A predator-based psychosocial stress animal model of PTSD in females: Influence of estrous phase and ovarian hormones

Abstract

Traumatized women are more likely than traumatized men to develop post-traumatic stress disorder (PTSD). Still, the inclusion of females in animal models of PTSD has largely been avoided, likely due to the variable hormone profile of female rodents. Because a valid animal model of PTSD that incorporates females is still needed, we examined the influence of estrous stage and ovarian hormones on the female rat response to a predator-based psychosocial stress model of PTSD. Female Sprague-Dawley rats were exposed to psychosocial stress or control conditions for 31 days. Stressed rats were given two cat exposures and daily social instability; control rats were handled daily. Beginning on Day 32, rats underwent physiological or behavioral testing. In Experiment 1, vaginal smears were collected on days of the first and second cat exposures and each day of behavioral testing to determine estrous stage. In Experiments 2 and 3, ovariectomized or sham control rats were exposed to stress or control conditions. Then, they were given behavioral testing (Exp 2), or their hearts were isolated and subjected to ischemia/reperfusion on a Langendorff isolated heart system (Exp 3). Chronic stress increased anxiety-like behavior, irrespective of estrous stage or ovariectomy condition. Ovariectomized females displayed greater startle responses and anxiety-like behavior than sham rats. Stress had no impact on myocardial sensitivity to ischemic injury; however, ovariectomized females exhibited greater ischemia-induced infarction than sham rats. These findings suggest that ovarian hormones may prevent anxiety-like behavior and be cardioprotective in non-stressed controls, but they do not interact with chronic stress to influence the development of PTSD-like sequelae in female rats.

Keywords: Animal model; Estrous; Ovariectomy; PTSD; Sex differences; Stress.

Fig. 1

Experimental timelines and procedures. In Experiment 1, rats in the stress group were exposed to a 31-day chronic stress paradigm that consisted of two cat exposures and daily social instability. Stressed rats were immobilized and exposed to an adult, female cat for 1 h on Days 1 (during the light cycle) and 11 (during the dark cycle), and throughout the 31-day paradigm, the cage mates of stressed rats were randomly changed on a daily basis. Rats in the no stress group remained in their home cages on Days 1 and 11 and remained with the same cage mate throughout the paradigm. On Day 32, all rats were tested for anxiety-like behavior on the elevated plus maze (EPM); on Day 33, all rats were tested for their acoustic startle responses; and, on Day 34, all rats were tested for anxiety-like behavior in an open field. Vaginal smears were collected from all rats on Days 1, 11, 32, 33, and 34 to determine the stage of estrous. In Experiment 2, rats underwent ovariectomy or sham surgery at 4 weeks of age, after which they were randomly assigned to stress or no stress groups. Four weeks after surgery, all rats underwent the same procedures as outlined in Experiment 1, except no vaginal smears were collected. In Experiment 3, rats underwent ovariectomy or sham surgery at 4 weeks of age, after which they were randomly assigned to stress or no stress groups. Four weeks after surgery, all rats underwent the same stress procedures as outlined in Experiment 2. On Day 32, all rats were tested for anxiety-like behavior on the EPM. On Day 33, all rats were anesthetized, and their hearts were removed and exposed to ischemia / reperfusion, as outlined in the methods.

Fig. 2

Representative images from each stage of estrous. A sterile micropipette was used to flush the vaginal cavity of female rats with distilled water (McLean et al., 2012). The collected sample was placed on a glass slide and allowed to dry at room temperature. The slides were then stained with crystal violet and examined under a microscope to determine the stage of estrous. The following guidelines were used to determine the stage of estrous: (a) diestrus (many leukocytes, occasional nucleated epithelial cells, no cornified cells); (b) proestrus (many nucleated epithelial cells, occasional cornified cells, and few leukocytes); (c) estrus (many cornified cells, few nucleated epithelial cells, no leukocytes); (d) metestrus (some cornified cells, nucleated epithelial cells, and leukocytes).

Fig. 3

Interactive influence of stress and estrous phase (at the time of testing) on female rat behavior. Stressed rats spent less time in the open arms of the EPM than non-stressed rats (a), which was not attributable to stress-induced alterations of general locomotor activity on the EPM (b). Stressed rats also spent less time in the center of the open field (c), exhibited less locomotor activity in the open field (d), and displayed fewer rearing episodes in the open field (e) than non-stressed rats. Data are presented as means ± SEM. Sample sizes for each estrous phase are indicated by the number under each bar. * p < 0.05 relative to no stress.

Fig. 4

Interactive influence of stress and ovariectomy on female rat behavior. Stressed rats, independent of surgery condition, spent less time in the open arms of the EPM (a), exhibited less locomotor activity in the open field (e), and displayed fewer rearing episodes in the open field (f) than non-stressed rats. Ovariectomized (OVX) rats demonstrated greater startle responses (c) and spent less time in the center of the open field (d) than sham rats. Data are presented as means ± SEM. * p < 0.05 relative to no stress; ** p < 0.05 relative to sham.

Fig. 5

Interactive influence of stress and ovariectomy on EPM behavior and myocardial sensitivity to ischemic injury. No significant effects were observed for the amount of time spent in the open arms of the EPM (a). OVX rats exhibited significantly larger infarct sizes than sham rats following ischemia (b). Images depict representative myocardial slices with white areas indicative of infarction (c). Data are presented as means ± SEM. * p < 0.05 relative to sham.