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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

Preservation of OHCs’ structural organization during culturing period. (A) Light micrograph of an acutely dissected OHC. (B) Same OHC as in (A) demonstrates well preserved CA1, CA3, and DG hippocampal regions at 8 DIV. Higher magnification of CA1 (C) and DG (D) regions of cresyl violet-stained OHC at 8 DIV also illustrate intact hippocampal cytoarchitecture. Serial imaging of PI-stained OHC at 1 (E), 5 (F), and 8 (G) DIV demonstrates recovery of slice from procedure-related cellular degeneration. Scale bars (A,B) 500 μm; (C,D) 50 μm; (E–G) 500 μm.
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Figure 2: Preservation of OHCs’ structural organization during culturing period. (A) Light micrograph of an acutely dissected OHC. (B) Same OHC as in (A) demonstrates well preserved CA1, CA3, and DG hippocampal regions at 8 DIV. Higher magnification of CA1 (C) and DG (D) regions of cresyl violet-stained OHC at 8 DIV also illustrate intact hippocampal cytoarchitecture. Serial imaging of PI-stained OHC at 1 (E), 5 (F), and 8 (G) DIV demonstrates recovery of slice from procedure-related cellular degeneration. Scale bars (A,B) 500 μm; (C,D) 50 μm; (E–G) 500 μm.

Mentions: Organotypic hippocampal slice cultures were grown for 8 days prior to blast exposure. Our data (Figure 2) together with the data from other groups (72, 73) demonstrated that this period is sufficient to allow slice procedure-related cellular degeneration to end. In addition, it has been demonstrated that by 7 DIV the majority of microglial cells return to the resting, ramified phenotype (74–76). At 8 DIV, OHCs were exposed to blast injury. Individual inserts with 4–6 OHCs were placed in 40-mm culture dishes containing 800 μl of serum-free medium, covered with Parafilm and sealed inside sterile plastic pouches (5 cm × 6.5 cm). Samples were placed on a rigid holder below the shock tube at 55° off axis. The distance from the end of the shock tube to the cultures was 22 cm. Samples were exposed to a single blast overpressure of 147 ± 18 kPa (low) or 278 ± 22 kPa (high). Side-on pressure was recorded using PCB113B28 (PCB Piezotronics, Depew, NY, USA) sensors located directly above the test sample at 10 MHz (National Instruments, Austin, TX, USA). The average duration and impulse for the low blast overpressure were 160.3 ± 15.7 μs and 10.39 ± 1.23 kPa × ms. The corresponding values for the high-blast group were 157.1 ± 8.2 μs and 18.10 ± 1.88 kPa × ms. Following blast exposure, under sterile conditions, inserts with OHCs were removed from the pouches and placed back in the incubator in fresh serum-free medium. Four different control groups were included in the studies. Incubator controls remained in the incubator throughout experimentation. Sham-exposed OHCs were prepared using an identical protocol, placed under the shock tube, but not exposed to the shockwave overpressure. In addition, low- and high-vibration control groups were used to determine the effect of mechanical vibration due to the firing of the shock tube. Vibration control OHCs were prepared using an identical protocol and placed on a separate rigid holder below the tube. This second holder was attached to the shock tube system, but located away from the shockwave. These OHCs were exposed to the system vibration, but not to the blast overpressure. Cell death attributable to mechanical vibration was quantified by comparing the vibration control group to the sham control group.


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)

Preservation of OHCs’ structural organization during culturing period. (A) Light micrograph of an acutely dissected OHC. (B) Same OHC as in (A) demonstrates well preserved CA1, CA3, and DG hippocampal regions at 8 DIV. Higher magnification of CA1 (C) and DG (D) regions of cresyl violet-stained OHC at 8 DIV also illustrate intact hippocampal cytoarchitecture. Serial imaging of PI-stained OHC at 1 (E), 5 (F), and 8 (G) DIV demonstrates recovery of slice from procedure-related cellular degeneration. Scale bars (A,B) 500 μm; (C,D) 50 μm; (E–G) 500 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Preservation of OHCs’ structural organization during culturing period. (A) Light micrograph of an acutely dissected OHC. (B) Same OHC as in (A) demonstrates well preserved CA1, CA3, and DG hippocampal regions at 8 DIV. Higher magnification of CA1 (C) and DG (D) regions of cresyl violet-stained OHC at 8 DIV also illustrate intact hippocampal cytoarchitecture. Serial imaging of PI-stained OHC at 1 (E), 5 (F), and 8 (G) DIV demonstrates recovery of slice from procedure-related cellular degeneration. Scale bars (A,B) 500 μm; (C,D) 50 μm; (E–G) 500 μm.
Mentions: Organotypic hippocampal slice cultures were grown for 8 days prior to blast exposure. Our data (Figure 2) together with the data from other groups (72, 73) demonstrated that this period is sufficient to allow slice procedure-related cellular degeneration to end. In addition, it has been demonstrated that by 7 DIV the majority of microglial cells return to the resting, ramified phenotype (74–76). At 8 DIV, OHCs were exposed to blast injury. Individual inserts with 4–6 OHCs were placed in 40-mm culture dishes containing 800 μl of serum-free medium, covered with Parafilm and sealed inside sterile plastic pouches (5 cm × 6.5 cm). Samples were placed on a rigid holder below the shock tube at 55° off axis. The distance from the end of the shock tube to the cultures was 22 cm. Samples were exposed to a single blast overpressure of 147 ± 18 kPa (low) or 278 ± 22 kPa (high). Side-on pressure was recorded using PCB113B28 (PCB Piezotronics, Depew, NY, USA) sensors located directly above the test sample at 10 MHz (National Instruments, Austin, TX, USA). The average duration and impulse for the low blast overpressure were 160.3 ± 15.7 μs and 10.39 ± 1.23 kPa × ms. The corresponding values for the high-blast group were 157.1 ± 8.2 μs and 18.10 ± 1.88 kPa × ms. Following blast exposure, under sterile conditions, inserts with OHCs were removed from the pouches and placed back in the incubator in fresh serum-free medium. Four different control groups were included in the studies. Incubator controls remained in the incubator throughout experimentation. Sham-exposed OHCs were prepared using an identical protocol, placed under the shock tube, but not exposed to the shockwave overpressure. In addition, low- and high-vibration control groups were used to determine the effect of mechanical vibration due to the firing of the shock tube. Vibration control OHCs were prepared using an identical protocol and placed on a separate rigid holder below the tube. This second holder was attached to the shock tube system, but located away from the shockwave. These OHCs were exposed to the system vibration, but not to the blast overpressure. Cell death attributable to mechanical vibration was quantified by comparing the vibration control group to the sham control group.

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