Large volume freezing in experimental hepatic cryosurgery. Avoidance of bleeding in hepatic freezing by an improvement in the technique.

In experiments on the dog's liver, large volumes of tissue were frozen by pouring liquid nitrogen onto the liver surface. There were no complications and no death from the procedure. Safety was produced by covering the liver surface with one to three layers of dacron velour cloth impregnated with a water-soluble jelly. The protective action of the gel-cloth was due to splinting of the liver capsule and to a more uniform and slower rate of freezing. The tissue became necrotic and over several months was replaced by dense fibrous tissue and ultimately resorbed. Although bleeding has been prevented, the problem of limitation of depth penetration remains as a hindrance to utilization in the treatment of hepatic tumors.     

The process of bone regeneration from devitalization to revitalization after pedicle freezing with immunohistochemical and histological examination in rabbits

The pedicle freezing procedure by liquid nitrogen is a method for the reconstruction of tumor-bearing bone after malignant tumor resection. However, the regenerative mechanism of bone after the pedicle freezing procedure is unclear. We investigated the complete process from devitalization to revitalization of bone after the pedicle freezing procedure in 13 rabbits. After osteotomy the 5 mm distal femurs were immersed in liquid nitrogen, and the specimens were divided into frozen area and sub-frozen area. The bilateral femurs were harvested for evaluation of bone regeneration by histological and immunohistochemical examination (VEGF, CD31, BMP-2 and Runx2) from 1 week to 52 weeks. The diameter of operating femurs was compared with contralateral femurs from 6 weeks to 52 weeks.No viable cells could be found from 1 to 8 weeks in the frozen area, and a mean 1.83 cm necrotic range were detected in the sub-frozen area. The periosteal reaction, massive fibrous tissue and immature bone matrix invaded from the normal area to the necrotic area from 12 weeks. Subsequently, the necrotic bone was gradually replaced by newly formed bone by creeping substitution, with endochondral and intramembrane bone formation. The diameter of frozen femurs was significantly larger than the contralateral femur at the same period from 8 weeks to 52 weeks (P < 0.01). All immunohistochemical factors were positively expressed in both areas at different time points. The active osteoblasts and microvessel migrated from marrow cavity and periosteum into dead bone. This study suggested that the frozen bone not only provides a scaffold but also possesses excellent osteoinductive properties.     

Large Ice Crystals in the Nucleus of Rapidly Frozen Liver Cells

Evidence in the literature shows that ice crystals that form in the nucleus of many rapidly cooled cells appear much larger than the ice crystals that form in the surrounding cytoplasm. We investigated the phenomenon in our laboratory using the techniques of freeze substitution and low temperature scanning electron microscopy on liver tissue frozen by liquid nitrogen plunge freezing. This method is estimated to cool the tissue at 1000°C/min. The results from these techniques show that the ice crystal sizes were statistically significantly larger in the nucleus than in the cytoplasm. It is our belief that this finding is important to cryobiology considering its potential role in the process of freezing and the mechanisms of damage during freezing of cells and tissues.     

液氮冷冻大鼠左冠状动脉前降支中上1／3所支配区域建立心肌梗死模型

目的探讨大鼠左冠状动脉前降支中上1／3所支配的区域液氮冷冻处理后对心肌形态学及心功能的影响,以建立适合移植干细胞再生修复心肌梗死研究的一种新的大鼠心肌梗死模型制作方法。方法80只雄性SD大鼠,随机分为3组即：冷冻组、结扎组、对照组。大鼠麻醉后,行气管插管连通动物呼吸机,打开胸廓暴露心脏,用特制的直径为0．6cm冷冻头置入液氮中冷冻降温后迅速冷冻大鼠左冠状动脉前降支中上1／3所支配的区域,或结扎左冠状动脉前降支中上1／3处。分别于处理后1d、3d、7d、14d、28d观察心脏病理组织学变化,并于处理28d后检测心功能的变化。结果在液氮冷冻大鼠心脏后,大鼠心肌组织出现凝固性坏死,继而有肉芽组织长人梗死灶内,最后形成疤痕。用液氮冷冻法可成功复制心肌梗死大鼠动物模型。与冠状动脉结扎法相比较,操作简单,手术时间短,死亡率低．心肌梗死面积变异小。结论液氮冷冻法作为一种复制心肌梗死模型的方法,有其自身的优势．可用于心肌梗死发生机制和干细胞治疗等方面的研究。     

Survival of Nippostrongylus brasiliensis larvae after freezing over liquid nitrogen

Survival of Nippostrongylus brasiliensis larvae after freezing over liquid nitrogen. International Journal for Parasitology4: 173–176. Third stage larvae of N. brasiliensis were frozen over liquid nitrogen and after storage for 7 days were thawed rapidly and inoculated subcutaneously into rats. When ensheathed larvae were used, none survived freezing as judged by motility and infectivity trials. Separate vials of exsheathed larvae survived freezing in proportions ranging from 10 to 64 per cent. Female worms, derived from frozen exsheathed larvae, had a normal complement of eggs in the uterus and both male and female worms had a normal histological appearance. Exsheathed larvae frozen in the presence of 10 per cent dimethylsulphoxide had the same survival rate as those frozen without the addition of cryoprotectant. The addition of 10 per cent glycerol adversely effected the survival of frozen exsheathed larvae.     

The effects of freezing, storage, and thawing on cell compartment integrity and ultrastructure

 The effects of slow freezing and thawing on enzyme compartmentalization and ultrastructure were studied in rat liver slices frozen in dry ice, isopentane/ethanol-dry ice, or liquid nitrogen, and stored at –80°C for 1–14 days. Non-frozen slices served as controls. Frozen liver slices were thawed in a Karnovsky fixative and processed for transmission electron microscopy (TEM). After all freezing protocols, the outer zone of frozen-thawed tissue was ultrastructurally very similar to that of non-frozen liver. Towards the center of the tissue, the ultrastructure progressively deteriorated. Comparison with 50-μm cryostat sections prepared for TEM showed that thawing and not freezing is the detrimental step for fair preservation of ultrastructure. After thawing, homogenization, and differential centrifugation, distribution patterns of soluble marker enzymes were analyzed (cytosol, lactate dehydrogenase; mitochondrial matrix, glutamate dehydrogenase; lysosomes, acid phosphatase). The enzyme activities were not affected by storage for 2 weeks and the activity distributions showed that protein leakage from compartments was only minimally increased in frozen-thawed tissue compared with that from non-frozen tissue, irrespective of the method of freezing. In conclusion, fairly large tissue slices (20×5×3 mm) may be frozen and stored at –80°C for biochemical, ultrahistochemical or ultrastructural studies. For ultrastructural analysis, only the periphery of the tissue slice should be used. Accepted: 12 May 1997     

Fast inverse prediction of the freezing front in cryosurgery

Cryosurgery has become a well-established technique for the ablation of undesirable tissues such as tumors and cancers. The motivation for this study is to improve the efficacy and safety of this technique. This study presents an inverse heat transfer method for monitoring the motion of the freezing front from a cryoprobe. With the help of a thermocouple inserted into the layer of diseased tissue, the inverse heat transfer method estimates simultaneously the blood perfusion rate and the thermal conductivities of both frozen and unfrozen tissues. This information is then fed to the Pennes bioheat equation that: (1) calculates the time-varying temperature distribution inside the layer of tissue and (2) predicts the motion of the freezing front. The effect of the most influential parameters on the inverse predictions is investigated. These parameters are (1) the initial guesses for the unknown Levenberg-Marquardt polynomial parameters of the thermo-physical properties; (2) the temperature of the cryoprobe; (3) the heat transfer coefficient of the impinging jet of liquid nitrogen; and (4) the noise on the temperature data recorded by the thermocouple probe. Results show that the proposed inverse method is a promising alternative to ultrasound and Magnetic Resonance Imaging (MRI) for monitoring the motion of the freezing front during cryosurgery. For all the cryogenic scenarios simulated, the predictions of the inverse model remain accurate and stable.     

Freezing characteristics of fresh water fish (Labeo rohita) by liquid nitrogen vapor

The lean fish Labeo rohita was subjected to liquid nitrogen vapor in a batch freezer to determine its freezing characteristics. The moisture content of the fish muscle tissue was about 75%. The initial and average freezing points were found to be − 1.34 and − 5.25 °C, respectively. The fish slab sample showed a decreased rate of temperature change in that region. The suggested cut-off freezing temperature was − 30 °C, corresponding to about 90% of total water frozen. Continued freezing below − 30 °C resulted in negligible freezing of residual liquid water in the sample. The effects of low-density polyethylene (LDPE) bags and aluminum foil on the freezing times and the overall heat transfer coefficients were sufficiently small that the authors recommended thicknesses of up to 0.1 and 0.5 mm for LDPE bags and aluminum foil, respectively. In these cases there was only 5% increase in freezing time compared to unpackaged fish samples.     

Vacuum Freeze Drying of Frozen Sections for Dry-Mounting, High-Resolution Autoradiography

Specimens no larger than 1.5 × 1.5 × 2 mm were frozen in liquid nitrogen and sectioned, while still frozen, with a refrigerated microtome. The frozen sections were dried in a vacuum, then pressed onto either Kodak NTB10 plates or onto slides which had been coated with Kodak NTB3 emulsion and dried. Radioactive mouse liver was used to test tissue preservation. Intestinal mucosa with Ha-labeled nuclei was used to test the quality of autoradiography. Good cytological detail was preserved in both tissues, with the autoradiographs interpretable at the cellular level.     

An Improved Method for In Situ Freezing of Cat Brain for Metabolic Studies

This study introduces a new method for rapid freezing of the cat brain. The method employed a Styrofoam box which was fitted around the head of the animal. Liquid nitrogen was poured into the box until the head was submerged. Temperature changes in three brain sites (ventral hypothalamus, the fourth ventricle, and the corpus callosum) and levels of labile carbohydrate metabolites (glycogen, glucose, ATP, P-creatine, and lactate) in five brain regions (cortex, thalamus, midbrain, cerebellum, and pons) frozen by the box method were compared with those frozen by a conventional cup method in which liquid nitrogen was poured into a hollow Styrofoam cup placed on top of the skull. The box method shortened the time of arrival of the freezing front and improved the freezing rate. The time required to bring the tissue to -20 degrees C was shortened, from 20 min at the ventral hypothalamus and 10-12 min at the fourth ventricle with the cup method, to less than 5 min at both sites with the box technique. Continued perfusion of brainstem prior to freezing was demonstrated. Levels of metabolites frozen by either method were similar. Lactate levels in any of the five brain regions studied by either method were not elevated, indicating no ischemic change.(ABSTRACT TRUNCATED AT 250 WORDS)     

Successful long-term cryopreservation of neonatal rat islet tissue

Neonatal rat pancreatic tissue was frozen to -196 degrees C using Me2SO as a cryoprotectant and a slow freezing rate to -70 degrees C followed by immersion in liquid nitrogen. Rapid thawing was used. Viability was demonstrated by successful transplantation to streptozotocin-induced diabetic recipients. Long-term preservation, up to 4 weeks, did not demonstrably affect viability. Cryopreservation techniques may afford a method for providing a diabetic recipient the opportunity to receive a large quantity of pooled islet tissue from well-matched donors.     

Nonrandom RNAseq gene expression associated with RNAlater and flash freezing storage methods

RNA sequencing is a popular next‐generation sequencing technique for assaying genome‐wide gene expression profiles. Nonetheless, it is susceptible to biases that are introduced by sample handling prior gene expression measurements. Two of the most common methods for preserving samples in both field‐based and laboratory conditions are submersion in RNAlater and flash freezing in liquid nitrogen. Flash freezing in liquid nitrogen can be impractical, particularly for field collections. RNAlater is a solution for stabilizing tissue for longer‐term storage as it rapidly permeates tissue to protect cellular RNA. In this study, we assessed genome‐wide expression patterns in 30‐day‐old fry collected from the same brood at the same time point that were flash‐frozen in liquid nitrogen and stored at ?80°C or submerged and stored in RNAlater at room temperature, simulating conditions of fieldwork. We show that sample storage is a significant factor influencing observed differential gene expression. In particular, genes with elevated GC content exhibit higher observed expression levels in liquid nitrogen flash‐freezing relative to RNAlater storage. Further, genes with higher expression in RNAlater relative to liquid nitrogen experience disproportionate enrichment for functional categories, many of which are involved in RNA processing. This suggests that RNAlater may elicit a physiological response that has the potential to bias biological interpretations of expression studies. The biases introduced to observed gene expression arising from mimicking many field‐based studies are substantial and should not be ignored.     

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.     

Cryopreservation of semen in the dog: use of ultra-freezers of -152 degrees C as a viable alternative to liquid nitrogen

This experimental work was carried out to validate the use of a -152 degrees C ultra-low temperature freezer to freeze and store canine semen. The semen of three dogs was pooled and processed to obtain a final dilution with a concentration of 100 x 10(6) spermatozoa/mL, glycerol at 5% and Equex at 0.5%. Then, four freezing protocols were tested to evaluate the cryosurvival of sperm at 1, 7, 30, 60 and 120 days after freezing: (I) semen was frozen and stored in liquid nitrogen; (II) semen was frozen in liquid nitrogen and stored in the ultra-low freezer at -152 degrees C; (III) semen was frozen in the vapour of liquid nitrogen and stored in the ultra-low freezer at -152 degrees C; (IV) semen was frozen and stored in the ultra-low freezer at -152 degrees C. Data were statistically analyzed by repeated measures analysis of variance to determine the effect of the freezing protocol and time on the sperm characteristics assessed. The percentages of sperm motility and of dead/live spermatozoa were similar throughout the experimental period, with no significant differences (P < 0.05) to be observed between four different freezing techniques tested. At 120 days after freezing, the percentage of abnormal cells and the percentage of sperm cells with abnormal acrosome were not significantly different between the freezing techniques. Although the number of dogs used was slightly low, in vitro results of this preliminary study showed that the use of ultra-freezers at -152 degrees C to freeze and store canine semen could be a viable alternative to liquid nitrogen.     

Freezing damage to isolated tomato fruit mitochondria as modified by cryoprotective agents and storage temperature

Isolated tomato (Lycopersicon esculentum var. Kc 146) fruit mitochondria could be stored successfully in the frozen state without a cryoprotective agent if the mitochondria were frozen quickly by immersion in liquid nitrogen and later thawed quickly at 30 C. Criteria of freezing damage were rate of respiration, adenosine diphosphate to oxygen ratio, and respiratory control ratio. Marked reduction in respiration and loss of respiratory control occurred when mitochondria were transferred from liquid nitrogen to −5, −10, or −18 C for 15 minutes prior to thawing at 30 C. Dimethylsulfoxide (5%) prevented freezing damage when mitochondria were incubated at −5 C but did not prevent freezing damage at −10 or −18 C. Isolated tomato mitochondria show promise as a model system for studying the nature of freezing damage and the mode of action of cryo-protective agents.     

Cryopreservation of monogonont rotifers

Development of techniques to maintain viable rotifer clones in a frozen state would preserve the genotype and reduce routine maintenance for those clones not being actively studied. To this end we have frozen Brachionus plicatilis in dimethyl sulfoxide at concentrations ranging from 6% to 18%. Survival rates decreased as the endpoint temperature was reduced from ?20 °C to ?45 °C, but did not decrease when the temperature was further reduced to ?196 °C (liquid nitrogen). Only 2% of the individuals survived freezing in liquid nitrogen.     

Experimental measurements and theoretical calculations on muscle and liver tissue during cryosurgery. I. Experiments on freezing of rabbit tissue.

Experiments on freezing of muscle and liver tissue of 113 rabbits were performed. The diameter on the frozen surface, and the thickness and the mass of the iceball were measured for the live and dead, body-temperature, animal. Four continuously cooled and six massive probes (2.5–20-mm diameter) were used with liquid nitrogen as the cooling agent. The following conclusions can be drawn: (1) with use of round probe-tips, the iceball has approximately spherical symmetry. However, the depth of the frozen tissue is about 15% smaller than the lateral extension on the visible surface. (2) For continuously cooled probes the diameter of the iceball in the steady state is about five times as large as the probe diameter. The maximal iceball diameter for massive probes is two times larger than the probe diameter. (3) The different blood circulation of liver and muscle tissue has an influence of only 10% on the size of the iceball. For clinical applications this difference is of little importance. (4) For live tissue the iceball is about 15% smaller than for body-temperature dead tissue. Thus, the main heat-transport process in tissue is heat condition.     

Long-term skin preservation by combined use of tissue culture and freezing techniques.

Simple but effective methods for shipping newly excised rabbit skin to a distant central laboratory for in vitro culture on a pigskin base, followed by freezing in a cryoprotective agent (DMSO) for frozen storage and subsequent reshipment to the originating laboratory while still frozen are described. At a suitable time the frozen tissue was rapidly thawed and transplanted to the autologous recipient rabbit. Of 12 cultures, seven indicated good to excellent cell growth and maturation on the host. The successful method combined the use of in vitro tissue culture and freezing to permit a central laboratory to grow the skin and ship it to the originating center for autografting at a convenient time.     

OPTIMAL FREEZING CONDITIONS FOR CEREBRAL METABOLITES IN RATS

Abstract— Optimal freezing conditions for metabolites were evaluated in 250-450 g rats. As a standard procedure, the brains were frozen in such a way that the blood pressure and arterial oxygenation were upheld during the freezing. The progression of the freezing front was determined by means of implanted thermocouples, and the interruption of the circulation by means of injections of carbon particles into the blood stream. The freezing gave rise to a rapid interruption of the circulation in the superficial cortical layer first reached by the freezing front well before the temperature reached 0°C. In deeper regions the progression of the freezing front was slower and interruption of the circulation occurred simultaneously with the freezing of the tissue. Measurements of labile cerebral metabolites, including phosphocreatine, ATP, ADP, AMP and lactate, failed to show signs of autolysis in the part of cortex which became unperfused at temperatures above zero. Since the energy state was identical in superficial cortical areas and in areas that did not freeze until after 40–90 s, it is concluded that the freezing technique gives optimal conditions for metabolites also in deep cerebral structures. Decapitation of unanaesthetized animals gave rise to large autolytic changes in the cerebral cortex. In unanaesthetized animals that were immersed in liquid nitrogen the changes were less marked and mainly affected the concentrations of phosphocreatine, ADP and lactate. When paralysed animals that were anaesthetized with N₂O were immersed in liquid nitrogen the only significant change from the control was a decrease in phosphocreatine content. The virtual absence of autolytic changes in this group of animals was not related to the anaesthesia since more pronounced changes were observed in phenobarbitone-anaesthetized rats immersed in the coolant. These differences could be explained by the fact that spontaneously breathing animals immersed in liquid nitrogen developed arterial hypoxia much faster than paralysed animals. It is concluded that an optimal metabolite pattern can only be obtained in anaesthetized animals, frozen with a method that was described by Kerr almost 40 years ago (Kerr , 1935). If unanaesthetized animals must be used, greater attention should be paid to the oxygenation of the blood during the freezing than to such factors as speed of freezing or depth of anaesthesia.     

Activity, stereospecificity, and stereoselectivity of microsomal enzymes in dependence on storage and freezing of rat liver samples

Liver microsomes are now one of the most widely used in vitro test systems for biotransformation studies of drugs, toxins, and other xenobiotics. The standard procedure of preparation of microsomes from fresh liver taken immediately after death of the animal is impossible in experiments with liver samples from human or wild animals and the choice of a relatively optimal way of liver storage is necessary in these cases. We studied the possibility of using the stereoselectivity and stereospecificity of biotransformation enzymes for evaluation of the changes in enzyme function dependent on tissue handling. Activity, stereospecificity, and stereoselectivity of several enzymes in microsomes prepared from fresh liver, frozen liver in liquid nitrogen, or ice-cooled liver were compared. The effect of storage period (2, 3, 5 h) on these parameters were also tested. Both freezing and cooling of liver change the native function of enzyme systems and could result in incorrect stereospecificity data for the microsomal metabolism. All parameters observed also differ in their dependence on period of ice cooled storage. As it is difficult to hold strictly to the same storage period, we recommend freezing liver in liquid nitrogen if the storage of liver is necessary. In projects comparing enzyme activities in human and laboratory animals the same freezing procedure of liver should be maintained before preparation of microsomes from all species.