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Changes in expression of neurorepair and neuroregenerative factors were examined after transient cerebral ischemia in relation to the effects of tissue plasminogen activator (tPA) and the free radical scavenger edaravone. Physiological saline or edaravone was injected twice during 90 min of transient middle cerebral artery occlusion (tMCAO) in rats, followed by the same saline or tPA at reperfusion. Sizes of the infarct and protein factors relating to neurorepair and neuroregeneration were examined at 4d after tMCAO. The protein factors examined were: a chondroitin sulfate proteoglycan neurocan, semaphorin type 3A (Sema3A), a myelin-associated glycoprotein receptor (Nogo receptor, Nogo-R), a synaptic regenerative factor (growth associated protein-43, GAP43), and a chemotropic factor netrin receptor (deleted in colorectal cancer, DCC). Two groups treated by edaravone only or edaravone plus tPA showed a reduction in infarct volume compared to the two groups treated by vehicle only or vehicle plus tPA. Immunohistochemistry and western blot analyses indicated that protein expression of neurocan, Sema3A, Nogo-R, GAP43, and DCC was decreased with tPA, but recovered with edaravone. Additive edaravone prevented the reductions of these five proteins induced by tPA. The present study demonstrates for the first time that exogenous tPA reduced protein factors involved in inhibiting and promoting axonal growth, but that edaravone ameliorated such damage in brain repair after acute ischemia., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We examined the neuroprotective effects amlodipine and/or atorvastatin in metabolic syndrome (MetS) Zucker fatty rats against transient (90 min) middle cerebral artery occlusion (MCAO). The rats were pretreated with vehicle, amlodipine, atorvastatin, or amlodipine plus atorvastatin for 28 days, and 24 hr after transient MCAO the infarct size was assessed via hematoxylin and eosin staining, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) and microtubule-associated protein 1 light chain 3 (LC3) expression were examined by immunohistochemistry to evaluate apoptosis and autophagy, respectively. Compared with the vehicle group, rats treated with amlodipine or atorvastatin alone showed a significant decrease in infarct volume (P < 0.01), which was further decreased in the amlodipine plus atorvastatin group (P < 0.001). Compared with the vehicle group, the numbers of TUNEL- and LC3-positive cells were markedly reduced by amlodipine or atorvastatin alone (P < 0.01) and further decreased by amlodipine plus atorvastatin (P < 0.001). The number of apoptotic TUNEL/autophagic LC3 double-positive cells was also significantly decreased with amlodipine or atorvastatin alone compared with vehicle (P < 0.01) and was further decreased by amlodipine plus atorvastatin (P < 0.001). These data suggest additive neuroprotective effects of combination amlodipine and atorvastatin treatment after acute ischemic stroke in MetS model Zucker rats. These effects are mediated, at least in part, via antiapoptotic and antiautophagic mechanisms. Further studies are now needed to expand these preliminary results to understand fully the mechanisms involved in the protective effects of amlodipine and atorvastatin against ischemic stroke., (Copyright © 2011 Wiley-Liss, Inc.)

Reactive oxygen species and their detrimental effects on the brain after transient ischemia have been implicated in the pathogenesis of the ischemic injury. The Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) system is currently recognized as the major cellular defense mechanism under oxidative stress, but the involvement of the Keap1-Nrf2 system in the ischemic brain injuries has not been fully investigated to date. In the present study, we investigated temporal changes of Keap1, Nrf2, and their downstream antioxidative proteins in post-ischemic mice brains with respect to spacial differences between the peri-infarct regions and the regions destined to infarct. In the peri-infarct regions, a steady level of Keap1 showed a decremental expression started at 2h of reperfusion after 60 min of transient middle cerebral artery occlusion (tMCAO). In contrast, Nrf2 began to show a significant increase at 2h with a peak at 8h of reperfusion after tMCAO. Both Keap1 and Nrf2 are mainly expressed in neuronal cells but not in glial cells. In the same peri-infarct region, downstream antioxidative proteins such as thioredoxin, glutathione, and heme oxygenase-1 showed significant increases at later time-points of 24-72 h of reperfusion after tMCAO. In the regions destined to infarct, a similar trend of expression changes to those in the peri-infarct regions was observed in Keap1, Nrf2, and 3 downstream antioxidative proteins with much less reactions. The changes found in this study suggest that the induced antioxidative stress proteins after cerebral ischemia may play an important endogenous neuroprotective response under oxidative stress after ischemic stroke., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Hepatocyte growth factor (HGF) and glial cell line-derived neurotrophic factor (GDNF) are strong neurotrophic factors. However, their potentials in neurogenesis, angiogenesis, synaptogenesis, and antifibrosis have not been compared. Therefore, we investigated these effects of HGF and GDNF in cerebral ischemia in the rat. Wistar rats were subjected to 90 min of transient middle cerebral artery occlusion (tMCAO). Immediately after reperfusion, HGF or GDNF was given by topical application. BrdU was injected intraperitoneally twice daily 1, 2, and 3 days after tMCAO. On 14 day, we histologically evaluated infarct volume, antiapoptotic effect, neurogenesis, angiogenesis, synaptogenesis, and antifibrosis. Both HGF and GDNF significantly reduced infarct size and the number of TUNEL-positive cells, but only HGF significantly increased the number of BrdU-positive cells in the subventricular zone, and 5'-bromo-2'-deoxyuridine -positive cells differentiated into mature neurons on the ischemic side. Enhancement of angiogenesis and synaptogenesis at the ischemic boundary zone was also observed only in HGF-treated rats. HGF significantly decreased the glial scar formation and scar thickness of the brain pia mater after tMCAO, but GDNF did not. Our study shows that both HGF and GDNF had significant neurotrophic effects, but only HGF can promote the neurogenesis, angiogenesis, and synaptogenesis and inhibit fibrotic change in brains after tMCAO., (© 2010 Wiley-Liss, Inc.)

Glial cell line-derived neurotrophic factor (GDNF) and hepatocyte growth factor (HGF) are strong neurotrophic factors, which function as antiapoptotic factors. However, the neuroprotective effect of GDNF and HGF in ameliorating ischemic brain injury via an antiautophagic effect has not been examined. Therefore, we investigated GDNF and HGF for changes of infarct size and antiapoptotic and antiautophagic effects after transient middle cerebral artery occlusion (tMCAO) in rats. For the estimation of ischemic brain injury, the infarct size was calculated at 24 hr after tMCAO by HE staining. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) was performed for evaluating the antiapoptotic effect. Western blot analysis of microtubule-associated protein 1 light chain 3 (LC3) and immunofluorescence analysis of LC3 and phosphorylated mTOR/Ser(2448) (p-mTOR) were performed for evaluating the antiautophagic effect. GDNF and HGF significantly reduced infarct size after cerebral ischemia. The amounts of LC3-I plus LC3-II (relative to beta-tubulin) were significantly increased after tMCAO, and GDNF and HGF significantly decreased them. GDNF and HGF significantly increased p-mTOR-positive cells. GDNF and HGF significantly decreased the numbers of TUNEL-, LC3-, and LC3/TUNEL double-positive cells. LC3/TUNEL double-positive cells accounted for about 34.3% of LC3 plus TUNEL-positive cells. This study suggests that the protective effects of GDNF and HGF were greatly associated with not only the antiapoptotic but also the antiautophagic effects; maybe two types of cell death can occur in the same cell at the same time, and GDNF and HGF are capable of ameliorating these two pathways.

Temporal and spatial differences and relationships of proteins relating to the ischemic penumbra were examined at 1, 3, 12, 24, and 48 h after 90 min of transient middle cerebral artery occlusion (tMCAO) in rats. 2, 3, 5-triphenyltetrazolium chloride (TTC) staining showed that the apparent infarction focus first appeared at 1h after tMCAO, which then largely matured at 24h. Immunohistochemistry and Western blot indicated no or trace levels of c-fos, hypoxia inducible factor-1 alpha (HIF-1 alpha), heat shock protein 70 (HSP70), and annexin V (A5) positive cells in the sham control brain. Expression of c-fos increased quickly and widely within and outside of the affected arterial territory (peak at 1h), and that of HIF-1 alpha reached the maximum at 12h in a smaller area than c-fos. HSP70 began to be induced during the first few hours after tMCAO, peaked at 24h, then decreased within 48 h, while A5 was slightly expressed at 3h, then gradually increased until 48 h. Double immunofluorescent analyses showed that the colocalization rates of c-fos/HIF-1 alpha, HIF-1 alpha/HSP70, HSP70/A5, and A5/TUNEL were 40.6%, 58.4%, 42.1% and 61.0%, respectively. These data suggest that multiple molecular penumbra exist after 90 min of tMCAO in the rat brain where several different proteins participate in different temporal and spatial expression patterns. Thus, there is a window for rescue of ischemic neural cells from 12 to 48 h after injury., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)