Nobuhiko Ohno

Histochemical analyses of living mouse liver under different hemodynamic conditions by “in vivo cryotechnique”

Although the morphology and molecular distribution in animal liver tissues have been examined using conventional preparation methods, the findings are always affected by the technical artifacts caused by perfusion-fixation and tissue-resection. Using “in vivo cryotechnique” (IVCT), we have examined living mouse livers with histochemical, immunohistochemical and ultrastructural analyses. In samples prepared by IVCT, widely open sinusoids with many flowing erythrocytes were observed under normal blood circulation, and their collapse or blood congestion was seen in ischemic or heart-arrested mice. In contrast, the sinusoidal cavities were artificially dilated by perfusion-fixation, and collapsed by immersion-fixation and quick-freezing (QF) methods of resected tissues. The immunoreactivity of serum albumin and immunoglobulin G and intensity of periodic acid–Schiff-staining in hepatocytes were well preserved with the QF method and IVCT. Furthermore, following tissue resection, serum proteins were rapidly translocated into hepatocytes as demonstrated by immunoreactions on QF tissues frozen 1 or 5xa0min after resection. Translocation was not observed in IVCT samples, indicating that IVCT could be useful to examine cell membrane permeability of hepatocytes under different pathological conditions. Both dynamic morphology and immunodistribution of soluble components in living mouse livers, reflecting their physiological and pathological states, can be precisely examined by IVCT with higher time-resolution.

Expression of protein 4.1G in Schwann cells of the peripheral nervous system

The membrane‐associated cytoskeletal proteins, including protein 4.1 family, play important roles in membrane integrity, protein targeting, and signal transduction. Although protein 4.1G (4.1G) is expressed ubiquitously in mammalian tissues, it can have very discrete distributions within cells. The present study investigated the expression and distributions of 4.1G in rodent sciatic nerve. Northern and Western blot analysis detected abundant 4.1G mRNA and protein in rat sciatic nerve extracts. Immunohistochemical staining with a 4.1G‐specific antibody and double immunolabeling with E‐cadherin, βIV spectrin, and connexin 32 detected 4.1G in paranodal loops, Schmidt‐Lanterman incisures, and periaxonal, mesaxonal, and abaxonal membranes of rodent sciatic nerve. Immunoelectron microscopy confirmed the immunodistribution of 4.1G in Schwann cells. In developing mouse sciatic nerves, 4.1G was diffusely distributed in immature Schwann cells and gradually localized at paranodes, incisures, and periaxonal and mesaxonal membranes during their maturation. These data support the concept that 4.1G plays an important role in the membrane expansion and specialization that occurs during formation and maintenance of myelin internodes in the peripheral nervous system.

Immunohistochemical detection of hypoxia in mouse liver tissues treated with pimonidazole using “in vivo cryotechnique”

To evaluate hypoxic cells in live mouse liver tissues, immunohistochemistry for protein adducts of reductively activated pimonidazole (PARaPi) was performed using the “in vivo cryotechnique (IVCT)” followed by freeze-substitution fixation. This method was used because cryotechniques have some merits for examining biological events in living animal organs with improved time-resolution compared to conventional perfusion and/or immersion chemical fixation. Pimonidazole was intraperitoneally injected into living mice, and then after various times of hypoxia, their livers were quickly frozen by IVCT. The frozen liver tissues were freeze-substituted in acetone containing 2% paraformaldehyde, and routinely embedded in paraffin wax. De-paraffinized sections were immunostained for PARaPi. In liver tissues of mice without hypoxia, almost no immunostained cells were detected. However, in liver tissues with 30xa0s of hypoxia, some hepatocytes in the pericentral zones were strongly immunostained. After 60xa0s of hypoxia, many hepatocytes were immunostained with various degrees of staining intensity in all lobular zones, indicating different reactivities of pimonidazole in the hepatocytes to hypoxia. At this time, the general immunoreactivity also appeared to be stronger around the central veins than other portal areas. Although many hepatocytes were immunostained for PARaPi in the liver tissues with perfusion fixation via heart, those with perfusion via portal vein were not immunostained. Thus, IVCT is useful to detect time-dependent hypoxic states with pimonidazole treatment in living animal organs.

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.

Immunohistochemical Detection of Phosphorylated Rhodopsin in Light-exposed Retina of Living Mouse with In Vivo Cryotechnique

The purpose of this study is to analyze the time-dependent molecular states of rhodopsin (Rho) phosphorylation in the specimens originating from eyeballs cryoimmobilized in situ in living animals. Whole eyeballs of living mice under various dark- and light-exposure conditions were quickly frozen using the in vivo cryotechnique with isopentane-propane cryogen cooled down in liquid nitrogen (−196C). The frozen whole-mount eyeballs were freeze substituted in acetone containing paraformaldehyde and embedded in paraffin wax. Deparaffinized sections were immunostained with anti-phosphorylated 334Ser Rho (P-Rho334) antibody. Immunoreactivity of P-Rho334 was specifically recognized in the outer segments of mouse retinas exposed to daylight. In the 12-h dark-adapted retinas, P-Rho334 immunoreactivity was completely eliminated. Moreover, in other retinas dark adapted for 12 or 36 hr and then exposed under the safety red light for 2 min, it was still barely recognized. Even in the eyeballs exposed to strong visible light for 10 sec, it was not detected. However, after 30, 60, and 180 sec of visible light exposure, P-Rho334 immunoreactivity was definitely recovered, similar to that under daylight condition. This is a new immunohistochemical approach to visualize the time-dependent Rho phosphorylation of living mice using the in vivo cryotechnique, in which changes could be detected within seconds following exposure to light.

Involvement of follicular basement membrane and vascular endothelium in blood-follicle barrier formation of mice revealed by 'in vivo cryotechnique'

The molecular sieve with size- and charge selectivity in ovarian follicles, the so-called blood-follicle barrier (BFB), was examined during follicular development under physiological conditions to reveal ovarian structures responsible for the BFB by using our in vivo cryotechnique (IVCT). Mouse ovary specimens were prepared with different methods including IVCT, immersion, or perfusion chemical fixation and quick-freezing following resection or perfusion. Their paraffin sections or cryosections were stained with hematoxylin-eosin or immunostained for serum proteins with different molecular weights: albumin, immunoglobulin (Ig) G1 heavy chain, inter-alpha-trypsin inhibitor (I alpha I), fibrinogen, and IgM. Their immunoreactivity was better preserved with IVCT. The immunostaining for albumin was clearly observed in blood vessels, interstitium, and developing follicles, but that of IgG1, I alpha I, or fibrinogen was significantly decreased inside the follicles. IgM was immunohistochemically decreased throughout the interstitium outside blood vessels. The immunoreactivities of IgG1 and IgM, as compared with albumin, were clearly changed along follicular basement membranes and around vascular endothelial cells respectively. These findings indicate that BFB functions throughout follicular development, and the follicular basement membrane and the vascular endothelium could play some significant roles in the permselectivity for such soluble proteins with intermediate and high molecular weight respectively.

Immunolocalization of serum proteins in living mouse glomeruli under various hemodynamic conditions by “in vivo cryotechnique”

Distribution of serum proteins in renal glomeruli is important for histopathology in medical and biological fields, but mechanisms of their passage through glomerular capillary loops (GCL) are still difficult to clarify. We have tried to visualize topographical changes of the serum proteins passing through GCL by “in vivo cryotechnique” in combination with immunohistochemistry. Albumin and immunoglobulin G (IgG), Ig kappa light chain and IgG1 heavy chain were mainly immunolocalized in GCL, but not colocalized with zonula occludens-1 (ZO-1) under normotensive condition. Under heart-arrest condition and in quick-frozen fresh tissues, albumin and kappa light chain were immunolocalized in Bowman’s space, indicating their passage caused by the stoppage of blood supply. However, under acute hypertensive condition, they were more clearly immunolocalized along basement membranes and in the Bowman’s space, indicating their increased passage through GCL. IgG was also more clearly localized in mesangial areas under acute hypertension, compared with that under the normotensive or heart-arrest condition. This study is the first direct visualization for glomerular passage of serum proteins under abnormal hemodynamic conditions by the “in vivo cryotechnique”, and the experimental protocol will be useful for morphofunctional examination of living mouse GCL and immunohistochemical analyses of dynamically changing proteins.

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.

Chondrosarcoma with Myxoid Change: A Study Using a Quick-freezing and Deep-etching Method

A middle-aged Japanese woman visited the Orthopedics Department of Nihon University Nerima Hikarigaoka Hospital complaining of pain in the left hip joint that had started approximately 8 months earlier. Following several examinations, including imaging diagnoses, an incisional biopsy demonstrated a malignant acetabular bone tumor, which was removed and examined by a quick-freezing and deep-etching (QF–DE) method, conventional electron microscopy, and light microscopy. Histologically, the tumor was a chondrosarcoma with marked myxoid changes. An interesting extracellular matrix was observed by the QF–DE method. The myxoid area consisted of a fine meshwork of proteoglycans (PG) without obvious aggrecans, which resembled that of PG usually present in the pericellular matrix of normal cartilage. Thin collagen fibrils with pleated surface structures of regular periodicity were also seen, which were sparsely distributed in wide areas except for the pericellular matrix. These collagen fibrils were of the type that are mainly located in the pericellular side of the territorial matrix in normal cartilage. A myxoid matrix consisting of thin collagen fibrils on the background of pericellular type PG suggested that the myxoid matrix in the chondrosarcoma resembled those of the pericellular and pericellular sides of the territorial matrices in normal cartilage.

Involvement of Follicular Basement Membrane and Vascular Endothelium in Blood-Follicle Barrier Formation of Mice

Blood-follicle barrier (BFB) in ovarian follicles is the molecular sieve selective for size and charge. By utilizing the “in vivo cryotechnique” (IVCT), ovarian structures responsible for the BFB were analyzed during development of follicles under physiological conditions. Immunoreactivity of mouse serum proteins was better preserved with IVCT compared with other conventional methods. Strong immunoreactivity of albumin was detected in blood vessels, interstitium, and developing follicles. There was a clear alteration of the immunostaining intensity of IgG1 between inside and outside of the follicular basement membranes. Immunoreactivity of IgM was significantly changed between inside and outside of the vascular endothelial cells. These results suggest that permselectivity of BFB for soluble proteins with intermediate molecular weights is dependent on the follicular basement membrane and the vascular endothelial cells could play significant roles in the permselectivity for soluble proteins with high molecular weight.