Direct arterial damage and neurovascular unit disruption by mechanical thrombectomy in a rat stroke model

Mechanical thrombectomy (MT) is a standard treatment for acute ischemic stroke that could cause hemorrhagic complications. We aimed to evaluate the pathology of MT-induced arterial damage and neurovascular unit (NVU) disruption in relation to tissue-type plasminogen activator (tPA) injection for acute ischemic stroke. We induced transient middle cerebral artery occlusion in male SHR/Izm rats for 2 hr. This was followed by reperfusion with/without tPA (3 mg/kg) and “rough suture” insertion that mimicked MT once or thrice (MT1 or MT3). Compared with the control group, the tPA + MT3 group presented with an increase in the cerebral infarct and hemorrhage with severer IgG leakage. Moreover, structural damage reaching the tunica media was detected in the MT3 and tPA + MT3 groups. The tPA + MT3 group presented with increased matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) expression with some MMP9-positive cells expressing a neutrophil marker myeloperoxidase. Furthermore, basal lamina detachment from astrocyte foot processes was observed in the tPA + MT1 and tPA + MT3 groups. These findings suggest that MT causes direct arterial damage, as well as VEGF and MMP9 upregulation, which results in NVU disruption and hemorrhagic complications in acute ischemic stroke, especially when combined with tPA.     

Profilin 1 knockdown prevents ischemic brain damage by promoting M2 microglial polarization associated with the RhoA/ROCK pathway

Microglial polarization to the anti-inflammatory M2 phenotype is essential in resolving neuroinflammation, making it a promising therapeutic strategy for stroke intervention. The actin cytoskeleton is known to be important for the physiological functions of microglia, including migration and phagocytosis. Profilin 1 (PFN1), an actin-binding protein, is involved in the dynamic transformation and reorganization of actin. However, the role of PFN1 in microglial polarization and ischemia/reperfusion injury is unclear. The role of PFN1 on microglial polarization was examined in vitro in BV2 microglial cells subjected to oxygen-glucose deprivation/reoxygenation (OGDR) and in vivo in male mice after transient middle cerebral artery occlusion (MCAO). Knockdown of PFN1 inhibited M1 microglial polarization and promoted M2 microglia polarization 48 hr after OGDR stimulation in BV2 cells and 7 days after MCAO-induced injury in male mice. RhoA/ROCK pathway was involved in the regulation of PFN1 during microglial polarization. Knockdown of PFN1 also significantly attenuated brain infarcts and edema, improved cerebral blood flow and neurological deficits in MCAO-injured mice. Inhibition of PFN1 effectively protected the brain against ischemia/reperfusion injuries by promoting M2 microglial polarization in vitro and in vivo.     

Peroxynitrite decomposition with FeTMPyP improves plasma-induced vascular dysfunction and infarction during mild but not severe hyperglycemic stroke

We investigated mechanisms by which circulating factors during hyperglycemic (HG) stroke affect cerebrovascular function and the role of peroxynitrite in stroke outcome. Middle cerebral arteries (MCAs) were isolated from male Wistar rats and perfused with plasma from rats that were hyperglycemic for 5 to 6 days by streptozotocin and underwent either MCA occlusion (HG MCAO) or Sham surgery (HG Sham) compared with MCA perfused with physiologic saline (No plasma). Myogenic responses and endothelial function were compared in untreated MCA (n=8/group) or with inhibitors of NADPH oxidase (apocynin; n=8), peroxynitrite (FeTMPyP; n=8) or endothelin-1 (ET-1)(A) (BQ-123; n=8). Finally, animals were treated in vivo before reperfusion after mild (<68% cerebral blood flow (CBF) decrease) or severe (>68% CBF decrease) MCAO with FeTMPyP (n=12) or vehicle (n=12) and CBF and infarction measured. The HG MCAO plasma increased tone in MCA versus No plasma (P<0.05) that was reversed by FeTMPyP, but not by apocynin or BQ-123. The HG Sham plasma also increased tone in MCA (P<0.05) that was reversed by BQ-123 only. In vivo, FeTMPyP was neuroprotective during mild, but not severe ischemia. These results show that circulating factors in plasma can affect cerebrovascular function through peroxynitrite generation and ET-1. In addition, peroxynitrite decomposition improves stroke outcome acutely during mild, but not severe HG ischemia.     

Acute Hyperglycemia Exacerbates Hemorrhagic Transformation after Embolic Stroke and Reperfusion with tPA: A Possible Role of TXNIP-NLRP3 Inflammasome

ObjectivesAcute hyperglycemia (HG) exacerbates reperfusion injury after stroke. Our recent studies showed that acute HG upregulates thioredoxin-interacting protein (TXNIP) expression, which in turn induces inflammation and neurovascular damage in a suture model of ischemic stroke. The aim of the present study was to investigate the effect of acute HG on TXNIP-associated neurovascular damage, in a more clinically relevant murine model of embolic stroke and intravenous tissue plasminogen activator (IV-tPA) reperfusion.Materials and methodsHG was induced in adult male mice, by intraperitoneal injection of 20% glucose. This was followed by embolic middle cerebral artery occlusion (eMCAO), with or without IV-tPA (10 mg/kg) given 3 h post embolization. Brain infarction, edema, hemoglobin content, expression of matrix metalloproteinase (MMP-9), vascular endothelial growth factor A (VEGFA), tight junction proteins (claudin-5, occluding, and zonula occludens-1), TXNIP, and NOD‐like receptor protein3 (NLRP3)-inflammasome activation were evaluated at 24 h after eMCAO.ResultsHG alone significantly increased TXNIP in the brain after eMCAO, and this was associated with exacerbated hemorrhagic transformation (HT; as measured by hemoglobin content). IV-tPA in HG conditions showed a trend to decrease infarct volume, but worsened HT after eMCAO, suggesting that HG reduces the therapeutic efficacy of IV-tPA. Further, HG and tPA-reperfusion did not show significant differences in expression of MMP-9, VEGFA, junction proteins, and NLRP3 inflammasome activation between the groups.ConclusionThe current findings suggest a potential role for TXNIP in the occurrence of HT in hyperglycemic conditions following eMCAO. Further studies are needed to understand the precise role of vascular TXNIP on HG/tPA-induced neurovascular damage after stroke.     

High-potassium preconditioning enhances tolerance to focal cerebral ischemia-reperfusion injury through anti-apoptotic effects in male rats

Imbalances between cellular K⁺ efflux and influx are considered to be involved in cerebral ischemia-reperfusion (I/R) injury. High-potassium pretreatment alleviates this injury, but the underlying molecular mechanism is unclear. In this study, we sought to investigate whether high-potassium preconditioning enhances cerebral tolerance to I/R injury through an anti-apoptotic mechanism. Adult male Sprague-Dawley rats were randomly divided into four groups (n = 40/group): a sham-operated group, normal saline group (3.2 ml/kg saline, intravenous (IV)), and low-dose and high-dose potassium chloride (KCl) groups (40 and 80 mg/kg KCl solution, IV, respectively). Subsequently, the rats underwent 90 min of middle cerebral artery occlusion (MCAO) followed by 24 hr of reperfusion (MCAO/R). Neurological deficit scores, 2,3,5-triphenyltetrazolium chloride (TTC) staining, hematoxylin and eosin staining, and TUNEL assay were used to assess neural injury. The expression of apoptotic proteins, brain potassium levels, mitochondrial function and oxidative stress were detected to explore the potential mechanism. After 24 hr of reperfusion, in both KCl treatment groups, neurological deficits and the cerebral infarct volume were reduced, and the apoptosis index of neurons was decreased. Furthermore, high-potassium preconditioning increased brain K⁺, adenosine triphosphate (ATP), cytochrome c oxidase (COX) levels, reduced malondialdehyde level, improved Na⁺/K⁺-ATPase, succinic dehydrogenase and superoxide dismutase activities, upregulated anti-apoptotic protein expression, and downregulated pro-apoptotic protein expression. This study suggests that high-potassium preconditioning enhanced cerebral tolerance to I/R injury in a rat MCAO/R model. The protective mechanism may involve apoptosis inhibition via preservation of intracellular K⁺ and improvement of mitochondrial function.     

Ultrastructural studies of the neurovascular unit reveal enhanced endothelial transcytosis in hyperglycemia-enhanced hemorrhagic transformation after stroke

AimsPre‐existing hyperglycemia (HG) aggravates the breakdown of blood–brain barrier (BBB) and increases the risk of hemorrhagic transformation (HT) after acute ischemic stroke in both animal models and patients. To date, HG‐induced ultrastructural changes of brain microvascular endothelial cells (BMECs) and the mechanisms underlying HG‐enhanced HT after ischemic stroke are poorly understood.MethodsWe used a mouse model of mild brain ischemia/reperfusion to investigate HG‐induced ultrastructural changes of BMECs that contribute to the impairment of BBB integrity after stroke. Adult male mice received systemic glucose administration 15 min before middle cerebral artery occlusion (MCAO) for 20 min. Ultrastructural characteristics of BMECs were evaluated using two‐dimensional and three‐dimensional electron microscopy and quantitatively analyzed.ResultsMice with acute HG had exacerbated BBB disruption and larger brain infarcts compared to mice with normoglycemia (NG) after MCAO and 4 h of reperfusion, as assessed by brain extravasation of the Evans blue dye and microtubule‐associated protein 2 immunostaining. Electron microscopy further revealed that HG mice had more endothelial vesicles in the striatal neurovascular unit than NG mice, which may account for their deterioration of BBB impairment. In contrast with enhanced endothelial transcytosis, paracellular tight junction ultrastructure was not disrupted after this mild ischemia/reperfusion insult or altered upon HG. Consistent with the observed increase of endothelial vesicles, transcytosis‐related proteins caveolin‐1, clathrin, and hypoxia‐inducible factor (HIF)‐1α were upregulated by HG after MCAO and reperfusion.ConclusionOur study provides solid structural evidence to understand the role of endothelial transcytosis in HG‐elicited BBB hyperpermeability. Enhanced transcytosis occurs prior to the physical breakdown of BMECs and is a promising therapeutic target to preserve BBB integrity.     

Progesterone is neuroprotective after transient middle cerebral artery occlusion in male rats

Progesterone (PROG) is a neurosteroid, possessing a variety of functions in the central nervous system. Exogenous PROG has been shown to reduce secondary neuronal loss in conjunction with attenuated brain edema after cerebral contusion and to reduce brain edema after focal cerebral ischemia. In the present study, we assessed the neuroprotective potential of PROG in a model of focal cerebral ischemia in the rat. Forty-eight male Wistar rats were randomly assigned to 4 groups, i.e. pretreatment with water soluble PROG, or dimethyl sulfoxide (DMSO) dissolved PROG, or DMSO as control or delayed treatment with DMSO dissolved PROG. Middle cerebral artery occlusion (MCAO) was induced by insertion of an intraluminal suture and reperfusion was performed by withdrawing the suture. Pretreatments were initiated 30 min before MCAO via intraperitoneal injection. Delayed treatment was initiated upon reperfusion following 2 h of MCAO. Infarct volume, body weight loss, and neurological deficit were measured 48 h after MCAO. Pre- and delayed treatment with DMSO dissolved PROG resulted in a 39% (P < 0.05) and 34% (P < 0.05) reduction in cerebral infarction, respectively, along with decreased body weight loss and improved neurological function as compared to control animals, whereas no statistically significant reduction in infarct volume by water soluble PROG was found. We demonstrated that administration of PROG to the male rat before or 2 hours after onset of MCAO reduces ischemic cell damage and improves physiological and neurological function 2 days after stroke. These results suggests potential therapeutic properties of PROG in the management of stroke.     

Mild carotid stenosis creates gradual,progressive, lifelong brain,and eye damage: An experimental laboratory rat model

In humans, carotid stenosis of 70% and above might be the cause of clinical symptoms such as transient ischemic attack and stroke. No clinical or animal studies have evaluated mild carotid occlusion, and few examined unilateral occlusion. Here, Westar rats underwent bilateral or unilateral carotid occlusion of 28–45%. Long-term effects were evaluated 9–11 months later. We conducted cognitive evaluation using spatial learning in a water maze and exploration behavior in an open field. Morphology of the brain was examined by MRI using diffusion-tensor imaging (DTI) and immunohistochemistry staining of the brain and eyes. Cognitive deficit was found in spatial memory and exploration behavior in both occluded groups. Brain and eyes histology presented severe damage in the bilateral group, compared to the unilateral one. DTI revealed an increase in mean diffusivity (MD) in the ventral thalamus and a decrease in fractional anisotropy in optic nerve and optic tract in bilateral rats, while unilateral rats showed only an increase in MD in the ventral pons. In those areas, a significant change in astrocytes, microglia, and number of apoptotic cells were found. Bilateral occlusion produced severe damage to both retinas, while unilateral occlusion produced damage mainly in the occluded side. We found that mild carotid stenosis, even in a unilateral occlusion, creates behavioral abnormalities presented by brain and eye histopathology. The results support our hypothesis that gradual formation of mild carotid stenosis along the life course leads to progressive damage that may create different degenerative diseases at a later age.     

Modification of kynurenine pathway via inhibition of kynurenine hydroxylase attenuates surgical brain injury complications in a male rat model

Neurosurgical procedures result in surgically induced brain injury (SBI) that causes postoperative complications including brain edema and neuronal apoptosis in the surrounding brain tissue. SBI leads to the release of cytokines that indirectly cause the stimulation of kynurenine 3-monooxygenase (KMO) and the release of neurotoxic quinolinic acid (QUIN). This study tested a KMO inhibitor, RO 61-8048, to prevent postoperative brain edema and consequent neuronal apoptosis in an in vivo model of SBI. A rodent model of SBI was utilized which involves partial resection of the right frontal lobe. A total of 127 Sprague-Dawley male rats (weight 275–325 g) were randomly divided into the following groups: Sham surgical group, SBI, SBI + DMSO, SBI + RO 61-8048 (10 mg/kg), SBI + RO 61-8048 (40 mg/kg), and SBI + RO 61-8048 (40 mg/kg) + KAT II inhibitor PF-04859989 (5 mg/kg). RO 61-8048 was administered by intraperitoneal injection after SBI. Postoperative assessment at different time points included brain water content (brain edema), neurological scoring, and western blot. SBI increased brain water content (ipsilateral frontal lobe), decreased neurological function, and increased apoptotic markers compared with sham animals. Treatment with RO 61-8048 (40 mg/kg) reduced brain water content and improved long-term neurological function after SBI. RO 61-8048 increased the expression of kynurenic acid while reducing QUIN and apoptotic markers in the surrounding brain tissue after SBI. These neuroprotective effects were reversed by PF-04859989. This study suggests KMO inhibition via RO 61-8048 as a potential postoperative therapy following neurosurgical procedures.     

厄贝沙坦对大鼠局灶性脑缺血再灌注后炎症反应的影响

目的观察厄贝沙坦对大鼠局灶性脑缺血再灌注后脑内及外周炎症反应的影响。方法采用改良Longa方法制备大鼠大脑中动脉阻塞(middle cerebralartery occlusion,MCAO)模型,于缺血90min再灌注后24h和72h进行梗死体积的测量,采用免疫组化和ELISA方法测量脑内和外周血的粘附分子。结果厄贝沙坦可以显著减少局灶性脑缺血再灌注后24h和72h的梗死体积(均P<0.01),改善神经功能(均P<0.01);降低脑内ICAM-1、VCAM-1的表达及其外周血浆中可溶性的形式sICAM-1、sVCAM-1蛋白的水平(均P<0.05)。结论厄贝沙坦可以降低粘附分子的表达,减少梗死体积,改善神经功能,对脑缺血再灌注起保护作用。     

Delayed matrix metalloproteinase inhibition reduces intracerebral hemorrhage after embolic stroke in rats

Hemorrhagic transformation (HT) and brain edema are life-threatening complications of recombinant tissue plasminogen activator (rt-PA)-induced reperfusion after ischemic stroke. The risk of HT limits the therapeutic window for reperfusion to 3 h after stroke onset. Pre-treatment with matrix metalloproteinase (MMP) inhibitors reduces HT and cerebral edema in experimental stroke. However, whether a delayed therapeutic intervention would be beneficial is unknown. In this study, 215 male Sprague–Dawley rats were subjected to embolic stroke and 75 rats were included in the final analysis. The animals were treated with the MMP inhibitor p-aminobenzoyl-gly-pro-d-leu-d-ala-hydroxamate before or after 3 or 6 h of ischemia. Animals were monitored for reperfusion and received rt-PA 6 h after ischemia onset. The results at 24 h showed that MMP inhibition 3 h after ischemia significantly decreased the degree of brain edema (17% of hemispheric enlargement in the treated group versus 24% in controls, P = 0.018), reduced the risk (OR = 0.163; 95% CI: 0.029 to 0.953) and gravity (0.09 versus 0.19 mg of parenchymal hemoglobin, P = 0.02) of intracerebral hemorrhage, and improved neurological outcome (20% of the treated animals had a slight deficit; all of the controls had a bad outcome, P < 0.05). Delaying MMP inhibition to 6 h after ischemia restricted the beneficial role of the treatment to a reduction in the risk of parenchymal hemorrhage (OR = 0.242; 95% CI: 0.060 to 0.989). Our results confirm the involvement of MMPs in HT and support the possibility of extending the therapeutic window for thrombolysis in stroke by administering a broad-spectrum MMP inhibitor after the onset of ischemia.     

Annexin A1 attenuates neuroinflammation through FPR2/p38/COX-2 pathway after intracerebral hemorrhage in male mice

Spontaneous intracerebral hemorrhage (ICH) is the deadliest stroke subtype and neuroinflammation is a critical component of the pathogenesis following ICH. Annexin A1-FPR2 signaling has been shown to play a protective role in animal stroke models. This study aimed to assess whether Annexin A1 attenuated neuroinflammation and brain edema after ICH and investigate the underlying mechanisms. Male CD-1 mice were subjected to collagenase-induced ICH. Annexin A1 was administered at 0.5 hr after ICH. Brain water content measurement, short-term and long-term neurobehavioral tests, Western blot and immnunofluorescence were performed. Results showed that Annexin A1 effectively attenuated brain edema, improved short-term neurological function and ameliorated microglia activation after ICH. Annexin A1 also improved memory function at 28 days after ICH. However, these beneficial effects were abolished with the administration of FPR2 antagonist Boc-2. Furthermore, AnxA1/FPR2 signaling may confer protective effects via inhibiting p38-associated inflammatory cascade. Our study demonstrated that Annexin A1/FPR2/p38 signaling pathway played an important role in attenuating neuroinflammation after ICH and that Annexin A1 could be a potential therapeutic strategy for ICH patients.     

An optimized dosing regimen of cimaglermin (neuregulin 1β3, glial growth factor 2) enhances molecular markers of neuroplasticity and functional recovery after permanent ischemic stroke in rats

Cimaglermin (neuregulin 1β3, glial growth factor 2) is a neuregulin growth factor family member in clinical development for chronic heart failure. Previously, in a permanent middle cerebral artery occlusion (pMCAO) rat stroke model, systemic cimaglermin treatment initiated up to 7 days after ischemia onset promoted recovery without reduced lesion volume. Presented here to extend the evidence are two studies that use a rat stroke model to evaluate the effects of cimaglermin dose level and dose frequency initiated 24 hr after pMCAO. Forelimb‐ and hindlimb‐placing scores (proprioceptive behavioral tests), body‐swing symmetry, and infarct volume were compared between treatment groups (n = 12/group). Possible mechanisms underlying cimaglermin‐mediated neurologic recovery were examined through axonal growth and synapse formation histological markers. Cimaglermin was evaluated over a wider dose range (0.02, 0.1, or 1.0 mg/kg) than doses previously shown to be effective but used the same dosing regimen (2 weeks of daily intravenous administration, then 1 week without treatment). The dose‐frequency study used the dose‐ranging study's most effective dose (1.0 mg/kg) to compare daily, once per week, and twice per week dosing for 3 weeks (then 1 week without treatment). Dose‐ and frequency‐dependent functional improvements were observed with cimaglermin without reduced lesion volume. Cimaglermin treatment significantly increased growth‐associated protein 43 expression in both hemispheres (particularly somatosensory and motor cortices) and also increased synaptophysin expression. These data indicate that cimaglermin enhances recovery after stroke. Immunohistochemical changes were consistent with axonal sprouting and synapse formation but not acute neuroprotection. Cimaglermin represents a potential clinical development candidate for ischemic stroke treatment. © 2015 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc.     

Expression of adiponectin receptors in the brain of adult zebrafish and mouse: Links with neurogenic niches and brain repair

Adiponectin and its receptors (adipor) have been initially characterized for their role in lipid and glucose metabolism. More recently, adiponectin signaling was shown to display anti-inflammatory effects and to participate in brain homeostasis and neuroprotection. In this study, we investigated adipor gene expression and its regulation under inflammatory conditions in two complementary models: mouse and zebrafish. We demonstrate that adipor1a, adipor1b, and adipor2 are widely distributed throughout the brain of adult fish, in neurons and also in radial glia, behaving as neural stem cells. We also show that telencephalic injury results in a decrease in adipor gene expression, inhibited by an anti-inflammatory treatment (Dexamethasone). Interestingly, adiponectin injection after brain injury led to a consistent decrease (a) in the recruitment of microglial cells at the lesioned site and (b) in the proliferation of neural progenitors, arguing for a neuroprotective role of adiponectin. In a comparative approach, we investigate Adipor1 and Adipor2 gene distribution in the brain of mice and demonstrated their expression in regions shared with fish including neurogenic regions. We also document Adipor gene expression in mice after middle cerebral artery occlusion and lipopolysaccharide injection. In contrast to zebrafish, these inflammatory stimuli do no impact cerebral adiponectin receptor gene expression in mouse. This work provides new insights regarding adipor expression in the brain of fish, and demonstrates evolutionary conserved distribution of adipor with mouse. This is the first report of adipor expression in adult neural stem cells of fish, suggesting a potential role of adiponectin signaling during vertebrate neurogenesis. It also suggests a potential contribution of inflammation in the regulation of adipor in fish.     

Topographic organization and possible function of the posterior optic tubercles in the brain of the desert locust Schistocerca gregaria

Migrating desert locusts, Schistocerca gregaria, are able to use the skylight polarization pattern for navigation. They detect polarized light with a specialized dorsal rim area in their compound eye. After multistage processing, polarization signals are transferred to the central complex, a midline‐spanning brain area involved in locomotor control. Polarization‐sensitive tangential neurons (TB‐neurons) of the protocerebral bridge, a part of the central complex, give rise to a topographic arrangement of preferred polarization angles in the bridge, suggesting that the central complex acts as an internal sky compass. TB‐neurons connect the protocerebral bridge with two adjacent brain areas, the posterior optic tubercles. To analyze the polarotopic organization of the central complex further, we investigated the number and morphologies of TB‐neurons and the presence and colocalization of three neuroactive substances in these neurons. Triple immunostaining with antisera against Diploptera punctata allatostatin (Dip‐AST), Manduca sexta allatotropin (Mas‐AT), and serotonin (5HT) raised in the same host species revealed three spatially distinct TB‐neuron clusters, each consisting of 10 neurons per hemisphere: cluster 1 and 3 showed Dip‐AST/5HT immunostaining, whereas cluster 2 showed Dip‐AST/Mas‐AT immunostaining. Five subtypes of TB‐neuron could be distinguished based on ramification patterns. Corresponding to ramification domains in the protocerebral bridge, the neurons invaded distinct but overlapping layers within the posterior optic tubercle. Similarly, neurons interconnecting the tubercles of the two hemispheres also targeted distinct layers of these neuropils. From these data we propose a neuronal circuit that may be suited to stabilize the internal sky compass in the central complex of the locust. J. Comp. Neurol. 523:1589–1607, 2015. © 2015 Wiley Periodicals, Inc.     

Micro‐RNA‐30a regulates ischemia‐induced cell death by targeting heat shock protein HSPA5 in primary cultured cortical neurons and mouse brain after stroke

Micro‐RNAs (miRs) have emerged as key gene regulators in many diseases, including stroke. We recently reported that miR‐30a protects N2A cells against ischemic injury, in part through enhancing beclin 1‐mediated autophagy. The present study explores further the involvement of miR‐30a in ischemia‐induced apoptosis and its possible mechanisms in primary cortical neurons and stroked mouse brain. We demonstrate that miR‐30a level is significantly decreased in cortical neurons after 1‐hr oxygen–glucose deprivation (OGD)/24‐hr reoxygenation. Overexpression of miR‐30a aggravated the OGD‐induced neuronal cell death, whereas inhibition of miR‐30a attenuated necrosis and apoptosis as determined by 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐di‐phenyl‐2H‐tetrazolium bromide, lactate dehydrogenase, TUNEL, and cleaved caspase‐3. The amount of HSPA5 protein, which is predicted to be a putative target of miR‐30a by TargetScan, could be reduced by pre‐miR‐30a, whereas it was increased by anti‐miR‐30a. Furthermore, the luciferase reporter assay confirmed that miR‐30a directly binds to the predicted 3′‐UTR target sites of the hspa5 gene. The cell injury regulated by miR‐30a in OGD‐treated cells could be aggravated by HSPA5 siRNA. We also observed an interaction of HSPA5 and caspase‐12 by coimmunoprecipitation and speculate that HSPA5 might be involved in endoplasmic reticulum stress‐induced apoptosis. In vivo, reduced miR‐30a increased the HSPA5 level and attenuated ischemic brain infarction in focal ischemia‐stroked mice. Downregulation of miR‐30a could prevent neural ischemic injury through upregulating HSPA5 protein expression, and decreased ER stress‐induced apoptosis might be one of the mechanisms underlying HSPA5‐mediated neuroprotection. © 2015 Wiley Periodicals, Inc.     

Mechanical reperfusion is associated with post-ischemic hemorrhage in rat brain

A major complication of recanalization therapy after an acute arterial occlusion in brain is hemorrhagic transformation (HT). Although it is known that prolonged ischemia is important in the development of HT, the role of reperfusion in ischemia–reperfusion induced HT is less well studied. To address the effect of reperfusion on HT, we assessed the incidence and severity of hemorrhage in rats after 5 h of middle cerebral artery occlusion (MCAO) followed by 19-hour reperfusion compared to rats with permanent occlusion (PMCAO) at the same 24-hour time point. The incidence and amount of hemorrhage, neurological function, and mortality rates were measured. MCAO (5 h) with 19-hour reperfusion was associated with a significantly higher incidence of cortical hemorrhage compared to PMCAO (81.8% vs 18.2%, p < 0.05). Hemorrhage scores were higher in the 5-hour MCAO/reperfusion group compared to PMCAO rats (17.6 ± 11.5 vs 2.4 ± 5.3 in cortex, 20.4 ± 4.6 vs 9.7 ± 4.5 in striatum, p < 0.01). Neurological function was worse in the ischemia–reperfusion group compared to PMCAO (p < 0.05) and mortality rates were insignificantly higher in the 5-hour MCAO/reperfusion group vs PMCAO group (54.5% vs18.1%; p < 0.08). The results suggest that reperfusion after prolonged ischemia is associated with increased hemorrhagic transformation and neurological deterioration as compared to permanent ischemia. Whether pharmacological treatments prior to reperfusion attenuate post-ischemic HT requires further study.     

The postnatal development of MT,V1, LGN,pulvinar and SC in prosimian galagos (Otolemur garnettii)

Considerable evidence supports the premise that the visual system of primates develops hierarchically, with primary visual cortex developing structurally and functionally first, thereby influencing the subsequent development of higher cortical areas. An apparent exception is the higher order middle temporal visual area (MT), which appears to be histologically distinct near the time of birth in marmosets. Here we used a number of histological and immunohistological markers to evaluate the maturation of cortical and subcortical components of the visual system in galagos ranging from newborns to adults. Galagos are representative of the large strepsirrhine branch of primate evolution, and studies of these primates help identify brain features that are broadly similar across primate taxa. The histological results support the view that MT is functional at or near the time of birth, as is primary visual cortex. Likewise, the superior colliculus, dorsal lateral geniculate nucleus, and the posterior nucleus of the pulvinar are well-developed by birth. Thus, these subcortical structures likely provide visual information directly or indirectly to cortex in newborn galagos. We conclude that MT resembles a primary sensory area by developing early, and that the early development of MT may influence the subsequent development of dorsal stream visual areas.     

Intracarotid injection of granulocyte-macrophage colony-stimulating factor induces neuroprotection in a rat transient middle cerebral artery occlusion model

Granulocyte-macrophage colony-stimulating factor (GM-CSF) was found to promote collateral flow in patients with coronary artery disease and also to induce arteriogenesis in a rat hypoperfusion brain model. Activated macrophages have been shown to induce vascular proliferation and play an important role in ischemic stroke. In this study, we examined the therapeutic effect of GM-CSF on the ischemic brain by activating microglia/macrophages. Rats were subjected to 1-h intraluminal middle cerebral artery occlusion (MCAO) and received an intracarotid injection of GM-CSF (5 ng) or saline immediately after reperfusion. Infarct volume, neurological function and histological findings were assessed 48 h later. An intracarotid injection of GM-CSF reduced the infarct volume and improved neurological function at 48 h after reperfusion. Histological analysis revealed that the number of activated microglia/macrophages to be increased and the number of apoptotic cells to be decreased in the area of the penumbra. These results suggest that intracarotid injection of GM-CSF may have a therapeutic effect on brain ischemia via activation of microglia/macrophages.     

Neurovascular and neuronal protection by E64d after focal cerebral ischemia in rats

Calpains and cathepsins are two families of proteases that play an important role in ischemic cell death. In this study, we investigated the effect of E64d, a mu-calpain and cathepsin B inhibitor, in the prevention of neuronal and endothelial apoptotic cell death after focal cerebral ischemia in rats. Rats underwent 2 hr of transient focal ischemia from middle cerebral artery occlusion (MCAO) and were sacrificed 24 hr later. E64d (5 mg/ kg intraperitoneally) was administered 30 min before MCAO. Assessment included neurological function, infarction volume, brain water content, blood-brain barrier permeability, histology, and immunohistochemistry. The E64d-treated rats had significant brain protection against ischemic damage. We observed a reduction of infarction volume, brain edema, and improved neurological scores in E64d-treated rats compared with the nontreated control. Furthermore, there was a remarkable reduction in both proteases and caspase-3 activation and apoptotic changes in both neurons and endothelial cells in E64d-treated rats. These results suggest that E64d protects the brain against ischemic/reperfusion injury by attenuating neuronal and endothelial apoptosis.