Atsushi Tsuchiya

Cerebral Ischemia and Angiogenesis

Angiogenesis occurs in a wide range of conditions. As ischemic tissue usually depends on collateral blood flow from newly produced vessels, acceleration of angiogenesis should be of therapeutic value to ischemic disorders. Indeed, therapeutic angiogenesis reduced tissue injury in myocardial or limb ischemia. In ischemic stroke, on the other hand, angiogenic factors often increase vascular permeability and thus may deteriorate tissue damage. In order to apply safely the therapeutic angiogenesis for ischemic stroke treatment, elucidating precise mechanism of brain angiogenesis is mandatory. In the present article, we review previous reports which investigated molecular mechanisms of angiogenesis. Endothelial cell mitogens, enzymes that degrade surrounding extracellular matrix, and molecules implicated in endothelial cells migration are induced rapidly in the ischemic brain. Their possible neuroprotective or injury exacerbating effects are discussed. Because therapeutic potential of angiogenic factors application had gained much attention, we here extensively reviewed relevant previous reports. In the future however, there is a need to consider angiogenesis in relation with regenerative medicine, as angiogenic factors sometimes possess neuron producing property.

Potentiation of neurogenesis and angiogenesis by G-CSF after focal cerebral ischemia in rats

Recently, granulocyte colony-stimulating factor (G-CSF) is expected to demonstrate beneficial effects on cerebral ischemia. Here, we showed the potential benefit of G-CSF administration after transient middle cerebral artery occlusion (tMCAO). Adult male Wistar rats received vehicle or G-CSF (50 microg/kg) subcutaneously after reperfusion, and were treated with 5-bromodeoxyuridine (BrdU, 50 mg/kg) once daily by the intraperitoneal route for 3 days after tMCAO. Nissl-stained sections at 7 days after tMCAO showed significant reduction of the infarction area (31%, P<0.01). At 7 days after tMCAO, BrdU plus NeuN double-positive cells increased by 43.3% in the G-CSF-treated group (P<0.05), and BrdU-positive endothelial cells were increased 2.29 times in the G-CSF-treated group, to a level as high as that in the vehicle-treated group (P<0.01), in the periischemic area. Our results indicate that G-CSF caused potentiation of neuroprotection and neurogenesis and is expected to have practical therapeutic potential in treating individuals after ischemic brain injury.

Decreased Focal Inflammatory Response by G-CSF May Improve Stroke Outcome After Transient Middle Cerebral Artery Occlusion in Rats

Recent studies have shown that administration of granulocyte colony‐stimulating factor (G‐CSF) is neuroprotective. However, the precise mechanisms of the neuroprotective effect of G‐CSF are not entirely known. We carried out 90‐min transient middle cerebral occlusion (tMCAO) of rats. The rats were injected with vehicle or G‐CSF (50 μg/kg) immediately after reperfusion and sacrificed 8, 24, or 72 hr later. 2,3,5‐Triphenyltetrazolium chloride (TTC) staining was carried out using brain sections of 72 hr, and immunohistochemistry was carried out with those of 8, 24, and 72 hr. TTC‐staining showed a significant reduction of infarct volume in the G‐CSF‐treated group (**P < 0.01). Immunohistochemistry showed a significant decrease of the number of cells expressing tumor necrosis factor‐α (TNF‐α) at 8–72 hr, transforming growth factor‐β (TGF‐β) and inducible nitric oxide synthase (iNOS) at 24 and 72 hr after tMCAO in the peri‐ischemic area (*P < 0.05 each). Our data suggest that the suppression of inflammatory cytokines and iNOS expression may be one mechanism of neuroprotection by G‐CSF.

Expression of netrin-1 and its receptors DCC and neogenin in rat brain after ischemia

It is very important to investigate the mechanism of axonal growth in the ischemic brain in order to consider a novel mean of therapy for stroke. Netrins are chemotropic factors for axon with chemoattractant or chemorepellant guidance activities, and deleted in colorectal cancer (DCC) and neogenin are receptors for netrins. In this study, we examined expressions of netrin-1, DCC, and neogenin in the brain after 90 min of transient middle cerebral artery occlusion (tMCAO). Netrin-1 was expressed in neurons at the peri-ischemic area with a peak at 14 days. DCC was expressed both in neurons and astrocytic feet with a peak at 14 days, though neogenin was expressed in endothelial cells at MCA territory with a peak at the same time point. These results suggest that netrin-1 is involved in the promotion of axonal growth. The expression of netrin-1 and DCC was overlapped both in the spatial and temporal patterns, indicating that DCC plays a role in netrin-1s axonal growth promoting effects. The location of neogenin positive cells differed from that of netrin-1 positive cells, thus its angiogenic activity may not have relevance with netrin-1.

Macrophage Infiltration, Lectin-Like Oxidized-LDL Receptor-1, and Monocyte Chemoattractant Protein-1 are reduced by Chronic HMG-CoA Reductase Inhibition

Statin reduces cerebrovascular events independent of its cholesterol lowering effect. We hypothesized that statin inhibits early atherosclerotic change in common carotid artery (CCA), and investigated its effect on lectin-like oxidized-LDL receptor-1 (LOX-1) and monocyte chemoattractant protein-1 (MCP-1) expression, both of which are early atherosclerotic markers. Stroke-prone spontaneous hypertensive rats (SHR-SP) of 8 weeks old were orally treated with vehicle or simvastatin (20mg/kg) daily. After 4 weeks of simvastatin or vehicle treatment, or 2 weeks of vehicle and 2 weeks of simvastatin treatment, CCA was removed. LOX-1 and MCP-1 expression as well as macrophage infiltration were histologically investigated. Lipid deposition was also investigated by Sudan III staining. Simvastatin groups showed significantly smaller amount of lipid deposition and LOX-1 and MCP-1 expression, independent of serum lipid levels. Macrophage infiltration was also decreased. Reduction of cerebrovascular events by statins may be brought by the direct inhibition of atherosclerotic change.

G-CSF enhances stem cell proliferation in rat hippocampus after transient middle cerebral artery occlusion

Granulocyte colony-stimulating factor (G-CSF) enhances the survival and stimulates the proliferation of neutrophil progenitors. Recently, the neurogenerative effect of G-CSF has been intensely investigated. In this study, we explored the possibility that G-CSF enhanced the cell proliferation in the rat dentate gyrus (DG) after focal cerebral ischemia, using a rat transient middle cerebral artery occlusion (tMCAO) model. At 7 days after tMCAO, the number of 5-bromodeoxyuridine (BrdU)-positive cells in the G-CSF-treated group was significantly increased compared with that in the vehicle-treated group in the ipsilateral SGZ (16.6+/-5.5/mm(2) in the vehicle-treated group versus 33.0+/-7.2/mm(2) in the G-CSF-treated group, **p<0.01) and in the ipsilateral GCL (14.2+/-2.8/mm(2) in the vehicle-treated group versus 21.0+/-3.8/mm(2) in the G-CSF-treated group, *p<0.05). This result showed the possibility of a neurogenerative role of G-CSF after tMCAO in rats.

Gelatin-Siloxane Hybrid Scaffolds with Vascular Endothelial Growth Factor Induces Brain Tissue Regeneration

In the brain after infarction or trauma, the tissue becomes pannecrotic and forms a cavity. In such situation, a scaffold is necessary to produce new tissue. In this study, we implanted a new porous gelatin-siloxane hybrid derived from gelatin and 3-(glycidoxypropyl) trimethoxysilane (gelatin-GPTMS) scaffolds into a brain defect, and investigated whether it makes a new brain tissue. In addition, vascular endothelial growth factor (VEGF) was added on gelatin-GPTMS scaffolds and its effect on tissue regeneration was examined. At 30 days after the implantation, the marginal territory of the scaffolds became occupied by newly formed tissue. Immunohistochemical analysis revealed that the new tissue was constituted by endothelial, astroglial and microglial cells, some of which were labeled for bromodeoxyuridine (BrdU). Addition of VEGF promoted numbers of these cells. Thus, combination of gelatin-GPTMS scaffolds and VEGF is preferable for brain regeneration.

Induction of platelet derived-endothelial cell growth factor in the brain after ischemia.

Abstract Objectives: Platelet derived-endothelial cell growth factor (PD-ECGF) is a highly potent angiogenic factor. Although angiogenesis plays an active role in pathophysiology of stroke, the expression pattern of this molecule in ischemic brain has not been investigated. In the present study, therefore, we investigated the change of PD-ECGF expression in the brain after ischemia. Methods: Using male Wistar rats, the right middle cerebral artery was occluded by a nylon thread for 90 minutes. The animals were decapitated 3 hours, 1, 4 and 10 days after the reperfusion, and frozen sections were prepared. We then performed immunohistochemistry for PD-ECGF and identified the cell phenotype which strongly expressed it by fluorescent double staining. Results: In the sham-operated brain, only small numbers of cells slightly expressed PD-ECGF. The number of positively stained cells increased at the peri-ischemic area from hour 3 of reperfusion. Not only small-sized cells but also large-sized cells became stained. The number of stained cells further increased, and peaked at day 4 for large-sized cells and at day 10 as to small-sized cells. Fluorescent double staining revealed that both large-sized and small-sized cells were neurons, indicating that neurons are the main source of PD-ECGF production in the ischemic brain. Discussion: PD-ECGF has a strong angiogenic property without vascular permeability increasing effect. This molecule may have a therapeutic potential for ischemic stroke treatment.

A unique case of limb-girdle muscular dystrophy type 2A carrying novel compound heterozygous mutations in the human CAPN3 gene

A unique sib pair afflicted by limb girdle muscular dystrophy type 2A (LGMD2A) is described showing a slowly progressive autosomal recessive type of muscular dystrophy with onset in the third and fourth decades. The patients had early asymmetric muscle involvement characterized by prominent biceps brachii atrophy with sparing of the knee extensors. Additional findings included elevation of serum creatine kinase level, myopathic EMG changes and dystrophic type of pathology on muscle biopsy. Asymmetrical wasting of muscles in the extremities exhibited uniform and highly selective CT imaging patterns. RNA and DNA analyses confirmed novel compound heterozygous mutations (R147X/L212F) in the human CAPN3 gene.