Bone Marrow Stromal Cells Alleviate Secondary Damage in the Substantia Nigra After Focal Cerebral Infarction in Rats.

Transplantation of bone marrow stromal cells (BMSCs) is a promising therapy for ischemic stroke. Previously, we had reported that the secondary degeneration occurred in the ipsilateral substantia nigra (SN) after permanent distal branch of middle cerebral artery occlusion (dMCAO) in Sprague-Dawley rats. However, whether BMSCs have neurorestorative effects on the secondary damage in the SN after focal cerebral infarction has not known. In this study, rats were subjected to dMCAO followed by intravenous administration of BMSCs 1 day later. We found that transplanted BMSCs survived and migrated to cortical infarct areas and ipsilateral SN. Furthermore, BMSCs promoted neurogenesis through proliferation and differentiation in the SN after dMCAO. Rats implanted with BMSCs showed significant improvement in their performance of modified neurological severity scores and adhesive-removal test. Engrafted BMSCs enhanced survival of dopaminergic neuron, reduced gliosis in the ipsilateral SN, and increased contents of dopamine (DA) and its metabolites in the ipsilateral striatum after dMCAO. With pseudorabies virus-152 as a retrograde tracer, we also demonstrated that BMSCs could effectively enhance the cortico-striatum-nigral connections. These results suggest that BMSCs transplantation exerts neurorestorative effects after cortical infarction through promoting endogenous neurogenesis, increasing contents of DA and its metabolites, alleviating the secondary neuronal damage in the SN, enhancing the cortico-striatum-nigral projections pathway, and finally improving the neurological functional outcome.

Inhibition of Cathepsins B Induces Neuroprotection Against Secondary Degeneration in Ipsilateral Substantia Nigra After Focal Cortical Infarction in Adult Male Rats.

Stroke is the leading cause of adult disability in the world. In general, recovery from stroke is incomplete. Accumulating evidences have shown that focal cerebral infarction leads to dynamic trans-neuronal degeneration in non-ischemic remote brain regions, with the disruption of connections to synapsed neurons sustaining ischemic insults. Previously, we had reported that the ipsilateral striatum, thalamus degenerated in succession after permanent distal branch of middle cerebral artery occlusion (dMCAO) in Sprague-Dawley (SD) rats and cathepsin (Cath) B was activated before these relay degeneration. Here, we investigate the role of CathB in the secondary degeneration of ipsilateral substantia nigra (SN) after focal cortical infarction. We further examined whether the inhibition of CathB with L-3-trans-(Propyl-carbamoyloxirane-2-carbonyl)-L-isoleucyl-L-proline methyl ester (CA-074Me) would attenuate secondary degeneration through enhancing the cortico-striatum-nigral connections and contribute to the neuroprotective effects. Our results demonstrated that secondary degeneration in the ipsilateral SN occurred and CathB was upregulated in the ipsilateral SN after focal cortical infarction. The inhibition of CathB with CA-074Me reduced the neuronal loss and gliosis in the ipsilateral SN. Using biotinylated dextran amine (BDA) or pseudorabies virus (PRV) 152 as anterograde or retrograde tracer to trace striatum-nigral and cortico-nigral projections pathway, CA-074Me can effectively enhance the cortico-striatum-nigral connections and exert neuroprotection against secondary degeneration in the ipsilateral SN after cortical ischemia. Our study suggests that the lysosomal protease CathB mediates the secondary damage in the ipsilateral SN after dMCAO, thus it can be a promising neuroprotective target for the rehabilitation of stroke patients.

Inhibition of Cathepsin B Alleviates Secondary Degeneration in Ipsilateral Thalamus After Focal Cerebral Infarction in Adult Rats.

Secondary degeneration in areas beyond ischemic foci can inhibit poststroke recovery. The cysteine protease Cathepsin B (CathB) regulates cell death and intracellular protein catabolism. To investigate the roles of CathB in the development of secondary degeneration in the ventroposterior nucleus (VPN) of the ipsilateral thalamus after focal cerebral infarction, infarct volumes, immunohistochemistry and immunofluorescence, and Western blotting analyses were conducted in a distal middle cerebral artery occlusion (dMCAO) stroke model in adult rats. We observed marked neuron loss and gliosis in the ipsilateral thalamus after dMCAO, and the expression of CathB and cleaved caspase-3 in the VPN was significantly upregulated; glial cells were the major source of CathB. Although it had no effect on infarct volume, delayed intracerebroventricular treatment with the membrane-permeable CathB inhibitor CA-074Me suppressed the expression of CathB and cleaved caspase-3 in ipsilateral VPN and accordingly alleviated the secondary degeneration. These data indicate that CathB mediates a novel mechanism of secondary degeneration in the VPN of the ipsilateral thalamus after focal cortical infarction and suggest that CathB might be a therapeutic target for the prevention of secondary degeneration in patients after stroke.

[A method for combining Fluoro-Jade B staining and immunofluorescent staining].

OBJECTIVE: To explore a method for combining Fluoro-Jade B (FJB) staining with immunofluorescent staining in rats with focal cortical infarction. METHOD: Permanent distal middle cerebral artery occlusion (dMCAO) was induced in rats by electrocoagulation. The rat models were randomized into two groups, and frozen sections of the brain tissues from each group were stained with FJB followed by immunofluorescent staining or in the reverse order. RESULTS: FJB staining followed by immunofluorescence staining clearly visualized both FJB-positive and immunofluorescence-positive cells in the frozen sections, but the staining protocol in the reverse sequence failed to clearly show the immunofluorescence-positive cells. CONCLUSION: FJB staining prior to immunofluorescence staining does not affect the staining effect of protein immunofluorescent staining and better visualizes the positive cells.

Related expressional change of HIF-1α to the neuroprotective activity of exendin-4 in transient global ischemia.

Transient global ischemia induces selective hippocampal pyramidal neuronal death. Under conditions of severe ischemic hypoxia, hypoxia-inducible factor-1α (HIF-1α) induces apoptosis. Exendin-4 (Ex-4), the glucagon-like peptide-1 receptor (GLP-1R) agonist, provides neuroprotection against brain damage after cerebral ischemia. We investigated the relationship between Ex-4 and HIF-1α by examining Ex-4-induced changes in HIF-1α expression in the gerbil hippocampus following global brain ischemia (in vivo) and in neuroblastoma cells (SH-SY5Y) and cortical primary neurons (in vitro). Twice-daily administration of Ex-4 (1 µg/kg) for 3 days after ischemia (30 min before and 30 min after ischemia on the day of surgery and 2 more days) decreased the number of Fluoro-Jade B-stained cells in the CA1 pyramidal region of the hippocampus of the ischemic brain. Western blot analysis indicated a significant decrease in HIF-1α expression in the ischemic compared with the Sham brain following Ex-4 treatment. These in-vivo results were confirmed in vitro in SH-SY5Y cells and primary cortical neurons treated with 100 nM of Ex-4 under hypoxic conditions (0.1%>O2). We found that Ex-4 decreased the HIF-1α expression in the SH-SY5Y cell line and primary cortical neurons under hypoxic conditions, and this effect was reversed by cotreatment with exendin (9-39), a GLP-1R antagonist. These results suggest that HIF-1α may be involved in the neuroprotective effect of Ex-4 in the hypoxia-damaged brain.

Neuroprotective effects of overexpressed cyclophilin B against Aß-induced neurotoxicity in PC12 cells.

Accumulated amyloid-ß (Aß) is a well-known cause of neuronal apoptosis in Alzheimer disease and functions in part by generating oxidative stress. Our previous work suggested that cyclophilin B (CypB) protects against endoplasmic reticulum (ER) stress. Therefore, in this study we examined the ability of CypB to protect against Aß toxicity. CypB is present in the neurons of rat and mouse brains, and treating neural cells with Aß(25-35) mediates apoptotic cell death. Aß(25-35)-induced neuronal toxicity was inhibited by the overexpression of CypB as measured by cell viability, apoptotic morphology, sub-G1 cell population, intracellular reactive oxygen species accumulation, activated caspase-3, PARP cleavage, Bcl-2 proteins, mitogen-activated protein kinase (MAPK) activation, and phosphoinositide 3-kinase (PI-3-K) activation. CypB/R95A PPIase mutants did not reduce Aß(25-35) toxicity. We showed that Aß(25-35)-induced apoptosis is more severe in a CypB knockdown model, confirming that CypB protects against Aß(25-35)-induced toxicity. Consequently, these findings suggest that CypB may protect against Aß toxicity by its antioxidant properties, by regulating MAPK and PI-3-K signaling, and through the ER stress pathway.

Voluntary exercise enhances survival and migration of neural progenitor cells after intracerebral haemorrhage in mice.

PRIMARY OBJECTIVE: This study explored the long-term effects of exercise on the proliferation, survival and migration of endogenous neural progenitor cells (NPCs) in the subventricular zone (SVZ) of the brain after intracerebral haemorrhage (ICH). RESEARCH DESIGN: ICH was induced by an injection of collagenase into the striatum. Animals in the voluntary running exercise group ran freely on a running wheel for 1, 3 and 6 weeks following the induction of ICH. METHODS AND PROCEDURE: Immunohistochemical labelling was performed to incorporate specific cell markers, such as Ki67 (proliferating cells), 5-bromodeoxyuridien (BrdU; surviving newborn cells) and doublecortin (DCX; neuroblasts or migrating cells). MAIN OUTCOMES AND RESULTS: Voluntary exercise for 3 and 6 weeks sustained more Ki67- or BrdU-immunostained cells in the SVZ after ICH than in the brains of sedentary mice. DCX-immunostained cells were more prominent in the striatum of the group that had exercised for 6 weeks compared to the time-matched sedentary group. Moreover, it was observed that proliferating green fluorescent protein (GFP)-positive cells that were infected with retrovirus were located more distally from the injection site in the exercise group than in the sedentary group. CONCLUSIONS: These data indicate that long-term exercise may enhance the proliferation and survival of NPCs and their migration toward injured areas, suggesting that exercise may contribute to neuronal injury recovery in cell-based therapies after ICH.

A novel method for olfactory bulbectomy using photochemically induced lesion.

We present the photochemically induced olfactory bulbectomy (P-bulbectomy) as a novel method to ablate the olfactory bulb thus inducing an animal model of depression. The photosensitizer Rose Bengal was injected through the tail vein and then the cool halogen light illuminated the skull region overlying of the olfactory bulb for 10 min. Two weeks after surgery, P-bulbectomy had completely removed olfactory bulb uniformly in all animals. P-bulbectomy induced typical depression-related behaviors such as hyperactivity in the open field test and an enhancement of immobility time and in the forced swimming test. Depression-related neurohistological phenomenon was also seen; reduction of choline-acetyltransferase-positive cell numbers in the medial septum and a decline in cell proliferation in the dentate gyrus of hippocampus. This study shows that P-bulbectomy may be a convenient and reproducible experimental method to produce an animal model of depression.

Voluntary exercise increases the new cell formation in the hippocampus of ovariectomized mice.

Voluntary exercise, such as running, can induce dramatic increases in adult hippocampal neurogenesis and improve learning and memory function. A recent report showed that exercise also improved memory problems in postmenopausal women. In this study, we examined whether voluntary running exercise could increase new cell formation in the hippocampus under menopausal conditions, modeled with ovariectomized (OVX) mice. Voluntary running exercise for 1 week significantly increased the number of bromodeoxyuridine (BrdU)- and Ki-67-immunoreactive cells in the hippocampus of mice 2 weeks after ovariectomy. In addition, 1 week of voluntary running exercise after 2 weeks in OVX mice increased the numbers of doublecortin- and calretinin-immunoreactive cells in the hippocampus. These data demonstrate that exercise may increase the birth of new cells in the hippocampus under estrogen-deprived conditions, suggesting that exercise may be helpful in improving brain function in climacteric women.