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A pig model of hepatic cryotherapy. In vivo temperature distribution during freezing and histopathological changes.

We aimed to assess the temperature distribution in the cryolesion during hepatic cryotherapy and the association with postoperative histological changes to optimise the technique and allow better preoperative planning. Hepatic cryolesions were produced in 22 pigs following laparotomy using a CMS-cryosystem and 8mm-AccuProbe-Cryoprobes. The temperature was measured in 1 min intervals at different distances from the probe during freezing. The animals were treated in 5 groups: (i) single freezing of 20 min; (ii) double freezing of 20 min each; (iii) single freezing of 40 min; (iv) single freezing of 20 min (n=4), histology at 1 week p.o., and (v) single freezing of 20 min and Pringle manoeuvre; [(i)-(iii) and (v): histology at 24 h p.o.]. The mean diameter of the -38 degrees C isotherm, i.e., the zone of effective treatment for colorectal metastases was 37 mm for group (i) with a mean iceball diameter of 59 mm and about 46 mm for groups (ii, iii, and v) with mean iceball diameters of 78, 75, and 75 mm, respectively. At 7 days postoperatively secondary necrosis was seen in the largest central part of the lesion, wherever temperatures of -15 degrees C or lower were achieved during cryosurgery. Under the hypothesis that -38 degrees C is the effective temperature for the destruction of colorectal liver metastases, a lesion of 37-mm diameter may be effectively treated with a single 8mm-AccuProbe-Cryoprobe and a 20 min single freeze cycle and a lesion of 46 mm may be effectively treated when a double freeze-thaw cycle of 20 min each, a single freeze cycle of 40 min, or a 20 min single freeze cycle with additional Pringle manoeuvre is used, when it is perfectly placed in the lesion.

Animals↗

Studies of a putative ice-binding motif in winter flounder skin-type anti-freeze polypeptide.

Winter flounder contains two distinct anti-freeze protein isoforms, which are the liver-type extracellular anti-freeze proteins and the skin-type intracellular anti-freeze protein. The skin-type anti-freeze proteins exhibit lower anti-freeze activities than the liver-type isoforms and this might be due to their lacking complete ice-binding motifs. One of the skin-type anti-freeze proteins, skin-type anti-freeze protein-3, does contain putative overlapping ice-binding motifs with the sequences '-K-DT-' and '-DT-K-'. Synthetic anti-freezes containing 0-3 repeats of the '-DT-K-' motif were tested for stability and activity. Loss of the single '-DT-K-' of skin-type anti-freeze protein-3 increases the anti-freeze activity and increasing the number of motifs to two or three lowers the activity. The decrease in activity with an increasing frequency of the motif correlates with a decrease in the helical content of these peptides at 0 degrees C.

Animals↗

Surface-induced denaturation of proteins during freezing and its inhibition by surfactants.

In this study, we found that the denaturation of proteins during freezing is closely related to surface-induced denaturation. Several proteins with varying sensitivities to freezing were tested, and the results were compared with susceptibilities to surface denaturation in unfrozen aqueous solution. Also, the influence of the surfactant Tween 80 on the denaturation of each of the proteins was examined during freeze-thawing, as were the effects of Tween 80 and several other surfactants on the stability of lactate dehydrogenase. Proteins formed insoluble precipitates when they were subjected to a quench cooling by dipping in liquid nitrogen, although freezing followed by supercooling caused less precipitation. A strong correlation (r = 0.99) was observed between the tendency of a protein to freeze denature and its tendency to surface denature. Also, the addition of small amounts of surface-active agents protected proteins from both freeze- and surface-induced denaturation. Freeze-induced denaturation of IL-1ra at the ice-water interface during freeze-drying was effectively prevented by adding a small amount of Tween 80. These results suggest that the denaturation of proteins during freeze-thawing can be ascribed primarily to the increase in the area of the ice-water interface during freezing.

Freezing↗

Oocyte freezing: here to stay?

Oocyte freezing is an established technology but, in contrast to embryo freezing, it has very limited application in clinical IVF programmes. Is there a chance that oocyte freezing will become an integrated routine in assisted reproductive technology? The delicate cytological architecture of the oocyte with a cold-sensitive spindle and a hardening zona have made the frozen oocyte 'unwanted' in assisted reproductive technology. Nevertheless, empirical improvements in freezing protocols and the use of ICSI for fertilization have led to an increasing number of live births. This mitigates against a simple ban on oocyte freezing. While efficiency of oocyte freezing can certainly be further improved by basic research, it is clear that there are humanitarian reasons for considering oocyte freezing as a future fully utilized assisted reproductive technology. The storage of the female genome as a particulate entity can provide an alternative in case of moral, ethical, legal or religious concerns about embryo freezing. Oocyte freezing can also offer hope for oocyte donation and preservation of fertility for women facing ovarian loss. The message is one of cautious optimism when looking for a place for oocyte freezing in routine assisted reproductive technology.

Animals↗

Effects of summer drought and winter freezing on stem hydraulic conductivity of Rhododendron species from contrasting climates.

We studied the limits to maximum water transport in three diffuse-porous evergreen shrubs exposed to frequent winter freeze-thaw events (Rhododendron maximum L. and R. catawbiense Michaux from the Appalachian Mountains) and to a severe summer drought (R. macrophyllum G. Don. from the Oregon Cascades). Percent loss of hydraulic conductivity (PLC), vulnerability curves to xylem embolism and freezing point temperatures of stems were measured over 2 years. Controlled freeze-thaw experiments were also conducted to determine the effect of thaw rate on PLC. During both years, native PLC was significantly higher in winter than in summer for R. macrophyllum. Seasonal changes in PLC were variable in both R. catawbiense and R. maximum. Only R. maximum plants growing in gaps or clearings showed higher PLC than understory plants. A rapid (2-4 day) natural recovery of high native PLC during the winter was observed in both R. maximum and R. macrophyllum. Compared with the bench-dehydration method, vulnerability curves based on the air-injection method consistently had less negative slopes and greater variation. Fifty percent PLC (PLC(50)) obtained from vulnerability curves based on the dehydration method occurred at -1.75, -2.42 and -2.96 MPa for R. catawbiense, R. maximum and R. macrophyllum, respectively. Among the study species, R. macrophyllum, which commonly experiences a summer drought, had the most negative water potential at PLC(50). In all species, stem freezing point temperatures were not consistently lower in winter than in summer. A single fast freeze-thaw event significantly increased PLC, and R. catawbiense had the highest PLC in response to freezing treatments. Recovery to control PLC values occurred if a low positive hydraulic pressure was maintained during thawing. Rhododendron macrophyllum plants, which commonly experience few freeze-thaw events, had large stem diameters, whereas plants of R. catawbiense, which had small stem diameters, suffered high embolism in response to a single freeze-thaw event. Both drought-induced and winter-induced embolism caused a significant reduction in hydraulic conductivity in all species during periods when drought or freeze-thaw events occurred in their native habitats. However, rapid recovery of PLC following freezing or drought maintained the species above their relatively low margins of safety for complete xylem dysfunction.

Climate↗

Tracheid diameter is the key trait determining the extent of freezing-induced embolism in conifers.

We tested the hypotheses that freezing-induced embolism is related to conduit diameter, and that conifers and angiosperms with conduits of equivalent diameter will exhibit similar losses of hydraulic conductivity in response to freezing. We surveyed the freeze-thaw response of conifers with a broad range of tracheid diameters by subjecting wood segments (root, stem and trunk wood) to a freeze-thaw cycle at -0.5 MPa in a centrifuge. Embolism increased as mean tracheid diameter exceeded 30 microm. Tracheids with a critical diameter greater than 43 microm were calculated to embolize in response to freezing and thawing at a xylem pressure of -0.5 MPa. To confirm that freezing-induced embolism is a function of conduit air content, we air-saturated stems of Abies lasiocarpa (Hook.) Nutt. (mean conduit diameter 13.7 +/- 0.7 microm) by pressurizing them 1 to 60 times above atmospheric pressure, prior to freezing and thawing. The air saturation method simulated the effect of increased tracheid size because the degree of super-saturation is proportional to a tracheid volume holding an equivalent amount of dissolved air at ambient pressure. Embolism increased when the dissolved air content was equivalent to a mean tracheid diameter of 30 microm at ambient air pressure. Our centrifuge and air-saturation data show that conifers are as vulnerable to freeze-thaw embolism as angiosperms with equal conduit diameter. We suggest that the hydraulic conductivity of conifer wood is maximized by increasing tracheid diameters in locations where freezing is rare. Conversely, the narrowing of tracheid diameters protects against freezing-induced embolism in cold climates.

Abies↗

Signal transduction, second messenger, and protein kinase responses during freezing exposures in wood frogs.

Changes in the percentage of adenosine 3', 5'-cyclic monophosphate (cAMP)-dependent protein kinase present as the active catalytic subunit (PKAc) and in the levels of the second messengers cAMP, guanosine 3',5'-cyclic monophosphate (cGMP), and D-myo-inositol 1,4,5-trisphosphate (IP3) were quantified in tissues of the freeze-tolerant wood frog Rana sylvatica over the course of freezing at -2.5 degrees C and thawing at 5 degrees C. Freezing exposure rapidly raised liver cAMP concentration and %PKAc (by 2- and 6-fold, respectively) within 2 min postnucleation; both peaked and stabilized between 5 and 60 min postnucleation but declined with longer freezing. Other organs also showed elevated PKAc during freezing, particularly skeletal muscle. By contrast, cGMP concentration was reduced in muscle and kidney after 24 h of freezing but rose after thawing in muscle. Liver also showed a twofold elevation of cGMP during thawing. The protein kinase C (PKC) second messenger, IP3, rose throughout freezing in liver, reaching levels 11-fold higher than control values after 24 h of freezing. IP3 was also elevated in brain after 4 and 8 h of freezing. The different patterns of cAMP, protein kinase A (PKA), and IP3 changes in liver suggest that, whereas cAMP and PKA clearly mediate the rapid activation of glucose output as a cryoprotectant, IP3 and PKC may be involved instead with metabolic responses that deal with the consequences of long-term freezing, such as ischemia resistance or cell volume control.

Animals↗

Seasonal changes in the freezing behavior of xylem ray parenchyma cells in four boreal hardwood species

The freezing behavior of xylem ray parenchyma cells in several boreal hardwood species, namely, Betula platyphylla, Populus canadensis, P. sieboldii, and Salix sachalinensis, was examined by differential thermal analysis (DTA), cryo-scanning electron microscopy (Cryo-SEM), and freeze-fracture replica electron microscopy. Although DTA profiles of samples harvested in summer and in winter suggested that the xylem ray parenchyma cells in all four species responded to freezing stress by extracellular freezing, Cryo-SEM showed clearly that the xylem ray parenchyma cells in all these species responded to freezing stress by shallow supercooling in summer and by extracellular freezing in winter. It is suggested that DTA failed to reveal the true freezing behavior of xylem ray parenchyma cells because of an overlap of temperature ranges between the high-temperature exotherm and the low-temperature exotherm and/or because of the limited extent of the LTE. The seasonal changes in freezing behavior of xylem ray parenchyma cells in all these boreal species, which are results of seasonal cold acclimation, support the hypothesis that a gradual shift of freezing behavior in xylem ray parenchyma cells from shallow supercooling in hardwood species that grow in tropical zones to extracellular freezing in hardwood species that grow in cold areas might be a result of the evolutionary adaptation of hardwood species to cold climates. Copyright 1999 Academic Press.

Journal Article↗

Wheeled and standard walkers in Parkinson's disease patients with gait freezing.

OBJECTIVES: Compare the efficacy of two walking assistance devices (wheeled walker and standard walker) to unassisted walking for patients with PD and gait freezing. BACKGROUND: Although numerous walking devices are used clinically, their relative effects on freezing and walking speed have never been systematically tested. METHODS: Nineteen PD patients (14 non-demented) walked under three conditions in randomized order: unassisted walking, standard walker, and wheeled walker. Patients walked up to three times in each condition through a standard course that included rising from a chair, walking through a doorway, straightway walking, pivoting, and return. Total walking time, freezing time and number of freezes were compared for the three conditions using mixed models (walking time) and Friedman's test (freezing). The wheeled walker was further studied by comparing the effect of an attached laser that projected a bar of light on the floor as a visual walking cue. RESULTS: Use of either type of device significantly slowed walking compared to unassisted walking. Neither walker reduced any index of freezing, nor the laser attachment offered any advantage to the wheeled walker. The standard walker increased freezing, and the wheeled walker had no effect on freezing. Among the non-demented subjects (n=14), the same patterns occurred, although the walking speed was less impaired by the wheeled walker than the standard walker in this group. CONCLUSIONS: Though walkers may stabilize patients and increase confidence, PD patients walk more slowly when using them, without reducing freezing. Because the wheeled walker was intermediate for walking time and does not aggravate freezing, if walkers are used for these subjects, this type of walker should be favored.

Aged↗

Cryopreservation of pronucleate mouse ova: slow versus ultrarapid freezing.

The usefulness of ultrarapid freezing of mouse pronucleate ova was investigated in comparison with slow, programmed freezing. Pronucleate mouse ova were frozen using an ultrarapid method in either 3.5 M dimethylsulphoxide (DMSO), 3.5 M propanediol (PROH) or a 1:1 mixture of both. After a brief exposure to the cryoprotectant, they were plunged into liquid nitrogen. Thawing was done at 37 degrees C and the cryoprotectant was rapidly diluted in a sucrose solution. Pronucleate ova were slowly cooled in a biological freezer using 1.5 M PROH as cryoprotectant. Thawing was done at room temperature and PROH was removed by multi-step dilutions. As control groups, pronucleate ova were either given no treatment, or exposed to PROH or DMSO without freezing and cultured in vitro. Ultrarapid freezing using 3.5 M DMSO as cryoprotectant resulted in rates of survival, cleavage and post-thaw development to blastocysts of 85, 89 and 37%, respectively. PROH as cryoprotectant, however, was inadequate in the ultrarapid freezing protocol and the combination of DMSO and PROH showed no further improvement. Slow, programmed freezing (using PROH) compared to ultrarapid freezing (using DMSO) resulted in similar rates of survival, cleavage and development of 80, 84, 20% and 92, 81 and 23% respectively. In conclusion, DMSO is a better cryoprotectant than PROH for ultrarapid freezing of pronucleate mouse ova, and ultrarapid freezing with 3.5 M DMSO is as effective as slow freezing with 1.5 M PROH, as evaluated by their subsequent development in vitro.

Animals↗

Value of a second freeze-thaw cycle in cryotherapy of common warts.

A study of open, randomized, parallel-group design was performed to investigate the impact of a second freeze-thaw cycle on the cure rate, at 3 months, from cryotherapy of common warts on the hands and feet. Cryotherapy was performed at 3-week intervals, and subjects were randomized to receive either one or two freeze-thaw cycles. In addition, all subjects used keratolytic wart paints throughout the study, and plantar warts were pared prior to freezing. Three hundred subjects were recruited. At 3 months, 124 were cured, 83 were not cured, and 93 had defaulted. Among those who did not default the cure rate was 57% from the single freeze technique, and 62% from the double freeze technique, a difference of 5% (P = 0.53, 95% CI-8.1-18.6). Separate analyses for subjects with warts on the hands and on the feet demonstrated no effect of double freezing on hand warts. In contrast, for plantar warts, the cure rate was 41% from single freezing and 65% for double freezing, a difference of 24% (P = 0.04, 95% CI 2.9-44.4). The use of a double freeze-thaw cycle confers little or no advantage over a single freeze in the treatment of hand warts, but may be considerably more effective for plantar warts.

Adult↗

The influence of rotational exercises on freezing in Parkinson's disease.

The advanced stage of Parkinson's disease (PD) is characterised by complex movement disturbances including freezing. Because freezing is resistant to drug therapy, there has recently been renewed interest in non-pharmacological treatment programmes. In this study the effect of rotational stimulation on freezing was investigated. Eight patients with idiopathic PD and freezing participated in the study. Switching from foot-lifting to 'stand up and walk' provoked freezing in all the tested patients. The mean OPMSP (Onset of Premotor Silence Period) in the EMG of the m. tibialis anterior during the 'foot-lifting' sessions preceding freezing was 124 msec. This value, together with a striking repetition of the EMG discharges in the m. tibialis anterior following the request to 'stand up and walk', was related to the inducement of freezing, as a decrease of 42 msec in the OPMSP (t = 2.61; p < or = 0.01) after rotational stimulation abolished freezing and the concomitant EMG disturbances. Rotational stimulation also reduced freezing frequency during daily life. Freezing periods fell to below 50% of the pre-treatment level the day following rotation (t = 5.58; p < or = 0.001). The OPMSP predicted the short and long-term effects of rotational exercises (R = 0.74; p < or = 0.03). The rather long-lasting effect of the stimulation suggests a possible modulation of neurochemical transmission. Further studies are required to shed more light on the point of action and to elucidate which neurotransmitter might be involved.

Aged↗

Hepatitis B vaccine freezing in the Indonesian cold chain: evidence and solutions.

OBJECTIVES: To document and characterize freezing temperatures in the Indonesian vaccine cold chain and to evaluate the feasibility of changes designed to reduce the occurrence of freezing. METHODS: Data loggers were used to measure temperatures of shipments of hepatitis B vaccine from manufacturer to point of use. Baseline conditions and three intervention phases were monitored. During each of the intervention phases, vaccines were removed progressively from the standard 2-8 degrees C cold chain. FINDINGS: Freezing temperatures were recorded in 75% of baseline shipments. The highest rates of freezing occurred during transport from province to district, storage in district-level ice-lined refrigerators, and storage in refrigerators in health centres. Interventions reduced freezing, without excessive heat exposure. CONCLUSIONS: Inadvertent freezing of freeze-sensitive vaccines is widespread in Indonesia. Simple strategies exist to reduce freezing - for example, selective transport and storage of vaccines at ambient temperatures. The use of vaccine vial monitors reduces the risk associated with heat-damaged vaccines in these scenarios. Policy changes that allow limited storage of freeze-sensitive vaccines at temperatures >2-8 degrees C would enable flexible vaccine distribution strategies that could reduce vaccine freezing, reduce costs, and increase capacity.

Cold Temperature↗

Cryopreservation of human spermatozoa: comparison of two cryoprotectants and two freezing methods.

A prospective experimental study was performed to compare sperm cryosurvival rate between two kinds of cyroprotective media: citrate-egg yolk-glycerol (CEG) and TEST-citrate-egg yolk-glycerol (TEST-EYG), and between two freezing methods: noncontrolled rate (NCR) freezing and computer controlled rate (CR) freezing. One hundred semen samples were included in the study. Each sample was divided into two parts, which were prepared as two CEG and two TEST-EYG mixture straws, then one straw from each mixture was frozen by the NCR-freezing method and the complementary straw was frozen by the CR-freezing method. After one week of cryostorage, the straws were thawed and the post-thaw sperm survival was assessed. As the results, the post-thaw sperm survival rate of the CEG media group was slightly higher than the TEST-EYG group in both the NCR-freezing method (51.2% vs 47.0%, p < 0.05) and the CR-freezing method (48.2% vs 43.4%, p < 0.05); and similarly; the NCR-freezing group yielded a slightly higher post-thaw sperm survival rate than the CR-freezing group in both the CEG media used (51.2% vs 48.2%, p < 0.05) and the TEST-EYG media used (47.0% vs 43.4%, p < 0.05). However, these did not have clinical significance because the differences were too small. In conclusion, the use of either CEG or TEST-EYG media, and either the NCR or CR-freezing method did not have clinical differences on the post-thaw sperm survival.

Adult↗

The influence of freeze duration on postfreeze recovery by caterpillars of Pyrrharctia isabella (Lepidoptera: Arctiidae): when is survival enough to qualify as recovery?

The effects of freeze exposure at -4.5 degrees C for as long as 6 weeks on long-term postfreeze survival and body fluid composition were investigated in caterpillars (woolly bears) of the arctiid moth Pyrrharctia isabella. Woolly bears routinely survived the initial postfreeze period with little difference between the 1-week (100%) and 6-week (95%) freeze treatments. Caterpillars in the latter treatment, however, reached the pupal stage almost one half as often as woolly bears in the 1-week freeze treatment. The success rate for adult emergence was not different for pupae from the two treatments (ca. 50%). Woolly bears responded to cold acclimation by accumulating glycerol to levels exceeding 300 mM although this was not augmented by extending the acclimation period to 6 weeks. There was a significant (P<0.05) rise in hemolymph [K+] during the first week of the freeze (23.4-37.8 mM), which then remained stable over the remainder of the 6-week freeze period. Hemolymph [Na+] did not change from the prefreeze level over the course of the freeze treatment. Body water content showed a modest rise during the course of the freeze treatment but the underlying cause for this change was uncertain. Prolonged freeze exposure had a major impact on long-term survival of the woolly bears but this was not reflected by any instability in body fluid composition. Moreover, short-term recovery was not an effective indicator of the tolerance of P. isabella caterpillars to prolonged freezing.

Adaptation, Physiological↗

Mechanism of freeze-thaw instability of aluminum hydroxycarbonate and magnesium hydroxide gels.

The effect of freeze-thaw cycles on the physical stability of aluminum hydroxycarbonate and magnesium hydroxide gels was studied. Coagulation following a freeze-thaw cycle, leading to the formation of visible aggregates, affected the content uniformity of both gels. The freeze-thaw cycles did not affect the crystal form or surface characteristics of the gels as determined by X-ray powder diffraction and point of zero charge, but caused a slight reduction in the rate of acid neutralization and a large increase in the rate of sedimentation. The greatest effect was observed after the first freeze-thaw cycle. While the duration of freezing was not a factor, the rate of freezing was important and was inversely related to the aggregate size. The aggregates which formed following a freeze-thaw cycle were not redispersed by shaking, but were reversed by ultrasonic treatment or homogenization. The adsorption of polymers or surface-active agents prior to freezing reduced and, in some cases, prevented the formation of aggregates. The physical instability produced by a freeze-thaw cycle was explained by the modified DLVO theory. The force exerted on the particles by the growing ice crystals forced the particles into the primary minimum, producing strong interparticle attraction. On thawing, simple agitation did not provide enough force to overcome the attractive force of the primary minimum. Adsorption of polymers or surface-active agents increased the steric repulsive force and prevented the particles from reaching the primary minimum.

Aluminum Hydroxide↗

Freeze-thaw effects on metabolic enzymes in wood frog organs.

To determine whether episodes of natural freezing and thawing altered the metabolic makeup of wood frog (Rana sylvatica) organs, the maximal activities of 28 enzymes of intermediary metabolism were assessed in six organs (brain, heart, kidney, liver, skeletal muscle, gut) of control (5 degrees C acclimated), frozen (24 h at -3 degrees C), and thawed (24 h back at 5 degrees C) frogs. The enzymes assessed represented pathways including glycolysis, gluconeo-genesis, amino acid metabolism, fatty acid metabolism, the TCA cycle, and adenylate metabolism. Organ-specific responses seen included (a) the number of enzymes affected by freeze-thaw (1 in gut ranging to 17 in heart), (b) the magnitude and direction of response (most often enzyme activities decreased during freezing and rebounded with thawing but, liver showed freeze-specific increases in several enzymes), and (c) the response to freezing versus thawing (enzyme activities in gut and kidney changed during freezing, whereas most enzymes in skeletal muscle responded to thawing). Overall, the data show that freeze-thaw implements selected changes to the maximal activities of various enzymes of intermediary metabolism and that these may aid organ-specific responses that alter fuel use during freeze-thaw, support cryoprotectant metabolism, and aid organ endurance of freeze-induced ischemia.

Acclimatization↗

Osmotic stress effects on the freezing tolerance of the antarctic nematode Panagrolaimus davidi.

The freezing and freezing survival of the Antarctic nematode Panagrolaimus davidi after exposure to solutions of different osmotic concentrations has been examined using a thermoelectric cooling stage and multi-specimen cooling block to see if there is any evidence that freeze-induced desiccation prevents inoculative freezing. The nematodes froze in all the test solutions used (up to 1138 mosmol.l-1) and at all cooling rates and nucleation temperatures tested. Freezing survival was at its maximum in 0.1 mol.l-1 NaCl in artificial tap water after 1 h exposure to the test solution and in artificial tap water after 24 h exposure. Hyperosmotic and hyposmotic stress adversely affected the nematodes' ability to survive freezing. In nonfrozen controls survival declined with increasing osmolality of the test solution. Measurements of the osmolality of water extracted from a variety of moss samples indicate that the nematodes are exposed to an osmotic concentration of about 9 mosmol.l-1 in their natural habitat. This is close to that of artificial tap water. Our experiments, and measurements of freeze concentration effects in the literature, indicate that freeze-induced desiccation is unlikely to prevent inoculative freezing and the survival of nematodes over the winter.

Animals↗