Acute death of astrocytes in blast-exposed rat organotypic hippocampal slice cultures

• Published in: PloS one 2017-03, Vol.12 (3), p.e0173167-e0173167
• Main Authors: Miller, Anna P, Shah, Alok S, Aperi, Brandy V, Kurpad, Shekar N, Stemper, Brian D, Glavaski-Joksimovic, Aleksandra
• Format: Article
• Language: English
• Subjects: Amino acids; Animals; Annexin V; Apoptosis; Astrocytes; Astrocytes - metabolism; Astrocytes - pathology; Biology; Biology and Life Sciences; Blast; Blast effect; Blast Injuries - etiology; Blast Injuries - metabolism; Blast Injuries - pathology; Brain; Brain injuries; Brain Injuries - etiology; Brain Injuries - metabolism; Brain Injuries - pathology; Brain research; Brain slice preparation; Calpain; Cell Death; Cell Membrane Permeability; Dextran; Disease Models, Animal; Excitatory Amino Acid Transporter 2 - genetics; Excitatory Amino Acid Transporter 2 - metabolism; Explosions; Exposure; Gene Expression; Glial fibrillary acidic protein; Glial Fibrillary Acidic Protein - metabolism; Glutamic acid transporter; Head injuries; Helium; Hippocampus; Iodides; Medicine and Health Sciences; Metabolism; Morphology; Mortality; Neurobiology; Neurodegeneration; Neurosciences; Neurosurgery; Overpressure; Post traumatic stress disorder; Propidium iodide; Proteolysis; Rats; Rodents; Shearing; Studies; Tissue Culture Techniques; Transporter; Traumatic brain injury; Veterans
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0173167

Blast traumatic brain injury (bTBI) affects civilians, soldiers, and veterans worldwide and presents significant health concerns. The mechanisms of neurodegeneration following bTBI remain elusive and current therapies are largely ineffective. It is important to better characterize blast-evoked cellular changes and underlying mechanisms in order to develop more effective therapies. In the present study, our group utilized rat organotypic hippocampal slice cultures (OHCs) as an in vitro system to model bTBI. OHCs were exposed to either 138 ± 22 kPa (low) or 273 ± 23 kPa (high) overpressures using an open-ended helium-driven shock tube, or were assigned to sham control group. At 2 hours (h) following injury, we have characterized the astrocytic response to a blast overpressure. Immunostaining against the astrocytic marker glial fibrillary acidic protein (GFAP) revealed acute shearing and morphological changes in astrocytes, including clasmatodendrosis. Moreover, overlap of GFAP immunostaining and propidium iodide (PI) indicated astrocytic death. Quantification of the number of dead astrocytes per counting area in the hippocampal cornu Ammonis 1 region (CA1), demonstrated a significant increase in dead astrocytes in the low- and high-blast, compared to sham control OHCs. However only a small number of GFAP-expressing astrocytes were co-labeled with the apoptotic marker Annexin V, suggesting necrosis as the primary type of cell death in the acute phase following blast exposure. Moreover, western blot analyses revealed calpain mediated breakdown of GFAP. The dextran exclusion additionally indicated membrane disruption as a potential mechanism of acute astrocytic death. Furthermore, although blast exposure did not evoke significant changes in glutamate transporter 1 (GLT-1) expression, loss of GLT-1-expressing astrocytes suggests dysregulation of glutamate uptake following injury. Our data illustrate the profound effect of blast overpressure on astrocytes in OHCs at 2 h following injury and suggest increased calpain activity and membrane disruption as potential underlying mechanisms.