Sei Saitoh

Extracellular space in mouse cerebellar cortex revealed by in vivo cryotechnique

Conventional methods of preparing tissue specimens for morphological investigation of the central nervous system suffer from inevitable artifacts caused by anoxia during the processing. In the present study we performed ultrastructural analyses of mouse cerebellar cortex using the in vivo cryotechnique (IVCr), which minimizes ischemic artifacts of target organs through direct cryofixation in vivo. In molecular and Purkinje cell layers of the mouse cerebellum prepared with IVCr, considerably large extracellular spaces (ECS) were detected among cellular profiles and synaptic clefts. The ECS obtained with IVCr without ischemia were larger than those obtained with IVCr after 8‐minute ischemia or a conventional quick‐freezing method with fresh resected tissues (FQF), but did not decrease with IVCr after 30‐second ischemia. By contrast, the parallel fibers observed with IVCr without ischemia were slightly smaller than those after 30‐second ischemia, and significantly smaller than those prepared with IVCr after 8‐minute ischemia or FQF. ECS were frequently preserved around synaptic clefts, although the rest were totally or partially enclosed with closely apposed glial processes. The estimated sizes of the ECS around synaptic clefts did not differ between the opened and enclosed synapses, suggesting that the opened synapses might be temporarily surrounded by glial sheaths dynamically extending or retracting throughout the perisynaptic ECS. These findings indicate IVCr to be useful for some morphological analyses of ECS in the central nervous system. The appreciable ECS around synapses would allow morphological and functional changes of neuronal and glial cells dynamically involved in synaptic remodeling or signal transduction. J. Comp. Neurol. 505:292–301, 2007.

Application of Cryobiopsy to Morphological and Immunohistochemical Analyses of Xenografted Human Lung Cancer Tissues and Functional Blood Vessels

Assessment of tissue specimens obtained with common immersion‐fixation followed by dehydration (IMDH) is affected by artifacts, which hinder precise evaluation of the histology and microenvironment of tumor tissues. The technical characteristics of cryobiopsy and in vivo cryotechnique (IVCT) where target organs are directly cryofixed in vivo are still unknown in practical examinations of tumor histopathology and microenvironment.

Conductive resins improve charging and resolution of acquired images in electron microscopic volume imaging

Recent advances in serial block-face imaging using scanning electron microscopy (SEM) have enabled the rapid and efficient acquisition of 3-dimensional (3D) ultrastructural information from a large volume of biological specimens including brain tissues. However, volume imaging under SEM is often hampered by sample charging, and typically requires specific sample preparation to reduce charging and increase image contrast. In the present study, we introduced carbon-based conductive resins for 3D analyses of subcellular ultrastructures, using serial block-face SEM (SBF-SEM) to image samples. Conductive resins were produced by adding the carbon black filler, Ketjen black, to resins commonly used for electron microscopic observations of biological specimens. Carbon black mostly localized around tissues and did not penetrate cells, whereas the conductive resins significantly reduced the charging of samples during SBF-SEM imaging. When serial images were acquired, embedding into the conductive resins improved the resolution of images by facilitating the successful cutting of samples in SBF-SEM. These results suggest that improving the conductivities of resins with a carbon black filler is a simple and useful option for reducing charging and enhancing the resolution of images obtained for volume imaging with SEM.

Recent Development of In Vivo Cryotechnique to Cryobiopsy for Living Animals

Various microscopic methods have been used to analyze the morphology and molecular distribution of cells and tissues. Using conventional procedures, however, ischemic or anoxic artifacts are inevitably caused by tissue-resection or perfusion-fixation. The in vivo cryotechnique (IVCT) was developed to overcome these problems, and was found to be useful with light microscopy for analyses of the distribution of water-soluble molecules without anoxic effects at high time resolution. But there are limitations to the application of IVCT, such as exposure of target organs of living small animals and immunoreactivity of lipid-soluble molecules owing to freeze-substitution with acetone. Recently, a new cryotechnique called ?cryobiopsy? has been developed, which enables one to obtain tissue specimens of large animals including humans without ischemia or anoxia, and has almost the same technical advantages as IVCT. Both IVCT and cryobiopsy complement other live-imaging techniques, and are useful for not only the morphological observation of cells and tissues under normal conditions, but also the preservation of all components in frozen tissue specimens. Therefore, morphofunctional information in vivo would be obtained by freeze-substituion for light or electron microscopy, and also by other analytical methods, such as freeze-fracture replication, X-ray microanalyses, or Raman microscopy. Considering the merits of both IVCT and cryobiopsy, their application should be expanded into other microscopic fields and also from experimental animal studies to clinical medicine.

Essential Function of Protein 4.1G in Targeting of Membrane Protein Palmitoylated 6 into Schmidt-Lanterman Incisures in Myelinated Nerves

ABSTRACT Protein 4.1G is a membrane skeletal protein found in specific subcellular structures in myelinated Schwann cells and seminiferous tubules. Here, we show that in the mouse sciatic nerve, protein 4.1G colocalized at Schmidt-Lanterman incisures (SLI) and the paranodes with a member of the membrane-associated guanylate kinase (MAGUK) family, membrane protein palmitoylated 6 (MPP6). Coimmunoprecipitation experiments revealed that MPP6 was interacting with protein 4.1G. In contrast to wild-type nerves, in 4.1G knockout mice, MPP6 was found largely in the cytoplasm near Schwann cell nuclei, indicating an abnormal protein transport. Although the SLI remained in the 4.1G knockout sciatic nerves, as confirmed by E-cadherin immunostaining, their shape was altered in aged 4.1G knockout nerves compared to their shape in wild-type nerves. In the seminiferous tubules, MPP6 was localized similarly to protein 4.1G along cell membranes of the spermatogonium and early spermatocytes. However, in contrast to myelinated peripheral nerves, the specific localization of MPP6 in the seminiferous tubules was unaltered in the absence of protein 4.1G. These results indicate that 4.1G has a specific role in the targeting of MPP6 to the SLI and the assembly of these subcellular structures.

Involvement of dynamin-2 in formation of discoid vesicles in urinary bladder umbrella cells

Umbrella cells (UCs) of the epithelium of the urinary bladder have the capacity to control bladder volume by regulating exocytosis/endocytosis of their intracellular discoid vesicles (DVs). Dynamin (Dyn) is a GTPase that promotes endocytic processes through scission of cell membranes. We have examined whether Dyn2, the most abundant Dyn form, is expressed in UCs and contributes to their endocytic actions. A specific antibody against Dyn2 was used to localize Dyn2 in human and rodent UCs by immunohistochemistry. To clarify the functional roles of Dyn2, mouse bladders were treated with a Dyn-GTPase inhibitor, dynasore, and its effects on their UC structure were assessed. Since uropathogenic Escherichia coli can be encased into UCs during infection, we used immunohistochemistry to determine whether bacteria-encasing compartments in the infected UCs were also enriched with Dyn2. Light microscopy showed that Dyn2 was abundantly expressed in UCs, especially near the apical cytoplasmic regions. By immunoelectron microscopy, Dyn2 was found on and around DV membranes in UCs. Ultrastructural analysis with a quick-freezing and deep-etching method confirmed these findings and revealed the existence of distinct Dyn2-bound microfilaments in close association with DV membranes. Dynasore treatment of bladders markedly reduced the number of DVs in UCs. In infected UCs, E. coli was encased in compartments enriched in Dyn2. Therefore, Dyn2 is highly enriched in UCs and mostly associated with membranes of DVs and microfilaments in the UCs. Pretreatment of bladders with dynasore inhibits E. coli invasion of UCs. Dyn2 thus contributes to the structural integrity of DVs and to the endocytic activity of UCs.

Interaction of Membrane Skeletal Protein, Protein 4.1B and p55, and Sodium Bicarbonate Cotransporter1 in Mouse Renal S1-S2 Proximal Tubules

Our recent studies demonstrated the localization of protein 4.1B, a member of the 4.1 skeletal membrane proteins, to the basolateral membranes of the S1-S2 renal proximal tubules. In the present studies, we investigated the presence of binding partners that could form a molecular complex with the 4.1B protein. Immunohistochemistry revealed the localization of p55, a membrane-associated guanylate kinase, and the sodium bicarbonate cotransporter1 (NBC1), to the basolateral membrane domain of S1-S2 in mouse renal proximal tubules. Using immunoprecipitation of kidney lysates with anti-p55 antibody, a positive band was blotted with anti-4.1B antibody. GST fusion proteins including the NBC1 and 4.1B regions were confirmed to bind with each other by electrophoresis after mixing. Both NBC1- and 4.1B-specific bands were detected in renal protein mixtures immunoprecipated by either anti-4.1B- or NBC1-specific antibodies. It is likely that NBC1, 4.1B, and p55 form a molecular complex in the basolateral membrane of the kidney S1-S2 proximal tubules. We propose that the 4.1B-containing membrane skeleton may play a role in regulating the Na+ and HCO3 - reabsorption in S1-S2 proximal tubules.

Immunoreactivity of Glutamate in Mouse Retina Inner Segment of Photoreceptors With In Vivo Cryotechnique

The purpose of this study was to clarify a previously controversial issue concerning glutamate (Glu) immunoreactivity (IR) in the inner segment (IS) of photoreceptors by using in vivo cryotechnique (IVCT) followed by freeze substitution (FS), which enabled us to analyze the cells and tissues reflecting living states. Eyeballs from anesthetized mice were directly frozen using IVCT. The frozen tissues were processed for FS fixation in acetone containing chemical fixatives, and embedded in paraffin. Deparaffinized sections were immunostained with an anti-Glu antibody. The strongest Glu-IR was obtained in the specimens prepared by FS with paraformaldehyde or a low concentration of glutaraldehyde, whereas no Glu-IR was obtained without the chemical fixatives. The Glu was immunolocalized in the IS, outer and inner plexiform and ganglion cell layers. Thus, the immunolocalization of Glu in the IS was clearly demonstrated using IVCT.

Permselectivity of blood-follicle barriers in mouse ovaries of the mifepristone-induced polycystic ovary model revealed by in vivo cryotechnique

Despite the potential association of polycystic ovary (PCO) syndrome with hemodynamic changes, follicular microenvironment and the involvement of blood follicle barriers (BFB), a histopathological examination has been hampered by artifacts caused by conventional preparation methods. In this study, mouse ovaries of a mifepristone-induced PCO model were morphologically and immunohistochemically examined by in vivo cryotechnique (IVCT), which prevents those technical artifacts. Ovarian specimens of PCO model mice were prepared by IVCT or the conventional perfusion fixation after s.c. injection of mifepristone. Their histology and immunolocalization of plasma proteins, including albumin (molecular mass, 69 kDa), immunoglobulin G (IgG, 150 kDa), inter-alpha-trypsin inhibitor (ITI, 220 kDa), fibrinogen (340 kDa), and IgM (900 kDa), were examined. In the PCO model, enlarged blood vessels with abundant blood flow were observed in addition to cystic follicles with degenerative membrana granulosa. The immunolocalization of albumin and IgM in the PCO model were similar to those in normal mice. Albumin immunolocalized in the blood vessels, interstitium or follicles, and IgM was mostly restricted within the blood vessels. In contrast, immunolocalization of IgG, ITI, and fibrinogen changed in the PCO model. Both IgG and ITI were clearly blocked by follicular basement membranes, and hardly observed in the membrana granulosa, though fibrinogen was mostly observed within blood vessels. These findings suggest that increased blood flow and enhanced selectivity of molecular permeation through the BFB are prominent features in the PCO ovaries, and changes in hemodynamic conditions and permselectivity of BFB are involved in the pathogenesis and pathophysiology of PCO syndrome.

Immunohistochemical Distribution of Serum Proteins in Living Mouse Heart with In Vivo Cryotechnique

In vivo cryotechnique (IVCT), which immediately cryofixes target organs in situ, was used to clarify the morphological features of beating heart tissue of living mice. IVCT was performed for diastolic heart tissue under the condition of monitoring with electrocardiogram (ECG). Other mouse hearts were prepared with conventional perfusion-fixation (PF-DH) or immersion-fixation followed by dehydration (IM-DH), and quick-freezing of resected heart tissues (FQF). Immunolocalizations of albumin, immunoglobulin G1 (IgG1), intravenously injected bovine serum albumin (BSA), and connexin 43 were examined after different intervals of BSA injection. In the case of IVCT, the exact stop time of beating mouse hearts was recorded by ECG, and open blood vessels with flowing erythrocytes were observed with less artificial tissue shrinkage than with conventional preparation methods. Both albumin and BSA were well preserved in intercalated discs and t-tubules of cardiomyocytes in addition to blood vessels and interstitial matrices. IgG1 was immunolocalized in interstitial matrices of heart tissues in addition to their blood vessels. At 4 hr after BSA injection, it was immunolocalized in the intercalated discs of cardiomyocytes and lost later at 8 hr. IVCT should prove to be more useful for the morphofunctional examination of dynamically changing heart tissue than conventional preparation methods.