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. 2017 Mar 6;12(3):e0173167.
doi: 10.1371/journal.pone.0173167. eCollection 2017.

Acute death of astrocytes in blast-exposed rat organotypic hippocampal slice cultures

Anna P Miller  1   2   3 , Alok S Shah  1   3 , Brandy V Aperi  1   3 , Shekar N Kurpad  1   2   3 , Brian D Stemper  1   3 , Aleksandra Glavaski-Joksimovic  1   2   3
Affiliations

Acute death of astrocytes in blast-exposed rat organotypic hippocampal slice cultures

Anna P Miller et al. PLoS One. .

Abstract

Blast traumatic brain injury (bTBI) affects civilians, soldiers, and veterans worldwide and presents significant health concerns. The mechanisms of neurodegeneration following bTBI remain elusive and current therapies are largely ineffective. It is important to better characterize blast-evoked cellular changes and underlying mechanisms in order to develop more effective therapies. In the present study, our group utilized rat organotypic hippocampal slice cultures (OHCs) as an in vitro system to model bTBI. OHCs were exposed to either 138 ± 22 kPa (low) or 273 ± 23 kPa (high) overpressures using an open-ended helium-driven shock tube, or were assigned to sham control group. At 2 hours (h) following injury, we have characterized the astrocytic response to a blast overpressure. Immunostaining against the astrocytic marker glial fibrillary acidic protein (GFAP) revealed acute shearing and morphological changes in astrocytes, including clasmatodendrosis. Moreover, overlap of GFAP immunostaining and propidium iodide (PI) indicated astrocytic death. Quantification of the number of dead astrocytes per counting area in the hippocampal cornu Ammonis 1 region (CA1), demonstrated a significant increase in dead astrocytes in the low- and high-blast, compared to sham control OHCs. However only a small number of GFAP-expressing astrocytes were co-labeled with the apoptotic marker Annexin V, suggesting necrosis as the primary type of cell death in the acute phase following blast exposure. Moreover, western blot analyses revealed calpain mediated breakdown of GFAP. The dextran exclusion additionally indicated membrane disruption as a potential mechanism of acute astrocytic death. Furthermore, although blast exposure did not evoke significant changes in glutamate transporter 1 (GLT-1) expression, loss of GLT-1-expressing astrocytes suggests dysregulation of glutamate uptake following injury. Our data illustrate the profound effect of blast overpressure on astrocytes in OHCs at 2 h following injury and suggest increased calpain activity and membrane disruption as potential underlying mechanisms.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Acute morphological changes and demise of astrocytes following blast exposure.
Representative confocal images acquired in the CA1 hippocampal region from sham controls (A, D), low-blast (B, E), and high-blast (C, F) OHCs that were fixed at 2 h following injury and stained with an anti-GFAP antibody (green), PI (red), and DAPI (blue). Shearing of the astrocytes (thin arrows) was detected in OHCs exposed to blast overpressure (B, C) while it was absent in the sham controls (A). Clasmatodendrosis (arrowheads) was also observed in the low- (E) and high-blast (F) groups, but it was very infrequent in the sham control group (D). At the same time point, only a few dead astrocytes were present in sham control OHCs (D) while significant number of dead astrocytes (thick arrows) was revealed in the low- (E) and high-blast (F) groups. (G) Schematic diagram of OHC, indicating approximate locations in the CA1 region (boxes) where images for quantification of dead astrocytes were taken. (H) Number of dead astrocytes per counting area in the CA1 hippocampal region at 2 h following injury was significantly higher in both the low- (*; P< 0.05; n = 5) and high-blast groups (*; P < 0.05; n = 5) compared to the sham control group (n = 5). Scale bars (A-C) 50 μm (D-F) 20 μm.
Fig 2
Fig 2. Limited early apoptotic death of astrocytes following blast exposure.
At 2 h following injury, Annexin V conjugated to Alexa 488 (green; A, E, I) was used to identify apoptotic cells in sham control (A-D), low-blast (E-H), and high-blast (I-L) groups. Samples were additionally labeled with the cell death marker PI (red; B, F, J), an antibody against GFAP (gray; C, G, K), and DAPI (blue). Overlay of Annexin V, PI, GFAP, and DAPI staining (D, H, L). Annexin V positive cells (arrow) were infrequent in all three experimental groups. Almost none of the observed Annexin V positive cells were co-labeled with GFAP. Scale bars 20 μm.
Fig 3
Fig 3. Calpain-mediated degradation of GFAP at 2 h following blast injury.
Proteins were isolated from sham control (Sham), low-blast (L-Blast), and high-blast (H-Blast) OHCs at 2 h post-injury and analyzed via western blot for expression of GFAP (A, B) or calpain (C). (A) The 38 kDa calpain associated GFAP-BDP was present in blast-injured OHCs, but not in corresponding sham control OHCs. (B) Following inhibition of calpain via calpeptin treatment, this GFAP-BDP at 38 kDa was not observed. (C) Calpain expression in OHCs exposed to blast overpressure compared to sham controls was not changed at this time point. (D) Densitometry analysis of GFAP 50 kDa/GAPDH ratio for 3 independent experiments revealed no significant (ns) differences among control and blast-exposed OHCs without or with calpeptin treatment.
Fig 4
Fig 4. Increased astrocytic membrane permeability at 2 h post injury.
Dex10 (green) and PI (red) labeled sham control (A), low-blast (B), or high-blast (C) OHCs were fixed 2 h following injury and further stained with an anti-GFAP antibody (gray). Dead astrocytes with increased membrane permeability, identified by overlap of GFAP, PI, and Dex10 staining (arrows), were only present in the low-blast (B) and high-blast (C) OHCs but not in the sham controls (A). Scale bars 20 μm.
Fig 5
Fig 5. Blast-induced loss of GLT-1-expressing astrocytes.
At 2 h following injury, sham control (A), low-blast (B), and high-blast (C) OHCs were stained using antibodies against GLT-1 (green), GFAP (gray), PI (red), and DAPI (blue). Dead astrocytes, identified by co-labeling of GFAP and PI (arrows) were also positive for GLT-1. (D) Representative immunoblot analyses of GLT-1 protein expression in sham control (Sham) and OHCs exposed to blast overpressure (L-Blast and H-Blast). (E) Densitometry analysis of GLT-1/GAPDH ratio for 3 independent experiments revealed no significant (ns) differences between sham control, low-blast, and high blast groups. Scale bars 20 μm.
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This work was supported by the Department of Neurosurgery, Medical College of Wisconsin and the VA Medical Research.