Astrocyte Reactivity Following Blast Exposure Involves Aberrant Histone Acetylation

Abstract

Blast induced neurotrauma (BINT) is a prevalent injury within military and civilian populations. The injury is characterized by persistent inflammation at the cellular level which manifests as a multitude of cognitive and functional impairments. Epigenetic regulation of transcription offers an important control mechanism for gene expression and cellular function which may underlie chronic inflammation and result in neurodegeneration. We hypothesize that altered histone acetylation patterns may be involved in blast induced inflammation and the chronic activation of glial cells. This study aimed to elucidate changes to histone acetylation occurring following injury and the roles these changes may have within the pathology. Sprague Dawley rats were subjected to either a 10 or 17 psi blast overpressure within an Advanced Blast Simulator (ABS). Sham animals underwent the same procedures without blast exposure. Memory impairments were measured using the Novel Object Recognition (NOR) test at 2 and 7 days post-injury. Tissues were collected at 7 days for Western blot and immunohistochemistry (IHC) analysis. Sham animals showed intact memory at each time point. The novel object discrimination decreased significantly between two and 7 days for each injury group (p < 0.05). This is indicative of the onset of memory impairment. Western blot analysis showed glial fibrillary acidic protein (GFAP), a known marker of activated astrocytes, was elevated in the prefrontal cortex (PFC) following blast exposure for both injury groups. Analysis of histone protein extract showed no changes in the level of any total histone proteins within the PFC. However, acetylation levels of histone H2b, H3, and H4 were decreased in both groups (p < 0.05). Co-localization immunofluorescence was used to further investigate any potential correlation between decreased histone acetylation and astrocyte activation. These experiments showed a similar decrease in H3 acetylation in astrocytes exposed to a 17 psi blast but not a 10 psi blast. Further investigation of gene expression by polymerase chain reaction (PCR) array, showed dysregulation of several cytokine and cytokine receptors that are involved in neuroinflammatory processes. We have shown aberrant histone acetylation patterns involved in blast induced astrogliosis and cognitive impairments. Further understanding of their role in the injury progression may lead to novel therapeutic targets.

Keywords: blast neurotrauma; brain injury; epigenetics; histone acetylation; neuroinflammation.

Figure 1

Representative 10 and 17 psi blast curve profiles from Advanced Blast Simulator (ABS). The curve is characterized by near instantaneous rise in pressure followed by an exponential decay and a brief negative phase before returning to ambient pressure. Animals were exposed to a single peak overpressure representing a free-field blast exposure.

Figure 2

(A) Heat map analysis of animal tracking following novel object recognition (NOR) test. Red represents increased time spent and blue represents minimal time spent during trial. Novel object location marked by white circle, familiar object was located in opposite corner. The heat map on the left shows increased time spent at the novel object which shows a discrimination index greater than 0.5 (intact memory). The heat map on the right shows approximately equal time spent at each object which is indicative of a discrimination index approximately equal to 0.5 (memory deficit). (B) Average discrimination index for each group at 2 and 7 days following injury. Across time points, each injury group shows a statistically significant decline in memory when compared to sham. *Indicates p < 0.05 when compared to sham. Data expressed as mean ± SEM. (N = 8/group).

Figure 3

(A) Western blot images created by Compass Software (ProteinSimple, Santa Clara, CA, USA) representing modified and total histone expression in the prefrontal cortex (PFC) for each treatment group. No differences in total histone expression was observed for any histone protein. (B) Levels of acetyl-H2b (AH2b), acetyl-H3 (AH3), and acetyl-H4 (AH4) were significantly decreased compared to sham. No differences were observed between injury groups. *Indicates p < 0.05. Data expressed as mean ± SEM. (N = 8/group).

Figure 4

Normalized nuclear histone deacetylase enzyme (HDAC) activity levels within the PFC. No significant changes in HDAC activity within the nucleus between any treatment groups. Data expressed as mean ± SEM. (N = 8/group).

Figure 5

Western blot analysis showed increased glial fibrillary acidic protein (GFAP) protein levels in the PFC of both injury groups. A normalized chemiluminescence value of one represents sham expression. This indicates astrogliosis occurring at the 7 day time point. No changes in the microglia marker Ionized Calcium-Binding Adapater Molecule 1 (IBA-1) were observed for either group. *Indicates p < 0.05. Data expressed as mean ± SEM. (N = 8/group).

Figure 6

(A) Representative images following immunohistochemistry (IHC) analysis. Green fluorescence represents GFAP expression while red represents AH3. In order to isolate, AH3 expression within astrocytes, only co-localized pixels were measured for analysis. (B) Decreased expression of AH3 was observed in astrocytes following 17 psi blast. *Indicates p < 0.05. Data expressed as mean ± SEM. (N = 8/group). (C) Images of astrocytes from sham and 17 psi groups taken at 63× to confirm co-localization and demonstrate observed changes in AH3 levels following blast exposure.

Figure 7

Changes in cytokine and cytokine receptor mRNA expression levels between sham and 17 psi blast group determined by polymerase chain reaction (PCR) array analysis. Only genes showing at least a two-fold increase or decrease are shown. Chemokine (C-X-C motif) receptor 5, 1 (CXCR5, CXCR1), Chemokine (C-C motif) ligand 12, 2, 21, 22, 4, 7 (CCL12, CCL2, CCL21, CCL22, CCL4, CCL7), Chemokine (C-C motif) receptor 2, 3 (CCR2, CCR3), C-reactive protein (CRP), Chemokine (C-X-C motif) ligand 2 (CXCL2), Interleukin 16, 36a (IL16, IL36a), Interleukin 1 receptor type 2 (IL1r2), Interleukin 2 receptor beta (IL2rβ), Lymphotoxin Alpha (LTα/TNF-β), Platelet factor 4 (PF4), Secreted phosphoprotein 1 (SPP). (N = 2/group).