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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

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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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.

Mentions: Live-cell imaging of IB4-labeled microglial cells at 4 and 24 h post-injury reveled that some of the microglial cells were co-labeled with PI implying blast-evoked microglial death (Figure 8). In addition, while the majority of the IB4-labeled microglial cells in the sham OHCs appeared as ramified – resting microglia, microglial cells in low- and high-blasted OHCs possessed mainly rounded morphology pertinent to their activation (Figure 8). Activation of microglial cells following blast exposure was also confirmed in Iba1 immunostained OHCs at 72 h post-injury (Figure 9). Quantification of Iba1 immunostained, activated microglial cells at 72 h post-injury in the CA1 ROI indicated that 57 ± 5% of microglial cells in the high-blast group were activated, which was significantly higher than 30 ± 4% of activated microglial cells in sham controls (P < 0.01; Figure 9). At the same time point, 36 ± 7% of microglial cells were activated in the low-blast group, which was also significantly different compared to the high-blast group (P < 0.05; Figure 9). The low-blast group demonstrated a trend toward increased percentage of activated microglial cells compared to the sham-injured OHCs (36 ± 7 vs. 30 ± 4%), although this difference did not reach statistical significance (Figure 9). Compared to the earlier time points following blast exposure, at 72 h post-injury a smaller number of microglial cells co-localized with the PI staining (Figures 8 and 9). However, at 72 h post-injury the total number of microglial cells per counting area in low- (P < 0.001) and high-blasted (P < 0.001) OHCs was significantly smaller compared to the sham-injured OHCs (Figure 9).


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)

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

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

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.
Mentions: Live-cell imaging of IB4-labeled microglial cells at 4 and 24 h post-injury reveled that some of the microglial cells were co-labeled with PI implying blast-evoked microglial death (Figure 8). In addition, while the majority of the IB4-labeled microglial cells in the sham OHCs appeared as ramified – resting microglia, microglial cells in low- and high-blasted OHCs possessed mainly rounded morphology pertinent to their activation (Figure 8). Activation of microglial cells following blast exposure was also confirmed in Iba1 immunostained OHCs at 72 h post-injury (Figure 9). Quantification of Iba1 immunostained, activated microglial cells at 72 h post-injury in the CA1 ROI indicated that 57 ± 5% of microglial cells in the high-blast group were activated, which was significantly higher than 30 ± 4% of activated microglial cells in sham controls (P < 0.01; Figure 9). At the same time point, 36 ± 7% of microglial cells were activated in the low-blast group, which was also significantly different compared to the high-blast group (P < 0.05; Figure 9). The low-blast group demonstrated a trend toward increased percentage of activated microglial cells compared to the sham-injured OHCs (36 ± 7 vs. 30 ± 4%), although this difference did not reach statistical significance (Figure 9). Compared to the earlier time points following blast exposure, at 72 h post-injury a smaller number of microglial cells co-localized with the PI staining (Figures 8 and 9). However, at 72 h post-injury the total number of microglial cells per counting area in low- (P < 0.001) and high-blasted (P < 0.001) OHCs was significantly smaller compared to the sham-injured OHCs (Figure 9).

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