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

Miller AP, Shah AS, Aperi BV, Budde MD, Pintar FA, Tarima S, Kurpad SN, Stemper BD, Glavaski-Joksimovic A - Front Neurol (2015)

Bottom Line: Using this model, we further characterized the cellular effects of the blast 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, Medical College of Wisconsin , Milwaukee, WI , USA ; Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin , Milwaukee, WI , USA ; Clement J. Zablocki Veterans Affairs Medical Center , Milwaukee, WI , USA.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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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.

Mentions: Following blast exposure, cell death visualized by PI staining was mainly present in the hippocampal neuronal layers including pyramidal cells of the CA1 and CA3 regions, and granule cells of the DG (Figure 3). Co-staining with PI and neuronal marker Tuj1 at 72 h post-injury further demonstrated that the majority of blast-killed cells were neurons that co-stained for PI and Tuj1 (Figure 6).


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

Miller AP, Shah AS, Aperi BV, Budde MD, Pintar FA, Tarima S, Kurpad SN, Stemper BD, Glavaski-Joksimovic A - Front Neurol (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4325926&req=5

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.
Mentions: Following blast exposure, cell death visualized by PI staining was mainly present in the hippocampal neuronal layers including pyramidal cells of the CA1 and CA3 regions, and granule cells of the DG (Figure 3). Co-staining with PI and neuronal marker Tuj1 at 72 h post-injury further demonstrated that the majority of blast-killed cells were neurons that co-stained for PI and Tuj1 (Figure 6).

Bottom Line: Using this model, we further characterized the cellular effects of the blast 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, Medical College of Wisconsin , Milwaukee, WI , USA ; Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin , Milwaukee, WI , USA ; Clement J. Zablocki Veterans Affairs Medical Center , Milwaukee, WI , USA.

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.

No MeSH data available.


Related in: MedlinePlus