Microtubular proteolysis in focal cerebral ischemia.

Calpain, a neutral protease activated by calcium, may promote microtubular proteolysis in ischemic brain. We tested this hypothesis in an animal model of focal cerebral ischemia without reperfusion. The earliest sign of tissue injury was observed after no more than 15 min of ischemia, with coiling of apical dendrites immunolabeled to show microtubule-associated protein 2 (MAP2). After 6 h of ischemia, MAP2 immunoreactivity was markedly diminished in the infarct zone. Quantitative Western analysis demonstrated that MAP2 was almost unmeasurable after 24 h of ischemia. An increase in calpain activity, shown by an antibody recognizing calpain-cleaved spectrin fragments, paralleled the loss of MAP2 immunostaining. Double-labeled immunofluorescent studies showed that intraneuronal calpain activity preceded evidence of MAP2 proteolysis. Perikaryal immunolabeling of tau protein became increasingly prominent between 1 and 6 h in neurons located within the transition zone between ischemic and unaffected tissue. Western blot experiments confirmed that dephosphorylation of tau protein occurred during 24 h of ischemia, but was not associated with significant loss of tau antigen. We conclude that focal cerebral ischemia is associated with early microtubular proteolysis caused by calpain.

Induction of PGH synthase and c-fos mRNA during early reperfusion of ischemic rat brain.

We examined the effect of reversible ischemia on the transcription of prostaglandin endoperoxide synthase (PGHS-1) and c-fos mRNA in rat cerebral cortex. The level of PGHS-1 mRNA climaxed after 30 min of ischemia whereas transcription of c-fos mRNA peaked after 60 min of postischemic reperfusion. We conclude that cerebral ischemia causes early transcription of PGHS-1, without modulation by the c-fos gene or its translated product.

Rapid expression of neuronal and inducible nitric oxide synthases during post-ischemic reperfusion in rat brain.

OBJECTIVE: To determine whether neuronal and inducible nitric oxide synthase (nNOS and iNOS) isoforms are expressed within cortical neurons during early reperfusion after focal cerebral ischemia. METHODS: Male spontaneously hypertensive rats underwent occlusion of the left middle cerebral artery for 2 h. Coronal brain sections with normal and ischemic cortex were obtained after 15 min or 1, 6 or 24 h of reperfusion. Immunohistochemical and double-label immunofluorescent techniques were used to confirm cellular identity and localize nNOS and iNOS. RESULTS: Immunoreactive nNOS was identified within isolated neurons in layer V of normal cortex. However, the number of nNOS-immunoreactive neurons in ischemic cortex rose markedly at 15 min and persisted for 24 h (P< or =0.001 at each time point when compared to normal cortex). Cells that were immunoreactive for nNOS appeared in perivascular clusters within ischemic brain at all sampling times. Immunoreactive iNOS was also expressed within neurons in ischemic cortex, peaking after 15 min of reperfusion (P< or =0.01). Although nNOS-immunoreactive neurons were observed in random numbers within normal tissue throughout reperfusion, iNOS-immunoreactive neurons increased steadily in the same region (P< or =0.05). CONCLUSIONS: Ischemic neurons become immunoreactive for both nNOS and iNOS during early reperfusion. Expression of iNOS immunoreactivity in unaffected neurons may reflect transcription of immediate early genes in response to stimulatory neurotransmission from ischemic cortex.

Glutamate receptor antagonists inhibit calpain-mediated cytoskeletal proteolysis in focal cerebral ischemia.

Excitatory amino acids may promote microtubular proteolysis observed in ischemic neuronal degeneration by calcium-mediated activation of calpain, a neutral protease. We tested this hypothesis in an animal model of focal cerebral ischemia without reperfusion. Spontaneously hypertensive rats were treated with 2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzo-(F)quinoxaline (NBQX), a competitive antagonist of the neuronal receptor for alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or cis-4-[phosphono-methyl]-2-piperidine carboxylic acid (CGS 19755), a competitive antagonist of the N-methyl-d-aspartate (NMDA) receptor. After treatment, all animals were subjected to permanent occlusion of the middle cerebral artery for 6 or 24 h. Infarct volumes measured in animals pretreated with CGS 19755 after 24 h of ischemia were significantly smaller than those quantified in ischemic controls. Rats pretreated with NBQX showed partial amelioration of cytoskeletal injury with preserved immunolabeling of microtubule-associated protein 2 (MAP 2) at 6 and 24 h and reduced accumulation of calpain-cleaved spectrin byproducts only at 6 h. Prevention of cytoskeletal damage was more effective after pretreatment with CGS 19755, as shown by retention of MAP 2 immunolabeling and significant restriction of calpain activity at both 6 and 24 h. Preserved immunolabeling of tau protein was observed at 6 and 24 h only in animals pretreated with CGS 19755. Western analysis performed on ischemic cortex taken from controls or rats pretreated with either NBQX or CGS 19755 suggested that loss of tau protein immunoreactivity was caused by dephosphorylation, rather than proteolysis. These results demonstrate a crucial link between excitotoxic neurotransmission, microtubular proteolysis, and neuronal degeneration in focal cerebral ischemia.

Permanent focal and transient global cerebral ischemia increase glial and neuronal expression of heme oxygenase-1, but not heme oxygenase-2, protein in rat brain.

Two heme oxygenase (HO) proteins have been identified to date; HO-1, a stress-induced protein, and HO-2, a constitutively expressed isoform. Recently, it was demonstrated that HO-1 mRNA expression is increased following transient global ischemia. The present study examined the effects of global and focal ischemia on HO-1 and HO-2 protein, using immunocytochemistry. Following 20 min of ischemia (rat 4 vessel occlusion model with hypotension) and 6 h of recirculation, increased HO-1 immunoreactivity was evident in hippocampal neurons. After 24 h of recirculation, HO-1 was observed in both hippocampal neurons and astroglial cells. By 72 h, expression was primarily glial and restricted to CA1 and CA3c. In addition to hippocampus, HO-1 was also evident in both neurons and glia in cerebral cortex and thalamus, and in striatal glial cells. Twenty-four hours following permanent focal ischemia, HO-1 immunoreactivity was observed in astroglial cells in the penumbra region surrounding the infarct. In contrast to HO-1, the pattern of HO-2 immunoreactivity was not altered following transient global or permanent focal ischemia. The increased expression of HO-1 following ischemia may confer protection against oxidative stress, but might also contribute to the subsequent neuronal degeneration.