RESUMO
The calpain family of cysteine proteases has a well-established causal role in neuronal cell death following acute brain injury. However, the relative contribution of calpain isoforms has not been determined in in vivo models. Identification of the calpain isoform responsible for neuronal injury is particularly important given the differential role of calpain isoforms in normal physiology. This study evaluates the role of m-calpain and micro-calpain in an in vivo model of global brain ischemia. Adeno-associated viral vectors expressing short hairpin RNAs targeting the catalytic subunits of micro- or m-calpain were used to knockdown expression of the targeted isoforms in adult rat hippocampal CA1 pyramidal neurons. Knockdown of micro-calpain, but not m-calpain, prevented calpain activity 72 h after 6-min transient forebrain ischemia, increased long-term survival and protected hippocampal electrophysiological function. These findings represent the first in vivo evidence that reducing expression of an individual calpain isoform can decrease post-ischemic neuronal death and preserve hippocampal function.
Assuntos
Isquemia Encefálica/fisiopatologia , Calpaína/genética , Sobrevivência Celular/genética , Hipocampo/fisiologia , Neurônios/fisiologia , Análise de Variância , Animais , Isquemia Encefálica/genética , Células Cultivadas , Dependovirus , Eletrofisiologia , Imunofluorescência , Vetores Genéticos/genética , Hipocampo/citologia , Masculino , Neurônios/citologia , Isoformas de Proteínas/genética , Interferência de RNA , RNA Interferente Pequeno/genética , RatosRESUMO
OBJECTIVE: Delayed neurodegeneration after transient global brain ischemia offers a therapeutic window for inhibiting molecular injury mechanisms. One such mechanism is calpain-mediated proteolysis, which peaks 24 to 48 hrs after transient forebrain ischemia in rats. This study tests the hypothesis that delayed calpain inhibitor therapy can reduce brain calpain activity and neurodegeneration after transient forebrain ischemia. DESIGN: Prospective randomized placebo-controlled animal trial. SETTING: University research laboratory. SUBJECTS: Adult male Long-Evans rats. INTERVENTIONS: Rats subjected to 10-min transient forebrain ischemia were randomized to intravenous infusion of calpain inhibitor CEP-3453 or vehicle beginning 22 hrs after injury. MEASUREMENTS AND MAIN RESULTS: In a dose-response study, a 60 mg/kg bolus followed by 30 mg/kg infusion was required to reduce postischemic brain calpain activity measured by Western blot of hippocampal homogenates at 48 hrs after injury. The same dosing protocol decreased degeneration of CA1 pyramidal neurons measured at 72 hrs after injury. CONCLUSIONS: These results suggest a causal role for calpains in delayed postischemic neurodegeneration, and demonstrate a broad therapeutic window for calpain inhibition in this model.
