Predegenerated nerve allografts versus fresh nerve allografts in nerve repair.

This study reevaluated the possibility of using predegenerated nerves as donor nerve allografts for nerve repair and compared the results of functional recovery to those obtained after standard, fresh nerve allograft repair. Twenty donor rats underwent a ligature/ section of the left sciatic nerve 4 weeks before nerve graft harvesting. Forty recipient rats underwent severing of the left sciatic nerve leaving a 15-mm gap between the nerve stumps. Graft repair was undertaken using either the predegenerated left sciatic nerve of the 20 donor rats (predegenerated group, 20 recipient rats) or the normal right sciatic nerve of the 20 donor rats (fresh group, 20 recipient rats). Recovery of function was assessed by gait analysis, electrophysiologic testing and histologic studies. Walking tracks measurements at 2 and 3 months, electromyography parameters at 2 and 3 months, peroperative nerve conduction velocity and nerve action potential amplitude measurements at 3 months, as well as assessments of myelinated nerve fiber density and surface of myelination showed that fresh and predegenerated nerve grafts induced a comparable return of function although there was some trend in higher electrophysiologic values in the predegenerated group. The only slight but significant difference was a larger mean nerve fiber diameter in the nerve segment distal to a predegenerated nerve graft compared to a fresh nerve graft. Although our study does not show a dramatic long-term advantage for predegenerated nerve grafts compared to fresh nerve grafts, their use as prosthetic material is encouraging.

A strategy for multiple immunophenotyping by image cytometry: model studies using latex microbeads labeled with seven streptavidin-bound fluorochromes.

Multiple immunophenotyping is aimed at identifying several cell populations in a single labeling procedure by their ability to bind combinations of specific labeled antibodies. The present work demonstrates the simultaneous discrimination by using image cytometry of aminomethylcoumarin acetate (AMCA), Lucifer yellow (LY), fluorescein isothiocyanate (FITC), R-phycoerythrin (PE), PE-Texas red tandem (Red613), peridinin-chlorophyll protein (PerCP), and allophycocyanin (APC), which were all bound to latex beads as streptavidin-conjugated fluorochromes. This has been the result of a step-by-step optimization of the several factors affecting the sensitivity and specificity of multiple immunofluorescence analysis. First, 14 streptavidin-conjugated fluorochromes were evaluated by using spectrofluorometry. A primary selection was then made of ten spectrally separable dyes that could be evaluated by using image cytometry. These dyes were bound to latex particles, and specific filter combinations were assembled to minimize crosstalk between fluorophores while preserving sufficient fluorescence intensity and counting statistics. Potential probe associations were then assessed by measuring the emissions of all fluorochromes that were detected by each filter combination. The resulting crosstalk matrix served as the basic tool both for final selection of the optimal filter combination and for dye set (composed, in this case, of the seven fluorochromes described above) and for mathematical correction of residual spectral overlap. Next, an image cytometry system was adapted to collect seven images of matched brightness with the selected combination of excitation/emission filters and dichroic mirrors. Finally, seven-parameter synthetic images were generated by digital image processing.

Factors regulating megakaryocyte progenitor commitment to polyploidization.

Recognizable megakaryocytes are polyploid cells generated by a clonogenic, diploid progenitor, termed CFU-MKC (colony forming unit, megakaryocyte). In order to quantify polyploidization, ploidy histograms of megakaryocytes determined by microphotometric or flow cytometric measurements of megakaryocyte DNA have generally been used. However these techniques provide no information on the rate of commitment of CFU-MKC to polyploidy. Using a technique of clonal analysis determining the distributions of the number of doublings (NbD) undergone by CFU-MKC before committing to polyploidization, the polyploidization probability of CFU-MKC could be derived. This probability was found to be a constant independent from CFU-MKC mitotic history, since NbD distributions are exponential functions characterized by a constant rate of decay per doubling. By studying the effects of growth factors on polyploidization probability, it was also shown that: (1) this parameter is negatively regulated by growth factors contained in poke-weed or WEHI conditioned media, as well as by erythropoietin; (2) commitment to polyploidization does not require prior CFU-MKC division; (3) bipotent erythroid-megakaryocyte progenitors have a lower polyploidization probability than CFU-MKC; (4) determination of polyploidization probability reflects the activity of growth factors with greater accuracy than megakaryocyte colony count.

Factors regulating megakaryocyte progenitor commitment to polyploidization.

Recognizable megakaryocytes are polyploid cells generated by a clonogenic, diploid progenitor, termed CFU-MKC (colony forming unit, megakaryocyte). In order to quantify polyploidization, ploidy histograms of megakaryocytes determined by microphotometric or flow cytometric measurements of megakaryocyte DNA have generally been used. However these techniques provide no information on the rate of commitment of CFU-MKC to polyploidy. Using a technique of clonal analysis determining the distributions of the number of doublings (NbD) undergone by CFU-MKC before committing to polyploidization, the polyploidization probability of CFU-MKC could be derived. This probability was found to be a constant independent from CFU-MKC mitotic history, since NbD distributions are exponential functions characterized by a constant rate of decay per doubling. By studying the effects of growth factors on polyploidization probability, it was also shown that: (1) this parameter is negatively regulated by growth factors contained in poke-weed or WEHI conditioned media, as well as by erythropoietin; (2) commitment to polyploidization does not require prior CFU-MKC division; (3) bipotent erythroid-megakaryocyte progenitors have a lower polyploidization probability than CFU-MKC; (4) determination of polyploidization probability reflects the activity of growth factors with greater accuracy than megakaryocyte colony count.

Purification, expansion, and multiple fluorochrome labeling of cord blood hemopoietic precursors: preliminary results.

CD34-positive cells were isolated from a total of 23 cords using CellPro Ceprate columns. AIS MicroCellector flasks, and panning. The cells were (1) expanded in serum-free culture supplemented with a variety of combinations of cytokines and (2) immunophenotyped using multiple fluorochrome labeling. The results indicated that the avidin column produced the highest purity of CD34-positive cells, and that immature blast cells could be expanded in serum-free culture. Preliminary results suggested that the four fluorochrome labeling technique may provide useful information on the lineage commitment of cord blood precursor and blast cells.