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Biomedical subjects

B Rubinsky

Publications and source records attributed to B Rubinsky.

At least 37 records · Page 2Linked to original sources
PubMed1994

Mechanical interactions between ice crystals and red blood cells during directional solidification.

Experiments in which red blood cells were frozen on a directional solidification stage under a microscope show that there is a mechanical interaction between ice crystals and cells in which cells are pushed and deformed by the ice crystals. The mechanical interaction occurs during freezing of cells in physiological saline and is significantly inhibited by the addition of 20% v/v glycerol to the solution. The addition of osmotically insignificant quantities of antifreeze proteins from the winter flounder or ocean pout to the physiological saline with 20% v/v glycerol generates strong mechanical interactions between the ice and the cells. The cells were destroyed during freezing in physiological saline, survived freezing in physiological saline with glycerol, and were completely destroyed by the addition of antifreeze proteins to the solution with glycerol. The difference in cell survival through freezing and thawing appears to be related, in part, to the habit of ice crystal growing in the suspension of red blood cells and the nature of mechanical interaction between the ice crystal and the cells. This suggests that mechanical damage may be a factor during cryopreservation of cells.

Animals↗
PubMed1994

MR imaging assisted temperature calculations during cryosurgery.

MRI has the potential of becoming an important imaging modality for monitoring the extent of the frozen region during cryosurgery. However, while the temperature history inside the frozen region is of utmost importance in determining the likely outcome of a cryosurgical procedure, it cannot be accessed directly through MRI because of the extremely low signal produced by the frozen region. We have developed a new MRI-assisted numerical technique that can calculate and display the temperature distribution in the frozen region on a standard MR image. The technique combines MR imaging data with a finite difference formulation of the energy equation. Here, the technique is described and experimental results that verify the technique are shown.

Algorithms↗
PubMed1994

1H magnetic resonance imaging of freezing and thawing in freeze-tolerant frogs.

Proton magnetic resonance imaging (MRI) of the processes of freezing and thawing in the wood frog Rana sylvatica provided noninvasive and real-time analysis of the mode of ice propagation through the body of a freeze-tolerant vertebrate. MRI revealed a directional movement of ice from the exterior inward that required several hours to reach completion. Freezing in core organs such as liver, which produces and exports cryoprotectant, and heart, which circulates it, was delayed and occurred well after the organs were surrounded by extraorgan ice. Natural thawing was a very different process; thawing began uniformly throughout the body, but core organs melted more rapidly than peripheral ones, an adaptation that may be key to the early restoration of heartbeat and breathing. The images presented demonstrate the sensitivity and power of MRI and its potential to become a critical monitoring technology in the development of cryopreservation techniques for mammalian organ explants.

Acclimatization↗
PubMed1994

Freeze tolerance in turtles: visual analysis by microscopy and magnetic resonance imaging.

Two visual techniques were used to analyze the patterns of natural freezing and thawing in freeze-tolerant hatchling painted turtles Chrysemys picta marginata. Directional solidification plus light microscopy of liver, heart, and skeletal muscle slices was used to compare freezing at -4 degrees C (a survivable temperature in vivo) and -20 degrees C (not survivable). At -4 degrees C tissues showed large amounts of ice in expanded extracellular and vascular spaces, occupying 36% (liver) and 61% (muscle) of total tissue volume. Cells at -4 degrees C were shrunken, but intracellular water remained; at -20 degrees C, however, cells showed little evidence of free water. Liver micrographs showed novel spherical shells of water associated with intracellular particles (apparently glycogen granules) suggesting that a noncolligative method of cell water retention was employed. Proton magnetic resonance imaging was used for noninvasive analysis of freezing and thawing in the intact animal. Images showed that freezing propagated in a directional manner through the body with ice formed first in extraorgan spaces (e.g., abdominal cavity, brain ventricles). However, thawing occurred uniformly throughout the body core, and organs melted more rapidly than the extraorgan ice surrounding them.

Acclimatization↗
PubMed1994

Temperature gradient osmometer and anomalies in freezing temperatures.

We have developed a new device that measures freezing and melting temperatures in nanoliter volume samples and can be used as a "freezing point osmometer" with a resolution many orders of magnitude greater than that of existing freezing point osmometers. Using this device we found anomalies in the depression of the freezing temperature and thermal hysteresis in aqueous solutions of hydrophilic amino acids, polyamino acids, and lectins. These anomalies would not have been possible to detect with currently used technology. The compounds that produce anomalies in freezing temperature were reported in the literature as having the ability to bind to cell membranes. This suggests a relation between a molecule's ability to bind to cell membranes and its anomalous freezing temperature depression. The new freezing point osmometer and our results could be important for studying and understanding organic molecules and their interaction with membranes and water.

Amino Acids↗
PubMed1993

Antifreeze glycoproteins increase solution viscosity.

A new microsensor that can analyze microliter volume samples was used to measure the viscosity of aqueous solutions of antifreeze glycoproteins as a function of temperature and concentration. The results show that at physiological concentrations which naturally occur in the fish, the antifreeze proteins significantly increase aqueous solution viscosity. The probability for ice nucleation is inversely proportional to viscosity. Therefore, the increased viscosity could explain, in part, reports on the beneficial effects of antifreeze glycoproteins during cryopreservation by vitrification. Reducing the probability for ice nucleation could be also beneficial for the survival of cold-water fish in their natural habitat. Millimolar concentrations of antifreeze proteins increase aqueous solution viscosity to values comparable with those of conventional cryoprotectants in molar concentrations.

Animals↗
PubMed1993

Transrectal ultrasound-guided percutaneous radical cryosurgical ablation of the prostate.

BACKGROUND: The two major treatments for prostate cancer, radical prostatectomy and radiation therapy, are associated with considerable morbidity and variable results. This article presents the preliminary results using percutaneous radical cryosurgical ablation under ultrasound guidance to treat prostate cancer. METHODS: The patient group consisted of all patients with localized prostate cancer who underwent cryosurgery between June 1, 1990 and May 1, 1992. Patients in Group 1 were treated by freezing of the tumor with two cryoprobes placed multiple times. Group 2 patients were treated by freezing of the tumor with five cryoprobes placed simultaneously. Cryoprobes (3 mm in diameter) were placed percutaneously with a transperineal approach. Cryoprobe placement and freezing were monitored using the transrectal ultrasound. RESULTS: Of the 55 patients (68 procedures) undergoing treatment, 23 have 3 months of follow-up with associated biopsy (Group 1, 8 patients; Group 2, 15 patients). In Group 1, three (37.5%) patients had residual disease. In Group 2, one (6.7%) patient had residual disease, whereas 14 (93.3%) patients did not. Combining both groups, 19 (82.6%) patients had no residual disease, whereas 4 (17.4%) patients had positive results on postoperative biopsy. Complications included rectal freezing, urethrorectal fistula, sloughing urethral tissue, impotence, perineal ecchymosis, penile edema, and ileus. CONCLUSIONS: Preliminary results indicate that percutaneous transperineal ultrasound-guided prostate cryosurgery may be an effective treatment for prostate cancer with minimal associated morbidity.

Biomarkers, Tumor↗
PubMed1993

Cryogenic protection of oocytes with antifreeze proteins.

Proteins belonging to a family of compounds known as "antifreeze proteins" interact with oocytes and protect the oolemma from damage at cryogenic temperatures. Experiments were performed with pig oocytes rapidly cooled to cryogenic temperatures in vitrifying solutions with and without antifreeze proteins. Four different types of antifreeze polypeptides and glycoproteins were tested. The integrity of the oolemma was examined with Fluorescein Diacetate (FDA) staining and morphological examinations. Results show that the pig oocyte oolemma is a primary site of injury during exposure to low temperatures and that all the different proteins have a similar ability to interact with and protect the oolemma. Our results may be important in developing solutions for long-term preservation of oocytes at cryogenic temperatures (cryopreservation).

Animals↗
PubMed1993

Monitoring cryosurgery in the brain and in the prostate with proton NMR.

This brief communication reports the results of preliminary studies performed to evaluate the feasibility of using NMR imaging to monitor the freezing of tissue during cryosurgery. Two tissues were studied, rabbit brain and dog prostate. NMR imaging of these tissues provided a clear distinction between frozen and unfrozen tissue and an accurate assessment of the extent of freezing in real time.

Animals↗
PubMed1993

A morphological study of cooling rate response in normal and neoplastic human liver tissue: cryosurgical implications.

The process of freezing in normal human livers and in human liver tumors was studied by freezing samples of these tissues with constant cooling rates and then examining the morphology of the frozen tissue, after freeze substitution, with the light microscope. Cooling rates varied from 2 degrees C/min up to approximately 2000 degrees C/min. It was observed that high cooling rates produce extensive intracellular ice in both normal and neoplastic liver. At slow rates of cooling, normal and neoplastic liver cells dehydrated and large extracellular ice crystals formed. Comparison of the frozen normal liver and the frozen malignant tumors shows that for the same rates of freezing, the tumor cells retain more cellular water and therefore show less susceptibility to dehydration at low rates of cooling. At slow cooling rates, the amount of cellular dehydration and consequent vascular and interstitial space engorgement changed with the type of tissue frozen. The greatest amount of dehydration occurred in normal human liver, followed by metastatic colon carcinoma and finally primary hepatocellular carcinoma. These results are important for cryosurgery since they suggest that malignant tissues have a different response to freezing than normal tissues.

Adenocarcinoma↗
PubMed1993

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 degrees 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.

Animals↗
PubMed1993

MRI-monitored cryosurgery in the rabbit brain.

The inability to observe the transient, irregular shape of the frozen region that develops during cryosurgery has inhibited the application of this surgical technique to the treatment of tumors in the brain and deep in visceral organs. We used proton NMR spin-echo and spoiled gradient-echo imaging to monitor the development of frozen lesions during cryosurgery in the rabbit brain and the resulting postcryosurgical changes up to 4 hr after freezing. Spoiled gradient-echo images (TE = 14 ms; TR = 50 ms) were acquired during freezing and thawing at a rate of 15 s/slice. Although the frozen region itself is invisible in MR images, its presence is distinguished easily from the surrounding unfrozen soft tissue because of the large contrast difference between frozen and unfrozen regions. T2-weighted spin-echo images (TE = 100 ms, TR = 2 s) obtained after thawing suggest that edema forms first at the margin of the region that was frozen (cryolesion) and then moves into the region's core. Histological examination showed complete necrosis in the cryolesion and a sharp transition to undamaged tissue at the margin of the lesion and its image. Blood-brain barrier (BBB) damage was investigated using gadolinium-DTPA. The region of edema in the T2-weighted spin-echo images was coincident with the area of BBB damage in the Gd-DTPA-enhanced T1-weighted spin-echo images (TE = 33 ms, TR = 400 ms) and both were distinguishable as areas of high signal relative to the surrounding normal tissue. The results of these experiments indicate that MR can both effectively monitor the cryosurgical freezing and thawing cycle and characterize the postcryosurgical changes in tissue during follow-up.

Animals↗
PubMed1993

Viscosity sensing with lamb-wave microsensor: dimethylsulfoxide solution viscosity as a function of temperature.

Recently, a new microsensor employing low-velocity ultrasonic Lamb waves was developed and demonstrated to be capable of measuring the viscosity of solutions in small volumes. The microsensor, when attached to a temperature-controlled stage, can measure viscosity as a function of temperature. In this investigation, the ultrasonic Lamb-wave oscillator is employed to experimentally measure the viscosity of dimethylsulfoxide (Me2SO) solutions as a function of temperature. The microsensor and the experimental procedure are described and results for 1M, 3M, and 5M Me2SO aqueous solutions are presented. Dimethylsulfoxide is a compound commonly employed as a cryoprotectant in cryopreservation, the low-temperature preservation of biological materials. The temperature dependence of viscosity obtained through this study can be used in determining the probability for ice nucleation in biological materials, a parameter of importance during cryopreservation.

Calibration↗
PubMed1992

Structural and functional similarity between fish antifreeze proteins and calcium-dependent lectins.

A cDNA for a type II antifreeze protein was isolated from liver of smelt (Osmerus mordax). The predicted protein sequence is homologous to that from sea raven (Hemitripterus americanus) and both show homology to a family of calcium-dependent lectins. Smelt and sea raven belong to taxonomic orders believed to have diverged prior to Cenozoic glaciation. Thus, type II antifreeze proteins appear to have evolved independently in these fish species from pre-existing calcium-dependent lectins. Sequence alignment of the antifreezes and the lectins suggest that these proteins adopt a similar fold, that the sea raven antifreeze has lost its Ca2+ binding sites, and the smelt antifreeze has retained one site. Experiments show that smelt antifreeze protein activity is responsive to Ca2+ but that of sea raven antifreeze protein is not. These results suggest that the type II fish antifreeze proteins and calcium-dependent lectins share a common ancestry, related folding structures, and functional similarity.

Amino Acid Sequence↗
PubMed1992

The cryoprotective effect of antifreeze glycopeptides from antarctic fishes.

Apparently vitrified cells and tissues often fail to survive, probably from damage from growth of microscopically invisible ice crystals. Special biological antifreezes from some polar fishes have been shown to adsorb to specific faces of ice crystals and inhibit crystal growth. Vitrification in the presence of antifreezes therefore may help enhance postvitrification viability of cells and tissues. We report here that the addition of fish antifreeze glycopeptides (AFGPs) to vitrifying solutions increases post-thaw viability in cultured immature pig oocytes and two-cell stage embryos of mice and pigs after rapid cooling to cryogenic temperatures. The criterion for viability is maturation to metaphase for the oocytes and the ability to develop into the four-cell stage for the pig embryo and the blastocyst stage for the mouse embryo. Without AFGPs, or with addition of antifreeze peptides (AFPs), the particular vitrifying solution and cooling/warming/culturing regime used in this study produced zero viability. In the presence of the AFGPs (40 mg/ml), survival of pig oocytes and embryos was increased to about 25%, and that of mouse embryos to 82%. Dose-response studies for the mouse embryos showed that the protective effect of AFGPs shows saturation kinetics and levels off at 20 mg/ml. The AFGPs appeared to preserve cell membrane structural integrity; however, an intact cell membrane did not always lead to viability. The absence of protective effect by AFPs suggests that protection by the AFGPs is unrelated to their common antifreeze property, i.e., inhibition of ice crystal growth, but probably results from interaction with and stabilization of the cell membranes unique to the AFGPs.

Animals↗
PubMed1992

An analytical study of cryosurgery in the lung.

The process of freezing in healthy lung tissue and in tumors in the lung during cryosurgery was modeled using one-dimensional close form techniques and finite difference techniques to determine the temperature profiles and the propagation of the freezing interface in the tissue. A thermal phenomenon was observed during freezing of lung tumors embedded in healthy tissue, (a) the freezing interface suddenly accelerates at the transition between the tumor and the healthy lung, (b) the frozen tumor temperature drops to low values once the freezing interface moves into the healthy lung, and (c) the outer boundary temperature has a point of sharp inflection corresponding to the time at which the tumor is completely frozen.

Algorithms↗
PubMed1992

Fish antifreeze proteins block Ca entry into rabbit parietal cells.

Many fish and insects have adapted to life at subfreezing temperatures by evolving so-called antifreeze proteins (AFP) that noncolligatively depress the freezing temperatures of aqueous solutions without affecting the melting temperature. AFP have been thought to function solely as antifreezes. Recently, however, we discovered that AFP also protect mammalian cells and organs from damage caused by exposure to hypothermic (above freezing) temperatures. It has been proposed that hypothermic damage is caused by changes in intracellular ionic content due to a reduction of active transport that is required to balance passive ion transport across cell membranes. Given this possibility, we tested whether AFP isolated from the Newfoundland ocean pout might reduce the Ca ion permeability of a mammalian cell, the rabbit gastric parietal cell, which has been particularly well studied in terms of Ca transport and signaling. Digital image processing of the Ca-sensitive fluorescent indicator fura-2 was used to measure intracellular free Ca in these cells. During stimulation with the cholinergic agonist carbachol, AFP inhibited passive Ca entry across the cell membrane without interfering with either the release of Ca from internal stores (indicating that the carbachol receptor and other signaling events were operational) or the normal active rates of Ca efflux from the cell (indicating that Ca pumping was also still intact). These results suggest that, in addition to their actual antifreeze properties, AFP may also help to confer cold tolerance in animals by preventing passive Ca entry into epithelial cells.

Action Potentials↗