Hatchling turtles survive freezing during winter hibernation.

Hatchlings of the painted turtle (Chrysemys picta marginata) are unique as the only reptile and highest vertebrate life form known to tolerate the natural freezing of extracellular body fluids during winter hibernation. Turtles survived frequent exposures to temperatures as low as -6 degrees C to -8 degrees C in their shallow terrestrial nests over the 1987-1988 winter. Hatchlings collected in April 1988 had a mean supercooling point of -3.28 +/- 0.24 degrees C and survived 24 hr of freezing at -4 degrees C with 53.4% +/- 1.98% of total body water as ice. Recovery appeared complete after 20 hr of thawing at 3 degrees C. However, freezing at -10.9 degrees C, resulting in 67% ice, was lethal. A survey of possible cryoprotectants revealed a 2- to 3-fold increase in glucose content of liver and blood and a 3-fold increase in blood glycerol in response to freezing. Although quantitatively low, these responses by spring turtles strongly indicate that these may be the winter-active cryoprotectants. The total amino acid pool of blood also increased 2.25-fold in freezing-exposed turtles, and taurine accounted for 52% of the increase. Most organs accumulated high concentrations of lactate during freezing, a response to the ischemic state imposed by extracellular freezing. Changes in glycogen phosphorylase activity and levels of glucose 6-phosphate and fructose 2,6-bisphosphate were also consistent with a dependence on anaerobic glycolysis during freezing. Studies of the molecular mechanisms of natural freeze tolerance in these turtles may identify protective strategies that can be used in mammalian organ cryopreservation technology.     

Survival and infectivity of Brugia malayi microfilariae after cryopreservation

Methods were studied for the cryopreservation of microfilariae of periodic Brugia malayi. RPMI-1640 tissue culture medium containing 6% dimethyl sulfoxide (DMSO) and 15% newborn calf serum was used as cryoprotectant. Samples were frozen slowly in the vapor phase of liquid nitrogen prior to emersion in liquid nitrogen (-196 degrees C). The freezing rate was -0.5 to -1.0 degrees C per minute, microfilariae remained viable for as long as, 212 and 375 days, survival rates were 94 to 98% and they were infective to Aedes togoi mosquitos. The infective larvae (L3) were obtained for 10-11 days after feeding at 28 degrees C room-temperature and the infection rate of L3 in test mosquitos was 22.4-30.6%. All DMSO should be removed from the freezing medium to restore microfilariae activity after freezing.     

An automatic device for freeze-clamping of cardiac tissue within a fraction of the contraction cycle.

An automatic quick-freeze clamping device has been developed. Opposed pneumatic pistons filled with aluminium caps previously cooled in liquid nitrogen are used to compress a portion (100 to 200 mg) of the myocardium to a 0.15 to 0.20 mm thick wafer, colling the tissue from 37 degrees C to -15 degrees C within 10 ms. The clamp is triggered electronically from the R-wave of the ECG. This tissue fixation by freezing within 10 ms is sufficiently rapid to study oscillations of myocardial metabolite levels during the contraction cycle of isolated perfused hearts of small mammals such as the rat and guinea pig whose rate is 4 to 5 beats per second.     

Stability of coagulation proteins in frozen plasma.

This study reports on the frozen stability of all commonly measured coagulation proteins in normal citrated plasma: activated partial thromboplastin time, prothrombin time (%), thrombin time and fibrinogen (Clauss); clotting assays for factors II, V, VII, VIII, IX, X, XI and XII; functional assays for protein C (clotting), protein S (clotting), antithrombin (chromogenic) and plasminogen (chromogenic); and immunological assays for von Willebrand factor and D-dimer. All these factors listed are stable for up to 3 months if frozen at -24 degrees C or lower. At -74 degrees C, all these factors (allowing for 10% variation) were stable for at least 18 months, most were stable for 24 months. The number of proteins showing > 5% variation over baseline after 6 months storage indicates that some decay does occur even at -74 degrees C. There was no clear advantage in snap freezing at -74 degrees C and then storing at -24 degrees C over both freezing and storing at -24 degrees C; therefore, the freezing process itself is not responsible for the loss of stability. The best stability, especially at -24 degrees C, was obtained when small samples (1 ml) were stored in screw-cap tubes with a minimum dead space. The decrease in stability of the coagulation proteins directly correlates with the effect of temperature and time.     

Studies of the Irradiation Protection Effect of Fetal Liver in Mice. I. Influence of the Gestational Age of the Donor Tissue

1. The gestational age of donor hematopoietic tissue appeared to have noinfluence on the 30-day mortality following homologous transplants in irradiated mice.

2. Recipients of second trimester homologous donor tissue had a late (90-day) mortality that was 16 per cent lower than that observed in animals receiving tissue from third trimester and neonatal donors, but statistical analysisshowed a low level of significance.

3. Irradiation alone appeared to cause a late mortality in non-injected irradiated animals and in isologously transplanted irradiated animals.

Submitted on February 13, 1961 Accepted on April 21, 1961     

New trends in gamete's cryopreservation

We developed new techniques to improve freezing and vitrification of sperm, oocytes and embryos. Our novel freezing technology is based on 'Multi-Thermal-Gradient' (MTG) freezing that is used for sperm. The freezing apparatus has the ability to control ice crystals propagation by changing thermal gradient or the liquid-ice interface velocity which optimizes ice crystals morphology during freezing of cells and tissue. Using this apparatus we were able to freeze bull, stallion, boar, ram, fowl and human sperm with normal post-thaw motility/pre-freezing motility of 70-100%. The vitrification method includes the cooling of nanoliter sample (the 'Minimum Drop Size' technique) in 'super-cooled' liquid nitrogen (-210 degrees C), which maximized cooling rate to the highest physically possible (24-130000 degrees C/min). Using this method we achieved very high survival of bovine oocytes and embryos. Vitrification of oocytes at the MII stage resulted with cleavage and blastocyst rate of 50 and 20%, respectively. The vitrification of in-vitro production (IVP) of bovine embryos allowed the production of a healthy calf after embryo-transfer carrying the name 'Zegugit' (in Hebrew: made from glass).     

Studies on Congenital Hemolytic Syndromes. IV. Gastrointestinal Absorption of Iron

1. Results of studies of gastrointestinal absorption of ferrous iron in normalchildren and those with heterozygous thalassemia were similar.

2. In one patient with absent erythropoiesis but severe anemia, no increasein the amount of iron absorbed was noted.

3. In sickle cell-hemoglobin C disease and hereditary spherocytosis havingonly slight anemia in the presence of increased erythropoiesis, normal amountsof iron were absorbed.

4. Patients with sickle cell anemia and thalassemia major in whom therewas active erythropoiesis and marked anemia absorbed abnormally largeamounts of iron. The amount absorbed by individuals with the latter diseasecould be reduced by administration of transfusions and concomitant suppression of erythropoiesis.

5. Usual values for serum iron and latent iron-binding capacity in severalcongenital hemolytic syndromes have been presented and their significancediscussed.

6. No specific effect on absorption was noted by increased or reducedamounts of tissue or serum iron or by reduced or increased latent iron-bindingprotein.

Submitted on June 20, 1961 Accepted on October 30, 1961     

Impact of reduced levels of protein C, free protein S and antithrombin in normal frozen plasma on the interpretation of patients' results

The objective of this study was to investigate the effect of freezing of normal plasma samples on protein C, free protein S (FPS) and antithrombin levels in order to determine its potential impact on the interpretation of the results of similarly frozen patients' samples. Protein C, FPS and antithrombin levels were measured by clotting-based test, by sandwich ELISA and by chromogenic assay, respectively, in 50 normal plasma samples prior to freezing, and after 2 and 4 weeks in parallel aliquots frozen at -25°C. The mean levels of the three proteins dropped significantly after a fortnight's freezing, protein C: 130.7-122.8% (P < 0.0246); FPS: 105.9-94.1% (P < 0.0016); antithrombin: 103.2-95.8% (P < 0.0001). The corresponding inter-assay coefficient of variances of the two sets of results were 8.9, 6.6 and 9.3%. Thereafter, only FPS declined significantly (84.3%) (P < 0.0001). In two of 48 and five of 48 cases at the end of 2 and 4 weeks, respectively, the levels of FPS values went below the lower limit of the normal range established from the 50 plasma samples. Freezing of plasma at -25°C for 24 h per se did not alter the levels of protein C and antithrombin and caused only a negligible change in FPS levels. Since 6, 4 and 14% of normal plasma samples would have been labeled as antithrombin, protein C and protein S deficient, respectively, had the tests been performed after 4 weeks of freezing, it is recommended that for correct interpretation of the results, laboratories should establish their reference ranges on normal samples frozen for the same period of time as the patients' samples.     

Surface crystallization of supercooled water in clouds

The process by which liquid cloud droplets homogeneously crystallize into ice is still not well understood. The ice nucleation process based on the standard and classical theory of homogeneous freezing initiates within the interior volume of a cloud droplet. Current experimental data on homogeneous freezing rates of ice in droplets of supercooled water, both in air and emulsion oil samples, show considerable scatter. For example, at -33 degrees C, the reported volume-based freezing rates of ice in supercooled water vary by as many as 5 orders of magnitude, which is well outside the range of measurement uncertainties. Here, we show that the process of ice nucleus formation at the air (or oil)-liquid water interface may help to explain why experimental results on ice nucleation rates yield different results in different ambient phases. Our results also suggest that surface crystallization of ice in cloud droplets can explain why low amounts of supercooled water have been observed in the atmosphere near -40 degrees C.     

Phosphatidylethanol in human organs and blood: a study on autopsy material and influences by storage conditions

OBJECTIVE: Phosphatidylethanol (PEth) is an abnormal phospholipid that is formed and accumulated in mammalian cells that have been exposed to ethanol. PEth has been proposed as a marker of ethanol abuse. This study was conducted to investigate the concentration of PEth in blood and organs obtained during the autopsy of alcoholics. In addition, we performed experiments on rat tissues and human blood to evaluate the effect of various storage conditions on PEth concentrations. METHODS: Human tissues and blood from alcoholics and controls were obtained at autopsy and frozen at -20 degrees C until extraction. Blood from healthy donors was incubated with ethanol for 24 hr and thereafter either extracted directly or stored at room temperature, stored at 4 degrees C, frozen at -20 degrees C, or frozen in liquid nitrogen and stored at -80 degrees C before extraction. Rats were given intraperitoneal injections of ethanol and then killed, either while still intoxicated or when sober. Rat organs were homogenized and extracted directly, after a period of storage, and/or after freezing at -20 degrees C. PEth concentration was analyzed using HPLC and verified by mass spectrometry. RESULTS: In all rat organs studied, PEth was formed during freezing at -20 degrees C with ethanol present. PEth concentrations of 9 to 205 mumol/liter were observed in the blood obtained at autopsy. The highest value was found in the case with the highest blood alcohol concentration (114 mmol/liter) at the time of death. In the experiments on human blood stored with ethanol present, PEth concentrations were not affected after 72 hr at 4 degrees C or after freezing in liquid nitrogen and storage at -80 degrees C for up to 144 hr but were slightly elevated after 24 hr at room temperature and at -20 degrees C. PEth was found in all organs obtained from the cadavers of alcoholics. Storage of organs at 4 degrees C for 24 hr with ethanol present had no effect on the PEth concentration. The PEth concentration was unaffected when no ethanol was present at the time of freezing. CONCLUSIONS: The rat experiments indicated that the very high PEth concentrations found in the organs of the alcoholics were probably largely formed while the organs were frozen at -20 degrees C. Our data suggest that tissue material from bodies that were exposed to ethanol must be stored properly to obtain reliable results from subsequent analysis for PEth. Tissue should not be frozen at -20 degrees C but instead stored refrigerated until extraction, preferably within hours of autopsy, or frozen in liquid nitrogen and stored at -80 degrees C. Blood samples that contain ethanol can be stored refrigerated for up to 72 hr or frozen in liquid nitrogen and stored at -80 degrees C without affecting PEth levels.     

Myocardial cyclic AMP determinations: the importance of rapidity of sample freezing

Using specially modified automatic freeze clamps studies were undertaken to determine the importance of rapid freeze clamping in the determination of myocardial cAMP content. Modification of the freeze clamps allowed the separation of the physical processes of clamping and freezing. When the tissue was clamped and maintained at 37 degrees C for periods in excess of 5 s before freezing cyclic AMP content increased by up to 100%. When clamped and held at room temperature (20 degrees C) increase were again observed if the freezing delay exceeded 5 s, however, the increases were not as great as those observed with 37 degrees C clamping. The biphasic time-dependency curve for tissue cAMP content v. clamping delay bears a close similarity to that observed following the onset of tissue ischemia suggesting perhaps that ischemia, secondary to mechanical clamping, rather than sympathetic stimulation, is the cause of the rise. It can be concluded that for reliable measurements of cAMP content tissue should be frozen within less than 5 s of sampling.     

Stability of human gallbladder bile: effect of freezing.

In the present study, the stability of the most essential biliary parameters of human gallbladder bile at -18 degrees C was examined over several months. In 12 patients with gallstone disease (10 female, two male; 52.1+/-13.3 years of age), bile was obtained through fine needle puncture of the gallbladder under local anesthetic. The concentrations of total lipids, cholesterol, phospholipids and bile acids, and the cholesterol saturation index and crystal appearance time were determined before and after freezing over a mean period of 4.38+/-2.9 months. Gallbladder bile obtained by fine needle puncture has proved to be of excellent quality. The total lipid concentration was unchanged before (8.30+/-4.16 g/dL) and after freezing (9.16+/-4.54 g/dL, P=0.6027). The biliary cholesterol, phospholipids and bile acid concentrations, and cholesterol saturation index showed no statistically significant differences before and after freezing. A significant difference arises in the context of subdivision of the group to the nucleation time. Before freezing, most patients had a nucleation time between five and eight days, which shortened to between one and four days after thawing (P=0.0100). The authors conclude that, with the exception of the nucleation time, human gallbladder bile can be stored at -18 degrees C for four months with stability of major lipid components.     

Articular cartilage preservation and storage. I. Application of tissue culture techniques to the storage of viable articular cartilage.

Articular cartilage slice explants were stored under various conditions, including freezing-thawing at various rates by using dimethyl sulfoxide (DMSO) as a cryoprotective agent, incubating in standard tissue culture medium (MEM Eagle:NCTC 135:15% fetal calf serum) in 5% CO2 and air at 4 degrees, 21 degrees, and 37 degrees C, and incubating in standard tissue culture medium containing 200 micrograms/ml alpha-tocopherol (vitamin E) at 37 degrees C after first ascertaining a dose-response curve of vitamin E. Results indicated that articular cartilage slice explants did not survive freezing or storage at 4 degrees and 21 degrees C as measured by 35S uptake. When stored at 37 degrees C in standard tissue culture in 5% CO2 and air, the slice explants remained viable for up to 60 days. The addition of alpha-tocopherol to the medium resulted in significantly less release of previously incorporated 35Sin stored cartilage slices and significantly less reduction of the amount of hexosamine present in the stored explants. alpha-Tocopherol in the medium also preserved safranin O staining. Thus, the application of tissue culture techniques to the storage of articular cartilage made it possible to preserve cartilage slice explants in a viable, biochemically "normal" state.     

Effect of cold acclimation on hemorheological behavior in rats with frostbite

The hemorheological behavior pre- and post-freezing and tissue survival area (TSA) of frostbitten hind feet in cold-acclimated rats (group CA) and the control (group C) were observed in order to go further into the mechanism of cold acclimation. The results indicated that blood viscosity and RBC aggregation tendency were lowered and RBC deformability improved obviously in CA rats as compared with those in group C (p<0.01). After the rats' hind feet were frozen, the hemorheological behavior in both group CA and C rats was changed abnormally and the changes were less serious in CA rats than those in C rats. The TSA of CA rats was much larger than that of C rats (p<0.01). The aforesaid results revealed that after rats were acclimated to cold, their resistance to freezing increased and their adaptive changes in hemorheological behavior occurred. The extent of abnormal changes in hemorheological behavior caused by freezing was reduced in CA rats, therefore the disorder microcirculation could be improved. These changes were beneficial to the frostbitten tissue to recovering and healing. So it is considered that in CA rats, the adaptive change in hemorheological behavior is one of the cause enhancing resistance to frostbite.     

Early and late functional and histopathological perturbations in the rabbit ear-artery following local cold injury.

BACKGROUND: These experiments aimed to study the in vivo short and long term neurovascular regeneration after frostbite. METHODS: The rabbit central ear-artery was used as the experimental model. The effects on the noradrenergic innervation of the artery were measured in isolated vascular ring segments the first day and 2, 3-4, and 8-10 or 10-20 weeks following freezing at -9 degrees C or -18 degrees C for 15 min with slow rewarming for 7 min at room temperature. RESULTS: Two days after freezing the sympathetic nerves were completely degenerated, as observed with glyoxylic acid-induced fluorescence. The vascular isometric tension responses to exogenous noradrenaline and endogenously released noradrenaline by electrical stimulation in vitro were abolished. A varying degree of necrosis of the vascular wall was observed. Two weeks after freezing at -18 degrees C in vitro responses to exogenous noradrenaline and electrical stimulation were still abolished, then gradually approaching control levels after 10-20 weeks of in vivo regeneration. Eight and 10 weeks after injury at -9 degrees C increased vascular tension responses to exogenous noradrenaline was found. In spite of a long regeneration period the total uptake and the spontaneous and K+ (75 mM) evoked releases of [3H]noradrenaline were persistently decreased after frostbite at -18 degrees C, but they were regenerated to control levels already 10-20 weeks after -9 degrees C. Regeneration of noradrenergic nerve function, expressed as [3H]noradrenaline uptake and release and responsiveness to electrical stimulation, expressed as vascular contraction, was slower than the regeneration of the vascular smooth muscle. Myointimal hyperplasia developed in response to -9 degrees C and -18 degrees C frostbite. The uptake and the K+ evoked release of [3H]noradrenaline were particularly sensitive parameters for autonomic nerve function. CONCLUSIONS: The present findings may demonstrate important neurovascular reactions to local frostbite and may explain human sequelae following frostbite.     

Studies on the Fate of Lymphocytes. I. Labeling Small Thymic Lymphocytes with Tritiated Thymidine

A procedure is described whereby 0.5 ml. suspensions containing 3-5 x 10⁸small lymphocytes per ml., nearly 50 per cent of which were labeled with H³thymidine, have been prepared from rat thymus. Repeated doses of thymidinetotaling 7-8 µc/Gm. and from two to three days were required to producethis percentage, and it is suggested that longer times would not be muchmore effective. Preliminary results indicate that cells of such suspensions injected in irradiated hosts are widely distributed, do not appear to disintegraterapidly or to be massively ingested by the macrophages of the reticuloendothelial system, and are still present in good condition one and two daysafter injection.

Submitted on June 29, 1961 Accepted on August 16, 1961     

Plasma atrial natriuretic peptide is unstable under most storage conditions.

BACKGROUND. Atrial natriuretic peptide (ANP) is a hormonal regulator of cardiovascular fluid volume. More than 1,000 scientific articles were written about ANP between 1987 and 1991. Because some articles hinted at problems with storing ANP, this study examined the effect of numerous techniques for storing and processing human ANP samples. METHODS AND RESULTS. Samples were obtained repeatedly from three patients, treated, and stored under a variety of conditions. Experiment 1 evaluated the effects of different preservatives at 35, 21, 14, 10, and 7 days before assay. Experiment 2 evaluated nonspecific binding of ANP to different storage tubes during 28 days of storage. Experiment 3 evaluated the effect of storage at -20 degrees C, -80 degrees C, and -196 degrees C for 1 month. ANP was very unstable, degrading as much as 30% after 3 days of storage and by more than 50% in 1 month even when stored at -80 degrees C. Only storage at -196 degrees C (in liquid nitrogen) kept ANP stable for 1 month. Extraction and lyophilization of the samples before freezing and assay within 7 days of freezing only partially minimized the amount of degradation. All other processing techniques had little effect on slowing the degradation of ANP. CONCLUSIONS. These findings raise disturbing questions about the interpretation of the substantial literature on ANP.     

Demonstration of HL-A Antigens on Human Kidney Cells by Means of the Micro-Immune Adherence Assay

Abstract. The presence of HL-A antigens on human kidney cells was investigated by means of the micro-immune adherence assay (MIAA). The MIAA was performed on kidney cells obtained from fresh kidneys and from kidneys stored at -96°C, on kidney cells maintained in culture, and on kidney cells stored in liquid nitrogen after programmed freezing.
Performed on fresh kidney cells, the MIAA has a reproducibility of 96–97%. Performed on cells from traditionally frozen kidneys, on cells maintained in culture, and on cells kept in liquid nitrogen after programmed freezing, the assay shows poor reproducibility and non-specific adherence occurs. This might partly explain the differences observed by other investigators in typing and cross-matching with lymphocytes on the one hand, and kidney cells on the other; for in these procedures kidney cells, usually from a tissue culture, are typed by means of the MIAA. Modern long-term kidney preserving techniques make it possible to perform the MIAA on fresh kidney cells for typing and cross-matching in kidney transplantation.     

Nonpolar density gradient ultracentrifugation in the direct determination of myocardial subcellular calcium

A new method is described for making direct measurements of compartmentalized subcellular stores of calcium in the isolated perfused dog heart. Cellular calcium was immobilized by freezing the myocardium in diastole with an extremely cold fluorocarbon fluid (-125 degrees C). All subsequent procedures were conducted under conditions which prevented ionic diffusion, either at temperatures well below the freezing point of water or in the absence of water. Sarcolemmal and mitochondrial enriched fractions were segregated from dessicated, homogenized, myocardial tissue by ultracentrifugation utilizing density-gradients composed of blends of silicone and halocarbon on fluids within which physiological salts are insoluble. The total calcium content of these isolated fractions were then determined by atomic absorption spectrophotometry. Ouabain and epinephrine were subsequently used to alter the contractility of the perfused hearts and such contractile alterations were then related to changes noted in the calcium activity of the isolated subcellular fractions. In this study the calcium levels of the enriched mitochondrial fractions were elevated by both ouabain and epinephrine, while the calcium levels of the enriched sarcolemmal fractions were elevated only by ouabain. The advantage of this segregative procedure is that it prevents artifactual intercompartmental calcium rearrangement and preserves calcium levels to those initially fixed in situ at the time of freezing.     

Reexamination of the effect of urinary kallikrein on renin release: evidence that kallikrein does not release renin but protects renin from destruction

Previously, we showed that superfusion of rat kidney slices by rat urinary kallikrein stimulated renin release. The resin measurements were performed on superfusion samples which we stored frozen at -20 C for 24 h. In this study we investigated the effect of freezing on the renin concentration of superfusion samples in the control period. The renin concentration measured immediately without freezing was 9.8 +/- 2.4 ng angiotensin I/10 ml . 3 h/mg tissue, while the concentration in the samples frozen for 24 h was 2.9 +/- 1.0 ng angiotensin I/10 ml . 3 h/mg. The renin concentration of the superfusion samples during the kallikrein perfusion period was the same as that of the nonfrozen control samples. It appeared, therefore, that kallikrein acted as if it stimulated renin release from kidney slices, when the renin was measured in frozen samples. To clarify this phenomenon, we added kallikrein, inactivated kallikrein, and albumin to superfusion samples of the control period and froze the samples for 24 h. After freezing, the renin concentration of the control samples decreased to about 20% of that of nonfrozen samples, except in those samples to which the various proteins were added. In these samples, the loss of renin activity was prevented. The addition of Trasylol, a specific inhibitor of kallikrein, blocked the protective effect of both kallikrein and albumin. These data suggest that the renin released into the superfusion media of kidney slices is destroyed by freezing and that kallikrein or BSA prevents this destruction. These data negate previous data indicating that kallikrein stimulates renin release.