Effects of Blast Overpressure on Neurons and Glial Cells in Rat Organotypic Hippocampal Slice Cultures

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.

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

“…However, we focused our analyses on the vigorous early astrocytic response to blast overpressure that was not previously reported. In our earlier studies we demonstrated robust activation of astrocytes at 72 h post injury, however at that time point we detected only a small number of dead astrocytes [34]. Though, corresponding to our current results, we observed dead microglial cells at 4 and 24 h post-injury [34].…”

“…In the present study we analyzed cellular changes in OHCs at 2 h following blast exposure, as under the same conditions we previously observed dramatic increase in cell death from 0 to 2 h post-injury [34]. At 2 h post-injury, in addition to dead astrocytes we also observed dead neurons and microglial cells (data not shown).…”

“…However, research from our group, in addition to the results of other experts in the field, suggests that a blast shock wave can transverse the cranium intact and generate tissue stress and strain leading to neuronal damage [24–29]. Correspondingly, data from in vitro bTBI models [30–33], including our recent findings [34], imply that blast overpressure can directly damage neurons and glial cells. In previous rat bTBI studies conducted by our [16, 28] and other groups [19, 35, 36], exposure to the peak overpressure magnitudes in the range of 100 to 450 kPa resulted in neurodegenerative changes and behavioral impairments.…”

“…OHCs were exposed to blast overpressures of approximately 150 and 280 kPa, that in our previous in vitro study evoked significant cell death [34]. Experimental designs similar to ours were implemented in several recent in vitro [32, 33, 119, 120, 123, 124] and in vivo bTBI studies [19, 122, 125, 126].…”

“…As previously demonstrated by data from our and other laboratories, this period is sufficient to allow recovery from any procedure-related degeneration [34, 91, 92]. An open-ended, helium-driven shock tube, built by our group, was used to generate overpressures of specific magnitudes and to produce injury in OHCs as described previously [34].…”