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PubMed1999

Freezing of testicular tissue as a minced suspension preserves sperm quality better than whole-biopsy freezing when glycerol is used as cryoprotectant.

Frozen-thawed testicular spermatozoa have been used successfully for ICSI, especially in cases of obstructive azoospermia with normal spermatogenesis. Fewer attempts, however, have been made to check whether these rather immature spermatozoa, in a different environment with several other cell types present, have cryobiological requirements other than those of ejaculated spermatozoa. This is the reason why the freezing protocols and cryoprotectants (glycerol) used for freezing testicular tissue are based on experience with semen freezing. This study aimed to assess whether cryosurvival and/or motility was influenced by freezing of testicular tissue either as an intact biopsy or as a shredded tissue suspension, when glycerol was used as cryoprotectant. Freezing of testicular tissue as a suspension preserved motility (type B + C) significantly better than freezing of whole biopsies (9.2% vs. 4.0%). Similar observations have been made for vitality (39.3% vs. 25.4%). Centrifugation on 50% Percoll in order to remove the cryoprotectant resulted in a huge loss of spermatozoa (or late spermatids) and should therefore be especially avoided in cases of testicular failure. On the basis of these observations, mincing of the testicular biopsies before freezing may be advocated. Testicular spermatozoa seem to be better preserved when frozen in suspension, at least when slowly permeating glycerol is used as a cryoprotectant.

Biopsy↗

PubMed1998

The cytoplasmic Cu,Zn superoxide dismutase of saccharomyces cerevisiae is required for resistance to freeze-thaw stress. Generation of free radicals during freezing and thawing.

The involvement of oxidative stress in freeze-thaw injury to yeast cells was analyzed using mutants defective in a range of antioxidant functions, including Cu,Zn superoxide dismutase (encoded by SOD1), Mn superoxide dismutase (SOD2), catalase A, catalase T, glutathione reductase, gamma-glutamylcysteine synthetase and Yap1 transcription factor. Only those affecting superoxide dismutases showed decreased freeze-thaw tolerance, with the sod1 mutant and the sod1 sod2 double mutant being most affected. This indicated that superoxide anions were formed during freezing and thawing. This was confirmed since the sod1 mutant could be made more resistant by treatment with the superoxide anion scavenger MnCl2, or by freezing in the absence of oxygen, or by the generation of a rho0 petite. Increased expression of SOD2 conferred freeze-thaw tolerance on the sod1 mutant indicating the ability of the mitochondrial superoxide dismutase to compensate for the lack of the cytoplasmic enzyme. Free radicals generated as a result of freezing and thawing were detected in cells directly using electron paramagnetic resonance spectroscopy with either alpha-phenyl-N-tert-butylnitrone or 5, 5-dimethyl-1-pyrroline-N-oxide as spin trap. Highest levels were formed in the sod1 and sod1 sod2 mutant strains, but lower levels were detected in the wild type. The results show that oxidative stress causes major injury to cells during aerobic freezing and thawing and that this is mainly initiated in the cytoplasm by an oxidative burst of superoxide radicals formed from oxygen and electrons leaked from the mitochondrial electron transport chain.

Copper↗

PubMedDate not supplied

Freezing impairment of male reproductive behaviors of the freeze-tolerant wood frog, Rana sylvatica.

The wood frog (Rana sylvatica), a temperate-zone anuran that overwinters within the frost zone, is adapted to tolerate the freezing and thawing of its tissues. Because the effects of freezing on complex neurobehavioral function are unknown and because R. sylvatica encounters subfreezing temperatures during its late-winter breeding season, we investigated the reproductive behaviors and physiology of male frogs after freezing (minimum body temperature, -2 degrees C) and postthaw recovery (4 degrees C). In tests simulating conditions at the breeding pool, these frogs, which otherwise behaved normally, exhibited reduced mate-searching effort and fewer assaults on mates and did not amplex females until 16-24 h after thawing. Although amplectic ability was ultimately restored in most frogs, they competed poorly for mates against never frozen controls. Further study suggested that the level of behavioral impairment depends on the severity of the freezing exposure. During freezing, tissues accumulated large quantities of the cryoprotectant glucose and desiccated extensively, responses that promote freezing survival. Freezing also caused marked hydroosmotic and metabolic perturbations, which may have impaired neuro-behavioral function, perhaps by interfering with the processing of audio, visual, and tactile stimuli. Individuals that encounter subfreezing temperatures shortly before arriving at the breeding pools may incur reduced reproductive success.

Acclimatization↗

PubMed1988

Ultrarapid embryo freezing: effect of dissolved gas and pH of the freezing solutions and straw irradiation.

While optimizing the ultrarapid embryo freezing procedure we noted that embryo survival was lowest when gas bubbles formed in the straws. Here we report the influences of gassing the freezing solutions with 5% CO2 in air, degassing the solution, the pH of the medium and straw irradiation on the survival and development in vitro of 2-cell mouse embryos. Embryos were ultrarapidly frozen in medium M2 containing 3 M dimethyl sulphoxide and 0.25 M sucrose. Significantly fewer embryos survived freezing and thawing in gassed solutions. The subsequent development of intact embryos to blastocysts was similar in gassed and non-gassed solutions. Survival and development of embryos frozen in solutions of pH 7.0, 7.6 or 8.0 was similar, but fewer embryos developed to blastocysts after freezing in media at pH 6.0. In straws sterilized by gamma irradiation before use, embryo survival after freezing and thawing was dramatically reduced compared with that seen when non-irradiated straws were used. With non-gassed solutions significantly more gas bubbles formed in irradiated straws than in non-irradiated straws. Although bubble formation was significantly reduced in all straws by using degassed freezing solutions, this did not improve embryo survival. We conclude that under conditions normally used for ultrarapid freezing, dissolved gas and pH have less effect on embryo viability than the use of irradiated straws.

Animals↗

PubMed1991

Comparison of freeze-blowing and funnel-freezing of rat brain for the measurement of cerebral glucose concentration in vivo.

The efficacy of funnel-freezing of rat brain to inactivate metabolic processes and preserve in vivo tissue glucose concentration was validated by comparing the results obtained by funnel-freezing with those obtained with freeze-blowing of brain. The arterial plasma glucose level was clamped at 9 mM in halothane-anesthetized rats to produce identical glucose levels in brain tissue prior to freeze fixation. In funnel-frozen and freeze-blown brains, tissue glucose concentrations were 2.47 +/- 0.05 and 2.47 +/- 0.06 mumol/g (means +/- SEM), respectively. Lactate levels in funnel-frozen brains were slightly but significantly higher than those in freeze-blown brains, i.e., 1.56 +/- 0.05 mumol/g versus 1.30 +/- 0.05 mumol/g (means +/- SEM; p less than 0.05). Regional analysis in funnel-frozen brains revealed that glucose concentrations in superficial and basal brain areas remained approximately equal at 2.30 +/- 0.1 mumol/g and 2.31 +/- 0.09 mumol/g (means +/- SEM), respectively. Our findings indicate that in the anesthetized rat, funnel-freezing of brain is suitable for the measurement of regional in vivo glucose concentrations.

Animals↗

PubMed1990

Immunoelectron microscopy of tissues processed by rapid freezing and freeze-substitution fixation without chemical fixatives: application to catalase in rat liver hepatocytes.

We report on the immunohistochemical demonstration of an enzyme at the electron microscopic level using specimens processed by rapid freezing and the freeze-substitution technique without the use of any chemical fixatives. Fresh rat liver tissue blocks were rapidly frozen by the metal contact method using liquid nitrogen, and were freeze-substituted with acetone without any chemical fixatives at -80 degrees C. Some of the freeze-substituted tissues were embedded in Lowicryl K4M at -20 degrees C; the others were returned to room temperature and embedded in Epok 812 at 60 degrees C. Ultra-thin sections were stained using anti-peroxisomal catalase antibody by the protein A-gold technique. The ultrastructure of the hepatocytes was very well preserved compared with that of conventionally processed tissues. The labeling for catalase was confined to peroxisomes. When the labeling density was compared among freeze-substituted tissues and conventionally processed tissues, that of freeze-substituted and Lowicryl K4M-embedded tissues was the most intense. These results show the usefulness of freeze-substituted tissues for immunohistochemical analysis of cell organelles.

Animals↗

PubMed1993

Freeze-drying of cephalothin sodium: granularly agglomerated crystallization during freezing. I.

Optimal conditions for the crystallization and subsequent freeze-drying of an aqueous cephalothin sodium (CET-Na) solution of supersaturated concentrations to obtain granular crystalline CET-Na have been discussed, as have the qualities of the product thus obtained. In general, CET-Na in supersaturated aqueous solution is barely recrystallized, even when in its frozen stage. Our previous report revealed that when the solution is kept at low temperatures for a long duration, the molecules begin to change structurally to as condensed a state as that of liquid crystals, and such change facilitates a spontaneous nucleation and seed-independent crystal growth in the frozen solution. These findings prompted the authors to investigate optimal CET-Na concentrations and thermal histories during the crystallization and subsequent freeze-drying process without seeding. It has subsequently been found out that optimization of the latter conditions gives granular agglomerated crystalline CET-Na contaminated with neither the amorphous nor the quasi-crystalline form. The optimized conditions are: 25-28% CET-Na concentrations; storage before the freezing process at 0 degrees C for 2 h, and subsequent storage at 20-25 degrees C for 1 h; cooling in the freezing process at a rate not faster than 0.5 degrees C/min; warming of the solution for facilitating crystallization prior to vacuum application for drying at -4 degrees C. Under these conditions, the freeze-dried product of CET-Na in granular form has been successfully obtained in a shorter freeze-drying cycle, exhibiting a faster reconstitution time than those of CET-Na prepared according to seeded crystallization followed by a conventional freeze-drying.

Cephalothin↗

PubMed2002

[Freezing injury of plant and oxidative stress--superoxide generation in freeze-thaw cycles of cabbage leaf tissues].

To test the hypothesis that oxidative stress is involved in inducing freezing injury of plants, nitroblue tetrazolium (NBT) was infiltrated into cabbage leaf tissues before they were frozen. NBT was reduced to formazans in freeze-thaw cycles. The ethanol-extractable mono-formazan was measured by a spectrophotometer, and used as a quantitative indicator of oxidative stress that developed during a freeze-thaw cycle. NBT per se as an oxidant increased the freezing injury slightly. When Me2SO as a cryoprotectant was infiltrated into leaf tissue, it apparently enhanced the freezing tolerance, and this is correlated to its ability in ameliorating oxidative stress, as indicated by the partial inhibition of NBT reduction. The results supported the hypothesis that freezing injury of plants is related to oxidative stress. The experiments indicated that it was possible that the reductant of NBT in the freeze-thaw cycle was superoxide anion free radical.

Brassica↗

PubMedDate not supplied

Multivariate metabolic profiling using 1H nuclear magnetic resonance spectroscopy of freeze-tolerant and freeze-intolerant earthworms exposed to frost.

Individuals of the freeze-tolerant earthworm, Dendrobaena octaedra, and four freeze-intolerant earthworm species (Dendrodrilus rubidus, Aporrectodea icterica, A. caliginosa, and A. longa) were frozen at -2 degree C. Control earthworms were exposed to +2 degree C. 1H nuclear magnetic resonance spectroscopy-based metabolic profiling in combination with multivariate pattern recognition methods (metabonomics) was used to produce a cross-species comparison. Several biochemical changes were detected as a result of freezing in all worm species, including an increase in relative free alanine concentrations, and an apparent conversion of adenosine to inosine. It was also possible to determine a number of biochemical changes that were unique to the freeze-tolerant species, D. octaedra. The most obvious difference was that, although all species showed an increase in glucose concentrations, the increase was largest in D. octaedra, and was coupled with a concomitant decrease in glycogen. This confirms that--like previously studied freeze-tolerant earthworm species--tolerance is effected by rapid glucose production from glycogen reserves. An additional difference noted was that succinate increased in all species on freezing, but the increase was least in D. octaedra. Furthermore there was no lactate accumulation in D. octaedra, whereas three of the other four species accumulated lactate. This indicates that anoxic metabolism was lowest in the freeze-tolerant species.

Animals↗

PubMed1990

Ultrastructure of human basophil leukocytes studied after spray freezing and freeze-substitution.

Normal human blood cells were prepared for transmission electron microscopy by spray freezing, freeze-substitution and embedment in Araldite. Spray freezing was found to damage many cells. Nevertheless, the method developed preserved some leukocytes well and spray frozen, freeze-substituted human basophils showed interesting differences from conventionally prepared cells. Surface vesicles and tubulovesicular structures, extragranular myelin figures, and membranous projections on putative thin membrane-bounded granules were all absent. Preservation of the basophilic granules was clearly improved and their membranes were usually intact. The bulk content of these granules did not look particulate, but showed a very fine texture. Intragranular myelin figure material was well preserved and often enclosed striking lucent areas. Charcot-Leyden crystals were clearly seen in basophils from some donors, always in well preserved granules. A fourth type of material, not seen in conventionally prepared human basophils, was also often observed in the granules. Large lucent vacuoles could be regularly distinguished from basophilic granules. The granules with partly concave outlines, seen in conventionally prepared human basophils to be associated with ill-defined lucent areas and irregularity or projections of the membrane, were absent. In freeze-substituted cells, images suggesting fusion of basophilic granules with each other were seen quite frequently. It is concluded that, while the techniques needed to be improved, rapid freezing and freeze-substitution afford a better understanding of the ultrastructure of human basophil leukocytes.

Adult↗

PubMed1991

Water Relations of Pachysandra Leaves during Freezing and Thawing : Evidence for a Negative Pressure Potential Alleviating Freeze-Dehydration Stress.

The evergreen herb Pachysandra terminalis becomes moderately frost-hardy in winter. The water relations of its frost-hardy leaves were studied during a freeze-thaw cycle. Leaf water potentials, measured by psychrometry at subfreezing temperatures, were identical with those of ice, indicating equilibrium freezing. Microscopic observations showed extracellular freezing of tissue water. As evidenced by thermal analysis, the freezing process starts with the crystallization of a minor volume which was identified as apoplasmic water. The following long-lasting exotherm indicated slow export of water from the protoplasts driven by extracellular crystallization. In partially frozen leaves, the fractions of liquid water were measured at several subfreezing temperatures by nuclear magnetic resonance spectroscopy. They were consistently greater than those calculated from the osmotic potentials of cellular fluid, and the differences increased with decreasing temperature. About 50% of the differences could be abolished by freeze-killing of the leaf and was thus ascribed to the effect of a (negative) pressure reinforcing the osmotic potential. The persistent part of the differences may have reflected a matric component. At -7 degrees C, the absolute values of both potentials were -1.7 megapascals each. The water relations of Pachysandra leaves clearly indicate nonideal equilibrium freezing where negative pressures and matric potentials contribute to the leaf water potential and thus alleviate freeze-dehydration of the tissue.

Journal Article↗

PubMed2004

[Freezing of gait or freezing of quadruped gait].

Parkinson's disease (PD) is frequently associated with freezing of gait. Patients with advanced PD learn to use tricks to relieve freezing of gait. Recently one of our patients informed us that he could overcome freezing of gait by crawling. Since crawling is a sort of quadruped gait that humans develop transiently, we wondered whether quadruped gait is preserved in PD. We recruited 16 patients with PD who had frequent and disabling freezing of gait. Under experimental conditions, after 12 hours without medication, seven patients developed biped freezing of gait. Of these seven patients, only two patients (the patients with the most severe freezing) also developed quadruped freezing of gait. This experiment suggests that bipedal gait is affected early in PD, while quadruped gait is preserved until late in this disease.

Aged↗

PubMed1984

Freeze drying and freeze substitution combined with low temperature-embedding. Preparation techniques for microprobe analysis of biological soft tissues.

An X-ray microanalytical and morphological investigation was carried out on rapidly frozen freeze-dried or freeze-substituted tissues. A comparison was made between different embedding and polymerisation procedures following freeze substitution and freeze drying. The investigation also included an analysis of specimens which had been infiltrated, embedded and polymerised by ultraviolet irradiation at low temperatures with Lowicryl-HM20. The method of freeze drying, followed by embedding and polymerisation at low temperatures in vacuo was found to give satisfactory results, comparable with more tedious and hazardous freeze substitution technique.

Animals↗

PubMed2006

The choice of a suitable oligosaccharide to prevent aggregation of PEGylated nanoparticles during freeze thawing and freeze drying.

In a previous study we have shown that the oligosaccharide inulin can prevent aggregation of poly(ethylene glycol) (PEG) coated plasmid DNA/cationic liposome complexes ("PEGylated lipoplexes") during freeze thawing and freeze drying [Hinrichs et al., 2005. J. Control. Release 103, 465]. By contrast, dextran clearly failed as stabilizer. These results were ascribed to the fact that inulin and PEG are compatible while dextran and PEG are not. In this study the stabilizing capacities of inulin and dextran (of various molecular weights) during freeze thawing and freeze drying of four different types of nanoparticles, each type with different amounts of PEG at their surface, were investigated. Freeze drying and freeze thawing of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/dioleoyl-phosphatidyl-ethanolamine (DOPE) liposomes and egg phosphatidyl choline (EPC)/cholesterol (CHOL) liposomes showed that inulins are excellent stabilizers even for highly PEGylated liposomes while (especially higher molecular weight) dextrans dramatically lost their stabilizing capacity when increasing the degree of PEGylation of the liposomes. The same results were obtained for plasmid DNA/DOTAP/DOPE complexes. Finally, both inulin and dextran could prevent full aggregation of plasmid DNA/polyethylenimine (PEI) complexes independent whether PEI was PEGylated or not. It is concluded that inulins are preferred as stabilizers over dextrans for various types of PEGylated nanoparticles due to their compatibility with PEG.

Cholesterol↗

PubMed1986

Microtubule structure studied by quick freezing: cryo-electron microscopy and freeze fracture.

Microtubules have been quickly frozen and examined by electron microscopy using several techniques: (1) freezing of a thin layer of solution by plunging into cryogen, followed by cryoelectron microscopy of the unstained vitrified samples; (2) freezing by the propane-jet method, followed by freeze fracturing and metal replication. The unstained frozen-hydrated microtubules show a structure in agreement with X-ray diffraction data; they differ from negatively stained particles mainly by the better preservation of cylindrical shape. Secondly, they reveal a supertwist of the profilaments that is not detected reliably by other methods. This allows a determination of the number of protofilaments and the polarity. The structural resolution of unstained microtubules is similar to that of stained ones (about 2-3 nm); it is limited by low contrast and lack of crystalline order. Propane-jet or cryo-block freezing followed by freeze fracturing reveals the structures of the inner and outer surfaces of the microtubule wall at a resolution of 4 nm or better. The outside is dominated by the longitudinal protofilaments whereas on the inside one observes tilted cross-striations. Although the freezing temperatures of the two methods are different (liquid nitrogen or helium) they yield similar results for the case of thin layers of protein solution.

Animals↗

PubMed1982

Quick-freezing and freeze-drying in preparation for high quality morphology and immunocytochemistry at the ultrastructural level: application to pancreatic beta cell.

Quick-freeze fixation and freeze-dry methods were used successfully to obtain ultrastructural localization of insulin in the pancreatic beta cell by the unlabeled antibody-enzyme technique. In unosmicated freeze-fixed and freeze-dried islets, insulin was specifically demonstrated over the dense core of the secretory granules. In osmicated freeze-fixed and freeze-dried islets, insulin antigenicity withstood the osmium tetroxide vapor treatment. In addition, the surrounding ultrastructural resolution of morphologic features was significantly improved, which allowed insulin to be localized not only in secretory granules, but also in intracellular membranous compartments, with a degree of confidence not heretofore possible. Extracellular sites of insulin positivity in the islet were also localized and possible exocytotic activity for showing insulin release was observed.

Animals↗

PubMed1993

Ultrastructure and X-ray microanalysis of epiphyseal growth cartilage of femoral head processed by rapid-freezing and freeze-substitution.

Epiphyseal growth cartilage of the femoral head obtained from Wistar rats was investigated after fixation by a rapid-freezing and freeze-substitution. Liquid helium was used in order to achieve a fast cooling rate without ice-crystal damage during the rapid freezing. Use of the rapid-freezing and freeze-substitution procedure provided better ultrastructural preservation of the chondrocyte than conventional chemical fixation methods. This procedure allowed a more reliable approach to electron probe analysis. X-ray microanalysis of the specimens confirmed that calcium is not detected in the initial matrix vesicles as a result of the freezing process. The results suggest that calcium release from precipitates occurs in the free state without any detectable formation of hydroxyapatite at the initial stage of calcification and that calcium is not tightly bound to the matrix vesicles.

Animals↗

PubMed1993

Ultrastructure of early chick embryo tissues after high pressure freezing and freeze substitution.

Early chick embryos (stages 11-29), or parts thereof, were frozen by the high pressure technique and subjected to a variety of freeze substitution protocols. In addition to hexadecene, unincubated albumen was used as a cryoprotectant to surround the specimens during the freezing event. The quality of freeze ranged from poor to very good in these highly aqueous specimens; very good freeze quality was observed minimally to depths of 30-40 microns. Freeze substitution was carried out with a custom-made aluminum chamber, automatic freeze substitution machine, or a combination of the two, in acetone containing 1) uranyl acetate; 2) acrolein and/or tannic acid; or 3) a sequence of 2), osmium, and glutaraldehyde. Excellent quality of morphology was cryopreserved with all solutions, given optimally cryoimmobilized tissue. Mitotic and pericentriolar microtubules and cytoskeletal elements were cryopreserved with all methods; membranous organelles were better preserved with osmium. Outstanding preservation of morphology of the ground substance of very hydrated embryonic cells, though limited in depth, was accomplished by high pressure cryoimmobilization and cryopreservation, demonstrating that the technique can be successfully applied to highly aqueous tissues.

Albumins↗