Canagliflozin Mitigates Acute Kidney Injury Secondary to Resuscitative Endovascular Balloon Occlusion of the Aorta in a Porcine Model of Hemorrhagic Shock.

OBJECTIVE: We investigated the potential of acute canagliflozin administration to mitigate acute kidney injury (AKI) and attenuate deleterious pro-inflammatory cytokine release in a clinically relevant swine model of severe renal ischemia reperfusion injury (IRI) induced by hemorrhage and aortic occlusion. BACKGROUND: Long-term canagliflozin use attenuates renal function decline and reduces AKI in diabetes mellitus and heart failure patients. Whilst several reports indicate prophylactic SGLT2 inhibition prevents AKI in IRI, the efficacy of acute administration on IRI and inflammation is not known. METHODS: Female swine (n=16) underwent controlled hemorrhage of 25% blood volume, followed by 90 min of aortic occlusion at the level of the renal ostia (via Resuscitative Endovascular Balloon Occlusion of the Aorta). A single 300 mg dose of oral canagliflozin or vehicle (saline) was delivered 5 mins into aortic occlusion. Hemodynamic monitoring, markers of renal function (serum creatinine, blood urea nitrogen, proteinuria and urinary neutrophil gelatinase-associated lipocalin) and serum cytokine concentrations (including interleukins: IL-1RA, IL-6, IL-8, IL-10, IL-18; and Tumor necrosis factor alpha) were analyzed after IRI, and during a 6h critical care phase. RESULTS: Compared to controls, animals receiving canagliflozin had less severe AKI, improved creatinine clearance, reduced proteinuria, and significantly lower tubular damage as evidenced by histopathology and urinary NGAL. Furthermore, the pro-inflammatory cytokine IL-6 was markedly attenuated without reduction in anti-inflammatory cytokines (IL-1RA and IL-10). CONCLUSIONS: A single dose of canagliflozin administered shortly into ischemic insult mitigates AKI and attenuates harmful pro-inflammatory cytokine release following trauma or surgery. These findings suggest a potential novel therapeutic role for canagliflozin in mitigating the effects of renal IRI worthy of further investigation.

Microbiome and Inflammasome Alterations Found During Radiation Dose Finding in a Sinclair Minipig Model of Gastrointestinal Acute Radiation Syndrome.

ABSTRACT: Both abdominal radiotherapy and a nuclear event can result in gastrointestinal symptoms, including acute radiation syndrome (GI-ARS). GI-ARS is characterized by compromised intestinal barrier integrity increasing the risk for infectious complications. Physiologically relevant animal models are crucial for elucidating host responses and therapeutic targets. We aimed to determine the radiation dose requirements for creating GI-ARS in the Sinclair minipig. Male, sexually mature swine were randomly divided into sham (n = 6) and three lower hemibody radiation dosage groups of 8, 10, and 12 Gy (n = 5/group) delivered using a linear accelerator-derived X-rays (1.9 Gy/min). Animals were monitored for GI-ARS symptoms for 14 days with rectal swab and blood collection at days 0-3, 7, 10, and 14 followed by necropsy for western blotting and histology. Dose-dependent increases in weight loss, diarrhea severity, and mortality (log-rank test, p = 0.041) were seen. Villi length was significantly reduced in all irradiated animals compared to controls (p < 0.001). Serum citrulline decreased and bacterial translocation increased post-irradiation compared to controls. Increased NLRP3 levels in post-mortem jejunum were seen (p = 0.0043) as well as increased IL-1ß levels in the 12 Gy group (p = 0.041). Radiation dose and survival were associated with significant gut microbial community shifts in beta diversity. Moreover, decedents had increased Porphyromonas, Campylobacter, Bacteroides, Parvimonas, and decreased Fusobacterium and decreased Aerococcus, Lactobacillus, Prevotella, and Streptococcus. Our novel Sinclair minipig model showed dose-dependent clinical symptoms of GI-ARS. These findings provide invaluable insights into the intricate interplay between GI-ARS, intestinal inflammation, and gut microbiota alterations offering potential targets for therapeutic and diagnostic interventions after radiation exposure.

Effects of combined ciprofloxacin and Neulasta therapy on intestinal pathology and gut microbiota after high-dose irradiation in mice.

Introduction: Treatments that currently exist in the strategic national stockpile for acute radiation syndrome (ARS) focus on the hematopoietic subsyndrome, with no treatments on gastrointestinal (GI)-ARS. While the gut microbiota helps maintain host homeostasis by mediating GI epithelial and mucosal integrity, radiation exposure can alter gut commensal microbiota which may leave the host susceptible to opportunistic pathogens and serious sequelae such as sepsis. To mitigate the effects of hematopoietic ARS irradiation, currently approved treatments exist in the form of colony stimulating factors and antibiotics: however, there are few studies examining how these therapeutics affect GI-ARS and the gut microbiota. The aim of our study was to examine the longitudinal effects of Neulasta and/or ciprofloxacin treatment on the gut microbiota after exposure to 9.5 Gy 60Co gamma-radiation in mice. Methods: The gut microbiota of vehicle and drug-treated mice exposed to sham or gamma-radiation was characterized by shotgun sequencing with alpha diversity, beta diversity, and taxonomy analyzed on days 2, 4, 9, and 15 post-irradiation. Results: No significant alpha diversity differences were observed following radiation, while beta diversity shifts and taxonomic profiles revealed significant alterations in Akkermansia, Bacteroides, and Lactobacillus. Ciprofloxacin generally led to lower Shannon diversity and Bacteroides prevalence with increases in Akkermansia and Lactobacillus compared to vehicle treated and irradiated mice. While Neulasta increased Shannon diversity and by day 9 had more similar taxonomic profiles to sham than ciprofloxacin-or vehicle-treated irradiated animals. Combined therapy of Neulasta and ciprofloxacin induced a decrease in Shannon diversity and resulted in unique taxonomic profiles early post-irradiation, returning closer to vehicle-treated levels over time, but persistent increases in Akkermansia and Bacteroides compared to Neulasta alone. Discussion: This study provides a framework for the identification of microbial elements that may influence radiosensitivity, biodosimetry and the efficacy of potential therapeutics. Moreover, increased survival from H-ARS using these therapeutics may affect the symptoms and appearance of what may have been subclinical GI-ARS.

Ferroptosis, Inflammation, and Microbiome Alterations in the Intestine in the Göttingen Minipig Model of Hematopoietic-Acute Radiation Syndrome.

Hematopoietic acute radiation syndrome (H-ARS) involves injury to multiple organ systems following total body irradiation (TBI). Our laboratory demonstrated that captopril, an angiotensin-converting enzyme inhibitor, mitigates H-ARS in Göttingen minipigs, with improved survival and hematopoietic recovery, as well as the suppression of acute inflammation. However, the effects of captopril on the gastrointestinal (GI) system after TBI are not well known. We used a Göttingen minipig H-ARS model to investigate captopril's effects on the GI following TBI (60Co 1.79 or 1.80 Gy, 0.42-0.48 Gy/min), with endpoints at 6 or 35 days. The vehicle or captopril (0.96 mg/kg) was administered orally twice daily for 12 days, starting 4 h post-irradiation. Ilea were harvested for histological, protein, and RNA analyses. TBI increased congestion and mucosa erosion and hemorrhage, which were modulated by captopril. GPX-4 and SLC7A11 were downregulated post-irradiation, consistent with ferroptosis at 6 and 35 days post-irradiation in all groups. Interestingly, p21/waf1 increased at 6 days in vehicle-treated but not captopril-treated animals. An RT-qPCR analysis showed that radiation increased the gene expression of inflammatory cytokines IL1B, TNFA, CCL2, IL18, and CXCL8, and the inflammasome component NLRP3. Captopril suppressed radiation-induced IL1B and TNFA. Rectal microbiome analysis showed that 1 day of captopril treatment with radiation decreased overall diversity, with increased Proteobacteria phyla and Escherichia genera. By 6 days, captopril increased the relative abundance of Enterococcus, previously associated with improved H-ARS survival in mice. Our data suggest that captopril mitigates senescence, some inflammation, and microbiome alterations, but not ferroptosis markers in the intestine following TBI.

Evaluation of novel hemostatic agents in a coagulopathic swine model of junctional hemorrhage.

BACKGROUND: Hemostatic dressings are used extensively in both military and civilian trauma to control lethal noncompressible hemorrhage. The ideal topical hemostatic agent would provide reliable hemostasis in patients with profound acidosis, coagulopathy, and shock. This study aimed to compare next-generation hemostatic agents against the current military standard in a translational swine model of vascular injury and coagulopathy. METHODS: Female Yorkshire swine were randomized to eight groups (total n = 63; control n = 14, per group n = 7) of hemostatic agents and included: QuikClot Combat Gauze (Teleflex, Morrisville, NC), which served as the control; BloodSTOP IX (LifeScience Plus, Mountain View, CA); Celox Rapid (Medtrade Product, Crewe, United Kingdom); ChitoSAM 100 (Sam Medical, Tualatin, OR); EVARREST Fibrin Sealant Patch (Ethicon, Raritan, NJ); TAC Wrapping Gauze (H&H Medical, Williamsburg, VA); ChitoGauze XR Pro (Tricol Biomedical, Portland, OR); and X-Stat 30 (RevMedX, Wilsonville, OR). Hemodilution via exchange transfusion of 6% hetastarch was performed to induce acidosis and coagulopathy. An arteriotomy was created, allowing 30 seconds of free bleeding followed by application of the hemostatic agent and compression via an external compression device. A total of three applications were allowed for continued/recurrent bleeding. All blood loss was collected, and hemostatic agents were weighed to calculate blood volume loss. Following a 180-minute observation period, angiography was completed to evaluate for technical complication and distal perfusion of the limb. Finally, the limb was ranged five times to assess for rebleeding and clot stability. RESULTS: All swine were confirmed coagulopathic with rotational thromboelastography and acidotic (pH 7.2 ± 0.02). BloodSTOP IX allowed a significant increase in blood loss and number of applications required to obtain hemostasis compared with all other groups. BloodSTOP IX demonstrated a decreased survival rate (29%, p = 0.02). All mortalities were directly attributed to exsanguination as a result of device failure. In surviving animals, there was no difference in extravasation. BloodSTOP IX had an increased rebleeding rate after ranging compared with QuikClot Combat Gauze ( p = 0.007). CONCLUSION: Most novel hemostatic agents demonstrated comparable efficacy compared with the currently military standard hemostatic dressing, CG.

Next-Generation REBOA (Resuscitative Endovascular Balloon Occlusion of the Aorta) Device Precisely Achieves Targeted Regional Optimization in a Porcine Model of Hemorrhagic Shock.

INTRODUCTION: Limitations such as time-dependent distal ischemia have slowed the adoption of resuscitative endovascular balloon occlusion of the aorta (REBOA) for noncompressible hemorrhage. Next-generation REBOA technologies may allow for controlled partial flow, known as targeted regional optimization, to reduce distal ischemia. We aimed to characterize the efficacy of one such catheter in a porcine model of lethal hemorrhagic shock. METHODS: Noncompressible hemorrhage from an iliac injury was induced in anesthetized swine (Sus scrofa) (70-90 kg), targeting 30% total blood volume. Animals were then randomized to partial aortic occlusion (PO) with targeted distal mean arterial pressure (MAP) of 35-40 mm of mercury (mm Hg) and complete aortic occlusion (CO) (n = 8 per group) for 90 min. All groups were then resuscitated during a two-h critical care (CC) phase, with flow rate and MAP recorded continuously at the distal infrarenal aorta and proximal carotid artery, and analyzed with two-way repeated measures analysis of variance with S-N-K post-hoc test. RESULTS: During aortic occlusion, MAP distal to the balloon was consistently maintained at 35.8 ± 0.3 mm Hg in the PO group compared to 27.1 ± 0.3 mm Hg in the CO group (P < 0.05), which also corresponded to higher flow rates (202.9 ± 4.8 mL/min PO versus 25.9 ± 0.8 mL/min CO; P < 0.05). MAP proximal to the balloon was significantly higher with CO versus PO (109.2 ± 2.3 mm Hg versus 85.2 ± 2.3 mm Hg; P < 0.05). During the CC phase, distal aortic flow and MAP were not significantly different between groups. However, creatinine returned to baseline levels by the end of the study in the PO group, but not the CO group. One animal died in the CO group, whereas none died in the PO group. CONCLUSIONS: This is the first examination of the next-generation pREBOA-PRO in a porcine model of lethal hemorrhagic shock. We show technical feasibility of this technique to precisely achieve targeted regional optimization without device failure or complication. The ability to titrate balloon inflation and thus distal flow/pressure may extend the therapeutic window of REBOA by mitigating distal ischemia.

Advanced partial occlusion controller allows for increased precision during targeted regional optimization in a porcine model of hemorrhagic shock.

BACKGROUND: 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. METHODS: 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. RESULTS: 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%, p < 0.0001). 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%, p < 0.0001). 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. CONCLUSION: 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.

Minimal Effects of Intravenous Administration of Xenogeneic Adipose Derived Stem Cells on Organ Function in a Porcine 40% TBSA Burn Model.

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.

A 30% incidence of renal cysts with varying sizes and densities in biomedical research swine is not associated with renal dysfunction.

BACKGROUND: Renal cystic disease arising from various etiologies results in fluid-filled cavities within the kidneys. Moreover, preexisting renal dysfunction has been shown to exacerbate multiple pathologies. While swine bred for biomedical research are often clinically inspected for illness/parasites, more advanced diagnostics may aid in uncovering underlying renal abnormalities. METHODS: Computed tomography was performed in 54 female prepubertal Yorkshire swine to characterize renal cysts; urine and blood chemistry, and histology of cysts were also performed. RESULTS: Digital reconstruction of right and left kidneys demonstrated that roughly one-third of the animals (17/54; 31%) had one or more renal cyst. Circulating biomarkers of renal function were not different between animals that had cysts and those that did not. Alternatively, urinary glucose (P = .03) was higher and sodium (P = .07) tended to be lower in animals with cysts compared to animals without, with no differences in protein (P = .14) or potassium (P = .20). Aspiration of cystic fluid was feasible in two animals, which revealed that the cystic fluid urea nitrogen (97.6 ± 28.7 vs 911.3 ± 468.2 mg/dL), potassium (29.8 ± 14.4 vs 148.2 ± 24.85 mmol/L), uric acid (2.55 ± 1.35 vs 11.4 ± 5.65 mg/dL), and creatinine (60.34 ± 17.26 vs 268.99 ± 95.79 mg/dL) were much lower than in the urine. Histology demonstrated a cyst that markedly compresses the adjacent cortex and is lined by a single layer of flattened epithelium, bounded by fibrous connective tissue which extends into the parenchyma. There is tubular atrophy and loss in these areas. CONCLUSION: This study provides valuable insight for future studies focusing on kidney function in swine bred for biomedical research.

The gut microbiome distinguishes mortality in trauma patients upon admission to the emergency department.

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 ß-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 ß-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). ß-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.

Impact of oral resuscitation on circulating and splenic leukocytes after burns.

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 IL17ß 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.

Point-of-Care Urinary Biomarker Testing for Risk Prediction in Critically Injured Combat Casualties.

BACKGROUND: Risk prediction is important during combat operations because resources are limited and triage decisions must be rapid and accurate. We evaluated 2 point-of-care urinary biomarker tests for risk prediction in combat casualties. STUDY DESIGN: This was an observational cohort study of critically injured military personnel admitted to Craig Joint Theater Hospital in Afghanistan from October 2012 to December 2013. We collected urine within 3 hours of admission and measured urinary biomarkers with NephroCheck and a neutrophil gelatinase-associated lipocalin dipstick (NGALds) to evaluate their ability to predict a combined end point of need for renal replacement therapy or death. Odds ratios (ORs) were calculated and receiver operator characteristic curves were generated for both tests. RESULTS: A total of 89 patients were included for analysis. The median Injury Severity Score was 18 and the combined end point occurred in 12 (13.5%) patients. NephroCheck was not associated with the combined end point (OR 1.56; 95% CI 0.81 to 3.03; p = 0.19) and the area under the curve of the receiver operator characteristic curve was 0.65. The NGALds was highly associated with the combined end point (OR 4.93; 95% CI 2.18 to 11.14; p < 0.001) and the area under the curve of the receiver operator characteristic curve was 0.84. The NGALds remained significantly associated with the combined end point in a logistic regression model that included Injury Severity Score as a covariate (OR 4.10; 95% CI 1.74 to 9.67; p = 0.001). CONCLUSIONS: Measurement of urinary biomarkers with an NGALds, but not NephroCheck, predicts poor outcomes in combat casualties. An NGALds is a simple urine dipstick that could be deployed to combat zones to prioritize aeromedical evacuation, help with triage decisions, and predict resource use.

A prospective study in severely injured patients reveals an altered gut microbiome is associated with transfusion volume.

BACKGROUND: Traumatic injury can lead to a compromised intestinal epithelial barrier and inflammation. While alterations in the gut microbiome of critically injured patients may influence clinical outcomes, the impact of trauma on gut microbial composition is unknown. Our objective was to determine if the gut microbiome is altered in severely injured patients and begin to characterize changes in the gut microbiome due to time and therapeutic intervention. METHODS: We conducted a prospective, observational study in adult patients (n = 72) sustaining severe injury admitted to a Level I Trauma Center. Healthy volunteers (n = 13) were also examined. Fecal specimens were collected on admission to the emergency department and at 3, 7, 10, and 13 days (±2 days) following injury. Microbial DNA was isolated for 16s rRNA sequencing, and α and ß diversities were estimated, according to taxonomic classification against the Greengenes database. RESULTS: The gut microbiome of trauma patients was altered on admission (i.e., within 30 minutes following injury) compared to healthy volunteers. Patients with an unchanged gut microbiome on admission were transfused more RBCs than those with an altered gut microbiome (p < 0.001). Although the gut microbiome started to return to a ß-diversity profile similar to that of healthy volunteers over time, it remained different from healthy controls. Alternatively, α diversity initially increased postinjury, but subsequently decreased during the hospitalization. Injured patients on admission had a decreased abundance of traditionally beneficial microbial phyla (e.g., Firmicutes) with a concomitant decrease in opportunistic phyla (e.g., Proteobacteria) compared to healthy controls (p < 0.05). Large amounts of blood products and RBCs were both associated with higher α diversity (p < 0.001) and a ß diversity clustering closer to healthy controls. CONCLUSION: The human gut microbiome changes early after trauma and may be aided by early massive transfusion. Ultimately, the gut microbiome of trauma patients may provide valuable diagnostic and therapeutic insight for the improvement of outcomes postinjury. LEVEL OF EVIDENCE: Prognostic and Epidemiological, level III.

Isolation and Characterization of Multipotent CD24+ Cells From the Renal Papilla of Swine.

Over 100,000 patients in the United States are currently waiting for a kidney transplant. With just over 10,000 cadaveric kidneys transplanted annually, it is of the utmost importance to optimize kidney viability upon transplantation. One exciting avenue may be xenotransplantation, which has rejuvenated interest after advanced gene editing techniques have been successfully used in swine. Simultaneously, acute kidney injury (AKI) is associated with high morbidity and mortality and currently lacks effective treatment. Animal models have been used extensively to address both of these issues, with recent emphasis on renal progenitor cells (RPCs). Due to anatomical similarities to humans we aimed to examine progenitor cells from the renal papillae of swine kidneys. To do this, RPCs were dissected from the renal papillae of healthy swine. Cell surface marker expression, proliferation, and differentiation of the RPCs were tested in vitro. Additionally, a mixed lymphocyte reaction was performed to examine immunomodulatory properties. RPCs displayed spindle shaped morphology with limited self-renewing capacity. Isolated RPCs were positive for CD24 and CD133 at early passages, but lost expression with subsequent passaging. Similarly, RPCs displayed myogenic, osteogenic, and adipogenic differentiation capacities at passage 2, but largely lost this by passage 6. Lastly, direct contact of RPCs with human lymphocytes increased release of IL6 and IL8. Taken together, RPCs from the papilla of porcine kidneys display transient stem cell properties that are lost with passaging, and either represent multiple types of progenitor cells, or a multipotent progenitor population. In instances of ischemic insult, augmentation of/with RPCs may potentiate regenerative properties of the kidney. While the use of swine for transplantation and ischemia studies confers obvious advantages, the populations of different progenitor cell populations within pig kidneys warrants further investigation. Ultimately, while gene editing techniques enhance the potential for xenotransplantation of organs or cells, the ultimate success of this strategy may be determined by the (dis)similarities of RPCs from different species.

Polytrauma independent of therapeutic intervention alters the gastrointestinal microbiome.

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.

Effect of Intravenous Fluid Volumes on the Adrenal Glucocorticoid Response After Burn Injury in Swine.

Severe thermal injury induces metabolic and physiological stress, prompting a disruption in the hypothalamic-pituitary-adrenal axis. The objective of this study was to evaluate potential confounding effects of Lactated Ringer's (LR) resuscitation on adrenal damage and cortisol production following burn. Anesthetized swine were instrumented with jugular catheters and sustained 40% TBSA burns from brass probes heated to 100°C. Animals recovered to consciousness and received IV fluid resuscitation with LR at two different volumes: 15 ml/kg/d (limited volume [LV], n = 6) or 2 ml/kg/%TBSA/d (modified Brooke [MB], n = 6). Nonburned animals (Sham) were both oral and IV fluid restricted (S-FR, n = 4) to induce stress. Computed tomography (CT) angiographies were performed at baseline (BL) and 48 hours postburn, while blood and urine samples were collected at BL, 6, 24, and 48 hours postburn, with euthanasia at 48 hours for adrenal harvesting. Urinary cortisol was elevated following burn/surgery in all animals and returned back to BL in S-FR (404 ± 48 pg/mg creatinine) but not MB (1332 ± 176 pg/mg creatinine; P = .005) or LV (1223 ± 335 pg/mg creatinine; P = .07) by 48 hours. Gene expression of cleavage enzymes (3ß-HSD, CYP17, CYP11, and CYP21) along the cortisol synthesis pathway showed minimal changes. Adrenal apoptosis (Terminal deoxynucleotidyl transferase dUTP nick-end labeling [TUNEL] staining) was greatest in the MB group (P ≤ .01) when compared to S-FR, partly due to elevations in c-Jun N-terminal kinase. Adrenal hemorrhaging was also greatest in MB animals, with no differences in tissue volume or wet-to-dry ratio. However, tissue levels of cytokines IL-1ß, IL-10, and IL-12 were greatest in LV. Burn injury elevates urinary cortisol and compromises adrenal gland integrity, which is affected by IV fluid volume.

Delivery of Allogeneic Adipose Stem Cells in Polyethylene Glycol-Fibrin Hydrogels as an Adjunct to Meshed Autografts After Sharp Debridement of Deep Partial Thickness Burns.

Harvesting of autografts results in donor site morbidities and is limited in scenarios such as large total body surface area burns. In these instances, coverage is increased by meshing grafts at the expense of delayed biologic closure. Moreover, graft meshing increases the likelihood of contraction and hypertrophic scarring, limits range of motion, and worsens cosmesis. Many tissue engineering technologies have touted the promise of adipose-derived stem cells (ASCs) for burn wounds. The primary objective of the current study was to determine feasibility and efficacy of in situ ASC delivery via PEGylated fibrin (FPEG) hydrogels as adjuncts to meshed split thickness skin grafts in a porcine model. Deep partial thickness burns were created on the dorsum of anesthetized Yorkshire pigs, and subsequently debrided on post-burn day 4. After debridement, wounds were treated with: split thickness skin grafts (STSG); meshed STSG (mSTSG); and mSTSG + FPEG with increasing doses of ASCs. We show that FPEG hydrogels can be delivered in situ to prevent the contraction seen after meshing of STSG. Moreover, ASCs delivered in FPEG dose-dependently increase blood vessel size which significantly correlates with CD31 protein levels. The current study reports a dual-action adjunct therapy to autografting administered in situ, wherein FPEG acts as both scaffolding to prevent contraction, and as a delivery vehicle for ASCs to accelerate angiogenesis. This strategy may be used to incorporate other biologics for generating tissue engineered products aimed at improving wound healing and minimizing donor sites or scarring. Stem Cells Translational Medicine 2018;7:360-372.

A model of recovery from inhalation injury and cutaneous burn in ambulatory swine.

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.

Quantitative long-term measurements of burns in a rat model using Spatial Frequency Domain Imaging (SFDI) and Laser Speckle Imaging (LSI).

BACKGROUND AND OJECTIVES: The current standard for diagnosis of burn severity and subsequent wound healing is through clinical examination, which is highly subjective. Several new technologies are shifting focus to burn care in an attempt to help quantify not only burn depth but also the progress of healing. While accurate early assessment of partial thickness burns is critical for dictating the course of treatment, the ability to quantitatively monitor wound status over time is critical for understanding treatment efficacy. SFDI and LSI are both non-invasive imaging modalities that have been shown to have great diagnostic value for burn severity, but have yet to be tested over the course of wound healing. METHODS: In this study, a hairless rat model (n = 6, 300-450 g) was used with a four pronged comb to create four identical partial thickness burns (superficial n = 3 and deep n = 3) that were used to monitor wound healing over a 28 days period. Weekly biopsies were taken for histological analysis to verify wound progression. Both SFDI and LSI were performed weekly to track the evolution of hemodynamic (blood flow and oxygen saturation) and structural (reduced scattering coefficient) properties for the burns. RESULTS: LSI showed significant changes in blood flow from baseline to 220% in superficial and 165% in deep burns by day 7. In superficial burns, blood flow returned to baseline levels by day 28, but not for deep burns where blood flow remained elevated. Smaller increases in blood flow were also observed in the surrounding tissue over the same time period. Oxygen saturation values measured with SFDI showed a progressive increase from baseline values of 66-74% in superficial burns and 72% in deep burns by day 28. Additionally, SFDI showed significant decreases in the reduced scattering coefficient shortly after the burns were created. The scattering coefficient progressively decreased in the wound area, but returned towards baseline conditions at the end of the 28 days period. Scattering changes in the surrounding tissue remained constant despite the presence of hemodynamic changes. CONCLUSIONS: Here, we show that LSI and SFDI are capable of monitoring changes in hemodynamic and scattering properties in burn wounds over a 28 days period. These results highlight the potential insights that can be gained by using non-invasive imaging technologies to study wound healing. Further development of these technologies could be revolutionary for wound monitoring and studying the efficacy of different treatments. Lasers Surg. Med. 49:293-304, 2017. © 2017 Wiley Periodicals, Inc.