Freeze-substitution: Origins and applications

Shiurba, Robert

doi:10.1016/s0074-7696(01)06019-3

Cited by 37 publications

(24 citation statements)

References 255 publications

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“…Although this value may be changed by increasing the sample size, it should be noted that the values obtained from micrographs generally depend on the analytical methods employed (Coggeshall and Lekan, 1996;Guillery and Herrup, 1997). Additionally, freeze-substitution is not free from morphological artifacts such as aggregation and shrinkage, although these artifacts are less extensive than those obtained with chemical fixation methods (Kellenberger, 1991;Shiurba, 2001). Therefore, we consider the changes in value in this study using the same analytical procedure with IVCr followed by freeze-substitution to have significant meaning, but further studies would be necessary to compare the results obtained by IVCr and those obtained with other methods.…”

Section: Ecs Revealed By Ivcrmentioning

confidence: 99%

Extracellular space in mouse cerebellar cortex revealed by in vivo cryotechnique

Ohno

Terada

Saitoh

et al. 2007

J of Comparative Neurology

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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.

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Section: Ecs Revealed By Ivcrmentioning

confidence: 99%

Extracellular space in mouse cerebellar cortex revealed by in vivo cryotechnique

Ohno

Terada

Saitoh

et al. 2007

J of Comparative Neurology

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“…These previous studies indicate that ovarian histology and the follicular microenvironment could be significantly changed in PCO syndrome and that the BFB could be involved in the functional changes through modulation of the components in follicular fluid. However, the knowledge about such changes in ovarian histology and BFB in the PCO syndrome are still limited, because conventional chemical fixation and dehydration often produces various morphological artifacts and molecular translocation caused by anoxia of tissue resection, artificial perfusion pressures with fixatives and dehydration shrinkage with organic solvents (Kellenberger 1991, Hippe-Sanwald 1993, Shiurba 2001, Zhou et al 2007a, 2007b.…”

Section: Introductionmentioning

confidence: 99%

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

Zhou

Ohno²,

Terada³

et al. 2008

Reproduction

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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-a-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. Reproduction (2008) 136 599-610

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“…Among them, the conventional preparation procedures, such as perfusion-or immersion fixation (IM) with chemical fixatives, often produce morphological artifacts caused by anoxia of tissue resection, artificial perfusion pressures with fixatives, and dehydration shrinkage with organic solvents (Kellenberger 1991, Hippe-Sanwald 1993, Shiurba 2001, Zhou et al 2007. For overcoming these problems, our 'in vivo cryotechnique' (IVCT) has been developed since 1996 to directly clarify functioning morphology and immunodistribution of molecular components in cells and tissues of living animal organs (Ohno et al 1996.…”

Section: Introductionmentioning

confidence: 99%

Involvement of follicular basement membrane and vascular endothelium in blood–follicle barrier formation of mice revealed by ‘in vivo cryotechnique’

Zhou¹,

Ohno²,

Terada³

et al. 2007

Reproduction

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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-a-trypsin inhibitor (IaI), 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, IaI, 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.

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Freeze-substitution: Origins and applications

Cited by 37 publications

References 255 publications

Extracellular space in mouse cerebellar cortex revealed by in vivo cryotechnique

Extracellular space in mouse cerebellar cortex revealed by in vivo cryotechnique

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

Involvement of follicular basement membrane and vascular endothelium in blood–follicle barrier formation of mice revealed by ‘in vivo cryotechnique’

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