A need for identifying ganglioside-positive cells with neuronal markers prompted us to establish a reliable method for double or triple immunostaining nervous tissues. Perfusion fixation with paraformaldehyde is typically performed for the routine immunostaining of various neuronal markers but is not suitable for immunostaining gangliosides. Acetone fixation of fresh cryosections is frequently used for ganglioside immunodetection; thus, we tested the effect of acetone treatment for unmasking the antigen epitope of gangliosides (acetone etching) on sections of paraformaldehyde-fixed nervous tissue from rats. Acetone etching significantly retrieved ganglioside immunoreactivity while preserving the immunoreactivity of neuronal markers. Various combinations of gangliosides and neuronal markers could be double-stained by the immunoenzyme method or triple-stained by the immunofluorescence method. This new method may provide additional information regarding the relationship between gangliosides and various neuronal markers from routinely paraformaldehyde-fixed nervous tissues, both freshly prepared specimens and those stocked in the laboratory.

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The present study was designed to examine the histochemical changes and occurrence of apoptosis in taste buds of rat circumvallate papillae following bilateral transection of the glossopharyngeal nerve. Following transection of the glossopharyngeal nerve, the number of taste buds was not altered until post-operative day 3 (PO3), but decreased significantly thereafter. The number of cells within a taste bud, however, decreased significantly from PO2. In normal, uninjured animals, approximately 15.4%, 9.0%, and 7.7% of taste bud cells were labeled with antibodies for phospholipase C β2 subunit (PLCβ2), a marker for type II cells, neural cell adhesion molecule (NCAM), a marker for type III cells, and Jacalin, a marker for type IV cells, respectively. Following gustatory nerve injury, the ratio of cells expressing markers of type III and type IV decreased gradually from PO2, and Jacalin-labeled taste bud cells disappeared on PO3. Under normal conditions, immunoreactivity for single-strand DNA (ssDNA), a marker of apoptosis, was detected in the nuclei of PLC β2-immunoreactive cells and cells showing no labeling for PLCβ2, NCAM, or Jacalin. On PO1, the number of taste bud cells showing ssDNA immunoreactivity increased to double that of normal uninjured animals; these ssDNA-immunoreactive cells were also labeled with NCAM and Jacalin as well as PLCβ2. The present results suggest that denervation of the gustatory nerve causes apoptosis in all types of taste bud cells, resulting in the rapid degeneration of taste buds.

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The present study introduces a novel method for the direct observation of histological paraffin sections by low vacuum scanning electron microscopy (LVSEM) with platinum blue (Pt-blue) treatment. Pt-blue was applied not only as a backscattered electron (BSE) signal enhancer but also as a histologically specific stain. In this method, paraffin sections of the rat tongue prepared for conventional light microscopy (LM) were stained on glass slides with a Pt-blue staining solution (pH 9) and observed in a LVSEM using BSE detector. Under LVSEM, overviews of whole sections as well as three-dimensional detailed observations of individual cells and tissues could be easily made at magnifications from ×40 to ×10,000. Each kind of cell and tissue observed in the section could be clearly distinguished due to the different yields of BSE signals, which depended on the surface structures and different affinities to Pt-blue. Thus, we roughly classified cellular and tissue components into three groups according to the staining intensity of Pt-blue observed by LM and LVSEM: 1) a strongly stained (deep blue by LM and brightest by LVSEM) group which included epithelial tissue, endothelium and mast cells; 2) a moderately stained (light blue and bright) group which included muscular tissue and nervous tissue; 3) an unstained or weakly stained (colorless and dark) group which included elastic fibers and collagen fibers. We expect that this method will prove useful for the three-dimensional direct observation of histological paraffin sections of various tissues by LVSEM with higher resolutions than LM.

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In the gastrointestinal musculature, interstitial cells of Cajal (ICC) distribute and regulate the gastrointestinal motility. Another type of mesenchymal cell, known as the fibroblast-like cell (FLC), has also been reported to be juxtaposed to the ICC. In this study, we examined the immunohistochemical properties of FLC in the murine gastrointestinal musculature using antibodies to small conductance Ca²⁺-activated K⁺ channel 3 (SK3), platelet-derived growth factor receptor α (PDGFRα), and CD34. SK3-immunopositive (SK3-ip) cells were observed in the musculature throughout the gastrointestinal tract. These SK3-ip cells were distinct from the ICC that were identified by c-Kit immunoreactivity. In the muscular layers, SK3-ip cells were bipolar in shape and were associated with the intramuscular ICC and nerve fiber bundles. In the myenteric layer multipolar-shaped SK3-ip cells encompassed the myenteric ganglia. SK3-ip cells in the subserosal plane formed a cellular network with their ramified processes. The distribution pattern of the SK3-ip cells in the ICC-deficient W^v/W^v mutant mice was similar to that in normal mice. We also demonstrated that SK3-ip cells showed the intense PDGFRα immunoreactivity that was previously examined in FLC. However, CD34 immunoreactivity, one of the markers of human FLC, was not observed in SK3-ip cells with the exception of subserosal FLC. Thus, our observations indicate that SK3- and PDGFRα-double immunopositive cells are FLC in the murine gastrointestinal musculature and behave as a basic cellular element throughout the gastrointestinal musculature.

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Nitric oxide (NO) has various roles in the skeletal musculature in both normal and pathological conditions. NO primarily activates soluble guanylate cyclase (sGC) and mediates subsequent intracellular signaling in target cells. We sought to identify the target cells of NO in the rat skeletal musculature, using subtypes of sGCα1 and sGCβ1 antibodies. Immunohistochemistry revealed that both antibodies stained the same cells with round or oval shapes, having several long processes. The sGC-immunopositive cells co-expressed NG2 chondroitin sulfate proteoglycan, a marker of pericytes. The sGC-immunopositive cells were associated with capillaries and formed cellular networks with elongated cytoplasmic processes. sGCα1 and sGCβ1 were not found in muscle sarcolemma that were stained by anti-dystrophin, or neuromuscular junctions, as detected by anti-synaptophysin. Based on these findings, we concluded that sGC immunoreactivity was specifically distributed in capillary pericytes. Pericytes in the skeletal musculature have been shown to be target cells of NO and are involved in the microvascular blood flow.

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The effects of transplanted embryonic spinal tissue on host motor nerve regeneration and target muscle reinervation were investigated in severed sciatic nerves of rats. The electromyogram (EMG) responses and number of motor end plates (MEP) in target muscles, number of nerve axons, and retrogradely labeled motor neurons were examined in transplantation-, anastomosis without transplantation-, and naïve groups of the animals. The EMG patterns of the transplantation group returned to nearly normal at the 8th week, but those of the anastomosis group did not. MEP counts in the transplantation group were significantly higher than in the anastomosis group. The myelinated axon counts and myelin sheath thickness in the transplantation group were significantly higher than those in the anastomosis group. The number of retrogradely labeled motor neurons was significantly higher in the transplantation group. We conclude that transplanted embryonic spinal tissue can promote both host motor nerve regeneration and target muscle reinnervation.

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