松浦 徹

Ischemic stroke is a major neurologic disorder and a leading cause of disability and death in the world. We compared neuroprotective effects of single or combination therapy of amlodipine (AM) and atorvastatin (AT) in such a metabolic syndrome model Zucker rat. The animals were pretreated with vehicle, AM, AT, or the combination of AM plus AT for 28days, and physical and serum parameters were analyzed, then 90min of transient middle cerebral artery occlusion (tMCAO), was performed followed by immunohistochemical analyses at 24h. Without affecting serum levels of lipids, adiponectin, and leptin, the combination therapy of AM plus AT ameliorated the post-ischemic brain weight increase. The single treatment with AM or AT itself exerted neuroprotective effects with reducing inductions of MMP-9 and AT2R, as well as with preserving collagen IV, and the combination therapy of AM plus AT showed a further synergistic benefit against acute ischemic neural damages. Single AT was more protective on these 3 molecules than single AM at this time point of 24h after tMCAO. Thus, the combination therapy with AM plus AT extended the neuroprotectives effect of single treatment with AM or AT on a part of neurovascular unit and a hypertension-related receptor.

Stroke is a major neurologic disorder and a leading cause of death in the world. We compared neuroprotective effects of single or combination therapy of amlodipine (AM) and atorvastatin (AT) in such a metabolic syndrome model Zucker rat after 90 min of transient middle cerebral artery occlusion (tMCAO). The animals were pretreated with vehicle, AM, AT, or the combination of AM plus AT for 28 days, and at 24h of tMCAO, infarct volume and immunohistochemical analyses were performed. The combination of AM plus AT treatment decreased the infarct volume stronger than each single treatment with AM or AT. The numbers of positive cells of oxidative stress markers such as 8-hydroxy-2'-deoxyguanosin (8-OHdG), 4-hydroxy-2-nonenal (4-HNE), and advanced end glycation products (AGE) and inflammation markers such as tumor necrosis factor alpha (TNF-α) and monocyte chemoattractant protein-1(MCP-1) decreased dramatically in the combination-treated group compared with single AM- or AT-treated group. The present study showed that single AM or AT treatment showed neuroprotective effects both with antioxidative and anti-inflammatory mechanisms, but combination therapy of AM plus AT presented a further synergistic benefit in acute ischemic neural damages.

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.

Apoptosis is one of the mechanisms contributing to neuronal degeneration in ischemic stroke. In vivo imaging of annexin V (A5) was performed at 12 hr, 24 hr, 48 hr, and 4 days after 90-min transient middle cerebral artery occlusion (tMCAO) in mice with a fluorescent protein Cy5.5. Immunohistochemistry for heat shock protein 70 (HSP70), A5, and TUNEL were also performed with brain sections after the tMCAO. In vivo fluorescence was strongly observed at 48 hr over the head, especially with removal of both head skin and skull bone. Zonal ex vivo fluorescent signals were surrounding the ischemic core, and double-positive cells with Cy5.5/exogenous A5 antibody were found in this area. HSP70 was observed at the peak time of 24 hr; A5 became detectable at 12 hr, with increasing numbers until 48 hr. The number of TUNEL-positive cells increased at 24 hr and retained the high level until 4 days, showing a dissociating temporal pattern with A5. Double-positive cells for A5/TUNEL reached their peak at 48 hr. All the data suggest that some cells still have a chance to be rescued for a long period after acute cerebral ischemia. The in vivo Cy5.5 fluorescence representing A5 signal spatially surrounding the ischemic core temporally detects an early-stage apoptosis after cerebral ischemia.

Recent studies have suggested that autophagy is involved in a neural death pathway following cerebral ischemia. In vivo detection of autophagy could be important for evaluating ischemic neural cell damage for human stroke patients. Using novel green fluorescent protein (GFP)-fused microtubule-associated protein 1 light chain 3 (LC3) transgenic (Tg) mice, in vivo imaging of autophagy was performed at 1, 3 and 6 d after 60 min transient middle cerebral artery occlusion (tMCAO). Ex vivo imaging of autophagy, testing of the autophagy inhibitor 3-methyladenine (3-MA), estern blot analysis, immunohistochemistry, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) and fluorescent analyses were performed on brain sections following tMCAO. In vivo fluorescent signals were detected above the ischemic hemisphere through the skull bone at 1, 3 and 6 d after tMCAO, with a peak at 1 d. Similar results were obtained with ex vivo fluorescence imaging. western blot analysis revealed maximum LC3-I and LC3-II expression at 1 d after tMCAO and fluorescence immunohistochemistry demonstrated that GFP-LC3-positive cells were primarily neuronal, not astroglial or microglial, cells. The number of GFP-LC3/TUNEL double-positive cells was greater in the periischemic area than in the core. These results provided evidence of in vivo autophagy detection, with a peak at 1 d, in a live animal model following cerebral ischemia. This novel technique could be valuable for monitoring autophagic processes in vivo in live stroke patients, as well as for clarifying the detailed role of autophagy in the ischemic brain, as well as in other neurological diseases.

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.