Blast exposure induces post-traumatic stress disorder-related traits in a rat model of mild traumatic brain injury

Abstract

Blast related traumatic brain injury (TBI) has been a major cause of injury in the wars in Iraq and Afghanistan. A striking feature of the mild TBI (mTBI) cases has been the prominent association with post-traumatic stress disorder (PTSD). However, because of the overlapping symptoms, distinction between the two disorders has been difficult. We studied a rat model of mTBI in which adult male rats were exposed to repetitive blast injury while under anesthesia. Blast exposure induced a variety of PTSD-related behavioral traits that were present many months after the blast exposure, including increased anxiety, enhanced contextual fear conditioning, and an altered response in a predator scent assay. We also found elevation in the amygdala of the protein stathmin 1, which is known to influence the generation of fear responses. Because the blast overpressure injuries occurred while animals were under general anesthesia, our results suggest that a blast-related mTBI exposure can, in the absence of any psychological stressor, induce PTSD-related traits that are chronic and persistent. These studies have implications for understanding the relationship of PTSD to mTBI in the population of veterans returning from the wars in Iraq and Afghanistan.

FIG. 1.

Nissl staining in the hippocampus (A,B) and neocortex (C,D) is shown from control (A,C) and 3×74.5 kPa blast-exposed (B,D) rats at 4.5 months post-blast exposure. No significant histological changes were noted. Scale bar=200 μm.

FIG. 2.

Shown are escape latencies over trials 1–4 on day 1 (A) of the Morris water maze or as composite values across 3 days of testing (B) as well as performance in a probe trial on day 4 (C). There were no statistically significant differences between the groups. Days to criteria (p=0.16, unpaired t-test) (D) and a plot of animals reaching criteria by day (p=0.18, log-rank test) (E) are shown for a win/shift eight arm radial maze task.

FIG. 3.

Blast-exposed and control rats were tested for 5 min in an elevated zero maze. Shown is time in motion (A), total distance moved (B), the latency to enter an open arm (C), and the latency to cross between two open arms (D, cross latency) as well as open arm entries (E), and time spent in open (F) and closed (G) arms. Values significantly different from controls are indicated by an asterisk (p<0.05, unpaired t-tests). Startle magnitude and sensory gating were examined in a prepulse inhibition assay. Shown in (H) are background readings (Pre), acoustic startle response (Pulse), startle following the prepulse, and the pulse-prepulse. Percent prepulse inhibition (PPI) is shown in (I). Asterisks indicate values significantly different from controls (p<0.05, unpaired t-tests). Error bars indicate the standard error of the mean (SEM) in all panels.

FIG. 4.

Activity of blast and control rats were assessed in an open field for 10 min pre-exposure, during a 10 min exposure to bedding soaked in cat urine, for 40 min immediately post-exposure and for 30 min at 3 days post exposure. Shown are move distance, move time, center entries, center time, center distance, and center rest time. Error bars indicate±standard error of the mean (SEM). Values significantly different from controls are indicated by an asterisk (p<0.05, unpaired t-test). Other statistical tests are discussed in the text.

FIG. 5.

Center time in an open field of blast-exposed and control rats was measured for 10 min before exposure (A,B), during a 10 min exposure to bedding soaked with cat urine (C,D), and for 40 min post-exposure. Values for center time for the first 10 min post-exposure (E) and in 5 min bins over the entire 40 min (F) are shown. Activity of blast-exposed and control rats was then measured for 30 min in an open field at 3 days post-predator scent exposure (G,H). Values significantly different from controls are indicated by an asterisk (p<0.05, unpaired t-test). Error bars indicate±standard error of the mean (SEM).

FIG. 6.

Light-side emergence latency (A), and latency to reach the center of the lighted side (B) are shown. Values significantly different from controls are indicated by an asterisk (p<0.05, unpaired t-test).

FIG. 7.

Contextual and cued fear conditioning were examined. During the training phase (A) each rat was placed inside the conditioning chamber for 2 min before the onset of the conditioned stimulus (an 80 dB tone) that lasted for 20 sec. A 2 sec footshock (0.7 mA) was delivered immediately after the termination of the conditioned stimulus. Freezing behavior was measured during minutes 0–2 of the training session (baseline), after the presentation of the tone and after the footshock. The test for contextual fear memory (B) was performed at 24 h by measuring freezing behavior during a 4 min test in the same conditioning chamber. Cued fear memory was tested another 24 h later (C). Each rat was placed in a novel context for 2 min and baseline freezing was measured, followed by exposure to the conditioned stimulus (tone) for 3 min. Asterisk indicates statistically significant difference (p<0.05, unpaired t-test) between blast-exposed and control groups. Error bars indicate ±standard error of the mean (SEM). Other statistical tests are discussed in the text.

FIG. 8.

Western blotting was performed on homogenates of amygdala (A) or hippocampus (B) from blast-exposed (B) and control (C) animals harvested at 8 months after blast exposure. The top panel in each set shows blotting with an antibody that recognizes stathmin 1 (Stmn1). In the lower panels the blots were reprobed for β-tubulin (β-tub) as a loading control. Representative blots are shown from experiments that were performed multiple times. In panels (C) and (D), levels of Stmn1 from the experiments in (A) and (B) are expressed as the ratio of Stmn1 to β-tubulin (± standard error of the mean [SEM]). Asterisk indicates p=0.01 versus control (unpaired t-test).