"In vivo cryotechnique" for paradigm shift to "living morphology" of animal organs

The morphological study has been one of the major approaches in medical and biological fields. For the last century, the conventional chemical fixation and alcohol dehydration were commonly used as an easy preparation method, but it was frequently pointed out that they usually yield many structural artifacts during their preparation processes. Although both conventional quick-freezing and high-pressure freezing methods, by which animal tissues are resected and frozen for physical fixation,can reduce such structural artifacts, the tissues have to be removed from living animal organs for the freezing. Therefore, such specimens are inevitably exposed to noxious stresses of anoxia and ischemia, exhibiting only dead morphological states of animal tissues without blood circulation. To the contrary, our "in vivo cryotechnique", by which all cells and tissues in animal bodies are cryofixed in vivo, can prevent such artifacts of resected specimens. By means of the cryotechnique, it is now possible to reveal the in vivo morphology of cells and tissues in living animal organs. Actually, it has been already applied to several animal organs, such as kidney, liver, intestine, cerebellum, eye ball, blood vessel, and joint cartilage, and brought new morphological findings, reflecting their physiological significance, which had been difficult to demonstrate by the conventional preparation methods. Moreover, its application to immunohistochemistry has also revealed more precise immunolocalizations of dynamically changing molecules in living animal organs, easily translocated by ischemic stresses and anoxia caused during the tissue resection. The "in vivo cryotechnique" allows us to perform novel morphological investigations of "living" morphological states, and develops new medical and biological fields with "living morphology" during this 21st century.Biomedical Reviews 2004; 15: 1-19

Histological Study and LYVE-1 Immunolocalization of Mouse Mesenteric Lymph Nodes with “In Vivo Cryotechnique”

The “in vivo cryotechnique” (IVCT) is a powerful tool to directly freeze living animal organs in order to maintain biological components in frozen tissues, reflecting their native states. In this study, mesenteric lymph nodes of living mice were directly frozen with IVCT, and we did morphological studies and immunohistochemical analyses on a hyaluronic acid receptor, LYVE-1. In lymph nodes, widely open lymphatic sinuses were observed, and many lymphocytes adhered to inner endothelial cells along subcapsular sinuses. The LYVE-1 was clearly immunolocalized at inner endothelial cells of subcapsular sinuses, as well as those of medullary sinuses. Conventional pre-embedding electron microscopy also showed LYVE-1 immunolocalization along both the apical and basal sides of cell membranes of inner endothelial cells. By triple-immunostaining for LYVE-1, smooth muscle actin, and type IV collagen, the LYVE-1 was immunolocalized only in the inner endothelial cells, but not in outer ones which were surrounded by collagen matrix and smooth muscle cells. Thus, the functional morphology of lymph nodes in vivo was demonstrated and LYVE-1 immunolocalization in inner endothelial cells of subcapsular sinuses suggests hyaluronic acid incorporation into lymph node parenchyma

Fine structure of OPCs observed by SBF-SEM

Oligodendrocyte precursor cells (OPC) arise from restricted regions of the central nervous system (CNS) and differentiate into myelin-forming cells after migration, but their ultrastructural characteristics have not been fully elucidated. This study examined the three-dimensional ultrastructure of OPCs in comparison with other glial cells in the early postnatal optic nerve by serial block-face scanning electron microscopy. We examined 70 putative OPCs (pOPC) that were distinct from other glial cells according to established morphological criteria. The pOPCs were unipolar in shape with relatively few processes, and their Golgi apparatus were localized in the perinuclear region with a single cisterna. Astrocytes abundant in the optic nerve were distinct from pOPCs and had a greater number of processes and more complicated Golgi apparatus morphology. All pOPCs and astrocytes contained a pair of centrioles (basal bodies). Among them, 45% of pOPCs extended a short cilium, and 20% of pOPCs had centrioles accompanied by vesicles, whereas all astrocytes with basal bodies had cilia with invaginated ciliary pockets. These results suggest that the fine structures of pOPCs during the developing and immature stages may account for their distinct behavior. Additionally, the vesicular transport of the centrioles, along with a short cilium length, suggests active ciliogenesis in pOPCs

Molecular cloning and sequencing of cDNA for rat cathepsin H Homology in pro-peptide regions of cysteine proteinases

AbstractA cDNA for rat cathepsin H was isolated and sequenced. The deduced protein comprising 333 amino acid residues is composed of a typical signal sequence (21 residues), a pro-peptide region (92 residues) and a mature enzyme region (220 residues). The amino acid sequence in the pro-peptide region, in particular, residues Phe-(−41) to Ser-(−29) of cathepsin H, is highly homologous to the pro-peptide regions of other cysteine proteinases. This homologous region may play a role in the processing of cysteine proteinases

Methodological Improvements With Conductive Materials for Volume Imaging of Neural Circuits by Electron Microscopy

Recent advancements in electron microscope volume imaging, such as serial imaging using scanning electron microscopy (SEM), have facilitated the acquisition of three-dimensional ultrastructural information of biological samples. These advancements help build a comprehensive understanding of the functional structures in entire organelles, cells, organs and organisms, including large-scale wiring maps of neural circuitry in various species. Advanced volume imaging of biological specimens has often been limited by artifacts and insufficient contrast, which are partly caused by problems in staining, serial sectioning and electron beam irradiation. To address these issues, methods of sample preparation have been modified and improved in order to achieve better resolution and higher signal-to-noise ratios (SNRs) in large tissue volumes. These improvements include the development of new embedding media for electron microscope imaging that have desirable physical properties such as less deformation in the electron beam and higher stability for sectioning. The optimization of embedding media involves multiple resins and filler materials including biological tissues, metallic particles and conductive carbon black. These materials alter the physical properties of the embedding media, such as conductivity, which reduces specimen charge, ameliorates damage to sections, reduces image deformation and results in better ultrastructural data. These improvements and further studies to improve electron microscope volume imaging methods provide options for better scale, quality and throughput in the three-dimensional ultrastructural analyses of biological samples. These efforts will enable a deeper understanding of neuronal circuitry and the structural foundation of basic and higher brain functions

Myelination and axonal electrical activity modulate the distribution and motility of mitochondria at CNS nodes of Ranvier

Energy production presents a formidable challenge to axons as their mitochondria are synthesized and degraded in neuronal cell bodies. To meet the energy demands of nerve conduction, small mitochondria are transported to and enriched at mitochondrial stationary sites located throughout the axon. In this study, we investigated whether size and motility of mitochondria in small myelinated central nervous system axons was differentially regulated at nodes, and whether mitochondrial distribution and motility are modulated by axonal electrical activity. The size/volume of mitochondrial stationary sites was significantly larger in juxtaparanodal/internodal axoplasm than in nodal/paranodal axoplasm. By 3-dimensional electron microscopy, we observed that axonal mitochondrial stationary sites were composed of multiple mitochondria of varying length, except at nodes where mitochondria were uniformly short and frequently absent altogether. Mitochondrial transport speed was significantly reduced in nodal axoplasm when compared to internodal axoplasm. Increased axonal electrical activity decreased mitochondrial transport and increased the size of mitochondrial stationary sites in nodal/paranodal axoplasm. Decreased axonal electrical activity had the opposite effects. In cerebellar axons of the myelin deficient rat, which contains voltage-gated Na(+) channel clusters but lacks paranodal specializations, axonal mitochondrial motility and stationary site size were similar at Na(+) channel clusters and other axonal regions. These results demonstrate juxtaparanodal/internodal enrichment of stationary mitochondria and neuronal activity-dependent dynamic modulation of mitochondrial distribution and transport in nodal axoplasm. In addition, the modulation of mitochondrial distribution and motility requires oligodendrocyte-axon interactions at paranodal specializations

Significance of measurement of tumor marker in primary breast cancer

We investigated a prognosis in the presence or absence of preoperative marker abnormality for 371 cases with primary breast cancer that we experienced in our department this time. 60 (16%) of 371 cases showed the abnormality of the tumor marker and 25 (41.7%) of 60 patients had a recurrence. The positive rate of the marker was 8.1% in CA 15 3, 6.7% in CEA, 4.1% in NCC ST 439, and each rate of recurrence was 56.7%, 48.0%, 33.3%. Rate of recurrence in the negative cases was 12.7%, 13.9, 15.0% respectively and recognized a significant difference statistically (p <0.001) . Of 11 cases (3.8%) shown CA 15 3 abnormal high level, 3 cases (27.2%) had recurrence when we examined in 0 3 metastases to lymph nodes according to markers. 281 cases (96.2%) was normal range in CA15 3. Only 15 cases (5%) had recurrence. It showed a significant difference statistically (p <0.05) . For the cases shown abnormality of the preoperative CA 15 3, careful serial observations are necessary

Flavones Prevent LPS-Induced Muscle Atrophy

Muscle atrophy is a complex process that occurs as a consequence of various stress events. Muscle atrophy-associated genes (atrogenes) such as atrogin-1/MAFbx and MuRF-1 are induced early in the atrophy process, and the increase in their expression precedes the loss of muscle weight. Although antioxidative nutrients suppress atrogene expression in skeletal muscle cells, the inhibitory effects of flavonoids on inflammation-induced atrogin-1/MAFbx expression have not been clarified. Here, we investigated the inhibitory effects of flavonoids on lipopolysaccharide (LPS)-induced atrogin-1/MAFbx expression. We examined whether nine flavonoids belonging to six flavonoid categories inhibited atrogin-1/MAFbx expression in mouse C2C12 myotubes. Two major flavones, apigenin and luteolin, displayed potent inhibitory effects on atrogin-1/MAFbx expression. The pretreatment with apigenin and luteolin significantly prevented the decrease in C2C12 myotube diameter caused by LPS stimulation. Importantly, the pretreatment of LPS-stimulated myoblasts with these flavones significantly inhibited LPS-induced JNK phosphorylation in C2C12 myotubes, resulting in the significant suppression of atrogin-1/MAFbx promoter activity. These results suggest that apigenin and luteolin, prevent LPS-mediated atrogin-1/MAFbx expression through the inhibition of the JNK signaling pathway in C2C12 myotubes. Thus, these flavones, apigenin and luteolin, may be promising agents to prevent LPS-induced muscle atrophy