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PubMed1998

Sex differences in effects of opioid blockade on stress-induced freezing behavior.

The present experiment examined the effects of naloxone on freezing behavior in male and female rats following stress and no-stress conditions. Twelve male and 12 female Wistar rats were exposed to 10 min of mild, unpredictable footshock stress and to a comparable no-stress condition. Immediately following stress or no-stress conditions, subjects were injected with naloxone or saline, and two independent observers measured freezing behavior. In male rats, naloxone potentiated freezing following stress but had no effect on freezing following no-stress. In females, naloxone did not affect freezing regardless of stress conditions. These results reveal a sex difference in effects of naloxone on freezing behavior and suggest that sex differences may exist with respect to the role of endogenous opioids under stress.

Analysis of Variance↗
PubMed1998

A computer controlled analysis of freezing behaviour.

The conditioned freezing response in rats has been much used both by psychologists and neuroscientists to investigate the behavioural effects of brain lesions and of changes in motivational state. The primary advantage of the freezing response is that it can be used without motivational manipulations such as food or water deprivation. Previously, freezing has been measured by a human observer either from video recordings or during the test sessions themselves. But these methods of data collection have potential disadvantages. In the present paper, we describe a new, computer controlled, automated procedure for assessing conditioned freezing. Each conditioning chamber contains a mini-video camera. Behaviour is analysed on-line by means of a programme which compares every two adjacent seconds of video tape to generate a screen representing the percentage difference between them. A difference of <0.05% (50 pixels) is classified as a freezing response. Experiments are described in which we measure conditioned freezing and its development over time, in response to contextual cues and to a discrete tone which had been paired with foot shock. We demonstrate our apparatus and methods of data analysis to be sensitive to: number of tone-shock pairings, rat strain and tone pre-exposure.

Animals↗
PubMed2000

Characterization and expression of cold-induced glutathione S-transferase in freezing tolerant Solanum commersonii, sensitive S. tuberosum and their interspecific somatic hybrids.

Glutathione S-transferases (GST) form a large family of non-photosynthetic enzymes known to function in detoxification of xenobiotics. We have cloned and characterized a novel, low temperature regulated GST, Solanum commersonii glutathione S-transferase (Scgst1), from a cold acclimated wild potato species S. commersonii and studied the level of its transcription in freezing tolerant and sensitive Solanum genotypes. Active oxygen species (AOS) were associated with the early steps of Scgst1 regulation since a strong mRNA signal was detected in hydrogen peroxide and salicylic acid treated plants. In experimental conditions where the formation of AOS is known to accelerate, such as excessive light at low temperature, significant accumulation of the transcript was observed in S. commersonii. Under similar experimental conditions, Scgst1 transcript did not accumulate in freezing sensitive S. tuberosum eventhough a single copy of the Scgst1 sequence was present in both species. Thus, Scgst1 in the S. tuberosum genome did not exhibit the same cold-induction properties as in S. commersonii. In comparison with the parental lines, the somatic hybrid SH9A (S. commersonii (+) S. tuberosum) had an interparental level of Scgst1 accumulation as well as freezing tolerance. The abundance of Scgst1 transcript thus correlated well with the freezing tolerance of the parental lines and the somatic hybrid SH9A. Increased GST enzyme activity was observed in S. commersonii and SH9A after 2 days of cold acclimation whereas the activity declined in S. tuberosum during the same period. Further studies of potato lines (S1) that were derived by selfing the somatic hybrid revealed a more complex relationship between freezing tolerance and Scgst1 expression level. In the S1 genotypes, the regulation of Scgst1 transcription resembled more that of S. tuberosum and was not directly related to their freezing tolerance. This could be due to the interaction of the two genomes in S1 genotypes as well as chromosomal rearrangements during meiosis.

Journal Article↗
PubMed1997

Freeze-drying of drug-free and drug-loaded solid lipid nanoparticles (SLN).

Solid lipid nanoparticles (SLN) of a quality acceptable for i.v. administration were freeze-dried. Dynasan 112 and Compritol ATO 888 were used as lipid matrices for the SLN, stabilisers were Lipoid S 75 and poloxamer 188, respectively. To study the protective effect of various types and concentrations of cryoprotectants (e.g. carbohydrates), freeze-thaw cycles were carried out as a pre-test. The sugar trehalose proved to be most effective in preventing particle growth during freezing and thawing and also in the freeze-drying process. Changes in particle size distribution during lyophilisation could be minimised by optimising the parameters of the lyophilisation process, i.e. freezing velocity and redispersion method. Lyophilised drug-free SLN could be reconstituted in a quality considered suitable for i.v. injection with regard to the size distribution. Loading with model drugs (tetracaine, etomidate) impairs the quality of reconstituted SLN. However, the lyophilisate quality is sufficient for formulations less critical to limited particle growth, e.g. freeze-dried SLN for oral administration.

Journal Article↗
PubMed2002

Effect of proline and arginine metabolism on freezing stress of Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the PUT1-encoded proline oxidase and the PUT2-encoded delta1-pyrroline-5-carboxylate dehydrogenase are required to convert proline to glutamate. We recently showed that a put1 disruptant accumulated higher levels of proline intracellularly and conferred higher resistance to freezing stress. Here, we determined the effect of put2 disruption on yeast cell viability under freezing stress. When grown on arginine as the sole nitrogen source, the put2 disruptant showed a significant decrease in cell viability after freezing despite the high proline and arginine contents. This result suggests that delta1-pyrroline-5-carboxylate or glutamate-gamma-semialdehyde, a proline catabolism intermediate, is toxic to yeast cells under freezing stress. In contrast, the survival rate of the wild-type and the put1-disruptant strains was found to increase after freezing in proportion to their arginine contents. This indicates that arginine has a cryoprotective function in yeast. Furthermore, the yeast cells accumulated proline as well as arginine in the vacuole, suggesting that there is a system for the transport of excess proline to the vacuole and that this vacuolar accumulation may be important in the freezing resistance of yeast cells.

Journal Article↗
PubMed2004

Vitrification versus programmable rate freezing of late stage murine embryos: a randomized comparison prior to application in clinical IVF.

A prospective randomized trial was performed to compare post-thaw development of murine blastocysts following programmable rate freezing and two methods of vitrification. Frozen 2-cell murine embryos (n = 429) thawed and cultured for 48 h, were randomly allocated by stage of development into four groups: control (not refrozen), programmable rate freezing (PR) in 0.25 ml straws, vitrification in flexible micropipettes by immersion in super-cooled (VSC) liquid nitrogen (LN2), and vitrification in flexible micropipettes by immersion in LN2 (VLN). Survival, developmental stage progression, presence or absence of an inner cell mass (ICM), and cell counts were recorded 24 h post-thaw. All measured outcomes were different between embryos from the control group and all freezing methods. Controlled-rate freezing resulted in the lowest total cell counts and fewest embryos with a distinct ICM. A higher percentage of embryos survived 24 h post-thaw, progressed to more advanced developmental stages and had higher total cell counts after VLN compared with PR. Moreover, fewer embryos, frozen by either PR or VSC, contained a detectable ICM compared with VLN. These data demonstrate that vitrification may be a better method for freezing murine blastocysts than PR, and may prove to be a superior method for freezing human blastocysts.

Animals↗
PubMed2006

Dynamics of fresh and freeze-dried strawberry and red onion by quasielastic neutron scattering.

The microscopic behavior of fresh and freeze-dried strawberry and red onion at different water contents (45 and 20 wt % water) has been investigated by quasielastic neutron scattering (QENS). To distinguish between the dynamics of the water and the biological material isotopic (H/D) substitution was used. The results show that all samples exhibit an onset of anharmonic motions on the experimental time scale (3-100 ps) at about 230-240 K. Above 250 K the dynamics is mainly of translational character and strongly dependent on the hydration level. The diffusion constant increases rapidly with increasing water content and at 280 K it is approximately 20% higher for the hydration water in freeze-dried strawberry than in freeze-dried red onion and around 2 orders of magnitude faster for the hydration water than for the biological material. Moreover, the diffusion constant of the biological part is about 50% faster in freeze-dried strawberry than in freeze-dried red onion. It was also found that the average relaxation time is slightly faster in fresh strawberry than in freeze-dried strawberry. From the results we can conclude that the water dynamics is not only promoting motions in the biological material, it is also affected by the structure (and possibly also the dynamics) of the biological material. Thus, the microscopic properties of the biological materials are interrelated with the properties of their hydration water.

Fragaria↗
PubMed2002

Cryopreservation of cat semen in straws: comparison of five different freezing rates.

The first pregnancies in domestic cats were obtained using semen frozen in pellets (Platz et al. 1978). Other freezing methods, vials (Lengwinant and Blottner 1994) or straws (Pope et al. 1991; Hay and Goodrowe 1993), have also been used. Pelleted freezing has often been the standard method (Howard 1986). Opinions about the freezing method are discordant; the best method for Pope et al. (1991) was using straws; in fact, the post-thaw motility and the percentage of normal acrosomes were of 44 +/- 4 and 62 +/- 3%, respectively, with straws and 11 +/- 3 and 26 +/- 4%, respectively, with pellets. According to Wood et al. (1993), there are no differences between the two methods, with a motility of 66.2% and a percentage of normal acrosomes of 28.6% for the pellet method and a motility of 67.0% and a percentage of normal acrosomes of 27.4% for the straw container. However, these two authors used two different freezing protocols. A high concentration of glycerol (i.e. 8%, vol/vol) damages cat semen (Zambelli 1994; Nelson et al. 1999), because of his toxicity to spermatozoa (Graham 1996); while a concentration of 4% is suggested (Zambelli 1994). Fast green FCF Bengal pink staining is often used to evaluate the acrosomal morphology (Wood et al. 1993; Zambelli et al. 1993). As there are no studies on the influence of freezing rate on motility and on acrosomal morphology, the aim of this study was to test five freezing rates in order to verify which is the best for the cryopreservation of cat semen in straws.

Acrosome↗
PubMed2001

The impact of the intensity of serial automated plasmapheresis and the speed of deep-freezing on the quality of plasma.

BACKGROUND: Data are lacking on the impact that the intensity of serial donor plasmapheresis has on the quality of source plasma. A study was conducted to examine the quality of source plasma produced by intensive plasmapheresis and slow deep-freezing and to compare it to source plasma manufactured by moderate plasmapheresis and rapid freezing. STUDY DESIGN AND METHODS: Seventy-five plasma samples from intensive plasmapheresis programs (Group 1) and 75 plasma units from moderate plasmapheresis programs (Group 2) were examined. The plasma had been deep-frozen either slowly at -30 degrees C in walk-in freezers (Group 1) or rapidly within 1 hour to a core temperature below -30 degrees C (Group 2). Determinations were made of the plasma levels of citrate; total protein; albumin; IgG; fibrinogen; factors II, V, VII, VIII, and IX; vWF; antithrombin; protein C; D-dimers; and prothrombin fragments 1+ 2. RESULTS: Plasma units of Group 2 contained substantially greater levels of citrate, IgG, FVIII, and FV than samples of Group 1 (p<0.0001). Plasma levels of total protein, albumin, and fibrinogen also were higher in Group 2 (p<0.0001, p = 0.007, and p = 0.006, respectively). Neither plasmapheresis intensity nor freezing procedure had any influence on the levels of factors II, VII, and IX, antithrombin, or protein C. There was no evidence of substantial coagulation activation in the plasma units of either group. However, higher FVIII clotting activity/chromogenic substrate activity ratios in rapidly frozen plasmas and a significant correlation between these ratios and prothrombin fragment 1+ 2 levels suggest that rapid freezing yields both more native FVIII and greater partial activation of FVIII. CONCLUSION: Source plasma collected from donors undergoing intensified plasmapheresis contains markedly lower levels of IgG than plasma units produced by moderate serial plasmapheresis. The combination of intensified plasmapheresis and slower freezing of source plasma results in substantially lower levels of FV and FVIII than does moderate plasmapheresis with rapid freezing. Prospective studies should establish the optimum conditions required for the safe and economic production of source plasma for fractionation.

Blood Banks↗
PubMed2002

The response of Anisakis larvae to freezing.

Anisakis third stage larvae utilize a variety of fish as intermediate hosts. Uncooked fish are rendered safe for human consumption by freezing. Larvae freeze by inoculative freezing from the surrounding medium but can survive freezing at temperatures down to -10 degrees C. This ability may be aided by the production of trehalose, which can act as a cryoprotectant, but does not involve recrystallization inhibition. Monitoring of fish freezing in commercial blast freezers and under conditions which simulate those of a domestic freezer, indicate that it can take a long time for all parts of the fish to reach a temperature that will kill the larvae. This, and the moderate freezing tolerance of larvae, emphasizes the need for fish to be frozen at a low enough temperature and for a sufficient time to ensure that fish are safe for consumption.

Animals↗
PubMed1990

A comparison of the freezing of human semen in the uncirculated vapour above liquid nitrogen and in a commercial semi-programmable freezer.

The cooling rates inside 0.25-ml semen straws filled with glycerol, egg yolk, citrate buffer were compared between a standard vapour freezing procedure and freezing in a Nicool LM-10 semi-programmable freezer. During vapour freezing, the cooling rate at the bottom of the straw was much faster than at the top and there was considerable variation between replicates. By contrast, position within the straw did not affect the cooling rate in the Nicool LM-10 procedure and replicates were more consistent. More motile spermatozoa survived the Nicool freezing procedure and their lateral head displacement was greater than vapour frozen spermatozoa. However the percentage of intact spermatozoa and their velocity was very similar after the two procedures. We conclude that the freezing procedure using the Nicool LM-10 provides a comparatively economical way to achieve consistent semen freezing for research studies.

Cell Membrane↗
PubMed1992

Relationship between freezing tolerance and shoot water relations of western red cedar.

Freezing tolerance and shoot water relations parameters of western red cedar (Thuja plicata Donn) seedlings were measured every 2 weeks from October 1989 to April 1990. Freezing tolerance, measured by freeze-induced electrolyte leakage, showed seasonal shifts in the temperature causing 50% foliage electrolyte leakage (LT(50)). The LT(50) value was -4 degrees C in October, it decreased to -20 degrees C in February and then increased to -6 degrees C in April. The foliage index of injury at -10 degrees C (II(-10)) also showed seasonal shifts from a high of 98% in October to a low of 18% in February followed by an increase to 82% in April. Osmotic potentials at saturation (Psi(s(sat))) and turgor loss point (Psi(s(tlp))) were, respectively, -1.07 and -1.26 MPa in October, -1.57 and -2.43 MPa in January, and -1.04 and -1.86 MPa in April. Dry weight fraction (DWF) increased and symplastic volume at full turgor (V(o)) decreased during the fall-winter acclimation phase, whereas DWF decreased and V(o) increased during the late winter-spring deacclimation phase. Relationships between seasonal patterns of freezing tolerance and shoot water relations parameters showed that LT(50) and II(-10) decreased linearly as Psi(s(tlp)) and V(o) decreased and DWF increased. There was no discernible difference in the relationship during fall acclimation or spring deacclimation. The freezing dehydration index at -10 degrees C (FDI(-10)) declined from 0.69 in November to 0.41 in February and increased to 0.56 in April. The value of II(-10) decreased linearly as FDI(-10) decreased, although a measurement made on actively growing spring foliage did not fit this relationship. The results indicate that seasonal changes in freezing tolerance of western red cedar are partially due to changes in tissue water content, symplastic volume, passive osmotic adjustment and FDI(-10).

Journal Article↗
PubMed1998

Freezing behaviors in leaf buds of cold-hardy conifers visualized by NMR microscopy.

1H-Nuclear magnetic resonance (NMR) microscopy was used to study freezing behavior in wintering leaf buds of Momi fir (Abies firma Sieb. et Zucc.) and Japanese red pine (Pinus densiflora Sieb. et Zucc.). The images acquired predominantly reflected the density of mobile (i.e., non-ice) protons mainly from unfrozen water. By comparing images taken at various subfreezing temperatures, we determined which tissues produced the high and low temperature exotherms detected by differential thermal analyses. Typical extra-organ freezing was successfully imaged in leaf buds of A. firma. The bud scales readily froze at -7 degrees C, but shoot primordia remained supercooled to -14 degrees C in December buds and to -21 degrees C in March buds. The size of supercooled shoot primordia was reduced with decreasing temperature, indicating a gradual decrease in water content of the shoot primordia. In contrast, the signal from shoot primordia of P. densiflora disappeared between -7 and -14 degrees C, corresponding to the high temperature exotherm at -8 degrees C, indicating extracellular freezing of the shoot primordia. The xylem and bark tissues readily froze at -7 degrees C in A. firma and between -7 and -14 degrees C in P. densiflora. We conclude that NMR microscopy can noninvasively provide more spatially specific information about freezing behavior in leaf buds than traditional methods such as differential thermal analysis. In particular, it allows the organized and harmonized freezing behaviors in complex organs to be visualized directly thereby revealing the diversity of mechanisms involved in freezing behaviors.

Journal Article↗
PubMed2001

Defensive freezing evoked by electrical stimulation of the periaqueductal gray: comparison between dorsolateral and ventrolateral regions.

Previous reports indicated that ventrolateral periaqueductal gray (vlPAG) plays a role in the expression of freezing behavior whereas dorsolateral periaqueductal gray (dlPAG) is involved on both freezing and active forms of defensive behaviors. In order to evaluate the role of each of these areas in the occurrence of defensive reactions, rats were electrically stimulated either in the dlPAG or vlPAG with different stimulus frequencies. Stepwise increases in the electrical stimulation of both dlPAG or vlPAG induced initially freezing and then a jumping response. Freezing induced by vlPAG stimulation had a tendency to disappear when the stimulation was turned off whereas freezing induced by dlPAG stimulation remained high in the absence of the stimulation. These results suggest that dlPAG and vlPAG are involved on defensive freezing probably through different neural circuitries.

Analysis of Variance↗
PubMed1993

Freeze-Induced Membrane Ultrastructural Alterations in Rye (Secale cereale) Leaves.

Freezing injury in protoplasts isolated from leaves of nonaccli-mated rye (Secale cereale cv Puma) is associated with the formation of the inverted hexagonal (HII) phase. However, in protoplasts from cold-acclimated rye, injury is associated with the occurrence of localized deviations in the fracture plane, a lesion referred to as the "fracture-jump lesion." To establish that these ultrastructural consequences of freezing are not unique to protoplasts, we have examined the manifestations of freezing injury in leaves of non-acclimated and cold-acclimated rye by freeze-fracture electron microscopy. At -10[deg]C, injury in nonacclimated leaves was manifested by the appearance of aparticulate domains in the plasma membrane, aparticulate lamellae subtending the plasma membrane, and by the frequent occurrence of the HII phase. The HII phase was not observed in leaves of cold-acclimated rye frozen to -35[deg]C. Rather, injury was associated with the occurrence of the fracture-jump lesion between the plasma membrane and closely appressed cytoplasmic membranes. Studies of the time dependence of HII phase formation in nonacclimated leaves indicated that freeze-induced dehydration requires longer times in leaves than in isolated protoplasts. These results demonstrate that the freeze-induced formation of the HII phase in nonacclimated rye and the fracture-jump lesion in cold-acclimated rye are not unique to protoplasts but also occur in the leaves from which the protoplasts are isolated.

Journal Article↗
PubMed1993

Ultrastructural Evidence That Intracellular Ice Formation and Possibly Cavitation Are the Sources of Freezing Injury in Supercooling Wood Tissue of Cornus florida L.

Although cellular injury in some woody plants has been correlated with freezing of supercooled water, there is no direct evidence that intracellular ice formation is responsible for the injury. In this study we tested the hypothesis that injury to xylem ray parenchyma cells in supercooling tissues is caused by intracellular ice formation. The ultrastructure of freezing-stress response in xylem ray parenchyma cells of flowering dogwood (Cornus florida L.) was determined in tissue prepared by freeze substitution. Wood tissue was collected in the winter, spring, and summer of 1992. Specimens were cooled from 0 to -60[deg]C at a rate of 5[deg]C h-1. Freezing stress did not affect the structural organization of wood tissue, but xylem ray parenchyma cells suffered severe injury in the form of intracellular ice crystals. The temperatures at which the ice crystals were first observed depended on the season in which the tissue was collected. Intracellular ice formation was observed at -20, -10, and -5[deg]C in winter, spring, and summer, respectively. Another type of freezing injury was manifested by fragmented protoplasm with indistinguishable plasma membranes and damaged cell ultrastructure but no evidence of intracellular ice. Intracellular cavitation may be a source of freezing injury in xylem ray parenchyma cells of flowering dogwood.

Journal Article↗
PubMed1995

Cold Acclimation of Arabidopsis thaliana (Effect on Plasma Membrane Lipid Composition and Freeze-Induced Lesions).

Maximum freezing tolerance of Arabidopsis thaliana L. Heyn (Columbia) was attained after 1 week of cold acclimation at 2[deg]C. During this time, there were significant changes in both the lipid composition of the plasma membrane and the freeze-induced lesions that were associated with injury. The proportion of phospholipids increased from 46.8 to 57.1 mol% of the total lipids with little change in the proportions of the phospholipid classes. Although the proportion of di-unsaturated species of phosphatidylcholine and phosphatidylethanolamine increased, mono-unsaturated species were still the preponderant species. The proportion of cerebrosides decreased from 7.3 to 4.3 mol% with only small changes in the proportions of the various molecular species. The proportion of free sterols decreased from 37.7 to 31.2 mol%, but there were only small changes in the proportions of sterylglucosides and acylated sterylglucosides. Freezing tolerance of protoplasts isolated from either nonacclimated or cold-acclimated leaves was similar to that of leaves from which the protoplasts were isolated (-3.5[deg]C for nonacclimated leaves; -10[deg]C for cold-acclimated leaves). In protoplasts isolated from nonacclimated leaves, the incidence of expansion-induced lysis was [less than or equal to]10% at any subzero temperature. Instead, freezing injury was associated with formation of the hexagonal II phase in the plasma membrane and subtending lamellae. In protoplasts isolated from cold-acclimated leaves, neither expansion-induced lysis nor freeze-induced formation of the hexagonal II phase occurred. Instead, injury was associated with the "fracture-jump lesion," which is manifested as localized deviations of the plasma membrane fracture plane to subtending lamellae. The relationship between the freeze-induced lesions and alterations in the lipid composition of the plasma membrane during cold acclimation is discussed.

Journal Article↗
PubMed1983

Osmotic Adjustment and the Development of Freezing Resistance in Fragaria virginiana.

Cold temperature acclimation in strawberry (Fragaria virginiana) leaves apparently involves the alteration of cellular osmotic properties. Alterations in leaf osmotic potential were closely correlated with alterations in soluble carbohydrate content of the leaf tissue and changing temperatures. Leaf starch content was inversely related to soluble carbohydrate levels, suggesting that starch is a partial source of osmoticum during osmotic adjustment associated with cold temperature stress. Free amino acid changes were more closely linked to senescence and growth processes while changes in ion content suggested a rapid mobilization of solutes at the onset of freezing temperatures. This was supported by changes in whole plant gradients in leaf osmotic potential before and after exposure to freezing temperatures. In terms of freezing resistance and the role of osmotic adjustment in the development of resistance, it was found that of all leaves undergoing osmotic adjustment only the younger leaves survived, suggesting an age-dependent component to freezing resistance in leaves. Freezing resistance appears to involve alterations in several cellular properties that act in concert to confer a hardy state of the tissue. Although osmotic adjustment may be an important component of the final combination of cellular properties, this study indicates that solute accumulation does not function alone to confer freezing resistance.

Journal Article↗