Prevention of JNK phosphorylation as a mechanism for rosiglitazone in neuroprotection after transient cerebral ischemia: activation of dual specificity phosphatase.

Rosiglitazone, a synthetic peroxisome proliferator-activated receptor-γ (PPARγ) agonist, prevents cell death after cerebral ischemia in animal models, but the underlying mechanism has not been clarified. In this study, we examined how rosiglitazone protects neurons against ischemia. Mice treated with rosiglitazone were subjected to 60 minutes of focal ischemia followed by reperfusion. Rosiglitazone reduced infarct volume after ischemia and reperfusion. We show that this neuroprotective effect was reversed with a PPARγ antagonist. Western blot analysis showed a significant increase in expression of phosphorylated stress-activated protein kinases (c-Jun N-terminal kinase (JNK) and p38) in ischemic brain tissue. Rosiglitazone blocked this increase. Furthermore, we observed that rosiglitazone increased expression of the dual-specificity phosphatase 8 (DUSP8) protein and messenger RNA in ischemic brain tissue. Dual-specificity phosphatase 8 is a mitogen-activated protein kinase phosphatase that can dephosphorylate JNK and p38. Another key finding of the present study was that knockdown of DUSP8 in primary cultured cortical neurons that were subjected to oxygen-glucose deprivation diminished rosiglitazone's effect on downregulation of JNK phosphorylation. Thus, rosiglitazone's neuroprotective effect after ischemia is mediated by blocking JNK phosphorylation induced by ischemia via DUSP8 upregulation.

Interleukin 6-preconditioned neural stem cells reduce ischaemic injury in stroke mice.

Transplantation of neural stem cells provides a promising therapy for stroke. Its efficacy, however, might be limited because of massive grafted-cell death after transplantation, and its insufficient capability for tissue repair. Interleukin 6 is a pro-inflammatory cytokine involved in the pathogenesis of various neurological disorders. Paradoxically, interleukin 6 promotes a pro-survival signalling pathway through activation of signal transducer and activator of transcription 3. In this study, we investigated whether cellular reprogramming of neural stem cells with interleukin 6 facilitates the effectiveness of cell transplantation therapy in ischaemic stroke. Neural stem cells harvested from the subventricular zone of foetal mice were preconditioned with interleukin 6 in vitro and transplanted into mouse brains 6 h or 7 days after transient middle cerebral artery occlusion. Interleukin 6 preconditioning protected the grafted neural stem cells from ischaemic reperfusion injury through signal transducer and activator of transcription 3-mediated upregulation of manganese superoxide dismutase, a primary mitochondrial antioxidant enzyme. In addition, interleukin 6 preconditioning induced secretion of vascular endothelial growth factor from the neural stem cells through activation of signal transducer and activator of transcription 3, resulting in promotion of angiogenesis in the ischaemic brain. Furthermore, transplantation of interleukin 6-preconditioned neural stem cells significantly attenuated infarct size and improved neurological performance compared with non-preconditioned neural stem cells. This interleukin 6-induced amelioration of ischaemic insults was abolished by transfecting the neural stem cells with signal transducer and activator of transcription 3 small interfering RNA before transplantation. These results indicate that preconditioning with interleukin 6, which reprograms neural stem cells to tolerate oxidative stress after ischaemic reperfusion injury and to induce angiogenesis through activation of signal transducer and activator of transcription 3, is a simple and beneficial approach for enhancing the effectiveness of cell transplantation therapy in ischaemic stroke.

Neural stem cells genetically modified to overexpress cu/zn-superoxide dismutase enhance amelioration of ischemic stroke in mice.

BACKGROUND AND PURPOSE: The harsh host brain microenvironment caused by production of reactive oxygen species after ischemic reperfusion injury offers a significant challenge to survival of transplanted neural stem cells (NSCs) after ischemic stroke. Copper/zinc-superoxide dismutase (SOD1) is a specific antioxidant enzyme that counteracts superoxide anions. We have investigated whether genetic manipulation to overexpress SOD1 enhances survival of grafted stem cells and accelerates amelioration of ischemic stroke. METHODS: NSCs genetically modified to overexpress or downexpress SOD1 were administered intracerebrally 2 days after transient middle cerebral artery occlusion. Histological and behavioral tests were examined from Days 0 to 28 after stroke. RESULTS: Overexpression of SOD1 suppressed production of superoxide anions after ischemic reperfusion injury and reduced NSC death after transplantation. In contrast, downexpression of SOD1 promoted superoxide generation and increased oxidative stress-mediated NSC death. Transplantation of SOD1-overexpressing NSCs enhanced angiogenesis in the ischemic border zone through upregulation of vascular endothelial growth factor. Moreover, grafted SOD1-overexpressing NSCs reduced infarct size and improved behavioral performance compared with NSCs that were not genetically modified. CONCLUSIONS: Our findings reveal a strong involvement of SOD1 expression in NSC survival after ischemic reperfusion injury. We propose that conferring antioxidant properties on NSCs by genetic manipulation of SOD1 is a potential approach for enhancing the effectiveness of cell transplantation therapy in ischemic stroke.

Minocycline-preconditioned neural stem cells enhance neuroprotection after ischemic stroke in rats.

Transplantation of neural stem cells (NSCs) offers a novel therapeutic strategy for stroke; however, massive grafted cell death following transplantation, possibly due to a hostile host brain environment, lessens the effectiveness of this approach. Here, we have investigated whether reprogramming NSCs with minocycline, a broadly used antibiotic also known to possess cytoprotective properties, enhances survival of grafted cells and promotes neuroprotection in ischemic stroke. NSCs harvested from the subventricular zone of fetal rats were preconditioned with minocycline in vitro and transplanted into rat brains 6 h after transient middle cerebral artery occlusion. Histological and behavioral tests were examined from days 0-28 after stroke. For in vitro experiments, NSCs were subjected to oxygen-glucose deprivation and reoxygenation. Cell viability and antioxidant gene expression were analyzed. Minocycline preconditioning protected the grafted NSCs from ischemic reperfusion injury via upregulation of Nrf2 and Nrf2-regulated antioxidant genes. Additionally, preconditioning with minocycline induced the NSCs to release paracrine factors, including brain-derived neurotrophic factor, nerve growth factor, glial cell-derived neurotrophic factor, and vascular endothelial growth factor. Moreover, transplantation of the minocycline-preconditioned NSCs significantly attenuated infarct size and improved neurological performance, compared with non-preconditioned NSCs. Minocycline-induced neuroprotection was abolished by transfecting the NSCs with Nrf2-small interfering RNA before transplantation. Thus, preconditioning with minocycline, which reprograms NSCs to tolerate oxidative stress after ischemic reperfusion injury and express higher levels of paracrine factors through Nrf2 up-regulation, is a simple and safe approach to enhance the effectiveness of transplantation therapy in ischemic stroke.

Oxidative stress in ischemic brain damage: mechanisms of cell death and potential molecular targets for neuroprotection.

Significant amounts of oxygen free radicals (oxidants) are generated during cerebral ischemia/reperfusion, and oxidative stress plays an important role in brain damage after stroke. In addition to oxidizing macromolecules, leading to cell injury, oxidants are also involved in cell death/survival signal pathways and cause mitochondrial dysfunction. Experimental data from laboratory animals that either overexpress (transgenic) or are deficient in (knock-out) antioxidant proteins, mainly superoxide dismutase, have provided strong evidence of the role of oxidative stress in ischemic brain damage. In addition to mitochondria, recent reports demonstrate that NADPH oxidase (NOX), an important pro-oxidant enzyme, is also involved in the generation of oxidants in the brain after stroke. Inhibition of NOX is neuroprotective against cerebral ischemia. We propose that superoxide dismutase and NOX activity in the brain is a major determinant for ischemic damage/repair and that these major anti- and pro-oxidant enzymes are potential endogenous molecular targets for stroke therapy.

Reperfusion and neurovascular dysfunction in stroke: from basic mechanisms to potential strategies for neuroprotection.

Effective stroke therapies require recanalization of occluded cerebral blood vessels. However, reperfusion can cause neurovascular injury, leading to cerebral edema, brain hemorrhage, and neuronal death by apoptosis/necrosis. These complications, which result from excess production of reactive oxygen species in mitochondria, significantly limit the benefits of stroke therapies. We have developed a focal stroke model using mice deficient in mitochondrial manganese-superoxide dismutase (SOD2-/+) to investigate neurovascular endothelial damage that occurs during reperfusion. Following focal stroke and reperfusion, SOD2-/+ mice had delayed blood-brain barrier breakdown, associated with activation of matrix metalloproteinase and high brain hemorrhage rates, whereas a decrease in apoptosis and hemorrhage was observed in SOD2 overexpressors. Thus, induction and activation of SOD2 is a novel strategy for neurovascular protection after ischemia/reperfusion. Our recent study identified the signal transducer and activator of transcription 3 (STAT3) as a transcription factor of the mouse SOD2 gene. During reperfusion, activation of STAT3 and its recruitment into the SOD2 gene were blocked, resulting in increased oxidative stress and neuronal apoptosis. In contrast, pharmacological activation of STAT3 induced SOD2 expression, which limits ischemic neuronal death. Our studies point to antioxidant-based neurovascular protective strategies as potential treatments to expand the therapeutic window of currently approved therapies.

The protective effect of early hypothermia on PTEN phosphorylation correlates with free radical inhibition in rat stroke.

We recently showed that intraischemic moderate hypothermia (30 degrees C) reduces ischemic damage through the Akt pathway after permanent distal middle cerebral artery occlusion in rats. The only Akt pathway component preserved by hypothermia is phosphorylated phosphatase and tensin homolog deleted on chromosome 10 (p-PTEN), which suggests that p-PTEN may have a central role in neuroprotection. Reactive oxygen species (ROS) are critically involved in mediating ischemic damage after stroke by interacting with signaling molecules, including Akt, PTEN, and delta-protein kinase C (PKC). We investigated the protective mechanisms of moderate hypothermia on these signaling proteins after transient focal ischemia in rats. Early moderate hypothermia (3 h) was administered 15 mins before reperfusion, and delayed moderate hypothermia (3 h) was applied 15 mins after reperfusion. Our results indicate that early hypothermia reduced infarction, whereas delayed hypothermia did not. However, both early and delayed hypothermia maintained levels of Mn-SOD (superoxide dismutase) and phosphorylated Akt and blocked delta-PKC cleavage, suggesting that these factors may not be critical to the protection of hypothermia. Nevertheless, early hypothermia preserved p-PTEN levels after reperfusion, whereas delayed hypothermia did not. Furthermore, ROS inhibition maintained levels of p-PTEN after stroke. Together, these findings suggest that phosphorylation levels of PTEN are closely associated with the protective effect of early hypothermia against stroke.

Effect of combined oral contraceptives on breast epithelial proliferation in young women.

The mammary gland undergoes morphologic changes during the menstrual cycle. Proliferation of normal breast epithelium is most extensive during the natural luteal phase. To determine the impact of one cycle of a combined oral contraceptive (COC) on breast homeostasis, we evaluated the proliferation index (PI), determined by KI-67 expression, in normal human mammary epithelial cells and correlated it with cellular proliferation in spontaneous menstrual cycles during the same period. Normal breast tissue samples were obtained from 82 patients randomized in two groups. Forty-two women in group A received one cycle of a COC (30 mug ethinyl estradiol and 150 mug levonorgestrel) administrated daily for 21 days, beginning on the first day of the menstrual cycle. Group B patients (n = 40) experienced a natural menstrual cycle. Menstrual cycle phase characterization was based on the date of the last period and subsequent menses and on progesterone serum levels obtained at the time of biopsy. The PI (number of Ki-67-positive nuclei per 1,000 epithelial cells), was significantly larger in group A (5.47 +/- 3.87), than in group B (3.27 +/- 3.24), p < 0.01. A cyclical variation of PI was observed in COC cycles. The rise in PI in the first week of the COC cycles was significantly higher than in the natural cycle (COC = 7.02 +/- 4.94; non-COC = 1.10 +/- 0.67; p < 0.0011). There was no significant difference between the two groups during the other weeks. Additionally, there was an inverse correlation between proliferation and chronological age, irrespective of the stage of the cycle. The PI of COC (p = 0.175) and natural cycles (p = 0.466) were not statistically different in younger patients. COC users have increased proliferative activity at the beginning of the menstrual cycle. This alteration in the pattern of proliferative activity may relate to the increased risk of breast cancer that has been associated with COCs.

Evaluating therapeutic targets for reperfusion-related brain hemorrhage.

OBJECTIVE: Early reperfusion after an ischemic stroke can cause blood-brain barrier injury with subsequent cerebral edema and devastating brain hemorrhage. These complications of early reperfusion, which result from excess production of reactive oxygen species, significantly limit the benefits of stroke therapies. In this article, we use a novel animal model that facilitates identification of specific components of the reperfusion injury process, including vascular injury and secondary brain damage, and allows assessment of therapeutic interventions. METHODS: Knock-out (KO) mice containing 50% manganese-superoxide dismutase activity (SOD2-KO) and transgenic mice overexpressing SOD2 undergo transient focal ischemia and reperfusion followed by assessment of infarct, edema, hemorrhage rates, metalloproteinase activation, and microvascular injury. RESULTS: SOD2-KO mice demonstrate delayed (>24h) blood-brain barrier breakdown associated with activation of matrix metalloproteinases, inflammation, and high brain hemorrhage rates. These adverse consequences are absent in wild-type littermates and minocycline-treated SOD2-KO animals. Increased hemorrhage rates also are absent in SOD2 overexpressors, which have reduced vascular endothelial cell death. Finally, we show that the tight junction membrane protein, occludin, is an early and specific target in oxidative stress-induced microvascular injury. INTERPRETATION: This model is ideal for studying ischemia/reperfusion-induced vascular injury and secondary brain hemorrhage and offers a unique opportunity to evaluate antioxidant-based neurovascular protective strategies as potential adjunct treatments to currently approved stroke therapies such as thrombolysis and endovascular clot retrieval.

Interferon-beta fails to protect in a model of transient focal stroke.

BACKGROUND AND PURPOSE: Compelling evidence supporting the role of inflammation in the development of cerebral infarction has focused attention on the potential of antiinflammatory treatment strategies for stroke. Interferon (IFN)-beta, an immunomodulatory agent approved for treatment of multiple sclerosis, is being evaluated in a phase I clinical trial in acute ischemic stroke. In the present study, we evaluated the effects of wild-type rat IFN-beta and its pegylated counterpart (PEG-IFN-beta) in a model of focal ischemia and reperfusion. METHODS: After 60 minutes of middle cerebral artery occlusion, rats (n=12/group) were treated with IV tail injections of 8 or 16 mug of IFN-beta in 300 muL of PBS once daily for 3 or 7 days or with IV or SC injections of PEG-IFN-beta for 1 day. The animals were assessed daily for weight and for neurological findings. Additional animals underwent complete hematology and chemistry profiles, as well as complete multiorgan necropsy studies. All of the brain tissue was evaluated for assessment of infarct areas, neutrophil infiltration, and presence of hemorrhagic transformations. RESULTS: IFN-beta and PEG-IFN-beta failed to protect against experimental ischemic brain injury as assessed by histopathology and neurological outcome. Furthermore, IFN-beta treatment was associated with significant weight loss and alterations in hematology and chemistry profiles. CONCLUSIONS: Our results suggest that additional preclinical studies are warranted.

Assessment of the proliferative, apoptotic and cellular renovation indices of the human mammary epithelium during the follicular and luteal phases of the menstrual cycle.

INTRODUCTION: During the menstrual cycle, the mammary gland goes through sequential waves of proliferation and apoptosis. In mammary epithelial cells, hormonal and non-hormonal factors regulate apoptosis. To determine the cyclical effects of gonadal steroids on breast homeostasis, we evaluated the apoptotic index (AI) determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining in human mammary epithelial cells during the spontaneous menstrual cycle and correlated it with cellular proliferation as determined by the expression of Ki-67 during the same period. METHODS: Normal breast tissue samples were obtained from 42 randomly selected patients in the proliferative (n = 21) and luteal (n = 21) phases. Menstrual cycle phase characterization was based on the date of the last and subsequent menses, and on progesterone serum levels obtained at the time of biopsy. RESULTS: The proliferation index (PI), defined as the number of Ki-67-positive nuclei per 1,000 epithelial cells, was significantly larger in the luteal phase (30.46) than in the follicular phase (13.45; P = 0.0033). The AI was defined as the number of TUNEL-positive cells per 1,000 epithelial cells. The average AI values in both phases of the menstrual cycle were not statistically significant (P = 0.21). However, the cell renewal index (CRI = PI/AI) was significantly higher in the luteal phase (P = 0.033). A significant cyclical variation of PI, AI and CRI was observed. PI and AI peaks occurred on about the 24th day of the menstrual cycle, whereas the CRI reached higher values on the 28th day. CONCLUSIONS: We conclude that proliferative activity is dependent mainly on hormonal fluctuations, whereas apoptotic activity is probably regulated by hormonal and non-hormonal factors.

Oxidative stress and neuronal death/survival signaling in cerebral ischemia.

It has been demonstrated by numerous studies that apoptotic cell death pathways are implicated in ischemic cerebral injury in ischemia models in vivo. Experimental ischemia and reperfusion models, such as transient focal/global ischemia in rodents, have been thoroughly studied and the numerous reports suggest the involvement of cell survival/death signaling pathways in the pathogenesis of apoptotic cell death in ischemic lesions. In these models, reoxygenation during reperfusion provides oxygen as a substrate for numerous enzymatic oxidation reactions and for mitochondrial oxidative phosphorylation to produce adenosine triphosphate. Oxygen radicals, the products of these biochemical and physiological reactions, are known to damage cellular lipids, proteins, and nucleic acids and to initiate cell signaling pathways after cerebral ischemia. Genetic manipulation of intrinsic antioxidants and factors in the signaling pathways has provided substantial understanding of the mechanisms involved in cell death/survival signaling pathways and the role of oxygen radicals in ischemic cerebral injury. Future studies of these pathways could provide novel therapeutic strategies in clinical stroke.

Akt/Bad signaling and motor neuron survival after spinal cord injury.

The serine-threonine kinase Akt is a cell survival signaling pathway that inactivates the proapoptotic BCL-2 family protein Bad and promotes cell survival in cerebral ischemia. Involvement of the Akt/Bad signaling pathway after spinal cord injury (SCI) is, however, uncertain. Our results showed that phospho-Akt (serine-473) and phospho-Bad (serine-136) were significantly upregulated at 1 day after SCI. In addition, phospho-Akt and phospho-Bad were colocalized in motor neurons that survived SCI and inhibition of PI3-K reduced expression of phospho-Akt and phospho-Bad. Dimerization of Bad with 14-3-3 in the cytosol was increased whereas Bad/Bcl-XL binding in the mitochondria was decreased after SCI. We further found that reduced oxidative stress by SOD1 overexpression in rats enhanced the expression of phospho-Akt, phospho-Bad, Bad/14-3-3 binding and reduced Bad/Bcl-XL binding after SCI, as compared to wild-type rats. We conclude that oxidative stress may play a role in modulating Akt/Bad signaling and subsequent motor neuron survival after SCI.

Superoxide dismutase 1 overexpression reduces MCP-1 and MIP-1 alpha expression after transient focal cerebral ischemia.

Proinflammatory cytokines and chemokines are quickly upregulated in response to ischemia/reperfusion (I/R) injury; however, the relationship between I/R-induced oxidative stress and cytokine/chemokine expression has not been elucidated. We investigated the temporal profile of cytokine and chemokine gene expression in transient focal cerebral ischemia using complementary DNA array technology. Among 96 genes studied, 10, 4, 11, and 5 genes were increased at 6, 12, 24, and 72 h of reperfusion, respectively, whereas, 4, 11, 8, and 21 genes, respectively, were decreased. To clarify the relationship between chemokines and oxidative stress, we compared the gene and protein expression of monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein-1 alpha (MIP-1 alpha) in wild-type (WT) mice and copper/zinc-superoxide dismutase (SOD 1) transgenic (Tg) mice. Monocyte chemoattractant protein-1 and MIP-1 alpha mRNA were significantly upregulated at 6 to 12 h of reperfusion. In the SOD 1 Tg mice, however, MCP-1 and MIP-1 alpha mRNA expression was significantly decreased 12 h postinsult. In the WT mice, MCP-1 and MIP-1 alpha protein expression peaked 24 h after onset of reperfusion determined by immunohistochemistry. In the SOD 1 Tg mice, MCP-1 and MIP-1 alpha immunopositive cells were reduced, as were concentrations of these proteins (measured by enzyme-linked immunosorbent assay) at 24 h of reperfusion. Our results suggest that MCP-1 and MIP-1 alpha expression is influenced by I/R-induced oxidative stress after transient focal stroke.

Matrix metalloproteinase-9 and myeloperoxidase expression: quantitative analysis by antigen immunohistochemistry in a model of transient focal cerebral ischemia.

BACKGROUND AND PURPOSE: Expression of matrix metalloproteinases (MMPs), proteolytic enzymes that degrade extracellular proteins, is altered after ischemia/reperfusion injury and may contribute to blood-brain barrier (BBB) breakdown. Neutrophils, a source of reactive oxygen species and MMP-9, infiltrate damaged tissue 6 to 24 hours after ischemia and have also been implicated in delayed secondary tissue damage. Here we examined the spatial-temporal relation between MMP-9 expression and neutrophil infiltration after stroke. METHODS: Knockout mice containing 50% manganese superoxide dismutase activity (SOD2-KOs), which are more susceptible to ischemic damage than wild-type (WT) littermates, underwent quantitative antigen (MMP-9, myeloperoxidase) immunohistochemistry (24 and 72 hours) analysis and protein expression by Western blotting (6, 12, 24, 48, and 72 hours) after transient focal cerebral ischemia. BBB breakdown was determined by Evans blue extravasation. RESULTS: There was a clear spatial relation between MMP-9 expression and Evans blue extravasation. MMP-9-positive cell and vessel counts for SOD2-KOs (72 hours) were significantly different from SOD2-KO (24 hours, P=0.004), WT (24 hours, P=0.01), and WT (72 hours, P=0.007) mice. In contrast, MMP-9-positive neutrophil counts were comparatively low and did not differ by time or animal type. MMP-9 expression was biphasic in SOD2-KOs but not in WT littermates, with a significant increase observed 6 to 12 hours after ischemic insult and again at 48 to 72 hours. SOD2-KOs showed increased MMP-9 expression compared with WT littermates at all time points studied (P< or =0.05). CONCLUSIONS: In this model, neutrophils are not the primary source of MMP-9 protein and thus are unlikely the key contributor to BBB breakdown observed in SOD2-KOs.

Neuronal death/survival signaling pathways in cerebral ischemia.

Cumulative evidence suggests that apoptosis plays a pivotal role in cell death in vitro after hypoxia. Apoptotic cell death pathways have also been implicated in ischemic cerebral injury in in vivo ischemia models. Experimental ischemia and reperfusion models, such as transient focal/global ischemia in rodents, have been thoroughly studied and the numerous reports suggest the involvement of cell survival/death signaling pathways in the pathogenesis of apoptotic cell death in ischemic lesions. In these models, reoxygenation during reperfusion provides a substrate for numerous enzymatic oxidation reactions. Oxygen radicals damage cellular lipids, proteins and nucleic acids, and initiate cell signaling pathways after cerebral ischemia. Genetic manipulation of intrinsic antioxidants and factors in the signaling pathways has provided substantial understanding of the mechanisms involved in cell death/survival signaling pathways and the role of oxygen radicals in ischemic cerebral injury. Future studies of these pathways may provide novel therapeutic strategies in clinical stroke.

Oxidative damage to the endoplasmic reticulum is implicated in ischemic neuronal cell death.

The endoplasmic reticulum (ER), which plays important roles in apoptosis, is susceptible to oxidative stress. Because reactive oxygen species (ROS) are robustly produced in the ischemic brain, ER damage by ROS may be implicated in ischemic neuronal cell death. We induced global brain ischemia on wild-type and copper/zinc superoxide dismutase (SOD1) transgenic rats and compared ER stress and neuronal damage. Phosphorylated forms of eukaryotic initiation factor 2 alpha (eIF2 alpha) and RNA-dependent protein kinase-like ER eIF2 alpha kinase (PERK), both of which play active roles in apoptosis, were increased in hippocampal CA1 neurons after ischemia but to a lesser degree in the transgenic animals. This finding, together with the finding that the transgenic animals showed decreased neuronal degeneration, indicates that oxidative ER damage is involved in ischemic neuronal cell death. To elucidate the mechanisms of ER damage by ROS, we analyzed glucose-regulated protein 78 (GRP78) binding with PERK and oxidative ER protein modification. The proteins were oxidatively modified and stagnated in the ER lumen, and GRP78 was detached from PERK by ischemia, all of which were attenuated by SOD1 overexpression. We propose that ROS attack and modify ER proteins and elicit ER stress response, which results in neuronal cell death.

Neurodegeneration in striatum induced by the mitochondrial toxin 3-nitropropionic acid: role of matrix metalloproteinase-9 in early blood-brain barrier disruption?

Blood-brain barrier (BBB) dysfunction is a potential mechanism involved in progressive striatal damage induced by the mitochondrial excitotoxin, 3-nitropropionic acid (3-NP). After activation by proteases and free radicals, matrix metalloproteinases (MMPs), particularly MMP-9 and -2, can digest the endothelial basal lamina leading to BBB opening. Using CD-1 mice, we show that MMP-9 expression by zymography is increased in the injured striatum compared with the contralateral striatum 2 hr after 3-NP injection [133.50 +/- 57.17 vs 50.25 +/- 13.56; mean +/- SD of optical densities in arbitrary units (A.U.); p < 0.005] and remains elevated until 24 hr (179.33 +/- 78.24 A.U.). After 4 hr, MMP-9 expression and activation are accompanied by an increase in BBB permeability. MMP inhibition attenuates BBB disruption, swelling, and lesion volume compared with vehicle-treated controls. There is a clear spatial relationship between MMP-9 expression and oxidized hydroethidine, indicating reactive oxygen species (ROS) production. Furthermore, transgenic mice that overexpress copper/zinc-superoxide dismutase (SOD1) show decreased lesion size and edema along with decreased immunoreactivity for MMP-9, compared with wild-type littermates (lesion: 38.8 +/- 15.1 and 53.3 +/- 10.3, respectively, p < or = 0.05; edema: 21.8 +/- 11.2 and 35.28 +/- 11, respectively, p < or = 0.05; MMP-9-positive cells: 352 +/- 57 and 510 +/- 45, respectively, p < or = 0.005), whereas knock-out mice deficient in SOD1 display significantly greater swelling (48.65 +/- 17; p < or = 0.05). We conclude that early expression and activation of MMP-9 by ROS may be involved in early BBB disruption and progressive striatal damage after 3-NP treatment.

Effects of cold injury-induced trauma in manganese superoxide dismutase-deficient mice.

Manganese superoxide dismutase (Mn-SOD, SOD2) is an inducible antioxidant localized to the mitochondria, which have been shown to be both the sites of superoxide anion (O(2)*-)) production and the target of free radical attacks. Knock-out mice with targeted disruption of Sod2 (SOD2-KO) are more susceptible to ischemic damage than their wild-type (WT) counterparts, showing increased loss of mitochondrial cytochrome c after trauma, but less apoptotic cell death in the first 24 h following controlled cortical injury. In this study, we sought to investigate whether oxidative stress plays a significant role in the development of secondary brain damage following cold injury-induced brain trauma (CIBT), a model of vasogenic edema. We first measured the levels of O(2)(*-) production 2 h after CIBT by means of in situ hydroethidine oxidation. We then examined lesion size, brain swelling, apoptosis by morphology and TUNEL-staining, neutrophil infiltration, and hemorrhage rates in both SOD2-KO and WT mice at 1, 3, and 7 days post-CIBT. We found no significant differences between SOD2-KO and WT littermates in any of the paradigms or endpoints studied. There was, however, a significant increase in hemorrhagic transformations in all animals that paralleled a robust inflammatory response at 3 days post insult compared with the 24-h endpoint. In the CIBT model used in this study, a 50% reduction in SOD2 activity did not appear to alter the injury response, suggesting that accumulation of free radicals does not play a significant role in secondary brain damage as previously thought with this particular model.

Effects of mild hypothermia on superoxide anion production, superoxide dismutase expression, and activity following transient focal cerebral ischemia.

Following a transient ischemic insult there is a marked increase in free radical (FR) production within the first 10-15 min of reperfusion and again at the peak of the inflammatory process. Hypothermia decreases lipid peroxidation following global ischemia, raising the possibility that it may act by reducing FR production early on and by maintaining or increasing endogenous antioxidant systems. By means of FR fluorescence, Western blot, immunohistochemistry, and enzymatic assay, we studied the effects of mild hypothermia on superoxide (O(-*)(2)) anion production, superoxide dismutase SOD expression, and activity following focal cerebral ischemia in rats. Mild hypothermia significantly reduced O(-*)(2) generation in the ischemic penumbra and corresponding contralateral region, but did not alter the bilateral SOD expression. SOD enzymatic activity in the ischemic core was slightly reduced in hypothermia-treated animals compared with normothermic controls. Our results suggest that the neuroprotective effect of mild hypothermia may be due, in part, to a reduction in neuronal and endothelial O(-*)(2) production during early reperfusion.