Virginia Research Day 2022

Medical Student Research Biomedical

14 Acute Mechanisms Of Sterile Tissue Injury Induced Vascular Leak, Ileus And Death

Christina B. Bagnati; Connor A. Schroeder; Mariko A. Locke; Nicholas J. Hale; Nicolas M. Du Fayet De La Tour; Gary D. Isaacs; Swapnil K. Sonkusare; Derese Getnet; Anthony J.M. Bauer Corresponding author: cbbagnati@liberty.edu

Liberty University College of Osteopathic Medicine, University of Virginia, Walter Reed Army Institute of Research, and Cross Biomedical

Background: Severe traumatic tissue injury from military combat, acts of terror, or civilian accidents is the leading cause of mortality worldwide in young adults. Although, damaged associated molecular pattern molecules released from injury sites cause a known delayed post-traumatic upregulation in pro- inflammatory immune responses, many of the mechanisms underlying the complications of blunt extremity trauma remain unknown. Therefore, we sought to elucidate the existence of a novel protein extract from damaged limb tissue that could trigger a non-inflammatory- induced microvascular leak, ileus, and acute death. Methods: A dorsal subcutaneous implanted, syngeneic donor bone-muscle matrix (BMX) or skeletal muscle matrix (MX) model of sterile peripheral tissue injury in mice was used and adjusted based on percentage of implanted BMX or MX to total body weight. Animals were studied 24 hours after resuscitation. Dexamethasone (5 mg/kg, i.p.) 1 hour before tissue implantation was used to isolate the inflammatory component of the post-trauma response. Gastrointestinal transit was assessed

by the oral administration of FITC-dextran 70 kDa MW (15 µl) with mice euthanized after 80 minutes. Vascular leak was assessed by confocal microscopy after i.v. microvascular labeling with isolectin B4-DyLight®594λ and FITC488λ-dextran. An Andor Dragonfly 200 high speed confocal system was used to visualize endothelial subcellular calcium sparklets within the microvasculature of intestinal whole-mounts of VE-cadherin (Cdh5- GCaMP8) C57Bl/6 mice. Cultured human umbilical vein endothelial cells (EGFP-HUVECs) were used to determine the direct effect of the tissue injury extract on acute changes in endothelial cell morphology. LC, HPLC-SEC and silver Western blotting techniques were used to characterize the “traumacidin” extract. Results: BMX 12.5%bw caused a significant delay in gastrointestinal transit, which was blocked with dexamethasone pretreatment. However, a transit delay was no longer blocked by dexamethasone upon increasing the BMX injury to 15%bw indicating the existence of an additional non-inflammatory trauma component. Confocal microscopy of ex vivo jejunal segments demonstrated a massive

intestinal microvascular leak of intravenous IB4-594λ/FITC-dextran (70 kDa, 100 µl) injected via the penile vein in both BMX and MX models, as well as after intravenous or direct jejunal loop application of the MX extract (200 µl). Confocal imaging of Cdh5- GCaMP8 endothelial subcellular calcium sparklets demonstrated an increase in calcium signaling within intact jejunal endothelial cells upon exposure to a trauma extract. HUVECs immediately contracted by 20% when exposed to 25 µl of the tissue injury extract. LC, HPLC- SEC and silver Western blotting techniques have isolated the “traumacidin” extract to a few proteins >100, but <300 kDa. Conclusions: Microvascular leak, endothelial cell dysfunction, ileus and acute death within minutes caused by the “traumacidin” extract is a relatively large skeletal muscle protein, which can be sequenced and identified. Therefore, this on-going study is elucidating the existence of a novel potent post-traumatic protein extract that if blocked could prevent death of the wounded warrior or civilian accident victim.

46