Dorcas P. O’Rourke

Biology and Diseases of Amphibians

Amphibians are commonly used in research, and are unique in that they typically represent a transition from early aquatic life forms to terrestrial adults. There are over 6200 species of living amphibians, with tremendous variation in morphology and behavior. This chapter presents an overview of amphibian biology, husbandry and diseases, with emphasis on species most commonly used in research, including Xenopus laevis, axolotls, leopard and bullfrogs, and dendrobatid frogs.

GPR4 deficiency alleviates intestinal inflammation in a mouse model of acute experimental colitis

GPR4 is a proton-sensing G protein-coupled receptor that can be activated by extracellular acidosis. It has recently been demonstrated that activation of GPR4 by acidosis increases the expression of numerous inflammatory and stress response genes in vascular endothelial cells (ECs) and also augments EC-leukocyte adhesion. Inhibition of GPR4 by siRNA or small molecule inhibitors reduces endothelial cell inflammation. As acidotic tissue microenvironments exist in many types of inflammatory disorders, including inflammatory bowel disease (IBD), we examined the role of GPR4 in intestinal inflammation using a dextran sulfate sodium (DSS)-induced acute colitis mouse model. We observed that GPR4 mRNA expression was increased in mouse and human IBD tissues when compared to control intestinal tissues. To determine the function of GPR4 in intestinal inflammation, wild-type and GPR4-deficient mice were treated with 3% DSS for 7days to induce acute colitis. Our results showed that the severity of colitis was decreased in GPR4-deficient DSS-treated mice in comparison to wild-type DSS-treated mice. Clinical parameters, macroscopic disease indicators, and histopathological features were less severe in the DSS-treated GPR4-deficient mice than the DSS-treated wild-type mice. Endothelial adhesion molecule expression, leukocyte infiltration, and isolated lymphoid follicle (ILF) formation were reduced in intestinal tissues of DSS-treated GPR4-null mice. Collectively, our results suggest GPR4 provides a pro-inflammatory role in the inflamed gut as the absence of GPR4 ameliorates intestinal inflammation in the acute experimental colitis mouse model.

Biology and Diseases of Reptiles

Since their emergence 310–320 million years ago, reptiles have evolved to be one of the most adaptive and remarkable groups of vertebrate animals on earth. Comprising over 9500 species, they can be found in diverse niches ranging from the arid Sahara desert to the tropical wetlands of the Amazon. Diverse reproductive physiologies and behaviors, along with adaptive characteristics such as parthenogenesis and development of venom glands for prey immobilization, make them attractive models for biomedical and basic biological research. This chapter provides information regarding biology, husbandry, and the diseases commonly affecting captive reptiles most frequently used in research.

Trypsin and rosmarinic acid reduce the toxicity of Micrurus fulvius venom in mice

Abstract Context. Antivenom is expensive and not always available, so alternative treatments are being investigated. Objective. The efficacy of trypsin or rosmarinic acid (RA) in treating Micrurus fulvius in a murine model is determined. Materials and methods. Design: randomized controlled blinded study. Subjects: Fifty mice (20–30 g). Study groups: Intraperitoneal injections of: 1) 2 mg/kg M. fulvius venom (approximately twice the LD50 for mice; n = 10); 2) 2 mg/kg M. fulvius venom incubated in vitro for 1 h prior to injection with RA at a 1:10 ratio (n = 17); 3) 2 mg/kg M. fulvius venom incubated in vitro for 1 h prior to injection with 1 mg of trypsin (n = 17); 4)1 mg trypsin IP without venom (n = 3); and 5) RA IP without venom (n = 3). Main outcome: time to toxicity (respiratory distress (< 25 breaths/min.), loss of spontaneous locomotor activity, or inability to upright self). Statistical analysis: Time to toxicity using Tukey–Kramer HSD; Survival to 4, 6, and 12 h using Chi-square analysis. Results. Onset of toxicity: venom + saline, 120.3 + 64.4 min; venom + rosmarinic acid, 238.1 ± 139.2 min (p = 0.15 relative to venom + saline); venom + trypsin, 319.7 + 201.0 min (p = 0.007 relative to venom + saline). Venom + trypsin but not venom + RA survival to 4 h was significant compared to venom + saline (p = 0.023). Two mice in the venom + trypsin group and one mouse in the venom + RA group survived to 12 h. Mice receiving trypsin without venom or RA without venom survived to 12 h without toxicity. Discussion. This work suggests that trypsin and RA may have efficacy in treatment M. fulvius envenomation. Conclusion. In vitro neutralization of M. Fulvius venom by trypsin justifies progressing to an in vivo model in future studies.

Long-term efficacy of pressure immobilization bandages in a porcine model of coral snake envenomation

BACKGROUND Pressure immobilization bandages delay mortality for 8 hours after coral snake envenomation, but long-term efficacy has not been established. OBJECTIVE The objective of this study is to determine the long-term efficacy of pressure immobilization bandages after coral snake envenomation in the absence of antivenom therapy. METHODS A randomized, observational pilot study was conducted. Ten pigs (17.3-25.6 kg) were sedated, intubated for 5 hours, and injected subcutaneously with 10 mg of lyophilized Micrurus fulvius venom resuspended in water. Pigs were randomly assigned to a control group (no treatment) or a treatment group (compression bandage and splint) approximately 1 minute after envenomation. Bandage pressure was not controlled. Pigs were monitored daily for 21 days for signs of respiratory depression, decreased oxygen saturations, and paralysis. In case of respiratory depression, pigs were humanely euthanized and time to death recorded. Statistical analysis was performed with Fisher exact test, Mann-Whitney U test, and Kaplan-Meier survival curve as appropriate. RESULTS Median survival time of control animals was 307 minutes compared with 1172 minutes in treated animals (P = .10). Sixty percent of pigs in the treatment group survived to 24 hours vs 0% of control pigs (P = .08). Two of the treatment pigs survived to the end point of 21 days but showed necrosis of the distal lower extremity. CONCLUSIONS Long-term survival after coral snake envenomation is possible in the absence of antivenom with the use of pressure immobilization bandages. The applied pressure of the bandage is critical to allowing survival without necrosis. Future studies should be designed to accurately monitor the pressures applied.

Abstract 5916: Proton-sensor GPR4 potentiates intestinal inflammation in the DSS-induced colitis mouse model

Inflammation and tissue acidosis are two factors that co-exist in inflammatory bowel disease (IBD) and can contribute to increased risk of colorectal cancer (CRC) development. GPR4 is a proton-sensing G protein-coupled receptor that can be activated by extracellular acidosis through several histidine residues and subsequently signal through downstream G-protein pathways. Recently, GPR4 has been shown to be activated by acidosis and can increase the expression of numerous inflammatory and stress response genes in vascular endothelial cells (ECs) and has functionally increased EC-leukocyte adhesion. Subsequently, genetic and small molecule approaches for the inhibition of GPR4 activity have reduced endothelial cell inflammation. In this study, we examined the role of GPR4 in intestinal inflammation using a dextran sulfate sodium (DSS)-induced colitis mouse model. We observed that GPR4 mRNA expression was increased in mouse and human IBD tissues when compared to control intestinal tissues. To determine the function of GPR4 in intestinal inflammation, wild-type and GPR4-deficient mice were treated with 3% DSS for acute and chronic time points for the induction of colitis. Our results showed that the severity of colitis was decreased in GPR4-deficient DSS-treated mice in comparison to wild-type DSS-treated mice. Clinical parameters, macroscopic disease indicators, and histopathological features were less severe in the DSS-treated GPR4-deficient mice than the DSS-treated wild-type mice. Inflammatory gene expression, endothelial adhesion molecule expression, leukocyte infiltration, and isolated lymphoid follicle (ILF) formation were reduced in intestinal tissues of DSS-treated GPR4-null mice. In summary, our results suggest GPR4 potentiates intestinal inflammation as the absence of GPR4 ameliorates intestinal inflammation in the DSS-induced colitis mouse model. Use of GPR4 inhibitors could prove a valuable therapeutic in the reduction of intestinal inflammation and subsequent CRC development. Note: This abstract was not presented at the meeting. Citation Format: Edward J. Sanderlin, Nancy R. Leffler, Kvin Lertpiriyapong, Qi Cai, Heng Hong, Vasudevan Bakthavatchalu, James G. Fox, Joani Z. Oswald, Calvin R. Justus, Elizabeth A. Krewson, Dorcas O’Rourke, Li V. Yang. Proton-sensor GPR4 potentiates intestinal inflammation in the DSS-induced colitis mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5916. doi:10.1158/1538-7445.AM2017-5916