Influence of post-traumatic stress disorder on neuroinflammation and cell proliferation in a rat model of traumatic brain injury

Abstract

Long-term consequences of traumatic brain injury (TBI) are closely associated with the development of severe psychiatric disorders, such as post-traumatic stress disorder (PTSD), yet preclinical studies on pathological changes after combined TBI with PTSD are lacking. In the present in vivo study, we assessed chronic neuroinflammation, neuronal cell loss, cell proliferation and neuronal differentiation in specific brain regions of adult Sprague-Dawley male rats following controlled cortical impact model of moderate TBI with or without exposure to PTSD. Eight weeks post-TBI, stereology-based histological analyses revealed no significant differences between sham and PTSD alone treatment across all brain regions examined, whereas significant exacerbation of OX6-positive activated microglial cells in the striatum, thalamus, and cerebral peduncle, but not cerebellum, in animals that received TBI alone and combined TBI-PTSD compared with PTSD alone and sham treatment. Additional immunohistochemical results revealed a significant loss of CA3 pyramidal neurons in the hippocampus of TBI alone and TBI-PTSD compared to PTSD alone and sham treatment. Further examination of neurogenic niches revealed a significant downregulation of Ki67-positive proliferating cells, but not DCX-positive neuronally migrating cells in the neurogenic subgranular zone and subventricular zone for both TBI alone and TBI-PTSD compared to PTSD alone and sham treatment. Comparisons of levels of neuroinflammation and neurogenesis between TBI alone and TBI+PTSD revealed that PTSD did not exacerbate the neuropathological hallmarks of TBI. These results indicate a progressive deterioration of the TBI brain, which, under the conditions of the present approach, was not intensified by PTSD, at least within our time window and within the examined areas of the brain. Although the PTSD manipulation employed here did not exacerbate the pathological effects of TBI, the observed long-term inflammation and suppressed cell proliferation may evolve into more severe neurodegenerative diseases and psychiatric disorders currently being recognized in traumatized TBI patients.

Figure 1. OX6 + expression in ipsilateral gray matter subcortical regions of TBI and TBI-PTSD rats.

Figures 1A–C represent quantitative data of estimated volumes (µm³) of OX6+ in A) cortex, B) striatum, and C) thalamus. Figures 1D–G represent OX6+ immunostaining of the ipsilateral side of cortex in D) sham-no PTSD, E) sham-PTSD, F) TBI-PTSD, G) TBI. Figures H-K represent OX6+ immunostaining of the ipsilateral side of striatum in H) sham-no PTSD, I) sham-PTSD, J) TBI-PTSD, K) TBI. Figures L-O represent OX6+ immunostaining of the ipsilateral side of thalamus in L) sham-no PTSD, M) sham-PTSD, N) TBI-PTSD, O) TBI. Note that in Figure 1C, N, the upregulation of activated microglia cells reach significance only for the group of chronic TBI combined with PTSD. Cortex, F_3,20 = 11.90,***p<0.0004; striatum, F_3,20 = 6.629, **p<0.0036; thalamus, F_3,20 = 5.999, ** p<0.0076. Scale bars for D, E, F, G, H, I, J, K, L, M, N, O are 1 µm.

Figure 2. OX6 + expression in ipsilateral subcortical white matter regions of TBI and TBI-PTSD rats.

Figures 2 A–C represent quantitative data of estimated volumes (µm³) of OX6+ in A) corpus callosum, B) cerebral peduncle, and C) fornix. Figures 2D–G represent OX6+ immunostaining of the ipsilateral side of corpus callosum in D) sham-no PTSD, E) sham-PTSD, F) TBI-PTSD, G) TBI. Figures 2 H–K represent OX6+ immunostaining of the ipsilateral side of cerebral peduncle in H) sham-no PTSD, I) sham-PTSD, J) TBI-PTSD, K) TBI. Figures2 L-O represent OX6+ immunostaining of the ipsilateral side of fornix in L) sham-no PTSD, M) sham-PTSD, N) TBI-PTSD, O) TBI. Corpus callosum, F_3,20 = 8.611, **p<0.0017, cerebral peduncle F_3,20 = 8.550, **p<0.002, fornix, F_3,20 = 8.368, **p<0.002. Scale bars for D, E, F, G, H, I, J, K, L, M, N, O are 1 µm.

Figure 3. OX6 + expression in white matter, granular cell layer and molecular layer of the cerebellum of TBI and TBI-PTSD rats.

Figure 3 A represents quantitative data of the estimated volumes (µm³) of OX6+ in three distinct regions of the cerebellum; white matter (WM), granular cell layer (GCL) and molecular layer (ML). Figures 3 B–F represent OX6+ immunostaining of the cerebellum in B) sham-no PTSD, C) sham-PTSD, D) TBI-PTSD, E) TBI. As shown in Figure 3 A, and in the photomicrographs B–E, there is no detectable upregulation of activated microglia cells in any of the examined cerebellar regions or across any of our treatment groups (sham-no PTSD, sham-PTSD, TBI-PTSD, and TBI alone); p>0.05. Scale bars for B-E are 1 µm.

Figure 4. H&E, cell proliferation Ki67+, and neuronal differentiation DCX+ expressions in the hippocampus of TBI and TBI-PTSD rats.

Figures 4 A–C represent quantitative data of A) total # of neurons in hippocampal CA3, B) estimated # of Ki67+ proliferating cells in the SGZ of the DG, and C) the estimated # of DCX+ migrating cells in the SGZ of the DG. Figures 4 D–G represent H&E staining of the ipsilateral hippocampal CA3 region in D) sham-no PTSD, E) sham-PTSD, F) TBI-PTSD, G) TBI. Figures 4 H-K represent Ki67+ immunostaining of the ipsilateral SGZ of the DG in H) sham-no PTSD, I) sham-PTSD, J) TBI-PTSD, K) TBI. Figures 4 L–O represent DCX+ immunostaining of the ipsilateral SGZ of the DG in L) sham-no PTSD, M) sham-PTSD, N) TBI-PTSD, O) TBI. CA3, F_3,8 = 13.570, **p<0.0017, SGZ Ki67, F_3,20 = 5.017, *p<0.0106, DCX, F_3,20 = 1.959 p<0.1512. Scale bars for D–G are 50 µm and H–O are 1 µm.

Figure 5. Cell proliferation Ki67+, and neuronal differentiation DCX+ expression in the SVZ of the lateral ventricle.

Figures 5 A–B represent quantitative data of A) total # of Ki67+ cells representing the number of cell proliferating in the SVZ, B) the estimated # of DCX+ migrating cells in the SVZ of the lateral ventricle. Figures 5 C–F represent Ki67+ immunostaining of the ipsilateral SVZ in C) sham-no PTSD, D) sham-PTSD, E) TBI, F) TBI-PTSD. Figures 5 G–J represent DCX+ immunostaining of the ipsilateral SVZ of the lateral ventricle in G) sham-no PTSD, H) sham-PTSD, I) TBI, J) TBI-PTSD. SVZ Ki67, F_3,20 = 7.863, **p<0.0012, SVZ DCX, F_3,20 = 0.324, ns p = 0.8076. Scale bars for C–J are 50 µm.