Abstract 2837: Delayed Calpain Inhibition Is Neuroprotective After Transient Global Brain Ischemia
Introduction/Hypothesis: The delay in neurodegeneration after transient global brain ischemia offers a potentially broad therapeutic window for inhibiting molecular injury mechanisms. One such mechanism, calpain-mediated proteolysis, peaks between 24 and 72 hours after transient forebrain ischemia (TFI) in rats. This study tests the hypothesis that calpain inhibition is neuroprotective when initiated 22 hours after TFI.
Methods: Male Long Evans rats (400 – 450 g) were anesthetized (halothane), mechanically ventilated, and instrumented for temperature and hemodynamic monitoring. Each underwent 10 minutes of TFI induced by reversible bilateral carotid occlusion and hypovolemic hypotension (MAP 30 mm Hg). Twenty-two hours after TFI, rats were block randomized (n=6/group) to receive either calpain inhibitor (CEP-3453 (Cephalon), 60 mg/kg IV bolus and 30 mg/kg IV infusion for 50 hours) or vehicle (normal saline IV bolus and infusion for 50 hours). Sham operated rats served as controls. Rats were euthanized 72 hours after injury and brains were processed for immunohistochemistry. Hippocampal CA1 sector calpain activity was analyzed by immunofluorescence using primary antibody that specifically detects calpain-cleaved alpha-spectrin (Ab38). Neurodegeneration was quantified by counting normal appearing Hoechst-stained neuronal nuclei in the hippocampal CA1 pyramidal layer. Ab38 immunofluorescence intensity and normal nuclei counts were compared between vehicle and CEP-3453 treatment groups using a two-tailed Student’s t-test (alpha error 0.05).
Results: Baseline and post-injury hemodynamic parameters and temperature were not significantly different between vehicle and CEP-3453 treatment groups. Relative to sham operated controls, mean CA1 sector Ab38 immunofluorescence increased by 263 ± 281% in vehicle treated rats and 68 ± 147% in CEP-3453 treated rats (p=0.17). Normal CA1 pyramidal layer nuclei averaged 10 ± 12% of control in vehicle treated rats and 50 ± 42% of control in CEP-3453 treated rats (p=0.047).
Conclusion: These results suggest a causal role for calpains in delayed post-ischemic neurodegeneration, and demonstrate a broad therapeutic window for calpain inhibition in this model of transient global brain ischemia.