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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

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).
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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).

Mentions: Our group has designed a compressed gas-driven, open-ended shock tube (Figure 1), which was used to expose OHCs to shockwave overpressure. The 7.5 cm-diameter, vertically oriented shock tube consists of a 17-cm driver section and a 152-cm driven section separated by a Mylar diaphragm. The driver section was pressurized with helium gas until exceeding the bursting pressure, at which time a shockwave was formed and traveled down the length of the driven section. Thickness of the Mylar membrane controlled bursting pressure and shockwave overpressure magnitude.


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)

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

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

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).
Mentions: Our group has designed a compressed gas-driven, open-ended shock tube (Figure 1), which was used to expose OHCs to shockwave overpressure. The 7.5 cm-diameter, vertically oriented shock tube consists of a 17-cm driver section and a 152-cm driven section separated by a Mylar diaphragm. The driver section was pressurized with helium gas until exceeding the bursting pressure, at which time a shockwave was formed and traveled down the length of the driven section. Thickness of the Mylar membrane controlled bursting pressure and shockwave overpressure magnitude.

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