Abolishing UCHL1's hydrolase activity exacerbates ischemia-induced axonal injury and functional deficits in mice.

Ubiquitin C-terminal hydrolase L1 (UCHL1) is a neuronal protein important in maintaining axonal integrity and motor function and may be important in the pathogenesis of many neurological disorders. UCHL1 may ameliorate acute injury and improve recovery after cerebral ischemia. In the current study, the hypothesis that UCHL1's hydrolase activity underlies its effect in maintaining axonal integrity and function is tested after ischemic injury. Hydrolase activity was inhibited by treatment with a UCHL1 hydrolase inhibitor or by employing knockin mice bearing a mutation in the hydrolase active site (C90A). Ischemic injury was induced by oxygen-glucose deprivation (OGD) in brain slice preparations and by transient middle cerebral artery occlusion (tMCAO) surgery in mice. Hydrolase activity inhibition increased restoration time and decreased the amplitude of evoked axonal responses in the corpus callosum after OGD. Mutation of the hydrolase active site exacerbated white matter injury as detected by SMI32 immunohistochemistry, and motor deficits as detected by beam balance and cylinder testing after tMCAO. These results demonstrate that UCHL1 hydrolase activity ameliorates white matter injury and functional deficits after acute ischemic injury and support the hypothesis that UCHL1 activity plays a significant role in preserving white matter integrity and recovery of function after cerebral ischemia.

Systemic treatment with ubiquitin carboxy terminal hydrolase L1 TAT protein ameliorates axonal injury and reduces functional deficits after traumatic brain injury in mice.

Traumatic brain injury (TBI) is often associated with axonal injury that leads to significant motor and cognitive deficits. Ubiquitin carboxy terminal hydrolase L1 (UCHL1) is highly expressed in neurons and loss of its activity plays an important role in the pathogenesis of TBI. Fusion protein was constructed containing wild type (WT) UCHL1 and the HIV trans-activator of transcription capsid protein transduction domain (TAT-UCHL1) that facilitates transport of the protein into neurons after systemic administration. Additional mutant proteins bearing cysteine to alanine UCHL1 mutations at cysteine 152 (C152A TAT-UCHL1) that prevents nitric oxide and reactive lipid binding of C152, and at cysteine 220 (C220A TAT-UCHL1) that inhibits farnesylation of the C220 site were also constructed. WT, C152A, and C220A TAT-UCHL1 proteins administered to mice systemically after controlled cortical impact (CCI) were detectable in brain at 1 h, 4 h and 24 h after CCI by immunoblot. Mice treated with C152A or WT TAT-UCHL1 decreased axonal injury detected by NF200 immunohistochemistry 24 h after CCI, but C220A TAT-UCHL1 treatment had no significant effect. Further study indicated that WT TAT-UCHL1 treatment administered 24 h after CCI alleviated axonal injury as detected by SMI32 immunoreactivity 7 d after CCI, improved motor and cognitive deficits, reduced accumulation of total and K48-linked poly-Ub proteins, and attenuated the increase of the autophagy marker Beclin-1. These results suggest that UCHL1 activity contributes to the pathogenesis of white matter injury, and that restoration of UCHL1 activity by systemic treatment with WT TAT-UCHL1 after CCI may improve motor and cognitive deficits. These results also suggest that farnesylation of the C220 site may be required for the protective effects of UCHL1.

Mutation of a Ubiquitin Carboxy Terminal Hydrolase L1 Lipid Binding Site Alleviates Cell Death, Axonal Injury, and Behavioral Deficits After Traumatic Brain Injury in Mice.

Ubiquitin carboxy terminal hydrolase L1 (UCHL1) is a protein highly expressed in neurons that may play important roles in the ubiquitin proteasome pathway (UPP) in neurons, axonal integrity, and motor function after traumatic brain injury (TBI). Binding of reactive lipid species to cysteine 152 of UCHL1 results in unfolding, aggregation, and inactivation of the enzyme. To test the role of this mechanism in TBI, mice bearing a cysteine to alanine mutation at site 152 (C152A mice) that renders UCHL1 resistant to inactivation by reactive lipids were subjected to the controlled cortical impact model (CCI) of TBI and compared to wild type (WT) controls. Alterations in protein ubiquitination and activation of autophagy pathway markers in traumatized brain were detected by immunoblotting. Cell death and axonal injury were determined by histological assessment and anti-amyloid precursor protein (APP) immunohistochemistry. Behavioral outcomes were determined using the beam balance and Morris water maze tests. C152A mice had reduced accumulation of ubiquitinated proteins, decreased activation of the autophagy markers Beclin-1 and LC3B, a decreased number of abnormal axons, decreased CA1 cell death, and improved motor and cognitive function compared to WT controls after CCI; no significant change in spared tissue volume was observed. These results suggest that binding of lipid substrates to cysteine 152 of UCHL1 is important in the pathogenesis of injury and recovery after TBI and may be a novel target for future therapeutic approaches.

Abolishing UCHL1's hydrolase activity exacerbates TBI-induced axonal injury and neuronal death in mice.

Ubiquitin (Ub) C-terminal hydrolase L1 (UCHL1) is a multifunctional protein that is expressed in neurons throughout brain at high levels. UCHL1 deletion is associated with axonal degeneration, progressive sensory motor ataxia, and premature death in mice. UCHL1 has been hypothesized to play a role in the pathogenesis of neurodegenerative diseases and recovery after neuronal injury. UCHL1 hydrolyzes Ub from polyubiquitinated (poly-Ub) proteins, but also may ligate Ub to select neuronal proteins, and interact with cytoskeletal proteins. These and other mechanisms have been hypothesized to underlie UCHL1's role in neurodegeneration and response to brain injury. A UCHL1 knockin mouse containing a C90A mutation (C90A) devoid of hydrolase activity was constructed. The C90A mouse did not develop the sensory and motor deficits, degeneration of the gracile nucleus and tract, or premature death as seen in UCHL1 deficient mice. C90A and wild type (WT) mice were subjected to the controlled cortical impact (CCI) model of traumatic brain injury (TBI), and cell death, axonal injury and behavioral outcome were assessed. C90A mice exhibited decreased spared tissue volume, greater loss of CA1 hippocampal neurons and greater axonal injury as detected using anti-amyloid precursor protein (APP) antibody and anti- non-phosphorylated neurofilament H (SMI-32) antibody immunohistochemistry after CCI compared to WT controls. Poly-Ub proteins and Beclin-1 were elevated after CCI in C90A mice compared to WT controls. Vestibular motor deficits assessed using the beam balance test resolved by day 5 after CCI in WT mice but not in C90A mice. These results suggest that the hydrolase activity of UCHL1 does not account for the progressive neurodegeneration and premature death seen in mice that do not express full length UCHL1. The hydrolase activity of UCHL1 contributes to the function of the ubiquitin proteasome pathway (UPP), ameliorates activation of autophagy, and improves motor recovery after CCI. Thus, UCHL1 hydrolase activity plays an important role in acute injury response after TBI.

Role of UCHL1 in axonal injury and functional recovery after cerebral ischemia.

Ubiquitin C-terminal hydrolase L1 (UCHL1) is a unique brain-specific deubiquitinating enzyme. Mutations in and aberrant function of UCHL1 have been linked to many neurological disorders. UCHL1 activity protects neurons from hypoxic injury, and binding of stroke-induced reactive lipid species to the cysteine 152 (C152) of UCHL1 unfolds the protein and disrupts its function. To investigate the role of UCHL1 and its adduction by reactive lipids in inhibiting repair and recovery of function following ischemic injury, a knock-in (KI) mouse expressing the UCHL1 C152A mutation was generated. Neurons derived from KI mice had less cell death and neurite injury after hypoxia. UCHL1 C152A KI and WT mice underwent middle cerebral artery occlusion (MCAO) or sham surgery. White matter injury was significantly decreased in KI compared with WT mice 7 d after MCAO. Histological analysis revealed decreased tissue loss at 21 d after injury in KI mice. There was also significantly improved sensorimotor recovery in postischemic KI mice. K63- and K48-linked polyubiquitinated proteins were increased in penumbra of WT mouse brains but not in KI mouse brains at 24 h post MCAO. The UCHL1 C152A mutation preserved excitatory synaptic drive to pyramidal neurons and their excitability in the periinfarct zone; axonal conduction velocity recovered by 21 d post MCAO in KI mice in corpus callosum. These results demonstrate that UCHL1 activity is an important determinant of function after ischemia and further demonstrate that the C152 site of UCHL1 plays a significant role in functional recovery after stroke.

In vivo transduction of neurons with TAT-UCH-L1 protects brain against controlled cortical impact injury.

Many mechanisms or pathways are involved in secondary post-traumatic brain injury, such as the ubiquitin-proteasome pathway (UPP), axonal degeneration and neuronal cell apoptosis. UCH-L1 is a protein that is expressed in high levels in neurons and may have important roles in the UPP, autophagy and axonal integrity. The current study aims to evaluate the role of UCH-L1 in post-traumatic brain injury (TBI) and its potential therapeutic effects. A novel protein was constructed that fused the protein transduction domain (PTD) of trans-activating transduction (TAT) protein with UCH-L1 (TAT-UCH-L1) in order to promote neuronal transduction. The TAT-UCH-L1 protein was readily detected in brain by immunoblotting and immunohistochemistry after i.p. administration in mice. TBI was induced in mice using the controlled cortical impact (CCI) model. TAT-UCH-L1 treatment significantly attenuated K48-linkage polyubiquitin (polyUb)-protein accumulation in hippocampus after CCI compared to vehicle controls, but had no effects on K65-linkage polyUb-protein. TAT-UCH-L1 treatment also attenuated expression of Beclin-1 and LC3BII after CCI. TAT-UCH-L1-treated mice had significantly increased spared tissue volumes and increased survival of CA3 neurons 21 d after CCI compared to control vehicle-treated mice. Axonal injury, detected by APP immunohistochemistry, was reduced in thalamus 24 h and 21 d after CCI in TAT-UCH-L1-treated mice. These results suggest that TAT-UCH-L1 treatment improves function of the UPP and decreases activation of autophagy after CCI. Furthermore, TAT-UCH-L1 treatment also attenuates axonal injury and increases hippocampal neuronal survival after CCI. Taken together these results suggest that UCH-L1 may play an important role in the pathogenesis of cell death and axonal injury after TBI.

Rosiglitazone attenuates inflammation and CA3 neuronal loss following traumatic brain injury in rats.

Rosiglitazone, a potent peroxisome proliferator-activated receptor (PPAR)-γ agonist, has been shown to confer neuroprotective effects in stroke and spinal cord injury, but its role in the traumatic brain injury (TBI) is still controversial. Using a controlled cortical impact model in rats, the current study was designed to determine the effects of rosiglitazone treatment (6 mg/kg at 5 min, 6 h and 24 h post injury) upon inflammation and histological outcome at 21 d after TBI. In addition, the effects of rosiglitazone upon inflammatory cytokine transcription, vestibulomotor behavior and spatial memory function were determined at earlier time points (24 h, 1-5 d, 14-20 d post injury, respectively). Compared with the vehicle-treated group, rosiglitazone treatment suppressed production of TNFα at 24 h after TBI, attenuated activation of microglia/macrophages and increased survival of CA3 neurons but had no effect on lesion volume at 21 d after TBI. Rosiglitazone-treated animals had improved performance on beam balance testing, but there was no difference in spatial memory function as determined by Morris water maze. In summary, this study indicates that rosiglitazone treatment in the first 24 h after TBI has limited anti-inflammatory and neuroprotective effects in rat traumatic injury. Further study using an alternative dosage paradigm and more sensitive behavioral testing may be warranted.

Protein disulfide isomerase as a novel target for cyclopentenone prostaglandins: implications for hypoxic ischemic injury.

Cyclooxygenase-2 (COX-2) is an important contributor to ischemic brain injury. Identification of the downstream mediators of COX-2 toxicity may allow the development of targeted therapies. Of particular interest is the cyclopentenone family of prostaglandin metabolites. Cyclopentenone prostaglandins (CyPGs) are highly reactive molecules that form covalent bonds with cellular thiols. Protein disulfide isomerase (PDI) is an important molecule for the restoration of denatured proteins following ischemia. Because PDI has several thiols, including thiols within the active thioredoxin-like domain, we hypothesized that PDI is a target of CyPGs and that CyPG binding of PDI is detrimental. CyPG-PDI binding was detected in vitro via immunoprecipitation and MS. CyPG-PDI binding decreased PDI enzymatic activity in recombinant PDI treated with CyPG, and PDI immunoprecipitated from neuronal culture treated with CyPG or anoxia. Toxic effects of binding were demonstrated in experiments showing that: (a) pharmacologic inhibition of PDI increased cell death in anoxic neurons, (b) PDI overexpression protected neurons exposed to anoxia and SH-SY5Y cells exposed to CyPG, and (c) PDI overexpression in SH-SY5Y cells attenuated ubiquitination of proteins and decreased activation of pro-apoptotic caspases. In conclusion, CyPG production and subsequent binding of PDI is a novel and potentially important mechanism of ischemic brain injury. We show that CyPGs bind to PDI, cyclopentenones inhibit PDI activity, and CyPG-PDI binding is associated with increased neuronal susceptibility to anoxia. Additional studies are necessary to determine the relative role of CyPG-dependent inhibition of PDI activity in ischemia and other neurodegenerative disorders.

Soluble epoxide hydrolase inhibitor trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid is neuroprotective in rat model of ischemic stroke.

Soluble epoxide hydrolase (sEH) diminishes vasodilatory and neuroprotective effects of epoxyeicosatrienoic acids by hydrolyzing them to inactive dihydroxy metabolites. The primary goals of this study were to investigate the effects of acute sEH inhibition by trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB) on infarct volume, functional outcome, and changes in cerebral blood flow (CBF) in a rat model of ischemic stroke. Focal cerebral ischemia was induced in rats for 90 min followed by reperfusion. At the end of 24 h after reperfusion rats were euthanized for infarct volume assessment by triphenyltetrazolium chloride staining. Brain cortical sEH activity was assessed by ultra performance liquid chromatography-tandem mass spectrometry. Functional outcome at 24 and 48 h after reperfusion was evaluated by arm flexion and sticky-tape tests. Changes in CBF were assessed by arterial spin-labeled-MRI at baseline, during ischemia, and at 180 min after reperfusion. Neuroprotective effects of t-AUCB were evaluated in primary rat neuronal cultures by Cytotox-Flour kit and propidium iodide staining. t-AUCB significantly reduced cortical infarct volume by 35% (14.5 ± 2.7% vs. 41.5 ± 4.5%), elevated cumulative epoxyeicosatrienoic acids-to-dihydroxyeicosatrienoic acids ratio in brain cortex by twofold (4.40 ± 1.89 vs. 1.97 ± 0.85), and improved functional outcome in arm-flexion test (day 1: 3.28 ± 0.5 s vs. 7.50 ± 0.9 s; day 2: 1.71 ± 0.4 s vs. 5.28 ± 0.5 s) when compared with that of the vehicle-treated group. t-AUCB significantly reduced neuronal cell death in a dose-dependent manner (vehicle: 70.9 ± 7.1% vs. t-AUCB0.1µM: 58 ± 5.11% vs. t-AUCB0.5µM: 39.9 ± 5.8%). These findings suggest that t-AUCB may exert its neuroprotective effects by affecting multiple components of neurovascular unit including neurons, astrocytes, and microvascular flow.

Prostaglandin D2 toxicity in primary neurons is mediated through its bioactive cyclopentenone metabolites.

Prostaglandin D2 (PGD2) is the most abundant prostaglandin in brain but its effect on neuronal cell death is complex and not completely understood. PGD2 may modulate neuronal cell death via activation of DP receptors or its metabolism to the cyclopentenone prostaglandins (CyPGs) PGJ2, Δ(12)-PGJ2 and 15-deoxy-Δ(12,14)-PGJ2, inducing cell death independently of prostaglandin receptors. This study aims to elucidate the effect of PGD2 on neuronal cell death and its underlying mechanisms. PGD2 dose-dependently induced cell death in rat primary neuron-enriched cultures in concentrations of ≥10µM, and this effect was not reversed by treatment with either DP1 or DP2 receptor antagonists. Antioxidants N-acetylcysteine (NAC) and glutathione which contain sulfhydryl groups that can bind to CyPGs, but not ascorbate or tocopherol, attenuated PGD2-induced cell death. Conversion of PGD2 to CyPGs was detected in neuronal culture medium; treatment with these CyPG metabolites alone exhibited effects similar to those of PGD2, including apoptotic neuronal cell death and accumulation of ubiquitinated proteins. Disruption of lipocalin-type prostaglandin D synthase (L-PGDS) protected neurons against hypoxia. These results support the hypothesis that PGD2 elicits its cytotoxic effects through its bioactive CyPG metabolites rather than DP receptor activation in primary neuronal culture.

COX2-derived primary and cyclopentenone prostaglandins are increased after asphyxial cardiac arrest.

BACKGROUND: Cyclopentenone prostaglandins have been identified as potential neurotoxic agents in the setting of hypoxia-ischemia. Cyclooxygenase-2 (COX-2), the upstream enzyme responsible for prostaglandin production is upregulated following hypoxic-ischemic brain injury. However, the temporal production and concentration of cyclopentenone prostaglandins has not been described following global brain ischemia. METHODS: Global brain ischemia was induced in rats by asphyxial cardiac arrest (ACA) followed by resuscitation. Rats were sacrificed between 24h and 7 days following resuscitation and their brains removed. Western blot, immunohistochemistry, and mass spectroscopy were performed. A cohort of rats was pretreated with the COX-2 inhibitor SC58125. RESULTS: COX-2 is induced in hippocampus at 24h following ACA. Multiple prostaglandins, including cyclopentenone prostaglandin species, are increased in hippocampus as 24h following ACA. Prostaglandin and cyclopentenone prostaglandin concentrations are returned to baseline at 3 and 7 days post-ischemia. The COX-2 inhibitor SC58125 completely abrogates the post-ischemic increase in prostaglandins and cyclopentenone prostaglandins. CONCLUSIONS: Prostaglandins, including cyclopentenone prostaglandins, are increased in ischemic brain, peak at 24h and can be attenuated by the COX-2 inhibitor SC58125. These data establish the presence of potentially neurotoxic cyclopentenone prostaglandins in post-ischemic brains, thus identifying a target and therapeutic window for neuroprotective therapies.

Increased generation of cyclopentenone prostaglandins after brain ischemia and their role in aggregation of ubiquitinated proteins in neurons.

The cyclopentenone prostaglandin (CyPG) J2 series, including prostaglandin J2 (PGJ2), Δ¹²-PGJ2, and 15-deoxy-∆¹²,¹4-prostaglandin J2 (15d-PGJ2), are active metabolites of PGD2, exerting multiple effects on neuronal function. However, the physiologic relevance of these effects remains uncertain as brain concentrations of CyPGs have not been precisely determined. In this study, we found that free PGD2 and the J2 series CyPGs (PGJ2, Δ¹²-PGJ2, and 15d-PGJ2) were increased in post-ischemic rat brain as detected by UPLC-MS/MS with 15d-PGJ2 being the most abundant CyPG. These increases were attenuated by pre-treating with the cyclooxygenase (COX) inhibitor piroxicam. Next, effects of chronic exposure to 15d-PGJ2 were examined by treating primary neurons with 15d-PGJ2, CAY10410 (a 15d-PGJ2 analog lacking the cyclopentenone ring structure), or vehicle for 24 to 96 h. Because we found that the concentration of free 15d-PGJ2 decreased rapidly in cell culture medium, freshly prepared medium containing 15d-PGJ2, CAY10410, or vehicle was changed twice daily to maintain steady extracellular concentrations. Incubation with 2.5 µM 15d-PGJ2, but not CAY10410, increased the neuronal cell death without the induction of caspase-3 or PARP cleavage, consistent with a primarily necrotic mechanism for 15d-PGJ2-induced cell death which was further supported by TUNEL assay results. Ubiquitinated protein accumulation and aggregation was observed after 96 h 15d-PGJ2 incubation, accompanied by compromised 20S proteasome activity. Unlike another proteasome inhibitor, MG132, 15d-PGJ2 treatment did not activate autophagy or induce aggresome formation. Therefore, the cumulative cytotoxic effects of increased generation of CyPGs after stroke may contribute to delayed post-ischemic neuronal injury.

Modification of ubiquitin-C-terminal hydrolase-L1 by cyclopentenone prostaglandins exacerbates hypoxic injury.

Cyclopentenone prostaglandins (CyPGs), such as 15-deoxy-Δ(12,14) -prostaglandin J(2) (15d-PGJ(2)), are active prostaglandin metabolites exerting a variety of biological effects that may be important in the pathogenesis of neurological diseases. Ubiquitin-C-terminal hydrolase L1 (UCH-L1) is a brain specific deubiquitinating enzyme whose aberrant function has been linked to neurodegenerative disorders. We report that [15d-PGJ(2)] detected by quadrapole mass spectrometry (MS) increases in rat brain after temporary focal ischemia, and that treatment with 15d-PGJ(2) induces accumulation of ubiquitinated proteins and exacerbates cell death in normoxic and hypoxic primary neurons. 15d-PGJ(2) covalently modifies UCH-L1 and inhibits its hydrolase activity. Pharmacologic inhibition of UCH-L1 exacerbates hypoxic neuronal death while transduction with a TAT-UCH-L1 fusion protein protects neurons from hypoxia. These studies indicate that UCH-L1 function is important in hypoxic neuronal death and that excessive production of CyPGs after stroke may exacerbate ischemic injury by modification and inhibition of UCH-L1.

The cyclooxygenase site, but not the peroxidase site of cyclooxygenase-2 is required for neurotoxicity in hypoxic and ischemic injury.

Cyclooxygenase-2 (COX-2) activity has been implicated in the pathogenesis of ischemic injury, but the exact mechanisms responsible for its toxicity remain unclear. Infection of primary neurons with an adenovirus expressing wild type (WT) COX-2 increased the susceptibility of neurons to hypoxia. Infection with an adenoviral vector expressing COX-2 with a mutation at the cyclooxygenase site did not increase susceptibility to hypoxia, whereas over-expression of COX-2 with a mutation in the peroxidase site produced similar susceptibility to hypoxia as WT COX-2. Primary neuronal cultures obtained from transgenic mice bearing a mutation in the COX-2 cylooxygenase site were protected from hypoxia. Mice with a mutation in the cyclooxygenase site had smaller infarctions 24 h after 70 min of middle cerebral artery occlusion than WT control mice. COX-2 activity had no effect on the formation of protein carbonyls. Ascorbate radicals were detected by electron paramagnetic resonance as a product of recombinant COX-2 activity and were blocked by COX-2 inhibitors. Similarly, formation of ascorbate radicals was inhibited in the presence of COX-2 inhibitors and in homogenates obtained from COX-2 null mice. Taken together, these results indicate that the cyclooxygenase activity of COX-2 is necessary to exacerbate neuronal hypoxia/ischemia injury rather than the peroxidase activity of the enzyme.

Prolonged opportunity for neuroprotection in experimental stroke with selective blockade of cyclooxygenase-2 activity.

The post-treatment effects of the selective cyclooxygenase (COX)-2 inhibitor, valdecoxib, were investigated in a rat model of temporary focal ischemia. Valdecoxib reduced basal brain prostaglandin E(2) concentrations at dosages that did not affect serum thromboxane B(2), consistent with a selective COX-2 effect. Temporary focal cerebral ischemia was produced in rats by middle cerebral artery occlusion for 90 min. There was increased expression of COX-2 protein detected by Western blot and immunocytochemistry within neurons in the ischemic cortex at 4 and 24 h after ischemia. Rats were treated with vehicle or valdecoxib 15 min before or 1.5, 3 and 6 h after cerebral ischemia. Rats were sacrificed and brain infarction volume determined 24 h after ischemia. Valdecoxib treatment was associated with a decrease in infarction volume when administered 15 min before, and 1.5 or 3 h but not 6 h after cerebral ischemia. There were no differences in physiological parameters during the procedure. Valdecoxib administered at 1.5 h after ischemia significantly reduced the concentrations of prostaglandin E(2) in ischemic penumbral cortex as compared to the vehicle-treated group and contralateral non-ischemic cortex. These results suggest that COX-2 inhibition with valdecoxib is effective when initiated both before and after middle cerebral artery occlusion.

Genetic disruption of cyclooxygenase-2 does not improve histological or behavioral outcome after traumatic brain injury in mice.

Increasing evidence suggests a role for cyclooxygenase-2 (COX-2) in traumatic brain injury (TBI). In the present study, the role of COX-2 in TBI was investigated using COX-2 gene-disrupted (COX-2 null) mice and wild-type (WT) controls that were subjected to the controlled cortical impact (CCI) model of TBI. There was increased expression of COX-2 in ipsilateral hippocampus in WT mice subjected to CCI. CCI resulted in a significant increase in prostaglandin E(2) concentrations in WT compared with COX-2 null hippocampi. There was a significant increase in TUNEL staining of CA1 neurons 24 hr after CCI in WT, but not in COX-2 null mice, compared with sham-operated controls, which is consistent with a protective role for COX-2 in the early phase of injury after TBI. However, there was no difference in lesion volume 21 days after CCI in COX-2 null and WT mice. COX-2 gene disruption did not alter Morris water maze performance. Taken together, these results suggest only a minor role for COX-2 activity in determining outcome after TBI in mouse.

Disruption of Bax protein prevents neuronal cell death but produces cognitive impairment in mice following traumatic brain injury.

Apoptosis contributes to delayed neuronal cell death in traumatic brain injury (TBI). To investigate if Bax plays a role in neuronal cell death and functional outcome after TBI, Bax gene disrupted (null) mice and wild-type (WT) controls were subjected to the controlled cortical impact (CCI) model of TBI. Motor function in WT and Bax null mice was evaluated using the round beam balance and the wire grip test on days 0-5. Spatial memory was assessed using a Morris Water Maze adopted for mice on days 14-18 post-injury. For histopathological analysis, animals were sacrificed 24 h and 21 days post-injury. In all three behavioral tests, the sham and TBI-injured Bax null mice performed significantly worse than their WT sham and TBI-injured counterparts. However, Bax null mice exhibited a higher percentage of surviving neurons in the CA1 and CA3 regions of hippocampus measured at 21 days post-injury. At 24 h after trauma, Bax null mice had fewer TUNEL positive cells in the CA1 and dentate regions of hippocampus as compared to WT mice, suggesting that deletion of the Bax gene ameliorates hippocampal cell death after TBI. Sham-operated Bax null mice had significantly greater brain volume as compared to WT mice. Thus, it is possible that Bax deficiency in the transgenic mice produces developmental behavioral effects, perhaps due to Bax's role in regulating cell death during development.

Neuronal cyclooxygenase-2 activity and prostaglandins PGE2, PGD2, and PGF2 alpha exacerbate hypoxic neuronal injury in neuron-enriched primary culture.

Cyclooxygenase-2 (COX-2) activity has been implicated in the pathogenesis of cerebral ischemia. To determine whether COX-2 activity within the neuron itself exacerbates hypoxic neuronal injury, neuron-enriched cultures were subjected to anoxia. Treatment with COX-2 selective antagonists decreased cell death. Neurons cultured from homozygous COX-2 gene disrupted mice were resistant to hypoxia compared to those of heterozygotes. Infection of primary neurons with AAV expressing COX-2 exacerbated cell death compared to neurons infected with enhanced green fluorescent protein (EGFP) control vector. Addition of PGE2, PGD2 or PGF2 alpha to the medium exacerbated injury, suggesting that the deleterious effects of COX-2 overexpression in hypoxia could be mediated by direct receptor mediated effects of prostaglandins. Overexpression of COX-2 did not increase expression of cyclin D1 or phosphoretinoblastoma protein (pRb), or cleavage of caspase 3 suggesting that this cell cycle mechanism does not mediate COX-2 toxicity in this model.

Cyclooxygenase-2 activity following traumatic brain injury in the developing rat.

Cyclooxygenase (COX) is the rate-limiting enzyme in the production of prostaglandins. COX-2, the predominant COX isoform in brain, is induced by synaptic activity. COX-2-generated prostaglandins are important regulators for a range of activities under physiologic conditions. However, under pathologic conditions, COX-2 activity can produce reactive oxygen species and toxic prostaglandin metabolites that can exacerbate brain injury. In this study, we examine the developmental production of COX-2 and test the ability of a COX-2 inhibitor, SC58125, to attenuate traumatic brain injury in developing rats. We show that constitutive COX-2 concentration is low (0.5-fold adult concentration) during the first postnatal week and then increases to 3-fold of adult levels between days 14-60. Controlled cortical impact (CCI) at postnatal day (PND) 17, but not PND 7, caused an additional 3-fold increase in COX-2 content and was associated with an increase in the COX-2 product PGE2. Treatment with the COX-2 inhibitor SC58125 in PND17 rats exposed to CCI attenuated the rise in PGE2 but did not attenuate lesion volume or improve performance in the Morris water maze.

Transgenic mice that overexpress the anti-apoptotic Bcl-2 protein have improved histological outcome but unchanged behavioral outcome after traumatic brain injury.

Increasing evidence suggests that apoptosis is a contributing factor to neuronal cell death in traumatic brain injury (TBI). There is increased expression, cleavage and activation of caspases as well as other proteins known to regulate apoptosis in neurons after TBI. These proteins include the proto-oncogene Bcl-2 which belongs to a family of proteins with both pro- and anti-apoptotic properties. To investigate the role of apoptosis in TBI and the importance of Bcl-2 protein on the severity and outcome of injury, Bcl-2 overexpressing transgenic and wild-type control mice were subjected to the controlled cortical impact model of TBI. There was no significant difference in the cleavage of caspase-3 or caspase-9 detected by Western blotting of hippocampal samples from transgenic or wild-type mice after TBI. Bcl-2 transgenic mice had smaller contusion volumes and increased numbers of surviving neurons in CA2 but not other regions of hippocampus compared to wild-type controls. By contrast, there was no difference in motor function determined by the round beam balance and wire grip tests between transgenic and wild-type mice after TBI. Cognitive function assessed by the Morris water maze was also not different between groups. These results suggest that overexpression of Bcl-2 is only partially neuroprotective and other members of this protein family may prove to be more important in protecting neurons from cell death.