Evaluation of delayed LNFPIII treatment initiation protocol on improving long-term behavioral and neuroinflammatory pathology in a mouse model of Gulf War Illness

Abstract

Chemical overexposures and war-related stress during the 1990-1991 Gulf War (GW) are implicated in the persisting pathological symptoms that many GW veterans continue to endure. These symptoms culminate into a disease known as Gulf War Illness (GWI) and affect about a third of the GW veteran population. Currently, comprehensive effective GWI treatment options are unavailable. Here, an established GWI mouse model was utilized to explore the (1) long-term behavioral and neuroinflammatory effects of deployment-related GWI chemicals exposure and (2) ability of the immunotherapeutic lacto-N-fucopentaose III (LNFPIII) to improve deficits when given months after the end of exposure. Male C57BL6/J mice (8-9 weeks old) were administered pyridostigmine bromide (PB) and DEET for 14 days along with corticosterone (CORT; latter 7 days) to emulate wartime stress. On day 15, a single injection of the nerve agent surrogate diisopropylfluorophosphate (DFP) was given. LNFPIII treatment began 7 months post GWI chemicals exposure and continued until study completion. A battery of behavioral tests for assessment of cognition/memory, mood, and motor function in rodents was performed beginning 8 months after exposure termination and was then followed by immunohistochemcal evaluation of neuroinflammation and neurogenesis. Within tests of motor function, prior GWI chemical exposure led to hyperactivity, impaired sensorimotor function, and altered gait. LNFPIII attenuated these motor-related deficits and improved overall grip strength. GWI mice also exhibited more anxiety-like behavior that was reduced by LNFPIII; this was test-specific. Short-term, but not long-term memory, was impaired by prior GWI exposure; LNFPIII improved this measure. In the brains of GWI mice, but not in mice treated with LNFPIII, glial activation was increased. Overall, it appears that months after exposure to GWI chemicals, behavioral deficits and neuroinflammation are present. Many of these deficits were attenuated by LNFPIII when treatment began long after GWI chemical exposure termination, highlighting its therapeutic potential for veterans with GWI.

Keywords: Behavior; Gulf war illness; LNFPIII; Neurogenesis; Neuroinflammation; Pesticides.

Fig. 1

Experimental and behavioral timelines. (A) Depicts the experimental timeline in which mice received GWI-related chemicals: pyridostigmine bromide (PB) and DEET (days 1–14, SC), corticosterone (days 8–14, via drinking water), and diisopropylfluorophosphate (DFP, day 15, IP). Lacto-N-fucopentaose III (LNFPIII, SC) treatment began 7 months after GWI exposure. (B) Is a detailed timeline of behavioral tests that were performed during months 8–10.

Fig. 2

Monthly Weights. Monthly weights were monitored and are presented for the start of study (0), post GWI exposure (0.5), start of LNFPIII treatment (6.5), prior to Radial Arm Maze (RAM) food restriction (7), during RAM food restriction (9), and end of study (11). Data are presented as mean ± SEM (n = 12–16/group). ^$$$ indicates significant main effect of time, p < 0.001. ^a indicates p < 0.05 from Vehicle.

Fig. 3

Long-term motor effects of prior GWI exposure and delayed LNFPIII treatment. Several motor effects of prior GWI exposure and LNFPIII treatment were evaluated by nest building (A); grip strength (B); sticker contact (C) in the sticker removal test; distance traveled (D) in the open field; descent time (E) in the pole test; and hindlimb base width (F), stride length (G), and interstep distance (H) in the gait test. Data are presented as mean ± SEM (n = 12–16/group). * and ** indicate significant main effect of GWI treatment, p < 0.05 and 0.01, respectively. ^$$$ indicates significant main effect of time, p < 0.001. ^# indicates significant main effect of LNFPIII treatment, p < 0.05. ^a and ^b indicate p < 0.05 from Vehicle-Vehicle and GWI-Vehicle, respectively.

Fig. 4

Long-term mood effects of prior GWI exposure and delayed LNFPIII treatment. Mood disturbances of prior GWI exposure and LNFPIII treatment were evaluated by several tests for anxiety-like and depressive-like behaviors: corner entries (A) in the open field; number of marbles buried (B) in the marble burying test; open arm time (C), latency to enter closed-arm (D), and stretch attends while in the closed arm (E) of the elevated zero maze; immobile and mobile time (F) and climbing attempts (G) in the swim test; average daily fluid intake (H) in the sucrose preference test. Data are presented as mean ± SEM (n = 12–16/group). *** indicates significant main effect of treatment, p < 0.001. ^$$$ indicates significant main effect of drink in the SP test, p < 0.001. ^ˆ indicates p < 0.10. ^a and ^aˆ indicate p < 0.05 or 0.10, respectively, from Vehicle-Vehicle. ^b and ^bˆ indicate p < 0.05 or 0.10, respectively, from GWI-Vehicle.

Fig. 5

Long-term cognition and memory effects of prior GWI exposure and delayed LNFPIII treatment. Cognition and memory were evaluated by several tests: novel preference index (NPI) (A) and total approaches % (B) in the novel object recognition test; acquisition phase time to target hole (TH) (C) and probe trial TH entries (D) in the Barnes Maze; and total errors in the Radial Arm Maze (RAM) foraging task (E) and Win-shift task (F). Additionally, improvement percentages from days 1–3 were calculated for the RAM foraging task (E). Data are presented as mean ± SEM (n = 12–16/group). * and ** indicate significance, p < 0.05 and 0.01, respectively. ^$$$ indicates significant main effect of time, p < 0.001. ^ˆ indicates p < 0.10. ^a and ^aˆ indicate p < 0.05 or 0.10, respectively, from Vehicle-Vehicle. ^b indicates p < 0.05 from GWI-Vehicle. ^c indicates p < 0.05 from days 1–3 in the RAM.

Fig. 6

GFAP and IBA-1 immunoreactivity in the hilus region of the hippocampus 11 months post-GWI exposure and delayed LNFPIII treatment. Immunoreactivity of (A) GFAP+ and (B) GFAP+/ASC + astrocytes and (C) IBA-1+ and (D) ASC+/IBA-1+ microglia were analyzed in ImageJ. Representative images taken at 40x are shown in panel (E). Data are presented as mean ± SEM, n = 4–6 mice per group. ^a and ^b indicate p ≤ 0.05 for Vehicle-Vehicle vs GWI-Vehicle and GWI-Vehicle vs GWI-LNFPIII, respectively. ^bˆ indicates p ≤ 0.10 for GWI-Vehicle vs GWI-LNFPIII.

Fig. 7

BrDU and DCX immunoreactivity in the hippocampus 11 months post-GWI exposure and delayed LNFPIII treatment. The number of (A) BrDU+/DCX+ and (B) BrDU + cells were counted in the hippocampus. Representative images taken at 40x are shown in panel (C). Data are presented as mean ± SEM, n = 4–6 mice per group. ^aˆ indicates p ≤ 0.10 from Vehicle-Vehicle.