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PubMed1990

Freeze-fracture scanning electron microscopy and comparative freeze-etching study of parallel fiber-Purkinje spine synapses of vertebrate cerebellar cortex.

The three dimensional surface morphology, topographical arrangement and intramembrane features of parallel fiber-Purkinje spine synapses of teleost fishes and Swiss albino mice cerebellar cortex have been complementarily studied with the scanning electron microscope (SEM) using the freeze-fracture method and with transmission electron microscope (TEM) by means of freeze-etching replica and thin-sectioning techniques. The SEM fractographs revealed for the first time the outer surface morphology of non-synaptic segments and synaptic varicosities of parallel fibers, which showed clusters of spheroidal synaptic vesicles joined by an amorphous substance. Freeze-etching preparations showed an 'en face' view of P face parallel fiber varicosity (presynaptic P face) characterized by a depressed synaptic active zone with exocytotic vesicle sites, round IMPs (7.5-15 nm), and rod particles (38.5-53 nm in length). Conversely the presynaptic E face exhibited a smooth surface, randomly distributed pits and very low density distribution of round IMPs. The P face of Purkinje spine membrane (postsynaptic P face) showed high density distribution of round or ovoid IMPs, 8-18.5 nm in diameter, and rod IMPs up to 79.2 nm in length. IMPs were also observed beneath the postsynaptic membrane (subsynaptic IMPs) corresponding to the localization of postsynaptic density. The E face of Purkinje spine membrane (postsynaptic E face) showed aggregation of round IMPs, 5.6-17.4 nm in diameter, at the synaptic active zone. Some of these IMPs, particularly the large ones, may morphologically represent the glutamate-receptor ion channels complex. The subsynaptic IMPs may correspond to protein kinases. Granular material and fine filaments were observed in the postsynaptic density. A better insight into the topographical relationship between pre- and postsynaptic structures was obtained of flat and invaginated spine synapses and beside a more realistic view of their glial ensheathment. The SEM fractographs and freeze-etching images were compared with corresponding TEM thin sections for orientation and proper identification of pre- and postsynaptic structures.

Animals↗

PubMed1985

Freeze-fracture studies of human blood platelets activated by thrombin using rapid freezing.

In this study the influence of thrombin activation on human blood platelets has been followed by freeze-fracturing electron microscopy using rapid freezing in order to catch the initial changes in shape and the morphological alterations during the process of exocytosis of secretory granules. We found that isolation of the platelets by itself leads to some degree of shape change, which made it impossible to study the resting discoid platelet by rapid freezing. Activation of the platelets by thrombin induced dilation of the "surface connecting system (SCS)" with formation of large vacuoles as a result of fusion of the secretory granules with SCS. No intermediary fusion stages or structures were observed even using rapid freezing. Volcano-like protrusions and the corresponding complementary pits were seen at the SCS. These structures were interpreted by us as fractures through protoplasmic channels crossing the SCS. These channels originate during the swelling of the SCS as a result of the fusion of secretory granules with the SCS.

Blood Platelets↗

PubMed1996

[The freeze-etching and freeze-fracturing technics and their use for studying cellular membranes in the central nervous system].

In the present review, both subkinds of the method for membrane splitting, namely freeze-etching and freeze-fracturing techniques, are presented. After a brief historical survey, a mechanism of their action is described. Special attention is paid to the consecutive stages in applying these techniques--fixation, fracturing, etching, replication, cleaning and observation. A new idea for a better cleaning of the replicas and an explanation of the different occurrence of intramembranous particles on the membrane fracture faces are proposed. Finally, the application of the freeze-etching and freeze-fracturing techniques to visualize the intramembranous structure of membranes in the central nervous system is outlined.

Animals↗

PubMed1998

Freeze-thaw immobilization of liposomes in chromatographic gel beads: evaluation by confocal microscopy and effects of freezing rate.

Biological membranes immobilized in chromatographic gel beads constitute a multifunctional affinity matrix. Membrane protein-solute interactions and drug partitioning into the lipid bilayers can conveniently be studied. By the use of confocal laser-scanning microscopy (CLSM) the distribution of immobilized model membranes in the beads has been visualized for the first time. Freeze-thaw-immobilized liposomes in Superdex 200 gel beads were situated in a thick shell surrounding a liposome-free core. The amount of phospholipids immobilized by freeze-thawing was dependent on the temperature in the cooling bath and the type of test tube used. A bath temperature of -25 degrees C gave higher immobilization yield than freezing at -75 or -8 degrees C did. Freeze-thawing in the presence of liposomes did not affect the gel bead shape or the refractive index homogeneity of the agarose network of the beads, as shown by confocal microscopy.

Chromatography↗

PubMed2000

Fine structure of transforming-type granules in mucous cells in the early postnatal rat parotid gland when processed by rapid freezing followed by freeze-substitution fixation.

The present study was designed to clarify the more precise ultrastructural feature of granules, especially mucous granules in the early postnatal rat parotid gland by using rapid freezing followed by freeze-substitution fixation. The parotid gland of Wistar rats (aged 0-10 days) was removed under anesthesia and immediately underwent cryofixation followed by substitution with osmium tetroxide. After fixation, the samples were embedded in Epon-Araldite, cut into ultrathin section, and then examined by transmission electron microscopy. Electron microscopy showed that the mucous granules of samples treated by freeze-substitution fixation had low electron density and were almost spherical in shape with a clear limiting membrane. By Day 5, granules that were a little more electron dense than mucous granules, granules with a more electron dense portion at their periphery, and heterogeneous granules with an internal highly electron dense portion were found. Moreover, cells containing both homogeneous granules with a high electron density similar to that of mature serous granules and heterogeneous granules were observed. These findings demonstrated that the morphology of the transforming-type mucous granules by chemical fixation in the previous study was an artifact and, as a result, strongly suggested that on the sequential morphological changes of transitional mucous/serous granules by rapid freezing method in the present study, the mucous cells in the early postnatal rat parotid gland undergo transformation to serous cells.

Animals↗

PubMed2000

Effect of vascular freezing on the histopathology of dissected small vessels in the rat: vascular freezing does induce intimal hyperplasia in arteries and veins.

Intimal hyperplasia is the primary response of a vessel wall after injury. It may be the single most significant factor affecting long-term patency. The purpose of this study was to find out whether freeze injury, inflicted on rat microvessels, would be followed by intimal hyperplasia. Toward this aim, we exposed the superficial femoral vessels in the rat. On one side they were frozen using liquid nitrogen spray. The other side was dissected as the control. Vessel segments, harvested immediately and after 1, 2, 3, and 5 months, were studied light microscopically for the occurrence and content of intimal hyperplasia. In the arteries a considerable intimal hyperplasia was found within a 4-week interval, persisting for at least 5 months, as a result of the freeze injury. In the veins, the intimal hyperplasia was much less marked but was nevertheless demonstrable. These findings are not in agreement with earlier studies, in which freezing of injured rat microarteries with liquid nitrogen spray was followed by complete regeneration of the vessel wall, without intimal hyperplasia taking place. The factors contributing to these differences are discussed. It is concluded that freezing of a vessel wall is followed by intimal hyperplasia, which is part of the normal healing process.

Animals↗

PubMed1982

Effects of freezing and thawing and storage by freezing on the biological and morphological properties of strain K-9 of Klebsiella pneumoniae and its variants.

Using strain K-9 of Klebsiella pneumoniae and its variants A, B and C, which possess a large, small, or extra-small capsule or are unencapsulated, and exhibit high, middle, or low mouse virulence or avirulence in the mouse, respectively, the effect of freezing and thawing on the protection and storage by freezing in 20% skim milk solution on their biological and morphological properties was investigated. Experimental results showed that these organisms were sensitive against freezing and thawing in the order variant C, the parent strain, variants A and B. 20% skim milk solution was the most favourable for survival of the organisms compared to trypticase soy broth and 10% glycerol. Their mouse virulence was similarly sensitive to freezing and thawing experiments although no alteration of their biochemical properties was observed. When the organisms were suspended in 20% skim milk solution and stored for 2 years at -70 degrees C, their mouse virulence was not altered. When morphological features were tested electron microscopically, cellular injuries such as vacuola or fissions of cell walls were observed in the variants but not in the parent strain. However, it was evident that conversion to unencapsulation from encapsulation occurred at a high frequency in the parent strain and in variant A when determined by the colonial morphologies in soft-agar medium.

Animals↗

PubMed2002

Freeze-drying of tert-butanol/water cosolvent systems: a case report on formation of a friable freeze-dried powder of tobramycin sulfate.

A case study is presented in which a tert-butanol (TBA)/water cosolvent system was found to be a useful means of producing freeze-dried tobramycin sulfate that readily forms a loose powder upon agitation in a specialized application in which a critical quality attribute is the ability to pour the sterile powder from the vial. Both formulation and processing variables are important in achieving acceptable physical properties of the cake as well as minimizing residual TBA levels. Liquid/liquid phase separation was observed above critical concentrations of both drug and TBA, resulting in a two-layered lyophilized cake with unacceptable appearance, physical properties, and residual TBA levels. However, the choice of tobramycin sulfate and TBA concentrations in the single-phase region of the phase diagram resulted in a lyophilized solid that can readily be poured from vials. Crystallization of TBA before drying is critical to achieving adequately low residual TBA levels, and this is reflected in the effect of thermal history of freezing on residual TBA levels, where rapid freezing results in incomplete crystallization of TBA and relatively high levels of residual solvent. Annealing at a temperature above T'(g) of the system after an initial freezing step significantly reduces the level of residual TBA. Secondary drying, even at increased temperature and for extended times, is not an effective method of reducing residual TBA levels.

Anti-Bacterial Agents↗

PubMed1984

Ciliated and microvillous structures of rat olfactory and nasal respiratory epithelia. A study using ultra-rapid cryo-fixation followed by freeze-substitution or freeze-etching.

The olfactory epithelium of the Sprague-Dawley rat showed structures which indicate that freeze-substitution after ultra-rapid cryo-fixation is a better method for its preservation than conventional fixation techniques. A new feature is that matrices of the distal parts of olfactory cilia range in their staining intensity from very dense to electron-lucent. Outlines of structures are smooth and membrane features can be clearly seen. The textures of mucus from olfactory and respiratory epithelia are distinctly different after freeze-fracturing and deep-etching following cryo-fixation. Olfactory cilia show no microtubule-attached axonemal structures. Cross-sectional diameters are smaller after freeze-substitution than after freeze-fracturing. Intramembranous particle densities are lower in nine regions of three cell types in cryo-fixed olfactory and respiratory epithelia than in those chemically fixed and cryoprotected. The fracture faces of membranes from etched, cryo-fixed cells have holes, a result which probably accounts for differences in particle density between cryo-fixed and chemically-fixed, cryo-protected cells. Particle diameters are usually the same using both methods. Densities of intramembranous particles and particles plus holes are highest in supporting cell processes, followed by endings and cilia of olfactory receptor cells, and are lowest in respiratory cilia. Particle densities at outer and inner surfaces are higher than those in either fracture face. Outer surfaces show a good correlation from region to region with densities summated over both fracture faces.

Animals↗

PubMed1994

Post-freeze recovery of peripheral nerve function in the freeze-tolerant wood frog, Rana sylvatica.

We investigated the restoration in peripheral nerve function and simple neurobehavioral reflexes in the freeze-tolerant wood frog (Rana sylvatica). Thirty-two specimens, allowed to freeze for 39 h and ultimately cooled to -2.2 degrees C, were sampled at various time intervals up to 60 h after thawing at 5 degrees C was initiated. The sciatic nerves of treated frogs were initially unresponsive to stimulation, but usually regained excitability within 5 h. Except for a slight reduction in nerve excitability, characteristics of the compound action potentials of treated frogs were indistinguishable from those of control frogs. Recovery times for the hindlimb retraction and righting reflexes were 8 h and 14 h, respectively. Concentrations of the cryoprotectant glucose increased 8.2-fold in the sciatic nerve and 10.5-fold in the underlying semimembranosis muscle of treated frogs, and remained elevated for at least 60 h after thawing was initiated. These organs lost 47.2% and 15.9%, respectively, of their water during freezing, but were rehydrated within 2 h of the onset of thawing. The accumulation of glucose and the withdrawal of tissue water apparently are cryoprotective responses which enable this species to survive freezing.

Action Potentials↗

PubMed1992

Enzyme histochemistry and immunohistochemistry with freeze-dried or freeze-substituted resin-embedded tissue.

Freeze-drying or freeze-substitution, combined with low-temperature resin-embedding, represents a new approach to the optimum preservation of tissue for enzyme histochemistry and immunohistochemistry. This method, which avoids tissue fixation, combines excellent tissue morphology with the preservation of enzyme activity and immunoreactivity and allows high-resolution enzyme histochemical and immunohistochemical studies to be performed. The activity of a wide range of enzymes can be demonstrated in sections of freeze-dried or freeze-substituted resin-embedded tissue. Enzymes are retained in situ with high activity, accurate localization and no diffusion. Immunohistochemical studies can also be performed on resin sections, and antigens--especially labile antigens--are immobilized in situ without denaturation and can be demonstrated with high sensitivity and accurately localized. This method allows the localization and distribution of enzymes and antigens to be studied in relation to excellent histological and cytological detail.

Animals↗

PubMedDate not supplied

Simplification of the methods for adding and removing glycerol during freeze-preservation of human red blood cells with the high or low glycerol methods: biochemical modification prior to freezing.

Simple methods have been developed for adding and removing glycerol during freeze-preservation with 20 per cent W/V glycerol at minus 150 C, or with 40 per cent W/V glycerol at minus 80 C. A one-step method with a 35 per cent W/V glycerol solution is used to prepare 20 per cent W/V glycerolized red blood cells, and a two-step method with a 57 per cent W/V glycerol solution is used to prepare 40 per cent W/V glycerolized red blood cells. The systems for washing glycerolized red blood cells have been simplified. This method consists of dilution of the thawed glycerolized red blood cells prior to recovery, followed by on-line dilution of these red blood cells with wash solutions during continuous flow centrifugation. This can be done in any of three commercially available washing systems, and they all use the same sodium chloride solutions. For the 40 per cent W/V glycerolized red blood cells, this process takes about 30 minutes and uses 2.2 to 3.2 liters of the sodium chloride solutions, whereas the 20 per cent W/V glycerolized red blood cells can be processed in about 20 minutes using 1.5 to 2.5 liters. After storage in CPD for three days at 4 C, red blood cells can be freeze-preserved with 40 per cent W/V glycerol at minus 80 C or with 20 per cent W/V glycerol at minus 150 C. When the thawed red blood cells are washed in the Fenwal Elutramatic, the IBM Blood Processor, or the Haemonetics Blood Processor and stored at 4 C in sodium chloride-glucose-phosphate for at least 24 hours before transfusion, they have excellent posttransfusion survival values and normal or slightly decreased oxygen transport function. Alternatively, these red blood cells can be rejuvenated before freeze-preservation so that their 2,3-DPG levels are increased and their affinity for oxygen is reduced. Red blood cells that are stored in CPD at 4 C for as long as 28 days can be rejuvenated with a solution containing pyruvate, inosine, glucose, phosphate, and adenine (PIGPA, Solution A) before freeze-preservation with 40 per cent W/V glycerol at minus 80 C. Any one of the above systems can be used to wash these red blood cells and they can be stored at 4 C in a sodium.

Adenine↗

PubMedDate not supplied

Effect of cooling and freezing, the two first steps of a freezing protocol, on the fertilizing ability of the rabbit sperm.

The effect of different phases of a freezing protocol on the fertilising ability of rabbit spermatozoa was evaluated. An extender containing 1.75 M DMSO and 0.05 M sucrose (final concentration) was used to freeze rabbit sperm. In the first experiment, the results obtained with fresh and cooled (5 degrees C for 45 min) sperm were compared; no differences were observed between fresh and cooled semen for any of the parameters studied: fertility rate (78% vs. 91% for fresh and cooled sperm, respectively), and normal embryos two days after insemination (6.8 vs. 8.5 normal embryos for fresh and cooled sperm). In the second experiment, the results obtained with fresh semen and sperm which had passed the first two steps of a freezing protocol (5 degrees C for 45 min and -30 degrees C for 30 min, and thawed at 50 degrees C for 15 s) were compared; the differences between them were obtained for fertility rate (94% vs. 61% for fresh and frozen sperm, respectively) and normal embryos two days after insemination (7.8 vs. 3.8 fresh and frozen sperm). These observations indicated that the differences in the results obtained with fresh and cryopreserved sperm were produced during the second step of the freezing protocol, and that apparently no toxic effect of DMSO was produced.

Animals↗

PubMed1996

Freeze-fracture enzyme cytochemistry: application of enzyme cytochemistry to freeze-fracture cytochemistry.

We present a novel freeze-fracture cytochemistry method based upon enzyme cytochemistry. By this method, freeze-fractured membranes are labeled with cerium as an enzyme cytochemical marker on replicas. The cerium capture method was suitable for freeze-fracture enzyme cytochemistry because the cerium phosphate reaction product is stable after replica-cleaning. As a model system, acid phosphatase, which is a well-known lysosomal marker, was detected on freeze-fractured membranes of lysosomes in the proximal tubular epithelium of the rat kidney. This technique should be a useful addition for analyzing the ultrastructure of biological membranes.

Acid Phosphatase↗

PubMed1999

The frog neuromuscular junction revisited after quick-freezing-freeze-drying: ultrastructure, immunogold labelling and high resolution calcium mapping.

Until now, most ultrastructural studies on the neuromuscular junction have been carried out on samples first exposed to chemical treatments--with fixatives and/or dehydration agents--that are known to induce, or to be inadequate to prevent, artefactual changes of the native state. We report here on the potential of a physical approach to the preparation of samples that combines quick-freezing and freeze-drying (with or without exposure to OsO4 vapours) followed by direct embedding of the samples in various resins. Thin sections from physically processed frog neuromuscular junctions, when compared to their chemically fixed counterparts, exhibit an overall excellent preservation, with the organelles retaining their native density and shape. These preparations were also investigated by electron spectroscopic imaging and electron energy loss spectroscopy, obtaining high resolution maps of native total calcium distribution within the nerve terminal. Finally, thin sections from analogously processed, however unfixed, preparations embedded in Lowicryl, were immunogold labelled before exposure to OsO4. Nerve-muscle preparations treated this way exhibited adequate preservation of ultrastructure and revealed the distribution of synaptophysin with high sensitivity and resolution. In conclusion, we provide an overview of the potential of the quick-freezing-freeze-drying approach in the study of the neuromuscular junction function.

Animals↗

PubMed2004

Heterosis in the freezing tolerance of crosses between two Arabidopsis thaliana accessions (Columbia-0 and C24) that show differences in non-acclimated and acclimated freezing tolerance.

Heterosis is broadly defined as the increased vigour of hybrids in comparison to their parents. In the model plant Arabidopsis thaliana, a significant heterosis effect on leaf-freezing tolerance was observed in the F(1) generation of a cross between the accessions Columbia-0 (Col) and C24. Parental Col plants were significantly more freezing-tolerant than C24 plants in both the acclimated and non-acclimated (NA) states. Mid-parent heterosis was observed in the F(1) plants, both in the basic tolerance of non-adapted plants and in freezing tolerance after cold acclimation. Best-parent heterosis, on the other hand, was only found after cold acclimation. The heterosis effect was reduced in the F(2) populations such that only mid-parent heterosis was evident. The leaf content of soluble sugars (fructose (Fru), glucose (Glc), sucrose (Suc) and raffinose (Raf)) increased dramatically in the F(1) plants after cold acclimation as compared to the parental lines. The content of proline (Pro), however, was only moderately increased in the F(1) plants under the same conditions. Correlation analyses showed that only Raf content was consistently related to leaf-freezing tolerance in both the acclimated and NA states. A quantification of mRNA levels in leaves of parental and F(1) lines using quantitative real-time RT-PCR showed no clear indication for an involvement of the investigated genes (CBF (C-repeat binding factor)1, CBF2, (cold-regulated protein (COR) 6.6, COR15a, COR15b, COR47 and COR78) in the heterosis effect.

Acclimatization↗

PubMed2004

Freezing-sensitive tomato has a functional CBF cold response pathway, but a CBF regulon that differs from that of freezing-tolerant Arabidopsis.

Many plants increase in freezing tolerance in response to low temperature, a process known as cold acclimation. In Arabidopsis, cold acclimation involves action of the CBF cold response pathway. Key components of the pathway include rapid cold-induced expression of three homologous genes encoding transcriptional activators, CBF1, 2 and 3 (also known as DREB1b, c and a, respectively), followed by expression of CBF-targeted genes, the CBF regulon, that increase freezing tolerance. Unlike Arabidopsis, tomato cannot cold acclimate raising the question of whether it has a functional CBF cold response pathway. Here we show that tomato, like Arabidopsis, encodes three CBF homologs, LeCBF1-3 (Lycopersicon esculentum CBF1-3), that are present in tandem array in the genome. Only the tomato LeCBF1 gene, however, was found to be cold-inducible. As is the case for Arabidopsis CBF1-3, transcripts for LeCBF1-3 did accumulate in response to mechanical agitation, but not in response to drought, ABA or high salinity. Constitutive overexpression of LeCBF1 in transgenic Arabidopsis plants induced expression of CBF-targeted genes and increased freezing tolerance indicating that LeCBF1 encodes a functional homolog of the Arabidopsis CBF1-3 proteins. However, constitutive overexpression of either LeCBF1 or AtCBF3 in transgenic tomato plants did not increase freezing tolerance. Gene expression studies, including the use of a cDNA microarray representing approximately 8000 tomato genes, identified only four genes that were induced 2.5-fold or more in the LeCBF1 or AtCBF3 overexpressing plants, three of which were putative members of the tomato CBF regulon as they were also upregulated in response to low temperature. Additional experiments indicated that of eight tomato genes that were likely orthologs of Arabidopsis CBF regulon genes, none were responsive to CBF overexpression in tomato. From these results, we conclude that tomato has a complete CBF cold response pathway, but that the tomato CBF regulon differs from that of Arabidopsis and appears to be considerably smaller and less diverse in function.

Acclimatization↗

PubMed1979

Understanding the relationship between lateral spread of freeze and depth of freeze.

The predictable relationship between lateral spread of freeze and depth of freeze is a keystone concept in the clinical evaluation of "depth dose" of cryosurgical procedures. This relationship can be readily determined and understood by carrying out simple experiments using potato models and checked directly by measurements on patients. When using specialized treatment methods such as the "cone-spray" technique with liquid nitrogen, it is necessary to limit the technique to properly selected cases and to spray the treated surfaces evenly if the predictable ratio of depth of freeze to lateral spread of freeze is to be used.

Animals↗