Optimized Fast-FISH with a-satellite probes: acceleration by microwave activation

It has been shown for several DNA probes that the recently introduced Fast-FISH (fluorescence in situ hybridization) technique is well suited for quantitative microscopy. For highly repetitive DNA probes the hybridization (renaturation) time and the number of subsequent washing steps were reduced considerably by omitting denaturing chemical agents (e.g., formamide). The appropriate hybridization temperature and time allow a clear discrimination between major and minor binding sites by quantitative fluorescence microscopy. The well-defined physical conditions for hybridization permit automatization of the procedure, e.g., by a programmable thermal cycler. Here, we present optimized conditions for a commercially available X-specific a-satellite probe. Highly fluorescent major binding sites were obtained for 74oC hybridization temperature and 60 min hybridization time. They were clearly discriminated from some low fluorescent minor binding sites on metaphase chromosomes as well as in interphase cell nuclei. On average, a total of 3.43 ± 1.59 binding sites were measured in metaphase spreads, and 2.69 ± 1.00 in interphase nuclei. Microwave activation for denaturation and hybridization was tested to accelerate the procedure. The slides with the target material and the hybridization buffer were placed in a standard microwave oven. After denaturation for 20 s at 900 W, hybridization was performed for 4 min at 90 W. The suitability of a microwave oven for Fast-FISH was confirmed by the application to a chromosome 1-specific a-satellite probe. In this case, denaturation was performed at 630 W for 60 s and hybridization at 90 W for 5 min. In all cases, the results were analyzed quantitatively and compared to the results obtained by Fast-FISH. The major binding sites were clearly discriminated by their brightness

Apoptosis of sensory neurons and satellite cells after sciatic nerve transection in C57BL/6J mice

The rate of axonal regeneration, after sciatic nerve lesion in adult C57BL/6J mice, is reduced when compared to other isogenic strains. It was observed that such low regeneration was not due just to a delay, since neuronal death was observed. Two general mechanisms of cell death, apoptosis and necrosis, may be involved. By using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) technique, we demonstrated that a large number of sensory neurons, as well as satellite cells found in the dorsal root ganglia, were intensely labeled, thus indicating that apoptotic mechanisms were involved in the death process. Although almost no labeled neurons or satellite cells were observed one week after transection, a more than ten-fold increase in TUNEL labeling was detected after two weeks. The results obtained with the C57BL/6J strain were compared with those of the A/J strain, which has a much higher peripheral nerve regeneration potential. In A/J mice, almost no labeling of sensory neurons or satellite cells was observed after one or two weeks, indicating the absence of neuronal loss. Our data confirm previous observations that approximately 40% of C57BL/6J sensory neurons die after sciatic nerve transection, and indicate that apoptotic events are involved. Also, our observations reinforce the hypothesis that the low rate of axonal regeneration occurring in C57BL/6J mice may be the result of a mismatch in the timing of the neurons need for neurotrophic substances, and production of the latter by non-neuronal cells in the distal stump.

Ectopic development of skeletal muscle induced by subcutaneous transplant of rat satellite cells

The present study analyzes the ectopic development of the rat skeletal muscle originated from transplanted satellite cells. Satellite cells (10(6) cells) obtained from hindlimb muscles of newborn female 2BAW Wistar rats were injected subcutaneously into the dorsal area of adult male rats. After 3, 7, and 14 days, the transplanted tissues (N = 4-5) were processed for histochemical analysis of peripheral nerves, inactive X-chromosome and acetylcholinesterase. Nicotinic acetylcholine receptors (nAChRs) were also labeled with tetramethylrhodamine-labeled alpha-bungarotoxin. The development of ectopic muscles was successful in 86% of the implantation sites. By day 3, the transplanted cells were organized as multinucleated fibers containing multiple clusters of nAChRs (N = 2-4), resembling those from non-innervated cultured skeletal muscle fibers. After 7 days, the transplanted cells appeared as a highly vascularized tissue formed by bundles of fibers containing peripheral nuclei. The presence of X chromatin body indicated that subcutaneously developed fibers originated from female donor satellite cells. Differently from the extensor digitorum longus muscle of adult male rat (87.9 ± 1.0 µm; N = 213), the diameter of ectopic fibers (59.1 µm; N = 213) did not obey a Gaussian distribution and had a higher coefficient of variation. After 7 and 14 days, the organization of the nAChR clusters was similar to that of clusters from adult innervated extensor digitorum longus muscle. These findings indicate the histocompatibility of rats from 2BAW colony and that satellite cells transplanted into the subcutaneous space of adult animals are able to develop and fuse to form differentiated skeletal muscle fibers.

Diversity among satellite glial cells in dorsal root ganglia of the rat

Peripheral glial cells consist of satellite, enteric glial, and Schwann cells. In dorsal root ganglia, besides pseudo-unipolar neurons, myelinated and nonmyelinated fibers, macrophages, and fibroblasts, satellite cells also constitute the resident components. Information on satellite cells is not abundant; however, they appear to provide mechanical and metabolic support for neurons by forming an envelope surrounding their cell bodies. Although there is a heterogeneous population of neurons in the dorsal root ganglia, satellite cells have been described to be a homogeneous group of perineuronal cells. Our objective was to characterize the ultrastructure, immunohistochemistry, and histochemistry of the satellite cells of the dorsal root ganglia of 17 adult 3-4-month-old Wistar rats of both genders. Ultrastructurally, the nuclei of some satellite cells are heterochromatic, whereas others are euchromatic, which may result from different amounts of nuclear activity. We observed positive immunoreactivity for S-100 and vimentin in the cytoplasm of satellite cells. The intensity of S-100 protein varied according to the size of the enveloped neuron. We also noted that vimentin expression assumed a ring-like pattern and was preferentially located in the cytoplasm around the areas stained for S-100. In addition, we observed nitric oxide synthase-positive small-sized neurons and negative large-sized neurons equal to that described in the literature. Satellite cells were also positive for NADPH-diaphorase, particularly those associated with small-sized neurons. We conclude that all satellite cells are not identical as previously thought because they have different patterns of glial marker expression and these differences may be correlated with the size and function of the neuron they envelope.

Implantation of muscle satellite cells overexpressing myogenin improves denervated muscle atrophy in rats

This study evaluated the effect of muscle satellite cells (MSCs) overexpressing myogenin (MyoG) on denervated muscle atrophy. Rat MSCs were isolated and transfected with the MyoG-EGFP plasmid vector GV143. MyoG-transfected MSCs (MTMs) were transplanted into rat gastrocnemius muscles at 1 week after surgical denervation. Controls included injections of untransfected MSCs or the vehicle only. Muscles were harvested and analyzed at 2, 4, and 24 weeks post-transplantation. Immunofluorescence confirmed MyoG overexpression in MTMs. The muscle wet weight ratio was significantly reduced at 2 weeks after MTM injection (67.17±6.79) compared with muscles injected with MSCs (58.83±5.31) or the vehicle (53.00±7.67; t=2.37, P=0.04 and t=3.39, P=0.007, respectively). The muscle fiber cross-sectional area was also larger at 2 weeks after MTM injection (2.63×103±0.39×103) compared with MSC injection (1.99×103±0.58×103) or the vehicle only (1.57×103±0.47×103; t=2.24, P=0.049 and t=4.22, P=0.002, respectively). At 4 and 24 weeks post-injection, the muscle mass and fiber cross-sectional area were similar across all three experimental groups. Immunohistochemistry showed that the MTM group had larger MyoG-positive fibers. The MTM group (3.18±1.13) also had higher expression of MyoG mRNA than other groups (1.41±0.65 and 1.03±0.19) at 2 weeks after injection (t=2.72, P=0.04). Transplanted MTMs delayed short-term atrophy of denervated muscles. This approach can be optimized as a novel stand-alone therapy or as a bridge to surgical re-innervation of damaged muscles.