Knockdown of the neuronal nitric oxide synthase gene retard the development of the cerebellar granule neurons in vitro.

The role of endogenous neuronal nitric oxide synthase (nNOS) gene in the development of cerebellar granule neurons (CGNs) is conflicting. Here, we tested the effect of antisense oligos (AS-ODN) on the endogenous nNOS gene and the development of the CGNs in vitro. The expression of nNOS increased in a development-dependent pattern both in terms of mRNA and protein. AS-ODN down-regulated nNOS gene, but in a posttranscriptional manner. Knockdown of nNOS protein decreased the viability of the CGNs from 7 to 13 days in culture (DIC). This activity of AS-ODN was mimicked by nNOS inhibitor I. The antagonist (nNOSi, MK-801, or ODQ) -induced decrease of cell viability was normalized by the provision of the sodium nitroprusside, an NO donor. This study provides direct evidence that endogenous nNOS, mainly by means of its principal product NO, plays an active role in sustaining the survival of developing CGNs at transition from differentiation to maturation.

A comparison of the use of adipose-derived and bone marrow-derived stem cells for peripheral nerve regeneration in vitro and in vivo.

BACKGROUND: To date, it has repeatedly been demonstrated that infusing bone marrow-derived stem cells (BMSCs) into acellular nerve scaffolds can promote and support axon regeneration through a peripheral nerve defect. However, harvesting BMSCs is an invasive and painful process fraught with a low cellular yield. METHODS: In pursuit of alternative stem cell sources, we isolated stem cells from the inguinal subcutaneous adipose tissue of adult Sprague-Dawley rats (adipose-derived stem cells, ADSCs). We used a co-culture system that allows isolated adult mesenchymal stem cells (MSCs) and Schwann cells (SCs) to grow in the same culture medium but without direct cellular contact. We verified SC phenotype in vitro by cell marker analysis and used red fluorescent protein-tagged ADSCs to detect their fate after being injected into a chemically extracted acellular nerve allograft (CEANA). To compare the regenerative effects of CEANA containing either BMSCs or ADSCs with an autograft and CEANA only on the sciatic nerve defect in vivo, we performed histological and functional assessments up to 16 weeks after grafting. RESULTS: In vitro, we observed reciprocal beneficial effects of ADSCs and SCs in the ADSC-SC co-culture system. Moreover, ADSCs were able to survive in CEANA for 5 days after in vitro implantation. Sixteen weeks after grafting, all results consistently showed that CEANA infused with BMSCs or ADSCs enhanced injured sciatic nerve repair compared to the acellular CEANA-only treatment. Furthermore, their beneficial effects on sciatic injury regeneration were comparable as histological and functional parameters evaluated showed no statistically significant differences. However, the autograft group was roundly superior to both the BMSC- or ADSC-loaded CEANA groups. CONCLUSION: The results of the present study show that ADSCs are a viable alternative stem cell source for treating sciatic nerve injury in lieu of BMSCs.

Bone marrow stromal and Schwann cells from adult rats can interact synergistically to aid in peripheral nerve repair even without intercellular contact in vitro.

To better understand the use of Schwann cells (SCs) and bone marrow stromal cells (BMSCs) together for nerve repair, we studied whether interactions between these two cell types by diffusible molecules can enhance their utility. In the present study, a co-culture system was established to allow BMSCs and SCs grow in the same culture medium but without physical contact. Before co-culture, the adult SCs were expanded until confluent. The adult BMSCs were cultured until P10 with CD29 and CD44 positive but CD45 negative. After 4 days in culture, > 80% of the BMSCs in the co-culture system showed both GFAP- and S-100-positive, but < 6% of the BMSCs in control culture system showed both GFAP- and S-100-positive. Meanwhile, 68.76% of the SCs in co-culture system showed S-100-positive, which was > 42.03% of the SCs in control culture system. Furthermore, the in vivo study also confirmed that differentiated BMSCs exert a more beneficial effect on repairing injured sciatic nerve function and axonal regeneration than undifferentiated BMSCs. These results indicate that the two most widely used cell types for promoting peripheral nerve regeneration may interact synergistically to aid their roles in peripheral nerve repair.