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. 2022 Jun 28;23(13):7185.
doi: 10.3390/ijms23137185.

The Role of Microglia in the (Mal)adaptive Response to Traumatic Experience in an Animal Model of PTSD

Kesem Nahum  1 , Doron Todder  2 , Joseph Zohar  3 , Hagit Cohen  1   2   4
Affiliations

The Role of Microglia in the (Mal)adaptive Response to Traumatic Experience in an Animal Model of PTSD

Kesem Nahum et al. Int J Mol Sci. .

Abstract

The present study investigates whether predator scent-stress (PSS) shifts the microglia from a quiescent to a chronically activated state and whether morphological alterations in microglial activation differ between individuals displaying resilient vs. vulnerable phenotypes. In addition, we examined the role that GC receptors play during PSS exposure in the impairment of microglial activation and thus in behavioral response. Adult male Sprague Dawley rats were exposed to PSS or sham-PSS for 15 min. Behaviors were assessed with the elevated plus-maze (EPM) and acoustic startle response (ASR) paradigms 7 days later. Localized brain expression of Iba-1 was assessed, visualized, and classified based on their morphology and stereological counted. Hydrocortisone and RU486 were administered systemically 10 min post PSS exposure and behavioral responses were measured on day 7 and hippocampal expression of Ionized calcium-binding adaptor molecule 1 (Iba-1) was subsequently evaluated. Animals whose behavior was extremely disrupted (PTSD-phenotype) selectively displayed excessive expression of Iba-1 with concomitant downregulation in the expression of CX3C chemokine receptor 1 (CX3CR1) in hippocampal structures as compared with rats whose behavior was minimally or partially disrupted. Changes in microglial morphology have also been related only to the PTSD-phenotype group. These data indicate that PSS-induced microglia activation in the hippocampus serves as a critical mechanistic link between the HPA-axis and PSS-induced impairment in behavioral responses.

Keywords: animal models; chemokine; immune system; microglia; post-traumatic stress disorder (PTSD).

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Study design—The effect of predator scent-stress (PSS) on corticosterone levels and behavioral responses. After habituation period of seven days, animals were exposure to PSS or sham-PSS for 15 min. Urine samples were collected 1–2 h after PSS exposure for corticosterone levels. Behaviors were assessed on day 7 using the elevated plus maze (EPM) and the acoustic startle response (ASR) assay. On day 8, rats were sacrificed, and their brains were collected for immunoreactivity analyses of Iba-1 and CRXCR1 in the hippocampus subregions.
Figure 2
Figure 2
The effect of the predator scent stress (PSS) paradigm on overall anxiety-like behavior and startle response: Three-dimensional parameters: The X-axis represents time spent in the open arms (min), the Y-axis represents acoustic startle amplitude, and the Z-axis—total exploration on the maze. The stress response was not homogeneous, and several subgroups were identifiable in the population. Circles (purple) represent the exposed group that exhibited a significant degree of anxiety-like and avoidant behaviors on the elevated plus-maze and a pattern of exaggerated startle responses with significantly reduced habituation 7 days after exposure (PTSD-like phenotype). Triangles (green) represent the exposed group that exhibited partial behavioral response patterns. Squares (gray) represent the exposed group that exhibited minimal behavioral disrupted. Diamonds (blue) represent the un-exposed control group.
Figure 3
Figure 3
Urine levels of corticosterone at 1–2 h after PSS-exposure or sham-PSS: All data represent the group means ± S.E.M.
Figure 4
Figure 4
The quantitative morphometric analysis of Iba-1 immunoreactivity (ir) in cells of the hippocampal subregions CA1 (a), CA3 (b), and DG (c) in unexposed controls (CONT) in animals with an extreme behavioral response (EBR) or partial response (PBR) to the PSS exposure, and in animals whose behavior was minimally affected by the stressor (MBR). All data represent the group means ± S.E.M.
Figure 5
Figure 5
Sholl-analysis for intersections per 3 mm radial unit distance from sham-PSS, EBR, PBR, and MBR animals. Results displayed as mean ± S.E.M. (a) CA1 hippocampal subregion at day 8 post PSS-exposure: * Control ≠ EBR and PBR, p < 0.04. # Control ≠ EBR, PBR and MBR, p < 0.03. ^ Control ≠ EBR, p = 0.04. ** EBR ≠ MBR, p < 0.04. ^^ PBR ≠ MBR, p < 0.03. ## Control ≠ MBR, p < 0.04. (b) CA3 hippocampal subregion at day 8 post PSS-exposure: * Control ≠ EBR and PBR, p < 0.04. # Control ≠ EBR, PBR and MBR, p < 0.03. ^ Control ≠ EBR, p = 0.04. ** EBR ≠ MBR, p < 0.04. ^^ PBR ≠ MBR, p < 0.03. ## Control ≠ MBR, p < 0.04. + Control ≠ PBR, p < 0.05. (c) DG hippocampal subregion at day 8 post PSS-exposure: * Control ≠ EBR and PBR, p < 0.04. # Control ≠ EBR, PBR and MBR, p < 0.03. ^ Control ≠ EBR, p = 0.04. ** EBR ≠ MBR, p < 0.04. ^^ PBR ≠ MBR, p < 0.03. ## Control ≠ MBR, p < 0.04.
Figure 6
Figure 6
Correlation analysis between hippocampal microglia measures and urine corticosterone levels. (a) CA1 hippocampal subregion and urine corticosterone, (b) CA3 hippocampal subregion and urine corticosterone and (c) DG hippocampal subregion and urine corticosterone.
Figure 7
Figure 7
Quantitative analysis of CX3CR1-ir cells in the CA1 (a), CA3 (b) and DG (c) hippocampal subregions at day 8 post PSS-exposure in animals with an extreme behavioral response (EBR) or partial response (PBR) to the PSS exposure, and in animals whose behavior was minimally affected by the stressor (MBR). All data represent the group means ± S.E.M.
Figure 8
Figure 8
Study design—Effects of high-dose hydrocortisone, RU486 (GC receptor antagonist-mifepristone) or saline immediately post-exposure on behavioral stress responses at day 7 post-exposure. After habituation period of seven days, animals were exposure to PSS or sham-PSS for 15 min. Animals were exposed to predator scent stress (PSS), treated for 1 day with hydrocortisone 25 mg/kg, saline or RU486 30 mg/kg and assessed behaviorally with the elevated plus maze (EPM) and acoustic startle response (ASR) tests at day 7. On day 8, rats were sacrificed, and their brains were collected for immunoreactivity analyses of Iba-1 in the hippocampus subregions.
Figure 9
Figure 9
High-dose hydrocortisone, saline or RU486 early post-exposure on behavioral stress responses at day 7 post-exposure: Three-dimensional parameters: The X-axis represents time spent in the open arms (min), the Y-axis represents acoustic startle amplitude (A.U), and the Z-axis—total exploration on the maze. Circles (purple) represent the exposed group that treated with Saline (n = 16), which exhibited a significant degree of anxiety-like and avoidant behaviors on the elevated plus-maze and a pattern of exaggerated startle responses with significantly reduced habituation 7 days after exposure (PTSD-like phenotype). Triangles (green) represent the exposed group that treated with Hydrocortisone 25 mg/kg (n = 16), which exhibited minimal or partial behavioral response patterns. Diamonds (blue) represent the sham-PSS-exposed control group (n = 10). Squares (orange) represent the exposed group that treated with RU486 30 mg/kg (n = 14), which exhibited extreme or partial behavioral response patterns.
Figure 10
Figure 10
Sholl-analysis for intersections per 3 mm radial unit distance from sham-PSS, PSS +Saline, PSS + Hydrocortisone 25 mg/kg and PSS + RU486 30 mg/kg groups. Results displayed as mean ± S.E.M. (a) CA1 hippocampal subregion at day 8 post PSS-exposure: # Sham-PSS ≠ PSS-Saline-p < 0.025. * Sham-PSS, PSS-Hydrocortisone ≠ PSS-Saline, PSS-RU486-p < 0.015. ^^ PSS-Hydrocortisone ≠ PSS-Saline, PSS-RU486-p = 0.03. ** Sham-PSS ≠ PSS-Hydrocortisone-p < 0.04. @ PSS-RU486 ≠ Sham-PSS, PSS-Hydrocortisone p < 0.03 (b) CA3 hippocampal subregion at day 8 post PSS-exposure: * Sham-PSS ≠ PSS-Saline, PSS-RU486-p < 0.05. ** PSS-Hydrocortisone ≠ PSS-RU486-p < 0.05. @ PSS-Hydrocortisone ≠, Sham-PSS, PSS-Saline, PSS-RU486-p < 0.045. ^ PSS-Hydrocortisone ≠ PSS-Saline, PSS-RU486-p = 0.05. (c) DG hippocampal subregion at day 8 post PSS-exposure: * Sham-PSS ≠ PSS-Saline, PSS-RU486-p < 0.04. # Sham-PSS ≠ PSS-Saline-p < 0.02.
Figure 11
Figure 11
Either neuroprotective or neurotoxic microglial function may be involved at different stages of the development of PTSD phenotype. (a) Well-adapted (MBR) phenotype. (b) PTSD-phenotype.
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