Flow Cytometry and Fluorescence Microscopy as Tools for Structural and Functional Analysis of Vacuoles Isolated from Yeast and Plant Cells.

A series of optimized protocols to isolate vacuoles from both yeast and plant cells, and to characterize the purified organelles at a functional and structural level, are described. For this purpose, we took advantage of the combined use of cell fractionation techniques with different fluorescence-based approaches namely flow cytometry, fluorescence microscopy and spectrofluorimetry. These protocols altogether constitute valuable tools for the study of vacuole structure and function, as well as for the high-throughput screening of drug libraries to identify new molecules that target the vacuole.

Effects of collagen membranes enriched with in vitro-differentiated N1E-115 cells on rat sciatic nerve regeneration after end-to-end repair.

Peripheral nerves possess the capacity of self-regeneration after traumatic injury but the extent of regeneration is often poor and may benefit from exogenous factors that enhance growth. The use of cellular systems is a rational approach for delivering neurotrophic factors at the nerve lesion site, and in the present study we investigated the effects of enwrapping the site of end-to-end rat sciatic nerve repair with an equine type III collagen membrane enriched or not with N1E-115 pre-differentiated neural cells. After neurotmesis, the sciatic nerve was repaired by end-to-end suture (End-to-End group), end-to-end suture enwrapped with an equine collagen type III membrane (End-to-EndMemb group); and end-to-end suture enwrapped with an equine collagen type III membrane previously covered with neural cells pre-differentiated in vitro from N1E-115 cells (End-to-EndMembCell group). Along the postoperative, motor and sensory functional recovery was evaluated using extensor postural thrust (EPT), withdrawal reflex latency (WRL) and ankle kinematics. After 20 weeks animals were sacrificed and the repaired sciatic nerves were processed for histological and stereological analysis. Results showed that enwrapment of the rapair site with a collagen membrane, with or without neural cell enrichment, did not lead to any significant improvement in most of functional and stereological predictors of nerve regeneration that we have assessed, with the exception of EPT which recovered significantly better after neural cell enriched membrane employment. It can thus be concluded that this particular type of nerve tissue engineering approach has very limited effects on nerve regeneration after sciatic end-to-end nerve reconstruction in the rat.

Use of PLGA 90:10 scaffolds enriched with in vitro-differentiated neural cells for repairing rat sciatic nerve defects.

Poly(lactic-co-glycolic acid) (PLGA) nerve tube guides, made of a novel proportion (90:10) of the two polymers, poly(L-lactide): poly(glycolide) and covered with a neural cell line differentiated in vitro, were tested in vivo for promoting nerve regeneration across a 10-mm gap of the rat sciatic nerve. Before in vivo testing, the PLGA 90:10 tubes were tested in vitro for water uptake and mass loss and compared with collagen sheets. The water uptake of the PLGA tubes was lower, and the mass loss was more rapid and higher than those of the collagen sheets when immersed in phosphate-buffered saline (PBS) solution. The pH values of immersing PBS did not change after soaking the collagen sheets and showed to be around 7.4. On the other hand, the pH values of PBS after soaking PLGA tubes decreased gradually during 10 days reaching values around 3.5. For the in vivo testing, 22 Sasco Sprague adult rats were divided into four groups--group 1: gap not reconstructed; group 2: gap reconstructed using an autologous nerve graft; group 3: gap reconstructed with PLGA 90:10 tube guides; group 4: gap reconstructed with PLGA 90:10 tube guides covered with neural cells differentiated in vitro. Motor and sensory functional recovery was evaluated throughout a healing period of 20 weeks using sciatic functional index, static sciatic index, extensor postural thrust, withdrawal reflex latency, and ankle kinematics. Stereological analysis was carried out on regenerated nerve fibers. Both motor and sensory functions improved significantly in the three experimental nerve repair groups, although the rate and extent of recovery was significantly higher in the group where the gap was reconstructed using the autologous graft. The presence of neural cells covering the inside of the PLGA tube guides did not make any difference in the functional recovery. By contrast, morphometric analysis showed that the introduction of N1E-115 cells inside PLGA 90:10 tube guides led to a significant lower number and size of regenerated nerve fibers, suggesting thus that this approach is not adequate for promoting peripheral nerve repair. Further studies are warranted to assess the role of other cellular systems as a foreseeable therapeutic strategy in peripheral nerve regeneration.

PLGA 90/10 and caprolactone biodegradable nerve guides for the reconstruction of the rat sciatic nerve.

The purpose of this study was to test in vivo two different nerve guides for promoting nerve regeneration across a 10-mm gap of the rat sciatic nerve: 1) one made of PLGA in a novel proportion (90:10) of the two polymers poly(L-lactide):poly(glycolide); 2) another made of (DL-lactide-epsilon-caprolactone) copolyester (Neurolac) tube, by comparing its healing efficacy with that of the more traditional methods of end-to-end nerve suture and autologous graft. Motor and sensory functional recovery were assessed throughout the healing period of 20 weeks, and the repaired nerves were processed for morphological and histomorphometrical analysis. Both motor and sensory functions improved significantly in all experimental nerve repaired groups. At the end of the 20-week follow-up, the end-to-end group showed better recovery of motor function when compared with the groups treated with guiding tubes. However, at this time point, the level of motor function in the Neurolac(R) and PLGA groups was similar to the one of the graft group. Nociception function also recovered faster in the end-to-end group compared with the Neurolac(R) and PLGA groups, and in this case, recovery was also delayed in the graft group. At the end of follow-up, nociception was similar in all experimental groups. Morphological and histomorphometrical analysis showed that axon regeneration occurred in both PLGA and Neurolac(R) experimental groups, with no significant differences in the total number of regenerated fibers, but disclosed a different pattern of degradation of the two types of tubes with larger biodegradation of PLGA material by the end of 20 weeks. These results suggest that both types of biomaterials are a good substrate for preparing tubular nerve guides, and their different pattern of degradation does not seem to influence the degree of nerve regeneration.

Evaluation of two biodegradable nerve guides for the reconstruction of the rat sciatic nerve.

The purpose of this study was to test in vivo two different nerve guides, one of PLGA made of a novel proportion (90:10) of the two polymers, Poly(L-lactide):Poly(glycolide), with (DL-lactide-epsilon-caprolactone) copolyester (Neurolac) tube, in promoting nerve regeneration across a 10 mm-gap of the rat sciatic nerve. Finally, end-to-end coaptation was performed. Motor and sensory functional recovery was assessed throughout the healing period of 20 weeks and the repaired nerves were processed for morphological analysis. Both motor and sensory functions improved significantly in all experimental nerve repair groups, although the rate and extent of recovery was significantly higher in the end-to-end group. No significant differences were detected in the comparison between the two types of tubes. Compatible with results of functional tests, morphological analysis showed that axon regeneration occurred in both PLGA and Neurolac experimental groups but disclosed a different pattern of degradation of the two types of tubes with larger biodegradation of PLGA material by the end of 20 weeks. These results suggest that both types of biomaterial are a good substrate for preparing tubular nerve guides and the different pattern of degradation does not seem to influence the degree of nerve regeneration.