Background and Purpose
Recent studies have demonstrated that neurotrophic factors promote neurogenesis following cerebral ischemia. However, as most of them do not pass through the blood-brain barrier and their half-lives are relatively short, continuous or repeated intracerebral or intraventricular infusion would be necessary. Meanwhile, intracerebral gene transfer can result in efficient local production of therapeutic molecules for a longer period by a single injection. Previous studies have indicated that intraventricular administration of heparin-binding epidermal growth factor-like growth factor (HB-EGF), a hypoxia-inducible neuroprotective protein, stimulates neurogenesis in the subventricular zone (SVZ) in both normal and ischemic brains. However, it is still unknown whether proliferating neuronal precursors can differentiate into mature neurons. Moreover, although the implication of HB-EGF in angiogenesis has been reported in vitro and in the cornea, there are few reports about the effects of HB-EGF on angiogenesis after cerebral ischemia. Here, we examined the efficacy of intraventricular injection of a recombinant adenovirus expressing HB-EGF (Ad-HB-EGF) on neurogenesis, angiogenesis and functional outcome after cerebral ischemia.
Methods
Transient focal ischemia was induced by the occlusion of the middle cerebral artery (MCAO) for 80 minutes with a nylon filament in 32 Wistar rats (250–300 g). Three days after MCAO, Ad-HB-EGF or Ad-LacZ as a control vector (n = 16, each) was injected into the lateral ventricle on the ischemic side. Bromodeoxyuridine (BrdU; 50 mg/kg) was injected intraperitoneally twice daily on the 6th and 7th day. On the 8th or 28th day after MCAO, we evaluated infarct volume, neurogenesis and angiogenesis histologically. Motor function was serially evaluated by the rotarod test after MCAO.
Results
Adenovirus-mediated gene expression was detected mainly in the ependymal cells at the periventricular area. The EGF-receptor was expressed by the neuronal stem/progenitor cells in the SVZ. There was no significant difference in infarct size between Ad-HB-EGF- and Ad-LacZ-treated rats on the 8th and 28th day after ischemia. Treatment with Ad-HB-EGF significantly increased the number of BrdU-positive cells in the SVZ on the 8th day, compared with Ad-LacZ (406 ± 110 vs 236 ± 92 cells/mm2, p < 0.05). In the Ad-HB-EGF-treated rats, some BrdU-positive cells differentiated into mature neurons in the striatum on the ischemic side on the 28th day. To the contrary, newborn mature neurons were seldom observed in the Ad-LacZ-treated rats. Increases in the number of BrdU/laminin-positive cells of microvessels at the peri-infarct striatum were also observed on the 8th day in Ad-HB-EGF-treated rats (36 ± 16 vs 6 ± 6 cells / mm2, p < 0.05). Treatment with Ad-HB-EGF significantly improved functional outcome on the 14th, 21st and 28th day after ischemia.
Conclusions
Our data demonstrated that gene therapy using Ad-HB-EGF contributes to functional recovery following ischemic stroke by promoting neurogenesis and angiogenesis.