bcl-2 Antisense treatment prevents induction of tolerance to focal ischemia in the rat brain.

In the rat, 60 minutes of transient ischemia to the middle cerebral artery results in infarction of the caudate putamen. Ischemic preconditioning with 20 minutes of transient focal ischemia produced tolerance (attenuated infarction volume) to 60 minutes of subsequent focal ischemia administered three days, five days, or seven days later. Western blots from tolerant caudate putamen demonstrated increased bcl-2 expression, maximum at 3 days and persisting through 7 days. Immunocytochemical examination found that bcl-2 was expressed in cells with both neuronal and nonneuronal morphology in striatum after preconditioning ischemia. bcl-2 antisense oligodeoxynucleotides (ODNs), bcl-2 sense ODNs, or artificial cerebrospinal fluid (CSF, vehicle) was infused into the lateral ventricle for the 72 hours between the 20-minute ischemic preconditioning and the 60-minute period of ischemia. Antisense ODN treatment reduced expression of bcl-2 in the striatum and blocked the induction of tolerance by preconditioning ischemia. Sense and CSF treatments had no effect on either bcl-2 expression or tolerance. In this model of induced tolerance to focal ischemia, bcl-2 appears to be a major determinant.

Suppression of endogenous bcl-2 expression by antisense treatment exacerbates ischemic neuronal death.

Previous studies have shown that overexpression of bcl-2 in transgenic mice or by viral vectors protects the brain against cerebral ischemia. However, it is not known whether bcl-2, which is endogenously expressed in response to ischemia, exerts a protective effect. To address this question, the authors blocked the endogenous expression of bcl-2 after ischemia using antisense oligodeoxynucleotides (ODN). Antisense, sense, scrambled ODN, or vehicles were infused in the lateral ventricle of the rat for 24 hours after 30 minutes of temporary middle cerebral artery occlusion. Twenty-four hours later the brains were removed and bcl-2 protein expression was assayed by Western blot. Antisense ODN, but not sense or scrambled ODN treatment, significantly inhibited bcl-2 protein expression after ischemia. Bcl-2 protein expression was also studied 24 hours after 60 minutes of temporary middle cerebral artery occlusion in vehicle and antisense ODN-treated rats. After 60 minutes of ischemia and vehicle treatment, bcl-2 was expressed in many neurons in the ventral cortical mantle and the medial striatum. After antisense ODN treatment there were few neurons in this region expressing bcl-2, instead most neurons TUNEL labeled. Treatment with the antisense ODN, but not sense ODN, increased infarction volume as determined by cresyl violet staining 72 hours after ischemia compared with vehicle controls. These results suggested that endogenously expressed bcl-2 promoted survival in ischemic neurons and was not simply an epiphenomenon in neurons already destined to live or die.

Apoptosis-suppressor gene bcl-2 expression after traumatic brain injury in rats.

Neuronal death after experimental traumatic brain injury (TBI) has features of both apoptosis and necrosis. Neurons in the peritrauma cortex, hippocampus, and dentate gyrus are particularly vulnerable. The apoptosis-suppressor gene bcl-2 is induced in brain after ischemia and epilepsy-induced injury and may serve to regulate neuronal death. We studied expression of bcl-2 mRNA and protein after experimental TBI in rats. To determine whether bcl-2 protein expression occurred in cells with evidence of apoptosis, triple-labeling studies were performed using (1) antibody against bcl-2, (2) bis-benzimide dye to examine gross nuclear morphology, and (3) terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling (TUNEL) to assess for DNA fragmentation. At 6 and 24 hr, bcl-2 mRNA was induced in ipsilateral peritrauma cortex, hippocampus, and dentate gyrus. By 72 hr the increase in bcl-2 mRNA was detected only in cortex. bcl-2 protein was induced at 8, 24, 72, and 168 hr in ipsilateral cortex and hippocampus. Cells expressing bcl-2 protein included neurons in the peritrauma cortex, hippocampus, hilus, and dentate gyrus. The gross nuclear morphology of neurons expressing bcl-2 appeared normal. Furthermore, biochemical evidence of DNA fragmentation, in a pattern characteristic of either apoptosis or necrosis, was seldom seen in neurons expressing bcl-2 protein (bcl-2 colocalized with TUNEL in 0-2% of TUNEL-positive cells observed). These data suggest that bcl-2 may play an important role in the regulation of neuronal death after TBI, and they support a role for bcl-2 as an inducible neuroprotective gene.

Interleukin-6 production in hemorrhagic shock is accompanied by neutrophil recruitment and lung injury.

Hemorrhagic shock (HS) initiates an inflammatory cascade that includes the production of cytokines and recruitment of neutrophils (PMN) and may progress to organ failure, inducing acute respiratory distress syndrome (ARDS). To examine the hypothesis that interleukin-6 (IL-6) contributes to PMN infiltration and lung damage in HS, we examined the lungs of rats subjected to unresuscitated and resuscitated HS for the production of IL-6 and activation of Stat3. Using semiquantitative RT-PCR, we found a striking increase in IL-6 mRNA levels only in resuscitated HS, with peak levels observed 1 h after initiation of resuscitation. Increased IL-6 protein expression was localized to bronchial and alveolar cells. Electrophoretic mobility shift assay of protein extracts from shock lungs exhibited an increase in Stat3 activation with kinetics similar to IL-6 mRNA. In situ DNA binding assay determined Stat3 activation predominantly within alveoli. Intratracheal instillation of IL-6 alone into normal rats resulted in PMN infiltration into lung interstitium and alveoli, marked elevation of bronchoalveolar lavage cellularity, and increased wet-to-dry ratio. These findings indicate that IL-6 production and Stat3 activation occur early in HS and may contribute to PMN-mediated lung injury, including ARDS after HS.

Increases in Bcl-2 and cleavage of caspase-1 and caspase-3 in human brain after head injury.

The bcl-2 and caspase families are important regulators of programmed cell death in experimental models of ischemic, excitotoxic, and traumatic brain injury. The Bcl-2 family members Bcl-2 and Bcl-xL suppress programmed cell death, whereas Bax promotes programmed cell death. Activated caspase-1 (interleukin-1beta converting enzyme) and caspase-3 (Yama/Apopain/Cpp32) cleave proteins that are important in maintaining cytoskeletal integrity and DNA repair, and activate deoxyribonucleases, producing cell death with morphological features of apoptosis. To address the question of whether these Bcl-2 and caspase family members participate in the process of delayed neuronal death in humans, we examined brain tissue samples removed from adult patients during surgical decompression for intracranial hypertension in the acute phase after traumatic brain injury (n=8) and compared these samples to brain tissue obtained at autopsy from non-trauma patients (n=6). An increase in Bcl-2 but not Bcl-xL or Bax, cleavage of caspase-1, up-regulation and cleavage of caspase-3, and evidence for DNA fragmentation with both apoptotic and necrotic morphologies were found in tissue from traumatic brain injury patients compared with controls. These findings are the first to demonstrate that programmed cell death occurs in human brain after acute injury, and identify potential pharmacological and molecular targets for the treatment of human head injury.

Caspase-3 mediated neuronal death after traumatic brain injury in rats.

During programmed cell death, activation of caspase-3 leads to proteolysis of DNA repair proteins, cytoskeletal proteins, and the inhibitor of caspase-activated deoxyribonuclease, culminating in morphologic changes and DNA damage defining apoptosis. The participation of caspase-3 activation in the evolution of neuronal death after traumatic brain injury in rats was examined. Cleavage of pro-caspase-3 in cytosolic cellular fractions and an increase in caspase-3-like enzyme activity were seen in injured brain versus control. Cleavage of the caspase-3 substrates DNA-dependent protein kinase and inhibitor of caspase-activated deoxyribonuclease and co-localization of cytosolic caspase-3 in neurons with evidence of DNA fragmentation were also identified. Intracerebral administration of the caspase-3 inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone (480 ng) after trauma reduced caspase-3-like activity and DNA fragmentation in injured brain versus vehicle at 24 h. Treatment with N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone for 72 h (480 ng/day) reduced contusion size and ipsilateral dorsal hippocampal tissue loss at 3 weeks but had no effect on functional outcome versus vehicle. These data demonstrate that caspase-3 activation contributes to brain tissue loss and downstream biochemical events that execute programmed cell death after traumatic brain injury. Caspase inhibition may prove efficacious in the treatment of certain types of brain injury where programmed cell death occurs.