Acute and Delayed Effects of Mechanical Injury on Calcium Homeostasis and Mitochondrial Potential of Primary Neuroglial Cell Culture: Potential Causal Contributions to Post-Traumatic Syndrome

Abstract

In vitro models of traumatic brain injury (TBI) help to elucidate the pathological mechanisms responsible for cell dysfunction and death. To simulate in vitro the mechanical brain trauma, primary neuroglial cultures were scratched during different periods of network formation. Fluorescence microscopy was used to measure changes in intracellular free Ca2+ concentration ([Ca2+]i) and mitochondrial potential (ΔΨm) a few minutes later and on days 3 and 7 after scratching. An increase in [Ca2+]i and a decrease in ΔΨm were observed ~10 s after the injury in cells located no further than 150–200 µm from the scratch border. Ca2+ entry into cells during mechanical damage of the primary neuroglial culture occurred predominantly through the NMDA-type glutamate ionotropic channels. MK801, an inhibitor of this type of glutamate receptor, prevented an acute increase in [Ca2+]i in 99% of neurons. Pathological changes in calcium homeostasis persisted in the primary neuroglial culture for one week after injury. Active cell migration in the scratch area occurred on day 11 after neurotrauma and was accompanied by a decrease in the ratio of live to dead cells in the areas adjacent to the injury. Immunohistochemical staining of glial fibrillary acidic protein and β-III tubulin showed that neuronal cells migrated to the injured area earlier than glial cells, but their repair potential was insufficient for survival. Mitochondrial Ca2+ overload and a drop in ΔΨm may cause delayed neuronal death and thus play a key role in the development of the post-traumatic syndrome. Preventing prolonged ΔΨm depolarization may be a promising therapeutic approach to improve neuronal survival after traumatic brain injury.

Insulin Diminishes Superoxide Increase in Cytosol and Mitochondria of Cultured Cortical Neurons Treated with Toxic Glutamate

“…We used the low-affinity calcium dye Fura-FF in the experiments because long-term exposure to high doses of Glu (100 µM) results in such a high intracellular calcium concentration ([Ca 2+ ] i ) that high-affinity probes, such as Fura-2/AM, are rapidly saturated with calcium and does not reflect true changes in [Ca 2+ ] i [ 5 , 35 ]. Fura-FF has been repeatedly used to measure [Ca 2+ ] i , including in experiments on Glu excitotoxicity [ 10 , 28 , 29 ]. Comparison of fluorescent images of neurons stained with Fura-FF and the Rh123 voltage-sensitive probe revealed a fundamental difference in the distribution of these dyes [ 28 ].…”

“…Comparison of fluorescent images of neurons stained with Fura-FF and the Rh123 voltage-sensitive probe revealed a fundamental difference in the distribution of these dyes [ 28 ]. In our recent work [ 10 ], we described in detail the changes in [Ca 2+ ] i in different phases of OCD and presented corresponding images proving that Fura-FF AM penetrates well into the cell and is fairly evenly distributed in the cytosol. Mitochondria in cells (in neurons in particular) are able to accumulate Fura-FF only when using concentrations of Fura-FF/AM that are 2 orders of magnitude higher than those that are usually used to load cells.…”

“…Fluorescence measurement results processing was performed as previously described in detail [ 10 ]. All data are presented as the mean ± standard error of the mean (SEM) and were performed with the GraphPad Prism software.…”

“…Any event that occurs downstream of the DCD onset is considered to influence the timing of cell death without altering its inevitability [ 8 ]. Long-lasting pathological changes in calcium homeostasis persisted in the surviving neurons after injury [ 10 ]. According to the data [ 4 , 6 , 9 ], DCD always occurs synchronously with a significant drop in the mitochondrial potential of the inner mitochondrial membrane (ΔΨm).…”