Depression following traumatic brain injury in mice is associated with down-regulation of hippocampal astrocyte glutamate transporters by thrombin.

Depression after traumatic brain injury (TBI) is common but the mechanisms by which TBI causes depression are unknown. TBI decreases glutamate transporters GLT-1 and GLAST and allows extravasation of thrombin. We examined the effects of thrombin on transporter expression in primary hippocampal astrocytes. Application of a PAR-1 agonist caused down-regulation of GLT-1, which was prevented by inhibition of Rho kinase (ROCK). To confirm these mechanisms in vivo, we subjected mice to closed-skull TBI. Thrombin activity in the hippocampus increased one day following TBI. Seven days following TBI, expression of GLT-1 and GLAST was reduced in the hippocampus, and this was prevented by administration of the PAR-1 antagonist SCH79797. Inhibition of ROCK attenuated the decrease in GLT-1, but not GLAST, after TBI. We measured changes in glutamate levels in the hippocampus seven days after TBI using an implanted biosensor. Stress-induced glutamate levels were significantly increased following TBI and this was attenuated by treatment with the ROCK inhibitor fasudil. We quantified depressive behavior following TBI and found that inhibition of PAR-1 or ROCK decreased these behaviors. These results identify a novel mechanism by which TBI results in down-regulation of astrocyte glutamate transporters and implicate astrocyte and glutamate transporter dysfunction in depression following TBI.

Stem cell factor and granulocyte colony-stimulating factor exhibit therapeutic effects in a mouse model of CADASIL.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a Notch3 dominant mutation-induced cerebral small vascular disease, is characterized by progressive degeneration of vascular smooth muscle cells (vSMCs) of small arteries in the brain, leading to recurrent ischemic stroke, vascular dementia and death. To date, no treatment can stop or delay the progression of this disease. Herein, we determined the therapeutic effects of stem cell factor (SCF) in combination with granulocyte colony-stimulating factor (G-CSF) (SCF+G-CSF) in a mouse model of CADASIL carrying the human mutant Notch3 gene. SCF+G-CSF was subcutaneously administered for 5 days and repeated 4 times with 1-4 month intervals. We found through water maze testing that SCF+G-CSF treatment improved cognitive function. SCF+G-CSF also attenuated vSMC degeneration in small arteries, increased cerebral blood vascular density, and inhibited apoptosis in CADASIL mice. We also discovered that loss of cerebral capillary endothelial cells and neural stem cells/neural progenitor cells (NSCs/NPCs) occurred in CADASIL mice. SCF+G-CSF treatment inhibited the CADASIL-induced cell loss in the endothelia and NSCs/NPCs and promoted neurogenesis. In an in vitro model of apoptosis, SCF+G-CSF prevented apoptotic cell death in vSMCs through AKT signaling and by inhibiting caspase-3 activity. These data suggest that SCF+G-CSF restricts the pathological progression of CADASIL. This study offers new insights into developing therapeutic strategies for CADASIL.

Rapid spread of a recently introduced virus (tomato yellow leaf curl virus) and its vector Bemisia tabaci (Hemiptera: Aleyrodidae) in Liaoning Province, China.

In Liaoning Province, China, tomato yellow leaf curl virus (TYLCV) was first detected in 2009 and in only four counties. To quantify the spread of TYLCV and to identify potential factors influencing its spread in Liaoning Province, we assayed for TYLCV within 1,055 whiteflies (Bemisia tabaci (Gennadius) complex) from 74 populations and 29 counties in 2011. The B. tabaci species of these individuals was determined based on molecular markers. TYLCV was found in 13 counties (Donggang, Liaoyang, Kazuo, Lingyuan, Heishan, Liaozhong, Kaiyuan, Taian, Dawa, Dashiqiao, Beizhen, Linghai, and Xingcheng) and was most frequently detected in the central plain. In addition, the percentage of whiteflies with TYLCV was significantly higher in B. tabaci Q than in B. tabaci B but was unrelated to the hosts (pepper, eggplant, tomato, cucumber, and kidney bean) on which the whiteflies had been collected. These results demonstrate that TYLCV has spread rapidly in Liaoning Province since its first detection and suggest that its spread is more closely associated with the introduction of B. tabaci Q than with the species of host plant. These findings also indicate that controls are now needed to reduce the further spread of TYLCV and that these controls should include the management of B. tabaci Q populations.

Novel pathological features and potential therapeutic approaches for CADASIL: insights obtained from a mouse model of CADASIL.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common condition of hereditary stroke and vascular dementia. CADASIL is caused by Notch3 mutation, leading to progressive degeneration of vascular smooth muscle cells (vSMCs) of the small arteries in the brain. However, the pathogenesis of CADASIL remains largely unknown, and treatment that can stop or delay the progression of CADASIL is not yet available. Using both wild type mice and transgenic mice carrying the human mutant Notch3 gene (CADASIL mice), we have recently characterized the pathological features of CADASIL and determined the therapeutic efficacy of two hematopoietic growth factors, stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) in CADASIL. Our findings have revealed novel pathological changes in the endothelium of cerebral capillaries and in the neural stem cells (NSCs). We have also observed the impairment of cognitive function in CADASIL mice. Moreover, SCF+G-CSF treatment improves cognitive function, inhibits Notch3 mutation-induced vSMC degeneration, cerebral blood bed reduction, cerebral capillary damage, and NSC loss, and increases neurogenesis and angiogenesis. Here we compile an overview of our recently published studies, which provide new insights into understanding the pathogenesis of CADASIL and developing therapeutic strategies for this devastating neurological disease.

Late exercise reduces neuroinflammation and cognitive dysfunction after traumatic brain injury.

Delayed secondary biochemical and cellular changes after traumatic brain injury continue for months to years, and are associated with chronic neuroinflammation and progressive neurodegeneration. Physical activity can reduce inflammation and facilitate recovery after brain injury. Here, we investigated the time-dependent effects, and underlying mechanisms of post-traumatic exercise initiation on outcome after moderate traumatic brain injury using a well-characterized mouse controlled cortical impact model. Late exercise initiation beginning at 5weeks after trauma, but not early initiation of exercise at 1week, significantly reduced working and retention memory impairment at 3months, and decreased lesion volume compared to non-exercise injury controls. Cognitive recovery was associated with attenuation of classical inflammatory pathways, activation of alternative inflammatory responses and enhancement of neurogenesis. In contrast, early initiation of exercise failed to alter behavioral recovery or lesion size, while increasing the neurotoxic pro-inflammatory responses. These data underscore the critical importance of timing of exercise initiation after trauma and its relation to neuroinflammation, and challenge the widely held view that effective neuroprotection requires early intervention.

The combination of stem cell factor and granulocyte-colony stimulating factor for chronic stroke treatment in aged animals.

BACKGROUND: Stroke occurs more frequently in the elderly population and presents the number one leading cause of persistent disability worldwide. Lack of effective treatment to enhance brain repair and improve functional restoration in chronic stroke, the recovery phase of stroke, is a challenging medical problem to be solved in stroke research. Our early study has revealed the therapeutic effects of stem cell factor (SCF) in combination with granulocyte-colony stimulating factor (G-CSF) (SCF+G-CSF) on chronic stroke in young animals. However, whether this treatment is effective and safe to the aged population remains to be determined. METHODS: Cortical brain ischemia was produced in aged C57BL mice or aged spontaneously hypertensive rats. SCF+G-CSF or equal volume of vehicle solution was subcutaneously injected for 7 days beginning at 3-4 months after induction of cortical brain ischemia. Using the approaches of biochemistry assays, flow cytometry, pathology, and evaluation of functional outcome, several doses of SCF+G-CSF have been examined for their safety and efficiency on chronic stroke in aged animals. RESULTS: All tested doses did not show acute or chronic toxicity in the aged animals. Additionally, SCF+G-CSF treatment in chronic stroke of aged animals mobilized bone marrow stem cells and improved functional outcome in a dose-dependent manner. CONCLUSIONS: SCF+G-CSF treatment is a safe and effective approach to chronic stroke in the aged condition. This study provides important information needed for developing a new therapeutic strategy to improve the health of older adults with chronic stroke.

Over-expression of HSP70 attenuates caspase-dependent and caspase-independent pathways and inhibits neuronal apoptosis.

HSP70 is a member of the family of heat-shock proteins that are known to be up-regulated in neurons following injury and/or stress. HSP70 over-expression has been linked to neuroprotection in multiple models, including neurodegenerative disorders. In contrast, less is known about the neuroprotective effects of HSP70 in neuronal apoptosis and with regard to modulation of programmed cell death (PCD) mechanisms in neurons. We examined the effects of HSP70 over-expression by transfection with HSP70-expression plasmids in primary cortical neurons and the SH-SY5Y neuronal cell line using four independent models of apoptosis: etoposide, staurosporine, C2-ceramide, and ß-Amyloid. In these apoptotic models, neurons transfected with the HSP70 construct showed significantly reduced induction of nuclear apoptotic markers and/or cell death. Furthermore, we demonstrated that HSP70 binds and potentially inactivates Apoptotic protease-activating factor 1, as well as apoptosis-inducing factor, key molecules involved in development of caspase-dependent and caspase-independent PCD, respectively. Markers of caspase-dependent PCD, including active caspase-3, caspase-9, and cleaved PARP were attenuated in neurons over-expressing HSP70. These data indicate that HSP70 protects against neuronal apoptosis and suggest that these effects reflect, at least in part, to inhibition of both caspase-dependent and caspase-independent PCD pathways.

Combined inhibition of cell death induced by apoptosis inducing factor and caspases provides additive neuroprotection in experimental traumatic brain injury.

Neuronal programmed cell death (PCD) contributes to delayed tissue damage after traumatic brain injury (TBI). Both caspase-dependent and caspase-independent mechanisms have been implicated, with the latter including apoptosis inducing factor (AIF). The peptidyl-proplyl isomerase Cyclophilin A (CypA) transports AIF from the cytosol to the nucleus, a key step for AIF-dependent cell death. We compared the effects of single versus combined inhibition of caspase and AIF pathways in a mouse controlled cortical impact (CCI) model, by examining the effects of CypA gene knockout (CypA(-/-)), caspase inhibition with a pan-caspase inhibitor (boc-aspartyl(OMe)-fluoromethylketone, BAF), or combined modulation. TBI caused caspase activation as well as translocation of AIF to the nucleus. Markers of caspase activation including caspase-specific fodrin cleavage fragments and number of FLIVO-positive cells were reduced in BAF-treated CypA(+/+) mice, whereas markers of AIF activation including AIF/H2AX interaction and AIF translocation to the nucleus were attenuated in CypA(-/-) mice. Each single intervention, (CypA(-/-) or BAF-treated CypA(+/+)) reduced the number of apoptotic cells (TUNEL-positive) in the cortex and improved long-term sensorimotor function; CypA(-/-) also attenuated microglial activation after injury. Importantly, BAF-treated CypA(-/-) mice, showed greater effects than either intervention alone on multiple outcomes including: reduction in TUNEL-positive cells, decrease in neuroinflammation, improved motor and cognitive recovery, and attenuation of lesion volume and neuronal loss in the hippocampus. Using two in vitro neuronal cell death models known to induce AIF-mediated PCD, we also showed that neurons from CypA(-/-) animals were protected and that effects were unrelated to caspase activation. These data indicate that AIF-mediated and caspase-dependent pathways contribute independently and in parallel to secondary injury after TBI, and suggest that combined therapeutic strategies directed at multiple PCD pathways may provide superior neuroprotection than those directed at single mechanisms.

Stem cell factor and granulocyte colony-stimulating factor promote neuronal lineage commitment of neural stem cells.

Stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) were originally discovered as growth factors for hematopoietic stem cells (HSCs). It has been well defined that SCF and G-CSF contribute to regulation of lineage commitment for HSCs. However, little is known about whether SCF and G-CSF play roles in the determination and differentiation of neural stem cells (NSCs). Here we demonstrate the novel function of SCF and G-CSF in controlling cell cycle and cell fate determination of NSCs. We also observe that SCF and G-CSF promote neuronal differentiation and inhibit astroglial differentiation at the early stage of differentiation. In addition, our research data reveal that SCF in combination with G-CSF has a dual function in promoting cell cycle exit and directing neuronal fate commitment at the stage of NSC dividing. This coordination effect of SCF+G-CSF on cell cycle arrest and neuronal differentiation is through enhancing neurogenin 1 (Ngn1) activity. These findings extend current knowledge regarding the role of SCF and G-CSF in the regulation of neurogenesis and provide insights into the contribution of hematopoietic growth factors to brain development and remodeling.

Stem cell factor and granulocyte colony-stimulating factor reduce ß-amyloid deposits in the brains of APP/PS1 transgenic mice.

INTRODUCTION: Alzheimer's disease (AD) is widely recognized as a serious public health problem and heavy financial burden. Currently, there is no treatment that can delay or stop the progressive brain damage in AD. Recently, we demonstrated that stem cell factor (SCF) in combination with granulocyte colony-stimulating factor (G-CSF) (SCF+G-CSF) has therapeutic effects on chronic stroke. The purpose of the present study is to determine whether SCF+G-CSF can reduce the burden of ß-amyloid deposits in a mouse model of AD. METHODS: APP/PS1 transgenic mice were used as the model of AD. To track bone marrow-derived cells in the brain, the bone marrow of the APP/PS1 mice was replaced with the bone marrow from mice expressing green fluorescent protein (GFP). Six weeks after bone marrow transplantation, mice were randomly divided into a saline control group and a SCF+G-CSF-treated group. SCF in combination with G-CSF was administered subcutaneously for 12 days. Circulating bone marrow stem cells (CD117+ cells) were quantified 1 day after the final injection. Nine months after treatment, at the age of 18 months, mice were sacrificed. Brain sections were processed for immunohistochemistry to identify ß-amyloid deposits and GFP expressing bone marrow-derived microglia in the brain. RESULTS: Systemic administration of SCF+G-CSF to APP/PS1 transgenic mice leads to long-term reduction of ß-amyloid deposition in the brain. In addition, we have also observed that the SCF+G-CSF treatment increases circulating bone marrow stem cells and augments bone marrow-derived microglial cells in the brains of APP/PS1 mice. Moreover, SCF+G-CSF treatment results in enhancement of the co-localization of bone marrow-derived microglia and ß-amyloid deposits in the brain. CONCLUSIONS: These data suggest that bone marrow-derived microglia play a role in SCF+G-CSF-induced long-term effects to reduce ß-amyloid deposits. This study provides insights into the contribution of the hematopoeitic growth factors, SCF and G-CSF, to limit ß-amyloid accumulation in AD and may offer a new therapeutic approach for AD.

Brain self-protection: the role of endogenous neural progenitor cells in adult brain after cerebral cortical ischemia.

Convincing evidence has shown that brain ischemia causes the proliferation of neural stem cells/neural progenitor cells (NSCs/NPCs) in both the subventricular zone (SVZ) and the subgranular zone (SGZ) of adult brain. The role of brain ischemia-induced NSC/NPC proliferation, however, has remained unclear. Here we have determined whether brain ischemia-induced amplification of the NSCs/NPCs in adult brain is required for brain self-protection. The approach of intracerebroventricular (ICV) infusion of cytosine arabinoside (Ara-C), an inhibitor for cell proliferation, for the first 7days after brain ischemia was used to block ischemia-induced NSC/NPC proliferation. We observed that ICV infusion of Ara-C caused a complete blockade of NSC/NPC proliferation in the SVZ and a dramatic reduction of NSC/NPC proliferation in the SGZ. Additionally, as a result of the inhibition of ischemia-induced NSC/NPC pool amplification, the number of neurons in the hippocampal CA1 and CA3 was significantly reduced, the infarction size was significantly enlarged, and neurological deficits were significantly worsened after focal brain ischemia. We also found that an NSC/NPC-conditioned medium showed neuroprotective effects in vitro and that adult NSC/NPC-released brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) are required for NSC/NPC-conditioned medium-induced neuroprotection. These data suggest that NSC/NPC-generated trophic factors are neuroprotective and that brain ischemia-triggered NSC/NPC proliferation is crucial for brain protection. This study provides insights into the contribution of endogenous NSCs/NPCs to brain self-protection in adult brain after ischemia injury.

alphaB-crystallin suppresses oxidative stress-induced astrocyte apoptosis by inhibiting caspase-3 activation.

alphaB-crystallin is a member of the small heat shock proteins, which is abundantly expressed in various vertebrate tissues including the central nervous system. In our previous report, we showed alphaB-crystallin induction in activated astrocytes in the postischemic brain and in H2O2-treated primary astrocyte cultures. To investigate the functional significance of alphaB-crystallin induction in astrocytes, we generated a stable C6 astroglioma cell line overexpressing alphaB-crystallin. In these cells, hydrogen peroxide-induced apoptosis was reduced by 60% compared to parent cells. Furthermore, the repression of alphaB-crystallin expression by alphaB-crystallin siRNA transfection suppressed this protective effect, indicating that alphaB-crystallin is responsible for the protection against H2O2-induced apoptosis in C6 astroglioma cells. Similar level of aggravation in H2O2-induced apoptosis was observed in primary astrocyte cultures when alphaB-crystallin expression was suppressed by alphaB-crystallin siRNA transfection, confirming the importance of alphaB-crystallin. In addition, the induction of caspase-3 activity after H2O2 treatment was markedly suppressed in alphaB-crystallin-overexpressing cells, and immunoprecipitation proved binding between alphaB-crystallin and partially processed caspase-3 (a p24 intermediate). These results indicate that alphaB-crystallin confers protection against hydrogen peroxide-induced astrocytes apoptosis in part by inhibiting caspase-3 activation.

The role of stem cell factor and granulocyte-colony stimulating factor in brain repair during chronic stroke.

Chronic stroke is a highly important but under-investigated scientific problem in neurologic research. We have reported earlier that stem cell factor (SCF) in combination with granulocyte-colony stimulating factor (G-CSF) treatment during chronic stroke improves functional outcomes. Here we have determined the contribution of bone marrow-derived cells in angiogenesis and neurogenesis, which are enhanced by SCF+G-CSF treatment during chronic stroke. Using bone marrow tracking, flow cytometry, 2-photon live brain imaging, and immunohistochemistry, we observed that the levels of circulating bone marrow stem cells (BMSCs) (CD34+/c-kit+) were significantly increased by SCF+G-CSF treatment. In addition, live brain imaging revealed that numerous bone marrow-derived cells migrate into the brain parenchyma in the treated mice. We also found that bone marrow-derived cells, bone marrow-derived endothelial cells, vascular density, and bone marrow-derived neurons were significantly augmented by SCF+G-CSF. It is interesting that, in addition to the increase in bone marrow-derived endothelial cells, the number of bone marrow-derived pericytes was reduced after SCF+G-CSF treatment during chronic stroke. These data suggest that SCF+G-CSF treatment can enhance repair of brain damage during chronic stroke by mobilizing BMSCs, and promoting the contribution of bone marrow-derived cells to angiogenesis and neurogenesis.

LEDGF binding to stress response element increases alphaB-crystallin expression in astrocytes with oxidative stress.

AlphaB-crystallin, known as a vertebrate lens protein, is a member of the small heat shock proteins (sHSP). AlphaB-crystallin is abundantly expressed in the vertebrate lens and striated muscles and it is also expressed constitutively in other tissues including the central nervous system (CNS). In our previous report, we showed alphaB-crystallin induction in activated astrocytes, which are enriched in the penumbra after transient focal cerebral ischemia. We also reported that alphaB-crystallin is significantly induced in astrocytes in the CA3 region of the hippocampus following KA-induced seizure. Here, we report that the expression of alphaB-crystallin is upregulated in H2O2-treated primary astrocyte cultures, which was prepared from newborn male Sprague-Dawley rats and that the proximal 408 bp of the alphaB-crystallin promoter harboring stress response element (STRE) is responsible for this induction. This effect of H2O2 was found to be virtually abolished by introducing mutations into STRE, and these mutations also impaired increased lens epithelial derived growth factor (LEDGF) binding to STRE after H2O2 treatment. Moreover, LEDGF was induced in primary astrocyte cultures after H2O2 treatment and alphaB-crystallin induction was significantly suppressed by transfecting small interfering RNA (siRNA) targeting LEDGF. Together these results indicate that the H2O2-induced upregulations of alphaB-crystallin in astrocytes are mediated by the LEDGF-STRE interaction on alphaB-crystallin promoter.

Hematopoietic growth factors pass through the blood-brain barrier in intact rats.

We have previously demonstrated that receptors for hematopoietic growth factors, stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF) are expressed in the neurons and the neural progenitor cells (NPCs) of the adult rat brain, and that systemic administration of SCF and G-CSF in the first week after induction of cortical brain ischemia (3 h-7 days post-ischemia) significantly improves functional outcome, augments NPC proliferation, and reduces infarct volume in rats. The purpose of the present study is to determine whether SCF and G-CSF pass through the blood-brain barrier (BBB) in intact rats. The growth factors were labeled with iodine (I(125)), a radioactive compound. I(125)-SCF and I(125)-G-CSF were intravenously administered and the concentrations of I(125)-SCF and I(125)-G-CSF in the blood plasma and the brain were determined at 10, 30, 60, and 120 min after injection. We observed that both SCF and G-CSF were slowly and continuously transported from the blood stream to the brain in the same rate. In addition, both immunofluorescent staining and Western blots showed that receptors for SCF and G-CSF were expressed in the capillaries of the adult rat brain, suggesting that SCF and G-CSF entry to the brain may be mediated via receptor-mediated transport, one of the endogenous transports in the BBB. These data indicate that both SCF and G-CSF were able to pass through the BBB in intact animals. This observation will help in further exploring the physiological role of peripheral SCF and G-CSF in the brain and therapeutic possibility to chronic stroke.

HMGB1, a novel cytokine-like mediator linking acute neuronal death and delayed neuroinflammation in the postischemic brain.

Cerebral ischemic injury proceeds with excitotoxicity-induced acute neuronal death in the ischemic core and with delayed damage processes in the penumbra. However, knowledge concerning the direct mediators that connect these two processes is limited. Here, we demonstrate that high-mobility group box 1 (HMGB1), a nonhistone DNA-binding protein, is massively released into the extracellular space immediately after ischemic insult and that it subsequently induces neuroinflammation in the postischemic brain. Short hairpin (sh)RNA-mediated HMGB1 downregulation in the postischemic brain suppressed infarct size, microglia activation, and proinflammatory marker induction, indicating that HMGB1 plays a crucial role in the inflammatory process. The proinflammatory cytokine-like function of extracellular HMGB1 was further verified in primary cortical cultures and microglial cultures. HMGB1 was found to accumulate in NMDA-treated primary cortical culture media, and supernatants collected from these cultures were found to trigger microglia activation, the hallmark of brain inflammation. Moreover, treatment with recombinant HMGB1 also induced microglial activation, but HMGB1-depleted supernatant produced by anti-HMGB1 antibody treatment or by HMGB1 shRNA expression did not, thus demonstrating the essential role of HMGB1 in microglial activation. Together, these results indicate that HMGB1 functions as a novel proinflammatory cytokine-like factor that connects excitotoxicity-induced acute damage processes and delayed inflammatory processes in the postischemic brain.

Co-induction of alphaB-crystallin and MAPKAPK-2 in astrocytes in the penumbra after transient focal cerebral ischemia.

alphaB-crystallin (alpha-BC), a member of the small heat-shock proteins (sHSP), is constitutively expressed in the vertebrate lens and in non-ocular tissues including the central nervous system (CNS). In this study we investigated the expression of alpha-BC in the rat brain after middle cerebral artery occlusion (MCAO). alpha-BC transcript and protein were transiently expressed 4 h after MCAO/reperfusion in the pyramidal neurons in the peri-infarct region of the ischemic hemisphere. Beginning 2 days after MCAO, significant alpha-BC induction appeared in reactive astrocytes in the penumbra, and this induction was sustained for several days. In addition, levels of MAPKAPK-2, one of the alpha-BC upstream kinases, and its phosphorylated form were upregulated gradually and peaked 4 days after ischemia/reperfusion injury. The immunohistochemical study indicated that alpha-BC was co-localized with MAPKAPK-2 and p-MAPKAPK-2. Furthermore, p38beta MAPK, an upstream kinase of MAPKAPK-2, which has been known to be involved in compensatory responses to stress, was also co-localized with alpha-BC in the penumbra. Our results suggest that the p38beta-dependent alpha-BC induction in neurons and astrocytes in the penumbra may play an important role in the postischemic brain.

Delayed genomic responses to transient middle cerebral artery occlusion in the rat.

Ischemic stress in the brain induces acute and massive neuronal death in the targeted area, which is followed by a second round of detrimental processes, called delayed neuronal death, in the neighboring areas. To test the hypothesis that transcriptional control plays a role in the pathophysiology of the postischemic brain, the genomic responses that occurred in the brain at 3, 6 and, 12 h, and 1, 2 or 4 days after transient middle cerebral artery occlusion (MCAO) were examined using a microarray harboring 5000 rat cDNAs. This analysis indicated that the number of up-regulated genes was gradually increased, along with the concomitant reduction in the number of down-regulated genes, until 12 h to 1 day after MCAO. It was followed by a delayed surge of down-regulated genes at 2 days after MCAO. Northern blots and immunohistological analysis confirmed the validity of these microarray data. We present a list of 85 genes that were up-regulated more than 2.3-fold between 12 h and 4 days after MCAO, which included 56 novel genes whose expression has not previously been implicated in ischemic pathophysiology. The list included genes involved in oxidative stress, inflammation, extracellular matrix (ECM), neuronal development and differentiation processes. Together these results suggest that the pathophysiology of the postischemic brain proceeds by the transcriptional activation of genes related to the process of delayed neuronal damage and/or recovery and repair.

Inhibition of delayed induction of p38 mitogen-activated protein kinase attenuates kainic acid-induced neuronal loss in the hippocampus.

The activation of p38 mitogen-activated protein kinase (MAPK) has been implicated in the pathological changes accompanying inflammatory and apoptotic processes of various cell types including neurons. In a kainic acid (KA)-induced mouse seizure model, p38 MAPK is induced in reactive astrocytes in the CA3 region of the hippocampus where severe neuronal loss occurs. Here we report the delayed and protracted activation of p38 MAPK in the CA3 region of the hippocampus of mice treated with KA. In this model, the inhibition of p38 MAPK isoforms by SB203580, a specific inhibitor, attenuated neuronal loss in the CA3 and CA1 regions of the hippocampus, which was accompanied by the suppression of the p38 MAPK activation as well as astrogliosis. Thus, the delayed and sustained induction of p38 MAPK plays a crucial role in the neuronal damage of KA-induced brain seizures.

Administration of the p38 MAPK inhibitor SB203580 affords brain protection with a wide therapeutic window against focal ischemic insult.

We have reported previously the delayed and differential induction of p38alpha and p38beta mitogen-activated protein kinases (MAPKs) in microglia and astrocytes, respectively, in brain after transient global ischemia. We report here the sustained induction and activation of p38alpha MAPK in activating microglia in rat brain after transient middle cerebral artery occlusion (MCAO). The intraventricular administration of SB203580, a p38 MAPK inhibitor, 30 min before MCAO reduced the infarct volume to 50% of the control, which was accompanied by the significant improvement of neurological deficits. More interestingly, the infarct volume was reduced to 72% and 77% when SB203580 was administered 6 hr and 12 hr after MCAO, respectively. The induction of various factors involved in inflammatory processes, such as inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and cyclooxygenase-2 (COX-2), was suppressed by the administration of SB203580 at 6 hr after MCAO. These results suggest that sustained activation of p38 MAPK pathway and p38 MAPK-associated inflammatory processes play a crucial role in postischemic brain.