N-cadherin mediates nitric oxide-induced neurogenesis in young and retired breeder neurospheres.

Neurogenesis may contribute to functional recovery after neural injury. Nitric oxide donors such as DETA-NONOate promote functional recovery after stroke. However, the mechanisms underlying functional improvement have not been ascertained. We therefore investigated the effects of DETA-NONOate on neural progenitor/stem cell neurospheres derived from the subventricular zone from young and retired breeder rat brain. Subventricular zone cells were dissociated from normal young adult male Wistar rats (2-3 months old) and retired breeder rats (14 months old), treated with or without DETA-NONOate. Subventricular zone neurosphere formation, proliferation, telomerase activity, and Neurogenin 1 mRNA expression were significantly decreased and glial fibrillary acidic protein expression was significantly increased in subventricular zone neurospheres from retired breeder rats compared with young rats. Treatment of neurospheres with DETA-NONOate significantly decreased neurosphere formation and telomerase activity, and promoted neuronal differentiation and neurite outgrowth concomitantly with increased N-cadherin and beta-catenin mRNA expression in both young and old neurospheres. DETA-NONOate selectively increased Neurogenin 1 and decreased glial fibrillary acidic protein mRNA expression in retired breeder neurospheres. N-cadherin significantly increased Neurogenin 1 mRNA expression in young and old neurospheres. Anti-N-cadherin reversed DETA-NONOate-induced neurosphere adhesion, neuronal differentiation, neurite outgrowth, and beta-catenin mRNA expression. Our data indicate that age has a potent effect on the characteristics of subventricular zone neurospheres; neurospheres from young rats show significantly higher formation, proliferation and telomerase activity than older neurospheres. In contrast, older neurospheres exhibit significantly increased glial differentiation than young neurospheres. DETA-NONOate promotes neuronal differentiation and neurite outgrowth in both young and older neurospheres. The molecular mechanisms associated with the DETA-NONOate modulation of neurospheres from young and older animals as well age dependent effects of neurospheres appear to be controlled by N-cadherin and beta-catenin gene expression, which subsequently regulates the neuronal differentiating factor Neurogenin expression in both young and old neural progenitor cells.

Human marrow stromal cell therapy for stroke in rat: neurotrophins and functional recovery.

OBJECTIVE: To test the effect of i.v.-injected human bone marrow stromal cells (hMSC) on neurologic functional deficits after stroke in rats. METHODS: Rats were subjected to transient middle cerebral artery occlusion and IV injected with 3 x 10(6) hMSC 1 day after stroke. Functional outcome was measured before and 1, 7, and 14 days after stroke. Mixed lymphocyte reaction and the development of cytotoxic T lymphocytes measured the immune rejection of hMSC. A monoclonal antibody specific to human cellular nuclei (mAb1281) was used to identify hMSC and to measure neural phenotype. ELISA analyzed neurotrophin levels in cerebral tissue from hMSC-treated or nontreated rats. Bromodeoxyuridine injections were used to identify newly formed cells. RESULTS: Significant recovery of function was found in rats treated with hMSC at 14 days compared with control rats with ischemia. Few (1 to 5%) hMSC expressed proteins phenotypic of brain parenchymal cells. Brain-derived neurotrophic factor and nerve growth factor significantly increased, and apoptotic cells significantly decreased in the ischemic boundary zone; significantly more bromodeoxyuridine-reactive cells were detected in the subventricular zone of the ischemic hemisphere of rats treated with hMSC. hMSC induced proliferation of lymphocytes without the induction of cytotoxic T lymphocytes. CONCLUSION: Neurologic benefit resulting from hMSC treatment of stroke in rats may derive from the increase of growth factors in the ischemic tissue, the reduction of apoptosis in the penumbral zone of the lesion, and the proliferation of endogenous cells in the subventricular zone.

Photodynamic therapy with photofrin in combination with Buthionine Sulfoximine (BSO) of human glioma in the nude rat.

High concentrations of cellular glutathione (GSH) within tumour cells may reduce the ability of photodynamic therapy (PDT) to selectively destroy tumour, consequently, a means of improving the therapeutic ratio of PDT in brain tumour is necessary. Therefore, we hypothesize that PDT in combination with Buthionine Sulfoximine (BSO), an agent which lowers cellular glutathione, can significantly enhance destruction of U87 and U251n tumour cells. PDT was performed using Photofrin as a photosensitiser in combination with BSO administration on male Fisher rats with intracerebral U87 and on non-tumour rats (administered at different optical doses in combination with Photofrin). In vitro experimentation utilising colony forming, cell cytotoxicity, and matrigel artificial basement membrane invasion assays showed significant enhancement of tumour kill and significant reduction of migration in tumour cells treated with BSO in combination with Photofrin PDT in comparison with individual therapies for both U87 and U251n cell lines. In vivo combination PDT-BSO treatment of U87 tumour rats exhibited significantly more tumour necrosis than individual treatments. In conclusion, our data suggests BSO enhances Photofrin PDT treatment of human glioma.

Photodynamic therapy with photofrin reduces invasiveness of malignant human glioma cells.

In this study we investigated the influence of Photofrin-based photodynamic therapy (PDT) on the migration of two human glioma cell lines in vitro. U87 and U25ln tumour cells were treated with Photofrin at various doses and subjected to a fixed optical (632 nm) dose of 100 mJ/cm(2). Photofrin cytotoxicity was determined using MTT and colony forming assays. Using a matrigel artificial basement membrane migration assay, we demonstrated that low doses of subcytotoxic PDT treatment, such as PDT with 2.5 micro g/ml Photofrin on U87 cells and 1 micro g/ml on U25ln cells, significantly ( p<0.001) inhibited in vitro migration of both cell lines. Furthermore, in a qualitative spheroid confrontation assay, subcytotoxic PDT of co-cultures between tumour spheroids and brain aggregates resulted in an absence of progressive tumour invasion and destruction of the brain aggregate. In conclusion, our data indicate that low-dose subcytotoxic PDT with Photofrin significantly inhibits invasiveness of U87 and U25ln cells.