Structural changes and impairment of function associated with freezing and thawing in muscle, nerve, and leucocytes

In some tissues, especially of skeletal muscle, leucocytes, and peripheral nerves, the effects of freezing to low temperatures, e.g., −78 or −150 °C, and thawing have been a total loss of function and a severe cellular structural disturbance. In the case of muscle, the fibers are converted during thawing into a highly condensed form, called thaw-rigor, in which the individual sarcomeres are shortened beyond a supercontracted state. This effect must be avoided if muscle is to be frozen and stored successfully. Leucocytes and especially the neutrophils are seriously affected by exposure to low temperature. The result is a general swelling of the cell and of its nucleus which is suggestive of damage to the membranes of the cell. Rat cutaneous nerves frozen and exposed to temperature below −15 °C showed after thawing a general disturbance of the myelin involving rupture and separaration of the lamellae. In the axoplasm the neurofilaments and neurotuabules are converted into small aggregates and the mitochondria appear to be swollen. Nerves frozen at −5 and some at −10 °C showed little change in structure, the myelin and axoplasm exhibiting a normal appearance. A check on viability by excitation from an electric stimulus indicated survival after exposure to −5 and in a few cases to −10 °C.     

Ultrastructural changes arising from freezing of leaf blade cells of rye (Secale cereale): an investigation using freeze-substitution

The survival at sub-zero temperatures of leaf blade cells of rye ( Secale cereale L. cv. Voima), which had not been cold acclimated, was determined by measuring the efflux of ninhydrin-positive substances: 50% of the cells were dead at −4°C (LT₅₀) and none survived at −12°C or below. Examination of ultrastructural changes during cold hardening and freezing injury requires frozen tissues prepared for transmission electron microscopy without thawing. Specimens were prepared from leaf blade segments at room temperature, −4°C or −12°C by plunge freezing at 3 m s⁻¹ into a cooling medium at −170°C followed by freeze-substitution in acetone with OsO₄ fixation. Comparisons of room temperature specimens were made with those prepared by chemical fixation using glutaraldehyde/paraformaldehyde/tannic acid. On freezing to −12°C, the cells were severely dehydrated and distorted, the vacuoles severely shrunken and the cytoplasm and mitochondria disorganized whereas the chloroplasts were little affected. On freezing to −4°C, some cells were as disorganized as those at −12°C, others were relatively intact, and some showed evidence of intracellular ice crystal formation.     

Artificial ice crystal formation in excised short shoots of Pinus silvestris L.

Controlled freezing methods adopted for Thermal Analysis (TA) and Differential Thermal Analysis (DTA), were used to investigate the capacity for subcooling short shoots of Pinus silvestris L. of various ages. Both methods showed that there were no differences in subcooling temperature along the length of the short shoots. Differences in age and water content have a significant effect on the freezing profiles. Short shoots of the current years growth were able to subcool to about −13°C. One-year-old short shoots subcooled to about −9°C. There were no apparent variations in subcooling temperatures from June to September. Rainfall or dry periods had no effect on subcooling temperatures. The freezing profiles of living material was characteristic and was not repeated during subsequent freezing cycles. Detection of ice crystal formation during refreezing experiments indicated whether short shoots were alive or dead.     

Cold acclimation of Ilex aquifolium under natural conditions with special regard to the photosynthetic apparatus

Frost resistance of leaves of holly ( Ilex aquifolium L.) increased from about −9°C in late summer to −24°C in mid-winter. The gradual rise in cold hardiness occurred when the minimum air temperature dropped to 0°C or below and was closely related to increase in the cellular sap concentration. Predominantly, the decrease in the osmotic potential of the cellular sap was caused by sugar accumulation, mainly of sucrose. The capacity of net photosynthesis of the leaves, as well as the total lipid and protein content and the proportion of individual lipids of the thylakoid membranes, did not significantly change during cold acclimation. The gradual shift towards desaturation in the fatty acids of the thylakoid lipids during the hardening period was neither correlated with alterations in the frost resistance nor did it affect the potential efficiency for various light-induced chloroplast membrane reactions such as linear photosynthetic electron transport, photophosphorylation and the proton gradient (ΔpH). It is suggested that in holly leaves reduction in cell volume changes during freeze-thawing and cryoprotection by sugars could play a dominant role for the increase in frost resistance. Seasonal changes in the degree of unsaturation of polar lipids of the thylakoids could contribute to maintain optimal functional efficiency of the membranes at low temperatures rather than to avoid freezing damage.     

Alterations in membrane transport properties by freezing injury in herbaceous plants:

Leakage of ions from a thawed tissue is a common phenomenon of freezing injury. This leakage is usually assumed to be due to loss of membrane semipermeability or membrane rupture by freezing injury. Freeze injured, yet living, onion (Allium cepa L.) epidermal cells were used to study alterations in cell membranes that result in leakage of ions. In spite of a large efflux of ions, freeze injured cells could be plasmolysed and they remained plasmolysed for several days just like the unfrozen control cells. Injured cells also exhibited protoplasmic streaming. Passive transport of KCl, urea and methyl urea across the cell membranes of injured and control cells was also studied. No difference could be detected for the transport rates of urea and methyl urea between control and injured cells. However, a dramatic increase in the transport rate of KCl was found for the injured cells. Depending upon the extent of initial freezing injury, an increase or a decrease in injury symptoms was found in the post-thaw period. During the progress of freezing injury, 10 days after thawing, a swelling of the protoplasm was seen in the irreversibly injured cells. In spite of this swelling, these cells could be plasmolysed. It appears that the high amount of K⁺ that leaks out into the extracellular water, due to freezing injury, causes protoplasmic swelling by replacing Ca²⁺ in the plasma membrane. We conclude that protoplasmic swelling is a sign of secondary injury. The results presented in this study show that membrane semipermeability is not completely lost and membrane rupture does not occur during the initial stage of freezing injury. In fact, the cells have the ability to repair damage depending upon the degree of injury. Our results show there are specific alterations in membrane semipermeability (e.g., transport of K⁺) which could be repaired completely depending on the degree of injury. These findings suggest that ion leakage due to freezing injury is due to alteration in the membrane proteins and not in the membrane lipids.     

THE COMBINED EFFECTS OF LOW TEMPERATURE AND SO2+NO2 POLLUTION ON THE NEW SEASON'S GROWTH AND WATER RELATIONS OF PICEA SITCHENSIS

Picea sitchensis (Bong.) Carr. seedlings were exposed to SO₂, NO₂ and SO₂+ NO₂ during dormancy in controlled environments, and were taken to night temperatures of 4, 0, −5, −10 and −15 °C in a freezer. Conditions in the freezer were carefully monitored during the low–temperature treatments. In two experiments, different photoenvironments and temperature regimes were imposed prior to the cold treatments, and different effects were observed. In the first, only limited frost hardiness was achieved and night temperatures of −15 °C were lethal. Temperatures of −5 and − 10 °C led to poor survival of lateral buds, particularly in plants exposed to 45 ppb SO₂. The poor bud break in plants exposed to SO₂ and to − 5 °C resulted in a loss of the effectiveness of this temperature as a chill requirement. Pressure-volume analysis showed that the shoots of plants exposed to NO₂ had greater elasticity (lower elastic moduli, e), so that loss of turgor occurred at lower relative water contents. In contrast, a hardening period (2 weeks in night/day temperatures of 3/10 °C and 8 h days at 50 μmol m⁻² s⁻¹ PAR) gave decreased elasticity and lower solute potentials of spruce shoots. In the second experiment, exposure to 30 ppb SO₂ and SO₂+ NO₂ led to slight, but consistent, increases in frost injury to the needles of plants frozen to − 5 and − 10 °C. The results suggest that the main interaction of low temperatures and winter pollutants may be on bud survival rather than on needle damage, but that effects are subtle, only occurring with certain combinations of pollutant dose and cold treatment.     

Increased ethylene synthesis enhances chilling tolerance in tomato

Freezing of nonacclimated protoplasts close to lethal temperatures induces alterations in the macromolecular organization of the plasma membrane but the significance of these structural changes in freezing injury is still uncertain. We therefore cooled non-acclimated protoplasts isolated from cultivars of winter rye ( Secale cereale L.) to two sub-zero temperatures using two different cooling rates and analyzed freeze-induced plasma membrane changes by freeze-fracture electron microscopy. When a high cooling rate was used a lipid phase transition was observed in 34% of the total membrane fracture faces of the protoplasts, while with a slow cooling rate it occurred only to a very small extent. Smooth, aparticulate lamellae were approximately three times more frequent at low than at high cooling rate. Lipid phase transition from lamellar to hexagonal_II (H_II) phase occurred at high cooling rate more frequently at −10°C than at −30°C in three cultivars. The results suggest that the greatly increased proportion of phase transition from bilayer to non-bilayer phase is an artifact caused by too fast a cooling rate of protoplasts. Furthermore, lateral phase separation of the plasma membrane with segregation of intramembrane particles and the appearance of membrane associated stacks of lipid lamellae, may cause cellular death by retarding the flow of intracellular water towards extracellular ice crystals formed during freezing.     

Changes in red cells following rapid freezing with extracellular cryoprotective agents

Cells rapidly frozen and thawed both in the presence and absence of extracellular cryoprotectants lose potassium in exchange for extracellular solute without hemolysis and with subsequent in vivo survival. There is some loss of 2,3-diphosphoglycerate from cells frozen with polyvinylpyrrolidine and hydroxyethyl starch as cryoprotectants. At least a portion of the cation exchange occurs following thawing, suggesting a transient loss of normal membrane semipermeability. The cryoprotectants reduce the freezing rate necessary for optimum recovery and may enhance the transient membrane permeability. They may also serve to stabilize membrane components against irreversible postthaw changes.     

Supercooling ability in apple seeds as affected by temperature-induced modifications in water relations in the seed parts

Prolonged storage of apple fruits ( Pyrus malus L. cv. Golden Delicious) at different temperatures (0, 12 and 35°C) decreased the water content in seed coats and endosperms, higher temperatures being much more effective than the lower (0°C) one. No effect of the temperature on the embryo hydration was found. However, a pronounced decrease in water potential in the embryos was observed during the first 9 weeks. The decrease was much faster and the water potential reached lower levels in embryos isolated from seeds pretreated with higher temperatures (12 or 35°C) than from cold-pretreated (0°C) material. Higher temperatures of fruit storage also resulted in a decreased permeability of the embryo membranes to electrolytes and sugars. At the same time, membrane permeability to water was not modified. It is proposed that the previously observed occurrence of the discontinuous type of freezing in apple seeds (Nguyen and Kacperska, Physiologia Plantarum (X): 000-000, 1989) is associated with the temperature-induced dehydration of seed coat and endosperm, whereas a higher super cooling ability of the high-temperature-pretreated embryos is due to a decrease in the free energy of water in the system, and to the effective protection of embryo cells against heterogenous ice nucleation. The changes in water potential showed a high negative correlation with the embryo phospholipid content determined in the other work (Nguyen et al. Plant Physiol. Bio chem. 25: 697–703, 1987). Therefore, it is proposed that changes in matrix potential play an important role in the regulation of the water potential in the embryo cells.     

Survival of the first stage larva of the metastrongyloid nematode Elaphostrongylus rangiferi under various conditions of temperature and humidity

First-stage larvae of E. rangiferi kept in water at 50°C died within 80 minutes, while at 6° the last larvae died between day 180 and 210. The time it took to reach 1_x= 0.5 (half of the larvae dead) at various temperatures between 6° and 50° was well described by the exponential function y = 614.6e^−0.15x, giving a value of 615 days to reach 1_x= 0.5 at 0°C. There was no clear decrease in the survival of larvae frozen at −20° in faeces and in water, and at −80° in faeces after 360 days. When subjected to repealed freezing and thawing, all larvae died within 77 days. When kept in air at RH = 20% and 22°C, all larvae died within 11 days, while when frozen (−20°C) in air at RH approx. 0%, 1_x stayed at approx. 0.5 from day 5 to day 16.     

Freezing and desiccation tolerance of imbibed canola seed

Depending on the environmental conditions, imbibed seeds survive subzero temperatures either by supercooling or by tolerating freezing-induced desiccation. We investigated what the predominant survival mechanism is in freezing canola ( Brassica napus cv. Quest) and concluded that it depends on the cooling rate. Seeds cooled at 3°C h⁻¹ or faster supercooled, whereas seeds cooled over a 4-day period to −12°C and then cooled at 3°C h⁻¹ to−40°C did not display low temperature exotherms. Both differential thermal analysis and nuclear magnetic resonance (NMR) spectroscopy confirmed that imbibed canola seeds undergo freezing-induced desiccation at slow cooling rates. The freezing tolerance of imbibed canola seed (LT₅₀) was determined by slowly cooling to −12°C for 48 h, followed with cooling at 3°C h⁻¹ to −40°C, or by holding at a constant −6°C (LD₅₀). For both tests, the loss in freezing tolerance of imbibed seeds was a function of time and temperature of imbibition. Freezing tolerance was rapidly lost after radicle emergence. Seeds imbibed in 100 μ M abscisic acid (ABA), particularly at 2°C, lost freezing tolerance at a slower rate compared with water-imbibed seeds. Seeds imbibed in water either at 23°C for 16 h, or 8°C for 6 days, or 2°C for 6 days were not germinable after storage at −6°C for 10 days. Seeds imbibed in ABA at 23°C for 24 h, or 8°C for 8 days, or 2°C for 15 days were highly germinable after 40 days at a constant −6°C. Desiccation injury induced at a high temperature (60°C), as with injury induced by freezing, was found to be a function of imbibition temperature and time.     

Ultrastructure-function correlative studies for cardiac cryopreservation. II. Hearts frozen to various temperatures without a cryoprotectant

Isolated rat hearts were perfused with balanced salt solution (BSS) for 20 min, sealed in a metal cannister, and cooled in a −20 °C acetone bath at a rate of 1 °C/min to one of four subzero core temperatures (−10, −12, −17, or −20 °C). Upon attainment of the desired temperature the hearts were rapidly thawed (40–50 ° C/min) and reperfused with BSS for an additional 20 min. Approximately half of the hearts cooled to −10 or −12 °C resumed spontaneous contractile activity after thawing. One of 16 hearts survived cooling to −17 °C, while no heart survived cooling to −20 °C. Nonfrozen controls gave a positive inotropic response to a standard test dose of ouabain; none of the thawed survivors did.     

Effect of a freeze-thaw cycle on properties of microsomal membranes from wheat

A freeze-thaw cycle to −12°C induced several physical and compositional changes in the microsomal membranes isolated from crown tissue of winter wheat (Triticum aestivum L. cv Frederick). Exposing 7-day-old, nonacclimated seedlings to a single freeze-thaw cycle prevented regrowth of the crown and resulted in increased membrane semipermeability. The phospholipid and protein content of microsomal membranes isolated from the crowns decreased by 70 and 50%, respectively. Microsomal membranes isolated after the lethal freeze-thaw stress, and liposomes prepared from total membrane lipids, exhibited greater microviscosity, measured by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. The number of free thiol groups per milligram membrane protein, measured using the specific fluorescent probe, N-dansylaziridine, decreased after freezing. In contrast, acclimated wheat seedlings which showed increased freezing tolerance, as indicated by survival and ion leakage, suffered almost no effects from the freeze thaw treatment as determined by measurements of membrane microviscosity, phospholipid content, protein content, or danzylaziridine fluorescence. An examination of membranes isolated from frozen tissue showed that most of the changes occurred during the freezing and not during the thawing phase.     

Cryobiology of neurospora crassa. I. Freeze response of Neurospora crassa conidia

Neurospora crassa conidia were frozen and thawed in water suspensions at various rates and with different minimum temperatures. Colony counts of the experimental conidia were compared with those of controls, which were taken as 100% survival. The data revealed that (1) survivals were near 100% after fast thaw (400 °/min) regardless of the freeze rate, (2) percentage of survival was inversely related to freeze rate when combined with slow thaw, (3) slow thaw (0.5 °/min) was damaging, and (3) the rates of freeze-thaw affected the system only in the −5 to −20 ° interval. The damaging freeze conditions were those which favor ice crystal growth. It is suggested that rupture of the membrane by ice crystals seems to be the plausible mechanism of damage in freezing and thawing N. crassa conidia.     

Identification of tissues showing the lowest tolerance to freezing in larvae of the rice stem borer, Chilo suppressalis

Abstract.  Even though overwintering larvae of the rice stem borer, Chilo suppressalis , are freeze-tolerant, they cannot survive below −30 °C. Furthermore, nondiapausing larvae cannot survive freezing. However, the cause of death due to freezing is unclear. To identify the cause of death by freezing in larvae, those tissues most injured by low temperatures are identified using the vital stain trypan blue. In overwintering larvae, the midgut of dead larvae stains blue, and remarkable colour density differences between dead and surviving larvae are observed in the midgut. In nondiapausing larvae incubated at −10 °C for several hours, the fat body of dead larvae is strongly stained. Furthermore, increases in mortality with treatment time correspond with increases in the area of the fat body stained. Sterile nondiapausing larvae with lower supercooling points, below −20 °C, do not freeze at −10 °C and survive the treatment. However, all the larvae die when subjected to inoculative freezing at −10 °C, and the fat body stains blue. These results suggest that the midgut in overwintering larvae and the fat body in nondiapausing larvae have the lowest tolerance to freezing.     

Measurement of Hippocampal Levels of Cellular Second Messengers Following In Situ Freezing

Abstract: The in situ freezing technique has been widely used to fix labile metabolites and cellular second messengers in cerebral cortex. In this study, we isolated specific brain regions at 0°C from coronal sections of frozen heads following in situ brain freezing and measured regional concentrations of labile metabolites and cellular messengers. These levels in the cortex were compared with those in cortical punches obtained at freezing temperature (less than −40°C) from the same in situ frozen brains and those of cortex dissected from decapitated animals. In both isoflurane- and pentobarbital-anesthetized animals, we observed that the levels of lactate, free fatty acids, inositol 1,4,5-trisphosphate, and diacylglycerol, as well as the proportion of protein kinase C associated with the membrane fraction, were similar in cortical punches taken at freezing temperature and those dissected at 0°C. However, with animals decapitated at room temperature, cortical and hippocampal levels of lactate, free fatty acids, and inositol 1,4,5-trisphosphate and the proportion of membrane protein kinase C were significantly higher than those of corresponding brain regions isolated at 0°C from in situ frozen brains ( p < 0.05). These results indicate that dissection of cortex and hippocampus at 0°C following in situ freezing will eliminate decapitation-induced production of artifacts and changes in the levels of cellular second messengers such as inositol 1,4,5-trisphosphate, diacylglycerol, and protein kinase C. The present technique, used in conjunction with in situ freezing, will fix cellular second messengers and labile metabolites in several regions of brain and may facilitate accurate characterization of molecular and cellular mechanisms underlying CNS function.     

The consequences of freezing temperatures followed by high irradiance on in vivo chlorophyll fluorescence and growth in Picea abies

Picea abies (L.) Karst. plants, propagated by cuttings, were subjected to one night of freezing temperatures (-5°C), high irradiance (1 200 or 1 800 μmol m⁻² s⁻¹), or freezing temperatures followed by high irradiance. The treatments were applied at bud burst, at time of shoot elongation, and when the shoots had ceased to elongate. The maximum quantum yield of photosynthesis, F_v/F_m, dry weight of branches and needles, and length and survival of shoots were measured. F_v/F_m and growth decreased after a night of freezing temperatures followed by high irradiance, at the time of bud burst and shoot elongation. High irradiance alone influenced F_v/F_m, but not growth. Freezing temperatures affected F_v/F_m, and growth at the time of shoot elongation. F₀ increased after a night of freezing temperatures and decreased with age of the current-year needles. It was concluded that the use of short-term measurements of chlorophyll fluorescence induction to predict changes in growth after a night of frost and subsequent high light was not a reliable method.     

Evaluation of freezing injury and freeze-thaw injury to membranes of Saskatoon serviceberry twigs by measuring HCN release

The influence of thawing on freeze-injured Saskatoon serviceberry ( Amelanchier alnifolia Nutt.) twigs was evaluated by refreezing freeze-thawed twigs and comparing the HCN release at -5°C fro these twigs to the HCN release at -5°C from twigs that had not been thawed. An effect of thawing depended on the physiological state of the twigs, on the degree of freezing stress, or on both. Manifestation of membrane injury does not have an absolute dependence on thawing. Post-thaw temperature influences manifestation of injury, since twigs warmed to 30°C released more HCN than twigs warmed to 1°C when refrozen to -5°C. Although thawing and post-thaw conditions can influence the magnitude of membrane injury, the critical event leading to injury occurs while plants are frozen.     

Stabilization of liposome bilayers to freezing and thawing: Effects of cryoprotective agents and membrane proteins

Lipid bilayer vesicles (liposomes) with and without glycoprotein incorporated into the membranes were tested for stability during freezing and thawing, in presence and absence of the cryoprotective agents (CPA) glycerol and dimethyl sulfoxide. Changes in turbidity and loss of energy transfer between fluorescent probes present in the bilayers were used to estimate membrane integrity.Freezing caused a 30 to 40% destruction of protein-free liposomes, in absence of CPA. CPA at 10 to 20% concentration prevented such losses, but at higher concentrations destabilized liposomes even without freezing. Protein-containing liposomes suffered no loss on freezing in absence or presence of CPA at moderate concentrations.Lowering of the storage temperature of frozen samples within the range of ?5 to ?27 °C increased the freeze damage. Slower rates of cooling and warming caused a slightly greater loss.The results are interpreted in terms of the liquid mosaic model for lipid bilayers. CPA at higher concentrations destabilize bilayers by dissolving phospholipids. At moderate concentrations, however, they prevent the damaging effect of dehydration of the lipid on freezing. Proteins appear to stabilize bilayers by providing increased hydration at the membrane surface, and by additional hydrophobic binding in the membrane interior.     

CO2 enrichment and development of freezing tolerance in Norway spruce

Plant growth and adaptation to cold and freezing temperatures in a CO₂-enriched atmosphere have received little attention despite their predicted effects on plant distribution and productivity. In this study we looked at the interaction between elevated CO₂ and development of freezing tolerance in Norway spruce ( Picea abies (L.) Karst.). First-year seedlings were grown under controlled conditions in an atmosphere enriched in CO₂ (70 Pa) for one simulated growth season. We measured shoot growth, registered the timing of growth cessation and bud set, measured needle net photosynthetic rate, and determined needle carbohydrate concentration (fructose+pinitol, glucose, sucrose, inositol, raffinose and starch). Freezing tolerance (LT₅₀) was determined after exposing whole seedlings to temperatures ranging from −6.5 to −36.0°C and scoring for visual needle browning. Elevated CO₂ did not affect height growth or the timing of growth cessation and bud set. The only statistically significant effects of CO₂ treatment were on seedling dry weight, percent dry matter and starch content. During the three weeks after growth cessation and bud set, freezing tolerance increased from −10 to −35°C, and there was a marked increase in all soluble sugars except inositol. However, neither freezing tolerance nor the concentration of soluble sugars was significantly influenced by elevated CO₂.