Abstract
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.
Introduction
Strokes are a major cause of death and result in a drastic reduction in the quality of life. New strategy for patients suffering from stroke is thus needed. Possible new strategies for treating ischemic strokes are broadly categorized into two groups: neuroprotection, which prevents damaged neurons from undergoing apoptosis in the acute phase of a stroke, and stem cell therapy, which allows for the repair of disrupted neuronal networks of newly born neurons in the chronic phase of a stroke (1). In this article, we focus on gene therapy delivering neurotrophic factor and free radical scavengers for neuroprotection, and on a bioaffinitive scaffold supporting neurosupplementation for stem cell therapy (Fig. 1).
Therapeutic strategy against ischemic stroke. Gene therapy can be applied not only for neuroprotection, but also for stem cell therapy. Increase of cerebral blood flow (CBF) and primary neuroprotection, in which free radical scavengers play an important role for essential neuroprotection. On the other hand, stem cell therapy is composed of two tactics: 1) activation of intrinsic neural stem cells, whose principal origin is the subventricular zone, and 2) transplantation of extrinsic neural stem cells. An appropriate scaffold is able to support this therapy.
Neurotrophic Factor and Gene Therapy
Development of neuronal and glial cells and their maintenance are under control of neurotrophic factors. Glial cell line-derived neurotrophic factor (GDNF) is a member of the transforming growth factor-β superfamily, and plays important roles in the survival of neurons under pathological conditions such as ischemia (16). We administrated GDNF into Wistar rats by placing spongel including GDNF in contact with the surface of the cerebral cortex, and sacrificed the animals at 24 h after permanent middle cerebral artery occlusion (MCAO). Both infarct size and brain edema were significantly reduced by topical application of GDNF (48% and 30%, respectively). Moreover, terminal deoxynucleotidyl transferase mediated dUTP-biotin in situ nick labeling (TUNEL) staining and immunoreactivities for caspases-1 and −3 were remarkably reduced with GDNF treatment. These data suggest that GDNF has the potential to reduce the infarct volume and brain edema through, at least partly, its antiapoptotic effect (12). To deliver neurotrophic factors more efficiently, we tried gene therapy using viral vectors. Among several viral vectors, we chose adenovirus vectors for the therapy, because these vectors efficiently infect both replicating and nonreplicating cells and its transient transgene expression seems to be favorable for stroke therapy. To examine a possible protective effect of exogenous GDNF gene expression against ischemic brain injury, a replication-defective adenoviral vector containing GDNF gene (Ad-GDNF) was directly injected into the cerebral cortex at 24 h before 90 min of MCAO in rats. 2,3,5-Triphenyltetrazolium chloride staining showed that infarct volume of the Ad-GDNF-treated group at 24 h after the MCAO was significantly smaller than that of the vehicle- or Ad-LacZ-treated group. The numbers of TUNEL, immunoreactive caspase-3, and cytochrome c-positive neurons induced in the ipsilateral cerebral cortex at 24 h after the MCAO were markedly reduced by the Ad-GDNF treatment. These results suggest that the successful exogenous GDNF gene transfer ameliorates ischemic brain injury after transient MCAO (13). Gene transfer to the ischemic brain may be a promising approach for treatment of ischemic penumbra.
Free Radical Scavengers
Plenty of free radicals are generated during an ischemic stroke. These can peroxidize lipid, protein, and DNA in various brain cells, and have been implicated in the pathogenesis of cerebral infarction (4). Free radicals are regarded as an important therapeutic target for improving the outcome of an ischemic stroke. Several studies using an animal model have reported that brain ischemic injury was reduced by using several free radical scavengers such as 2,2,5,7,8-pentamethyl-6-hydroxychromane (PMC) (10), disodium 4-[(tert-butylimino)-methyl] benzene-1,3-disulfonate N-oxide (NXY-059) (14), and 3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186) (2). In large human clinical trials, NXY-059 initially showed a reduction in disability following a stroke (SAINT I) (15), but eventually failed to represent the initial effect (SAINT II) (21). In contrast, we originally reported that MCI-186 ameliorated brain edema in the ischemic brain (2), and have already applied this to a human clinical setting in Japan, and shown the effect of reducing the disability in stroke patients (24). Recently, we have found new free radical scavengers, especially from preexisting drugs applied for different diseases [e.g., calcium channel blockers (CCBs) for hypertension] or endogenous effectors that humans originally possess (e.g., biliverdin), because these are expected to be applied to a clinical setting in the near future.
Several CCBs have a dihydropyridine ring reducing oxidative stress (27). In order to clarify whether CCBs have an antioxidative property against cerebral ischemia, we treated Wistar rats with a CCB, azelnidipine (1 mg/kg), subjected the rats to 90-min MCAO, and estimated its effects on infarct volume and oxidative damage. Treatment with azelnizipine reduced both infarct volume and brain edema, compared with the vehicle. The expression of oxidative stress markers, such as HEL, 4-HNE, AGE, and 8-OHdG, were significantly decreased by azelnizipine treatment. These results suggested that azelnizipine had a neuroprotective effect in the ischemic brain, which originated from its antioxidative property (18).
Biliverdin, one of the by-products of heme degradation, is also reported to possess a cytoprotective effect against oxidative stress (22). To estimate its effect against ischemic injury, biliverdin was administered intraperioneally after 90-min MCAO. Biliverdin treatment significantly reduced infarct volume of the cerebral cortices. Ethidium staining at 4 h after MCAO revealed that superoxide production in the cerebral cortex was significantly reduced by biliverdin treatment. Moreover, biliverdin treatment decreased the number of stained cells when oxidative injury markers 4-HNE and 8-OHdG were used. These data indicated that biliverdin administration ameliorated ischemic brain injury through an antioxidant mechanism (5).
Stem Cell Therapy
To supply new neurons into the damaged brain after a stroke, two tactics are proposed. One is the activation of intrinsic neural stem cells (19, 23, 26). The other is the transplantation of extrinsic neural stem cells (8). In this article, we are focusing on intrinsic neural stem cells. In a normal brain, glial fibrillary acidic protein (GFAP)-expressing cells in the subventricular zone (SVZ) of the lateral ventricles include neural stem cells that give rise to olfactory bulb neurons only (3, 7, 17). To clarify whether neural stem cells supply new neurons to areas injured by cerebral ischemia, several study groups have performed region-specific cell labeling and long-term tracing experiments using a Cre-loxP system. SVZ-derived neuroblasts were found to migrate towards the injured striatum after MCAO. Long-term tracing study showed that the SVZ-derived neuroblasts differentiated into mature neurons in the striatum, in which they expressed neuronal-specific nuclear protein and formed synapses with neighboring striatal cells (26). These data demonstrate that the SVZ plays an important role as a cell source in neuronal regeneration after a stroke.
Recent studies reported that the scaffold is an important factor for intrinsic or extrinsic stem cells to survive in necrotic brain tissue forming a cavity in the injured brain (9, 11, 25). We thus studied scaffolds by providing an appropriate environment to stem cells, and developed novel methods using a porous gelatin-siloxane hybrid derived from the integration of gelatin and 3-(glycidoxypropyl) trimethoxysilane (20). This porous hybrid implanted into a defective part of the cerebral cortex and maintained at the same site for 60 days retained the integrity of the brain's shape. Marginal cavities of the scaffolds remained occupied by newly formed tissue, where newly produced endothelial, astroglial, and microglial cells were observed with bromodeoxyuridine double positivity. In addition, those cells increased in a dose-dependent manner following the addition of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) (1, 6). These results suggest that this new porous gelatin-siloxane hybrid has biocompatibility in a lesion of the central nervous system, and thus provided a potential scaffold for cell migration and angiogenesis.
In this article, we briefly highlighted recent progress in the development of these distinct new strategies for the treatment of damaged brains following a stroke. To realize more effective therapies for patients suffering from stroke, it is important to combine these strategies in the acute or the chronic phase following a stroke.