Core body temperature as adjunct to endpoint determination in murine median lethal dose testing of rattlesnake venom

Abstract

Median lethal dose (LD50) testing in mice is the 'gold standard' for evaluating the lethality of snake venoms and the effectiveness of interventions. As part of a study to determine the murine LD50 of the venom of 3 species of rattlesnake, temperature data were collected in an attempt to more precisely define humane endpoints. We used an 'up-and-down' methodology of estimating the LD50 that involved serial intraperitoneal injection of predetermined concentrations of venom. By using a rectal thermistor probe, body temperature was taken once before administration and at various times after venom exposure. All but one mouse showed a marked, immediate, dose-dependent drop in temperature of approximately 2 to 6°C at 15 to 45 min after administration. The lowest temperature sustained by any surviving mouse was 33.2°C. Surviving mice generally returned to near-baseline temperatures within 2 h after venom administration, whereas mice that did not survive continued to show a gradual decline in temperature until death or euthanasia. Logistic regression modeling controlling for the effects of baseline core body temperature and venom type showed that core body temperature was a significant predictor of survival. Linear regression of the interaction of time and survival was used to estimate temperatures predictive of death at the earliest time point and demonstrated that venom type had a significant influence on temperature values. Overall, our data suggest that core body temperature is a useful adjunct to monitoring for endpoints in LD50 studies and may be a valuable predictor of survival in venom studies.

Figure 1.

Core body temperature over time. For all venoms, green lines represent surviving mice, orange lines represent nonsurviving mice, and pink lines represent averaged temperatures of 6 saline-control mice. For Western Diamondback and Northern Pacific venoms, green lines also represent a dose of 0.6 LD₅₀, and orange lines represent a dose of 1.8 LD₅₀. For Southern Pacific venom, solid green lines represent a dose of 0.3 LD₅₀, solid orange lines represent a dose of 3.1 LD₅₀, and dashed lines (green or orange) represent an LD₅₀ dose. The asterisk denotes the lowest body temperature of any surviving mouse of any venom type (33.2 °C). Error bars on the saline-control (pink) line represent the SEM.

Figure 2.

Graphs of linear regression models predicting temperature from the interaction of time and survival. Graphs show linear regression of temperatures over time for surviving and nonsurviving mice for each venom and combined venoms.

Figure 3.

Clinical signs, photographs, and temperatures of select mice. A. Normal mouse. Temperature before venom injection, 37.3 °C.B. Nonsurviving mouse:dose, 3LD₅₀;temperature at 15 min after injection, 35.6 °C. Eyes open, intermittent sniffing, minimal voluntary movement, cervical ventroflexion, intermittent mild skin and body twitch, moderate dyspnea (decreased rate, increased effort), short bout of mouth and abdominal grooming. C. Surviving and nonsurviving mice at 30 min after injection. C1. Surviving mouse; LD₅₀ dose; temperature, 35.9 °C. C2. Nonsurviving mouse: dose, 3LD₅₀; temperature, 33.7 °C. Marked piloerection present in both mice; dyspnea (decreased rate, increased effort) in both mice, with C1 (mild to moderate) less dyspneic than C2 (severe). C2 shows eye squint or grimace, mouth grooming, cervical ventroflexion, and pinned back ears. D. Examples of typical intermittent behavior after venom injection in a surviving mouse, LD₅₀ dose. Immediately through 2 h after injection, D1 and D2 show abdominal and mouth grooming. D3 shows abdominal pressing, body stretching, and tail elevation,which we considered to be signs of acute abdominal pain and discomfort, similar to the behavior in the standardized writhing test.^,,, E. Progression of clinical signs in a high-dose (3LD₅₀), nonsurviving mouse. E1 is at 1 h after injection; temperature, 32.6 °C. Mouse is sternal with body and nose touching floor, eyes nearly shut, no voluntary movement but weakly responsive to stimuli (moderate resistance when handled), extremities pale, significant piloerection, marked abdominal respiratory effort. E2 is at 2 h after injection;temperature, 29.5 °C. Mouse is sternal with body and head flat on cage floor, eyes shut, pale extremities, no voluntary movement but responsive to stimuli (mild to moderate resistance when handled but tires easily), slow shallow breathing at rest, open-mouth breathing. E3 is at 3 h after injection; temperature, 27.6 °C. Mouse is sternal with body and head lying flat on wet food pile (many mice preferred to lie on or near wet food provided on the cage floor), very weakly responsive to stimuli, moderate piloerection, pale extremities, slow shallow breathing, ears pinned back, open-mouth breathing, intermittent gasping; mouse euthanized. F. Surviving mouse: LD₅₀ dose; 6 h after injection; temperature, 35.4 °C. At several hours after injection, many mice exhibited this characteristic presentation, embodied by minimal to no voluntary movement, marked piloerection, cervical ventroflexion, mouth slightly open, mildly squinted eyes, and pinned-back ears.