Limits...
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

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

License
getmorefigures.php?uid=PMC4325926&req=5

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.

Mentions: Organotypic hippocampal slice cultures’ ultrastructural organization was well preserved throughout the culture period as evaluated by phase-contrast microscopy and cresyl violet staining (Figure 2). In accordance with previously described characteristics of interface slice cultures (91), after 8 DIV OHCs thinned due to the spread of tissue, but the major hippocampal regions (CA1, CA3, and DG) were well conserved with clearly visible boundaries (Figures 2A,B). Maintenance of typical hippocampal cytoarchitecture in OHCs was also demonstrated with cresyl violet staining at 8 DIV (Figures 2C,D). In addition, using a PI uptake assay and serial imaging at 1, 5, and 8 DIV, we have confirmed results from previous studies (72, 73) that 7 days is a sufficient period to allow OHCs to recover from procedure-related cellular degeneration (Figures 2E–G). Moreover, the low level of PI staining (Figures 3 and 4) and LDH release (Figure 5) observed in incubator and sham controls throughout the experiment confirmed good vitality of OHCs in our experiments.


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)

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

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

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.
Mentions: Organotypic hippocampal slice cultures’ ultrastructural organization was well preserved throughout the culture period as evaluated by phase-contrast microscopy and cresyl violet staining (Figure 2). In accordance with previously described characteristics of interface slice cultures (91), after 8 DIV OHCs thinned due to the spread of tissue, but the major hippocampal regions (CA1, CA3, and DG) were well conserved with clearly visible boundaries (Figures 2A,B). Maintenance of typical hippocampal cytoarchitecture in OHCs was also demonstrated with cresyl violet staining at 8 DIV (Figures 2C,D). In addition, using a PI uptake assay and serial imaging at 1, 5, and 8 DIV, we have confirmed results from previous studies (72, 73) that 7 days is a sufficient period to allow OHCs to recover from procedure-related cellular degeneration (Figures 2E–G). Moreover, the low level of PI staining (Figures 3 and 4) and LDH release (Figure 5) observed in incubator and sham controls throughout the experiment confirmed good vitality of OHCs in our experiments.

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