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Burn shock and acute fluid resuscitation continue to spark intense interest and debate among burn clinicians. Following a major burn injury, fluid resuscitation of burn shock is life-saving, but paradoxically can also be a source of increased morbidity and mortality because of the unintended consequence of systemic edema formation. Considerable research over the past two decades has been devoted to understanding the mechanisms of edema formation, and to develop strategies to curb resuscitation fluids and limit edema development. Recognition of burn endotheliopathy—injury to the endothelium’s glycocalyx layer—is one of the most important recent developments in our understanding of burn shock pathophysiology. Newer monitoring approaches and resuscitation endpoints, along with alternative resuscitation strategies to crystalloids alone, such as administration of albumin, or plasma, or high-dose ascorbic acid, have had mixed results in limiting fluid creep. Clear demonstration of improvements in outcomes with all of these approaches remains elusive. This comprehensive review article on burn shock and acute resuscitation accompanies the American Burn Association’s State of the Science meeting held in New Orleans, LA on November 2 to 3, 2021 and the Proceedings of that conference published in this journal.

Adipose stem cells (ASCs) have shown therapeutic promise for various conditions, including burn injury. While ASCs have immunomodulatory properties, concerns exist over pro-coagulant activity after intravenous (IV) administration. In the present study, we examined IV human ASC delivery in terms of coagulation, organ function, and inflammation in a 40% total body surface area (TBSA) swine burn model. Anesthetized female Yorkshire swine were burned and randomized to receive 15 ml/kg Lactated Ringer’s containing: no ASCs; a low dose (5 × 105 ASCs/kg); or a high dose (5 × 106 ASCs/kg). For biochemical analysis, blood was collected at baseline (BL), 3, 6, 12, and 24 h post-burn, while kidney and liver tissue was collected post-euthanasia. A significant, but transient, effect of ASCs was seen on prothrombin times and INR, wherein low doses revealed slight hypercoagulation. Burns increased partial thromboplastin time, fibrinogen, and d-dimer levels, which was unchanged with ASC administration. ASCs tended to exacerbate increases in bilirubin at 3 h, but this didn’t reach statistical significance. A significant effect of ASCs on creatinine and BUN was seen, wherein low doses elevated levels at 24 h (creatinine, P = 0.0012; BUN, P = 0.0195). Hepatic and renal TUNEL staining were similar for all groups. A dose-dependent decrease in IL-8 was observed, while low doses significantly increased IL-1RA at 3h (P = 0.050), IL-12 at 12h (P = 0.021) and IL-6 at 24 h post-burn (P = 0.035). IV administration of xenogeneic ASCs slightly increased coagulation, but effects on burn-induced renal and hepatic dysfunction effects were minimal. Despite some significant immunomodulation, organ dysfunction effects were modest. Collectively, this study provides evidence to be skeptical about xenogeneic ASC administration in regards to burns.

Various studies have reported discordant results on the magnitude and direction of burn-induced coagulopathy (BIC), which has recently been associated with multiple organ dysfunction syndrome (MODS) and death. The increased mechanistic understanding of BIC is due, in part, to novel assays that have expanded the armamentarium beyond traditional tests like PT and aPTT. Still, BIC is a dynamic process, and the progression is difficult to define in the thermally-injured. To this end, we aimed to enhance the understanding of burn-induced coagulation abnormalities by employing functional assessments of platelet aggregation, viscoelastic kinetics, and thrombin generation in an extensive burn model in swine. Anesthetized Yorkshire pigs sustained 40% total body surface area (TBSA) full-thickness contact burns and recovered in metabolic cages. Blood was collected at baseline (BL), as well as 6, 24, and 48 h after injury. A significant effect of burn (P 0.0001) was seen on platelets, with mild thrombocytopenia apparent at 24 h. While slight decreases in aPTT were not significant, rotational thromboelastometry (ROTEM) analysis revealed hypercoagulation 6 and 24 h after burn by a decreased clotting time. Maximum clot firmness increased after burn, but was not statistically significant until 48 h. Hypercoagulation was not supported by platelet aggregation, as the response to ADP was greatly and persistently diminished, and the response to collagen was unchanged. Endogenous thrombin potential was significantly reduced at 6 and 24 h after burn (P 0.0001), and also correlated with a number of ROTEM parameters and collagen-induced platelet aggregation. In contrast, PT was not correlated with other measured parameters. Taken together, novel coagulation parameters may be more sensitive than PT in characterizing coagulopathy in the setting of burns. The data presented herein makes initial strides to report the natural history of several of these variables over time in a large animal model of extensive burns, indicating early hypercoagulability followed by hypocoagulation. Future work will elucidate the effects of standard of care.

While urinary output (UOP) remains the primary endpoint for titration of intravenous fluid resuscitation, it is an insufficient indicator of fluid responsiveness. Although advanced hemodynamic monitoring (including arterial pulse wave analysis [PWA]) is of recent interest, the validity of PWA-derived indices in burn resuscitation extremes has not been established. The goal of this paper is to test the hypothesis that PWA-derived cardiac output (CO) and stroke volume (SV) indices as well as pulse pressure variation (PPV) and systolic pressure variation (SPV) can play a complementary role to UOP in burn resuscitation. Swine were instrumented with a Swan-Ganz catheter for reference CO and underwent a 40% TBSA burns with varying resuscitation paradigms, and were monitored for 24 hours in an ICU setting under mechanical ventilation. The longitudinal changes in PWA-derived indices were investigated, and resuscitation adequacy was compared as determined by UOP vs PWA indices. The results indicated that PWA-derived indices exhibited trends consistent with reference CO and SV measurements: CO and SV indices were proportional to reference CO and SV, respectively (CO: postcalibration limits of agreement [LoA] = ±24.7 [ml/min/kg], SV: postcalibration LoA = ±0.30 [ml/kg]) while PPV and SPV were inversely proportional to reference SV (PPV: postcalibration LoA = ±0.32 [ml/kg], SPV: postcalibration LoA = ±0.31 [ml/kg]). The results also indicated that PWA-derived indices exhibited notable discrepancies from UOP in determining adequate burn resuscitation. Hence, it was concluded that the PWA-derived indices may have complementary value to UOP in assessing and guiding burn resuscitation.

Background Hemodynamic aberrations after severe burns are treated with aggressive intravenous (IV) fluid resuscitation however, oral resuscitation has been proposed in resource poor scenarios. Previously we have shown that animals receiving oral fluid following burns were able to recover kidney function. However, immune function such as circulating and splenic immune cell populations after oral or intravenous fluid administration was not examined. Herein, we perform a follow up analysis of splenic tissue and plasma from the previous animal study to examine the splenic response following these resuscitation strategies after burn injury. Methods Eighteen anesthetized Yorkshire swine receiving 40%TBSA contact burns were randomized to receive either: (1) no fluids (Fluid Restricted; negative control), (2) 70 mL/kg/d Oral Rehydration Salt solution (Oral), or (3) 2 mL/kg/%TBSA/d of lactated Ringer’s solution IV. Blood was drawn for blood cell analysis, and CT scans were performed before and 48 h post-burn, at which point spleens were harvested for histological, Western blot, and RT-PCR analyses. Results Splenic artery diameter decreased by −0.97 ± 0.14 mm in fluid-restricted animals, while IV led to an increase of 0.68 ± 0.30 mm. No significant differences were detected in white and red pulp. IV fluids reduced the population of splenic monocytes (CD163; P = 0.001) and neutrophils (MPO protein; P = 0.13), as well as cytokines IL-8 (P = 0.003), IFN-γ (P = 0.11) and TNFα (P = 0.05). Additionally, withholding IV fluids consistently decreased the expression of FoxP3, CCR6, and IL17β in spleen, suggesting a shift in T-cell phenotype with IV resuscitation. Conclusions The route of fluid administration has a minor influence on the changes in circulating and splenic leukocytes post-burn in the acute phase. Further research is needed to help guide resuscitation approaches using immunologic markers of splenic function following burns.

BACKGROUND Traumatic injury can lead to a compromised intestinal epithelial barrier, decreased gut perfusion, and inflammation. While recent studies indicate that the gut microbiome (GM) is altered early following traumatic injury, the impact of GM changes on clinical outcomes remains unknown. Our objective of this follow-up study was to determine if the GM is associated with clinical outcomes in critically injured patients. METHODS We conducted a prospective, observational study in adult patients (N = 67) sustaining severe injury admitted to a level I trauma center. Fecal specimens were collected on admission to the emergency department, and microbial DNA from all samples was analyzed using the Quantitative Insights Into Microbial Ecology pipeline and compared against the Greengenes database. α-Diversity and β-diversity were estimated using the observed species metrics and analyzed with t tests and permutational analysis of variance for overall significance, with post hoc pairwise analyses. RESULTS Our patient population consisted of 63% males with a mean age of 44 years. Seventy-eight percent of the patients suffered blunt trauma with 22% undergoing penetrating injuries. The mean body mass index was 26.9 kg/m. Significant differences in admission β-diversity were noted by hospital length of stay, intensive care unit hospital length of stay, number of days on the ventilator, infections, and acute respiratory distress syndrome (p < 0.05). β-Diversity on admission differed in patients who died compared with patients who lived (mean time to death, 8 days). There were also significantly less operational taxonomic units in samples from patients who died versus those who survived. A number of species were enriched in the GM of injured patients who died, which included some traditionally probiotic species such as Akkermansia muciniphilia, Oxalobacter formigenes, and Eubacterium biforme (p < 0.05). CONCLUSION Gut microbiome diversity on admission in severely injured patients is predictive of a variety of clinically important outcomes. While our study does not address causality, the GM of trauma patients may provide valuable diagnostic and therapeutic targets for the care of injured patients. LEVEL OF EVIDENCE Prognostic and epidemiological, level III.

Background The endothelial glycocalyx controls vascular permeability, cellular signaling, blood-endothelial cell adhesion, extravasation, and transmission of shear stress signals. Burn injury compromises integrity of this layer increasing vascular permeability, which is further exacerbated by large volumes of (intravenous) crystalloids. We have shown that enteral resuscitation is able to reverse burn-induced acute kidney injury (AKI), and herein, we present a follow-up examination of the integrity of the glycocalyx layer and its relationship with renal dysfunction after burn injury. Materials and methods Anesthetized Yorkshire pigs sustained 40% total body surface area full-thickness contact burns and recovered in metabolic cages for one of three treatments: no fluids (oral or intravenous); (n = 6), ad libitum water (n = 6), or volume-matched oral rehydration solution (ORS; n = 6) for 48 h. Urine and blood were collected at baseline (BL), 6, 12, 24, 32, and 48 h after burn at which point kidneys were harvested. Results In no fluid and water groups (but not ORS), plasma levels of glycosaminoglycans (GAGs) were elevated after burn (P ≤ 0.031). Syndecan-1 was elevated by 6 h after burn in all animals, but levels declined by 24 h with enteral fluids. Urinary GAGs in the no-fluid group were elevated after burn. No differences among treatments were detected in syndecan-1 levels, or glomerular lectin within the kidney. Conclusions Collectively, these data demonstrate that ORS prevented increases in circulating GAGs. Furthermore, an inexpensive and simple method for detecting GAGs provides a sensitive measure of endotheliopathy after burn.

The acute systemic inflammatory response syndrome (SIRS) and multiorgan dysfunction (MOD) that occur in large burn injuries may be attributed, in part, to immunosuppressive responses such as decreased lymphocytes. However, the mitochondrial bioenergetics of lymphocytes after severe burn injury are poorly understood. The purpose of this study was to examine mitochondrial function of lymphocytes following severe burns in a swine model. Anesthetized Yorkshire swine ( n = 17) sustained 40% total body surface area full-thickness contact burns. Blood was collected at pre-injury (Baseline; BL) and at 24 and 48 h after injury for complete blood cell analysis, flow cytometry, cytokine analysis, and ficoll separation of intact lymphocytes for high-resolution mitochondrial respirometry analysis. While neutrophil numbers increased, a concomitant decrease was found in lymphocytes ( P + or CD8+ lymphocytes. No changes in immune cell population were observed from 24 h to 48 h post-injury. IL 12-23 decreased while a transient increase in IL 4 was found from BL to 24h ( P

While extensive research using animal models has improved the understanding of acute kidney injury (AKI), this knowledge has not been translated into effective treatments. Many promising interventions for AKI identified in mice and rats have not been validated in subsequent clinical trials. As a result, the mortality rate of AKI patients remains high. Inflammation plays a fundamental role in the pathogenesis of AKI, and one reason for the failure to translate promising therapeutics may lie in the profound difference between the immune systems of rodents and humans. The immune systems of large animals such as swine, nonhuman primates, sheep, dogs and cats, more closely resemble the human immune system. Therefore, in the absence of a basic understanding of the pathophysiology of human AKI, large animals are attractive models to test novel interventions. However, there is a lack of reviews on large animal models for AKI in the literature. In this review, we will first highlight differences in innate and adaptive immunities among rodents, large animals, and humans in relation to AKI. After illustrating the potential merits of large animals in testing therapies for AKI, we will summarize the current state of the evidence in terms of what therapeutics have been tested in large animal models. The aim of this review is not to suggest that murine models are not valid to study AKI. Instead, our objective is to demonstrate that large animal models can serve as valuable and complementary tools in translating potential therapeutics into clinical practice.

Targeted regional optimization (TRO), a partial resuscitative endovascular balloon occlusion of the aorta strategy, may mitigate distal ischemia and extend the window of effectiveness for this adjunct. An automated device may allow greater control and precise regulation of flow past the balloon, while being less resource-intensive. The objective of this study was to assess the technical feasibility of the novel advanced partial occlusion controller (APOC) in achieving TRO at multiple distal pressures.Female swine (n = 48, 68.1 ± 0.7 kg) were randomized to a target distal mean arterial pressure (MAP) of 25 mm Hg, 35 mm Hg, or 45 mm Hg by either manual (MAN) or APOC regulation (n = 8 per group). Uncontrolled hemorrhage was generated by liver laceration. Targeted regional optimization was performed for 85 minutes, followed by surgical control and a 6-hour critical care phase. Proximal and distal MAP and flow rates were measured continuously.At a target distal MAP of 25 mm Hg, there was no difference in the MAP attained (APOC: 26.2 ± 1.05 vs. MAN: 26.1 ± 1.78 mm Hg) but the APOC had significantly less deviance (10.9%) than manual titration (14.9%, p0.0001). Similarly, at a target distal MAP of 45 mm Hg, there was no difference in mean pressure (44.0 ± 0.900 mm Hg vs. 45.2 ± 1.31 mm Hg) but APOC had less deviance (9.34% vs. 11.9%, p0.0001). There was no difference between APOC and MAN in mean (34.6 mm Hg vs. 33.7 mm Hg) or deviance (9.95% vs. 10.4%) at a target distal MAP of 35 mm Hg, respectively. The APOC made on average 77 balloon volume adjustments per experiment compared with 29 by manual titrations.The novel APOC consistently achieved and sustained precisely regulated TRO across all groups and demonstrated reduced deviance at the 25 mm Hg and 45 mm Hg groups compared with manual titration.

Targeted regional optimization (TRO) describes partial resuscitative endovascular balloon occlusion of the aorta strategy that allows for controlled distal perfusion to balance hemostasis and tissue perfusion. This study characterized hemodynamics at specific targeted distal flow rates in a swine model of uncontrolled hemorrhage to determine if precise TRO by volume was possible.Anesthetized swine were subjected to liver laceration and randomized into TRO at distal flows of 300 mL/min (n = 8), 500 mL/min (n = 8), or 700 mL/min (n = 8). After 90 min, the animals received damage control packing and were monitored for 6 h. Hemodynamic parameters were measured continuously, and hematology and serologic labs obtained at predetermined intervals.During TRO, the average percent deviation from the targeted flow was lower than 15.9% for all cohorts. Average renal flow rates were significantly different across all cohorts during TRO phase (P 0.0001; TRO300 = 63.1 ± 1.2; TRO500 = 133.70 ± 1.93; TRO700 = 109.3 ± 2.0), with the TRO700 cohort having less renal flow than TRO500. The TRO500 and TRO700 average renal flow rates inverted during the intensive care unit phase (P 0.0001; TRO300 = 86.20 ± 0.40; TRO500 = 148.50 ± 1.45; TRO700 = 181.1 ± 0.70). There was higher blood urea nitrogen, creatinine, and potassium in the TRO300 cohort at the end of the experiment, but no difference in lactate or pH between cohorts.This study demonstrated technical feasibility of TRO as a strategy to improve outcomes after prolonged periods of aortic occlusion and resuscitation in the setting of ongoing solid organ hemorrhage. A dose-dependent ischemic end-organ injury occurs beginning with partial aortic occlusion that progresses through the critical care phase, with exaggerated effect on renal function.

The mechanisms by which lower limb ischemia/reperfusion induces acute kidney injury (AKI) remain largely uncharacterized. We hypothesized that tourniquet-induced lower limb ischemia/reperfusion (TILLIR) would inhibit mitochondrial function in the renal cortex. We used a murine model to show that TILLIR of the high thigh regions inflicted time-dependent AKI as determined by renal function and histology. This effect was associated with decreased activities of mitochondrial complexes I, II, V and citrate synthase in the kidney cortex. Moreover, TILLIR reduced mRNA levels of a master regulator of mitochondrial biogenesis PGC-1α, and its downstream genes NDUFS1 and ATP5o in the renal cortex. TILLIR also increased serum corticosterone concentrations. TILLIR did not significantly affect protein levels of the critical regulators of mitophagy PINK1 and PARK2, mitochondrial transport proteins Tom20 and Tom70, or heat-shock protein 27. TILLIR had no significant effect on mitochondrial oxidative stress as determined by mitochondrial ability to generate reactive oxygen species, protein carbonylation, or protein levels of MnSOD and peroxiredoxin1. However, TILLIR inhibited classic autophagic flux by increasing p62 protein abundance and preventing the conversion of LC3-I to LC3-II. TILLIR increased phosphorylation of cytosolic and mitochondrial ERK1/2 and mitochondrial AKT1, as well as mitochondrial SGK1 activity. In conclusion, lower limb ischemia/reperfusion induces distal AKI by inhibiting mitochondrial function through reducing mitochondrial biogenesis. This AKI occurs without significantly affecting PINK1-PARK2-mediated mitophagy or mitochondrial oxidative stress in the kidney cortex.

OBJECTIVE Gut dysbiosis, an imbalance in the gut microbiome, occurs after trauma, which may be ameliorated with transfusion. We hypothesized that gut hypoperfusion following trauma causes dysbiosis and that whole blood (WB) resuscitation mitigates these effects. METHODS Anesthetized rats underwent sham (S; laparotomy only, n = 6); multiple injuries (T; laparotomy, liver and skeletal muscle crush injuries, and femur fracture, n = 5); multiple injuries and 40% hemorrhage (H; n = 7); and multiple injuries, hemorrhage, and WB resuscitation (R; n = 7), which was given as 20% estimated blood volume from donor rats 1 hour posttrauma. Baseline cecal mesenteric tissue oxygen (O2) concentration was measured following laparotomy and at 1 hour and 2 hours posttrauma. Fecal samples were collected preinjury and at euthanasia (2 hours). 16S rRNA sequencing was performed on purified DNA, and diversity and phylogeny were analyzed with QIIME (Knight Lab, La Jolla, CA; Caporaso Lab, Flagstaff, AZ) using the Greengenes 16S rRNA database (operational taxonomic units; 97% similarity). α and β diversities were estimated using observed species metrics. Permutational analysis of variance was performed for overall significance. RESULTS In H rats, an average decline of 36% ± 3.6% was seen in the mesenteric O2 concentration at 1 hour without improvement by 2 hours postinjury, which was reversed following resuscitation at 2 hours postinjury (4.1% ± 3.1% difference from baseline). There was no change in tissue O2 concentration in the S or T rats. β Diversity differed among groups for all measured indices except Bray-Curtis, with the spatial median of the S and R rats more similar compared with S and H rats (p < 0.05). While there was no difference in α diversity found among the groups, indices were significantly correlated with mesenteric O2 concentration. Members of the family Enterobacteriaceae were significantly enriched in only 2 hours. CONCLUSION Mesenteric perfusion after trauma and hemorrhage is restored with WB resuscitation, which influences β diversity of the gut microbiome. Whole blood resuscitation may also mitigate the effects of hemorrhage on intestinal dysbiosis, thereby influencing outcomes.

Introduction Urine output (UOP) still remains the primary endpoint utilized as a surrogate for cardiac output (CO) and adequacy of perfusion during burn resuscitation. The role of arterial blood pressure (BP) waveform as a guiding tool for burn resuscitation has not been rigorously explored. As we move toward developing and validating novel endpoint to complement UOP, we compared the potential of pulse wave analysis (PWA) of arterial BP waveforms for estimating cardiac output (CO) and stroke volume (SV) in a large animal model of 40% total body surface area (TBSA) burns with varying resuscitation paradigms. Methods Anesthetized swine were instrumented and hemorrhaged 15% of their blood volume, and sustained a 40% TBSA full-thickness contact burn with aluminum billets. Animals were kept in a surgical ICU setting overnight, during which anesthesia was maintained with a combination of propofol, ketamine, and fentanyl. Animals were randomized to 3 different intravenous fluid (lactated Ringer’s, LR) levels: under resuscitation with no IV fluids, adequate resuscitation protocolized with a clinical decision support tool, or over resuscitation with a starting rate of 500mL/hour. We computed 20 surrogate measures of CO and SV (10 each) via PWA of arterial BP, and calibrated them to reference CO via thermo-dilution and SV on a subject-by-subject basis. Surrogate performance was quantified in terms of correlation coefficient and root-mean-squared error (RMSE) relative to reference CO and SV. Results Animals received 0±0, 4.4±1.6, and 9.1±1.3 mL/kg/% TBSA in the under, adequate, and over resuscitation groups (p=0.0036), respectively. Among the ten surrogate measures of CO and SV, 4 surrogates had positive proportionality to reference CO and SV consistently. The best surrogate measure of CO and SV was [mean BP-diastolic BP]/[heart period] and [mean BP-diastolic BP], respectively, which yielded r value of 0.82+/-0.19 and RMSE of 0.23+/-0.14 lpm for CO and r value of 0.85+/-0.17 and RMSE of 2.7+/-2.0 ml for SV (Fig. 1). Conclusions The initial results suggest that PWA-based surrogates of CO and SV have the potential to track reference CO and SV in extremes of resuscitation post-burn. The current model produces under-resuscitated, adequately resuscitated, and over-resuscitated animals, making it possible to rigorously examine the efficacy of PWA-based tracking of CO and SV in a wide spectrum of burn resuscitation scenarios. Hence, PWA may provide metrics useful for developing surrogates of CO and SV as novel treatment endpoints of burn resuscitation that indicate perfusion and volume status of burn injury patients.

Introduction Optimized fluid resuscitation of burn patients is a clinical care challenge as both under- and over- resuscitation have deleterious consequences. The gold-standard endpoint guiding burn resuscitation is urinary output (UO), which is known to have limited efficacy. We investigated the potential of the dynamic indices of fluid responsiveness derived from arterial blood pressure (BP) waveforms in conveying information about burn resuscitation. In particular, we investigated pulse pressure variation (PPV) and systolic pressure variation (SPV), which have been shown to be valuable in a number of other indications. Methods We conducted a retrospective analysis of arterial BP waveform data acquired from six anesthetized and mechanically-ventilated pigs (33±5 kg weight and 40% total burned surface area) which were instrumented for hemodynamic monitoring for 24 hours. The animals were either under-, over-, or adequately-resuscitated (guided by a burn resuscitation decision support system), with two animals in each group. PPV and SPV were calculated on an hourly basis. Fluid responsiveness thresholds of 15% and 6% were used respectively for PPV and SPV, as per literature. Results All of the animals experienced an immediate rise in PPV and SPV following the injury (PPV and SPV start from large values as seen in Fig. 1 and Fig. 2). In the under-resuscitated group, PPV and SPV increased above the threshold, reaching maximum values in the last eight hours (PPV: 49.8±20%, SPV: 24.7±3.6%), indicating severe hypovolemia. In the over-resuscitated group, PPV and SPV decreased below the threshold, reaching their minimum in the last eight hours (PPV: 8.7±3.6%, SPV: 4.1±1.9%), indicating major hypervolemia. In the adequately-resuscitated group, PPV and SPV maintained closer to the threshold throughout the duration of the experiment, and at the end, PPV was 15.6±4.2% and SPV was 6.2±2.6%. Conclusions Our initial results suggest that PPV and SPV may help distinguish under-, adequately-, and over-resuscitated burn patients, and potentially complement UO in the hemodynamic assessment of the burn injury patients.

Introduction While recent reports underscore the significance of the gut microbiome (GM) in health and disease, its importance in burn outcomes remains unclear. Moreover, aggressive intravenous (IV) fluid resuscitation of patients may alter intestinal flora. Herein, we describe GM changes following a large burn in swine randomized to different volumes of IV Lactated Ringers' (LR). Methods Anesthetized Yorkshire swine sustained 40% Total body surface area full-thickness burns and were randomized to different volumes of IV LR: none (n = 3), 15 mL/kg/day (Low; n = 6), or 80 mL/kg/day (High; n = 6). At baseline and days 1 and 2, fecal swabs were collected for 16 s rDNA sequencing. Proximal jejunum was collected immediately after euthanasia (day 2) for western blot, histopathology, and cytokine analyses. Results Burns produced significant shifts in β-diversity and non-significant reductions in α-diversity that did not recover regardless of treatment group. Burn-induced increases in Proteobacteria and decreases in Firmicutes were attenuated by IV fluids in a dose-dependent manner, and also correlated with α-diversity. IV fluids caused a dose-dependent increase in Bacteroides and prevented a transient increase in the opportunistic pathogen Haemophilus parainfluenzae. While high volumes of IV fluids increased intestinal Hsp70 levels (p = 0.0464), they reduced SGLT1 (p = 0.0213) and caspase3 (p = 0.0139) levels. IV fluids elicited a non-specific cytokine response however Bacteroidetes levels correlated with intestinal IL18 levels (p = 0.0166, R = 0.4201). Conclusions We present the first report on the gut microbiome in a porcine burn model, and present data to suggest that IV fluids may influence GM and gut functional proteins following a burn. Overall, burn injury results GM diversity shifts, which may expose diagnostic and/or therapeutic targets to improve outcomes.

Volumetric muscle loss (VML) occurs after severe orthopedic trauma and results in loss of muscle fibers and function that can leave patients permanently disabled. Although animals models of VML are useful to test possible therapeutic strategies, the pathophysiological characteristics of remaining skeletal muscle and changes in metabolism are not thoroughly understood. Herein, alterations of neuromuscular function, muscle fiber morphology, myosin heavy chain expression, and myofiber mitochondrial respiration were evaluated in an adult Yorkshire swine VML injury model. VML injured animals showed reduced peak isometric strength ( P < 0.05) and a shift toward smaller muscle fibers independent of fiber type ( P < 0.001). The muscle remaining after VML had a greater distribution of type I fibers and lower distribution of type II fibers ( P < 0.001). Skeletal muscle mitochondrial state 2 and state 3, reflecting complex I respiration, increased after injury ( P < 0.05) with a consistent trend to display higher oxygen flux per milligram of tissue. However, this was largely driven by increased mitochondrial content after VML which was associated with higher mitochondrial fission (FIS-1 protein levels). This study demonstrates an underlying perturbation of oxidative metabolism within the remaining musculature following surgical creation of an isolated, sterile VML injury in a porcine model that may be influential to the development of insidious pathophysiology and regenerative and rehabilitative therapies. NEW & NOTEWORTHY The natural injury sequela of volumetric muscle loss (VML) and associated pathophysiology of the remaining muscle is still incompletely understood. Herein we demonstrate a chronic muscle function deficit, with an increase in type I muscle fibers and parallel increase in oxidative capacity of remaining skeletal muscle. It is possible that the alteration in oxidative capacity after VML could largely be due to heightened mitochondrial activity and an increase in mitochondrial abundance.

While clinical examination is needed for burn severity diagnosis, several emerging technologies aim to quantify this process for added objectivity. Accurate assessments become easier after burn progression, but earlier assessments of partial thickness burn depth could lead to earlier excision and grafting and subsequent improved healing times, reduced rates of scarring/infection, and shorter hospital stays. Spatial Frequency Domain Imaging (SFDI), Laser Speckle Imaging (LSI) and thermal imaging are three non-invasive imaging modalities that have some diagnostic ability for noninvasive assessment of burn severity, but have not been compared in a controlled experiment. Here we tested the ability of these imaging techniques to assess the severity of histologically confirmed graded burns in a swine model. Controlled, graded burn wounds, 3 cm in diameter were created on the dorsum of Yorkshire pigs (n=3, 45–55kg) using a custom-made burn tool that ensures consistent pressure has been employed by various burn research groups. For each pig, a total of 16 burn wounds were created on the dorsal side. Biopsies were taken for histological analysis to verify the severity of the burn. Clinical analysis, SFDI, LSI and thermal imaging were performed at 24 and 72 hours after burn to assess the accuracy of each imaging technique. In terms of diagnostic accuracy, using histology as a reference, SFDI (85%) and clinical analysis (83%) performed significantly better that LSI (75%) and thermography (73%) 24 hours after the burn. There was no statistically significant improvement from 24 to 72 hours across the different imaging modalities. These data indicate that these imaging modalities, and specifically SFDI, can be added to the burn clinicians’ toolbox to aid in early assessment of burn severity.

Healing of burn wounds is necessary for survival; however tracking progression or healing of burns is an inexact science. Recently, the relationship of mortality and wound healing has been documented with a software termed WoundFlow. The objective of the current study was to confirm various factors that impact burn wound healing, as well as to establish a timeline and rate of successful healing. A retrospective analysis was performed on adults (n=115) with at least 20% TBSA burn that had at least two computer-based wound mappings. The % open wound (%OW) was calculated over time to document healing trajectory until successful healing or death. Only 2% of patients in the group with successful wound healing died. A decrease in the %OW of 0.8 (IQR: 0.7-1.1) was associated with survival. Disparities in wound healing trajectories between survivors and non-survivors were distinguishable by 2weeks post-injury (P

Resuscitative endovascular balloon occlusion of the aorta (REBOA) allows for effective temporization of exsanguination from non-compressible hemorrhage (NCTH) below the diaphragm. However, the therapeutic window for aortic occlusion is time-limited given the ischemia-reperfusion injury generated. Significant effort has been put into translational research to develop new strategies to alleviate the ischemia-reperfusion injury and extend the application of endoaortic occlusion. Targeted regional optimization (TRO) is a partial REBOA strategy to augment proximal aortic and cerebral blood flow while targeting minimal threshold of distal perfusion beyond the zone of partial aortic occlusion. The objective of TRO is to reduce the degree of ischemia caused by complete aortic occlusion while providing control of distal hemorrhage. This review provides a synopsis of the concept of TRO, pre-clinical, translational experiences with TRO and early clinical outcomes. Early results from TRO strategies are promising; however, further studies are needed prior to large-scale implementation into clinical practice.

Chartered by the U.S. Congress in 1961, the Armed Forces Radiobiology Research Institute (AFRRI) is a Joint Department of Defense (DoD) entity with the mission of carrying out the Medical Radiological Defense Research Program in support of our military forces around the globe. In the last 60 years, the investigators at AFRRI have conducted exploratory and developmental research with broad application to the field of radiation sciences. As the only DoD facility dedicated to radiation research, AFRRI's Medical Radiobiology Advisory Team provides deployable medical and radiobiological subject matter expertise, advising commanders in the response to a U.S. nuclear weapon incident and other nuclear or radiological material incidents. AFRRI received the DoD Joint Meritorious Unit Award on February 17, 2004, for its exceptionally meritorious achievements from September 11, 2001 to June 20, 2003, in response to acts of terrorism and nuclear/radiological threats at home and abroad. In August 2009, the American Nuclear Society designated the institute a nuclear historic landmark as the U.S.'s primary source of medical nuclear and radiological research, preparedness and training. Since then, research has continued, and core areas of study include prevention, assessment and treatment of radiological injuries that may occur from exposure to a wide range of doses (low to high). AFRRI collaborates with other government entities, academic institutions, civilian laboratories and other countries to research the biological effects of ionizing radiation. Notable early research contributions were the establishment of dose limits for major acute radiation syndromes in primates, applicable to human exposures, followed by the subsequent evolution of radiobiology concepts, particularly the importance of immune collapse and combined injury. In this century, the program has been essential in the development and validation of prophylactic and therapeutic drugs, such as Amifostine, Neupogen®, Neulasta®, Nplate® and Leukine®, all of which are used to prevent and treat radiation injuries. Moreover, AFRRI has helped develop rapid, high-precision, biodosimetry tools ranging from novel assays to software decision support. New drug candidates and biological dose assessment technologies are currently being developed. Such efforts are supported by unique and unmatched radiation sources and generators that allow for comprehensive analyses across the various types and qualities of radiation. These include but are not limited to both 60Co facilities, a TRIGA® reactor providing variable mixed neutron and γ-ray fields, a clinical linear accelerator, and a small animal radiation research platform with low-energy photons. There are five major research areas at AFRRI that encompass the prevention, assessment and treatment of injuries resulting from the effects of ionizing radiation: 1. biodosimetry; 2. low-level and low-dose-rate radiation; 3. internal contamination and metal toxicity; 4. radiation combined injury; and 5. radiation medical countermeasures. These research areas are bolstered by an educational component to broadcast and increase awareness of the medical effects of ionizing radiation, in the mass-casualty scenario after a nuclear detonation or radiological accidents. This work provides a description of the military medical operations as well as the radiation facilities and capabilities present at AFRRI, followed by a review and discussion of each of the research areas.

Background Patients with severe burn injury (over 20% of the total body surface area) experience profound hypermetabolism which significantly prolongs wound healing. Adipose-derived stem cells (ASCs) have been proposed as an attractive solution for treating burn wounds, including the potential for autologous ASC expansion. While subcutaneous adipocytes display an altered metabolic profile post-burn, it is not known if this is the case with the stem cells associated with the adipose tissue. Methods ASCs were isolated from discarded burn skin of severely injured human subjects (BH, n = 6) and unburned subcutaneous adipose tissue of patients undergoing elective abdominoplasty (UH, n = 6) and were analyzed at passages 2, 4, and 6. Flow cytometry was used to quantify ASC cell surface markers CD90, CD105, and CD73. Mitochondrial abundance and reactive oxygen species (ROS) production were determined with MitoTracker Green and MitoSOX Red, respectively, while JC-10 Mitochondrial Membrane Potential Assays were also performed. Mitochondrial respiration and glycolysis were analyzed with a high-resolution respirometer (Seahorse XFe24 Analyzer). Results There was no difference in age between BH and UH (34 ± 6 and 41 ± 4 years, respectively, P = 0.49). While passage 2 ASCs had lower ASC marker expression than subsequent passages, there were no significant differences in the expression between BH and UH ASCs. Similarly, no differences in mitochondrial abundance or membrane potential were found amongst passages or groups. Two-way ANOVA showed a significant effect (P P = 0.035) but not UH ASCs. On the contrary, basal glycolysis significantly decreased in BH ASCs (P = 0.011) with subsequent passaging, but not UH ASCs. Conclusions In conclusion, ASCs from burned individuals become increasingly oxidative and less glycolytic upon passaging when compared to ASCs from unburned patients. This increase in oxidative capacities was associated with ROS production in later passages. While the autologous expansion of ASCs holds great promise for treating burned patients with limited donor sites, the potential negative consequences of using them require further investigation.

While visual clinical impression is the standard of care for burn severity assessment, accuracy improves after the injury progresses, leading to patients waiting days for a diagnosis. Spatial Frequency Domain Imaging (SFDI) has shown the potential to assess burn grade as early as 24 hours post-burn. Here, we compared SFDI and Laser Speckle Imaging (LSI) at 24 and 72 hours after burn injury to clinical burn severity diagnosis. Three patients with burns ranging in severity were imaged. Both techniques showed an ability to categorize burn severity 24 hours after the initial injury, well before the outcome was determined by a clinician at 72 hours or later.

Adequate resuscitation is critical for burn wound patients where the affected total body surface area (TBSA) exceeds 20%, as fluid loss leads to organ failure, shock, and patient mortality. Protocols have been developed to combat fluid loss, however excess fluids can also lead to compartment syndromes. Here, we used spatial frequency domain imaging (SFDI), a non-contact wide field imaging technique to measure in-vivo water changes in a 40 % TBSA porcine burn model. In this pilot study one pig received intravenous fluids according to the Parkland formula on top of enteral fluid resuscitation, while a second pig received no fluids during the experiment. Unburned regions of skin were imaged with SFDI from 3 to 22 hours post-burn. This imaging technique uses structured illumination, projected at multiple spatial frequencies and wavelengths, to measure tissue reflectance, which is used to obtain the reduced scattering and absorption coefficients. Water fraction is based upon absorption in the near-infrared spectrum at 971 nm, where water has a high extinction coefficient. SFDI measurements of superficial tissue hydration showed increased water fraction for the pig that received fluid resuscitation (+17%), and a decrease for the pig that received no fluids (-5%). Analysis showed decreased scattering measured in the pig that received fluids (+13%), suggesting increased interstitial water pressure. These preliminary results are consistent with systemic hydration, as estimated based on CT-measured subcutaneous fat thickness. SFDI may, therefore offer the possibility of non-invasively monitoring fluid resuscitation of large TBSA patients.

On June 17 to 18, 2019, the American Burn Association, in conjunction with Underwriters Laboratories, convened a group of experts on burn resuscitation in Washington, DC. The goal of the meeting was to identify and discuss novel research and strategies to optimize the process of burn resuscitation. Patients who sustain a large thermal injury (involving >20% of the total body surface area [TBSA]) face a sequence of challenges, beginning with burn shock. Over the last century, research has helped elucidate much of the underlying pathophysiology of burn shock, which places multiple organ systems at risk of damage or dysfunction. These studies advanced the understanding of the need for fluids for resuscitation. The resultant practice of judicious and timely infusion of crystalloids has improved mortality after major thermal injury. However, much remains unclear about how to further improve and customize resuscitation practice to limit the morbidities associated with edema and volume overload. Herein, we review the history and pathophysiology of shock following thermal injury, and propose some of the priorities for resuscitation research. Recommendations include: studying the utility of alternative endpoints to resuscitation, reexamining plasma as a primary or adjunctive resuscitation fluid, and applying information about inflammation and endotheliopathy to target the underlying causes of burn shock. Undoubtedly, these future research efforts will require a concerted effort from the burn and research communities.

While visual assessment by a clinician is the standard of care for burn severity evaluations, new technologies at various stages of development are attempting to add objectivity to this practice by quantifying burn severity. Assessment accuracy generally improves after the burn injury has progressed, but early assessments that correctly identify superficial partial and deep partial burns have the potential to lead to more prompt treatments and shorter recovery times. To date, Spatial Frequency Domain Imaging (SFDI) has only been used in animal models of burns, but has shown the potential to categorize burns accurately at earlier time points. Here we examine the potential for SFDI to assess burn severity in clinical patients. We also utilize Laser Speckle Imaging (LSI), an FDA cleared non-invasive imaging technology that typically measures blood perfusion in order to evaluate burns in clinical patients. We present a case series of two patients, both with partial thickness burns of varying severity. Partial thickness burns are often difficult for clinicians to categorize based on visual appearance alone. SFDI and LSI were both performed on each patient at approximately 24 and 72 h after their respective burn incidents. Each technique was able to render spatially resolved information that enabled improved assessment accuracy for each burn. This represents the first publication of SFDI applied to clinical burn patients after being successfully utilized in animal models, and highlights the potential for SFDI as a feasible tool for the timely categorization of burn severity. Keywords: Burns, Clinical, Humans, Spatial Frequency Domain Imaging, Laser Speckle Imaging

Background This study characterizes the gastrointestinal (GI) microbiome in a pre-clinical polytrauma hemorrhage model. Methods Rats (n = 6) were anesthetized, hemorrhaged 20% of their blood volume, and subjected to a femur fracture and crush injuries to the small intestine, liver, and limb skeletal muscle without resuscitation. Fecal samples were collected pre-injury and 2 h post-injury. Purified DNA from the samples underwent 16s rRNA sequencing for microbial quantification. Bacterial diversity analysis and taxonomic classification were performed. Results Following injury, the gut microbial composition was altered with a shift in beta diversity and significant differences in the relative abundance of taxa. The relative abundance of the families Lachnospiraceae and Mogibacteriaceae was increased at 2 h, while Barnesiellaceae and Bacteroidaceae were decreased. Alpha diversity was unchanged. Conclusions The GI microbiome is altered in rats subjected to a polytrauma hemorrhage model at 2 h post-injury in the absence of antibiotics or therapeutic interventions.

Introduction While visual assessment is the standard for burn severity evaluation, new technologies are attempting to increase objectivity. Burn depth assessment accuracy improves over time as the appearance changes, but earlier diagnosis potentially leads to prompt treatment and shorter recovery. Laser Speckle Imaging (LSI), FDA approved for grading burns, is typically done 72hrs after injury. Spatial Frequency Domain Imaging (SFDI), a novel technology only tested in animal burn models, uses multiple wavelengths of patterned light to quantify tissue absorption (hemodynamics) and scattering (structure). SFDI has shown the potential to differentiate burns as early as 24hrs postburn by measuring scattering (µs’) changes due to collagen damage. Here, we examine SFDI burn severity assessment in clinical patients, and compare against LSI and clinical assessment. Methods 5 burn cases were imaged with LSI and SFDI approximately 24 and 72hrs postburn. The clinician was blinded to imaging results and made diagnoses on their standard of care. Results One patient received no surgical intervention, one received excision and xenograft, and two received excision and autograft. An autograft was recommended for one patient, but not performed due to preexisting conditions. SFDI measurements at 24hrs showed low µs’ values in regions that eventually required autografts, and high µs’ in burns that did not. While clinical decisions were made no earlier than 72hrs postburn, µs’ maps at 24hrs postburn were able to illustrate severity in these regions. In 2 of 3 autograft cases, LSI was unable to determine the severity until 72hrs postburn. Conclusions Here, we compared SFDI to LSI in a clinical setting to quantify burn wound severity. Similar to animal models, SFDI was demonstrated to accurately characterize severity earlier than the perfusion-based LSI. This is likely due to sensitivity of µs’ measured by SFDI to the structural changes in damaged collagen (i.e., zone of coagulation) that occur immediately postburn.

Introduction Adipose stem cells (ASCs) have shown therapeutic promise for inflammatory conditions that beget multi organ dysfunction, including burns. While ASCs have immunomodulatory properties, some studies have brought up safety concerns of increased pro-coagulant activity such as pulmonary microvascular thrombi formation after intravenous (IV) administration of ASCs. In the present study, the aims are two-fold: 1) to verify if IV administration of human ASCs promotes coagulation and 2) to determine if human ASCs affect organ function in a 40% total body surface area (TBSA) swine burn model. Methods Female Yorkshire swine (39.63 ± 8.26kg) were anesthetized and subjected to 40% TBSA full thickness contact burns according to a formerly established model. After recovery from anesthesia, animals were randomized to receive 15ml/kg Lactated Ringer’s Solution containing: 1- no ASCs; 2- a low dose (5x105 ASCs/kg), or 3- a high dose (5x106 ASCs/kg) over a 15-minute period as a bolus. Blood was collected at baseline (BL) and 3, 6, 12, and 24h post burn to determine the effect of ASCs on organ function and coagulation status. At 24h post-burn, animals were humanely euthanized, and kidney and liver tissue was collected for histological and Western blot analyses. Data is presented as mean ± SEM, and statistical significance was set at p< 0.05. Results The high dose of ASCs significantly increased the circulating number of monocytes starting at 12 hours. Two-way ANOVA revealed a significant effect of ASCs on both prothrombin times (PT) and international normalized ratio (INR) (1.03 ± 0.04, 0.93 ± 0.03, and 1.02 ± 0.04 for no, low and high ASC groups, respectively at 24 hours). There were no differences in partial thromboplastin time, fibrinogen, or d-dimer levels. Both doses of ASCs briefly exacerbated burn-induced increases in total bilirubin at 3 hours (0.062 ± 0.025mg/dL, 0.148 ± 0.060mg/dL, and 0.211 ± 0.086mg/dL in no, low, and high ASC groups, respectively). ASCs did not alter urine output; yet, there was a significant effect of ASCs on creatinine. Western blotting revealed a rise in caspase expression in the liver of animals receiving a low dose of ASCs, while there was no difference in caspase expression in kidneys. Conclusions We show that IV administration of xenogeneic ASCs produces minimal changes in coagulation status and renal and hepatic dysfunction. Modest changes in the extrinsic coagulation pathway were dose-dependent, while exacerbation of liver dysfunction was brief and normalized after administration of ASCs was completed. We cannot rule out that continuous infusion of ASCs would not have a cumulative effect on organ dysfunction.

Inhalation injury commonly accompanies thermal injury, increasing the likelihood of mortality and multiple organ dysfunction (MOD). Large animal models have given important insight into the pathophysiology of this injury; however recapitulating late MOD has remained difficult. The current report describes experiments using a smoke inhalation and burn model, with follow-up of ambulatory swine for 14days with bronchoscopy, CT scanning, and bronchoalveolar lavage fluid (BALF)/blood collection. Clinically, animals cleared airway damage in the first several days after-injury. This was mirrored with erythematous airways on day 2 after-injury, which resolved by the end of the experiment, as did parenchymal damage seen on CT. An initial rise in the protein content of BALF immediately after-injury was followed by a dramatic increase in the concentration of leukocytes. Circulating neutrophils increased while lymphocytes decreased; both correlated with cell counts in BALF. IL8 levels in BALF increased 30-fold and remained elevated throughout the experiment. IL1ra increased circulation immediately after-injury, and afterwards in BALF. Other cytokines (TNFα, IL12) transiently increased in BALF (and decreased in circulation) on day 2. Taken together, these results display a remarkable capability for the lungs to recover in the absence of intubation, with further evidence of the role of cytokines such as IL8 and IL1ra. The possible exacerbating effects of clinical practices such as ventilation and bronchoscopies should be considered.