Progressive Cognitive and Post-Traumatic Stress Disorder-Related Behavioral Traits in Rats Exposed to Repetitive Low-Level Blast

Abstract

Many military veterans who experienced blast-related traumatic brain injuries (TBI) in the conflicts in Iraq and Afghanistan currently have chronic cognitive and mental health problems including post-traumatic stress disorder (PTSD). Besides static symptoms, new symptoms may emerge or existing symptoms may worsen. TBI is also a risk factor for later development of neurodegenerative diseases. In rats exposed to repetitive low-level blast overpressure (BOP), robust and enduring cognitive and PTSD-related behavioral traits develop that are present for at least one year after blast exposure. Here we determined the time-course of the appearance of these traits by testing rats in the immediate post-blast period. Three cohorts of rats examined within the first eight weeks exhibited no behavioral phenotype or, in one cohort, features of anxiety. None showed the altered cued fear responses or impaired novel object recognition characteristic of the fully developed phenotype. Two cohorts retested 36 to 42 weeks after blast exposure exhibited the expanded behavioral phenotype including anxiety as well as altered cued fear learning and impaired novel object recognition. Combined with previous work, the chronic behavioral phenotype has been observed in six cohorts of blast-exposed rats studied at 3–4 months or longer after blast injury, and the three cohorts studied here document the progressive nature of the cognitive/behavioral phenotype. These studies suggest the existence of a latent, delayed emerging and progressive blast-induced cognitive and behavioral phenotype. The delayed onset has implications for the evolution of post-blast neurobehavioral syndromes in military veterans and its modeling in experimental animals.

Introduction

Traumatic brain injury (TBI) is common in military life. Military personnel deployed to combat zones are especially prone to such injuries, leading to TBI as a major cause of combat-related disability.¹ Public awareness of military-related TBI increased recently because of the conflicts in Iraq and Afghanistan.² Military-related TBIs occur through various mechanisms. Certain types of TBI, however, are relatively unique to the military, the most prominent being TBI related to blast injury. Indeed, exposures to mortars, artillery shells, and improvised explosive devices (IEDs) constituted the major cause of TBI in Iraq and Afghanistan.^2–4

A history of TBI is identified frequently in veterans seeking treatment at Department of Veterans Affairs (VA) mental health clinics.⁵ TBI has been linked to mental health problems including anxiety, depression, impulsivity, insomnia, and suicidality.^6,7 A striking feature in the most recent veterans from Iraq and Afghanistan has been the overlap between a history of blast-related mild TBI (mTBI) and clinical symptoms consistent with a diagnosis of post-traumatic stress disorder (PTSD).²

Symptoms that follow mild TBI (mTBI) often resolve. Neurological symptoms, however, can persist and evolve into a chronic post-concussion syndrome that can last for years. Alongside static symptoms, new symptoms may develop or existing symptoms may worsen.^8,9 TBI is also a risk factor for the later development of neurodegenerative diseases.¹⁰

Cognitive and PTSD-related behavioral traits develop in rats exposed to repetitive low-level blast, that are present for at least one year after exposure. This chronic phenotype has been described in several previous reports.^11–14 The current study aimed to evaluate the effect of repetitive blast overpressure (BOP) exposure on expression of these behavioral phenotypes in a longitudinal design over weeks to months to capture the temporal development of the phenotype.

Here we show that these traits develop in a delayed manner, typically absent in the first eight weeks after blast exposure but being consistently present three months and longer after exposure. These studies suggest the existence of a progressive blast-induced cognitive and behavioral phenotype. Its delayed onset has implications for laboratory modeling of post-blast neurobehavioral syndromes, an important step in the discovery and development of effective interventions. The delayed onset also has implications for the nature of post-blast neurobehavioral syndromes in military veterans.

Methods

Animals

A total of 118 adult male Long Evans hooded rats (250–350 g; 10 weeks of age; Charles River Laboratories International, Wilmington, MA) were used. The study protocol was reviewed and approved by the Institutional Animal Care and Use Committees of the Walter Reed Army Institute of Research (WRAIR)/Naval Medical Research Center and the James J. Peters VA Medical Center. Experiments were conducted in compliance with the Animal Welfare Act and per the principles set forth in 2011 in the Guide for Care and Use of Laboratory Animals from the Institute of Laboratory Animals Resources, National Research Council, National Academy Press.

BOP exposure

Rats were exposed to overpressure injury using a shock tube, which simulates the effects of air blast exposure under experimental conditions. The shock tube has a 0.32-m circular diameter and is a 5.94 m-long steel tube divided into a 0.76-m compression chamber separated from a 5.18-m expansion chamber. The compression and expansion chambers are separated by polyethylene terephthalate Mylar TM sheets (Du Pont, Wilmington, DE) that control the peak pressure generated. The peak pressure at the end of the expansion chamber was determined with piezoresistive gauges designed specifically for pressure-time (impulse) measurements (Model 102M152, PCB, Piezotronics, Depew, NY).

Individual rats were anesthetized using an isoflurane gas anesthesia system consisting of a vaporizer, gas lines, and valves and an activated charcoal-scavenging system adapted for use with rodents. Rats were placed into a polycarbonate induction chamber, which was closed and immediately flushed with 5% isoflurane mixture in air for 2 min. Rats were placed into a cone-shaped plastic restraint device and then placed in the shock tube.

Movement was restricted further during the blast exposure using 1.5 cm diameter flattened rubber tourniquet tubing. Three tourniquets were spaced evenly to secure the head region, the upper torso, and lower torso while the animal was in the plastic restraint cone. The end of each tubing was threaded through a toggle and run outside of the exposure cage where it was tied to firmly affix the animal and prevent movement during the blast overpressure exposure without restricting breathing.

Rats were assigned randomly to sham or blast conditions and were placed in the shock tube lying prone with the plane representing a line from the tail to the nose of the body in line with the longitudinal axis of the shock tube with the head placed more upstream. The total length of time under anesthesia including placement in the shock tube and execution of the blast procedure was typically less than 3 min. Blast-exposed animals received 74.5 kPa (equivalent to 10.8 psi, duration 4.8 msec, impulse 175.8 kPa*msec) exposures administered one exposure per day for three consecutive days. Further details of the physical characteristics of the blast wave are described in Ahlers and associates.¹⁵ Control (sham) exposed animals were treated identically including receiving anesthesia and being placed in the blast tube but did not receive a blast exposure.

Within 10 days after the last blast or sham exposure, animals were transported in a climate-controlled van from the WRAIR to the James J. Peters VA Medical Center (Bronx, NY,). Animals left in the morning from the WRAIR and arrived in the afternoon of the same day at the James J. Peters VA Medical Center, where all other procedures were performed.

Animal housing

Animals were housed at a constant 70–72^oF temperature with rooms on a 12:12 h light cycle with lights on at 7 AM. All subjects were housed individually in standard clear plastic cages equipped with Bed-O'Cobs laboratory animal bedding (The Andersons, Maumee, OH) and EnviroDri nesting paper (Sheppard Specialty Papers, Milford, NJ). Access to food and water was ad libitum. Subjects were housed on racks in random order to prevent rack position effects. Cages were coded to allow maintenance of blinding to groups during behavioral testing. All cohorts received the same treatment and handling.

Behavioral testing

Three cohorts of blast-exposed rats and controls were studied. The same protocols were used in each cohort with only the time of testing after blast exposure varying. The number of animals per cohort and the exact timing of testing of each cohort in relationship to blast exposure is described in Table 1.

Table 1.

Time following Blast Exposure when Behavioral Testing Was Conducted

Task	Cohort 1	Cohort 2	Cohort 3
N (tested early)	20 blast and 18 control	20 blast and 20 control	20 blast and 20 control
N (tested late)	8 blast and 8 control	12 blast and 12 control	Not tested
Elevated zero maze
Early	1 week	3 weeks	5 weeks
Late	36 weeks	40 weeks	Not performed
Light dark escape
Early	2 weeks	3 weeks	4 weeks
Late	36 weeks	40 weeks	Not performed
Novel object recognition
Early	2 weeks	4 weeks	6 weeks
Late	Not performed	41 weeks	Not performed
Fear conditioning
Early	5 weeks	5 weeks	8 weeks
Late	36 weeks	42 weeks	Not performed

Elevated zero maze (EZM)

The apparatus consisted of a circular black Plexiglas runway 121.92 cm in diameter and raised 76 cm off the floor (San Diego Instruments, San Diego, CA). The textured runway itself was 5.08 cm across and divided equally into alternating quadrants of open runway enclosed only by a 1.27 cm lip and closed runway with smooth 15.24 cm walls. All subjects received a 5-min trial beginning in a closed arc of the runway. During each trial, subjects were allowed to move freely around the runway, with all movement tracked automatically by a video camera placed on the ceiling directly above the maze.

Data were analyzed by ANYMAZE (San Diego Instruments, San Diego CA) yielding measures of total movement time and distance for the entire maze, as well as time spent and distance traveled in each of the individual quadrants. From the quadrant data, measures of total open and closed arc times, latency to enter an open arc, total open arm entries, and latency to completely cross an open arc between two closed arcs were calculated. Subject position was determined by centroid location.

Light/dark emergence (LD)

A light/dark emergence task was run in Versamax activity cages with opaque black Plexiglas boxes enclosing the left half of the interiors so that only the right sides were illuminated. Animals began in the dark side and were allowed to explore freely for 10 min with access to the left (light) side through an open doorway located in the center of the monitor. Subject side preference and emergence latencies were tracked by centroid location with all movement automatically tracked and quantified. Light-side emergence latency, time to reach the center of the lighted side (light side center latency), and percent total light-side duration were calculated from beam breaks. All equipment was wiped clean between tests.

Novel object recognition (NOR)

Rats were habituated to the arena (90 cm length × 60 cm width × 40 cm height) for 20 min, 24 h before training. On the training day, two identical objects were placed on opposite ends of the empty arena, and the rat was allowed to explore the objects freely for 7 min. After a 1-h delay, during which the rat was held in its home cage, one of the two familiar objects was replaced with a novel one, and the rat was allowed to explore freely the familiar and novel object (NO) for 5 min to assess short-term memory (STM). After a 24-h delay, during which the rat was held in its home cage, one of the two familiar objects was replaced with a novel one different from the ones used during the STM. The rat was allowed to explore freely the familiar and NO for 5 min to assess long-term memory (LTM).

Raw exploration times for each object were expressed in seconds. Object exploration was defined as sniffing or touching the object with the vibrissae or when the animal's head was oriented toward the object with the nose placed at a distance of less than 2 cm from the object. All sessions were recorded by video camera (Sentech, Carrollton, TX) and analyzed with ANYMAZE software (San Diego Instruments). In addition, offline analysis by an investigator blind to the blast-exposed status of the animals was performed. Objects to be discriminated were of different size, shape, and color and were made of plastic or metal material. The objects consisted of a 330 mL soda can, a metal box, a cup, and a plastic tube. All objects were cleaned with 70% ethanol between trials.

Contextual and cued fear conditioning

Sound-attenuated isolation cubicles (Coulbourn Instruments, Holliston, MA) were utilized. Each cubicle was equipped with a grid floor for delivery of the unconditioned stimulus (US) and overhead cameras. All aspects of the test were controlled and monitored by the Freeze Frame conditioning and video tracking system (Actimetrics, Coulbourn Instruments). During training the chambers were scented with almond extract, lined with white paper towels, had background noise generated by a small fan, and were cleaned before and between trials with 70% ethanol.

Each subject was placed inside the conditioning chamber for 2 min before the onset of a conditioned stimulus (CS; an 80 dB, 2 kHz tone), which lasted for 20 sec with a coterminating 2-sec footshock (0.7 mA; US). A total of three tone/shock pairings were administered with the first/second and second/third separated by 1 min. Each rat remained in the chamber for an additional 40 sec after the third CS-US pairing before being returned to its home cage.

Freezing was defined as a lack of movement (except for respiration) in each 10-sec interval. Minutes 0–2 during the training session were used to measure baseline freezing. Contextual fear memory testing was performed 24 h after the training session by measuring freezing behavior during a 4-min test in the conditioning chamber under conditions identical to those of the training session with the exception that no footshock or tone (CS or US) was presented.

Animals were returned to their home cage for another 24 h at which time cued conditioning was tested. To create a new context with different properties, the chambers were free of background noise (fan turned off), lined with blue paper towels, scented with lemon extract, and cleaned before and during all trials with isopropanol. Each subject was placed in this novel context for 2 min, and baseline freezing was measured, followed by exposure to the CS (20-sec tone) at 120 and 290 sec.

Statistical analysis

Values are expressed as mean ± standard error of the mean. Comparisons were performed using repeated-measures ANOVA, or unpaired t-tests. When repeated-measures analysis of variance (ANOVA) was used, sphericity was assessed using the Mauchly test. If the assumption of sphericity was violated (p < 0.05, Mauchly test), significance was determined using the Greenhouse-Geisser correction. Statistical tests were performed using the program GraphPad Prism 8.0 (GraphPad Software) or SPSS v26 (IBM).

Results

A variety of cognitive and PTSD-related behavioral traits^11–14,16 develop in rats exposed to low-level repetitive BOP injuries using 74.5 kPa exposures delivered one per day for three consecutive days (3  × 74.5 kPa). Multiple cohorts of rats exposed to this protocol have been tested 3–4 months or more after a single sequence of blast exposures and have been observed consistently to exhibit a PTSD-related behavioral phenotype.^11–14,16

Here rats were tested in the immediate post-blast period in tasks found previously to be most informative (EZM, LD emergence, NOR, and contextual/cued fear conditioning).^11–14 We tested three cohorts of rats within eight weeks of blast exposure. Two cohorts were retested in a subset of tasks at 36–42 weeks after blast exposure. Table 1 shows the tests performed and timing of each test in relation to the last blast exposure. Table 2 summarizes the results.

Table 2.

Summary of Behavioral Test Findings

Behavioral test	Cohort 1 (this study)	Cohort 2 (this study)	Cohort 3 (this study)	Elder et al. 11	Perez-Garcia et al. 13	Perez-Garcia et al. 12 Cohort 1	Perez-Garcia et al. 12 Cohort 2
Elevated zero maze
Early	Anxiety (1 week)	Normal (3 weeks)	Normal (5 weeks)	Not tested	Not tested	Not tested	Not tested
Late	Anxiety (36 weeks)	Anxiety (40 weeks)	Not tested	Anxiety (24 weeks)	Anxiety (30 weeks)	Anxiety (13 weeks)	Anxiety (13 weeks)
Light dark escape
Early	Normal (2 weeks)	Normal (3 weeks)	Normal (4 weeks)	Not tested	Not tested	Not tested	Not tested
Late	Anxiety (36 weeks)	Normal (40 weeks)	Not tested	Anxiety (25 weeks)	Anxiety (29 weeks)	Anxiety (12 weeks)	Anxiety (12 weeks)
Novel object recognition
Early
Novel object preference	Yes (2 weeks)	Yes (4 weeks)	Yes (6 weeks)	Not tested	Not tested	Not tested	Not tested
Discrimination index	Normal (2 weeks)	Decreased in LTM but not STM (4 weeks)	Decreased in STM and LTM (6 weeks)	Not tested	Not tested	Not tested	Not tested
Exploration time	Decreased (2 weeks)	Normal (4 weeks)	Decreased (6 weeks)	Not tested	Not tested	Not tested	Not tested
Late					Impaired 31 weeks
Novel object preference	Not tested	No (41 weeks)	Not tested	Not tested	Yes (31 weeks)	Impaired 14–15 weeks and when retested 4 weeks later	Impaired 14–15 weeks and when retested 4 weeks later
Discrimination index	Not tested	Decreased in STM and LTM (41 weeks)	Not tested	Not tested	Decreased in LTM but not STM (31 weeks)
Exploration time	Not tested	Decreased (41 weeks)	Not tested	Not tested	Decreased (41 weeks)
Fear conditioning
Early	Normal (5 weeks)	Normal (5 weeks)	Normal (8 weeks)	Not tested	Not tested	Not tested	Not tested
Late	Cued fear freezing increased (36 weeks)	Cued fear freezing increased (42 weeks)	Not tested	Cued fear freezing increased (25 weeks)	Cued fear freezing increased (35 weeks)	Cued fear freezing increased (16 weeks)	Cued fear freezing increased (16 weeks)

Abnormal results are indicated in bold.

Testing of Cohort 1

When examined one week after BOP exposure, cohort 1 exhibited an anxiety phenotype in an EZM (Fig. 1A), making fewer open arm entries (p < 0.001, unpaired t test) as well as spending less time in the open arms (p < 0.0001) and exhibiting a longer latency to cross to a second open arm (cross arm latency; p < 0.01). By contrast, at two weeks after exposure, cohort 1 did not show changes in a LD escape task (Fig. 1C) with blast-exposed rats exhibiting a similar latency to the light center as well as spending similar time on the light side when compared with corresponding behaviors among the individuals in the control (sham exposed) group.

FIG. 1.

Elevated zero maze (EZM) and light/dark (LD) testing of cohort 1. Blast-exposed (n = 20) and control (n = 18) rats from cohort 1 were tested in an EZM and LD emergence task within two weeks of blast exposure (A and C). A subset (8 blast and 8 control) was retested at 36 weeks after blast exposure (B and D). Shown for the EZM (A and B) are time in motion (Move Time), distance moved (Move Distance), latency to enter an open arm, open arm entries, time spent in the open arms as well as the latency to cross into the second open arm (cross arm latency). In the LD task (C and D), total distance moved, latency to reach the light center, entries into the light center, as a well as time spent in the light center, total time spent on the light side, and total distance traveled on the light side are shown. Error bars indicate the standard error of the mean. Asterisks indicate values significantly different from controls (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired t tests).

When retested at 36 weeks after blast exposure, however, in addition to anxiety in the EZM (Fig. 1B), cohort 1 exhibited an anxiety phenotype in the LD escape task with blast-exposed spending less time on (p < 0.05) and traveling shorter distances on the light side (Fig. 1D; p < 0.05), changes that were not observed in the immediate post-blast period (Fig. 1C).

When tested in NOR at two weeks after blast exposure (Fig. 2A), both controls and blast-exposed rats in cohort 1 spent more time exploring the NO compared with the previously experienced familiar object (FO) when presented 1 h (STM; p < 0.01) or 24 h (long-term memory, LTM; p < 0.01) later. Blast-exposed rats also showed no differences from controls when a discrimination index was calculated (Fig. 2B) relating the relative tendency to explore the NO versus FO. Blast-exposed rats differed from controls only in that they spent less total time exploring the objects (Fig. 2C) during the training (p < 0.05) and LTM sessions (p < 0.05). Cohort 1 was not retested in NOR at a later time point.

FIG. 2.

Novel object recognition (NOR) testing of cohort 1. Blast-exposed (n = 20) and control (n = 18) rats from cohort 1 were tested in a NOR task two weeks after blast exposure. Panel A shows time spent exploring the objects (OB1 and OB2) during the training session as well as exploration of the previously presented familiar object (FO) compared with the novel object (NO) when presented 1 h (short-term memory, STM) or 24 h (long-term memory, LTM) later. Panels B and C show the discrimination index (B) and total time spent exploring the objects (C) during the indicated sessions. Asterisks indicate values significantly different from controls (*p < 0.05, **p < 0.01, ****p < 0.0001, unpaired t tests). Further discussion of statistical testing can be found in the text.

At five weeks after exposure, the blast-exposed rats in cohort 1 exhibited normal fear learning in a contextual/cued fear-learning task (Fig. 3A). Blast-exposed and control groups responded similarly in the training phase with increased freezing after the pairing of the tone with the shock (F _{1, 33} = 52.720, p < 0.001; repeated measures ANOVA) but without between group differences (F _{1, 33} = 1.198, p = 0.282). Both groups froze similarly in the contextual phase when reintroduced into the training chamber 24 h later when no tone or shock was presented (F _{1, 36} = 0.003; p = 0.95). They also responded similarly in the cued phase when the tone alone was presented another 24 h later in a novel environment (Fig. 3A).

FIG. 3.

Testing of cohort 1 in fear learning. Blast-exposed (n = 20) and control (n = 18) rats from cohort 1 were tested in a fear conditioning (FC) paradigm five weeks after blast exposure (A). A subset (8 blast and 8 control) was retested at 36 weeks (B). Results are shown for the training phase, contextual fear memory, which was tested 24 h after training, and cued fear memory, which was tested another 24 h later. A repeated measures analysis of variance of freezing during the training sessions revealed a significant within-subjects effect of freezing for baseline versus tone (F _{1, 33} = 52.720, p < 0.001 for 5 weeks; F _{1, 11} = 54.363, p < 0.001 for 36 weeks) but no effect of freezing*condition (F _{1, 33} = 0.311, p = 0.581, 5 weeks; F _{1, 11} = 2.828, p = 0.121, 36 weeks). A test of between-subject effects revealed no significant group differences during the training sessions (F _{1, 33} = 1.198, p = 0.282, 5 weeks; F _{1, 11} = 1.976, p = 0.121, 36 weeks). There were no differences between blast-exposed and control groups in the contextual testing (F _{1, 36} = 0.003, p = 0.95 for 5 weeks; F _{1, 14} = 0.313, p = 0.585 for 36 weeks). In the cued phase testing, at early and late time points both blast and control groups in cohort 1 responded with freezing after presentation of the tone (F _{2.1, 65.3} = 12.766, p < 0.001, freezing*condition F _{2.1, 65.3} = 0.608, p = 0.556 for 5 weeks; F _{3, 27} = 8.999, p < 0.001, freezing*condition F _{3, 27} = 1.838, p = 0.164 for 36 weeks). While there were no between-group differences at 5 weeks (F _{1, 31} = 1.270, p = 0.268), at 36 weeks, the blast-exposed exhibited increased freezing compared with the controls (F _{1, 9} = 9.556, p = 0.013). Error bars in all panels indicate the standard error of the mean. Asterisks indicate values significantly different from controls at individual time points (*p < 0.05, unpaired t test).

At 36 weeks post-blast (Fig. 3B), however, while the blast-exposed rats responded similarly in the training and contextual phases, they exhibited exaggerated freezing in the cued phase of the task (F _{1, 9} = 9.556, p = 0.013), compared with the identical group when studied at five weeks post-blast.

Testing of Cohort 2

Cohort 2 showed no abnormalities in EZM or LD in the immediate post-blast period (Fig. 4A,4C). When cohort 2 was retested 40 weeks after exposure (Fig. 4B), however, blast exposed rats exhibited anxiety in the EZM, moving less (p < 0.05), making fewer open arm entries (p < 0.01), spending less time in the open arms (p < 0.05), and exhibiting a longer cross arm latency (p < 0.05). Blast-exposed rats of cohort 2 did not show abnormalities in a LD task at 40 weeks (Fig. 4D).

FIG. 4.

Elevated zero maze (EZM) and light/dark (LD) testing of cohort 2. Blast-exposed (n = 20) and control (n = 20) rats from cohort 2 were tested in an EZM and LD emergence task three weeks after blast exposure (A and B). A subset (12 blast and 12 control) was retested at 40 weeks (C and D). Shown for the EZM (A and C) are time in motion (Move Time), distance traveled (Move Distance), the latency to enter an open arm, open arm entries, time spent in the open arms as well as the latency to cross between two open arms (cross arm latency). In the LD task (B and D), total distance moved, latency to reach the light center, entries into the light center, as a well as time spent in the light center, total time spent on the light side, and total distance traveled on the light side are shown. Error bars indicate the standard error of the mean. Asterisks indicate values significantly different from controls (*p < 0.05, **p < 0.01, unpaired t tests).

When cohort 2 was tested in NOR at four weeks after exposure, blast-exposed and control groups spent equal time exploring the objects (Fig. 5D). Time exploring the NO compared with the previously presented FO whether presented 1 h (STM) or 24 h (LTM) later (Fig. 5A) was not different between blast-exposed and control groups. A discrimination index (Fig. 5C) did not show differences between blast-exposed and control groups in the STM testing but was lower in the blast-exposed group in the LTM testing (p < 0.05).

FIG. 5.

Novel object recognition (NOR) testing of cohort 2. Blast-exposed (n = 20) and control (n = 20) rats from cohort 2 were tested in a NOR task at four weeks after blast exposure (A,C,D). A subset (12 blast and 12 control) was retested at 41 weeks (B,E,F). Panels A and B show time spent exploring the objects (OB1 and OB2) during the training session as well as exploration of the previously presented familiar object (FO) compared with the novel object (NO) when presented 1 h (short-term memory, STM) or 24 h (long-term memory, LTM) later. Panels C–F show the discrimination index (C and E) and total time spend exploring the objects (D and F) during the indicated sessions. Error bars indicate the standard error of the mean. Asterisks indicate values significantly different from controls (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired t tests).

By contrast at 41 weeks, rats exposed to the BOP treatment no longer recognized the unfamiliar object as novel, and spent no more time exploring the NO than the FO in either the STM or LTM sessions (Fig. 4B). The lack of preference for the NO was reflected in a lower discrimination index (Fig. 5E) for the blast-exposed group in both the STM (p < 0.05) and LTM (p < 0.01) testing. Blast-exposed animals spent less total time exploring the objects in all three testing sessions at 42 weeks (Fig. 5F; p < 0.01). Blast-exposed rats in cohort 2 also exhibited increased freezing in the cued phase of a contextual/cued fear-learning task at 42 weeks (Fig. 6B; F _{1, 18} = 4.669, p = 0.044) but not at five weeks (Fig. 6A; F _{1, 36} = 0.654 p = 0.424) post-blast.

FIG. 6.

Testing of fear learning in cohort 2. Blast-exposed (n = 20) and control (n = 20) rats from cohort 2 were tested in a fear conditioning paradigm four weeks after blast exposure (A). A subset (12 blast and 12 control) was retested at 41 weeks (B). Results are shown for the training phase, contextual fear memory, which was tested 24 h after training, and cued fear memory, which was tested another 24 h later. A repeated-measures analysis of variance of freezing during the training sessions revealed a significant within-subjects effect of freezing for baseline versus post-tone/shock (F _{3.856, 142.689} = 62.642, p < 0.001 for 5 weeks; F _{6, 132} = 12.993, p < 0.001 for 42 weeks). The interaction effect of freezing*condition was significant at five weeks (F _{3.865, 142.689} = 3.514, p = 0.010) and at 42 weeks (F _{6. 132} = 3.359, p = 0.004). A test of between-subject effects revealed no significant group differences during the training sessions at five weeks (F _{1, 37} = 3.161, p = 0.084), but a significant effect at 42 weeks (F _{1, 22} = 5.898, p = 0.024) with the blast-exposed freezing more. There were no differences between blast and control groups in the contextual testing (F _{1, 38} = 0.297; p = 0.589 for 5 weeks; F _{1, 22} = 0.776, p = 0.388 for 42 weeks). In the cued phase testing, both early and late time points responded similarly with freezing after presentation of the tone (F _{3, 108} = 3.544, p = 0.017, freezing*condition F _{3, 108} = 1.252, p = 0.295 for 5 weeks; F _{7, 126} = 3.558, p = 0.002, freezing*condition F _{7, 126} = 0.477, p = 0.850 for 42 weeks). While there was no difference between blast-exposed and controls at 5 weeks (F _{1, 36} = 0.654, p = 0.424), at 41 weeks, blast-exposed exhibited increased freezing compared with controls (F _{1, 18} = 4.669, p = 0.044). Error bars indicate the standard error of the mean. Asterisks indicate values significantly different from controls at individual time points (*p < 0.05, **p < 0.01, unpaired t tests).

Testing of Cohort 3

Cohort 3 was only tested in the immediate post-blast period. In LD testing at four weeks after blast exposure, the blast-exposed group moved more (i.e., traversed a longer total distance; p < 0.01) but made a similar number of light center entries and explored the light side for a similar amount of time (Fig. 7A). Cohort 3 showed no abnormalities in EZM testing at five weeks (Fig. 7B).

FIG. 7.

Elevated zero maze (EZM) and light/dark (LD) testing of cohort 3. Blast-exposed (n = 26) and control (n = 13) rats were tested in a LD emergence task and an EZM and at four weeks or five weeks after blast exposure. In the LD task (A), total distance moved, latency to reach the light center, entries into the light center, as a well as time spent in the light center, total time spent on the light side, and total distance traveled on the light side are shown. For the EZM (B) total distance moved (Move Distance), time in motion (Move Time), latency to enter an open arm, open arm entries, time spent in the open arms as well as the latency to cross into the second open arm (cross arm latency) are shown. There were no statistically significant differences between the blast-exposed and control.

When tested in NOR at six weeks after blast exposure (Fig. 8), both controls and blast-exposed rats of cohort 3 spent more time exploring the NO compared with the previously presented FO when presented 1 h (STM; p < 0.05) or 24 h (LTM; p < 0.01) later (Fig. 8A). When a discrimination index was calculated, however, blast-exposed subjects spent relatively less time investigating the NO compared with the FO (Fig. 8B) in both the STM (p < 0.01) and LTM (p < 0.05) sessions. Blast-exposed rats also spent less total time exploring the two objects during the training (p < 0.05) and LTM (p < 0.05) sessions (Fig. 8C). Thus, while the blast-exposed rats of cohort 3 recognized the unfamiliar object as novel, they exhibited subtle changes in exploratory behavior at six weeks after blast exposure. Cohort 3 showed no abnormalities in contextual/cued fear learning at eight weeks after blast overpressure exposure (Fig. 8D).

FIG. 8.

Testing of novel object recognition (NOR) and fear learning in cohort 3. Blast-exposed (n = 26) and control (n = 13) rats were tested in NOR and a cued/contextual fear conditioning (FC) task at six weeks (NOR) or eight weeks (FC) after blast exposure. In the NOR task (A), time is shown spent exploring the objects (OB1 and OB2) during the training session as well as exploration of the previously presented familiar object (FO) compared with the novel object (NO) when presented 1 h (short-term memory, STM) or 24 h (long-term memory, LTM) later. Panels B and C show the discrimination index (B) and total time spend exploring the objects (C) during the indicated sessions. Results from contextual and cued fear conditioning testing (D) are shown for the training phase, contextual fear memory, which was tested 24 h after training, and cued fear memory, which was tested another 24 h later. A repeated-measures analysis of variance of freezing during the training sessions revealed a significant within-subjects effect of freezing for baseline versus post-tone/shock (F _{3.075, 101.472} = 26.205, p < 0.001) with an interaction effect of freezing*condition (F _{3.075, 101.472} = 3.175, p = 0.026). A test of between- subject effects, however, revealed no significant group differences during the training sessions (F _{1, 33} = 3.369, p = 0.075). There were no differences between blast and control groups in the contextual testing (F _{1, 37} = 0.514; p = 0.478). In the cued phase testing, both control and blast-exposed responded similarly with freezing after presentation of the tone (F _{2.647, 100.587} = 17.400, p < 0.001, freezing*condition F _{2.647, 100.587}, p = 0.684). There were no between-subjects effects (F _{1, 38} = 0.609, p = 0.440). Asterisks indicate values significantly different from controls (*p < 0.05, **p < 0.01, ****p < 0.0001, unpaired t tests).

Discussion

Blast-related mechanisms have long constituted an important element of combat-related TBI and were the major cause of TBI in Iraq and Afghanistan.^3,4,10,17 Estimates are that at least 10–20% of veterans returning from these conflicts had a TBI during deployment.^2–4,17 There are also concerns over the potential adverse consequences of subclinical blast exposures.^18–20 This form of blast exposure, now being referred to as military occupational blast exposure, is common for many service members in combat as well as non-combat settings.¹⁸ Whether occupational repetitive low-level blast exposures may cause later health problems is unknown but is a subject of current concern.^18,21

This study utilized an animal model that was designed to mimic a blast-related human mild TBI or subclinical blast exposure. Studies using this system established that exposures up to 74.5 kPa (equivalent to 10.8 psi), while representing a level of blast that is transmitted to brain,²² produce no gross neuropathological effects, and histological examination of the lung shows no hemorrhage or other pathology.¹⁵ Because blast-related TBI may involve a combination of injuries related to effects of the primary blast wave as well as damage from rotational/acceleration injury,^23,24 during the BOP exposures, head motion is restricted to minimize rotation/acceleration injury. The lack of evidence for coup/contrecoup injuries or brain tissue damage generally on histology supports the mild nature of the injury and the lack of significant rotation/acceleration injury.^11,15

The 74.5 kPa exposures also fall within a range that Song and colleagues²⁴ have identified as representing a mild blast injury in rodents. Because multiple blast exposures were common among veterans returning from Iraq and Afghanistan,²⁵ in most experiments (including the present), we have used a design in which rats received three 74.5-kPa exposures delivered one exposure per day on three consecutive days.

Rats subjected to this repetitive low-level blast exposure have chronic cognitive impairments and behavioral traits that are still present one year after blast exposure.¹⁴ These traits have been documented using a battery of well-established behavioral paradigms (EZM, LD emergence, fear conditioning, NOR, NO localization) that have established a chronic anxiety related phenotype as well as altered fear responses and impaired recognition memory.¹⁴ These traits model the enduring neurobehavioral syndromes that veterans often have after blast exposure.²⁵

While these traits are PTSD related, this is not a model of PTSD because the blast exposures occur under general anesthesia and no added psychological stressor is present. Rather, the model suggests that blast exposure alone without a psychological stressor can induce PTSD-related traits, a condition we have referred to as blast-induced “PTSD.”¹⁴ If rats in this model are subjected to a single predator scent exposure eight months after the last blast exposure, however, additional anxiety-related changes develop that persist,¹⁶ suggesting that blast exposure not only induces PTSD-related traits, but also sensitizes the brain to react abnormally to subsequent psychological stressors. These observations have implications for how the mental health disorders that follow blast exposure are conceptualized—in particular, the role of physical vs. psychological trauma in military veterans who carry a diagnosis of PTSD and have a history of blast-related TBI.²⁶

The major finding of this study is that this behavioral phenotype is not fully present initially but rather develops and evolves over months. Three cohorts of rats examined within the first eight weeks after blast exposure exhibited no behavioral phenotype or at most an incomplete form of the chronic phenotype that is seen later. The timing of testing for the three cohorts (Table 1) began with testing in the immediate blast period (cohort 1); when the behavioral phenotype was not seen in cohort 1, we moved to a slightly later time point for cohort 2. After cohort 2 failed to exhibit the phenotype, we moved to a still later time point for cohort 3. When cohorts 1 and 2 were retested 36 to 42 weeks after the initial blast exposure, both exhibited the expanded behavioral phenotype including anxiety as well as impaired NOR and altered cued fear learning.

A progressive behavioral phenotype could be reflected in the appearance of a trait that was not seen previously or in an increase across time in the magnitude of an outcome that was present previously. Table 2 and Fig. 9 summarize the behavioral testing in this study along with testing of four additional cohorts studied previously at 3–4 months or longer after blast exposure.^11–13 In measures of anxiety, only cohort 1 studied here showed features of anxiety at the early time point (1 week). This cohort exhibited features of anxiety in an EZM at one week after blast exposure. At one week after blast exposure, however, this cohort did not show the anxiety phenotype in a LD escape task. Yet, these identical subjects showed features of anxiety in both EZM and LD when retested at 36 weeks.

FIG. 9.

Timeline of experiments and appearance of behavioral phenotype. Cohorts 1–3 refer to those in the present studies. L/D, light/dark; NOR, novel object recognition; PTSD, post-traumatic stress disorder. Color image is available online.

Cohort 2 did not show anxiety in EZM or LD early at three weeks but showed anxiety in EZM when tested at 40 weeks. Cohort 3, which was only tested in the acute phase (4–5 weeks), showed no anxiety in EZM or LD. By contrast, four additional cohorts tested in previous studies at three months or later all showed an anxiety phenotype in both EZM and LD (Table 2). Overall, these data demonstrate that anxiety in blast-exposed rats evolves, becoming more apparent at later time points. This is broadly consistent with studies in veterans with blast-related mTBI who exhibit, among other neurological and neuropsychological symptoms, self-endorsed anxiety that increases over time.^9,27

The NOR testing revealed the evolving nature of the cognitive deficit. Three cohorts tested within six weeks of blast exposure all showed a preference for the NO over the FO in both testing at 1 h (STM) and 24 h (LTM) suggesting intact memory for object recognition. Two of the three cohorts, however, were impaired when a discrimination index was calculated, which reflects the preference for exploring the NO compared with the FO. A reduced discrimination index can be viewed as either representing a subtle memory deficit or an alteration in novelty preference.²⁸ Whichever interpretation is applied, cohorts 2 and 3 exhibited a lowered discrimination index in STM (cohort 2) or STM and LTM (cohort 3).

Total time spent exploring the objects was also decreased in two of the three cohorts (1 and 3). Thus, all three cohorts exhibited at least subtle evidence of altered NOR behavior when tested within six weeks of blast exposure. By contrast, when cohort 2 was retested at 41 weeks, the subjects had progressed in deficits in this task so that no preference for the NO versus the FO could be detected. This group also exhibited an altered discrimination index in both STM and LTM and less exploratory behavior, a phenotype similar to that observed in two additional cohorts of blast-exposed rats studied previously at four months after blast exposure.¹²

The delayed appearance of changes in fear learning were the most striking of the blast-induced behavioral phenotypes we explored. When examined five months or longer post-exposure, blast-exposed rats have typically exhibited normal contextual fear memory but shown exaggerated freezing to a tone presented in the cued phase.^11–13 All three cohorts studied showed no abnormalities in fear learning when tested at eight weeks or less. Of the two cohorts that were retested later, however, both showed increased cued phase freezing at 36 or 42 weeks after blast exposure. An altered cued phase freezing was not observed in any of the three cohorts examined at eight weeks or less after blast exposure, and when current and previously published work^11–13 are examined in aggregate, six of six cohorts showed increased freezing at four months or more.

Collectively, when viewed in the context of previously published work in four other cohorts of rats (Table 2),^11–13 these results suggest an altered behavioral phenotype that is incomplete at fewer than eight weeks after blast but consistently present after 3–4 months or longer (Fig. 9). The domains differed in that features of anxiety were inconsistently present early but were a consistent feature at later time points.

The NOR testing revealed a cognitive domain abnormality that progressed from relatively subtle deficits at early post-blast evaluation times to clear impairments by later evaluation times. By contrast, altered cued fear responses likely represent features that are truly delayed in their appearance, not being present early but emerging as a consistent feature that manifests with passing time after exposure to the traumatic event.

Many examples in the literature describe specific behavioral phenotypes that mimic human disease states. Few of these describe progressive features, however. Transgenic mice have been used successfully to model progressive behavioral and motor disabilities associated with Alzheimer's disease or alterations in the dopaminergic system.^29–32 Recently, transgenic rats have been developed that recapitulate the pathological hallmarks of Alzheimer's disease showing an age-dependent buildup of amyloid-β that correlated with worsening behavioral performance in the open field as well as progressive deficits in performance on learning and memory tasks.³³ TBI has been reported to enhance tau-related pathology after controlled cortical impact injury in transgenic mice harboring Alzheimer's disease-related mutations.^34,35

In wild type rats, one study has reported a progressive loss of cortical neurons over a six-month period accompanied by appearance of increasing numbers of phospho-tau immunoreactive neurons after a fluid percussion injury, but in that study, behavior was not assessed.³⁶ To our knowledge, the work presented herein is the first to describe a progressive behavioral phenotype in an animal model of blast injury.

The delayed onset of the behavioral phenotype has implications for the nature of post-blast neurobehavioral syndromes in military veterans as well as its laboratory modeling. A TBI is generally considered a monophasic illness in which a maximum deficit appears shortly after injury with recovery occurring to variable degrees. A TBI also confers a risk for the later development of neurodegenerative diseases, however.² In the model studied here, there seems to be a critical period (Fig. 9) between about eight weeks and 3–4 months after blast exposure, during which the full behavioral phenotype develops.

Studies in veterans returning from Iraq and Afghanistan have found that up to 40% of those who experience an mTBI will have three or more post-concussive symptoms at three months after injury.^37–40 Besides static symptoms, new symptoms may also develop.^8,9 Of note, Mac Donald and colleagues⁹ conducted a prospective longitudinal study of 50 active-duty US military personnel who had concussive blast injury in Afghanistan and compared them with combat-deployed controls. Between one and five years of follow-up, worsening of symptoms and a decline in overall global functioning was observed in more than 70% of the blast-injured service members, a decline nearly completely driven by worsening PTSD and depression.⁹

The degree to which the worsening observed by Mac Donald and coworkers⁹ is driven by blast-related mechanisms versus purely psychological factors is unclear. Altered tau processing has been observed frequently in experimental animal models of blast injury,^41–65 and the rat model under study here also exhibits a chronic tauopathy.⁶⁶ Along with tau-related pathology, a distinctive type of interface astroglial scarring at the gray/white matter interface has been described in human post-mortem material, a lesion that appears to be specific to blast injury.⁶⁷

Vascular and inflammatory factors have been widely implicated as contributing to the pathophysiology of blast injury in experimental animals.^{43,54,68–70} Vascular effects have been particularly prominent in the model under study here,^71–73 although chronic inflammation has not been a prominent feature.⁷⁴ Which (if any) of these blast-related mechanisms and structural pathologies can form a basis for the delayed appearance of the behavioral phenotype is unclear.

Conclusion

Through a series of experiments conducted with seven cohorts of rats, we have confirmed that repetitive blast exposures induce an anxiety-related behavior and the development of chronic PTSD-like behavioral traits. Future experiments will be needed to describe the biochemical and anatomic changes that may underlie the mechanistic basis for the observed phenotype.

Whatever its basis, however, the animal studies reported here suggest the existence of a progressive blast-induced behavioral phenotype. The delayed onset of the behavioral phenotype has implications for the nature of post-blast related neurobehavioral syndromes in humans and for their laboratory modeling in animals. Evidence from both humans and animals suggest that in military veterans, these syndromes may have a neurodegenerative basis.

Authors' Contributions

GPG, RDG, MAG, RA, UK, PRH, DGC, SG, STA, and GE designed research. STA designed the blast exposures. GPG, GMP, AOP, DP, RA, and UK performed research. GPG and GE analyzed data. GPG, RDG, MAG, PRH, DGC, SG, STA, and GE participated in the drafting and revising of the manuscript. All authors reviewed and edited the manuscript.

Funding Information

This work was supported by the Department of Veterans Affairs, Veterans Health Administration, Rehabilitation Research and Development Service Awards 1I01RX000996 (GE), 1I01RX002660 (GE), 1I21RX003019 (GE), 1I01RX000684 (SG), and 1I01RX002333 (SG), the Department of Veterans Affairs Office of Research and Development Medical Research Service 1I01BX004067 (GE) and 1I01BX002311 (DC), Department of Defense work unit number 0000B999.0000.000.A1503 (STA), the Alzheimer's Drug Discovery Foundation (SG) and by NIA P50 AG005138 and P30 AG066514 both to Mary Sano (SG, PRH).

Author Disclosure Statement

No competing financial interests exist.