Dufour glands of Apis mellifera and Melipona bicolor were studied under light and transmission electron microscopy, using the cytochemical techniques of mercury bromophenol blue for protein detection, imidazole-buffered osmium tetroxide selective staining of unsaturated lipids, lanthanum nitrate for intercellular junction identification and zinc-iodide-osmium tetroxide for cytoplasmic endomembrane visualization. The results in both species corroborated the lipid nature of the gland secretion and showed in A. mellifera the poverty of the synthetic machinery in the worker gland cells in comparison with the queen, as expected by previous biochemical analyses. The pathway of the exogenous compounds of the secretion is intracellular, since substances can penetrate the cell folds and intercellular junctions, but their access to the gland lumen is barred by the apical intercellular junctions.

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In human breast cancer, overexpression of HER2 protein (score 3+) is a good indication for herceptin (trastuzumab) administration and can expect a favorable prognosis. In this study, we examined the correlation between the expression of HER2 protein and the status of mRNA amplification in human breast cancer tissues prepared using laser assisted microdissection (LAM) technique.
Twenty breast cancers were selected for this study. The surgical specimens were obtained under informed consent. Fresh tumor tissues were cut into no more than 10×5×5 mm sections, and frozen immediately in hexane/dry-ice acetone. Eight-micrometer thick sections were cut and mounted on a membrane film-coated slide glass, fixed in methanol and stained with toluidine blue. Microdissection was carried out using a LAM instrument (LS 337 Laser Scissors^TM, Cell Robotics Inc. USA). RNA was extracted from the microdissected tumor tissues by means of guanidium thiocyanate method, and transcribed to cDNA. HER2 and GAPDH (internal control) mRNAs were analyzed by real time polymerase chain reaction (PCR) based on a fluorogenic TaqMan method. Amplification of HER2 mRNA was standardized with the copy number of GAPDH mRNA. Immunohistochemistry (IHC) for HER2 protein was carried out using LSAB (labeled streptavidine biotin) method.
Regarding the status of HER2 mRNA amplification, we obtained 4 cases of invalid score and 4 cases of distinct and unequivocal amplification. The status of HER2 mRNA amplification of these 8 cases was well correlated with the expression of HER2 protein evaluated as score 0 and 3+. The cases evaluated as score 1+ and 2+ in the immunohistochemistry were quite heterogeneous and showed a relatively wide range of HER2 amplification.
A combination of LAM technique and real time PCR is a powerful and reliable method for the quantitative analysis of mRNA. Our results indicated that the groups of HER2 score 1+ and 2+ include candidates for herceptin therapy.

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Glial and vascular cell proliferation is known to be induced in several neurological disorders of the central nervous system. However, whether leptomeningeal cells proliferate in response to neuronal disorders including brain infarction or hemorrhage, remains unknown. We assessed ³H-thymidine incorporation into proliferating leptomeningeal cells in delayed neuronal death following a transient forebrain ischemia for 5 min in gerbils; the CA1 hippocampal neurons died 2-6 days after the ischemia. The number of ³H-labeled leptomeningeal cells started to increase 2 days after ischemia and reached 5 times that of the sham-operated animals 6 days after ischemia. The proliferative response was suppressed to the baseline level at 8 days after ischemia. The total number of leptomeningeal cells (³H-labeled and unlabeled cells) also started to increase 2 days after ischemia and reached 1.4 times at 4-8 days after that. These observations indicated that leptomeningeal cells begin to proliferate in response to ischemia-induced neuronal death in the central nervous system.

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Gastrointestinal stromal tumor (GIST), immunohistochemically characterized by c-kit protein (CD117) expression, is known to be resistant to chemotherapy and radiotherapy when it is malignant. Recently, Gleevec (STI571) has been introduced for the therapy of metastatic GISTs, and appropriate evaluation of c-kit protein has thus been clinically relevant to confirm the indication of Gleevec therapy. However, we often experienced the instability of immunohistochemical staining for c-kit protein. The aim of the present study is to establish a reproducible immunohistochemical procedure for c-kit protein detection in paraffin sections. Regarding the fixation period, formalin fixation for 24 to 72 hours gave the best result among several fixation conditions. For the purpose of epitope retrieval, hydrated heating in EDTA solution, pH 8.0, employing a pressure cooker gave the most reproducible immunostaining result. For visualization of the reaction products, diaminobenzidine solution available from DakoCytomation Co. was the most sensitive. Under these conditions, c-kit protein immunoreactivity was clearly observed in 20 (95%) of 21 GISTs, routinely processed for histologic preparation. Two tumors were proven to be false negative by the routine diagnostic procedure.

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The distribution of peripherin mRNA in adult rat retinal ganglion cells revealed by in situ hybridization was relevant to the distribution of immunoreactivity to anti-peripherin. Peripherin positive ganglion cells began to differentiate from the 13th gestational day. These peripherin positive ganglion cells expressed α-internexin from the early phase of differentiation and majority of the peripherin positive ganglion cells completed their migration by the 17th gestational day. The optic nerve layer was gradually enlarged during the 15-17th gestational day. However, unlike adult retina, neurofilament triplets were not revealed in the prenatal optic nerve. A different group of neurons with α-internexin without peripherin increased during the 15-19th gestational day and neurons of the internal nuclear layer seemed to transiently express α-internexin because they were seen in the internal nuclear layer above horizontal cells on the 19th gestational day. Although horizontal cells were clearly identified in the retina on the 19th gestational day, calbindin 28KD was not revealed in this stage.

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The aim of the present study was to investigate the quantity and quality of hydroxyapatite (HA) in the zone of calcified cartilage of articular cartilage and underlying subchondral bone by Fourier transform infrared microscopy (FTIRM). We hypothesized that the amount of HA deposited in osteoarthritis (OA) might differ from normal cartilage, and that such differences might be significant in the pathology of OA. The tibial epiphysis of the male Hartley strain for the spontaneous OA model and the control Strain 13 guinea pigs (12-month old) were examined. The mineral:matrix ratio was determined by integrating areas of υ1, υ3 bands at 900-1200 cm^-1 to Amid 1 band at 1585-1725 cm ^-1. The CO₃:PO₄ ratio was determined by integrating areas of υ2 carbonate band at 850-900 cm^-1 to υ1, υ3 bands. In Hartley strain, the mean mineral:matrix ratio of the medial side was significantly lower than the lateral side at the zone of the calcified cartilage and subchondral bone. The mean CO₃:PO ₄ ratio was significantly higher in the medial side than the lateral one at the zone of the calcified cartilage and subchondral bone. In contrast, the results obtained from Strain 13 showed no significant differences between the two sides. Our results showed diminished deposition and increased maturation of HA in Hartley strain. These findings may correlate with the pathological process of knee OA, and indicate that FTIRM is a useful tool for determining the variability of HA in OA.

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We investigated the histological and immunohistochemical changes within and around the cervical intervertebral discs in symptomatic myeloradiculopathy and myelopathy patients. A total of 163 cervical intervertebral discs, 100 herniated discs and 63 spondylotic discs, were harvested en bloc during anterior decompressive surgeries and histological examination was performed, focusing on nucleus pulposus, annulus fibrosus, cartilaginous endplate, and ligamentous enthesis. There was a close relationship between MRI grade and histological degenerative changes. Histological features of the herniated intervertebral discs were the presence of granulation tissue, newly-developed blood vessels and infiltration of CD68-positive cells, which surrounded the herniated tissue in the outer layer of the annulus fibrosus. Marked vascular invasion was detected in 67% of herniated discs and 54% of spondylotic discs. Immunohistochemically, chondrocytes positive for matrix metalloproteinase (MMP)-3, tumor necrosis factor (TNF)-α, basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) were detected in both herniated and spondylotic discs. Our results indicate that the neoangiogenesis process is associated with overexpression of bFGF and VEGF, and suggest that intervertebral disc degradation is enhanced by macrophage infiltration, which is regulated by MMP-3 and TNF-α. The expression of these proteins may be enhanced by the tear of annulus fibrosus.

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Semiquantitative histological evaluation was performed on inner ears from Mongolian gerbils to study vestibular damages using ototoxic drugs. Comparisons were made between animals receiving five daily injections of gentamicin/gelfoam slurry, streptomycin/gelfoam slurry, saline/gelfoam slurry and noninjected controls. For gentamicin treated ears, when percent decrease of nuclei in sensory cell layer was compared, the damage to the sensory cells was significantly more severe in the posterior crista than in the superior and lateral crista from two of three gerbils. For streptomycin treated ears, when percent decrease of nuclei in sensory cell layer was compared, the damage to the posterior crista was similar to superior and lateral crista. These results indicate that our models are useful for detecting different vestibulotoxicity among three cristae.

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Age-related changes in astrocytes, tanycytes and the microvasculature were investigated in the median eminence (ME) of the anterior hypothalamo-hypophyseal system of 2- and 24-month old rats by means of immunocytochemistry and scanning electron microscopy. A computer-assisted image processing system showed a significant increase of the glial fibrillary acidic protein (GFAP) immunoreactivity in the internal layer (IL) and the perivascular region of the external layer (EL) of the ME with age. However, no remarkable changes in either layer could be detected employing vimentin immunoreactivity. An immunoelectron microscopic study revealed that the number of the GFAP-immunoreactive processes markedly increased with age in regions surrounding the capillaries of the EL. Therefore, it may be considered that, in both layers, the astrocytic cell bodies and associated glial processes exhibit a significant increase with age. Investigation of the microvasculature under a scanning electron microscope demonstrated the development and the sinusoidal progress of the external capillary plexus with age, but these changes were not observed in the internal capillary network of the ME. In conclusion, the results of the present study showed a close correlation between the increase of the GFAP-immunoreactive astrocytes and the development of the external capillary plexus. In addition, there was a close correlation between the increase in the GFAP-immunoreactive astrocytes and the sinusoidal progress.

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To clarify the clinical significance of hepatocyte growth factor (HGF) and its specific receptor c-Met in colorectal cancer, we performed serological analysis of HGF in 108 patients who underwent colon cancer surgery and 200 age-matched control subjects, and immunohistochemical analysis of HGF and c-Met in surgically resected samples in 50 of the 108 patients. Serological analysis demonstrated that preoperative serum HGF level was significantly higher in the cancer group than in the control group (p<0.0001), and increased in patients with advance of depth of invasion (p=0.0173), liver metastasis (p=0.0024), and tumor stage (p=0.0069). Immunohistochemical analysis demonstrated the strong expression and colocalization of HGF and c-Met in the cancer cells. Tissue HGF staining score was increased in patients with advance of venous invasion (p=0.0092), whereas tissue c-Met staining score was decreased in patients with advance of lymphatic invasion (p=0.0234), liver metastasis (p=0.0176), and serum HGF level over 300 pg/mL (p=0.018). The present results suggest that the verification of both preoperative serum HGF level and tissue HGF and c-Met levels contributes to the prognostic evaluation of tumor progression in the preoperative period. Furthermore, it is likely that HGF oversupply-associated c-Met downregulation occurs in colorectal cancer via autocrine, paracrine and endocrine mechanisms.

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