Mood and memory deficits in a model of Gulf War illness are linked with reduced neurogenesis, partial neuron loss, and mild inflammation in the hippocampus

Abstract

Impairments in mood and cognitive function are the key brain abnormalities observed in Gulf war illness (GWI), a chronic multisymptom health problem afflicting ∼25% of veterans who served in the Persian Gulf War-1. Although the precise cause of GWI is still unknown, combined exposure to a nerve gas prophylaxis drug pyridostigmine bromide (PB) and pesticides DEET and permethrin during the war has been proposed as one of the foremost causes of GWI. We investigated the effect of 4 weeks of exposure to Gulf war illness-related (GWIR) chemicals in the absence or presence of mild stress on mood and cognitive function, dentate gyrus neurogenesis, and neurons, microglia, and astrocytes in the hippocampus. Combined exposure to low doses of GWIR chemicals PB, DEET, and permethrin induced depressive- and anxiety-like behavior and spatial learning and memory dysfunction. Application of mild stress in the period of exposure to chemicals exacerbated the extent of mood and cognitive dysfunction. Furthermore, these behavioral impairments were associated with reduced hippocampal volume and multiple cellular alterations such as chronic reductions in neural stem cell activity and neurogenesis, partial loss of principal neurons, and mild inflammation comprising sporadic occurrence of activated microglia and significant hypertrophy of astrocytes. The results show the first evidence of an association between mood and cognitive dysfunction and hippocampal pathology epitomized by decreased neurogenesis, partial loss of principal neurons, and mild inflammation in a model of GWI. Hence, treatment strategies that are efficacious for enhancing neurogenesis and suppressing inflammation may be helpful for alleviation of mood and cognitive dysfunction observed in GWI.

Figure 1

Exposure of adult rats to Gulf war illness-related chemicals results in increased depressive- and anxiety-like behavior, and addition of mild stress during the period of exposure exacerbates the detrimental effects of chemicals on mood function. (a1) A rat undergoing forced swim test (FST) is shown. (a2) The bar chart demonstrates that rats exposed to chemicals (CHEM group) and chemicals and stress (CHEM+STRESS group) spend greater amount of time in floating (a measure of depressive-like behavior) in FST in comparison with vehicle-treated control rats (VEH group). (b1, b2) Bar charts show that exposure of rats to CHEM or CHEM+STRESS results in considerably fewer entries into open arms and reduced open arm dwell times (indices of anxiety-like behavior) in an elevated plus maze (EPM) test in comparison with rats exposed to VEH. *p<0.05; **p<0.01; ***p<0.001. NS, not significant.

Figure 2

Exposure of adult rats to Gulf war illness-related chemicals results in spatial learning impairments. (a1) The bar chart compares average swim speeds of animals treated with vehicle (VEH group), chemicals (CHEM group), and chemicals and stress (CHEM+STRESS group) during spatial learning. (a2), Learning curves (using swim path length values to reach the hidden platform) over seven daily sessions in a water maze test (WMT) among the three groups are compared. Repeated measures ANOVA reveal that animals in all three groups show ability for spatial learning (p<0.0001). However, two-way RM-ANOVA with Bonferroni multiple comparisons post hoc test revealed that swim path lengths were significantly longer in CHEM and CHEM+STRESS groups than the VEH group in the third learning session (p<0.05–0.01). (a3) Inferior swim path efficiency values for animals exposed to CHEM or CHEM+STRESS during the last five sessions of learning in comparison with the VEH-treated group is illustrated. (a4) The swim path lengths in the thigmotactic zone (TZ) are comparable among the three groups. Exposure of adult rats to Gulf war illness-related chemicals results in spatial memory impairments. The cartoon in (b1) depicts the various quadrants of the water maze pool, the target area (TA, denoting the actual position of the platform), the platform area (PA, denoting an annulus covering the position of the platform as well as immediate surrounding regions), and the platform quadrant (PQ, denoting the quadrant of the pool where the platform was located) during learning sessions. NE-Q, northeast quadrant; NW-Q, northwest quadrant; SE-Q, southeast quadrant; SW-Q, southwest quadrant. (b2–e3) The results of a 30-s probe (memory retrieval) test conducted 24 h after the last learning session. (b2–b4) The swim path lengths and (c1–c3) dwell times of rats in the PQ vis-à-vis the other three quadrants are compared in VEH-treated (b2, c1), CHEM-treated (b3, c2), or CHEM+STRESS-treated (b4, c3) groups. Note that rats treated with VEH display longer swim path length and greater dwell time in the PQ vis-à-vis the other quadrants, implying successful memory retrieval. However, rats treated with CHEM or CHEM+STRESS display similar swim path lengths and dwell times in all quadrants, suggesting impairments in spatial memory retrieval function. (d1–e3) Bar charts compare swim path length values in PQ (d1), dwell time in PQ (d2), and swim path lengths for first entry to TA (e1), PA (e2), and PQ (e3) among the three groups. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. NS, not significant.

Figure 3

Exposure of adult rats to Gulf war illness-related chemicals reduces hippocampal neurogenesis in the immediate postexposure period, and the addition of mild stress during the period of chemical exposure exacerbates the detrimental effects on neurogenesis. (a1–c1) Examples of newly born cells labeled through 5′-bromodeoxyuridine (BrdU) injections (indicated by arrows) in the subgranular zone–granule cell layer (SGZ-GCL) of animals treated with vehicle (VEH, a1), chemicals (CHEM, b1), or chemicals and stress (CHEM+STRESS, c1). (a2, b2, c2) Magnified views of some of the BrdU+ newly born cells from (a1), (b1), and (c1), respectively, are shown. (d) The bar chart shows that the numbers of BrdU+ newly born cells in the SGZ-GCL are reduced in animals treated with CHEM or CHEM+STRESS in comparison with VEH-treated control animals. (e1–e4) Examples of BrdU+ newly born cells that differentiate into doublecortin (DCX)-positive neurons are shown. (f) The bar chart shows that percentages of BrdU+ newly born cells that differentiate into DCX+ neurons are comparable among the three groups. (g) The bar chart shows that net neurogenesis is decreased in animals treated with CHEM or CHEM+STRESS in comparison with VEH-treated control animals. (h1–j1) The distribution of DCX+ newly born neurons between VEH-treated (h1), CHEM-treated (i1), and CHEM+STRESS-treated (j1) groups is compared. (h2, i2, j2) Magnified views of some DCX+ newly born neurons from (h1), (i1), and (j1), respectively, are shown. (k) The bar chart shows that the total number of DCX+ newly born neurons is decreased in animals treated with CHEM or CHEM+STRESS in comparison with VEH-treated animals. DH, dentate hilus; ML, molecular layer. Scale bar: a1, b1, c1, h1, i1, j1=200 μm; a2, b2, c2, h2, i2, j2=50 μm; e1–e4=5 μm. ***p<0.001.

Figure 4

Exposure of adult rats to Gulf war illness-related chemicals reduces hippocampal neurogenesis even at 4 months after the exposure and the addition of mild stress during the period of exposure exacerbates the detrimental effects of chemicals on neurogenesis. (a1–c1) Examples of newly born cells (arrows) labeled through 5′-bromodeoxyuridine (BrdU) injections (indicated by arrows) in the subgranular zone–granule cell layer (SGZ-GCL) of animals treated with vehicle (VEH, a1), chemicals (CHEM, b1), or chemicals and stress (CHEM+STRESS, c1). (a2, b2, c2) Magnified views of boxed regions from (a1), (b1), and (c1), respectively, are shown. (d1) An orthogonal view of a BrdU+ newly born cell that differentiated into neuron-specific nuclear antigen (NeuN)-positive mature neuron. (d2) A three-dimensional view of the newly born neuron from (d1). (D3) An orthogonal view of a BrdU+ newly born cell that did not differentiate into NeuN+ neuron. (e1–e3) Bar charts compare the number of newly added BrdU+ cells to the SGZ-GCL (e1), percentage of BrdU+ newly added cells that differentiated into mature NeuN+ neurons (e2), and net hippocampal neurogenesis (e3) between the VEH-, CHEM-, and CHEM+STRESS-treated groups. Note that although the percentages of BrdU+ newly born cells that differentiate into NeuN+ neurons are comparable among the three groups (e2), the extent of addition of newly born cells (e1) and net neurogenesis (e3) are reduced in animals treated with CHEM or CHEM+STRESS in comparison with VEH-treated control animals. (f1–h1) The distribution of DCX+ newly born neurons between VEH-treated (h1), CHEM-treated (i1), and CHEM+STRESS-treated (j1) groups is compared. (f2, g2, h2) Magnified views of some DCX+ newly born neurons from (f1), (g1), and (h1), respectively, are shown. (i) The bar chart shows that the total number of DCX+ newly born neurons is decreased in animals treated with CHEM or CHEM+STRESS in comparison with VEH-treated animals. Scale bar: a1, b1, c1, f1, g1, h1=200 μm; a2, b2, c2, f2, g2, h2=50 μm; d1–d3=10 μm. *p<0.05; **p<0.01; ***p<0.001.

Figure 5

Exposure of adult rats to Gulf war illness-related chemicals or chemicals and stress does not kill neural stem cells (NSCs) but induce alterations in NSC proliferative behavior. (a1–a3) The distribution of Sox-2+ cells in the subgranular zone–granule cell layer (SGZ-GCL) of animals treated with vehicle (VEH, a1), chemicals (CHEM, a2), or chemicals and stress (CHEM+STRESS, a3) is shown. (b1–b3) Examples of Sox-2+ cells that lack S-100β (putative NSCs, indicated by arrows) and Sox-2+ cells that express S-100β (mature astrocytes, indicated by curved arrows) in the SGZ. (c1, c2) Bar charts demonstrate that numbers of both Sox-2+ cells (c1) and Sox-2+ cells that lack S-100β (putative NSCs; c2) are comparable among the three groups in the SGZ. (d1–e3) Examples of Sox-2+ cells expressing Ki-67 (ie, proliferating NSCs, indicated by arrows) in the SGZ. Curved arrows show Sox-2+ cells that lack Ki-67 expression. (f1, f2) Bar charts show that animals treated with CHEM or CHEM+STRESS display reduced percentages as well as numbers of Sox-2+ cells expressing Ki-67 in comparison with VEH-treated control animals. Scale bar: A1–A3=25 μm; b1–b3, d1–d3, e1–e3=20 μm. *p<0.05; ***p<0.001.

Figure 6

Exposure of adult rats to Gulf war illness-related chemicals or chemicals and stress induces partial neuron loss in the hippocampus. (a1, b1, c1) The distribution of neuron-specific nuclear antigen (NeuN)-positive neurons in different layers of the hippocampus of animals treated with vehicle (VEH, a1), chemicals (CHEM, b1), or chemicals and stress (CHEM+STRESS, c1). (a2–c4) Magnified views of regions of the dentate gyrus (a2, b2, c2), CA1 subfield (a3, b3, c3), and CA3 subfield from (a1), (b1), and (c1). (d1–d4) Bar charts demonstrate that animals treated with CHEM or CHEM+STRESS display some loss of neurons in the granule cell layer (d1), dentate hilus (d2), CA1 pyramidal cell layer (d3), and CA3 pyramidal cell layer (d4) in comparison with VEH-treated control animals. (e1–e4) Bar charts compare volumes of the dentate gyrus (e1), CA1 subfield (e2), CA3 subfield (e3), and the entire hippocampus including fimbria (e4) among the three groups of animals. Scale bar: a1, b1, c1=500 μm; a2, b2, c2=200 μm; a3–c4=100 μm. *p<0.05; **p<0.01; ***p<0.001.

Figure 7

Exposure of adult rats to Gulf war illness-related chemicals or chemicals and stress induces microglial and astrocyte reactivity in the hippocampus. (a1–c4) The occurrence of ED-1+ activated microglia in regions such as fimbria (F) and the hippocampal fissure (HF) in animals treated with CHEM (b1, b2) or CHEM+STRESS (c1–c4) in contrast to their absence in animals treated with VEH (a1, a2). (b1–c4) Inserts illustrate the magnified view of the morphology of activated microglia indicated by arrows. (d) The bar chart compares the number of ED-1+ activated microglia between CHEM and CHEM+STRESS groups. (e1–g3) The distribution and morphology of GFAP+ astrocytes in the dentate gyrus (upper row), CA1 subfield (middle row), and CA3 subfield (lower row) between animals treated with VEH (e1–e3), CHEM (f1–f3) or CHEM+STRESS (g1–g3) is compared. Note the presence of hypertrophied GFAP+ astrocytes in hippocampal regions of animals treated with CHEM (f1–f3) or CHEM+STRESS (g1–g3) but not in animals treated with VEH (e1–e3). Scale bar: e1–g3=100 μm. (h1–h4) Bar charts compare area fraction of GFAP+ structures among the three groups in the dentate gyrus (h1), CA1 subfield (h2), CA3 subfield (h3), and the entire hippocampus (h4). *p<0.05; **p<0.01; ***p<0.001. DH, dentate hilus; GCL, granule cell layer; CA1-SR, CA1 stratum radiatum; CA3-SR, CA3 stratum radiatum.