calpain and Cerebral-Infarction

Ischemic stroke is a leading cause of long lasting disability in humans and oxidative stress an important underlying cause. The present study aims to determine the effect of short term (seven-days) administration of high dosage grape seed and skin extract (GSSE 2.5 g/kg) on ischemia/reperfusion (I/R) injury in a rat model of global ischemia. Ischemia was induced by occlusion of the common carotid arteries for 30 min followed by one-hour reperfusion on control or GSSE treated animals. I/R induced a drastic oxidative stress characterized by high lipid and protein oxidation, a drop in antioxidant enzyme defenses, disturbed transition metals as free iron overload and depletion of copper, zinc and manganese as well as of associated brain enzyme activities as glutamine synthetase and lactate dehydrogenase. I/R also induced NO and calcium disruption and an increase in calpain activity, a calcium-sensitive cysteine protease. Interestingly, almost all I/R-induced disturbances were prevented by GSSE pretreatment as oxidative stress, transition metals associated enzyme activities, brain damage size and histology. Owing to its antioxidant potential, high dosage GSSE protected efficiently the brain against ischemic stroke and should be translated to humans.

Topics: Animals; Antioxidants; Brain; Brain Ischemia; Calcium; Calpain; Cerebral Infarction; Female; Malondialdehyde; Nitrites; Oxidoreductases; Plant Extracts; Rats; Rats, Wistar; Reactive Oxygen Species; Reperfusion Injury; Seeds

Taurine, an abundant amino acid in the nervous system, is reported to reduce ischemic brain injury in a dose-dependent manner. This study was designed to investigate whether taurine protected brain against experimental stroke through affecting mitochondria-mediated cell death pathway. Rats were subjected to 2-h ischemia by intraluminal filament, and then reperfused for 22 h. It was confirmed again that taurine (50 mg/kg) administered intravenously 1 h after ischemia markedly improved neurological function and decreased infarct volume at 22 h after reperfusion. In vehicle-treated rats, the levels of intracellular ATP and the levels of cytosolic and mitochondrial Bcl-xL in the penumbra and core were markedly reduced, while the levels of cytosolic Bax in the core and mitochondrial Bax in the penumbra and core were enhanced significantly. There was a decrease in cytochrome C in mitochondria and an increase in cytochrome C in the cytosol of the penumbra and core. These changes were reversed by taurine. Furthermore, taurine inhibited the activation of calpain and caspase-3, reduced the degradation of αII-spectrin, and attenuated the necrotic and apoptotic cell death in the penumbra and core. These data demonstrated that preserving the mitochondrial function and blocking the mitochondria-mediated cell death pathway may be one mechanism of taurine's action against brain ischemia.

Topics: Adenosine Triphosphate; Animals; bcl-2-Associated X Protein; bcl-X Protein; Brain Ischemia; Calpain; Caspase 3; Cell Death; Cerebral Infarction; Cytochromes c; Disease Models, Animal; Dose-Response Relationship, Drug; Male; Mitochondria; Nervous System Diseases; Rats; Rats, Sprague-Dawley; Stroke; Taurine

Two intracellular cysteine proteases (calpains and caspases) and inducible nitric oxide synthase (iNOS) participate in the ischemic brain injury. In vitro nitric oxide (NO) regulates calpain and caspase-3 activation. The present study investigated whether aminoguanidine (AG), an iNOS inhibitor, protected brain against experimental stroke through inhibiting calpain and caspase-3 activation. Rats received 1h ischemia by intraluminal filament, then, reperfused for 23 h (R 23 h). AG (100 mg/kg) was administered intraperitoneally 5 min before ischemia. Our data showed that treatment with AG markedly improved neurological deficit, reduced brain swelling, decreased infarct volume, and attenuated the necrotic cell death in ischemic penumbra and core, and apoptotic cell death in penumbra at R 23 h. Enzymatic studies demonstrated the significant inhibition of the activities of mu- and m-calpain and caspase-3, and Western blot analysis revealed marked increases in the levels of MAP-2 and spectrin in penumbra and core in AG-treated rats versus vehicle-treated rats. AG also significantly enhanced the calpastatin levels in core, although it had no significant effects on that in penumbra. These data demonstrate that inhibiting calpain and caspase-3 activation is one mechanism of AG against experimental stroke, suggesting that NO produced by iNOS may be involved in calpain- and caspase-3-mediated ischemic cell death, at least in part.

Topics: Animals; Apoptosis; Biotransformation; Blotting, Western; Brain; Brain Edema; Calcium-Binding Proteins; Calpain; Caspase 3; Cerebral Infarction; Cytosol; Enzyme Activation; Guanidines; Male; Microtubule-Associated Proteins; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Recovery of Function; Spectrin; Stroke

Poly (ADP-ribose) polymerase (PARP) is a nuclear repair enzyme whose role is widely depicted in various physiological and pathological processes. In the present study, we wanted to check the status of PARP and the role of various cell death proteases involved in apoptotic and non-apoptotic forms of cell death during transient focal cerebral ischemia in rat model. The activation of these proteases can result in the production of PARP fragments which can be treated as specific signature fragments to the particular protease involved in the pathology and hence the type of cell death.. In the ischemic samples, we observed activation of calpain, cathepsin-b, caspase-3, and granzyme-b which were known to act on and cleave PARP to produce specific signature fragments by Western blot and immunohistochemical analysis. Cresyl violet staining showed the presence of apoptotic and necrotic cell deaths. Further we observed interaction of AIF and gra-b with PARP in double immunofluorescence and co-immunoprecipitation experiments.. Activation of calpains, cathepsin-b, caspase-3, and granzyme-b correlated with either apoptotic or necrotic cell deaths in cresyl violet staining. The appearance of PARP signature fragments gives a clear idea on the involvement of particular protease in the pathology. Appearance of signature fragments like 89- and 50-kDa indicates the involvement of apoptotic and necrotic cell death in the pathology. Further interaction of AIF and gra-b with PARP also indicates the involvement of non-apoptotic modes of cell death during the pathology of focal cerebral ischemia.

Topics: Animals; Apoptosis Inducing Factor; Brain; Brain Ischemia; Calpain; Caspases; Cathepsin B; Cell Death; Cerebral Infarction; Granzymes; Male; Neuropsychological Tests; Peptide Hydrolases; Poly(ADP-ribose) Polymerases; Random Allocation; Rats; Rats, Wistar

Calpastatin, a naturally occurring protein, is the only inhibitor that is specific for calpain. A novel blood-brain barrier (BBB)-permeant calpastatin-based calpain inhibitor, named B27-HYD, was developed and used to assess calpain's contribution to neurological dysfunction after stroke in rats. Postischemic administration of B27-HYD reduced infarct volume and neurological deficits by 35% and 44%, respectively, compared to untreated animals. We also show that the pharmacologic intervention has engaged the intended biologic target. Our data further demonstrates the potential utility of SBDP145, a signature biomarker of acute brain injury, in evaluating possible mechanisms of calpain in the pathogenesis of stroke and as an adjunct in guiding therapeutic decision making.

Topics: Animals; Blood-Brain Barrier; Brain; Calcium-Binding Proteins; Calpain; Cerebral Infarction; Cysteine Proteinase Inhibitors; Disease Models, Animal; Male; Peptide Fragments; Rats; Rats, Wistar; Spectrin

Impact-induced head injury in infants results in acute focal contusions and traumatic axonal injury (TAI) that are associated with chronic holohemispheric cortical and white matter atrophy and may contribute to poor outcome in brain-injured children less than 4 years of age. Contusive brain trauma in postnatal day (PND) 11 or PND 17 rat pups, ages neurologically equivalent to a human infant and toddler, respectively, leads to cortical tissue loss and white matter atrophy which are associated with cognitive deficits. In adult models of brain trauma and in brain-injured humans, acute and sustained activation of the calpain family of calcium-activated neutral proteases has been implicated in neuronal death and TAI. PND 11 or PND 17 rat pups were subjected to closed head injury over the left hemisphere using the controlled cortical impact device and sacrificed at 6 h, 24 h or 3 days. Hemorrhagic contusions and tissue tears in the cortex and white matter were visible at 6 h, and neuronal loss was evident by 3 days. Calpain activation was observed in cell soma and dendrites of injured neurons at 6 h, and in degenerating dendrites and atrophic neurons at 24 h after injury at both ages. Axonal accumulation of amyloid precursor protein, indicative of TAI, was observed in the corpus callosum and lateral aspects of the white matter below the site of impact, and in the thalamus in PND 11 rats only. Intra-axonal calpain activation was observed to a limited extent in the corpus callosum and subcortical white matter tracts in both brain-injured PND 11 and PND 17 rats. Collectively, these results provide evidence that calpain activation may participate in neuronal loss in the injured cortex, but may not contribute to the pathogenesis of TAI following contusive brain trauma in the immature rat.

Topics: Aging; Amyloid beta-Protein Precursor; Animals; Animals, Newborn; Axons; Biomarkers; Brain; Brain Injuries; Calpain; Cell Death; Cerebral Infarction; Dendrites; Diffuse Axonal Injury; Disease Models, Animal; Nerve Degeneration; Rats; Up-Regulation

Stroke patients often experience a significant temporal delay between the onset of ischemia and the time to initiation of therapy. Thus, there is a need for neuroprotectants with a long therapeutic window of opportunity. The efficacy of a potent, central nervous system-penetrating calpain inhibitor (MDL 28,170) was evaluated in a temporary model of focal cerebral ischemia to determine the window of opportunity for intracellular protease inhibition.. An ex vivo brain protease inhibition assay established pharmacodynamic dosing parameters for MDL 28,170. Middle cerebral artery (MCA) occlusion was accomplished by advancing a monofilament through the internal carotid artery to the origin of the MCA. Postmortem infarct volumes were determined by quantitative image analysis of triphenyltetrazolium-stained brain sections.. Maximal inhibition of brain protease activity was observed 30 minutes after injection of MDL 28,170 with an estimated pharmacodynamic half-life of 2 hours. MDL 28,170 caused a dose-dependent reduction in infarct volume when administered 30 minutes after MCA occlusion. A window of opportunity study was conducted to determine the maximal delay between the onset of ischemia and the initiation of efficacious therapy. MDL 28,170 reduced infarct volume when therapy was delayed for 0.5, 3, 4, and 6 hours after the initiation of ischemia. The protective effect of MDL 28,170 was lost after an 8-hour delay.. These data indicate that the therapeutic window of opportunity for calpain inhibition is at least 6 hours in a reversible focal cerebral ischemia model. This protection is observed despite the lethal hypoxic and excitotoxic challenge, suggesting that calpain activation may be an obligatory, downstream event in the ischemic cell death cascade.

Topics: Animals; Brain; Calpain; Carotid Artery, Internal; Cell Death; Cerebral Arterial Diseases; Cerebral Infarction; Coloring Agents; Cysteine Proteinase Inhibitors; Dipeptides; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Activation; Half-Life; Hypoxia; Image Processing, Computer-Assisted; Ischemic Attack, Transient; Male; Neuroprotective Agents; Neurotoxins; Rats; Rats, Wistar; Tetrazolium Salts; Time Factors

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.

Topics: Animals; Blotting, Western; Calpain; Cerebral Infarction; Cytoskeleton; Excitatory Amino Acid Antagonists; Immunohistochemistry; Ischemic Attack, Transient; Male; Microtubule-Associated Proteins; Pipecolic Acids; Quinoxalines; Rats; Rats, Inbred SHR; Receptors, Glutamate; Spectrin; Synaptic Transmission; tau Proteins

The effect of a novel neuroprotective compound, NS-7 [4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy)pyrimidine hydrochloride], on ischemia-induced fodrin breakdown was examined both in vitro and in vivo. The fodrin breakdown was measured by western blot followed by a densitometric analysis. In slices of the rat cerebral cortex, a pronounced fodrin breakdown was observed under hypoxic and hypoglycemic conditions. The enhancement of fodrin breakdown was completely blocked by omission of extracellular Ca2+ and significantly inhibited by calpain inhibitors such as E-64 and calpain inhibitor-I, thereby suggesting that the fodrin breakdown induced by hypoxia/hypoglycemia is due to the activation of Ca2+-stimulated neutral protease calpain. NS-7 (1-30 microM) produced a concentration-dependent inhibition of hypoxia/hypoglycemia-induced fodrin breakdown. In rats with unilateral middle cerebral artery occlusion (MCAO), a pronounced fodrin breakdown was observed in the cerebral cortex and striatum, although the time course for the development of the fodrin breakdown was much slower in the cerebral cortex than in the striatum. NS-7 (0.5 mg/kg i.v.), when injected immediately after MCAO, suppressed not only the fodrin breakdown but also the infarction in the cerebral cortex. From these results it is suggested that inhibition of calpain activation is implicated in the neuroprotective action of NS-7.

Topics: Animals; Arterial Occlusive Diseases; Brain Ischemia; Calcium; Calpain; Carrier Proteins; Cerebral Cortex; Cerebral Infarction; Enzyme Activation; Glucose; Hypoglycemia; Hypoxia; Male; Microfilament Proteins; Neostriatum; Nerve Tissue Proteins; Neuroprotective Agents; Organ Culture Techniques; Oxygen; Piperazines; Pyrimidines; Rats; Rats, Sprague-Dawley

Ischemia-induced elevation of intracellular calcium triggers a cascade of events which is considered to play a major role in neuronal death. One candidate to participate in this process is the calcium-sensitive protease, calpain. This protease is activated by calcium, and is capable of degrading critical cytoskeletal and regulatory proteins. In order to further elucidate the role of calpain in focal ischemic damage, the present study investigated the proteolysis of spectrin, a preferred substrate for calpain, in response to transient focal ischemia. Ischemia was induced by occluding reversibly both carotid arteries and the left middle cerebral artery for three hours in Sprague-Dawley rats. Western blotting techniques were used to identify and quantify the amounts of spectrin breakdown products (BDPs) in neocortical samples from the area destined for infarction, the peri-infarct area, and the contralateral hemisphere. Substantial increases in spectrin proteolysis were observed within the first few hours of ischemia in the areas that will undergo infarction. The increase in spectrin BDPs in these areas reached a plateau around the end of the 3 h ischemic period. In the peri-infarct zone, the levels of spectrin BDPs increased in a biphasic manner. A small to moderate increase was observed by the second hour of ischemia, followed by a larger increase between the 6th and 24th hours post-ischemia. The contralateral neocortex showed a significant increase in BDPs at 2 h after the initiation of ischemia. A smaller increase in BDPs was observed thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Analysis of Variance; Animals; Calcium; Calpain; Cerebral Cortex; Cerebral Infarction; Cytoskeletal Proteins; Enzyme Activation; Ischemic Attack, Transient; Male; Rats; Rats, Sprague-Dawley; Reference Values; Reperfusion; Spectrin; Time Factors

Experiments were conducted to determine whether a potent, reversible calpain inhibitor could reduce the cortical ischemic brain damage associated with focal ischemia in the rat. AK275 (Z-Leu-Abu-CONH-CH2CH3), the active isomer of the diastereomeric mixture, CX275, was employed in conjunction with a novel method of perfusing drug directly onto the infarcted cortical surface. This protocol reduced or eliminated numerous, nonspecific pharmacokinetic, hemodynamic, and other potentially confounding variables that might complicate interpretation of any drug effect. Focal ischemia was induced using a variation of the middle cerebral artery occlusion method. These studies demonstrated a reliable and robust neuroprotective effect of AK275 over the concentration range of 10 to 200 microM (perfused supracortically at 4 microliters/h for 21 h). Moreover, a 75% reduction in infarct volume was observed when initiation of drug treatment was delayed for 3 h postocclusion. Our data further support an important role of calpain in ischemia-induced neuropathology and suggest that calpain inhibitors may provide a unique and potentially powerful means of treating stroke and other ischemic brain incidents.

Topics: Animals; Body Temperature; Brain; Brain Damage, Chronic; Brain Ischemia; Calpain; Cerebral Infarction; Dipeptides; Dose-Response Relationship, Drug; Male; Rats; Rats, Sprague-Dawley; Reperfusion; Time Factors

Excessive elevation of intracellular calcium and uncontrolled activation of calcium-sensitive events are believed to play a central role in ischemic neuronal damage. Calcium-activated proteolysis by calpain is a candidate to participate in this form of pathology because it is activated under ischemic conditions and its activation results in the degradation of crucial cytoskeletal and regulatory proteins. The present studies examined the effects of a cell-penetrating inhibitor of calpain on the pathological outcome after transient focal ischemia in the brain.. Twenty-five male Sprague-Dawley rats were divided into four groups: a saline-treated group, a vehicle-treated group, and two calpain inhibitor-treated groups (Cbz-Val-Phe-H; 30-mg/kg and 60-mg/kg cumulative doses). Ischemia was induced by occluding the left middle cerebral artery and both common carotid arteries for 3 hours followed by reperfusion. Animals were killed 72 hours after surgery, and quantitative measurements of infarction volumes were performed using histological techniques. Eight additional rats were killed 30 minutes after ischemia and examined for the extent of proteolysis using immunoblot techniques. A final group of 12 animals was decapitated after injection of vehicle or calpain inhibitor, and the proteolytic response was measured after 60 minutes of total ischemia.. Rats treated with Cbz-Val-Phe-H exhibited significantly smaller volumes of cerebral infarction than saline-treated or vehicle-treated control animals. Intravenous injections of cumulative doses of 30 mg/kg or 60 mg/kg of Cbz-Val-Phe-H were effective in reducing infarction, edema, and calcium-activated proteolysis. The proteolytic response to postdecapitation ischemia was also reduced by the calpain inhibitor.. These results demonstrate the neuroprotective effect of a cell-penetrating calpain inhibitor when administered systemically. The findings suggest that targeting intracellular, calcium-activated mechanisms, such as proteolysis, represents a viable therapeutic strategy for limiting neurological damage after ischemia.

Topics: Analysis of Variance; Animals; Brain Edema; Calpain; Cerebral Infarction; Dipeptides; Disease Models, Animal; Ischemic Attack, Transient; Male; Rats; Rats, Sprague-Dawley