluid resuscitation risked precipitating florid congestive heart failure. Our data suggest that great caution must be exercised during fluid resuscitation of the blunt pulmonary trauma patient. The views expressed are those of the authors and do not reflect the official policy or position of the Department of the Army, Department of Defense, or the U.S. Government.

Combined renal and common hepatic artery denervation as a novel approach to reduce cardiometabolic risk: technical approach, feasibility and safety in a pre-clinical model

BackgroundCardiovascular and metabolic regulation is governed by neurohumoral signalling in relevant organs such as kidney, liver, pancreas, duodenum, adipose tissue, and skeletal muscle. Combined targeting of relevant neural outflows may provide a unique therapeutic opportunity for cardiometabolic disease.ObjectivesWe aimed to investigate the feasibility, safety, and performance of a novel device-based approach for multi-organ denervation in a swine model over 30 and 90 days of follow-up.MethodsFive Yorkshire cross pigs underwent combined percutaneous denervation in the renal arteries and the common hepatic artery (CHA) with the iRF Denervation System. Control animals (n = 3) were also studied. Specific energy doses were administered in the renal arteries and CHA. Blood was collected at 30 and 90 days. All animals had a pre-terminal procedure angiography. Tissue samples were collected for norepinephrine (NEPI) bioanalysis. Histopathological evaluation of collateral structures and tissues near the treatment sites was performed to assess treatment safety.ResultsAll animals entered and exited the study in good health. No stenosis or vessel abnormalities were present. No significant changes in serum chemistry occurred. NEPI concentrations were significantly reduced in the liver (− 88%, p = 0.005), kidneys (− 78%, p < 0.001), pancreas (− 78%, p = 0.018) and duodenum (− 95%, p = 0.028) following multi-organ denervation treatment compared to control animals. Histologic findings were consistent with favourable tissue responses at 90 days follow-up.ConclusionsSignificant and sustained denervation of the treated organs was achieved at 90 days without major safety events. Our findings demonstrate the feasibility of multi-organ denervation using a novel iRF Denervation System in a single procedure.

Reston virus causes severe respiratory disease in young domestic pigs

Reston virus (RESTV), an ebolavirus, causes clinical disease in macaques but has yet only been associated with rare asymptomatic infections in humans. Its 2008 emergence in pigs in the Philippines raised concerns about food safety, pathogenicity, and zoonotic potential, questions that are still unanswered. Until today, the virulence of RESTV for pigs has remained elusive, with unclear pathogenicity in naturally infected animals and only one experimental study demonstrating susceptibility and evidence for shedding but no disease. Here we show that combined oropharyngeal and nasal infection of young (3- to 7-wk-old) Yorkshire cross pigs with RESTV resulted in severe respiratory disease, with most animals reaching humane endpoint within a week. RESTV-infected pigs developed severe cyanosis, tachypnea, and acute interstitial pneumonia, with RESTV shedding from oronasal mucosal membranes. Our studies indicate that RESTV should be considered a livestock pathogen with zoonotic potential.

Comparison of the efficacy of silver-based antimicrobial burn dressings in a porcine model of burn wounds

PurposeA variety of silver-based antimicrobial dressings are available on the market and are commonly used to prevent infection. Such prophylaxis is particularly important in treating burns, yet there is a paucity of evidence confirming the efficacy of commercially available dressingsin vivo. We describe here an in vivo porcine model of burns, which we use to test the antimicrobial efficacy of three common wound dressings and a control. ProceduresDomestic Yorkshire-cross pigs were medicated for pain management before inflicting burns with a heated brass rod. The wounds were artificially challenged with a mixture of two pathogens commonly associated with burn wound infection:Staphylococcus aureus and Pseudomonas aeruginosa. The following dressing materials were sutured in place: gauze, nanocrystalline silver, silver-plated nylon, and polyethylene/polyester coated with high-oxidation silver salts. After 1 and 3 days, the wounds were assessed for erythema, swelling, and re-epithelialization, tissue was biopsied to determine the recovery of the challenge microorganisms, and histology was performed. We also examined the number of microorganisms present on the dressings themselves. ResultsHistology indicated that 30 s was sufficient to produce burns extending into the deep dermal layer. After 3 days, nanocrystalline silver and silver-plated nylon led to slightly reduced swelling relative to simple gauze, although none of the dressings significantly affected erythema or wound re-epithelialization. All the dressings led to decreased recovery of the challenge organisms from the burn tissue, relative to simple gauze. However, the magnitude of the reduction was greatest for nanocrystalline silver (log10 reduction = 4–5); additionally, only nanocrystalline silver gave a statistically significant decrease (P = 0.02). Notably, the antimicrobial effect for all dressings was reduced by Day 3 relative to Day 1. Similar trends were observed for microbial retention on the dressings themselves. ConclusionNanocrystalline silver-based wound dressings generally outperformed silver-plated nylon and high-oxidation silver salts in thisin vivo model of burn wounds. Relative to prophylactic use, it may be advisable to change the dressings more frequently when treating an infected wound.

Renal Function, Potassium Homeostasis, and Vasopressin Regulation During Acute Hypotensive Stress in a Pig Model of Hemorrhagic Shock

Vasopressin (VP) can regulate potassium excretion in conditions of low urine flow. In severe hemorrhagic shock, the use of pharmacologic doses of VP has advantages over other pressor agents as it can increase urine output. We hypothesized that the physiological response to the rapid rise in plasma potassium (pK+) within minutes of the onset of hemorrhage would involve increased VP synthesis and VP‐mediated maintenance of renal potassium excretion in the face of severely decreased urine flow. In this study we examined the effect of the VP response on renal potassium handling during acute shock stress.Anesthetized, mechanically ventilated Yorkshire cross pigs (body weight 8.8±0.2 kg, n=44) were instrumented with vascular catheters and cardiac Swan Ganz catheter for hemodynamic assessments, and a bladder catheter for continuous urine collection. After baseline measurements, pigs were divided into control (n=14) or hemorrhage (n=30) groups. Hemorrhagic shock was induced by arterial blood withdrawal over 30 minutes (3 ml/kg/min for 7 minutes followed by a rate of 1 ml/kg/min until a goal oxygen debt of over 60 ml/kg was achieved with total shed volume of about 30 ml/kg and decrease in blood pressure by 50%). Blood samples for electrolytes and VP measurements were obtained at 10 minute intervals up to 60 minutes after the start of hemorrhage. Hemodynamics were assessed before resuscitation and up to 4 hours after hemorrhage. Six of the 30 hemorrhaged animals did not survive the acute blood loss beyond the first hour of hemorrhage, so we compared the results of survivors (n=24) to that of non‐survivors (n=6) to determine if altered renal fluid and electrolyte handling may have contributed to non‐survival.Non‐survivors had a higher (p&lt;0.05) pK+ (7.0±0.7 mEq/L) at the end of hemorrhage than survivors (5.4±0.2 mEq/L) vs no change from baseline in controls (4.5±0.1 mEq/L). There was no change in plasma VP in controls whereas hemorrhage increased VP in survivors and non‐survivors (126±14 to 275±41 and 80±18 to 476±211 pg/ml, p&lt;0.05, respectively). Thus, a deficit in VP synthesis and release was not responsible for the fatal hyperkalemia in non‐survivors. Indeed, in hemorrhaged animals VP mRNA, V1aR and V1bR expression in the hypothalamus and pituitary were increased compared to controls. Controls exhibited no change in renal fluid and electrolyte handling. Hemorrhage decreased urine flow (21.0±2.4 to 8.9±1.9 and 20.8±5.8 to 4.0±1.6 ul/min/kg, in survivors and non‐survivors, respectively) along with a decrease in glomerular filtration (p&lt;0.05). Osmotic clearance decreased and was accompanied by increased free water clearance in hemorrhage. Fractional excretion of potassium remained constant. A significantly higher fractional excretion of water distinguished non‐survivors from survivors. A difference in the renal tubular handling of fluid may hinder effective potassium excretion after acute hypotensive stress, and hence results in exacerbated hyperkalemia in non‐survivors despite adequate VP circulating levels.Support or Funding InformationThe views expressed are those of the authors and do not reflect the official policy or position of the Department of the Army, Department of Defense, or the U.S. Government.

Reference Intervals for and the Effects of Sample Handling and Sex on Rotational Thromboelastometry in Healthy Adult Pigs.

Accurate assessment of coagulation in porcine studies is essential. We sought to establish normal values for porcine rotational thromboelastometry (ROTEM) according to the American Society for Veterinary Clinical Pathology guidelines and to assess the effects of various preanalytical parameters on those measurements. Healthy Yorkshire-cross pigs (n = 81; 46 males and 35 females) were anesthetized. By using a 18-gauge needle attached to a vacuum phlebotomy tube, blood was acquired from the cranial vena cava. Tubes were filled in the following order: evacuation clot tube, EDTA tube, heparin tube, and 2 citrate tubes. The citrate tubes were randomly assigned to 30 min with or without constant agitation on a rocker. The following parameters were reported according to the manufacturer's recommendations: clotting time, clot formation time, α, (tangent to the clot formation curve when the clot firmness is 20 mm), clot firmness after 10 and 20 min, maximal clot firmness, maximum lysis, and lysis indexes at 30 and 45 min. Reference intervals were reported as mean ± 2 SD (parametric distribution) or 2.5th and 97.5th percentile of the population's results (nonparametric distribution). The effects of sex, sampling order, and agitation on ROTEM results were analyzed through linear regression. Neither sex nor sample agitation influenced any of the ROTEM parameters. Combined reference intervals were established for each ROTEM parameter by pooling data from the nonagitated tubes for both male and female pigs. This study is the first to establish ROTEM reference intervals from a large number of male and female adult Yorkshire-cross pigs and to provide a detailed description of preanalytical sample processing.

Vasopressin Receptor Expression Reveals Role of Endogenous Vasopressin in Ameliorating Acute Lung Injury in Pig Models of Pulmonary Hypertension

Acute lung injury (ALI) that evolves into acute respiratory distress syndrome (ARDS) is a critical care management challenge with 30–40% mortality. Despite the beneficial ability of pharmacological doses of vasopressin (VP)to elevate blood pressure without worsening pulmonary hypertension (PHN) in severe septic shock ARDS, circulating VP levels (not necessarily indicative of local tissue VP action) remain relatively low and it is unclear whether VP has a physiological role in maintaining pulmonary blood flow and oxygenation in ARDS. We hypothesized that if endogenous VP has an important role in ameliorating ALI, vascular VP receptor mRNA expression will increase within hours after initial ALI insult in order to replenish spent receptors that are degraded when activated by endogenous VP.Anesthetized Yorkshire cross pigs(n=36, 7.9 ± 0.2 kg body weight) were used to examine pulmonary hemodynamics in ALI without the confounding effects of systemic hypotension. PHN was induced by either disrupting the lung alveolar‐capillary interface with oleic acid (OA; 0.1 ml/kg iv, n=14) or by causing endothelial dysfunction with endotoxin (ETX; up to 35,000 units iv, n=16). ALI pigs had elevated pulmonary to systemic vascular resistance ratios (PVR/SVR: OA = 0.48 ± 0.07 and ETX = 0.48 ±0.04) compared to controls (0.20 ± 0.01, n=6). Mean arterial pressure was constant (60±4 mm Hg in all groups). ALI was managed with supplemental oxygen with or without mesenchymal stem cells to facilitate lung tissue repair. Within 2 hours after treatment, PVR/SVR began to return towards baseline in the ETX group but remained elevated in the OA group. This provided a range of pulmonary vasoconstriction states to compare against changes in plasma VP, pituitary VP content, and the mRNA expression of VP, V1aR, V1bR, and V2R receptors in pulmonary, carotid, renal, celiac, and mesenteric arteries, and aorta collected12 to 16 hours after initial ALI. Stepwise multiple regression revealed that PVR/SVR at 2–4 hours of established lung injury was positively correlated with V1aR expression in the aorta and mesenteric artery and V2R expression in the pulmonary and renal arteries, and negatively correlated with V2R expression in the aorta and celiac arteries (r2=0.88, p&lt;0.0001). Results are consistent with endogenous VP responding to pulmonary hypertension in ALI via a pulmonary V2R mediated vasodilatory counter effect, in the face of V1aR mediated vasoconstriction in other vascular beds, to maintain pulmonary blood flow and improve blood oxygenation.Support or Funding InformationThis project was supported by the US Army Medical Command. The views expressed in this abstract are those of the authors and do not reflect the official policy or position of the Department of the Army, Department of Defense, or the U.S. GovernmentThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Acellular Biological Scaffold Repair Does Not Overcome the Pathobiology of Volumetric Muscle Loss Injury

Volumetric muscle loss (VML) resulting from extremity trauma presents chronic and persistent functional deficits, restricted joint range of motion, and fibrosis, which ultimately manifest disability. Currently, there is a clinical need for therapies aimed at addressing the debilitating outcomes due to VML injuries. Recent investigations have examined the use of commercially available biological scaffolds as a regenerative medicine approach for VML. However, there is a paucity of large animal data to advance clinical translation. We hypothesized that repair of VML injuries with an acellular biologic scaffold would improve function, specifically neuromuscular strength following injury. To test this hypothesis, female Yorkshire Cross pigs (n=10) were randomized to sham or an ~20% VML injury to the peroneous tertius muscle, and injuries were left non‐repaired, or surgically repaired with an acellular biologic scaffold derived from either porcine small intestinal submucosa (SIS) or urinary bladder matrix (UBM) using standard clinical surgical implantation procedures. Analysis of muscle volume using CT imaging and muscle function via peroneal nerve stimulation was conducted through 12 weeks post‐injury. At this terminal time‐point, full thickness biopsy samples were used for histological analysis and sequencing of RNA extracted from the injured muscle tissue. All studies were conducted in compliance with the Animal Welfare Act, the Implementing Animal Welfare Regulations and in accordance with the principles of the Guide for the Care and Use of Laboratory Animals. Unexpected adverse advents occurred in 80% of the VML injuries repaired with the SIS scaffold. Both the repair with SIS and UBM resulted in a significant fibrotic response and increased volume of the anterior compartment. However, this volume could not be attributed to increased muscle, as histologic analysis of the muscle indicated sub‐physiologic islands of muscle fibers (~50 fibers). To understand the quality of muscle, peak muscle torque was normalized to the CT‐derived volume of the anterior compartment at 10 weeks post‐injury. Collectively in the non‐ and ECM‐repaired limbs there was ~45% deficit compared to sham operated limbs. Additionally, in both the non‐ and ECM‐repaired limbs there was an ~30% deficit in absolute peak isometric torque through 12 weeks post‐injury. Tissue level RNA sequencing analysis of the non‐repaired and surgically repaired samples compared to Sham controls revealed increases in expression of sets of genes associated with inflammatory signaling and fibrogenesis and downregulation of myogenic repair gene sets. Quantitative comparison of the transcriptomes from surgically repaired samples against the non‐repaired samples indicated high degrees of correlation amongst the differentially expressed genes. These results indicate the gene expression programs induced from a VML insult are not appreciably augmented when treated with surgical repair of an acellular biologic scaffold. Collectively, these data indicate that VML results in extensive and continued disruption of the remaining musculature and that ECM repair does not ameliorate the resulting pathobiology.

Autologous Minced Muscle Grafts Improve Muscle Strength in a Porcine Model of Volumetric Muscle Loss Injury.

The traumatic loss of muscle tissue, defined as volumetric muscle loss (VML) injury, has no definitive therapy. The purposes of this study were: (1) to develop a porcine model of VML and (2) to investigate autologous minced muscle grafts (1-mm pieces of muscle) as a potential therapeutic. Minced grafts were evaluated because they have promoted fiber regeneration and functional recovery in rat VML models and do not require US Food and Drug Administration approval for clinical use. In 5 female Yorkshire-cross pigs, ≈5 g (≈20%) of tissue was excised from the peroneous tertius muscle (≈3 × 3 × 1.5-cm defect) of each leg. The defect in one leg was treated with autologous minced grafts derived from the contralateral leg. Maximal isometric tetanic strength assessments of the dorsiflexor muscles (ie, the peroneous tertius muscle) were performed before and biweekly up to 12 weeks postinjury. VML injury resulted in a -43.5% ± 7.2% strength deficit 12 weeks postinjury in nonrepaired legs. Autologous minced muscle graft repair significantly improved strength over 12 weeks (32% strength increase 12 weeks postinjury vs. nonrepaired muscles with a remaining -27.8% ± 7.0% strength deficit; P < 0.001). Nonrepaired muscles developed extensive fibrosis and presented no evidence of muscle fiber regeneration within the defect area. Minced graft-treated muscles presented areas of putative de novo muscle fiber regeneration within the defect area, although extensive fibrotic tissue deposition was also present. Autologous minced muscle grafts partially restored neuromuscular strength in a novel porcine model of VML.

Vasopressin‐Altered Glycemic Control May Lead to Metabolic Disorders of Severe Hemorrhagic Shock

Metabolic disorders associated with trauma and hemorrhage typically follow an early hyperglycemic reflex aimed at increasing brain blood glucose in the face of oxygen debt. The hyperglycemic reflex is believed to be initiated by hypoxia‐induced carotidbody stimulation via a vasopressin (VP) V1a receptor mechanism. We hypothesized that elevated circulating VP levels released in response to severe shock contribute not only to restoring blood pressure, but may also prolong altered glycemic control. Thus, we examined VP influence on glucose regulation after severe oxygen debt from hemorrhagic shock using Yorkshire cross pigs (n=29, body weight 31.1+0.3 kg). Rapid hemorrhage (30 ml/kg bloodshed over 20 minutes) was induced to cause a 50% drop in mean arterial pressure and an oxygen debt of 68+5 ml/kg. Blood glucose levels rose from 124+10 mg/dl at baseline to 174+10mg/dl after hemorrhage (p&lt;0.01), with accompanying increases in VP, epinephrine, glucagon, and insulin. Relationships between blood glucose, VP, epinephrine, glucagon, insulin, pancreatic blood flow and liver blood flow were examined. Multiple regression analysis of glycemic control from baseline through the end of hemorrhage indicated that glucose levels were strongly associated with VP and epinephrine, and inversely related to glucagon which correlated with VP (p&lt;0.01). VP and pancreatic blood flow remained negatively correlated in all phases of baseline, hemorrhage and resuscitation with exogenous VP (30 ng/kg/min i.v., n=7). Thus pancreatic blood flow was significantly lower (p&lt;0.01) with VP‐treated pigs (0.14+0.02 ml/kg/min) than in pigs that were resuscitated with normal saline where pancreatic blood flow (0.36+0.04ml/kg/min, n=10) did not further decrease. Exogenous VP during resuscitation also stimulated liver blood flow, and seemed to keep glucagon from decreasing despite the decreased pancreatic blood flow. VP did not affect insulin levels. Results suggest that VP mediates a hyperglycemic response in severe hemorrhagic shock via influence on glucagon secretion. If hyperglycemia continues beyond this acute response to hemorrhage, long‐term metabolic disorders could develop which would lead to tissue oxidative damage and contribute to trauma‐induced multisystem organ failure. Strategies for critical care management of hemorrhagic shock need to incorporate understanding of VP‐induced metabolic alterations of glycemic control.Support or Funding InformationThis project was supported by U.S. Army Medical Research and Materiel Command Congressionally Directed Medical Research Program grant #DAMD17‐03‐1‐0072. The views expressed in this abstract are those of the authors and do not reflect the official policy or position of the Department of the Army, Department of Defense, or the U.S. Government.

Vasopressin Receptor Regulation in Maintaining Potassium Homeostasis in a Sus scrofa Model of Hemorrhagic Shock

We previously showed that elevated potassium (K)levels seen with severe hemorrhage appear to stimulate enhanced vasopressin(VP) release, and that in turn, elevated VP levels help correct hyperkalemia by increasing renal tubular K secretion. We thus tested whether brain VP receptors controlling vasopressin release were regulated synergistically with kidney VP receptors whichmodulated renal K excretion. We hypothesized that brain VP receptor up‐regulation may enable positive feedback for K‐stimulated augmentation of VP responsiveness in hemorrhage, and that renal VP receptor expression would reflect VP receptor mediated action on renal K excretion. Thus, in anesthetized Yorkshire cross pigs (n=35) we compared plasma VP (pVP) and plasmaK (pK) before, at the end of hemorrhage (30 ml/kg over 20 minutes), and after resuscitation, against mRNA expression of V1aR, V1bR, and V2R receptors in brain and kidney tissues collected after resuscitation. Multiple regression analysis showed that increased pVP at the end of hemorrhage positively correlated (p&lt;0.05) with expression of V1aR and V1bR in the hypothalamus, V1bR in the anterior pituitary, and V2R in the renal cortex. Plasma K levels at the end of resuscitation were negatively correlated (p&lt;0.05) with renalcortex V2R expression. In a different group of control animals that did not undergo hemorrhage, pVP and pK remained unchanged throughout. Results suggest that an up‐regulation of V1a and V1b receptors in the brain contributed to an amplified VP response in hemorrhage. In addition, VP appears to stimulate renal V2R expression to lower pK levels and maintain K homeostasis via a renal V2R‐mediated negative feedback mechanism.Support or Funding InformationThis project was supported by U.S. Army Medical Resarch and Materiel Command Congressionally Directed Medical Research Program grant #DAMD17‐03‐1‐0072. The views expressed in this abstract are those of the authors and do not reflect the official policy or position of the Department of the Army, Department of Defense, or the U.S. Government.

Full Enteral Feeding during Severe Hypotension does not Adversely Affect Gastrointestinal Microcirculation in a Porcine (Sus scrofa) Model of Endotoxic Shock

Introduction: The use of enteral nutrition in septic shock is controversial during periods of significant hypotension due to concern of possible metabolic stimulation of an ischemic gastrointestinal system. In this study, using a piglet model of endotoxic shock, we tested the hypothesis that full enteral feeding during shock would not adversely affect gastrointestinal microcirculation. Methods: Yorkshire cross pigs were either fed or non-fed during endotoxic shock and compared against controls. Severe hypotension was induced by administration of E. coli. Hemodynamics and microcirculatory blood flow (measured via colored microspheres) were compared throughout the experiment. Post-mortem biopsies of the stomach, small bowel, and large bowel were examined from representative pigs in each group. Results: Despite a sustained decrease in MAP with ETX exposure, regional blood flow (RBF) to the stomach was maintained throughout the experiment. RBF to the small intestine dropped, and remained low, with lowered MAP. The large intestine followed the small intestine pattern but with consistency issues within the data. Post-mortem biopsies revealed normal stomach tissue in both groups. Both fed and non-fed ETX animals showed mild to moderate erosion in the small and large bowel but there was no significant effect of feeding compared to not feeding. Conclusion: While this study is limited in duration, it does appear that initially there is no harm to the microcirculation or integrity of the gastrointestinal tract with addition of feeding during severe septic shock. Results indicate that feeding during severe hypotension, which would help maintain nutrition, should be considered.

Selection of non-steroidal anti-inflammatory drug and treatment regimen for sulfur mustard-induced cutaneous lesions

The inflammatory process plays an important role in sulfur mustard (HD) injury and HD pathogenesis, suggesting that anti-inflammatory treatments applied as soon as possible following HD injury may reduce tissue damage and accelerate healing. This study used the HD dermal weanling swine model to investigate the efficacy of two non-steroidal anti-inflammatory drugs, capsaicin and diclofenac, when applied in combination with the steroid, clobetasol. The therapeutic regimen was also investigated with respect to initiation of treatment post-exposure, frequency and duration. Yorkshire-cross pigs were randomly assigned to experimental groups, corresponding to all combinations of treatment (capsaicin with clobetasol or diclofenac with clobetasol), onset time (1, 2 or 4 h post-exposure), treatment duration (1, 3 or 5 days) and frequency of applications (2, 3 or 4 per day). For each animal, two sites on the ventral abdomen were exposed to 400 μL of neat HD for 8 min to achieve superficial dermal (SD) lesions and two sites were exposed to 400 μL neat HD for 30 min to achieve deep dermal (DD) lesions. Each treatment regimen was tested against a SD and a DD injury. Untreated SD and DD lesion sites served as within-animal controls. Assessments, up to one week post-challenge, included digital photographs, clinical assessments (lesion size measurements and modified Draize scoring), transepidermal water loss (TEWL), reflectance colorimetry and histopathologic evaluations that included an estimate for depth of injury and wound healing parameters. Diclofenac plus clobetasol treatment resulted in significant reductions in lesion contracture and modified Draize scores, increased barrier function (decreased TEWL), and increased healing as determined by histopathology for both SD and DD injury when compared with untreated sites and sites treated with capsaicin plus clobetasol. An increased duration of treatment from 1 to 5 days was most commonly associated with decreased clinical assessment and histopathological severity scores. Therefore, a combination of diclofenac and clobetasol application, when administered for at least five days, shows promise in ameliorating HD-induced lesions.

Vasopressin decreases pulmonary-to-systemic vascular resistance ratio in a porcine model of severe hemorrhagic shock.

Vasopressors are gaining renewed interest as treatment adjuncts in hemorrhagic shock. The ideal vasoconstrictor will increase systemic blood pressure without increasing pulmonary vascular resistance (PVR), which hinders pulmonary perfusion and exacerbates hypoxemia. However, the selectivity of pressors for pulmonary versus systemic vasoconstriction during hemorrhage has not been characterized. The purpose of this study was to test the hypothesis that vasopressin (VP) has distinct effects on pulmonary versus systemic hemodynamics, unlike the catecholamine vasopressors norepinephrine (NE) and phenylephrine (PE). Anesthetized and ventilated pigs were assigned to resuscitation with saline only (n = 7) or saline with VP (n = 6), NE (n = 6), or PE (n = 6). Animals were hemorrhaged to a target volume of 30 mL/kg and a mean arterial pressure of 35 mmHg. One hour after the start of hemorrhage, animals were resuscitated with saline up to one shed blood volume, followed by either additional saline or a vasopressor. Hemodynamics and oxygenation were measured hourly for 4 h after the start of hemorrhage. Vasopressin increased systemic vascular resistance (SVR) while sparing the pulmonary vasculature, leading to a 45% decrease in the PVR/SVR ratio compared with treatment with PE. Conversely, NE induced pulmonary hypertension and led to an increased PVR/SVR ratio associated with decreased oxygen saturation. Phenylephrine and crystalloid had no significant effect on the PVR/SVR ratio. Sparing of pulmonary vasoconstriction occurs only with VP, not with administration of crystalloid or catecholamine pressors. The ability of VP to maintain blood oxygenation indicates that VP may prevent hypoxemia in the management of hemorrhagic shock.