A Chronic Longitudinal Characterization of Neurobehavioral and Neuropathological Cognitive Impairment in a Mouse Model of Gulf War Agent Exposure

Abstract

Gulf War Illness (GWI) is a chronic multisymptom illness with a central nervous system component that includes memory impairment as well as neurological and musculoskeletal deficits. Previous studies have shown that in the First Persian Gulf War conflict (1990-1991) exposure to Gulf War (GW) agents, such as pyridostigmine bromide (PB) and permethrin (PER), were key contributors to the etiology of GWI. For this study, we used our previously established mouse model of GW agent exposure (10 days PB+PER) and undertook an extensive lifelong neurobehavioral characterization of the mice from 11 days to 22.5 months post exposure in order to address the persistence and chronicity of effects suffered by the current GWI patient population, 24 years post-exposure. Mice were evaluated using a battery of neurobehavioral testing paradigms, including Open Field Test (OFT), Elevated Plus Maze (EPM), Three Chamber Testing, Radial Arm Water Maze (RAWM), and Barnes Maze (BM) Test. We also carried out neuropathological analyses at 22.5 months post exposure to GW agents after the final behavioral testing. Our results demonstrate that PB+PER exposed mice exhibit neurobehavioral deficits beginning at the 13 months post exposure time point and continuing trends through the 22.5 month post exposure time point. Furthermore, neuropathological changes, including an increase in GFAP staining in the cerebral cortices of exposed mice, were noted 22.5 months post exposure. Thus, the persistent neuroinflammation evident in our model presents a platform with which to identify novel biological pathways, correlating with emergent outcomes that may be amenable to therapeutic targeting. Furthermore, in this work we confirmed our previous findings that GW agent exposure causes neuropathological changes, and have presented novel data which demonstrate increased disinhibition, and lack of social preference in PB+PER exposed mice at 13 months after exposure. We also extended upon our previous work to cover the lifespan of the laboratory mouse using a battery of neurobehavioral techniques.

Keywords: Gulf War; mouse model; neurobehavior; neuropathology; permethrin (PER); pyridostigmine bromide (PB).

Figure 1

No locomotor impairment and anxiety-like behaviors were observed at 11 days-post acute exposure to PB+PER. (A) Perimeter Duration (s), (B) Cumulative Distance Traveled Per Time Interval (cm), (C) Immobile Duration (s), (D) Mobile Duration (s), (E) Inner Circle Duration (s), and (F) Inner Circle Frequency (#) were examined for exposed and control mice during the Open Field Test.

Figure 2

PB+PER exposed mice demonstrated disinhibition, 13 months post exposure, as revealed by Elevated Plus Maze testing. EPM testing revealed that PB+PER mice spent significantly less time in the closed arms (A), and a significantly larger proportion of time in the open arms (B) as compared to their controls. No differences were noted between the two groups when examining (C) number of visits to the Closed Arms (#). PB+PER mice visited the open arms (#) more times as compared to the control mice (D). In addition, exposed mice spent more time in the center as compared to the control mice (E). Therefore, the exposed mice exhibited reduced anxiety-like behaviors. No differences were noted between the two groups when examining the number of visits to the center area (the junction between the closed and the open arms) (F). Furthermore, EPM testing revealed that there were no differences between exposed and control mice when examining (G) Cumulative distance moved (cm) and (H) Velocity (cm/s). ^*p < 0.05; ^**p < 0.01; ^***p < 0.005.

Figure 3

PB+PER exposed mice showed a lack of social preference at 13 months post exposure to GW agents. When examining (A) Sociability, the number of visits to either the empty cage or stranger 1, the control mice exhibited a slight trend toward an increased number of visits to the stranger 1 mouse vs. the empty cage, although it did not reach statistical significance, it did exemplify normal behaviors, demonstrating preference for the novel mouse (stranger 1). However, the PB+PER exposed mice showed a lack of social preference, by visiting the empty cage and the stranger 1 an equal number of times. When examining (B) Social Interaction, the time (s) the animals spent with either the empty cage or stranger 1, the control mice spent significantly more time with stranger 1 as compared to the empty cage. However, the PB+PER exposed mice showed a lack of social preference spending an equal amount of time with either the empty cage or the stranger 1. When examining (C) Social Novelty, the number of visits to either stranger 1 (familiar mouse) or stranger 2 (novel mouse), the control mice did not indicate a strong preference for one or the other, likewise, no differences in social novelty were evident when examining PB+PER exposed. In addition, when examining (D) Social Memory, as indicated by the time spent with either stranger 1 or stranger 2, the control mice appeared to spent slightly more time with the stranger 2, however, those differences did not reach statistical significance, the PB+PER exposed mice did not show a strong preference for the novel mouse (stranger 2) over the familiar mouse (stranger 1). When examining (E) Social Interaction—Proximity (s), the control mice showed a statistically significant preference to spent more time with stranger 1 as compared to the empty cage, demonstrating normal healthy social behaviors. However, the PB+PER mice a lack of social preference, demonstrated by a similar amount of time spent between stranger 1 and the empty cage. When examining (F) Social Memory—Proximity (s), the control mice showed a trend to spend more time with stranger 2 as compared to stranger 1, indicative of normal healthy social behaviors, although those differences did not reach statistical significance. However, the PB+PER mice once again showed a lack of social preference, demonstrated by an similar amount of time spent between stranger 1 and stranger 2. ^*p < 0.05.

Figure 4

Acute exercise may improve working and reference memory in exposed mice during RAWM acquisition testing, 13 months post exposure. When examining (A) the Goal Arm Frequency (#), the number of times the mice entered the goal arm, and (B) the duration at the goal arm (s), no overall differences were observed between control and PB+PER mice. When examining (C) the cumulative distance traveled, a difference was noted on Day 4 between control and PB+PER mice, where PB+PER mice outperformed the control mice. No differences were noted when examining their (D) velocities by day. In addition, when examining (E) the number of working memory errors made by either the exposed mice or the control mice over the 5 day testing period at 13 months post exposure, differences were observed on day 2, where PB+PER mice made more errors as compared to controls. However, PB+PER exposed mice seemed to improve (make less errors) as they continued to advance through the acquisition days (E,F). When examining (F) the number of reference memory errors, significant differences were observed between the two groups on days 4 and 5, where PB+PER mice performed better/on par with their control counterparts. Overall these data suggest that learning abilities are intact in PB+PER exposed mice, and that acute exercise may improve working and reference memory in PB+PER exposed mice. ^*p < 0.05; ^**p < 0.01.

Figure 5

No overall behavioral differences were observed in exposed mice during BM testing at 22.5 months post-exposure to PB+PER. Control and exposed mice behaved similarly when we examined (A) cumulative distance traveled to the target hole, (B) escape latency, and (C) velocity, over a 4-day period. In addition, when the mice were examined 24-h after the last acquisition trial, by a single probe (day 5), the PB+PER exposed mice appeared to travel a longer (D) cumulative distance to reach the TH as compared to controls. When examining the (E) primary error rate (#), PB+PER exposed mice appeared to make more mistakes as compared to controls. Although there were apparent trends, these differences failed to reach statistical significance.

Figure 6

No anxiety-like behaviors were observed in the PB+PER exposed mice, 22.5 months post exposure, as revealed by EPM testing. EPM testing revealed that PB+PER exposed mice spent a similar amount of time in (A) the closed arms, and a similar amount of time in the open arms (B) as compared to their controls. No differences were noted between the two groups when examining (C) number of visits to the closed arms (#) and (D) the number of visits to the open arms. No differences were noted between the two groups when examining (E) cumulative distance traveled (cm) and (F) velocity (cm/s).

Figure 7

Three Chamber testing revealed that PB+PER exposed mice exhibit normal sociability and social interaction behaviors, 22.5 months post exposure to GW agents. When examining (A) Sociability, the number of visits to either the empty cage or stranger 1, the control mice exhibited a slight trend toward an increased number of visits to the stranger 1 mouse as compared to the empty cage. In addition, PB+PER exposed mice demonstrated preference for the novel mouse (stranger 1) over the empty cage. When examining (B) Social Interaction, the time (s) the animals spent with either the empty cage or stranger 1, the control mice appeared to spend more time with stranger 1 as compared to the empty cage, although those differences did not reach statistical significance. Similarly, the PB+PER exposed mice continued to demonstrate a clear preference for the novel mouse (stranger 1) as compared to the empty cage. When examining (C) Social Novelty, the number of visits to either stranger 1 (familiar mouse) or stranger 2 (novel mouse), the control mice did not indicate a strong preference for one or the other, likewise, no differences in social novelty were evident when examining PB+PER exposed mice. In addition, when examining (D) Social Memory, as indicated by the time spent with either stranger 1 or stranger 2, the control mice did not demonstrate a strong preference to spend more time stranger 2 over stranger 1. Likewise, the PB+PER exposed mice did not show a strong preference for the novel mouse (stranger 2) over the familiar mouse (stranger 1). When examining (E) Social Interaction—Proximity (s), the control mice showed a trend indicating increased preference to spent more time with stranger 1 as compared to the empty cage, demonstrating normal healthy social behaviors. Likewise, the PB+PER mice showed a statistically significant preference to spent more time with stranger 1 as compared to the empty cage. When examining (F) Social Memory—Proximity (s), both the control and the PB+PER exposed mice did not show a strong preference for the novel mouse (stranger 2) over the familiar mouse (stranger 1).

Figure 8

PB+PER exposure altered astrocytic activation in the cerebral cortices of mice, 22.5 months post exposure. PB+PER exposure did not significantly alter astrocytic activation in the hippocampi of exposed mice (B,D) compared to controls (A,C) at 22.5 months post exposure. PB+PER exposure significantly increased astrocytic activation in the cerebral cortices (F,H) of exposed mice, as compared to controls (E,G) at 22.5 months post exposure. Representative images used 10X (A,B,E,F), and 40X (C,D,G,H) objectives (scale bars represent 100 and 20 μm, respectively). Histograms depict the quantification of the GFAP stain in the hippocampi and cerebral cortices from control and exposed mice, as % Area per microscopic field. ^*p < 0.05.

Figure 9

PB+PER exposure did not alter microglial levels in hippocampi and cortices of mice, 22.5 months post-exposure. The IBA-1 stain showed no differences between exposed (B,D) and control (A,C) mice in the hippocampi and the cerebral cortices of exposed (F,H) and control (E,G) animals. Insets in (G) and (H) represent magnified images of ramified and resting microglia in both control and PB+PER exposed mice, respectively, at 22.5 months post exposure to GW agents. Representative images used 10X (A,B,E,F), and 40X (C,D,G,H) objectives, scale bars represent 100 and 20 μm, respectively. Histograms depict the quantification of the IBA-1 stain in the hippocampi and cerebral cortices from control and exposed mice as % Area per microscopic field.

Figure 10

No differences were observed in neurogenesis in mice, 22.5 months post exposure. PB+PER exposure did not alter neurogenesis in the dentate gyrus (DG) region in exposed animals at 22.5 months post exposure (B) as compared to controls (A). The depicted micrographs were taken using 20X (A,B), scale bars represent 50 μm. Histograms depict the manual quantification of the doublecortin (DCX) stain in the dentate gyri, as total number of cells within the DG.

Figure 11

No differences were observed in SYP staining in cerebral cortices and the CA3 regions of exposed mice, ~22.5 months post-exposure. No differences were observed in SYP staining in the CA3 region of exposed mice (B) vs. controls (A). No differences were observed in SYP staining in the cerebral cortices of exposed mice (D) vs. controls (C) (Student t-test, p = 0.84). Representative images were taken at 40X magnification (scale bars represent 20 μm). Inset depicts positive SYP staining showing dark brown pre-synaptic vesicles stained within the cell soma (see inset in C). Histograms depict the quantification of the SYP stain in the hippocampi and cerebral cortices, as % Area per microscopic field.

Figure 12

No alterations in cell morphology detected 22.5 months post exposure to PB+PER. Nissl staining revealed no gross morphological changes in nuclei/cell body of pyramidal neurons post exposure to PB+PER (A–D). Similarly, the majority of cells in the hippocampi and cerebral cortices of PB+PER exposed mice (F,H) as compared to controls (E,G) were free from damaged and swollen axons and degenerated neurons when compared to a positive control (PSAPP mouse model of Alzheimer's Disease; see inset in E). Representative images were taken at 40X magnification (scale bar represents 20 μm).