In the canine prostate, glandular epithelial cells showed uniformly intense nuclear staining for androgen receptor (AR). AR was also localized in the nuclei of the fibro-muscular stromal cells. Immunoreactivity of 5 alpha-reductase type I was positive in most glandular epithelial cells. No fibro-muscular stromal cells were stained. Immunolocalization of 5 alpha-reductase type II was clearly detected in the interacinar fibro-muscular stromal cells, but not in the glandular epithelial cells. After treatment with antiandrogen, chlormadinone acetate (CMA) on canine spontaneous benign prostatic hyperplasia (BPH), glandular atrophy was evident. The interacinar fibro-muscular stroma was prominent. The nuclear staining for AR in both epithelial and stromal cells was remarkably decreased. Furthermore, the immunoreaction for 5 alpha-reductase type I in most glandular epithelial cells was negative or very weak. The immunoreaction of 5 alpha-reductase type II in the interacinar fibro-muscular stromal cells was negative or very weak. The decreased AR-immunostaining may be explained by the decrease in the number of AR and it may play a key role in the down-regulation of 5 alpha-reductase gene expression by CMA. The atrophy after treatment with CMA may be due to shrinkage of both glandular and stromal compartments in the prostate tissue.
The procedures recently developed in our laboratory to observe three-dimensional structures of cell organelles in thick histochemical and cytochemical specimens by means of high voltage electron microscopy are reviewed. Various tissues obtained from the organs of experimental animals in vivo such as rat and mouse liver, intestine and pancreas; or cultured cells, either primary cultures from animal tissues or established cell lines maintained in tissue culture laboratory such as Wistar rat hepatocytes or CHO-K1 cells cultured under normal or experimental conditions, were prefixed in buffered 2.5% glutaraldehyde, stained with various histochemical and cytochemical reactions and postfixed in 1% osmium tetroxide. The reactions used were acid phosphatase, cytochrome oxidase, glucose-6-phosphatase, thiamine pyrophosphatase, DAB, ZIO, PA-TCH-SP reactions, silver impregnation, immunostaining and radioautography. After the reactions the tissues in vivo were embedded in epoxy resin and thick sectioned at 0.5-2.0 μm, while the whole mount cultured cells in vitro were dried in a critical point dryer. All the specimens were observed in JEOL JEM-4000EX or Hitachi H-1250M high voltage electron microscopes at 400-1000 kV. By tilting the specimens, stereo-pair micrographs were recorded and they were observed with stereoscopes or further analyzed with an image analyzer and observed with anaglyph type glasses. The results showed that the stereo-pair images obtained from thick specimens revealed 3-D ultrastructures and relationships. The procedure is useful to analyze 3-D and 4-D ultrastructures of cells and tissues.
Metallothionein (MT) proteins are encoded by a family of genes containing at least 10 functional isoforms. Computational structural analysis has helped to better understand the structure of this protein. The MT protein is easily demonstrated by immunohistochemistry and immunoelectron microscopy. The advantage of immunohistochemistry is that it allows cellular localization of the proteins in fresh paraffin-embedded and archived specimens. MT proteins detected by immunohistochemistry can also be correlated with histopathological features of the tissue sections. There have been several reports on the immunohistochemical detection of the MT protein in a variety of tumors. However, as immunohistochemical methods are unable to distinguish the different MT isoforms, reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization of mRNA transcripts have enabled the analyses of all known functional MT isoforms. Molecular biology techniques could therefore complement immunohistochemistry in the analysis of MT expression from transcription to translation.
Taste is one of the most important senses that allows for human survival. Recent electrophysiological investigations have revealed some information about the somatotopy of the rat nucleus tractus solitarius (NTS). The aim of this study was to assess rat NTS somatotopy using morphological techniques. Each animal was anesthetized and a retrograde axonal tracer, horseradish peroxidase, was injected intra- and submucosally into the anterior portion or the middle and posterior portions of the left half of rat tongue. After 24-48 hr, each rat was anesthetized again and perfusion-fixed. The brainstem was removed and cut in 50 μm-thick sections using a vibrotome. The sections were subjected to a tetramethylbenzidine-peroxidase reaction, and were then assessed under light-microscopy. The horseradish peroxidase that was deposited in the anterior third of the tongue labeled neuron cell bodies in the rostrofrontolateral region of the ipsilateral NTS, while that deposited in the middle and posterior thirds of the tongue labeled neuron cell bodies in the caudoposteromedial region of the ipsilateral NTS. There was no cross-projection. The morphological findings were in accord with the electrophysiological results in the literature. Retrograde labeling revealed a distinct pattern of neural projection that corresponded to the anterior portion, and the middle and posterior portions of the tongue.
The rostral ventrolateral medulla (RVLM) is considered to be a major center for the regulation of sympathetic and cardiovascular activities. Several clinical studies have indicated a possible causal relationship between vasculoneural contact of the RVLM and essential hypertension. We previously reported that pulsatile compression of the ventral surface of the unilateral RVLM with a pulsating probe in anesthetized (with urethane) and artificially ventilated rats elevates arterial pressure, heart rate, and sympathetic nerve activity . However, we have not investigated how and what kind of neurons are activated by compression. Therefore, we performed compression experiments in rats and examined the expression of c-fos, a marker of neuronal activation. The data indicate that after the compression of the ventral surface of the RVLM, c-fos mRNA was increased in the rostral and caudal ventrolateral medulla and the number of neurons containing specifically induced Fos-protein was considerably larger. They also suggest that the Fos-positive neurons also contain tyrosine hydroxylase, indicating that mechanical pressure (compression)-stimulated neural excitation occurred in the A1/C1 catecholamine-neurons and the specific area in the ventral surface of the RVLM is sensitive to compression.
Osteopontin (OPN), a secreted adhesive glycoprotein, is significantly overexpressed in a variety of malignant tumors. Moreover, increased levels of OPN have been detected in the blood of patients with metastatic carcinoma. On immunohistochemical examination, OPN was shown to be expressed in papillary thyroid carcinoma. To investigate the mechanisms of the induction of OPN expression in papillary thyroid carcinoma, we examined the expression of c-src and OPN in 30 cases of papillary thyroid carcinoma by Northern blotting, Western blottimg, in situ hybridization and immunohistochemistry. Each of 5 cases studied by Northern and Western blotting analyses revealed substantial increases of c-src and OPN mRNA and protein levels in tumor areas as compared with surrounding normal thyroid tissues. Moreover, signals of c-src and OPN mRNA were detected in both infiltrating mononuclear cells and tumor cells by in situ hybridization. Immunohistochemical localizations of c-Src and OPN proteins were the same as those of transcripts described above. These results suggested that the induction of the expression of OPN invasiveness in papillary thyroid carcinoma was one of the important downstream events of increased c-Src expression.
The distribution of γ-aminobutyrate (GABA) and its synthesizing enzyme glutamate decarboxylase (GAD) in the epiphyseal growth plate of the rat long bone was studied by immunohistochemistry. GABA, GAD65 and GAD67 immunoreactive chondrocytes were found in hypertrophic zones of the growth plate, but not in reserve and proliferative zones. In the hypertrophic zone, GABA immunoreactivity was more intense in the maturation zone than in the degenerative zone and the zone of provisional calcification. Patterns of immunoreactivity for GAD65 and GAD67 were almost comparable to those of GABA. In addition, GAD65 and GAD67 mRNAs were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). These results suggest that GABA, which is synthesized de novo in chondrocytes of the growth plate may have certain functional roles in skeletal growth.
In human thyroid carcinoma, there is no evidence that the activity of NO synthase and the contribution of endothelial NOS (eNOS) have been determined. To determine the relationship between NOS and human thyroid carcinomas, we assayed eNOS expression and assessed the activity of NOS in thyroid papillary carcinomas (TPC), benign thyroid tumors, and normal thyroid tissues. We investigated the involvement of NO in TPC using an assay system to measure NOS activity by high performance liquid chromatography (HPLC), which could be applied to determine citrulline enrichment. The TPC showed high levels of NOS activity. Moreover, histochemical staining with NADPH-diaphorase revealed a positive reaction in the TPC samples, localized to the blood vessel endothelium. These cells were much less stained, however, in normal thyroid tissue. When we incubated these samples with an anti-eNOS antibody, we observed staining of the TPC samples, again localized to the blood vessel endothelium cells, but much less than in the normal thyroid samples. RT-PCR amplification using eNOS specific primers revealed that eNOS mRNA was abundant in the TPC samples, but was much less expressed in normal thyroid. These results show that NOS activity exists in TPC and that it is associated with malignancy.