Fluorescence in situ hybridization (FISH) represents the newest methodological approach for testing for genetic alterations in individual cells. FISH can detect gene amplification both in metaphase chromosomes and in interphase nuclei. FISH allows the determination of the amplification levels and amplification patterns: clustered signals and multiple scattered signals found by FISH correspond respectively to amplicons in homogeneous staining regions and in double-minute chromosomes found in conventional cytogenetics. Using dual-colour FISH, differentiation of low level amplification from increased gene number by polysomy is also possible. FISH can detect gene amplification not only in isolated nuclei and imprinted cells, but also in formalin-fixed paraffin-embedded tissues, and is therefore a potent tool to detect gene amplification in archival solid tumor specimens. A direct correlation can be made with the morphology, so that tissue localization of such genetic events as tumor heterogeneity and tumor progression is feasible. Furthermore, in combination with immunohistochemistry, direct correlation with the oncogene amplification and the oncoprotein overexpression are possible. Although there have been numerous FISH studies examining oncogene amplifications using clinical materials, at the present time the most important clinical application of FISH is the identification of breast cancer patients eligible for the new adjuvant therapy by humanized monoclonal antibody to c-erbB-2 protein (trastuzumab).
The recent developments in fluorescent-probes and laser technologies have provided us opportunities to observe the biological functions in living organs and tissues under a microscope. Although single-photon excitation confocal microscopy is an indispensable tool for observing these specimens, the observable depth of this microscopy is still not enough to analyze the functions of whole parts of organs in living organisms. To observe the deeper parts, we have attempted to develop new real-time two-photon excitation microscopy equipped with a microlens-arrayed multipinhole scanning device. Using this real-time two-photon microscope, we examined the dynamics of the intracellular calcium concentration ([Ca2+] i) in the cardiac myocytes in the rat whole heart to understand their physiological functions and mechanisms. We could observe Ca 2+ waves on the surface in the whole heart. However, we still need higher-energy laser power for two-photon excitation and visualization of [Ca2+]i dynamics in the deeper parts of the whole heart.
We sought to immunohistochemically localize types I, II and III collagen in various regions of the temporomandibular joint (TMJ) in two female rhesus monkeys at different growth stages: juvenile and late-adolescence. In the condylar cartilage, staining for types I and III collagen was weak in the fibrous layer, intense in the pre-cartilaginous layer, and faint in the cartilaginous layer, whereas that for type II collagen was limited to the cartilaginous layer. The glenoid fossa showed moderate staining for type I collagen and weak staining for type II collagen, indicating that this tissue consists of chondroid bone, which is an intermediary between bone and cartilage. TMJ discs showed more intense staining for types I and III collagen in their periphery than in their centers. Staining for type II collagen was not detected throughout the disc. The posterior attachments showed rather weak staining for type I collagen and relatively intense staining for type III collagen. These results suggest that there are regional differences in the phenotypic expression of the three major collagen components in the growing monkey TMJ, which probably provide specific biomechanical resistance.
Although the subcellular localization of estrogen receptor α (ERα) is generally recognized as in the nucleus, some studies have shown a cytoplasmic distribution. We used a time-lapse imaging technique to examine intracellular and intranuclear localizations of ERα using a green fluorescent protein (GFP)-ERα chimera protein in living COS-1 cells of different external milieu in a cultured condition. Transfected cells were treated with the ERα agonist, estradiol and the partial antagonist, tamoxifen, to investigate the ligand specific changes of distribution of GFP-ERα in a single cell. GFP-ERα was accumulated in the nucleus even in the absence of ligand exposure. Cells showed diffuse fine-grained fluorescence of GFP-ERα in the nucleus without ligands. These results were confirmed by using anti-ERα antibody immunocytochemistry. GFP-ERα gradually and clearly formed small clusters or more punctate patterns in the nuclei of cells after ligand incubation for 60 min, while some of the transfected cells showed a finely diffuse pattern. The manner of nuclear redistribution did not depend on the type of ligand. The present study showed that GFP-ERα is restricted to the nucleus and accumulates with estradiol and tamoxifen, suggesting that accumulation of ER α in the nucleus could be necessary for ERα to function with ligands.
A neuronal tracer, WGA-HRP, was injected into the rat parotid gland (PG), submandibular gland (SMG), and sublingual gland (SLG) to label the superior cervical ganglion (SCG) neurons, and the number, size of soma, dendritic arborization and immunoreactivities (IR) of vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY) were investigated. The number and size of neurons projecting into SMG were greater than those of neurons projecting into the PG or SLG. The PG and SLG projecting neurons did not show significant differences in their number and size of soma, even the weight of the SLG was much smaller than the other two glands. The total length of dendrites of the SMG projecting neurons was greater than those of the PG or SLG projecting neurons. PG and SLG projecting neurons showed no significant difference in the total length of their dendrites. PG and SLG projecting neurons with NPY-IR were larger in number than SMG projecting neurons. SLG projecting neurons with VIP-IR were greater in number than neurons projecting into the other two glands. Our results indicate the differences in morphology and neuropeptide immunoreactivities among the SCG neurons which may be due to the differences in the neuronal functions and the participation of neurotrophic factors.
The distribution of histamine (HIS)-containing neurons was studied by the avidin-biotin peroxidase complex (ABC) method, and compared with that of the monoaminergic and aromatic L-amino acid decarboxylase-positive neurons (D neurons) in the laboratory shrew (Suncus murinus) brain. In the laboratory shrew brain, HIS-immunoreactive (IR) neurons were strictly confined to the mammillary area. Packed clusters of HIS-IR neurons were localized to four regions: the dorsal and ventral premammillary nucleus, lateral mammillary nucleus and the posterior hypothalamic nucleus. HIS-IR nerve fibers were found mainly in the diencephalon and telencephalon. In particular, dense histaminergic innervation was observed in the preoptic area, bed nucleus of the stria terminalis and suprachiasmatic nucleus. Some HIS-IR nerve fibers were detected in the paraventricular nucleus, anterior hypothalamic nucleus, lateral septal nucleus and cerebral cortex. Few HIS-IR nerve fibers were found in the caudate nucleus, medial septal nucleus, colliculus, hippocampus and locus coeruleus. Comparison of the distribution of HIS-IR neurons with those of monoaminergic neurons and D neurons in the laboratory shrew brain revealed the following findings, which are unique to the laboratory shrew: 1) The distribution of HIS IR neurons was localized to a specific area. 2) Although HIS-IR nerve fibers were densely distributed in the median eminence in the rat brain, only a small number of HIS-IR nerve fibers were observed in this area in the laboratory shrew.
Using behavioral, immunohistochemical and pharmacological studies, we report here that large thoracic burn injuries remotely induce hindpaw hyperalgesia during the healing stage. During 2-3 weeks after thoracic burn injury when the skin was regenerating from the wound, we observed by formalin test that the number of flinching behaviors significantly increased and simultaneously we observed by von Frey test that rats developed mechano-hyperalgesia in the foot. In the dorsal horn of the lumbar spinal cord in burn injured rats, c-Fos expression was significantly augmented after plantar formalin injection. The expression of μ-opioid receptor in burn injured rats was significantly decreased compared with that in sham operated rats. The expression of substance P and CGRP in the lumbar dorsal horn was not different between burn and sham operated animals. We also observed that intrathecal administration of glutamate receptor antagonists (MK801 and CNQX) but not cyclooxigenase-2 antagonist (NS-398) reversed the threshold of von Frey test on the foot up to the control level at 2 weeks after injury. Collectively, we analyzed a new pain model showing foot hyperalgesia after thoracic burn injury and demonstrated that neurotransmission of glutamate was enhanced at the lumbar spinal cord level by immunocytochemistry and intrathecal administration of NMDA and non NMDA antagonists. Although the precise mechanism of how remote hyperalgesia at the healing stage developed in this model remains to be confirmed, substances such as trophic factors released from the regenerating skin may cause systemic hyperalgesia including in the foot.