Vulnerability of central neurons to secondary insults after in vitro mechanical stretch.
Mild traumatic brain injuries are of major public health significance. Neurons in such injuries often survive the primary mechanical deformation only to succumb to subsequent insults. To study mechanisms of vulnerability of injured neurons to secondary insults, we used an in vitro model of sublethal mechanical stretch. Stretch enhanced the vulnerability of the neurons to excitotoxic insults, causing nuclear irregularities, DNA fragmentation, and death suggestive of apoptosis. However, the DNA degradation was not attributable to classical (caspase mediated) or caspase-independent apoptosis. Rather, it was associated with profound stretch-induced mitochondrial dysfunction and the overproduction of reactive oxygen species (ROS). Sublethally stretched neurons produced surprisingly high levels of ROS, but these in isolation were insufficient to kill the cells. To be lethal, the ROS also needed to combine with nitric oxide (NO) to form the highly reactive species peroxynitrite. Peroxynitrite was not produced after stretch alone and arose only after combining stretch with an insult capable of stimulating NO production, such as NMDA or an NO donor. This explained the exquisite sensitivity of sublethally stretched neurons to a secondary NMDA insult. ROS scavengers and NO synthase (NOS) inhibitors prevented cell death and DNA degradation. Moreover, inhibiting neuronal NOS activation by NMDA using peptides that perturb NMDA receptor-postsynaptic density-95 interactions also reduced protein nitration and cell death, indicating that the reactive nitrogen species produced were neuronal in origin. Our data explain the mechanism of enhanced vulnerability of sublethally injured neurons to secondary excitotoxic insults and highlight the importance of secondary mechanisms to the ultimate outcome of neurons in mild neurotrauma.
Arundine
Mark
M
Toronto Western Hospital Research Institute, Toronto, Ontario, M5T 2S8 Canada,.
Aarts
Michelle
M
Lau
Anthony
A
Tymianski
Michael
M
D018750:6-Cyano-7-nitroquinoxaline-2,3-dione / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis; D001930:Brain Injuries / Q000473:pathology; D002154:Calpain / Q000502:physiology; D002467:Cell Nucleus / Q000473:pathology; D002478:Cells, Cultured / Q000378:metabolism / Q000473:pathology; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004247:DNA / Q000032:analysis; D053938:DNA Fragmentation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D020287:In Situ Nick-End Labeling; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000187:drug effects / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology; D016202:N-Methylaspartate / Q000494:pharmacology / Q000633:toxicity; D009474:Neurons / Q000378:metabolism / Q000473:pathology*; D009498:Neurotoxins / Q000494:pharmacology / Q000633:toxicity; D009569:Nitric Oxide / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000494:pharmacology / Q000633:toxicity; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D018384:Oxidative Stress; D030421:Peroxynitrous Acid / Q000378:metabolism; D011166:Porphyrins / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D016194:Receptors, N-Methyl-D-Aspartate / Q000187:drug effects / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013314:Stress, Mechanical; D013482:Superoxide Dismutase / Q000494:pharmacology
D004791:Enzyme Inhibitors; D018691:Excitatory Amino Acid Antagonists; D009498:Neurotoxins; D020030:Nitric Oxide Donors; D011166:Porphyrins; D017382:Reactive Oxygen Species; D016194:Receptors, N-Methyl-D-Aspartate; D011993:Recombinant Fusion Proteins; C480958:tetrakis(4-benzoic acid)porphyrin; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D016202:N-Methylaspartate; D018750:6-Cyano-7-nitroquinoxaline-2,3-dione; D004247:DNA; D019001:Nitric Oxide Synthase; D013482:Superoxide Dismutase; D002154:Calpain; D003546:Cysteine Endopeptidases