Shoko Nagotani

Tridermal tumorigenesis of induced pluripotent stem cells transplanted in ischemic brain.

Stroke is a major neurologic disorder. Induced pluripotent stem (iPS) cells can be produced from basically any part of patients, with high reproduction ability and pluripotency to differentiate into various types of cells, suggesting that iPS cells can provide a hopeful therapy for cell transplantation. However, transplantation of iPS cells into ischemic brain has not been reported. In this study, we showed that the iPS cells fate in a mouse model of transient middle cerebral artery occlusion (MCAO). Undifferentiated iPS cells (5 × 105) were transplanted into ipsilateral striatum and cortex at 24 h after 30 mins of transient MCAO. Behavioral and histologic analyses were performed at 28 day after the cell transplantation. To our surprise, the transplanted iPS cells expanded and formed much larger tumors in mice postischemic brain than in sham-operated brain. The clinical recovery of the MCAO+iPS group was delayed as compared with the MCAO+PBS (phosphate-buffered saline) group. iPS cells formed tridermal teratoma, but could supply a great number of Dcx-positive neuroblasts and a few mature neurons in the ischemic lesion. iPS cells have a promising potential to provide neural cells after ischemic brain injury, if tumorigenesis is properly controlled.

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.

Implantation of a new porous gelatin-siloxane hybrid into a brain lesion as a potential scaffold for tissue regeneration

For brain tissue regeneration, any scaffold for migrated or transplanted stem cells with supportive angiogenesis is important once necrotic brain tissue has formed a cavity after injury such as cerebral ischemia. In this study, a new porous gelatin–siloxane hybrid derived from the integration of gelatin and 3-(glycidoxypropyl) trimethoxysilane was implanted as a three-dimensional scaffold into a defect of the cerebral cortex. The porous hybrid implanted into the lesion remained at the same site for 60 days, kept integrity of the brain shape, and attached well to the surrounding brain tissues. Marginal cavities of the scaffolds were occupied by newly formed tissue in the brain, where newly produced vascular endothelial, astroglial, and microglial cells were found with bromodeoxyuridine double positivity, and the numbers of those cells were dose-dependently increased with the addition of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). Extension of dendrites was also found from the surrounding cerebral cortex to the newly formed tissue, especially with the addition of bFGF and EGF. The present study showed that a new porous gelatin–siloxane hybrid had biocompatibility after implantation into a lesion of the central nervous system, and thus provided a potential scaffold for cell migration, angiogenesis and dendrite elongation with dose-dependent effects of additive bFGF and EGF.

Reduction of cerebral infarction in stroke-prone spontaneously hypertensive rats by statins associated with amelioration of oxidative stress

Background and Purpose— This study aimed to clarify the effect of statins on spontaneous stroke and to examine the antioxidative effect in artificial transient middle cerebral artery occlusion (tMCAO). Methods— Stroke-prone spontaneous hypertensive rats (SHR-SP) were treated with pitavastatin, atorvastatin, simvastatin, or vehicle for 4 weeks. Physiological parameters, serum lipids, and infarct volumes were examined. The markers for oxidative stresses on lipids and DNA were immunohistochemically detected in vehicle-treated or simvastatin-treated SHR-SP with tMCAO. Results— Atorvastatin and simvastatin decreased infarct volumes, with simvastatin most effective. Simvastatin significantly reduced immunoreactivities for oxidative stress markers for lipids and DNA in neurons after tMCAO. Conclusions— The results suggest that the antioxidative properties of statins may be implicated in their beneficial effects against neuronal damage in cerebral ischemia.

The protective effect of dantrolene on ischemic neuronal cell death is associated with reduced expression of endoplasmic reticulum stress markers.

The endoplasmic reticulum (ER) plays an important role in ischemic neuronal cell death. In order to determine the effect of dantrolene, a ryanodine receptor antagonist, on ER stress response and ischemic brain injury, we investigated changes in ER stress-related molecules, that is phosphorylated form of double-stranded RNA-activated protein kinase (PKR)-like ER kinase (p-PERK), phosphorylated form of eukaryotic initiation factor 2alpha (p-eIF2alpha), activating transcription factor-4 (ATF-4), and C/EBP-homologous protein (CHOP), as well as terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) in the peri-ischemic area and ischemic core region of rat brain after transient middle cerebral artery occlusion (MCAO). In contrast to the cases treated with vehicle, the infarct volume and TUNEL-positive cells were significantly reduced at 24 h of reperfusion by treatment with dantrolene. The immunoreactivities for p-PERK, p-eIF2alpha, ATF-4, and CHOP were increased at the ischemic peripheral region after MCAO, which were partially inhibited by dantrolene treatment. The present results suggest that dantrolene significantly decreased infarct volume and provided neuroprotective effect on rats after transient MCAO by reducing ER stress-mediated apoptotic signal pathway activation in the ischemic area.

Neural precursor cells division and migration in neonatal rat brain after ischemic/hypoxic injury.

Ischemia/hypoxia (I/H) causes severe perinatal brain disorders such as cerebral palsy. The neonatal brain possesses much plasticity, and to enhance new cell production would be an innovative means of therapy for such disorders. In order to elucidate the dynamic changes of neural progenitor cells in the neonatal brain after ischemia, we investigated new cells production in the subventricular zone and subsequent migration of these cells to the injured area. Newly produced cells were confirmed by incorporation of bromodeoxyuridine (BrdU), and attempt for differentiation was investigated by immunohistochemistry for molecular markers of each cellular lineage. In the sham-control brain, there were many BrdU-labeled cells which gradually decreased as the animal becomes older. Many of these cells were oligodendroglial progenitor or microglial cells. Although there were only few neuronal cells labeled for BrdU in the sham-control, they dramatically increased after I/H. They were located at just beneath the subventricular zone where the progenitor cells reside and to the injured area, indicating that newly produced cells migrated to the infarct region and differentiated into neuronal precursor cells in order to compensate the lost neural cells. We found that BrdU-labeled astroglial, oligodendroglial progenitor, and microglial cells were also increased after I/H, suggesting that they also play active roles in recovery. Progenitor cells may have potential for treating perinatal brain disorders.

Expression of neurocan after transient middle cerebral artery occlusion in adult rat brain

Neurocan is one of the major chondroitin sulfate proteoglycans in the nervous tissues. The expression and proteolytic cleavage of neurocan are developmentally regulated in the normal rat brain, and the full-length neurocan is detected in juvenile brains but not in normal adult brains. Recently, some studies showed that the full-length neurocan was detectable even in the adult brain when it was exposed to mechanical incision or epileptic stimulation. In the present study, we demonstrated by Western blot analysis that the full-length neurocan transiently appeared in the peri-ischemic region of transient middle cerebral artery occlusion (tMCAO) in adult rat with a peak level at 4 days after tMCAO. Immunohistochemical analysis showed that a clear positive signal of neurocan was observed 4 days after tMCAO in the peri-ischemic region of cerebral cortex and caudate, where cells strongly positive in GFAP expression were also distributed. These results indicate that accumulation of the full-length neurocan produced by reactive astrocytes may be one of the processes for tissue repair and reconstruction of neural networks after focal brain ischemia as well.

Vascular protection and restorative therapy in ischemic stroke

Possible strategies for treating stroke include: 1) thrombolytic therapy with tissue plasminogen activator (tPA): restoring cerebral blood flow in the acute phase of ischemic stroke but sometimes causing hemorrhagic transformation (HT); 2) stem cell therapy: the repair of disrupted neuronal networks with newly born neurons in the chronic phase of ischemic stroke. Firstly, we estimated the vascular protective effect of a free radical scavenger, edaravone, in the tPA-treated rat model of middle cerebral artery occlusion. Edaravone prevented dramatically decreased the hemorrhagic transformation and improved the neurologic score and survival rate of tPA-treated rats. Secondly, we attempted to restore brain tissue using a novel biomaterial, polydimethysiloxane-tetraethoxysilane (PDMS-TEOS) hybrid with or without vascular endothelial growth factor (VEGF), and we could show that implantation of a PDMS-TEOS scaffold with VEGF might be effective for treating old brain infarction or trauma. In the future, we will combine these strategies to develop more effective therapies for treatment of strokes.

Gene and stem cell therapy in ischemic stroke.

Possible strategies for treating ischemic stroke include neuroprotection (preventing injured neurons from undergoing apoptosis in the acute phase of cerebral ischemia) and stem cell therapy (the repair of disrupted neuronal networks with newly born neurons in the chronic phase of cerebral ischemia). First, we estimated the neuroprotective effect of glial cell line-derived neurotrophic factor (GDNF) by administration of GFNF protein. GDNF protein showed a direct protective effect against ischemic brain damage. Pretreatment of animals with adenoviral vector containing GDNF gene (Ad-GDNF) 24 h before the subsequent transient middle cerebral artery occlusion (MCAO) effectively reduced infarcted volume. Secondly, we studied the neuroprotective effect of a calcium channel blocker, azelnidipine, or a by-product of heme degradation, biliverdin. Both azelnidipine and biliverdin had a neuroprotective effect in the ischemic brain through their antioxidative property. Lastly, we developed a restorative stroke therapy with a bioaffinitive scaffold, which is able to provide an appropriate platform for newly born neurons. In the future, we will combine these strategies to develop more effective therapies for treatment of strokes.

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.