大野 伸彦

The transport mechanism of soluble molecules throughout the interstitial matrix is closely associated with human tumor behavior in vivo. However, the examination of soluble components in histological architectures has been hampered by artifacts caused during conventional tissue preparation. In this study, the immunodistribution of intrinsic and extrinsic serum components in tumor tissues was examined in xenografted human tumor cells using 'in vivo cryotechnique' (IVCT) and cryobiopsy, where target tissues are directly cryofixed in vivo. Human lung cancer cells were subcutaneously injected into the dorsal flank of nude mice, and paraffin sections and cryosections of produced tumors were prepared with different methods. Immunolocalization of serum proteins, including albumin, immunoglobulin G (IgG) and IgM, as well as intravenously injected bovine serum albumin (BSA) was examined. Their immunodistribution was more clearly observed in the interstitium by both IVCT and cryobiopsy than conventional methods. IgM was immunolocalized within blood vessels, whereas albumin and IgG were observed in the tumor interstitium. Moreover, intravenously injected bovine serum albumin exhibited leakage from the blood capillaries into surrounding connective tissues in 24 h, but it gradually diffused to the interstitium of the tumor masses during 3 days. These results suggest that molecular leakage from blood capillaries varies significantly in different areas of developing tumors, and that small serum proteins, but not large ones, were abundantly immunolocalized in the tumor interstitium. Both IVCT and cryobiopsy were found to be useful for immunohistochemical studies of soluble molecules in tumors with blood circulation, and may therefore be helpful for further histopathological analyses.

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.

Immunohistochemical analyses on local distributions of serum proteins in living mouse kidneys are usually difficult to examine with conventional preparation methods. By using our "in vivo cryotechnique" combined with freeze-substitution, we have checked immunolocalizations of the serum proteins in nephrons of bovine serum albumin (BSA)-overload mice, and compared them with those obtained by the conventional preparation methods. In two days of daily BSA-injected mice, the immunolocalization of BSA could be observed in Bowman's space and urinary tubules with their overt proteinuria, where another endogenous mouse albumin was similarly immunolocalized. The leakage of BSA and mouse albumin in Bowman's space and their reabsorption into proximal tubules were detected in 55% of nephrons, where no leakage of immunoglobulin G1 (IgG1) was detected. However, the leakage of IgG1, in addition to BSA and mouse albumin, was detected in the other nephrons. By carefully examining immunolocalizations of BSA and IgG1, they were obviously different from those obtained by the conventional preparation methods without normal blood circulation into the kidneys. The immunolocalizations of both BSA and mouse serum proteins could be directly analyzed with the "in vivo cryotechnique", suggesting that functional damage to glomerular filtration barriers are different at early stages of the BSA-overload mouse model, depending on each nephron of living mice.

When all biological materials in cells and tissues of living animal organs are quickly and promptly frozen, immunolocalization of their components and structural features in situ is necessary to understand their in vivo functioning states. However, these direct morphological analyses were difficult to achieve by conventional chemical fixation methods during the last century. A new cryofixation method, named the "in vivo cryotechnique", in which the normal blood circulation in living animals is always retained at the moment of freezing, has become a powerful tool to visualize the real native morphology of cells and tissues with functional meaning. The "in vivo cryotechnique" can usually be combined with a wide range of subsequent preparation techniques, and can thereby enable us to perform various direct analyses on biological samples, reflecting the physiological functions of living animal organs.

The in vivo injection of cadmium (Cd) was reported to induce blood-testis barrier disruption, and assumed to be an experimental model to examine junctional structures in seminiferous tubules. The purpose of this study is to investigate time-dependent changes of albumin permeability in the normal or Cd-treated mouse testis by our "in vivo cryotechnique" with immunohistochemistry, reflecting tight junctional (TJ) barriers of Sertoli cells. The albumin in the seminiferous tubules was firstly immobilized by the cryotechnique, in which normal blood circulation was always kept. The cryofixed testicular tissues were then processed for freeze-substitution, and embedded in the paraffin wax. Serial sections were immunostained by anti-mouse albumin antibody with peroxidase immunostaining, and also stained with hematoxylin-eosine (HE) for morphological observation. In normal seminiferous tubules, the immunoreaction products were localized around peritubular myoid cells and between Leydig cells, as well as in blood vessels. They were also localized as arch-like patterns around some spermatogonia in basal compartments of seminiferous tubules. Twenty-four and 48 hrs after Cd-treatment, some enlarged spaces and vesicular formations in the seminiferous epithelium were observed on the HE-stained sections. The albumin immunolocalization was detected not only in the basal compartments, but also in the adluminal compartments between Sertoli cells and germ cells. Thus, the structural disruptions of inter-Sertoli TJ barriers could be clearly demonstrated by the "in vivo cryotechnique".

The purpose of this study is to visualize topographical changes of serum proteins, albumin and immunoglobulin, passing through mouse glomerular capillary loops and their reabsorption in renal proximal tubules by immunohistochemistry in combination with our "in vivo cryotechnique". The "in vivo cryotechnique" was performed on left mouse kidneys under normotensive, experimentally acute hypertensive and heart-arrest conditions. The cryofixed tissues by the technique were routinely processed for freeze-substitution. Serial deparaffinized sections were stained with hematoxylin-eosine and immunostained with anti-mouse albumin, immunoglobulin G (IgG), kappa or lambda light chain and IgG1 heavy chain antibodies. Under the normotensive and heart-arrest conditions, albumin and IgG were clearly immunolocalized in blood vessels and slightly in apical cytoplasmic parts of some proximal tubules. Under the acute hypertensive condition, the albumin and kappa or lambda light chains, but not IgG1 heavy chain, were strongly immunolocalized in the apical cytoplasm of almost all proximal tubules. This study is the first in vivo visualization for glomerular passage of serum proteins and their transtubular absorption. Thus, the "in vivo cryotechnique" with freeze-substitution can be used for clarifying not only the functional morphology of living animal cells, but also in situ immunohistochemical localization of their components.

The purpose of this study was to examine time-dependent topographical changes of leaking proteins from blood vessels in the mouse cerebellum to assess the effect of normal blood circulation on the blood-brain barrier (BBB). The distribution of leaking serum proteins was immunohistochemically examined by various cryotechniques including our "in vivo cryotechnique". The cryofixed cerebellar tissues were processed for the freeze-substitution method, and finally embedded in the common paraffin wax. Serial de-paraffinized sections were immunostained by anti-mouse immunoglobulin-G (IgG) or albumin antibody. By combination of the "in vivo cryotechnique", in which normal blood flow into the cerebellum was always kept in vivo, with the freeze-substitution method, serum IgG and albumin were clearly localized inside of cerebellar blood vessels. To examine abnormal leakage of blood vessels as a model of anoxia, some cerebellar tissues were partially removed from brains in the mouse skull and quickly frozen in the isopentane-propane within a minute. In such resected cerebellar tissues, serum IgG and albumin were diffusely immunostained in large areas around the blood capillaries, probably because of easy leakage of the serum components through the immediately changed BBB. To the contrary, no serum protein could be identified outside blood capillaries under living conditions of the anesthetized mice. The present combination method, both "in vivo cryotechnique" and freeze-substitution, for immunohistochemistry enabled us to examine the in vivo localization of serum components in mouse brains due to alteration of the BBB. (C) 2004 Elsevier B.V. All rights reserved.