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. 2015 Feb 12:6:20.
doi: 10.3389/fneur.2015.00020. eCollection 2015.

Effects of blast overpressure on neurons and glial cells in rat organotypic hippocampal slice cultures

Anna P Miller  1 , Alok S Shah  2 , Brandy V Aperi  2 , Matthew D Budde  2 , Frank A Pintar  1 , Sergey Tarima  3 , Shekar N Kurpad  1 , Brian D Stemper  2 , Aleksandra Glavaski-Joksimovic  1
Affiliations

Effects of blast overpressure on neurons and glial cells in rat organotypic hippocampal slice cultures

Anna P Miller et al. Front Neurol. .

Abstract

Due to recent involvement in military conflicts, and an increase in the use of explosives, there has been an escalation in the incidence of blast-induced traumatic brain injury (bTBI) among US military personnel. Having a better understanding of the cellular and molecular cascade of events in bTBI is prerequisite for the development of an effective therapy that currently is unavailable. The present study utilized organotypic hippocampal slice cultures (OHCs) exposed to blast overpressures of 150 kPa (low) and 280 kPa (high) as an in vitro bTBI model. Using this model, we further characterized the cellular effects of the blast injury. Blast-evoked cell death was visualized by a propidium iodide (PI) uptake assay as early as 2 h post-injury. Quantification of PI staining in the cornu Ammonis 1 and 3 (CA1 and CA3) and the dentate gyrus regions of the hippocampus at 2, 24, 48, and 72 h following blast exposure revealed significant time dependent effects. OHCs exposed to 150 kPa demonstrated a slow increase in cell death plateauing between 24 and 48 h, while OHCs from the high-blast group exhibited a rapid increase in cell death already at 2 h, peaking at ~24 h post-injury. Measurements of lactate dehydrogenase release into the culture medium also revealed a significant increase in cell lysis in both low- and high-blast groups compared to sham controls. OHCs were fixed at 72 h post-injury and immunostained for markers against neurons, astrocytes, and microglia. Labeling OHCs with PI, neuronal, and glial markers revealed that the blast-evoked extensive neuronal death and to a lesser extent loss of glial cells. Furthermore, our data demonstrated activation of astrocytes and microglial cells in low- and high-blasted OHCs, which reached a statistically significant difference in the high-blast group. These data confirmed that our in vitro bTBI model is a useful tool for studying cellular and molecular changes after blast exposure.

Keywords: blast injury; cell death; hippocampus; in vitro model; organotypic slice culture; traumatic brain injury.

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Figures

Figure 1
Figure 1
Open-ended helium-driven shock tube. (A) The shock tube consists of driver and driven sections separated by a Mylar membrane that bursts at a specific pressure to create a blast overpressure of a predetermined magnitude. For blast injury, culture dishes containing serum-free medium and Millicell inserts with OHCs were sealed inside sterile plastic pouches and placed below the tube and out of the blast wind. Peak overpressures were recorded by pressure sensor placed above the culture dish with OHCs. (B) Representative pressure profile from OHCs exposed to high-blast overpressure (~280 kPa). (C) Representative pressure profile from OHCs exposed to low blast overpressure (~150 kPa).
Figure 2
Figure 2
Preservation of OHCs’ structural organization during culturing period. (A) Light micrograph of an acutely dissected OHC. (B) Same OHC as in (A) demonstrates well preserved CA1, CA3, and DG hippocampal regions at 8 DIV. Higher magnification of CA1 (C) and DG (D) regions of cresyl violet-stained OHC at 8 DIV also illustrate intact hippocampal cytoarchitecture. Serial imaging of PI-stained OHC at 1 (E), 5 (F), and 8 (G) DIV demonstrates recovery of slice from procedure-related cellular degeneration. Scale bars (A,B) 500 μm; (C,D) 50 μm; (E–G) 500 μm.
Figure 3
Figure 3
Cell death in OHCs after blast exposure. Following the 8 DIV recovery period from dissection, OHCs were exposed to a 150 kPa (low) or 280 kPa (high) blast overpressure or were sham-injured. (A–E) Representative micrographs of sham OHCs over a time course of 72 h, demonstrating low levels of dead PI-stained cells (white) throughout the experiment. In OHCs exposed to a low (F–J) or high blast (K–O) overpressure, dead cells (white) were observed as early as 2 h following injury and the damage intensified at later time points. The CA1, CA3, and the DG hippocampal regions [outlined in red in (O)] appear particularly vulnerable to the blast in both high and low groups. Scale bars 500 μm.
Figure 4
Figure 4
Cell death quantification in CA1, CA3, and DG hippocampal regions following blast injury. OHCs were exposed to overpressures of 150 kPa (low; n = 29) and 280 kPa (high; n = 13–17) and cell death was assessed at multiple time points following blast exposure using PI uptake assay. Data from the blasted groups were compared with the following control groups: sham-injured OHCs (n = 35–38), incubator controls (n = 38–39), low-vibration controls (n = 6–7), and high-vibration controls (n = 5). Quantitative analysis of blast-evoked cell death was performed by measuring the percent area of CA1 (A), CA3 (B), and DG (C) regions with PI staining above the threshold. Results are expressed as values and confidence interval predicted by the linear statistical model. *P < 0.05; **P < 0.01, ***P < 0.001.
Figure 5
Figure 5
Lactate dehydrogenase release in response to blast damage. Measurements of LDH released into the culture medium up to 72 h following exposure to high or low blast overpressures indicated a significant difference in the amount of LDH released into the medium between high and low blast at 2 and 24 h, and a significant difference between blasted sections and shams at all time points post-injury. *P < 0.05, ***P < 0.001, blast compared to sham-injured OHCs, #P < 0.05, ##P < 0.01, high- compared to low-blast group. n = 5–8 wells per each experimental group (each well equivalent to 5 slices).
Figure 6
Figure 6
Neurons in OHCs were particularly vulnerable to blast injury. Representative confocal images of CA1 region of sham-injured (A), low (B), and high blast-exposed OHCs (C) at 72 h following injury. Sections were co-stained against neuronal marker Tuj1 (green) and PI (red). Confocal images with the overlay of PI and Tuj1 staining demonstrated good viability of neurons in the sham OHCs (A), and significant number of killed neurons in blast-exposed OHCs (B,C) that co-expressed Tuj-1 and PI (arrows). Scale bars 25 μm.
Figure 7
Figure 7
Astrocyte activation in blast-exposed OHCs. Representative images of CA1 region from sham-injured (A), low-blast (B), and high-blast OHCs (C) that were fixed at 72 h following blast exposure and stained with anti-GFAP (green), PI (red), and the nuclear counter stain (DAPI). (A) OHCs maintained low level of astrocyte activation and PI staining at 72 h following sham injury. Activated astrocytes, as visualized by increased GFAP expression, hypertrophy, and thicker processes, were observed both in low- (B) and high-blast (C) groups at 72 h following blast exposure. (D) Quantification of GFAP staining demonstrated significant increase in GFAP MPI in OHCs exposed to high-blast compared to sham-injured OHCs (***P < 0.001) and OHCs exposed to the low blast (#P < 0.05). Scale bars 50 μm.
Figure 8
Figure 8
Live microglia imaging in OHCs following blast exposure. Microglial cells in OHCs were labeled with IB4 (green) and images of CA1 region were captured at 4 h (A–C) and 24 h (D–F) following sham (A,D) or blast injury (B,C,E,F). OHCs maintained low level of microglial activation and PI staining (red) at 4 h (A) and 24 h (D) following sham injury. Dead microglial cells that were co-labeled with IB4 and PI (arrows) were observed in low- (B) and high- (C) blast group at 4 h post-injury. Even more prominent microglial death was detected in low- (E) and high- (F) blasted OHCs at 24 h post-injury. Activation-induced change in microglia morphology from ramified to rounded was also observed in blasted OHCs (B,C,E,F). Scale bars 50 μm.
Figure 9
Figure 9
Quantification of activated microglia and total microglia number per counting area in blast-exposed OHCs. OHCs were fixed at 72 h post-injury and stained with Iba1 (green), PI (red), and DAPI counter stain (blue). Representative confocal images of CA1 region of sham-injured (A) and blasted OHCs (B,C). Sham-injured sections (A) showed ramified, resting microglia (arrows). Low-blast (B) and high-blast (C) OHCs demonstrate increased number of activated, rounded, amoeboid microglia (arrowheads). Scale bars (A–C) 50 μm. (D) Quantification of activated microglia within ROI in CA1 area revealed significantly higher percentage of activated microglia in high-blast OHCs compared to sham controls (**P < 0.01) and compared to low-blast sections (#P < 0.05). (E) Quantification of total number of microglial cells per counting area in CA1 region demonstrated significant decrease in OHCs exposed to low (***P < 0.001) and high blast (***P < 0.001) compared to the sham-injured OHCs. n = 5–9 sections per each experimental group.
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