Minzhi Chen

Caspase‐3 Mediated Neuronal Death After Traumatic Brain Injury in Rats

Abstract: 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.

Early Detection of DNA Strand Breaks in the Brain After Transient Focal Ischemia: Implications for the Role of DNA Damage in Apoptosis and Neuronal Cell Death

Abstract: Using in situ DNA polymerase I‐mediated biotin‐dATP nick‐translation (PANT) and terminal deoxynucleotidyl‐transferase‐mediated dUTP nick end‐labeling (TUNEL), we investigated the evolution of DNA strand breaks, a marker of DNA damage, in rat brain after 1 h of middle cerebral artery occlusion and various durations of reperfusion. DNA single‐strand breaks (SSBs) detected by PANT were present in neurons after as little as 1 min of reperfusion. Numbers of neurons containing an SSB increased progressively in the ischemic core but decreased in the ischemic penumbra after 1 h of reperfusion. DNA double‐strand breaks (DSBs) detected by TUNEL were first seen in neurons after 1 h of reperfusion, and their numbers then increased progressively in the ischemic core, with a regional distribution similar to that of SSBs. However, the number of SSB‐containing cells was greater than that of DSB‐containing cells at all time points tested. SSB‐containing cells detected within the first hour of reperfusion were exclusively neuronal and exhibited normal nuclear morphology. At 16–72 h of reperfusion, many SSB‐ and DSB‐containing cells, including both neurons and astrocytes, showed morphological changes consistent with apoptosis. Gel electrophoresis of DNA isolated from the ischemic core showed DNA fragmentation at 24 h, when both SSBs and DSBs were present, but not at 1 h, when few DSBs were detected. These results suggest that damage to nuclear DNA is an early event after neuronal ischemia and that the accumulation of unrepaired DNA SSBs may contribute to delayed ischemic neuronal death, perhaps by triggering apoptosis.

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‐1β 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.—Clark, R. S. B., Kochanek, P. M., Chen, M., Watkins, S. C., Marion, D. W., Chen, J., Hamilton, R. L., Loeffert, J. E., Graham, S. H. Increases in Bcl‐2 and cleavage of caspase‐1 and caspase‐3 in human brain after head injury. FASEB J. 13, 813–821 (1999)

Inducible nitric oxide synthase is an endogenous neuroprotectant after traumatic brain injury in rats and mice

Nitric oxide (NO) derived from the inducible isoform of NO synthase (iNOS) is an inflammatory product implicated both in secondary damage and in recovery from brain injury. To address the role of iNOS in experimental traumatic brain injury (TBI), we used 2 paradigms in 2 species. In a model of controlled cortical impact (CCI) with secondary hypoxemia, rats were treated with vehicle or with 1 of 2 iNOS inhibitors (aminoguanidine and L-N-iminoethyl-lysine), administered by Alzet pump for 5 days and 1.5 days after injury, respectively. In a model of CCI, knockout mice lacking the iNOS gene (iNOS–/–) were compared with wild-type (iNOS+/+) mice. Functional outcome (motor and cognitive) during the first 20 days after injury, and histopathology at 21 days, were assessed in both studies. Treatment of rats with either of the iNOS inhibitors after TBI significantly exacerbated deficits in cognitive performance, as assessed by Morris water maze (MWM) and increased neuron loss in vulnerable regions (CA3 and CA1) of hippocampus. Uninjured iNOS+/+ and iNOS–/– mice performed equally well in both motor and cognitive tasks. However, after TBI, iNOS–/– mice showed markedly worse performance in the MWM task than iNOS+/+ mice. A beneficial role for iNOS in TBI is supported.

Inducible nitric oxide synthase expression in cerebrovascular smooth muscle and neutrophils after traumatic brain injury in immature rats

The inflammatory response after traumatic brain injury (TBI) includes cytokine production, leukocyte infiltration, and microglial activation. Production of nitric oxide by inducible nitric oxide synthase (iNOS) occurs during acute inflammation outside of the CNS and in models of cerebral ischemia, and therefore may contribute to the inflammatory response after TBI. The purpose of this study was to localize and define the time course of iNOS expression after TBI in the immature rat. Immature Wistar rats (age 3.5-4.5 wk) were anesthetized and subjected to percussive trauma to the right parietal cortex. Nontraumatized rats were used as controls (n = 7). At 2, 24, 48, or 168 h (n = 3/group) posttrauma rats were killed by perfusion fixation. Brains were removed, frozen, sectioned, immunostained with antibodies against iNOS and glial fibrillary acidic protein (GFAP, a marker specific for astrocytes), and imaged using fluorescent detection systems. There was no detectable expression of iNOS in control brains. At 2 h, minimal cerebrovascular iNOS expression was seen in the peritrauma area. At 24 and 48 h, there was marked peritrauma cerebrovascular iNOS expression that appeared to be restricted to vascular smooth muscle cells and infiltrated leukocytes. Further dual-immunolabeling showed that the leukocytes expressing iNOS were predominantly neutrophils. At 168 h, iNOS expression was no longer detectable. iNOS was not detectable in GFAP-positive cells. The prominent expression of iNOS protein after TBI in cerebrovascular smooth muscle cells and infiltrated neutrophils suggests that iNOS may play a role in cerebrovascular disturbances and secondary brain injury after trauma.

Detection of single- and double-strand DNA breaks after traumatic brain injury in rats: comparison of in situ labeling techniques using DNA polymerase I, the Klenow fragment of DNA polymerase I, and terminal deoxynucleotidyl transferase.

DNA damage is a common sequela of traumatic brain injury (TBI). Available techniques for the in situ identification of DNA damage include DNA polymerase I-mediated biotin-dATP nick-translation (PANT), the Klenow fragment of DNA polymerase I-mediated biotin-dATP nick-end labeling (Klenow), and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). While TUNEL has been widely utilized to detect primarily double-strand DNA breaks, the use of PANT to detect primarily single-strand DNA breaks and Klenow to detect both single- and double-strand DNA breaks has not been reported after TBI. Accordingly, coronal brain sections from naive rats and rats at 0, 0.5, 1, 2, 6, 24, and 72 h (n = 3-5/group) after controlled cortical impact with imposed secondary insult were processed using the PANT, Klenow, and TUNEL methods. Cells with DNA breaks were detected by PANT in the ipsilateral hemisphere as early as 0.5 h after injury and were maximal at 6 h (cortex = 66.3+/-15.8, dentate gyrus 58.6+/-12.8, CA1 = 15.8+/-5.9, CA3 = 12.8+/-4.2 cells/x 400 field, mean +/- SEM, all p < 0.05 versus naive). Cells with DNA breaks were detected by Klenow as early as 30 min and were maximal at 24 h (cortex = 56.3+/-14.3, dentate gyrus 78.0+/-16.7, CA1 = 25.8+/-4.7, CA3 = 29.3+/-15.1 cells/x 400 field, all p < 0.05 versus naive). Cells with DNA breaks were not detected by TUNEL until 2 h and were maximal at 24 h (cortex = 47.7+/-21.4, dentate gyrus 63.0+/-11.9, CA1 = 5.6+/-5.4, CA3 = 6.9+/-3.7 cells/x 400 field, cortex and dentate gyrus p < 0.05 versus naive). Dual-label immunofluorescence revealed that PANT-positive cells were predominately neurons. These data demonstrate that TBI results in extensive DNA damage, which includes both single- and double-strand breaks in injured cortex and hippocampus. The presence of multiple types of DNA breaks implicate several pathways in the evolution of DNA damage after TBI.

Alterations in inducible 72‐kDa heat shock protein and the chaperone cofactor BAG‐1 in human brain after head injury

The stress response in injured brain is well characterized after experimental ischemic and traumatic brain injury (TBI); however, the induction and regulation of the stress response in humans after TBI remains largely undefined. Accordingly, we examined injured brain tissue from adult patients (n = 8) that underwent emergent surgical decompression after TBI, for alterations in the inducible 72‐kDa heat shock protein (Hsp70), the constitutive 73‐kDa heat shock protein (Hsc70), and isoforms of the chaperone cofactor BAG‐1. Control samples (n = 6) were obtained postmortem from patients dying of causes unrelated to CNS trauma. Western blot analysis showed that Hsp70, but not Hsc70, was increased in patients after TBI versus controls. Both Hsp70 and Hsc70 coimmunoprecipitated with the cofactor BAG‐1. The 33 and 46, but not the 50‐kDa BAG‐1 isoforms were increased in patients after TBI versus controls. The ratio of the 46/33‐kDa isoforms was increased in TBI versus controls, suggesting negative modulation of Hsp70/Hsc70 protein refolding activity in injured brain. These data implicate induction of the stress response and its modulation by the chaperone cofactor and Bcl‐2 family member BAG‐1, after TBI in humans.