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.
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.
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.
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