S100A6 (calcyclin) is a novel marker of neural stem cells and astrocyte precursors in the subgranular zone of the adult mouse hippocampus

S100A6 (calcyclin), an EF‐hand calcium binding protein, is considered to play various roles in the brain, for example, cell proliferation and differentiation, calcium homeostasis, and neuronal degeneration. In addition to some limbic nuclei, S100A6 is distributed in the rostral migratory stream, one of the major neurogenic niches of the adult brain. However, the potential involvement of S100A6 in adult neurogenesis remains unclear. In this study, we aimed to elucidate the role of S100A6 in the other major neurogenic niche, the subgranular zone of the dentate gyrus in the adult mouse hippocampus. Immunofluorescent multiple labeling showed that S100A6 was highly expressed in neural stem cells labeled by sex determining region Y‐box 2, brain lipid‐binding protein protein and glial fibrillary acidic protein. S100A6+ cells often extended a long process typical of radial glial morphology. In addition, S100A6 was found in some S100β+ astrocyte lineage cells. Interestingly, proliferating cell nuclear antigen was detected in a fraction of S100A6+/S100β+ cells. These cells were considered to be lineage‐restricted astrocyte precursors maintaining mitotic potential. On the other hand, S100A6 was rarely seen in neural lineage cells labeled by T‐box brain protein 2, doublecortin, calretinin and calbindin D28K. Cell fate‐tracing experiment using BrdU showed that the majority of newly generated immature astrocytes were immunoreactive for S100A6, while mature astrocytes lacked S100A6 immunoreactivity. Administration of S100 protein inhibitor, trifluoperazine, caused a reduction in production of S100β+ astrocyte lineage cells, but had no impact on neurogenesis. Overall, our data provide the first evidence that S100A6 is a specific marker of neural stem cells and astrocyte precursors, and may be especially important for generation of astrocytes in the adult hippocampus. © 2013 Wiley Periodicals, Inc.     

Long‐term accumulation of microglia with proneurogenic phenotype concomitant with persistent neurogenesis in adult subventricular zone after stroke

Neural stem cells (NSCs) in the adult rat subventricular zone (SVZ) generate new striatal neurons during several months after ischemic stroke. Whether the microglial response associated with ischemic injury extends into SVZ and influences neuroblast production is unknown. Here, we demonstrate increased numbers of activated microglia in ipsilateral SVZ concomitant with neuroblast migration into the striatum at 2, 6, and 16 weeks, with maximum at 6 weeks, following 2 h middle cerebral artery occlusion in rats. In the peri‐infarct striatum, numbers of activated microglia peaked already at 2 weeks and declined thereafter. Microglia in SVZ were resident or originated from bone marrow, with maximum proliferation during the first 2 weeks postinsult. In SVZ, microglia exhibited ramified or intermediate morphology, signifying a downregulated inflammatory profile, whereas amoeboid or round phagocytic microglia were frequent in the peri‐infarct striatum. Numbers of microglia expressing markers of antigen‐presenting cells (MHC‐II, CD86) increased in SVZ but very few lymphocytes were detected. Using quantitative PCR, strong short‐ and long‐term increase (at 1 and 6 weeks postinfarct) of insulin‐like growth factor‐1 (IGF‐1) gene expression was detected in SVZ tissue. Elevated numbers of IGF‐1‐expressing microglia were found in SVZ at 2, 6, and 16 weeks after stroke. At 16 weeks, 5% of microglia but no other cells in SVZ expressed the IGF‐1 protein, which mitigates apoptosis and promotes proliferation and differentiation of NSCs. The long‐term accumulation of microglia with proneurogenic phenotype in the SVZ implies a supportive role of these cells for the continuous neurogenesis after stroke. © 2008 Wiley‐Liss, Inc.     

Human embryonic neural stem cell transplantation increases subventricular zone cell proliferation and promotes peri‐infarct angiogenesis after focal cerebral ischemia

Neurogenesis and angiogenesis are two important processes that may contribute to the repair of brain injury after stroke. This study was designed to investigate whether transplantation of human embryonic neural stem cells (NSCs) into cortical peri‐infarction 24 h after ischemia effects cell proliferation in the subventricular zone (SVZ) and angiogenesis in the peri‐infarct zone. NSCs were prepared from embryonic human brains at 8 weeks gestation. Focal cerebral ischemia was induced by permanent occlusion of the middle cerebral artery of adult rats. Animals were randomly divided into two groups (n = 30, each) at 24 h after ischemia: NSC‐grafted and medium‐grafted groups. Toluidine blue staining and 5′‐bromo‐2′‐deoxyuridine (BrdU) or von Willebrand factor (vWF) immunohistochemistry were performed at 7, 14 and 28 days after transplantation. NSC transplantation increased the number of BrdU‐positive cells in the ischemic ipsilateral SVZ compared with the medium control at 7 days (P < 0.01). This difference in SVZ cell proliferation persisted at 14 days (P < 0.01), but was not significant at 28 days (P > 0.05). In addition, angiogenesis, as indicated by BrdU and vWF staining in cortical peri‐infarct regions, was augmented by 46% and 65% in NSC‐grafted rats versus medium‐grafted rats at 7 and 14 days, respectively (P < 0.05). However, this increase became non‐significant at 28 days (P > 0.05). Our results indicate that NSC transplantation enhances endogenous cell proliferation in the SVZ and promotes angiogenesis in the peri‐infarct zone, even if it is performed in the acute phase of ischemic injury.     

Chain formation and glial tube assembly in the shift from neonatal to adult subventricular zone of the rodent forebrain

The subventricular zone (SVZ) is regarded as an embryonic germinal layer persisting at the end of cerebral cortex neurogenesis and capable of generating neuronal precursors throughout life. The two distinct compartments of the adult rodent forebrain SVZ, astrocytic glial tubes and chains of migrating cells, are not distinguishable in the embryonic and early postnatal counterpart. In this study we analyzed the SVZ of mice and rats around birth and throughout different postnatal stages, describing molecular and morphological changes which lead to the typical structural arrangement of adult SVZ. In both species studied, most changes occurred during the first month of life, the transition being slightly delayed in mice, in spite of their earlier development. Important modifications affected the glial cells, eventually leading to glial tube assembly. These changes involved an overall reorganization of glial processes and their mutual relationships, as well as gliogenesis occurring within the SVZ which gives rise to glial cell subpopulations. The neuroblast cell population remained qualitatively quite homogeneous throughout all the stages investigated, changes being restricted to the relationships among cells and consequent formation of chains at about the third postnatal week. Electron microscopy showed that chain formation is not directly linked to glial tube assembly, generally preceding the occurrence of complete glial ensheathment. Moreover, chain and glial tube formation is asymmetric in the medial/lateral aspect of the SVZ, being inversely related. The attainment of an adult SVZ compartmentalization, on the other hand, seems linked to the pattern of expression of adhesion and extracellular matrix molecules.     

Proliferation, migration and differentiation of neuronal progenitor cells in the adult mouse subventricular zone surgically separated from its olfactory bulb

The subventricular zone of the adult mammalian forebrain contains progenitor cells that, by migrating along a restricted pathway called the ‘rostral migratory stream’ (RMS), add new neurons to the olfactory bulb throughout life. To determine the influence of the olfactory bulb on the development of these progenitor cells, we performed lesions that interrupt this pathway and separate the olfactory bulb from the rest of the forebrain. By labelling cells born at several survival times after the lesions with the thymidine analogue bromodeoxyuridine (BrdU), we found that disconnection from the bulb influences the rate of BrdU incorporation by the progenitor cells. The number of labelled cells in lesioned mice was almost half that found in control mice. In the disconnected migratory pathway, the number of neurons expressing calretinin was increased indicating that neuronal differentiation was enhanced: newly born neurons occurred within and around the RMS, most of them expressed calretinin and left the pathway starting about 2 weeks after the lesion. Thereafter, these neurons preserving their phenotype, spread for long distances, and accumulated ectopically in dorsal regions of the anterior olfactory nucleus and the frontal cortex. Finally, transplantation of adult subventricular cells into the lesioned pathway showed that the lesion neither prevents neuronal migration nor alters its direction. Thus, although the olfactory bulb appears to regulate the pace of the developmental processes, its disconnection does not prevent the proliferation, migration and phenotypic acquisition of newly generated bulbar interneurons that, since they cannot reach their terminal domains, populate some precise regions of the lesioned adult forebrain.     

Transplantation of human neural precursor cells in Matrigel scaffolding improves outcome from focal cerebral ischemia after delayed postischemic treatment in rats

Transplantation of neural cells is a potential approach for stroke treatment, but disruption of tissue architecture may limit transplant efficacy. One strategy for enhancing the ability of transplants to restore brain structure and function is to administer cells together with biomaterial scaffolding. We electrocoagulated the distal middle cerebral artery in adult rats and, 3 weeks later, injected one of the following into the infarct cavity: artificial cerebrospinal fluid, Matrigel scaffolding, human embryonic stem cell-derived neuronal precursor cells, scaffolding plus cells, or cells cultured in and administered together with scaffolding. Five weeks after transplantation, the latter two groups showed ∼50% and ∼60% reductions, respectively, in infarct cavity volume. Rats given cells cultured in and administered together with scaffolding also showed (1) survival and neuronal differentiation of transplanted cells shown by immunostaining for neuronal marker proteins and cleaved caspase-3, and by patch-clamp recording, 8 weeks after transplantation and (2) improved outcome on tests of sensorimotor and cognitive functions, 4 to 9 weeks after transplantation. These results indicate that transplantation of human neural cells together with biomaterial scaffolding has the potential to improve the outcome from stroke, even when treatment is delayed for several weeks after the ischemic event.     

Calorie restriction activates new adult born olfactory‐bulb neurones in a ghrelin‐dependent manner but acyl‐ghrelin does not enhance subventricular zone neurogenesis

The ageing and degenerating brain show deficits in neural stem/progenitor cell (NSPC) plasticity that are accompanied by impairments in olfactory discrimination. Emerging evidence suggests that the gut hormone ghrelin plays an important role in protecting neurones, promoting synaptic plasticity and increasing hippocampal neurogenesis in the adult brain. In the present study, we investigated the role of ghrelin with respect to modulating adult subventricular zone (SVZ) NSPCs that give rise to new olfactory bulb (OB) neurones. We characterised the expression of the ghrelin receptor, growth hormone secretagogue receptor (GHSR), using an immunohistochemical approach in GHSR‐eGFP reporter mice to show that GHSR is expressed in several regions, including the OB but not in the SVZ of the lateral ventricle. These data suggest that acyl‐ghrelin does not mediate a direct effect on NSPC in the SVZ. Consistent with these findings, treatment with acyl‐ghrelin or genetic silencing of GHSR did not alter NSPC proliferation within the SVZ. Similarly, using a bromodeoxyuridine pulse‐chase approach, we show that peripheral treatment of adult rats with acyl‐ghrelin did not increase the number of new adult‐born neurones in the granule cell layer of the OB. These data demonstrate that acyl‐ghrelin does not increase adult OB neurogenesis. Finally, we investigated whether elevating ghrelin indirectly, via calorie restriction (CR), regulated the activity of new adult‐born cells in the OB. Overnight CR induced c‐Fos expression in new adult‐born OB cells but not in developmentally born cells, whereas neuronal activity was absent following re‐feeding. These effects were not present in ghrelin^?/? mice, suggesting that adult‐born cells are uniquely sensitive to changes in ghrelin mediated by fasting and re‐feeding. In summary, ghrelin does not promote neurogenesis in the SVZ and OB; however, new adult‐born OB cells are activated by CR in a ghrelin‐dependent manner.     

Cellular localization of epidermal‐type and brain‐type fatty acid‐binding proteins in adult hippocampus and their response to cerebral ischemia

This study aimed at an analysis of expression of epidermal‐type and brain‐type fatty acid‐binding proteins (E‐FABP and B‐FABP, also called FABP5 and FABP7, respectively) in adult hippocampus and their potential value as neuroprotective factors after ischemic brain damage in monkey model. The immunostaining and Western blotting results show that FABP5 was mainly expressed in neurons, whereas FABP7 was primarily expressed in astrocytes and progenitors of the subgranular zone (SGZ). Interestingly, FABP5 expression in neurons increased in cornu Ammonis 1 (CA1) and remains stable within dentate gyrus (DG) after ischemia; FABP7 expression increased within both CA1 and SGZ. This indicates a potential role for FABP5 and FABP7 in intracellular fatty acid transport within different neural cells. The change in FABP5–7 expression within CA1 and DG of the adult postischemic hippocampus was compatible with previous findings of downregulation in CA1 neurons and upregulation in SGZ progenitor cells after ischemia. Altogether, the present data suggest that polyunsaturated fatty acids, such as docosahexaenoic acid, may act via FABP5 or 7 to regulate adult postischemic hippocampal neuronal antiapoptosis or neurogenesis in primates. © 2009 Wiley‐Liss, Inc.     

MicroRNA-29b is a therapeutic target in cerebral ischemia associated with aquaporin 4

MicroRNA-29b (miR-29b) is involved in regulating ischemia process, but the molecular mechanism is unclear. In this work, we explored the function of miR-29b in cerebral ischemia. The level of miR-29b in white blood cells was evaluated in patients and mice after ischemic stroke. Brain infarct volume and National Institute of Health stroke scale (NIHSS) scores were analyzed to determine the relationship between miR-29b expression and the severity of stroke. The relationship of miR-29b and aquaporin-4 (AQP4) was further studied in mice. We found that miR-29b was significantly downregulated in stroke patients (P<0.05). MiR-29b level negatively associated with NIHSS scores (r=−0.349, P<0.01) and brain infarct volume (r=−0.321, P<0.05). In ischemic mice, miR-29b in the brain and blood were both downregulated (r=0.723, P<0.05). MiR-29b overexpression reduced infarct volume (49.50±6.55 versus 35.48±2.28 mm³, P<0.05), edema (164±4% versus 108±4%, P<0.05), and blood–brain barrier (BBB) disruption compared with controls (15±9% versus 7±3%, P<0.05). Aquaporin-4 expression greatly decreased after miR-29b overexpression (28±7% versus 11±3%, P<0.05). Dual-luciferase reporter system showed that AQP-4 was the direct target of miR-29b (P<0.05). We concluded that miR-29b could potentially predict stroke outcomes as a novel circulating biomarker, and miR-29b overexpression reduced BBB disruption after ischemic stroke via downregulating AQP-4.     

Pre-stroke DNA immunization against neurite growth inhibitors is beneficial to the recovery from focal cerebral ischemia in rats

BACKGROUND: Inhibitory signals, i.e. neurite growth inhibitors (NGIs), presenting on central nervous system (CNS) myelin have been shown to play a crucial role in inhibiting lesioned axonal sprouting and leading to less functional recovery. Vaccines targeting NGIs may provide multifactorial protection against brain insults by overcoming the inhibitory effects of these NGIs and boosting the immune repair mechanisms of body. OBJECTIVE: To evaluate the effect of pre-stroke DNA immunization against NGIs on the rehabilitation for sensorimotor function of rat models of focal cerebral ischemia. DESIGN: A completely randomized design, and controlled experiment. SETTING: Brain Injury Research Laboratory, Department of Neurosurgery, National Neuroscience Institute, Singapore. MATERIALS: Sixty adult male Sprague-Dawley rats ranging in age from 45 to 120 days and in body mass from 180 to 250 g were provided by the Animal Center of Department of Anatomy, Faculty of Medicine, National University of Singapore. pcDNA3.1(+)-neurite growth inhibitors (pcDNA-NGIs) a gift was provided by Dr. Xiao from the Department of Clinical Research, Singapore General Hospital, Singapore. METHODS: The experiment was carried out at Brain Injury Research Laboratory, Department of Neurosurgery, National Neuroscience Institute, Singapore from August 2003 to April 2005. ① The involved rats were randomized into 3 groups: model group (group A), pcDNA3.1(+) group (group B) and pcDNA-NGIs group (group C), with 20 rats in each group. Left focal cerebral ischemia was permanently induced through middle cerebral artery occlusion with the assistance of an operating microscope. Successful middle cerebral artery occlusion was determined by a 20% decrease to baseline in the ipsilateral cerebral blood flow. 100 μg of pcDNA-NGIs eluted in phosphate-buffered saline (PBS) was intramuscularly injected into the tibial muscle once a week before middle cerebral artery occlusion for 6 weeks in group C. As control, pcDNA3.1 (+) was also administrated in the same way in group B and nothing was administrated in group A. ② The modified neurological severity score (mNSS), a composite of motor, sensory, reflex and balance tests, was used to test the sensorimotor deficit. The mNSS was graded on 0–18, i.e. normal score was 0, maximal deficit score was 18, and 1 point was warded for the inability to perform the tasks or the lack of a tested reflex. ③ The newly generated axons of corticorubral projection were traced by stereotaxic guided injection of 100 g/L biotinylated dextran amine (BDA). Rats were sacrificed two weeks after tracing, and cryostat coronal sections of midbrains (30 μm) were reacted to BDA according to the manufacturer's instruction by the free-floating method. Images were captured on a DM RXA2 LEICA Microscope with a Spot Digital Camera system (Germany), and the numbers of labeled axons on the denervated side in four standard coronal sections including the red nucleus were manually quantified. MAIN OUTCOME MEASURES: ① The number of newly generated axons of corticorubral projection; ② The improvement of the sensorimotor deficit. RESULTS: All the involved 60 rats entered the final analysis. ① The number of newly generated axons of corticorubral projection of rats: Only ipsilateral axons of corticorubral projiction were noted with little evidence of fibers crossing to the contralateral red nucleus in rats of groups A and B. More BDA-positive fibers crossing the midline and terminating in the contralateral red nucleus in appropriate target areas mirroring the non-differentiated red nucleus were found in rats of group C. Quantitative analysis showed that BDA-labelled axons in the denervated side of rats in group C were more than those in group B (P < 0.05). ② The improvement of the sensorimotor deficit: At two weeks after middle cerebral artery occlusion, significant improvement in sensorimotor deficit was found in rats of group C. There was significant difference of improvement in sensorimotor deficit of rats between group C and group B or group A at eight and 10 weeks after middle cerebral artery occlusion (P < 0.05). CONCLUSION: Pre-stroke DNA immunization against NGIs led to increased sensorimotor recovery following focal cerebral ischemia and compensatory newly growth of axons from corticorubral projection.     

Poststroke DNA immunization against neurite growth inhibitors is beneficial to the recovery from local cerebral ischemia in rats

BACKGROUND： Inhibitory signals, i.e. neurite growth inhibitors （NGIs）, presenting on central nervous system （CNS） myelin have been shown to play a crucial role in inhibiting lesioned axonal sprouting and leading to less functional recovery. Vaccines targeting NGIs may provide multifactorial protection against brain insults by overcoming the inhibitory effects of these NGIs and boosting the body＇s immune repair mechanisms. OBJECTIVE： To evaluate the effect of poststroke DNA immunization against NGIs on the rehabilitation for sensorimotor function of rat models of local cerebral ischemia. DESIGN： Completely randomized grouping design, and controlled experiment. SETTING： Brain Injury Research Laboratory, Department of Neurosurgery, National Neuroscience Institute, Singapore. MATERIALS： Sixty adult male Sprague-Dawley rats ranging in age from 45 to 120 days and in weight from 180 to 250 grams were provided by Animal Center of Department of Anatomy, Faculty of Medicine, National University of Singapore. pcDNA3.1（＋）-neurite growth inhibitors （pcDNA-NGIs） a gift was provided by Dr. Xiao from Department of Clinical Research, Singapore General Hospital, Singapore. METHODS： The experiment was carried out at Brain Injury Research Laboratory, Department of Neurosurgery, National Neuroscience Institute, Singapore from August 2003 to April 2005. （1）The involved rats were randomized into 3 groups： pcDNA-NGIs group （group A）, pcDNA3.1 （＋） group （group B） and model group （group C）, with 20 rats in each group. Left focal cerebral ischemia （FCI） was permanently induced through middle cerebral artery occlusion （MCAO） with the assistance of an operating microscope. Successful MCAO was determined by a 20% decrease to baseline in the ipsilateral cerebral blood flow. 100 μg of pcDNA-NGIs eluted in phosphate-buffered saline （PBS） was intramuscularly injected into the tibial muscle once a week after MCAO for 6 weeks in group A. As control, pcDNA3.1 （＋） was also administrated in the same way in group B and nothing was administrated in group C. （2） The modified neurological severity score （mNSS）, a composite of motor, sensory, reflex and balance tests, was used to test the sensorimotor deficit. The mNSS was graded on a scale of 0 - 18, i.e. normal score was 0, maximal deficit score was 18, and 1 point was warded for the inability to perform the tasks or the lack of a tested reflex. （3） The newly generated axons of corticorubral projection were traced by stereotaxic guided injection of 100 g/L biotinylated dextran amine. Rats were sacrificed two weeks after tracing, and cryostat coronal sections of midbrains （30μm） were reacted to BDA according to the manufacturer＇s instruction by the free-floating method. Images were captured on a DM RXA2 LEICA Microscope with a Spot Digital Camera system （Germany）, and the numbers of labeled axons on the denervated side in four standard coronal sections including the red nucleus were manually quantified. MAIN OUTCOME MEASURES： （1） The number of newly generated axons of corticorubral projection. （2）The improvement in sensorimotor deficit. RESULTS： All the involved 60 rats entered the stage of final analysis. （1） The number of newly generated axons of corticorubral projection of rats： Only ipsilateral axons of CRP were noted with little evidence of fibers crossing to the contralateral red nucleus in rats of groups B and C. More BDA-positive fibers crossing the midline and terminating in the contralateral red nucleus in appropriate target areas mirroring the non-differentiated red nucleus were found in rats of group A. Quantitative analysis showed that BDA-labeled axons in the denervated side of rats in group A were more than those in group B （P 〈 0.05）. （2） Improvement in sensorimotor deficit of rats： At 2 weeks after immunization, significant improvement in sensorimotor deficit was found in rats of group A. There were significant differences of improvement in sensorimotor deficit of rats between group A and group B or group C at 12 and 14 weeks after immunization （P 〈 0.05）. CONCLUSION： （1） Poststroke DNA immunization against NGIs leads to increased sensorimotor recovery following FCI and compensatory newly growth of axons from corticorubral projection.     

Poststroke DNA immunization against neurite growth inhibitors is beneficial to the recovery from local cerebral ischemia in rats

BACKGROUND: Inhibitory signals, i.e. neurite growth inhibitors (NGIs), presenting on central nervous system (CNS) myelin have been shown to play a crucial role in inhibiting lesioned axonal sprouting and leading to less functional recovery. Vaccines targeting NGIs may provide multifactorial protection against brain insults by overcoming the inhibitory effects of these NGIs and boosting the body's immune repair mechanisms. OBJECTIVE: To evaluate the effect of poststroke DNA immunization against NGIs on the rehabilitation for sensorimotor function of rat models of local cerebral ischemia. DESIGN: Completely randomized grouping design, and controlled experiment. SETTING: Brain Injury Research Laboratory, Department of Neurosurgery, National Neuroscience Institute, Singapore. MATERIALS: Sixty adult male Sprague-Dawley rats ranging in age from 45 to 120 days and in weight from 180 to 250 grams were provided by Animal Center of Department of Anatomy, Faculty of Medicine, National University of Singapore. pcDNA3.1(+)-neurite growth inhibitors (pcDNA-NGIs) a gift was provided by Dr. Xiao from Department of Clinical Research, Singapore General Hospital, Singapore. METHODS: The experiment was carried out at Brain Injury Research Laboratory, Department of Neurosurgery, National Neuroscience Institute, Singapore from August 2003 to April 2005. ①The involved rats were randomized into 3 groups: pcDNA-NGIs group (group A), pcDNA3.1 (+) group (group B) and model group (group C), with 20 rats in each group. Left focal cerebral ischemia (FCI) was permanently induced through middle cerebral artery occlusion (MCAO) with the assistance of an operating microscope. Successful MCAO was determined by a 20% decrease to baseline in the ipsilateral cerebral blood flow. 100 μg of pcDNA-NGIs eluted in phosphate-buffered saline (PBS) was intramuscularly injected into the tibial muscle once a week after MCAO for 6 weeks in group A. As control, pcDNA3.1 (+) was also administrated in the same way in group B and nothing was administrated in group C. ② The modified neurological severity score (mNSS), a composite of motor, sensory, reflex and balance tests, was used to test the sensorimotor deficit. The mNSS was graded on a scale of 0–18, i.e. normal score was 0, maximal deficit score was 18, and 1 point was warded for the inability to perform the tasks or the lack of a tested reflex. ③ The newly generated axons of corticorubral projection were traced by stereotaxic guided injection of 100 g/L biotinylated dextran amine. Rats were sacrificed two weeks after tracing, and cryostat coronal sections of midbrains (30 μm) were reacted to BDA according to the manufacturer's instruction by the free-floating method. Images were captured on a DM RXA2 LEICA Microscope with a Spot Digital Camera system (Germany), and the numbers of labeled axons on the denervated side in four standard coronal sections including the red nucleus were manually quantified. MAIN OUTCOME MEASURES: ① The number of newly generated axons of corticorubral projection. ②The improvement in sensorimotor deficit. RESULTS: All the involved 60 rats entered the stage of final analysis. ① The number of newly generated axons of corticorubral projection of rats: Only ipsilateral axons of CRP were noted with little evidence of fibers crossing to the contralateral red nucleus in rats of groups B and C. More BDA-positive fibers crossing the midline and terminating in the contralateral red nucleus in appropriate target areas mirroring the non-differentiated red nucleus were found in rats of group A. Quantitative analysis showed that BDA-labeled axons in the denervated side of rats in group A were more than those in group B (P < 0.05). ② Improvement in sensorimotor deficit of rats: At 2 weeks after immunization, significant improvement in sensorimotor deficit was found in rats of group A. There were significant differences of improvement in sensorimotor deficit of rats between group A and group B or group C at 12 and 14 weeks after immunization (P < 0.05). CONCLUSION: ① Poststroke DNA immunization against NGIs leads to increased sensorimotor recovery following FCI and compensatory newly growth of axons from corticorubral projection.     

Exosomes containing miR-451a is involved in the protective effect of cerebral ischemic preconditioning against cerebral ischemia and reperfusion injury

AimTo study the role of exosomes in the protective effect of cerebral ischemic preconditioning (cerebral‐IPC) against cerebral I/R injury.MethodMouse models of cerebral‐IPC and MCAO/R were established as described previously, and their behavioral, pathological, and proteomic changes were analyzed. Neuro‐2a subjected to OGD/R were treated with exosomes isolated from the plasma of sham‐operated and cerebral‐IPC mice. The differentially expressed miRNAs between exosomes derived from sham‐operated (S‐exosomes) and preconditioned (IPC‐exosomes) mice were identified through miRNA array, and their targets were identified through database search. The control and OGD/R cells were treated with the IPC‐exosomes, miRNA mimic or target protein inhibitor, and their viability, oxidative, stress and apoptosis rates were measured. The activated pathways were identified by analyzing the levels of relevant proteins.ResultsCerebral‐IPC mitigated the cerebral injury following ischemia and reperfusion, and increased the number of plasma exosomes. IPC‐exosomes increased the survival of Neuro‐2a cells after OGD/R. The miR‐451a targeting Rac1 was upregulated in the IPC‐exosomes relative to S‐exosomes. The miR‐451a mimic and the Rac1 inhibitor NSC23766 reversed OGD/R‐mediated activation of Rac1 and its downstream pathways.ConclusionCerebral‐IPC ameliorated cerebral I/R injury by inducing the release of exosomes containing miR‐451a.     

Mild cerebral hypoxia–ischemia produces a sub-acute transient inflammatory response that is less selective and prolonged after a substantial insult

Cerebral ischemia initiates various injurious processes including neuroinflammatory responses such as activation of microglia and increases in cytokine and nitric oxide release. Evidence primarily from in vitro studies, indicates that neuroinflammatory effects can be either beneficial or harmful, possibly related to stimulus strength. We investigated using in vivo models, the effect of a mild or substantial cerebral hypoxia–ischemia on: cerebral microglial/macrophage activation (ED1), pro-inflammatory cytokines (tumor necrosis factor-alpha), nitrosative stress (nitrotyrosine) and permanent brain damage. A mild insult produced a transient (1–2 days post) increase in activated microglia/macrophages within subcortical white and not gray matter but transiently increased cytokine or nitrotyrosine expression in cortex and not white matter. There was also prolonged scattered cell death in cortex and white matter over weeks along with loss of myelin/axons and cortical atrophy at 4 weeks post-insult. In contrast, a substantial insult produced white and gray matter necrosis, cyst formation and atrophy, along with increases in tumor necrosis factor and nitrotyrosine staining within both white and gray matter starting at 1–2 days post-insult. Microglial/macrophage staining was increased starting at 1-week post a substantial insult and remained elevated for weeks thereafter.Thus, a transient neuroinflammatory response occurs following a mild insult whereas prolonged scattered cell death occurs for weeks, particularly in white matter. Insult severity also affects the progression of the neuroinflammatory response, which is prolonged after a substantial insult. Effective therapy will need to be customized for insult severity and timing; and, monitoring the injury processes with imaging or biomarkers may help guide treatment.     

Recombinant tissue plasminogen activator induces blood-brain barrier breakdown by a matrix metalloproteinase-9-independent pathway after transient focal cerebral ischemia in mouse

The role of the inducible matrix metalloproteinase (MMP)-9 in blood-brain barrier (BBB) disruption after ischemic stroke is well accepted. Recombinant tissue plasminogen activator (r-tPA) is the only approved thrombolytic treatment of ischemic stroke but r-tPA is potentially neurotoxic. Vasogenic edema after r-tPA treatment has been linked with an increase in cerebral MMP-9. However, because cerebral ischemia clearly increases the levels of endogenous tPA, the consequence of additional r-tPA may be questionable. In this study, wild type and MMP-9 knockout mice were subjected to 90 min transient middle cerebral artery occlusion and treated with 10 mg/kg r-tPA. At 24 h after occlusion, BBB permeability, hemispheric enlargement, collagen and laminin degradation as well as cerebral infarction were increased in both wild type and MMP-9 knockout treated animals as compared with non-treated animals. Mortality was increased in wild type but reduced in knockout treated mice. Cerebral MMP-9 concentration was not modified by r-tPA. However, pre-treatment with p-aminobenzoyl-gly-pro-D-leu-D-ala-hydroxamate, a broad-spectrum MMP inhibitor, counteracted the effects of r-tPA on the neurovascular unit and decreased mortality in both wild type and knockout mice. MMP inhibition did not modify cerebral infarction in r-tPA-treated animals. Our results suggest that r-tPA toxicity is mainly independent of MMP-9 after transient middle cerebral artery occlusion but could involve some other MMPs. Additionally, our results support the hypothesis of a dissociation between r-tPA-dependent mechanisms of BBB breakdown and cerebral infarction. Due to the importance of r-tPA in thrombolytic treatment of ischemic stroke patients, the MMPs that could participate in r-tPA-induced BBB disruption should be further characterized.