Although these cytoskeletal components are localized throughout the axon, there is a unique cytoskeleton associated with the most proximal segment of the axon. However, the consequence of nervous system injury for this specialized cytoskeleton has not been explored.The AIS functions as both a physiological and physical bridge between axonal and somatodendritic domains.Similarly, IV spectrin mutant mice develop ataxia, tremors, and deafness. Morphologically the AIS is the transition zone from the soma to the axon compartments, and the AIS cytoskeleton functions to maintain neuronal polarity and proper axonal trafficking. For example, loss of ankG causes axons to acquire molecular and structural characteristics of dendrites including spines. Together, these observations emphasize the importance of the AIS cytoskeleton in normal nervous system function.Since genetic loss of the AIS cytoskeleton disrupts neuronal function and organization, we considered whether nervous system injury also disrupts this essential axonal domain.Embryonic cortices were dissected and collected in HBSS at C.The cell suspension was left to settle for min, and the subsequent supernatant was filtered through a m cell strainer and gml laminin at cells mm dish.Neurons were incubated in a humidified CO incubator at C.Cells were fed every dby replacing half the media with fresh maintenance media.Once in the chamber, half the media was taken out of each well and stored for future use. Control cultures were washed times in BSS containing mM glucose and maintained at C in normoxic conditions for up to h.Cultures were subsequently removed from the chamber and media was replaced with half conditioned media and half fresh maintenance media for up to h.Following deprivation, fresh drug was added to the maintenance media at the appropriate concentration.PI was added to cultures hafter deprivation at gml.For the majority of the quantification, three fields of view were collected per coverslip. In some cases, PI was quantified using ten fields of view.For quantification of in vitro immunofluorescence, the same fields of view used for PI quantification were used for quantifying AIS immunofluorescence. In a few cases, immunostaining was performed using brains from perfused animals.Briefly, anesthetized animals were perfused with PFA.Brains were dissected and postfixed in PFA for hfollowed by equilibration in sucrose, sectioning, and immunolabeling.In vivo immunofluorescence was quantified using five fields collected for each ipsilateral and contralateral cortex of each animal.A IV spectrinpositive AIS was defined as a fluorescent segment of at least m in length.Rat or mouse brain homogenates were prepared from freshly dissected tissue.The resulting supernatant was used for protein estimation using the BCA method were aliquoted into separate tubes for each condition.The animals were awakened from anesthesia to confirm neurological deficits associated with occlusion.After min, the animals were briefly reanesthetized for removal of the suture.Animals were killed for analyses at various times postocclusion. Separate nonsurvival cohorts were performed to confirm equivalency of blood flow J.During all surgical procedures, animals were maintained at C.Immunofluorescence was performed as described above. Each hemisphere was homogenized separately in icecold. To ensure equal protein loading, protein concentrations were estimated from the resulting supernatant and resolving sample buffer was added to a final concentration of mgml.