Ischemic Rat Brain Research Articles

Early Inhibition of MMP Activity in Ischemic Rat Brain Promotes Expression of Tight Junction Proteins and Angiogenesis During Recovery

In cerebral ischemia, matrix metalloproteinases (MMPs) have a dual role by acutely disrupting tight junction proteins (TJPs) in the blood-brain barrier (BBB) and chronically promoting angiogenesis. Since TJP remodeling of the neurovascular unit (NVU) is important in recovery and early inhibition of MMPs is neuroprotective, we hypothesized that short-term MMP inhibition would reduce infarct size and promote angiogenesis after ischemia. Adult spontaneously hypertensive rats had a transient middle cerebral artery occlusion with reperfusion. At the onset of ischemia, they received a single dose of the MMP inhibitor, GM6001. They were studied at multiple times up to 4 weeks with immunohistochemistry, biochemistry, and magnetic resonance imaging (MRI). We observed newly formed vessels in peri-infarct regions at 3 weeks after reperfusion. Dynamic contrast-enhanced MRI showed BBB opening in new vessels. Along with the new vessels, pericytes expressed zonula occludens-1 (ZO-1) and MMP-3, astrocytes expressed ZO-1, occludin, and MMP-2, while endothelial cells expressed claudin-5. The GM6001, which reduced tissue loss at 3 to 4 weeks, significantly increased new vessel formation with expression of TJPs and MMPs. Our results show that pericytes and astrocytes act spatiotemporally, contributing to extraendothelial TJP formation, and that MMPs are involved in BBB restoration during recovery. Early MMP inhibition benefits neurovascular remodeling after stroke.

Combination Therapy With Insulin‐like Growth Factor‐1 and Hypothermia Synergistically Improves Outcome After Transient Global Brain Ischemia in the Rat

Hypothermia has a well-established neuroprotective effect and offers a foundation for combination therapy for brain ischemia. The authors evaluated the effect of combination therapy with insulin-like growth factor-1 (IGF-1) and hypothermia on brain structure and function in the setting of global brain ischemia and reperfusion in rats. Male Sprague-Dawley rats were randomly assigned to groups by a registrar. Animals were subjected to 8 minutes of global brain ischemia using bilateral carotid occlusion and systemic hypotension, followed by 7 days (Stage I dose studies) or 28 days (Stage II outcome studies) of reperfusion. Sham controls were subjected to surgery, but not ischemia. Stage II animals were randomized to no treatment, IGF-1 at the dose determined in Stage I, hypothermia (32°C for 4 hours), or a combination of IGF-1 and hypothermia. Stage II animals underwent 21 days of spatial memory testing. At 7 days (Stage I) or 28 days (Stage II), brains were harvested for counting of CA1 neurons. The primary Stage II outcome was a neurologic outcome index computed as the ratio of viable CA1 neurons per 300-μm field to the number of days to reach success criteria on the memory task. Stage I experiments confirmed the neuroprotective effect of the hypothermia protocol and IGF-1 at a dose of 0.6 U/kg. Stage II studies suggested that early neuroprotection with hypothermia and IGF-1 was not well maintained to 28 days and that combination therapy was more beneficial than either IGF-1 or hypothermia alone. Median and interquartile ranges (IQRs) of viable neurons per 300-μm field were 114 (IQR = 99.5 to 136) for sham, three (IQR = 2 to 4.8) for untreated ischemia, four (IQR = 3 to 70.25) for ischemia treated with IGF-1 alone, 25 (IQR = 3 to 70) for ischemia treated with hypothermia alone, and 78 (IQR 47.3 to 97.5) for ischemia treated with combination therapy. Days to memory success criteria were 13.6 (IQR = 11.5 to 15.5 days) for sham, 23.5 (IQR = 20 to 25.5 days) for untreated ischemia, 17.5 (IQR = 15.5 to 25.5 days) for ischemia treated with IGF-1, 15 (IQR = 14.5 to 21 days) for ischemia treated with hypothermia, and 13.5 (IQR = 12.25 to 18.5 days) for ischemia treated with combination therapy. Neurologic outcome indices were 8.5 (IQR = 7.4 to 9.5) for sham, 0.14 (IQR = 0.08 to 0.2) for untreated ischemia, 0.18 (IQR = 0.17 to 4.6) for ischemia treated with IGF-1, 0.7 (IQR = 0.2 to 4.8) for ischemia treated with hypothermia, and 5.7 (IQR = 3.3 to 6.2) for ischemia treated with combination therapy. Statistically significant differences in neuron counts, days to memory test criteria, and outcome index were found between sham and untreated ischemic animals. Of the three treatment regimens, only combination therapy showed a statistically significant difference from the untreated ischemic group for neuronal salvage (p = 0.02), days to criteria (p = 0.043), and outcome index (p = 0.014). Combination therapy with IGF-1 (0.6 U/kg) and therapeutic hypothermia (32°C for 4 hours) at the onset of reperfusion synergistically preserves CA1 structure and function at 28 days after 8 minutes of global brain ischemia in healthy male rats.

Complexity in differentiating the expression of truncated or matured forms of MMP-2 and MMP-9 through zymography in rat brain tissues after acute ischaemic stroke

Matrix metalloproteinases (MMPs) play an important role in the pathogenesis of ischaemic stroke. In particular, the mature forms of MMPs 2 and 9 have similar sizes and share gelatine as a common substrate. Both MMPs are upregulated in ischaemic stroke and play detrimental roles during stroke pathogenesis. Throughout this study, we demonstrated that pro-MMP-2 and pro-MMP-9 from ischaemic rat brain tissue homogenate is detected either through immunoblotting or zymography because of the remarkable size difference between these enzymes (72 versus 95kDa, respectively). However, the mature MMP-2 and MMP-9 cannot be discriminated through zymography because of the almost identical sizes of these forms (66 and 67kDa, respectively). The use of gelatine zymography on ischaemic rat brain tissue homogenate revealed a 65-kDa MMP band, corresponding to the heterogeneous band of mature MMP-2 and/or MMP-9. Furthermore, we also detected mature MMPs of 65kDa generated from both recombinant human MMP-2 and MMP-9. Using a pull down assay in rat brain tissue homogenate with gelatine–agarose beads, we showed increased activities for both the pro and mature forms of MMP-2 and MMP-9. However, we could not determine the origin of the respective mature MMPs from the heterogeneous band. Thus, in this study, we demonstrated that the identification and quantification of mature MMP-2 and MMP-9 could not be achieved using zymography alone. Therefore, the development of a reliable technique to identify and measure the respective MMPs is needed to test new stroke therapies targeting MMP-2 and MMP-9.

Increased Expression of Neurotrophin 4 Following Focal Cerebral Ischemia in Adult Rat Brain with Treadmill Exercise

Neurotrophin 4 (NT-4) belongs to the family of neurotrophic factors, and it interacts with the tyrosine kinase B (trkB) receptor. NT-4 has neuroprotective effects following cerebral ischemia. Its role might be similar to brain-derived neurotrophic factor (BDNF), because both interact with trkB. Exercise also improves neural function by increasing neurotrophic factors. However, expression profiles of NT-4 in the brain during exercise are unknown. Here, we assessed the expressions of NT-4 and its receptor, trkB, following cerebral ischemia and hypothesized that exercise changes the expressions of NT-4 and trkB. Results showed that in a permanent middle cerebral artery occlusion rat model, ischemia decreased NT-4 and trkB expression. Immunohistochemistry showed their immunoreactivities around the region of the ischemic area. Treadmill exercise changed the expression of NT-4, which increased in the contralateral hemisphere in rats with ischemic injury. TrkB also showed similar patterns to its neurotophins. The change in NT-4 suggested that exercise might have primed NT4 production so that further injury causes slightly greater increases in NT4 compared with non-exercise controls.

Expression of MCP‐1 and fractalkine on endothelial cells and astrocytes may contribute to the invasion and migration of brain macrophages in ischemic rat brain lesions

Some macrophages expressing NG2 chondroitin sulfate proteoglycan (NG2) and the macrophage marker Iba1 accumulate in the ischemic core of a rat brain subjected to transient middle cerebral artery occlusion (MCAO) for 90 min. These cells are termed BINCs (for brain Iba1(+) /NG2(+) cells) and may play a neuroprotective role. Because BINCs are bone marrow-derived cells, they are able to invade ischemic tissue after the onset of an ischemic insult. In this study, chemokine-based mechanisms underlying the invasion of BINCs or their progenitor cells were investigated. We found that isolated BINCs expressed mRNA encoding CCR2 and CX3CR1 at high levels. Cultured astrocytes expressed mRNA encoding their ligands, MCP-1 and fractalkine. Recombinant MCP-1 and/or fractalkine, as well as astrocytes, induced the migration of BINCs in vitro. mRNA for MCP-1, fractalkine, CCR2, and CX3CR1 was expressed in the ischemic core during the acute phase of the ischemic event. Immunohistochemical studies revealed that vascular endothelial cells and astrocytic endfeet expressed MCP-1 and fractalkine, respectively, in the ischemic core during the acute phase. CCR2(+) /Iba1(+) monocytes attached to the inside of the vascular wall at 1 day postreperfusion (dpr), and there were CCR2(+) /CX3CR1(+) macrophage-like cells in the parenchyma in the ischemic lesion core at 2 dpr, which may be the progenitors for BINCs. These results suggest that CCR2(+) monocytes are first attracted to the ischemic lesion by MCP-1(+) endothelial cells and migrate toward fractalkine(+) astrocytic endfeet through the disrupted blood-brain barrier. Thus, chemokines may play a critical role in the accumulation of neuroprotective BINCs. © 2013 Wiley Periodicals, Inc.

Exercise Therapy Downregulates the Overexpression of TLR4, TLR2, MyD88 and NF-κB after Cerebral Ischemia in Rats.

Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) are considered to mediate the inflammatory reaction of cerebral ischemia injury, and exercise can inhibit the activity of the Toll-like receptor signaling pathway in the peripheral blood of humans. Although physical exercise has been demonstrated to be neuroprotective in both clinical and laboratory settings, the underlying mechanism remains unclear. To clarify this critical issue, this study investigated the effects of treadmill training on the recovery of neurological function and the expression of TLR2 and TLR4 and their main downstream targets, nuclear factor-kappaB (NF-κB) and myeloid differentiation factor 88 (MyD88), in the ischemic rat brain after middle cerebral artery occlusion-reperfusion (MCAo/R). Rats were divided into seven groups: sham control without MCAo/R and five, nine and 16 days post-ischemic exercise or non-exercise. The neurological function and infarct volume were measured, and reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were used to detect the expression of TLR2, TLR4, NF-κB and MyD88 in ischemic brain tissue. The results indicated that treadmill training promoted functional recovery and reduced the overexpression of TLR2, TLR4, NF-κB and MyD88 in rat brain tissue after ischemia, a finding that may have implications for understanding the mechanism of exercise therapy after brain ischemia and indicating new therapeutic strategies for the pharmacological modulation of TLR signaling.

Abstract TP112: Post-conditioning with Isoflurane Reduces Ischemia-induced Hemorrhage and Brain Injury Following MCAO in Rats: A Longitudinal Study with MRI

The objective of this study was to use multimodal MR imaging to evaluate the effect of isoflurane on short-term and long-term neuronal tissue damage and neurological deficits after focal brain ischemia in rats and correlate the improvement with the behavioral outcome. Male Wistar/Kyoto rats were divided into 4 experimental groups; Control (Group I, n=5), Sham (Group II, n=5), MCAO (Group III, n=20) and MCAO with isoflurane treatment (Group IV, n=20). Animals were anesthetized with 5% isoflurane and maintained on 2% isoflurane during surgery. MCA was transiently occluded (90 min) using intra-luminal thread. After surgery, group IV was kept under 2% isoflurane for 90 min period. Other groups were allowed to recover immediately after the surgery. Animals were subjected to MRI to measure, infract volume, edema and blood flow in 8 in vivo imaging sessions performed at 6, 12, 24, 48, 72h (short term) and 7, 14 and 21d (long term) post MCAO. Anatomical, diffusion and arterial spin labeling (ASL) were performed with 7T MR Scanner. The following parameters were used in DTI; TE/TR=23/5000 ms, diffusion directions 30, b=100, 850, and 1850 s/mm2, FOV 4.0 x 4.0 cm, 12 slices with thickness 1 mm, and matrix 128 x 128. Standard ASL sequence was used to calculate the regional blood flow. Diffusion images were processed to generate diffusion parameter maps. Mean diffusion parameters calculated in the infarct regions were manually drawn on T2 maps. Behavioral improvement was measured in terms of latency on rotarod and beam walk. Rats post-conditioned with isoflurane did not show hemorrhage during short term or long term post MCAO, while non-conditioned rat develop hemorrhage after short term post MCAO. Lesion in the cortical area was noticed since 6h post MCAO. However, cortical lesion was significantly reduced in the treatment group. Post-conditioned rats had significantly higher latency on rotarod and lesser number of foot faults on beam walk test measured on day 3,7,14 and 21 of MCAO, implying isoflurane mediated functional protection. Our results for the first time, at the longitudinal level show that maintaining MCAO rats under isoflurane environment before reperfusion offers enhanced neuroprotection and recovery from behavioral dysfunction through reduced brain hemorrhage.

Abstract TP258: Oleic Acid Decreased Brain Infarct Volume In Middle Cerebral Artery Occlusion Rats By Peroxisome Proliferator-Activated Receptor γ Activation

OA, a monounsaturated ω-9 fatty acid, is most abundant in all dietary fats and oils, especially olive oil, canola oil and high-oleic sunflower and safflower oil. OA was reported to have an anti-inflammatory effect by inhibiting reactive oxygen species (ROS), p38 MAPK, and Akt/IKK/NF-κB signaling pathways in LPS-stimulated BV2 microglia. The accumulation of FFAs after cerebral ischemia has been thought to contribute to injury-associated secondary brain damage by direct and indirect mechanisms. However, the role of OA has not been paid attention or known for exacerbating neuronal cell death in ischemic conditions. We hypothesized that OA may exert important role on the neuroprotective effect in ischemic rat brain and this effect exert through PPARγ activation. The infarct volume of systemic administration of OA in transient middle cerebral artery occlusion (MCAo) rat was measured after pretreatment of the PPARγ antagonist, GW9662 in order to determine whether PPARγ activation is critical for this effect, and the effect was compared with non-treated group. The expression of COX-2 and MMP-9 was investigated and the induction of HO-1 was also investigated by immunohistochemistry, RT-PCR and zymography in order to confirm the mechanism via activation of PPARγ. OA decreased infarct size induced by MCAo as late as 3 h after the injury, and the effect of OA was inhibited by the pretreatment of GW9662, a PPARγ antagonist. OA also inhibited the COX-2 and MMP-9 expression and induced HO-1 gene in MCAo rats. In conclusion, OA significantly reduces the infarct volume of MCAo rats through anti-inflammatory and anti-oxidative effect by acting, at least partly, as a PPARγ agonist. Given that this compound was administered as late as 3 h after the onset of the ischemic damage, OA might be very useful for stroke therapy.

Müller Glia Cells Activation in Rat Retina After Optic Nerve Injury: Spatiotemporal Correlation with Transcription Initiation Factor IIB

Transcription initiation factor IIB (TFIIB) is an ideal factor to localize core promoters and plays a central role in the assembly of the pre-initiation complex. Previous studies showed that the assembly of TFIIB played an important role in rat ischemic brain injury. To elucidate the expression and possible functions of TFIIB in retina lesion and repair, we performed an optic nerve crush (ONC) model in adult rats. Western blot analysis and immunohistochemistry showed a significant upregulation of TFIIB in retina after ONC. Immunofluorescent labeling indicated that TFIIB was localized mainly in the Müller glia cells (MGCs); colocalization of TFIIB and proliferating cell nuclear antigen (PCNA) in the injured retina suggested that TFIIB might participate in MGCs proliferation. In addition, we also examined the expression of the retinal progenitor markers (Nestin and Pax6) whose changes were correlated with the expression of TFIIB. In vitro, we induced MGCs differentiation with brain nerve growth factor (BNGF) and found that TFIIB expression was increased in the differentiated process, which was collected with the expression of PCNA, Nestin, and Pax6. Additionally, knocking TFIIB down with siRNA inhibited the expression of PCNA, Nestin, and Pax6. Collectively, we hypothesized ONC-induced upregulation of TFIIB in the retina was associated with MGCs activation and differentiation.

Brain-derived neurotrophic factor blood levels in two models of transient brain ischemia in rats

We monitored possible influence of transient focal and global brain ischemia on BDNF blood level. In both models noticeable fluctuation of BDNF concentration mainly in reperfusion was observed. During the first 90 min, BDNF in total blood and in blood cells continuously decreased in both models but plasma BDNF raised at 40 min and peaked at 90 min of reperfusion. Our data confirm the impact of transient brain ischemia on BDNF levels in the circulatory system, suggest blood cells as a possible source of BDNF and demonstrate the interdependence of blood compartments and physiological state of an affected organism.

Expression of somatostatine in ischemia of rat brain

This study used the immunohistochemical method to follow the expression of cytoplasmatic protein somatostanin in the course of ischemia of rat brain. The aim of the study was to define all the areas of expression of somatostain and to show the protein distribution on the map. All the sections of telencephalon, diencephalon and midbrain were studied in resistant, and transitory ischemia, which enabled us to observe the reaction of neurons to an ischemic attack or to repeated attacks. The results of this study show that there is a difference in the reaction between the resistant and transitory ischemia groups of rats, especially in the parietofrontal cortex, area amygdaloidea anterior, clastrum, nc. reuniens and nc. suprachiasmaticus. The mapping shows the reaction in the structures of motor, sensitive and sensory cortex, mostly in the laminae I/Ill and V/VI, hippocampus- gyrus dentatus and CA1, CA2, CA3, endopiriform nucleus, paraventricular and periventricular nucleus of hypothalamus, corpus amygdaloideum, claustrum and caudoputamen. The more primitive sections of the brain - rhinencephalon, also showed a reaction, which led us to conclude that both newer and older brain structures reacted immunohistochemically. Histological data showed that small neurons are most commonly found while the second most common are big pyramidal cells of multipolar and bipolar type, with the different body shape. Our findings have confirmed the results of rare studies that dealt with these issues, and offered a precise and detailed map of cells expressing somatostanin in the rat brain following ischemic attack.

Reversible Loss of N-Acetylaspartate After 15-Min Transient Middle Cerebral Artery Occlusion in Rat: A Longitudinal Study with In Vivo Proton Magnetic Resonance Spectroscopy

It is generally accepted that N-acetylaspartate (NAA) can be used a biochemical marker for assessing neuronal viability/integrity after cerebral ischemia. However, this view has recently been questioned based on observations showing that after a photothrombotic permanent ischemia the acute decline of NAA in the infracted regions, where massive neuronal loss persists, is reversible over time. In this study, we measured the longitudinal changes of NAA and total creatine (Cr) in ischemic rat brain after a 15-min transient middle cerebral artery occlusion (MCAO) by in vivo (1)H magnetic resonance spectroscopy. The results showed that the levels of NAA and total Cr in the ischemic lesion decrease significantly at 1 day post-ichemia, followed by spontaneous recovery to the control levels by 2 weeks and remained stable thereafter up to 16 weeks. The normalization of NAA and total Cr levels was associated histologically with persisted neuronal loss up to 90 % in the ischemic core, and accompanied by marked reactive astrocytic responses occurring with a similar time course. The absolute T(2) relaxation time in the ischemic lesion increased during acute phase, and declined afterwards during subacute and chronic phases of 15-min MCAO. The delayed decreases of T(2) in the ischemic lesion might be associated with deposition of paramagnetic species, such as manganese and iron originated from chronic inflammation, vascular degradation and/or hemorrhagic transformation. The results of this study give further support to the hypothesis that the recovery of NAA after cerebral ischemia might have contributions from reactive glia cells, and caution the use of NAA as a specific neuronal marker during the chronic stage of cerebral ischemia.

Effects of skilled and unskilled training on functional recovery and brain plasticity after focal ischemia in adult rats

Stroke is a leading cause of morbidity and mortality worldwide. Recovery of motor function after stroke can be modified by post-injury experience, but most of surviving patients exhibit persistence of the motor dysfunctions even after rehabilitative therapy. In this study we investigated if skilled and unskilled training induce different motor recovery and brain plasticity after experimental focal ischemia. We tested this hypothesis by evaluating the motor skill relearning and the immunocontent of Synapsin-I, PSD-95 and GFAP (pre and post-synaptic elements, as well as surrounding astroglia) in sensorimotor cortex of both hemispheres 6 weeks after endothelin-1-induced focal brain ischemia in rats. Synapsin-I and PSD-95 levels were increased by skilled training in ischemic sensorimotor cortex. The content of GFAP was augmented as a result of focal brain ischemia in ischemic sensorimotor cortex and that was not modified by rehabilitation training. Unexpectedly, animals remained permanently impaired at the end of motor/functional evaluations. Significant modifications in protein expression were not observed in undamaged sensorimotor cortex. We conclude that skilled motor activity can positively affect brain plasticity after focal ischemia despite of no functional improvement in conditions here tested.

Effects of metabotropic glutamate mGlu5 receptor antagonist on tyrosine phosphorylation of NMDA receptor subunits and cell death in the hippocampus after brain ischemia in rats

Tyrosine phosphorylation of the N-methyl-D-aspartate (NMDA) receptor appears to be associated with the regulation of the receptor's ion channel. This study focused on the effect of a metabotropic glutamate mGlu5 receptor antagonist on tyrosine phosphorylation of NMDA receptor subunits and cell death in the hippocampal CA1 region after transient global ischemia and sought to explore their mechanisms. Pretreatment with the mGlu5 receptor antagonist reduced cell death in the hippocampal CA1 region on day 3 after the transient ischemia. Transient ischemia increased the tyrosine phosphorylation of NMDA receptor subunits, which are a major target of Src family tyrosine kinases. Therefore, we investigated the effect of the antagonist on tyrosine phosphorylation of the NMDA receptor subunits after transient ischemia. Tyrosine phosphorylation of the NR2A subunit, but not that of the NR2B one, was inhibited by the mGlu5 receptor antagonist. The administration of the antagonist also attenuated the increase in the amount of active form of Src after the reperfusion. We further demonstrated that the administration of a Src-family kinase inhibitor prevented cell death in the hippocampal CA1 region and attenuated the increase in the tyrosine phosphorylation of the NMDA receptor subunits after the reperfusion. These findings suggest that mGlu5 receptor in the hippocampal CA1 region after transient ischemia is involved in the activation of Src and subsequent tyrosine phosphorylation of NMDA receptor subunits, which actions may contribute to alterations of properties of the NMDA receptor and may be related to pathogenic events leading to neuronal cell death.

Endogenous CNTF mediates stroke-induced adult CNS neurogenesis in mice

Focal brain ischemia in adult rats rapidly and robustly induces neurogenesis in the subventricular zone (SVZ) but there are few and inconsistent reports in mice, presenting a hurdle to genetically investigate the endogenous neurogenic regulators such as ciliary neurotrophic factor (CNTF). Here, we first provide a platform for further studies by showing that middle cerebral artery occlusion in adult male C57BL/6 mice robustly enhances neurogenesis in the SVZ only under very specific conditions, i.e., 14days after a 30min occlusion. CNTF expression paralleled changes in the number of proliferated, BrdU-positive, SVZ cells. Stroke-induced proliferation was absent in CNTF−/− mice, suggesting that it is mediated by CNTF. MCAO-increased CNTF appears to act on C cell proliferation and by inducing FGF2 expression but not via EGF expression or Notch1 signaling of neural stem cells in the SVZ. CNTF is unique, as expression of other gp130 ligands, IL-6 and LIF, did not predict SVZ proliferation or showed no or only small compensatory increases in CNTF−/− mice. Expression of tumor necrosis factor-α, which can inhibit neurogenesis, and the presence of leukocytes in the SVZ were inversely correlated with neurogenesis, but pro-inflammatory cytokines did not affect CNTF expression in cultured astrocytes. These results suggest that slowly up-regulated CNTF in the SVZ mediates stroke-induced neurogenesis and is counteracted by inflammation. Further pharmacological stimulation of endogenous CNTF might be a good therapeutic strategy for cell replacement after stroke as CNTF regulates normal patterns of neurogenesis and is expressed almost exclusively in the nervous system.

MiR-210 activates notch signaling pathway in angiogenesis induced by cerebral ischemia

The compensatory angiogenesis that occurs after cerebral ischemia increases blood flow to the injured area and limits extension of the ischemic penumbra. In this way, it improves the local blood supply. Fostering compensatory angiogenesis is an effective treatment for ischemic cerebrovascular disease. However, angiogenesis in the adult organism is a complex, multi-step process, and the mechanisms underlying the regulation of angiogenesis are not well understood. Although Notch signaling reportedly regulates the vascularization process that occurs in ischemic tissues, little is known about the role of Notch signaling in the regulation of ischemia-induced angiogenesis after ischemic stroke. Recent research has indicated that miR-210, a hypoxia-induced microRNA, plays a crucial role in regulating the biological processes that occur in blood vessel endothelial cells under hypoxic conditions. This study was undertaken to investigate the role of miR-210 in regulating angiogenesis in response to brain ischemia injury and the role of the Notch pathway in the body's response. We found miR-210 to be significantly up-regulated in adult rat ischemic brain cortexes in which the expression of Notch1 signaling molecules was also increased. Hypoxic models of human umbilical vein endothelial cells (HUVE-12) were used to assess changes in miR-210 and Notch1 expression in endothelial cells. Results were consistent with in vivo findings. To determine the molecular mechanisms behind these phenomena, we transfected HUVE-12 cells with miR-210 recombinant lentiviral vectors. We found that miR-210 overexpression caused up-regulation of Notch1 signaling molecules and induced endothelial cells to migrate and form capillary-like structures on Matrigel. These data suggest that miR-210 is involved in the regulation of angiogenesis in response to ischemic injury to the brain. Up-regulation of miR-210 can activate the Notch signaling pathway, which may contribute to angiogenesis after cerebral ischemia.

Resveratrol prevents CA1 neurons against ischemic injury by parallel modulation of both GSK‐3β and CREB through PI3‐K/Akt pathways

Accumulating evidence indicates that resveratrol potently protects against cerebral ischemia damage due to its oxygen free radicals scavenging and antioxidant properties. However, cellular mechanisms that may underlie the neuroprotective effects of resveratrol in brain ischemia are not fully understood yet. This study aimed to investigate the potential association between the neuroprotective effect of resveratrol and the apoptosis/survival signaling pathways, in particular the glycogen synthase kinase 3 (GSK-3β) and cAMP response element-binding protein (CREB) through phosphatidylinositol 3-kinase (PI3-K)-dependent pathway. An experimental model of global cerebral ischemia was induced in rats by the four-vessel occlusion method for 10 min and followed by different periods of reperfusion. Nissl staining indicated extensive neuronal death at 7 days after ischemia/reperfusion. Administration of resveratrol by i.p. injections (30 mg/kg) for 7 days before ischemia significantly attenuated neuronal death. Both GSK-3β and CREB appear to play a critical role in resveratrol neuroprotection through the PI3-K/Akt pathway, as resveratrol pretreatment increased the phosphorylation of Akt, GSK-3β and CREB in 1 h in the CA1 hippocampus after ischemia/reperfusion. Furthermore, administration of LY294002, an inhibitor of PI3-K, compromised the neuroprotective effect of resveratrol and decreased the level of p-Akt, p-GSK-3β and p-CREB after ischemic injury. Taken together, the results suggest that resveratrol protects against delayed neuronal death in the hippocampal CA1 by maintaining the pro-survival states of Akt, GSK-3β and CREB pathways. These data suggest that the neuroprotective effect of resveratrol may be mediated through activation of the PI3-K/Akt signaling pathway, subsequently downregulating expression of GSK-3β and CREB, thereby leading to prevention of neuronal death after brain ischemia in rats.

Early steps of microglial activation are directly affected by neuroprotectant FK506 in both in vitro inflammation and in rat model of stroke

Neuroprotective and/or neuroregenerative activity of FK506, its derivatives, and to a lesser extent cyclosporin A (CsA) in animal models of neurodegenerative diseases of different etiology have been reported. Here, we verified a hypothesis that the most likely mechanism of their neuroprotective action is inhibition of the early steps of inflammatory activation of microglia by interference with mitogen-activated protein kinase (MAPK) signaling. The effect of immunosuppressants on lipopolysaccharide (LPS)-induced changes in morphology, proliferation, and motility of rat primary microglial cultures was evaluated. FK506 and CsA directly inhibited LPS-induced microglia activation and inflammatory responses. While both drugs efficiently reduced the expression of iNOS and the release of nitric oxide, only FK506 strongly inhibited the expression of Cox-2 and secretion of the mature form of IL-1β. FK506 strongly reduced LPS-induced activation of MAPK, and its downstream signaling crucial for inflammatory responses. Comparative analysis of global gene expression in rat ischemic brains and in LPS-stimulated microglial cultures revealed many genes and signaling pathways regulated in the same way in both systems. FK506 treatment blocked a majority of genes induced by an ischemic insult in the cortex, in particular inflammatory/innate immunity and apoptosis-related genes. Microglia-mediated inflammation is considered as one of the most important components of brain injury after trauma or stroke; thus, effective and multifaceted blockade of microglial activation by FK506 has clinical relevance and potential therapeutic implications.Electronic supplementary materialThe online version of this article (doi:10.1007/s00109-012-0925-9) contains supplementary material, which is available to authorized users.

Human Albumin Prevents 6-Hydroxydopamine-Induced Loss of Tyrosine Hydroxylase in In Vitro and In Vivo

Human albumin has recently been demonstrated to protect brain neurons from injury in rat ischemic brain. However, there is no information available about whether human albumin can prevent loss of tyrosine hydroxylase (TH) expression of dopaminergic (DA) neurons induced by 6-hydroxydopamine (6-OHDA) toxicity that is most commonly used to create a rat model of Parkinson's disease (PD). In the present study, two microliters of 1.25% human albumin were stereotaxically injected into the right striatum of rats one day before or 7 days after the 6-OHDA lesion in the same side. D-Amphetamine-induced rotational asymmetry was measured 7 days, 3 and 10 weeks after 6-OHDA lesion. We observed that intrastriatal administration of human albumin significantly reduced the degree of rotational asymmetry. The number of TH-immunoreactive neurons present in the substantia nigra was greater in 6-OHDA lesioned rats following human albumin-treatment than non-human albumin treatment. TH-immunoreactivity in the 6-OHDA-lesioned striatum was also significantly increased in the human albumin-treated rats. To examine the mechanisms underlying the effects of human albumin, we challenged PC12 cells with 6-OHDA as an in vitro model of PD. Incubation with human albumin prevented 6-OHDA-induced reduction of cell viability in PC12 cell cultures, as measured by MTT assay. Furthermore, human albumin reduced 6-OHDA-induced formation of reactive oxygen species (ROS) and apoptosis in cultured PC12 cells, as assessed by flow cytometry. Western blot analysis showed that human albumin inhibited 6-OHDA-induced activation of JNK, c-Jun, ERK, and p38 mitogen-activated protein kinases (MAPK) signaling in PC12 cultures challenged with 6-OHDA. Human albumin may protect against 6-OHDA toxicity by influencing MAPK pathway followed by anti-ROS formation and anti-apoptosis.

Increase of 20-HETE Synthase after Brain Ischemia in Rats Revealed by PET Study with 11C-Labeled 20-HETE Synthase-Specific Inhibitor

20-Hydroxyeicosatetraenoic acid (20-HETE), an arachidonic acid metabolite known to be produced after cerebral ischemia, has been implicated in ischemic and reperfusion injury by mediating vasoconstriction. To develop a positron emission tomography (PET) probe for 20-HETE synthase imaging, which might be useful for monitoring vasoconstrictive processes in patients with brain ischemia, we synthesized a (11)C-labeled specific 20-HETE synthase inhibitor, N'(4-dimethylaminohexyloxy)phenyl imidazole ([(11)C]TROA). Autoradiographic study showed that [(11)C]TROA has high-specific binding in the kidney and liver consistent with the previously reported distribution of 20-HETE synthase. Using transient middle cerebral artery occlusion in rats, PET study showed significant increases in the binding of [(11)C]TROA in the ipsilateral hemisphere of rat brains after 7 and 10 days, which was blocked by co-injection of excess amounts of TROA (10 mg/kg). The increased [(11)C]TROA binding on the ipsilateral side returned to basal levels within 14 days. In addition, quantitative real-time PCR revealed that increased expression of 20-HETE synthase was only shown on the ipsilateral side on day 7. These results indicate that [(11)C]TROA might be a useful PET probe for imaging of 20-HETE synthase in patients with cerebral ischemia.