Cyclosporin-A treatment attenuates delayed cytoskeletal alterations and secondary axotomy following mild axonal stretch injury.
Following central nervous system trauma, diffuse axonal injury and secondary axotomy result from a cascade of cellular alterations including cytoskeletal and mitochondrial disruption. We have examined the link between intracellular changes following mild/moderate axonal stretch injury and secondary axotomy in rat cortical neurons cultured to relative maturity (21 days in vitro). Axon bundles were transiently stretched to a strain level between 103% and 106% using controlled pressurized fluid. Double-immunohistochemical analysis of neurofilaments, neuronal spectrin, alpha-internexin, cytochrome-c, and ubiquitin was conducted at 24-, 48-, 72-, and 96-h postinjury. Stretch injury resulted in delayed cytoskeletal damage, maximal at 48-h postinjury. Accumulation of cytochrome-c and ubiquitin was also evident at 48 h following injury and colocalized to axonal regions of cytoskeletal disruption. Pretreatment of cultures with cyclosporin-A, an inhibitor of calcineurin and the mitochondrial membrane transitional pore, reduced the degree of cytoskeletal damage in stretch-injured axonal bundles. At 48-h postinjury, 20% of untreated cultures demonstrated secondary axotomy, whereas cyclosporin A-treated axon bundles remained intact. By 72-h postinjury, 50% of control preparations and 7% of cyclosporin A-treated axonal bundles had progressed to secondary axotomy, respectively. Statistical analyses demonstrated a significant (p < 0.05) reduction in secondary axotomy between treated and untreated cultures. In summary, these results suggest that cyclosporin-A reduces progressive cytoskeletal damage and secondary axotomy following transient axonal stretch injury in vitro.
Staal
J A
JA
NeuroRepair Group, Menzies Research Institute, University of Tasmania, Hobart, Tasmania, Australia.
Dickson
T C
TC
Chung
R S
RS
Vickers
J C
JC
D000818:Animals; D001370:Axonal Transport / Q000187:drug effects; D001369:Axons / Q000187:drug effects*; D019771:Axotomy; D002478:Cells, Cultured; D016572:Cyclosporine / Q000494:pharmacology*; D045304:Cytochromes c / Q000378:metabolism; D003599:Cytoskeleton / Q000187:drug effects / Q000502:physiology*; D004622:Embryo, Mammalian; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D019579:Neocortex / Q000166:cytology; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000473:pathology*; D010477:Perfusion / Q000009:adverse effects; D051381:Rats; D017208:Rats, Wistar; D013314:Stress, Mechanical; D013997:Time Factors
D004791:Enzyme Inhibitors; D009419:Nerve Tissue Proteins; D016572:Cyclosporine; D045304:Cytochromes c