No abstract

Energy deficit after traumatic brain injury (TBI) may alter ionic homeostasis, neurotransmission, biosynthesis, and cellular transport. Using an in vitro model for TBI, we tested the hypothesis that stretch-induced injury alters mitochondrial membrane potential (⌬ m ) and ATP in astrocytes and neurons. Astrocytes, pure neuronal cultures, and mixed neuronal plus glial cultures grown on Silastic membranes were subjected to mild, moderate, and severe stretch. After injury, ⌬ m was measured using rhodamine-123, and ATP was quantified with a luciferinluciferase assay. In astrocytes, ⌬ m dropped significantly, and ATP content declined 43-52% 15 min after mild or moderate stretch but recovered by 24 h. In pure neurons, ⌬ m declined at 15 min only in the severely stretched group. At 48 h postinjury, ⌬ m remained decreased in severely stretched neurons and dropped in moderately stretched neurons. Intracellular ATP content did not change in any group of injured pure neurons. We also found that astrocytes and neurons release ATP extracellularly following injury. In contrast to pure neurons, ⌬ m in neurons of mixed neuronal plus glial cultures declined 15 min after mild, moderate, or severe stretch and recovered by 24 -48 h. ATP content in mixed cultures declined 22-28% after mild to severe stretch with recovery by 24 h. Our findings demonstrate that injury causes mitochondrial dysfunction in astrocytes and suggest that astrocyte injury alters mitochondrial function in local neurons.

No abstract

In our previous studies, we have shown that in vitro biaxial strain (stretch) injury of neurons in neuronal plus glial cultures increases intracellular free calcium ([Ca(2+)](i)) and decreases mitochondrial membrane potential (deltapsi(m)). The goal of this study was to determine whether strain injury, without the addition of exogenous agents, causes glutamate release, and whether NMDA receptor antagonists affect the post-strain injury rise in [Ca(2+)](i) and decrease in deltapsi(m). [Ca(2+)](i) and deltapsi(m) were measured using the fluorescent indicators fura-2 AM and rhodamine-1,2,3 (rh123). Strain injury of neuronal plus glial cultures caused an immediate 100-200 nM elevation in neuronal [Ca(2+)]i and a decline in neuronal deltapsi(m) by 15 min post-injury. Pretreatment with the NMDA receptor antagonist MK-801 (10 microM) attenuated the [Ca(2+)](i) elevation after mild, but not moderate and severe injury. MK-801 pretreatment reduced the decline in deltapsi(m) after mild and moderate, but not after severe injury. The NMDA receptor antagonist D-2-amino-5-phosphonopentanoic acid (APV; 100 microM) had effects similar to MK-801. Simultaneous measurement of [Ca(2+)](i) and deltapsi(m) demonstrated a significant correlation and a temporal relationship between [Ca(2+)](i) elevation and depression of deltapsi(m). We conclude that NMDA receptor stimulation contributes to some of the changes in [Ca(2+)](i) and deltapsi(m) after less severe strain injury. However, after more pronounced injury other mechanisms appear to be more involved.