calpain and triphenyltetrazolium

Ischemic stroke is one of the leading causes of morbidity and mortality. Treatment options are limited and only a minority of patients receive acute interventions. Understanding the mechanisms that mediate neuronal injury and death may identify targets for neuroprotective treatments. Here we show that the aberrant activity of the protein kinase Cdk5 is a principal cause of neuronal death in rodents during stroke. Ischemia induced either by embolic middle cerebral artery occlusion (MCAO) in vivo or by oxygen and glucose deprivation in brain slices caused calpain-dependent conversion of the Cdk5-activating cofactor p35 to p25. Inhibition of aberrant Cdk5 during ischemia protected dopamine neurotransmission, maintained field potentials, and blocked excitotoxicity. Furthermore, pharmacological inhibition or conditional knock-out (CKO) of Cdk5 prevented neuronal death in response to ischemia. Moreover, Cdk5 CKO dramatically reduced infarctions following MCAO. Thus, targeting aberrant Cdk5 activity may serve as an effective treatment for stroke.

Topics: Animals; Calpain; Cell Death; Corpus Striatum; Cyclin-Dependent Kinase 5; Disease Models, Animal; Estrogens; Female; Glial Fibrillary Acidic Protein; Hypoxia; In Vitro Techniques; Infarction, Middle Cerebral Artery; Male; Mice, Knockout; Nerve Tissue Proteins; Nervous System Diseases; Neurons; Phosphotransferases; Rats; Rats, Sprague-Dawley; Tetrazolium Salts; Time Factors; Tissue Plasminogen Activator

Calpains and cathepsins are two families of proteases that play an important role in ischemic cell death. In this study, we investigated the effect of E64d, a mu-calpain and cathepsin B inhibitor, in the prevention of neuronal and endothelial apoptotic cell death after focal cerebral ischemia in rats. Rats underwent 2 hr of transient focal ischemia from middle cerebral artery occlusion (MCAO) and were sacrificed 24 hr later. E64d (5 mg/ kg intraperitoneally) was administered 30 min before MCAO. Assessment included neurological function, infarction volume, brain water content, blood-brain barrier permeability, histology, and immunohistochemistry. The E64d-treated rats had significant brain protection against ischemic damage. We observed a reduction of infarction volume, brain edema, and improved neurological scores in E64d-treated rats compared with the nontreated control. Furthermore, there was a remarkable reduction in both proteases and caspase-3 activation and apoptotic changes in both neurons and endothelial cells in E64d-treated rats. These results suggest that E64d protects the brain against ischemic/reperfusion injury by attenuating neuronal and endothelial apoptosis.

Topics: Analysis of Variance; Animals; Apoptosis; Brain Edema; Brain Infarction; Brain Ischemia; Calpain; Caspase 3; Caspases; Cathepsins; Endothelium, Vascular; Functional Laterality; Gene Expression; Immunohistochemistry; In Situ Nick-End Labeling; Infarction, Middle Cerebral Artery; Leucine; Male; Neurologic Examination; Neurons; Neuroprotective Agents; Phosphopyruvate Hydratase; Rats; Rats, Sprague-Dawley; Tetrazolium Salts

Studies of ischemia/reperfusion (I/R) injury and preconditioning have shown that ion homeostasis, particularly calcium homeostasis, is critical to limiting tissue damage. However, the relationship between ion homeostasis and specific cell death pathways has not been investigated in the context of I/R. Previously we reported that calpain cleaved Bid in the absence of detectable caspase activation (1). In this study, we have shown that an inhibitor of the sodium/hydrogen exchanger prevented calpain activation after I/R. Calpain inhibitors prevented cleavage of Bid as well as the downstream indices of cell death, including DNA strand breaks, creatine kinase (CK) release, and infarction measured by triphenyl tetrazolium chloride (TTC) staining. In contrast, the broad spectrum caspase inhibitor IDN6734 was not protective in this model. To ascertain whether mitochondrial dysfunction downstream of these events was a required step, we utilized a peptide corresponding to residues 4-23 of Bcl-x(L) conjugated to the protein transduction domain of HIV TAT (TAT-BH4), which has been shown to protect mitochondria against Ca2+-induced deltaPsi(m) loss (2). TAT-BH4 attenuated CK release and loss of TTC staining, demonstrating the role of mitochondria and a pro-apoptotic Bcl-2 family member in the process leading to cell death. We propose the following pathway. (i) Reperfusion results in sodium influx followed by calcium accumulation. (ii) This leads to calpain activation, which in turn leads to Bid cleavage. (iii) Bid targets the mitochondria, causing dysfunction and release of pro-apoptotic factors, resulting in DNA fragmentation and death of the cell. Ischemia/reperfusion initiates a cell death pathway that is independent of caspases but requires calpain and mitochondrial dysfunction.

Topics: Animals; BH3 Interacting Domain Death Agonist Protein; Blotting, Western; Calcium; Calpain; Carrier Proteins; Coloring Agents; Creatine Kinase; DNA; DNA Fragmentation; Heart; Male; Microscopy, Fluorescence; Mitochondria; Models, Biological; Necrosis; Protein Binding; Rabbits; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tetrazolium Salts; Time Factors

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