Osteopontin is a mediator of the lateral migration of neuroblasts from the subventricular zone after focal cerebral ischemia

We and others have shown that focal cerebral ischemia induces lateral migration of neuroblasts from the ipsilateral subventricular zone (SVZ) to the ischemic striatum. The signaling pathways underlying this phenomenon are not fully understood. The present study examined the role of osteopontin (OPN) in post-ischemic lateral migration of neuroblasts. Focal ischemia was induced by transient middle cerebral artery occlusion in adult spontaneous hypertensive rats. The expression of OPN in the ischemic brain was evaluated by immunohistochemistry, which showed that an up-regulation of OPN expression in the ipsilateral striatum at day 3, 7, 14 and 1 month of reperfusion with a peak at day 7. Double staining showed co-localization of OPN with ED1⁺ macrophages/microglia in the ischemic regions. Inhibition of OPN activity by infusing a neutralizing antibody against OPN into the ischemic striatum significantly decreased the area covered with doublecortin⁺ neuroblasts in the ipsilateral striatum. In vitro, OPN treatment did not affect the proliferation of neural progenitors, but induced an increased trans-well and radial migration of neural progenitors. The cultured neural progenitors expressed the OPN receptors CD44 and integrin β₁. Blockade of the CD44 receptor had no effects on OPN mediated trans-well and radial migration of neural progenitors. However, blockade of integrin β₁ receptor abolished the migration of neural progenitors in the absence or the presence of OPN. These results suggest that up-regulated expression of OPN produced by macrophages/microglia in the ischemic brain is an attractant and inducer for the lateral migration of neuroblasts from the SVZ to the injured region.     

Persistent migration of neuroblasts from the subventricular zone to the injured striatum mediated by osteopontin following intracerebral hemorrhage

We investigated intracerebral hemorrhage (ICH)-induced lateral migration of neuroblasts and the mechanism underlying this migration. ICH model was induced by collagenase injection into the striatum of adult wild-type and osteopontin (OPN) knockout mice. In the wild-type mice, the lateral migration of neuroblasts from the ipsislateral subventricular zone (SVZ) towards the hematoma started at day 3 and continued up to day 28 after ICH. In addition to migrating towards the hematoma, neuroblasts also migrated to the area of ipsilateral striatum remote to the hematoma. The migrating neuroblasts were closely associated with activated astrocytes and blood vessels in the injured striatum. Following ICH, the expression of OPN was up-regulated in the ipsilateral striatum from day 1 to day 28. In vitro , OPN treatment did not affect the proliferation of neural progenitors, but enhanced the trans-well and radial migration of neural progenitors. In vivo , OPN deficiency did not affect the proliferation of neural progenitors in the SVZ. However, following ICH a significant decrease in lateral neuroblast migration was observed in the OPN knockout mice compared with the wild-type mice. These results suggest that increased OPN expression in the injured striatum plays a significant role in the lateral migration of neuroblasts following ICH.     

Indomethacin treatment reduces microglia activation and increases numbers of neuroblasts in the subventricular zone and ischaemic striatum after focal ischaemia

Neuroblasts from the subventricular zone (SVZ) migrate to striatum following stroke, but most of them die in the ischaemic milieu and this can be related to exacerbated microglial activation. Here, we explored the effects of the non-steroidal anti-inflammatory indomethacin on microglial activation, neuronal preservation and neuroblast migration following experimental striatal stroke in adult rats. Animals were submitted to endothelin-1 (ET-1)-induced focal striatal ischaemia and were treated with indomethacin or sterile saline (i.p.) for 7 days, being perfused after 8 or 14 days. Immunohistochemistry was performed to assess neuronal loss (anti-NeuN), microglial activation (anti-Iba1, ED1) and migrating neuroblasts (anti-DCX) by counting NeuN, ED1 and DCX-positive cells in the ischaemic striatum or SVZ. Indomethacin treatment reduced microglia activation and the number of ED1⁺ cells in both 8 and 14 days post injury as compared with controls. There was an increase in the number of DCX⁺ cells in both SVZ and striatum at the same survival times. Moreover, there was a decrease in the number of NeuN⁺ cells in indomethacin-treated animals as compared with the control group at 8 days but not after 14 days post injury. Our results suggest that indomethacin treatment modulates microglia activation, contributing to increased neuroblast proliferation in the SVZ and migration to the ischaemic striatum following stroke.     

Specific expression of galanin in the peri-infarct zone after permanent focal cerebral ischemia in the rat

Galanin (Gal) is a neuropeptide with supposed neurotrophic-like action. In the present study, expression of Gal has been investigated in the core and peri-infarct zone at 1, 4, 24 and 72 h after middle cerebral artery occlusion (MCAo) in the rat. Three days after MCAo a small but consistent number of morphological intact Gal-positive neuronal cells were observed in the peri-infarct zone. Gal-positive cells were barely detectable in the infarct and peri-infarct zone at 24 h. No Gal immunopositive cells were detected in brain subjected to 1 and 4 h of ischemia. Gal immunoreactivity was also detected in myelinated fibers 4 and 24 h after focal ischemia. Gal may be a peptide with neurotrophic and plasticity functions under stress conditions.     

TLR2 has a detrimental role in mouse transient focal cerebral ischemia

A significant up-regulation of Toll-like-receptor (TLR) mRNAs between 3 and 48 h reperfusion time after induction of transient focal cerebral ischemia for 1h was revealed by applying global gene expression profiling in postischemic mouse brains. Compared to TLR4 and TLR9, TLR2 proved to be the most significantly up-regulated TLR in the ipsilateral brain hemisphere. TLR2-protein was found to be expressed mainly in microglia in the postischemic brain tissue, but also in selected endothelial cells, neurons, and astrocytes. Additionally, TLR2-related genes with pro-inflammatory and pro-apoptotic capabilities were induced. Therefore we hypothesized that TLR2-signaling could exacerbate the primary brain damage after ischemia. Two days after induction of transient focal cerebral ischemia (1h), we found a significant decrease of the infarct volume in TLR2 deficient mice compared to wild type mice (75+/-5 vs. 42+/-7 mm(3)). We conclude that TLR2 up-regulation and TLR2-signaling are important events in focal cerebral ischemia and contribute to the deterioration of ischemic damage.     

AMD3100对局灶脑缺血/再灌注大鼠大脑皮质血管再生的影响

目的探讨AMD3100阻断SDF-1/CXCR4轴后,对局灶脑缺血/再灌注大鼠缺血半暗带血管再生的影响。方法将SD大鼠随机分为假手术组(S组)、模型组(IR组)、AMD3100组(IRA组)、生理盐水组(IRN组)。采用线栓法制备大鼠局灶脑缺血/再灌注模型,缺血2h后将IR、IRA和IRN组分为再灌注12h,1、3和7d四个亚组。HE染色观察局灶脑缺血/再灌注后大脑皮质病理变化。免疫组化法检测CD31在缺血半暗带表达。荧光定量PCR检测外周血中AC133mRNA表达。结果与IRN组比较,IRA 12h外周血中AC133mRNA显著升高,第1d升高达峰值(P0.01),IRA 3dAC133mRNA表达比IRA1d显著减少(P0.05);与IRN组比较,IRA组CD31阳性血管密度在第1d无显著变化(P0.05),第3和7d血管密度显著减少(P0.01);IRA 7d梗死区由大量坏死神经细胞和泡沫细胞填充,坏死较严重。结论持续注射AMD3100能动员干/祖细胞快速进入外周血,但可能抑制局灶脑缺血/再灌注大鼠缺血半暗带血管再生,加重梗死区坏死。     

CD44 deficiency in mice protects brain from cerebral ischemia injury

CD44 is a transmembrane glycoprotein known to be involved in endothelial cell recognition, lymphocyte trafficking, and regulation of cytokine gene expression in inflammatory diseases. In the present study, we demonstrated that the expression of CD44 mRNA was induced in a mouse model of cerebral ischemia. A potential role of CD44 in ischemic brain injury was investigated using CD44-deficient (CD44-/-) mice. Over 50% (p < 0.05, n = 14) and 78% (p < 0.05, n = 10) reduction in ischemic infarct was observed in CD44-/- mice compared with that of wild-type mice following transient (30 min ischemia) and permanent (24 h) occlusion of the middle cerebral artery (MCAO), respectively. Similarly, significant improvement was observed in neurological function in CD44-/- mice as evidenced by spontaneous and forced motor task scores. The marked protection from ischemic brain injury in CD44-/- mice was associated with normal physiological parameters, cytokine gene expression, astrocyte and microglia activation as compared with wild-type mice. However, in CD44-/- mice, significantly lower expression of soluble interleukin-1beta protein was noted after brain ischemia. Our data provide new evidence on the potential role of CD44 in brain tissue in response to ischemia and may suggest that this effect might be associated with selective reduction in inflammatory cytokines such as interleukin-1beta.     

Notch1 signaling modulates neuronal progenitor activity in the subventricular zone in response to aging and focal ischemia

Neurogenesis diminishes with aging and ischemia‐induced neurogenesis also occurs, but reduced in aged brain. Currently, the cellular and molecular pathways mediating these effects remain largely unknown. Our previous study has shown that Notch1 signaling regulates neurogenesis in subventricular zone (SVZ) of young adult brain after focal ischemia, but whether a similar effect occurs in aged normal and ischemic animals is unknown. Here, we used normal and ischemic aged rat brains to investigate whether Notch1 signaling was involved in the reduction of neurogenesis in response to aging and modulates neurogenesis in aged brains after focal ischemia. By Western blot, we found that Notch1 and Jagged1 expression in the SVZ of aged brain was significantly reduced compared with young adult brain. Consistently, the activated form of Notch1 (Notch intracellular domain; NICD) expression was also declined. Immunohistochemistry confirmed that expression and activation of Notch1 signaling in the SVZ of aged brain were reduced. Double or triple immunostaining showed that that Notch1 was mainly expressed in doublecortin (DCX)‐positive cells, whereas Jagged1 was predominantly expressed in astroglial cells in the SVZ of normal aged rat brain. In addition, disruption or activation of Notch1 signaling altered the number of proliferating cells labeled by bromodeoxyuridine (BrdU) and DCX in the SVZ of aged brain. Moreover, ischemia‐induced cell proliferation in the SVZ of aged brain was enhanced by activating the Notch1 pathway and was suppressed by inhibiting the Notch1 signaling. Reduced infarct volume and improved motor deficits were also observed in Notch1 activator–treated aged ischemic rats. Our data suggest that Notch1 signaling modulates the SVZ neurogenesis in aged brain in normal and ischemic conditions.     

Neuroprotective effect of curcumin in middle cerebral artery occlusion induced focal cerebral ischemia in rats

Free radical induced neuronal damage is implicated in cerebral ischemia reperfusion (IR) injury and antioxidants are reported to have neuroprotective activity. Several in vitro and in vivo studies have proved the antioxidant potential of curcumin and its metabolites. Hence, in the present study the neuroprotective potential of curcumin was investigated in middle cerebral artery occlusion (MCAO) induced focal cerebral IR injury. 2 h of MCAO and 22 h of reperfusion resulted in the infarct volume of 210.39 +/- 31.25 mm3. Administration of curcumin 100 and 300 mg/kg, i.p. 30 min. after MCAO produced 37.23 +/- 5.10% and 46.39 +/- 10.23% (p < 0.05) reduction in infarct volume, respectively. Ischemia induced cerebral edema was reduced in a dose dependent manner. Curcumin at 300 mg/kg, i.p. produced 50.96 +/- 6.04% reduction in edema (p < 0.05) volume. Increase in lipid peroxidation after MCAO in ipsilateral and contralateral hemisphere of brain was observed, which was reduced by curcumin (300 mg/kg, i.p.)-treatment. Decrease in superoxide dismutase and glutathione peroxidase activity was observed in ipsilateral hemisphere of MCAO animal. Curcumin-treatment (300 mg/kg, i.p.) prevented IR injury mediated fall in glutathione peroxide activity. Peroxynitrite measured using rhodamine123 fluorescence and anti-nitrotyrosine immunofluorescence indicated increased peroxynitrite formation after IR insult. Curcumin-treatment reduced peroxynitrite formation and hence the extent of tyrosine nitration in the cytosolic proteins. These results suggest the neuroprotective potential of curcumin in cerebral ischemia and is mediated through its antioxidant activity.     

Discovery of new small molecules that influence neuroblast cell migration from the subventricular zone

Using SVZ (subventricular zone) tissue explants from one-day-old mice, we investigated the activity of new amino aromatic disulfide analogues and polyazamacrocycles on the migration of SVZ cells (neuroblasts). We found that among the tested analogues, non-peptidic disulfide derivative 8 significantly decreases the migration of neuroblasts from SVZ cells, and antagonized the stimulating activity of disulfide cyclic peptide 1. Discovery of compounds 1 and 8 constitutes new chemical tools which could be used to understand the mechanism of neuroblast migration during neurogenesis and eventually to identify specific genes involved in the neurogenesis.     

电针对局灶脑缺血／再灌注模型大鼠缺血海马区血管再生的影响及其机制

目的：探讨电针促进局灶脑缺血/再灌注后缺血海马区血管再生的机制。方法180只雄性SD大鼠随机分为假手术组、模型组、电针组、CXCR4特异性拮抗剂AMD3100药物组、AMD3100＋电针组。线栓法制备右侧局灶脑缺血/再灌注模型。取大鼠“百会”穴（ GV 20）及左侧“四关”穴（合谷LI 4/太冲LR 3）为电针穴位,刺激时间为30 min/d。采用逆转录聚合酶链反应法（ RT-PCR）检测各组缺血海马区SDF-1α、CXCR4 mRNA表达,免疫荧光双标法检测CD34＋VEGFR2＋EPCs源性血管的表达。结果与假手术组比较,模型组与电针组SDF-1α、CX-CR4 mRNA表达明显增高（P＜0.05）,其中电针组各时间点相对模型组增高更为显著（P＜0.05）。 AMD3100＋电针组缺血海马SDF-1α、CXCR4 mRNA表达在再灌注后1 d时明显高于电针组（ P＜0.05）,但后逐渐下降,7 d时明显低于电针组（ P＜0.01）。与模型组比较,电针组再灌注3 d、7 d海马CD34＋VEGFR2＋EPCs源性血管表达明显增多（ P＜0.05）。与电针组比较,AMD3100＋电针组再灌注后7 d CD34＋VEGFR2＋EPCs源性血管表达明显下降（ P＜0.01）。 CD34＋VEGFR2＋血管表达变化与SDF-1α的表达变化显著相关（R＝0.784,P＜0.01）。结论电针可通过上调局灶脑缺血/再灌注大鼠缺血海马区SDF-1α/CXCR4的表达,促进血管再生。     

Integrin-linked kinase is a positive mediator of L6 myoblast differentiation

Overexpression of ILK in L6 myoblasts results in increased ILK kinase activity, stimulating myotube formation and induction of biochemical differentiation markers. Expression of a dominant negative ILK mutant, ILK(E359K), inhibits endogenous ILK activation and L6 differentiation. Cell cycle analysis of ILK(E359K) cells cultured in serum-free conditions indicates significant apoptosis (11-19% sub-diploid peak) which is not seen in insulin treated cells. Expression of ILK variants does not have significant effects on S-phase transit, however. Known targets of ILK, PKB/Akt or glycogen synthase kinase 3beta are not obviously involved in ILK-induced L6 differentiation. Insulin-stimulated phosphorylation of PKB at Ser473 is unimpaired in the ILK(E359K) cells, suggesting that PKB is not a myogenic target of ILK. Inhibition of GSK3beta by LiCl blocks L6 myogenesis, indicating that ILK-mediated inhibition of GSK3beta is not sufficient for differentiation. Our data do suggest that a LiCl-sensitive interaction of ILK is important in L6 myoblast differentiation.     

Identification and characterization of neuroblasts in the subventricular zone and rostral migratory stream of the adult human brain

It is of great interest to identify new neurons in the adult human brain, but the persistence of neurogenesis in the subventricular zone (SVZ) and the existence of the rostral migratory stream (RMS)-like pathway in the adult human forebrain remain highly controversial. In the present study, we have described the general configuration of the RMS in adult monkey, fetal human and adult human brains. We provide evidence that neuroblasts exist continuously in the anterior ventral SVZ and RMS of the adult human brain. The neuroblasts appear singly or in pairs without forming chains; they exhibit migratory morphologies and co-express the immature neuronal markers doublecortin, polysialylated neural cell adhesion molecule and βIII-tubulin. Few of these neuroblasts appear to be actively proliferating in the anterior ventral SVZ but none in the RMS, indicating that neuroblasts distributed along the RMS are most likely derived from the ventral SVZ. Interestingly, no neuroblasts are found in the adult human olfactory bulb. Taken together, our data suggest that the SVZ maintains the ability to produce neuroblasts in the adult human brain.     

Bone-marrow cell therapy induces differentiation of radial glia-like cells and rescues the number of oligodendrocyte progenitors in the subventricular zone after global cerebral ischemia

The subventricular zone (SVZ) is recognized as one of the neurogenic regions in the adult mammalian central nervous system and the presence of cells that share similar characteristics with developmental radial glia, the radial glia-like cells (RGLCs) has been demonstrated in this region. In this study, we investigated whether and how SVZ cells respond to global ischemia and/or to the intravenous transplant of bone-marrow mononuclear cells (BMMCs). Adult rats were subjected to bilateral common carotid ligation (BCCL) and after 1 day 2 × 10⁷ BMMCs or saline injection. The BMMC transplant stimulated a transitory increase in the proliferation of SVZ cells in the BCCL group. We observed a significant increase in the number of RGLCs 3 days after ischemia, in both BCCL and BCCL + BMMC groups. However, this increase persisted in the subsequent days only in BCCL animals that received the transplant. BMMC transplantation also inhibits the reduction of NG2-positive oligodendrocyte progenitors in the SVZ observed in the BCCL group. Interestingly, brain-derived neurotrophic factor (BDNF) expression was up-regulated in the SVZ in the treated animals, but not in the other groups. These data thus suggest that BMMC transplantation modulates the phenotype of RGLCs/progenitors in the SVZ and could have a protective role after ischemia.     

Effect of Sargentodoxa cuneata total phenolic acids on focal cerebral ischemia reperfusion injury rats model

Objective

Explore the possible protective effect of Sargentodoxa cuneata total phenolic acids on cerebral ischemia reperfusion injury rats.

双侧迷走神经和颈总动脉联合切断法建立急性脑缺血大鼠模型

目的建立一种操作简单的急性脑缺血动物模型。方法取雄性Wistar大鼠40只,体重200～230g,手术前禁食12 h,自由饮水,随机分为对照组A、B、C组及模型D组,共4组,每组10只。即A组:假手术组,仅切开颈部两侧皮肤,分离双侧颈总动脉和迷走神经,不切断,然后缝合;B组:仅切断双侧颈部迷走神经;C组:结扎并切断双侧颈总动脉(CCA);D组:联合组,即结扎并切断双侧颈总动脉,同时切断双侧颈部迷走神经。观察各组大鼠手术后的脑缺血症状,记录各组大鼠在8h内的死亡情况,超过8h死亡的动物按8h计,计算死亡率和死亡时间。结果 A组大鼠没有脑缺血症状,无死亡;B组大鼠无脑缺血症状,呼吸变慢变深,心率血压上升,但无死亡;C组大鼠部分出现脑缺血症状,眼睑下垂,活动能力低下,自发运动减少,有些大鼠术后自发运动增加,在8 h内无死亡;D组大鼠大多数出现较为明显的脑缺血症状,在8 h内全部死亡。结论采取同时结扎并切断大鼠双侧颈总动脉和双侧颈部迷走神经的方法,可以建立急性脑缺血大鼠动物模型,此方法具有手术简单,成功率高,术后动物缓慢死亡的特点。     

Effects of pretreatment with PACAP on the infarct size and functional outcome in rat permanent focal cerebral ischemia

PACAP exerts neuroprotective effects under various neurotoxic conditions in vitro. In vivo, it reduces brain damage after global and transient focal ischemia. The present study investigated whether PACAP has neuroprotective effects when applied before the onset of permanent ischemia. Rats were given bolus injections of PACAP38 intracerebroventricularly, and then underwent permanent middle cerebral artery occlusion. The results show that 2 μg of PACAP significantly reduced the infarct size measured 12 and 24 h after the onset of ischemia. No further reduction was obtained by a 7-day pretreatment. PACAP also ameliorated certain sensorimotor deficits. Our present study provides further evidence for the neuroprotective effects of PACAP, and implies that it might be a promising preventive therapeutic agent in ameliorating ischemic brain damage.