benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Infarction--Middle-Cerebral-Artery

Necroptosis contributes to ischemia-induced brain injury. Tumor necrosis factor (TNF) receptor associated factor 2 (TRAF2) has been reported to suppress necroptotic cell death under several pathological conditions. In this study, we investigated the role of TRAF2 in experimental stroke using a mouse middle cerebral artery occlusion (MCAO) model and in vitro cellular models. TRAF2 expression in the ischemic brain was assessed with western blot and real-time RT-PCR. Gene knockdown of TRAF2 by lentivirus was utilized to investigate the role of TRAF2 in stroke outcomes. The expression of TRAF2 was significantly induced in the ischemic brain at 24 h after reperfusion, and neurons and microglia were two of the cellular sources of TRAF2 induction. Striatal knockdown of TRAF2 increased infarction size, cell death, microglial activation and the expression of pro-inflammatory markers at 24 h after reperfusion. TRAF2 expression and necroptosis were induced in mouse primary microglia treated with conditioned medium collected from neurons subject to oxygen and glucose deprivation (OGD) and in TNFα-treated mouse hippocampal neuronal HT-22 cells in the presence of the pan-caspase inhibitor Z-VAD. In addition, TRAF2 knockdown exacerbated microglial cell death and neuronal cell death under these conditions. Moreover, pre-treatment with a specific necroptosis inhibitor necrostatin-1 (nec-1) suppressed the cell death exacerbated by TRAF2 knockdown in the brain following MCAO, indicating that TRAF2 impacted ischemic brain damage through necroptosis mechanism. Taken together, our results demonstrate that TRAF2 is a novel regulator of cerebral ischemic injury.

Topics: Amino Acid Chloromethyl Ketones; Animals; Brain; Cell Hypoxia; Cells, Cultured; Culture Media, Conditioned; Disease Models, Animal; GTPase-Activating Proteins; Infarction, Middle Cerebral Artery; Male; Mice; Mice, Inbred ICR; Microglia; Necroptosis; Protein Kinases; Reperfusion Injury; RNA Interference; RNA, Small Interfering; TNF Receptor-Associated Factor 2; Tumor Necrosis Factor-alpha

Necroptosis, a novel type of programmed cell death, is involved in stroke-induced ischemic brain injury. Although studies have sought to explore the mechanisms of necroptosis, its signaling pathway has not yet to be completely elucidated. Thus, we used oxygen-glucose deprivation (OGD) and middle cerebral artery occlusion (MCAO) models mimicking ischemic stroke (IS) conditions to investigate mechanisms of necroptosis. We found that OGD and MCAO induced cell death, local brain ischemia and neurological deficit, while zVAD-fmk (zVAD, an apoptotic inhibitor), GSK'872 (a receptor interacting protein kinase-3 (RIP3) inhibitor), and combined treatment alleviated cell death and ischemic brain injury. Moreover, OGD and MCAO upregulated protein expression of the triggers of necroptosis: receptor interacting protein kinase-1 (RIP1), RIP3 and mixed lineage kinase domain-like protein (MLKL). The upregulation of these proteins was inhibited by GSK'872, combination treatments and RIP3 siRNA but not zVAD treatment. Intriguingly, hypoxia-inducible factor-1 alpha (HIF-1α), an important transcriptional factor under hypoxic conditions, was upregulated by OGD and MCAO. Similar to their inhibitory effects on aforementioned proteins upregulation, GSK'872, combination treatments and RIP3 siRNA decreased HIF-1α protein level. These findings indicate that necroptosis contributes to ischemic brain injury induced by OGD and MCAO and implicate HIF-1α, RIP1, RIP3, and MLKL in necroptosis.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Brain Injuries; Brain Ischemia; Cell Line; Down-Regulation; Glucose; GTPase-Activating Proteins; Hypoxia-Inducible Factor 1, alpha Subunit; Infarction, Middle Cerebral Artery; Male; Mice, Inbred C57BL; Necrosis; Oxygen; Receptor-Interacting Protein Serine-Threonine Kinases; Reperfusion Injury; RNA, Small Interfering

It has been reported that ischemic insult increases the formation of autophagosomes and activates autophagy. However, the role of autophagy in ischemic neuronal damage remains elusive. This study was taken to assess the role of autophagy in ischemic brain damage. Focal cerebral ischemia was introduced by permanent middle cerebral artery occlusion (pMCAO). Activation of autophagy was assessed by morphological and biochemical examinations. To determine the contribution of autophagy/lysosome to ischemic neuronal death, rats were pretreated with a single intracerebral ventricle injection of the autophagy inhibitors 3-methyl-adenine (3-MA) and bafliomycin A1 (BFA) or the cathepsin B inhibitor Z-FA-fmk after pMCAO. The effects of 3-MA and Z-FA-fmk on brain damage, expression of proteins involved in regulation of autophagy and apoptosis were assessed with 2,3,5-triphenyltetrazolium chloride (TTC) staining and immunoblotting. The results showed that pMACO increased the formation of autophagosomes and autolysosomes, the mRNA and protein levels of LC3-II and the protein levels of cathepsin B. 3-MA, BFA and Z-FA-fmk significantly reduced infarct volume, brain edema and motor deficits. The neuroprotective effects of 3-MA and Z-FA-fmk were associated with an inhibition on ischemia-induced upregulation of LC3-II and cathepsin B and a partial reversion of ischemia-induced downregulation of cytoprotective Bcl-2. These results demonstrate that ischemic insult activates autophagy and an autophagic mechanism may contribute to ischemic neuronal injury. Thus, autophagy may be a potential target for developing a novel therapy for stroke.

Topics: Adenine; Amino Acid Chloromethyl Ketones; Animals; Autophagy; Biomarkers; Brain; Brain Ischemia; Cathepsin B; Cysteine Proteinase Inhibitors; Humans; Infarction, Middle Cerebral Artery; Lysosomes; Male; Neurons; Neuroprotective Agents; Phagosomes; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Signal Transduction

A series of isoquinoline-1,3,4-trione derivatives were identified as novel and potent inhibitors of caspase-3 through structural modification of the original compound from high-throughput screening. Various analogues (2, 6, 9, 13, and 14) were synthesized and identified as caspase inhibitors, and the introduction of a 6-N-acyl group (compound 13) greatly improved their activity. Some of them showed low nanomolar potency against caspase-3 in vitro (for example, for 6k, IC50 = 40 nM) and significant protection against apoptosis in a model cell system. Additionally, compound 13f demonstrated a dose-dependent decrease in infarct volume in the transient MCA occlusion stroke model. The present small-molecule caspase-3 inhibitor with novel structures different from structures of known caspase inhibitors revealed a new direction for therapeutic strategies directed against diseases involving abnormally up-regulated apoptosis.

Topics: Animals; Apoptosis; Arterial Occlusive Diseases; Caspase 3; Caspase Inhibitors; Caspases; Drug Design; Humans; Infarction, Middle Cerebral Artery; Isoquinolines; Jurkat Cells; Male; Rats; Rats, Sprague-Dawley; Structure-Activity Relationship; Succinates

Recent data indicate that the neuroprotective efficacy of isoflurane is not sustained. Delayed neuronal death, mediated in part by apoptosis, contributes to the gradual increase in the size of the infarction. These data suggest that isoflurane may not be able to inhibit delayed neuronal death. The prevention of apoptosis by a caspase inhibitor might provide neuroprotection in addition to that provided by isoflurane. The current study was conducted to determine whether isoflurane-mediated neuroprotection can be made more durable with the administration of z-VAD-fmk, a nonspecific caspase inhibitor.. Fasted Wister rats were allocated to awake-zVAD, awake-vehicle, isoflurane-zVAD, or isoflurane-vehicle groups (n = 16/group). Animals were subjected to focal ischemia for 60 min by filament occlusion of the middle cerebral artery. In the awake groups, isoflurane was discontinued after occlusion of the middle cerebral artery. In the isoflurane groups, isoflurane anesthesia was maintained at 1.5 minimum alveolar concentration during occlusion of the middle cerebral artery. Before and after ischemia, daily injections of z-VAD-fmk or vehicle were administered into the lateral cerebral ventricle for 14 days. Neurologic assessment was performed 14 days after ischemia. The volume of cerebral infarction and the number of intact neurons in the periinfarct cortex were determined by image analysis of hematoxylin and eosin-stained coronal brain sections.. Infarction volume was less in the isoflurane-zVAD group (23 +/- 11 mm, mean +/- SD) than in isoflurane-vehicle, awake-vehicle, and awake-zVAD groups (82 +/- 31, 86 +/- 31, and 59 +/- 25 mm, respectively; P < 0.05). In comparison with the awake-vehicle and isoflurane-vehicle groups, the administration of z-VAD-fmk significantly decreased infarction volume (P < 0.05). The infarction volume between the awake-vehicle and isoflurane-vehicle groups was not different. The number of intact neurons within the periinfarct cortex was significantly less in the awake-vehicle group than in the other three groups (P < 0.05). The isoflurane-zVAD group demonstrated better neurologic function than the awake-vehicle group (P < 0.05).. These findings are consistent with the premise that ongoing delayed neuronal death, in part mediated by apoptosis, contributes to the progression of cerebral infarction during the recovery period, and its inhibition can provide sustained neuroprotection.

Topics: Amino Acid Chloromethyl Ketones; Anesthetics, Inhalation; Animals; Blood Gas Analysis; Brain; Brain Ischemia; Caspase Inhibitors; Enzyme Inhibitors; Hemodynamics; Infarction, Middle Cerebral Artery; Isoflurane; Male; Neurons; Neuroprotective Agents; Rats; Rats, Wistar

Focal ischemia in the cerebral cortex results in acute and delayed cell death in the ischemic cortex and non-ischemic thalamus. We examined the hypothesis that neurons in ischemic and non-ischemic regions died from different mechanisms; specifically, we tested whether a mixed form of cell death containing both necrotic and apoptotic changes could be identified in individual cells. Focal barrel cortex ischemia in rats was induced by occlusion of small branches of the middle cerebral artery (MCA) corresponding to the barrel cortex, local blood flow was measured by quantitative autoradiography. Cell death was visualized by 2,3,5-triphenyltetrazolium chloride (TTC) staining, hematoxylin-eosin (H&E) staining, the terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and caspase-3 staining 1 to 10 days after the ischemia. Electron microscopy was used for ultrastructural examination. Cell death occurred in the ipsilateral cortex 24 h after ischemia, followed by selective neuronal death in the ventrobasal (VB) thalamus 3 days later. TUNEL positive neurons were found in these two regions, but with striking morphological differences, designated as type I and type II TUNEL positive cells. The type I TUNEL positive cells in the ischemic cortex underwent necrotic changes. The type II TUNEL positive cells in the thalamus and the cortex penumbra region represented a hybrid death, featured by concurrent apoptotic and necrotic alterations in individual cells, including marked caspase-3 activation, nuclear condensation/fragmentation, but with swollen cytoplasm, damaged organelles and deteriorated membranes. Cell death in the thalamus and the cortex penumbra were attenuated by delayed administration of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (Z-VAD-FMK). Our data suggest that TUNEL staining should be evaluated with morphological changes, the hybrid death but not typical apoptosis occurs in the penumbra region and non-ischemic thalamus after cerebral ischemia.

Topics: Amino Acid Chloromethyl Ketones; Analysis of Variance; Animals; Apoptosis; Autoradiography; Brain Ischemia; Caspase 3; Caspases; Cell Count; Cerebral Cortex; Functional Laterality; Infarction, Middle Cerebral Artery; Injections, Intraventricular; Microscopy, Electron; Necrosis; Neurons; Neuroprotective Agents; Rats; Rats, Wistar; Regional Blood Flow; Staining and Labeling; Tetrazolium Salts; Thalamus; Time Factors

The present study investigated the activation of extracellular-signal-regulated kinase (ERK) and the potential role of interleukin-1 beta (IL-1beta) in the brain's response to focal brain ischemia in the permanent middle cerebral artery occlusion (pMCAO) model. Phosphorylated ERK p44 and p42 were increased time-dependently and significantly 18- and 28-fold, respectively, at 24-h post-pMCAO. Similarly, IL-1beta protein levels were significantly increased with the peak at 24 h in the lesioned core of the ischemic hemisphere compared to the contralateral side. Previous studies using various stimuli have shown ERK-dependent IL-1 induction. The results from our study suggest that this relation may also exist in vivo in ischemic brain tissue. Based on the progressive nature of IL-1 induction, we hypothetized that inhibition of interleukin-converting enzyme (ICE) could provide an extended time-window for neuroprotection. Therefore, we applied N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD x fmk), an ICE blocker 3 or 6 h after pMCAO. Reductions of infarct volume, however, were not observed. Taken together with previous results, where we showed protective activity of zVAD x fmk when given immediately after pMCAO, we conclude that the time window for zVAD x fmk is less than 3 h.

Topics: Amino Acid Chloromethyl Ketones; Animals; Brain Ischemia; Caspase 1; Caspase Inhibitors; Enzyme Activation; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Immunoblotting; Infarction, Middle Cerebral Artery; Interleukin-1; JNK Mitogen-Activated Protein Kinases; Kinetics; Ligation; Magnetic Resonance Imaging; Male; MAP Kinase Kinase 4; Middle Cerebral Artery; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Rats; Rats, Inbred F344

These studies have addressed the role of caspase-3 activation in neuronal death after cerebral ischemia in different animal models. The authors were unable to show activation of procaspase-3 measured as an induction of DEVDase (Asp-Glu-Val-Asp) activity after focal or transient forebrain ischemia in rats. DEVDase activity could not be induced in the cytosolic fraction of the brain tissue obtained from these animals by exogenous cytochrome c/dATP and Ca2+. However, the addition of granzyme B to these cytosolic fractions resulted in a significant activation of DEVDase, confirming that the conditions were permissive to analyze proteolytic cleavage of the DEVD-AMC (7-amino-4-methyl-coumarin) substrate. Consistent with these findings, zVal-Ala-Asp-fluoromethylketone administered after focal ischemia did not have a neuroprotective effect. In contrast to these findings, a large increase in DEVDase activity was detected in a model of hypoxic-ischemia in postnatal-day-7 rats. Furthermore, in postnatal-day-7 animals treated with MK-801, in which it has been suggested that excessive apoptosis is induced, the authors were unable to detect activation of DEVDase activity but were able to induce it in vitro by the addition of cytochrome c/dATP and Ca2+ to the cytosolic fraction. Analysis of cytochrome c distribution did not provide definitive evidence for selective cytochrome c release in the permanent focal ischemia model, whereas in the transient model a small but consistent amount of cytochrome c was found in the cytosolic fraction. However, in both models the majority of cytochrome c remained associated with the mitochondrial fraction. In conclusion, the authors were unable to substantiate a role of mitochondrially derived cytochrome c and procaspase-3 activation in ischemia-induced cell death in adult brain, but did see a clear induction of caspase-3 in neonatal hypoxia.

Topics: Amino Acid Chloromethyl Ketones; Animals; Animals, Newborn; Brain; Brain Ischemia; Caspase 3; Caspases; Cell Death; Cytochrome c Group; Dizocilpine Maleate; Enzyme Activation; Granzymes; Humans; Infarction, Middle Cerebral Artery; Male; Mice; Mice, Inbred C57BL; Neurons; Neuroprotective Agents; Peptide Hydrolases; Protein Precursors; Rats; Rats, Sprague-Dawley; Rats, Wistar; Serine Endopeptidases

Marrow stromal cells (MSCs) transplantation into brain has been employed to treat experimental ischemia. However, MSCs undergo apoptosis and few survive in the ischemic brain. We test the hypotheses that coadministration of bone marrow cells (BMCs) with a cell-permeable inhibitor of caspases, Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD), into the ischemic boundary zone (IBZ) of brain promotes BMCs survival and improve outcome. Experimental groups consist of: 24 h after MCAo, either phosphate-buffered saline (PBS, n=4), dead BMC (n=4), fresh BMC (n=10), Z-VAD only (n=4), or BMC with Z-VAD (n=6) were intracerebrally injected. BMCs were harvested from donor adult rats labeled with bromodeoxyuridine (BrdU). Rats were subjected to an adhesive-removal somatosensory and motor-rotarod functional tests before MCAo and at 1 and 7 days after MCAo. Rats treated with a combination of Z-VAD and BMCs exhibited significant improvement in the adhesive-removal test at 7 days compared with the control group (combined MCAo+PBS and MCAo+dead BMC) (p<0.01), and the numbers of BrdU-BMC increased (p<0.05) and apoptotic cells decreased (p<0.05) compared with BMC alone transplantation. Our data suggest that intracerebral coadministration of BMC with Z-VAD enhances the survival of grafted BMC and improves neurological functional recovery after MCAo.

Topics: Amino Acid Chloromethyl Ketones; Animals; Behavior, Animal; Bone Marrow Transplantation; Caspase Inhibitors; Cell Count; Cysteine Proteinase Inhibitors; Disease Models, Animal; Drug Administration Routes; Graft Survival; Infarction, Middle Cerebral Artery; Male; Neuroprotective Agents; Rats; Rats, Wistar; Recovery of Function; Stromal Cells; Treatment Outcome