The effect of temperature at which slow cooling is terminated and of thawing rate on the survival of one-cell mouse embryos frozen in dimethyl sulfoxide or 1,2-propanediol solutions

We investigated the slow freezing of one-cell mouse embryos with either dimethyl sulfoxide (Me2SO) or 1,2-propanediol (PROH) as the cryoprotectant. One-cell embryos, collected from superovulated C57BL/6J x CBA/Ca females were exposed to 1.5 M solutions of either Me2SO or PROH. The embryos were cooled at 0.3 degrees C/min to temperatures between -10 degrees and -80 degrees C before being plunged into LN2 and then warmed at either 20 degrees C/min or 450 degrees C/min. Survival was expressed as the percentage of hatching or hatched blastocysts per frozen-thawed embryo. When the slow cooling was in 1.5 M PROH, the temperature at which survival rates after slow thawing began to increase was -35 degrees C (52.6 +/- 5.2% survival). For slow cooling in 1.5 M Me2SO this temperature was -50 degrees C (45.0 +/- 2.9% survival). The addition of sucrose to the 1.5 M PROH solution raised the temperature at which survival rates after slow thawing began to increase to -30 degrees C (54.8 +/- 3.7% survival). If slow cooling was stopped at high subzero temperatures, embryos survived better after rapid thawing than slow thawing. If slow cooling was stopped at low subzero temperatures, the survival rate was not dependent on the thawing rate if freezing was done in 1.5 M PROH. When freezing was in Me2SO solutions and to subzero temperatures of -60 degrees and -80 degrees C, slow thawing gave better survival than rapid thawing. The addition of sucrose to the Me2SO freezing solution restored the survival rates at -60 degrees and -80 degrees C. These results indicate that high rates of survival may be obtained from one-cell mouse embryos by a rapid or a slow thawing procedure, as has been found for other developmental stages. The results also indicate that PROH provides superior protection compared to Me2SO against freezing-thawing damage and that the addition of sucrose to the freezing solutions prior to freezing improves the overall survival rates. Embryos that survived freezing and developed in culture implanted and formed normal fetuses at rates similar to those of nonfrozen control embryos (60% vs 68% and 53% vs 58%, respectively.     

Effects of various low temperatures, cryoprotective agents and cooling rates on the survival, fertilizability and development of frozen-thawed mouse eggs

Frozen mouse eggs were examined to determine the effects of low temperatures, concentration of cryoprotective agents and cooling rates on their survival, fertilizability in vitro and subsequent development. Dimethyl sulfoxide administered at 1.5 M concentration was found to be the most effective cryoprotective agent. Upon thawing, 51% and 56% of the eggs appeared to be normal after having been cooled at 0.33 degrees C/min to -30 degrees C and -50 degrees C, but only 18% of the eggs appeared to be normal after having been cooled at the same rate to -75 degrees C. When eggs were cooled at 0.33 degrees C/min to -45 degrees C and the cooling rate increased to 1 degree C/min from -45 degrees C to -75 degrees C, 44% and 72% appeared normal upon thawing. Of the normal eggs fertilized in vitro from C3H mice, 65% cleaved to the 2-cell stage and 24% of the 2-cell eggs developed into blastocysts. Following the transfer of 17 blastocysts into three recipient mice, one mouse delivered three normal young.     

The role of the house fly (Musca domestica) in the dissemination of hookworm

The effect of cooling rate, warming rate, and duration of phase transition upon survival of frozen canine kidneys was investigated. In the present study, 11 kidneys out of 14 rapidly cooled (2--4degreesC/min) to --22degreesC and thawed (70--110degreesC/min) were viable following contralateral nephrectomy. The serum creatinine and BUN levels rose to a maximum of 8.4 and 30 mg%, respectively, on the eighth day post-contralateral nephrectomy. Average survival time was 10 days; however, two of the dogs in this group were allowed to survive, one for 3 months and one for over 2 years. Eight kidneys out of 16 slowly cooled (0.25-1.0degreesC/min) and either rapidly or slowly warmed (20-30degreesC/min) had function to produce small amounts of urine; however, they did not survive more than 5 days after contralateral nephrectomy. Cooling rates of 0.1 and 10degreesC/min were too harmful to the kidney to have renal function after reimplantation. The minimum renal cell damage as assessed by LDH and GOT in the post-freeze perfusate was found in the 2-4degreesC/min cooling rate following rapid warming (70degrees-110degreesC/min). Correlation of the duration of phase transition time to renal cell damage was linear for LDH and GOT (r=0.93). This result suggests that the duration of phase transition time also is an important factor during the freezing process, affecting post-thaw survival of canine kidneys.     

Caffeine and other predictors of bone density among pre- and perimenopausal women

Cryopreservation has proved to be a highly successful method for long-term storage of viable embryos. The objective of this study on rat blastocysts was to define conditions for their cryopreservation. Three cryoprotectants, dimethyl sulfoxide, glycerol, and propanediol/sucrose, were compared in two cooling programs (to -30 or -80 degrees C) and two thawing protocols. The cooling was followed by immersion in liquid nitrogen. Programmed thawing was at the rate of 8 degrees C per minute; fast thawing consisted of direct exposure of the frozen embryos to the ambient laboratory temperature. The survival after the freeze/thaw was assessed from the post-thaw embryo morphology and ability to develop into apparently normal offspring in uteri of foster mothers (embryonic survival). The best method for preservation of rat blastocysts proved to be programmed cooling to -80 degrees C followed by fast thawing with glycerol as cryoprotectant (embryonic survival of 28.1%). In all the experimental groups, the proportion of embryos with good to excellent preservation of morphology was high. With dimethyl sulfoxide, after programmed cooling to -80 degrees C, embryonic survival was 9.9% (programmed thawing) and 17.5% (fast thawing). No embryos survived after programmed cooling to -30 degrees C. However, when the cryoprotectant was propanediol/sucrose, no difference was observed between programmed cooling to -80 degrees C with either method of thawing and programmed cooling to -30 degrees C and fast thawing (12.3, 6.2, and 8.0%, respectively).     

Comparison of the different strategies for cryopreserving and storage of the bone marrow CD34+ cells. Possibility of unprogrammed rate freezing and storage at -80 degrees C mechanical freezer

We have compared the efficiency of the different strategies allowing for a long term storage of a human CD34+ bone marrow cells. Accordingly, the aliquots of CD34+ cells isolated from bone marrow were frozen using: controlled rate freezing equipment, or freezer unprogrammed in a -80 degrees C mechanical freezer. After freezing, CD34+ cells were subsequently stored for one month in a liquid nitrogen tank at -196 degrees C or in mechanical freezer at -80 degrees C. We have found that both the viability and the recovery of clonogeneic progenitors of CD34+ cells samples stored at different temperature were similar. Therefore, regarding the costs and simplicity, we recommend the unprogrammed freezing and storage of human CD34+ cells at -80 degrees C in a mechanical freezer as a convenient, inexpensive, and reliable method for storing marrow for transplantation. This data also indirectly indicate that the aliquots of the CD34+ cells can be shipped frozen on dry ice (-80 degrees C), and that these cells will maintain viability under this conditions. Furthermore, in this study we have confirmed validity of our earlier observation that human CFU-Meg progenitors are more sensitive to cryopreservation.     

Reactive oxygen species generation by seminal cells during cryopreservation

OBJECTIVES: To test the hypothesis that conventionally used procedures for semen cryopreservation may cause an increase in the production of reactive oxygen species (ROS) by sperm or by seminal leukocytes, which may contribute to poor sperm function following cryopreservation. METHODS: Eighteen semen specimens with normal parameters from healthy male donors 22 to 40 years of age were each divided into two portions. The first portion was combined 1:1 with Test Yolk Buffer-Glycerol Freezing Medium and was frozen by gradual cooling into liquid nitrogen (-196 degrees C). The second portion was washed and the cells were resuspended in Sperm Washing Medium (SWM) and incubated at room temperature to serve as controls. After a period of treatment, frozen samples were thawed and semen cells were washed and resuspended in SWM. ROS generation by semen cells from each treatment group was measured on a luminometer. Sperm motility, sperm viability, and sperm membrane integrity were also measured in both control and freeze-thaw samples. To further assess ROS generation by semen cells during the cooling process, aliquots of washed semen cells and purified polymorphonuclear leukocytes (PMNs) were incubated separately at different temperature conditions (37 degrees C, 22 degrees C, 4 degrees C, and -20 degrees C). ROS activity in each treatment group was measured and compared with each other. RESULTS: In both semen cells and PMNs, ROS activity increased significantly during the cooling process. The highest ROS levels were recorded in both groups when cooled to 4 degrees C. The ROS levels were extremely low in samples cooled to -20 degrees C and in freeze-thaw samples, probably due to marked loss of cell viability. CONCLUSIONS: Gradual reduction of temperature during the process of semen cryopreservation can cause a significant ROS generation by semen cells. ROS is particularly elevated during cooling if the semen sample is contaminated by more than 0.5 x 10(6) leukocytes. Removal of leukocytes from semen samples or treatment with antioxidants prior to cryopreservation may improve sperm viability and function.     

Cryopreservation and culture of human corneal keratocytes

PURPOSE: To assess the effects of two different concentrations of albumin in a cryoprotective solution and two freezing methods on human corneal keratocyte ctyopreservation. METHODS: Isolated keratocytes were used for cryopreservation. Solutions of 10% dimethylsulfoxide with either 2% or 10% human albumin were used as cryoprotective agents. Cells either were transferred directly into a -80 degrees C freezer (freezing rate, 2 degrees C/min) or were cooled in a programmed freezer (1 degrees C/min until -40 degrees C and then 10 degrees C/min), which resulted in four different cryopreservation protocols. Cells were stored at -80 degrees C, then were thawed at 37 degrees C, and subsequently were cultured. Keratocytes were studied by means of trypan blue staining, growth assay, apoptosis assays, transmission electron microscopy, and immunochemistry. RESULTS: The percentage of cells that were alive after thawing ranged from 80% to 99% by trypan blue staining and from 45% to 60% by flow cytometry. The ratio of the number of living cells at the end of primary culture after cryopreservation to that before cryopreservation was significantly (P=0.04) higher after direct transfer into the -80 degrees C freezer than after controlled-rate freezing, whereas the albumin concentration had no significant influence on this ratio (P=0.45). The percentage of apoptotic cells was significantly higher after cryopreservation than in the control group of noncryopreserved cells; more than 5% 24 hours after thawing. Cryopreservation did not modify the keratocyte ultrastructure. Fibroblast growth factor dramatically decreased the serum-induced cell expression of alpha smooth muscle actin, whereas cryopreservation had no influence on this cell expression. CONCLUSIONS: A freeze-thaw trauma, which was related to cryopreservation-induced cell apoptosis, was revealed during primary culture after thawing. Direct transfer into the -80 degrees C freezer resulted in better postcryopreservation growth in the culture than controlled-rate freezing. A change in albumin concentration from 2% to 10% did not affect the results.     

Mild posttraumatic hypothermia reduces mortality after severe controlled cortical impact in rats

The effect of posttraumatic hypothermia (brain temperature controlled at 32 degrees C for 4 h) on mortality after severe controlled cortical impact (CCI) was studied in rats. Four posttraumatic brain temperatures were compared: 37 degrees C (n = 10), 36 degrees C (n = 4), 32 degrees C (n = 10), and uncontrolled (UC; n = 6). Rats were anesthetized and subjected to severe CCI (4.0-m/s velocity, 3.0-mm depth) to the exposed left parietal cortex. At 10 min posttrauma the rats were cooled or maintained at their target brain temperature, using external cooling or warming. Brain temperature in the UC group was recorded but not regulated, and rectal temperature was maintained at 37 +/- 0.5 degrees C. After 4 h, rats were rewarmed over a 1-h period to 37 degrees C, extubated, and observed for 24 h. In the 37 and 36 degree C groups, 24-h mortality was 50% (37 degrees C = 5/10, 36 degrees C = 2/4). In the 32 degree C group, 24-h mortality was 10% (1/10). In the UC group, brain temperature was 35.4 +/- 0.6 degrees C during the 4-h treatment period and 24-h mortality was 0% (0/6). Mortality was higher in groups with brain temperatures > or = 36 degrees C versus those with brain temperatures < 36 degrees C (50 vs. 6%, respectively; p < 0.05). Additionally, electroencephalograms (EEG) were recorded in subsets of each temperature group and the percentage of time that the EEG was suppressed (isoelectric) was determined. Percentage of EEG suppression was greater in the hypothermic (32 degrees C, n = 6; UC, n = 4) groups than in the normothermic (36 degrees C, n = 3; 37 degrees C, n = 6) groups (23.3 +/- 14.3 vs. 1.2 +/- 3.1%, respectively; p < 0.05). Posttraumatic hypothermia suppressed EEG during treatment and reduced mortality after severe CCI. The threshold for this protective effect appears to be a brain temperature < 36 degrees C. Thus, even mild hypothermia may be beneficial after severe brain trauma.     

Postintubation tracheal stenosis

Cryopreservation techniques for umbilical cord blood (UCB) have been based on methods established for marrow (BM) and peripheral blood progenitor cells (PBPC) with varying degrees of success. The aim of this study was to optimise cryopreservation of UCB haemopoietic cells based on sound cryopreservation principles. UCB samples were cryopreserved with different combinations of DMSO and hydroxyethyl starch (HES) by a variety of freezing protocols. After cooling at 1 degree C/min in solutions containing 4% HES and various concentrations of DMSO there was a dramatic fall in CD34+ recovery from 85.4% (s.d. 28.4) to 12.2% (s.d. 10.0) as DMSO concentration was reduced from 5 to 2.5%. Varying HES concentration in solutions containing 5% DMSO did not have a significant effect on CD34+ cell recovery. Increasing cooling rate from 1 to 10 degrees C/min significantly reduced CD34+ recovery (P < 0.0001) while increasing DMSO concentration up to 10% had little effect (P = 0.8, two-way ANOVA). Good recovery of UCB CD34+ cells can be achieved with 5-10% DMSO at a controlled cooling rate of 1 degrees C/min. There was a significant difference (P < 0.0001) in the apparent recovery of CD34+ cells between paired aliquots thawed in the presence (recovery = 76.8%, s.d. 26.0) and absence (32.5%, s.d. 18.7) of DNase. In conclusion, conditions for cryopreserving UCB for clinical banking that yield optimal recovery of CD34+ cells have been established.     

Cryopreservation of unfertilized mouse oocytes: the effect of replacing sodium with choline in the freezing medium

Although embryo cryopreservation has become commonplace in many species, effective methods are not available for routine freezing of unfertilized eggs. Cryopreservation-induced damage may be caused by the high concentration of sodium ions in conventional freezing media. This study investigates the effect of a newly developed low-sodium choline-based medium (CJ2) on the ability of unfertilized, metaphase II mouse eggs to survive cryopreservation and develop to the blastocyst stage in vitro. Specifically, the effects of cooling to subzero temperatures, thawing rate, LN2 plunge temperature, and equilibration with a low-sodium medium prior to freezing are examined. In contrast to cooling to 23, 0, or -7.0 degreesC in a sodium-based freezing medium (ETFM), cooling in CJ2 had no significant negative effect on oocyte survival or development. Oocytes frozen in CJ2 survived plunging into LN2 from -10, -20, or -33 degreesC at significantly higher rates than oocytes frozen in ETFM. With the protocol used (1.5 M PrOH, 0.1 M sucrose, -0.3 C/min, plunging at -33 degreesC) rapid thawing by direct submersion in 30 degreesC water was more detrimental to oocyte survival than holding in air for 30 or 120 s prior to transfer to water. Equilibration of unfertilized oocytes with a low-sodium medium prior to cryopreservation in CJ2 significantly increased survival and blastocyst development. These results demonstrate that the high concentration of sodium in conventional freezing media is detrimental to oocyte cryopreservation and show that choline is a promising replacement. Reducing the sodium content of the freezing medium to a very low level or eliminating sodium altogether may allow oocytes and other cells to be frozen more effectively.     

Rapid cooling contracture with cold cardioplegia

BACKGROUND: Cold cardioplegia can induce rapid cooling contracture. The relations of cardioplegia-induced cooling contracture to myocardial temperature or myocyte calcium are unknown. METHODS: Twelve crystalloid-perfused isovolumic rat hearts received three 2-minute cardioplegic infusions (1 mmol/L calcium) at 4 degrees, 20 degrees, and 37 degrees C in random order, each followed by 10 minutes of beating at 37 degrees C. Finally, warm induction of arrest by a 1-minute cardioplegic infusion at 37 degrees C was followed by a 1-minute infusion at 4 degrees C. Indo-1 was used to measure the intracellular Ca2+ concentration in 6 of these hearts. Additional hearts received hypoxic, glucose-free cardioplegia at 4 degrees or 37 degrees C. RESULTS: After 1 minute of cardioplegia at 4 degrees, 20 degrees, and 37 degrees C, left ventricular developed pressure rose rapidly to 54% +/- 3%, 43% +/- 3%, and 18% +/- 1% of its prearrest value, whereas the intracellular Ca2+ concentration reached 166% +/- 23%, 94% +/- 4%, and 37% +/- 10% of its prearrest transient. Coronary flow was 5.7 +/- 0.2, 8.7 +/- 0.3, and 12.6 +/- 0.6 mL/min, respectively. Warm cardioplegia induction at 37 degrees C reduced left ventricular developed pressure and [Ca2+]i during subsequent 4 degrees C cardioplegia by 16% (p = 0.001) and 34% (p = 0.03), respectively. Adenosine triphosphate and phosphocreatine contents were lower after 4 degrees C than after 37 degrees C hypoxic, glucose-free cardioplegia. CONCLUSIONS: Rapid cooling during cardioplegia increases left ventricular pressure, [Ca2+]i and coronary resistance, and is energy consuming. The absence of rapid cooling contracture may be a benefit of warm heart operations and warm induction of cardioplegic arrest.     

Cryopreservation of equine embryos with glycerol plus sucrose and glycerol plus 1,2-propanediol

Six or 7-day-old equine embryos were divided into 4 groups; Group 1, n = 15, Day 7 embryos destined for immediate transfer; Group 2, n = 15, Day 6 embryos destined for deep-freezing with glycerol plus sucrose as cryoprotectant; Group 3, n = 10, Day 6 embryos destined for deep-freezing with glycerol plus 1,2-propanediol as cryoprotectant and Group 4, n = 3, fresh embryos destined for ultrastructural analysis. All the frozen/thawed embryos were transferred to recipient mares, except 3 embryos in Group 3 that were subjected to ultrastructural analysis. After thawing the cryoprotectants were removed by successive dilutions in PBS + 15% v:v fetal calf serum (FCS) containing decreasing concentrations of the cryoprotectants. Pregnancy was diagnosed ultrasonographically in 53.3%, 13.3% and 0% of the mares in Groups 1, 2 and 3 respectively. Ultrastructural analysis showed differences between frozen/thawed and fresh embryos. In the former, embryonic cells were deformed and showed dilation of the intercellular and perivitelline spaces, a decrease of desmosome number in the junctional complexes, few microvilli on the apical surface of the trophectoderm and an almost total absence of pinocytotic vesicles. Most of the mitochondria showed regions containing dilation and irregularities on the cristae, which appeared electron-dense. The results obtained with Groups 2 and 3 embryos showed that the cryoprotectants employed were not effective in protecting the embryos against damage during freezing and thawing. Indeed, the ultrastructural changes observed in the Group 3 embryos explained the absence of any established pregnancies in this group of mares.     

Cryopreservation of expanded mouse blastocysts by vitrification in ethylene glycol-based solutions

Experiments were conducted to find optimal conditions for obtaining high survival of expanded mouse blastocysts after vitrification. The vitrification solutions used were designated EFS20, EFS30 and EFS40, and contained 20%, 30% and 40% ethylene glycol, respectively, diluted in PB1 medium containing 30% Ficoll plus 0.5 mol sucrose l-1. In the toxicity test of the solutions and each cryoprotectant, ethylene glycol was found to be toxic to embryos. For vitrification, expanded blastocysts were exposed to the vitrification solutions at 10, 20 or 25 degrees C for various periods; they were then cooled rapidly in liquid nitrogen, after which they were warmed rapidly. When the embryos were directly exposed to EFS40 at 20 degrees C for 2 min before vitrification, 66% of them re-expanded during 48 h of post-warming culture. The re-expansion rates decreased when exposure time was shortened (0.5 min), when exposure temperature was lowered (10 degrees C), or when embryos were vitrified in EFS20 and EFS30, although these conditions should be less toxic. When embryos had been pretreated in a dilute (10-20%) ethylene glycol solution for 5 min, followed by short exposure (0.5 min) to EFS40 at 20 degrees C, post-vitrification survival rate increased to 83-84%; furthermore, the rate reached 94% when the temperature was increased to 25 degrees C. Expanded blastocysts cryopreserved by this two-step method developed into live young as well as control embryos after transfer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ambient temperature effects on thermoregulation and endurance in anticholinesterase-treated rats

In sedentary animals, physostigmine (PH) administration resulted in a decreased core temperature that is ambient temperature (Ta) dependent. PH administration in rats exercising on a treadmill (26 degrees C, 50% rh, 11m/min, 6 degrees incline) decremented endurance and increased rate of rise of core temperature (heating rate, HR). This study was undertaken to examine the effects of Ta on the endurance and thermoregulatory decrements of PH-treated running rats. Adult male rats (510-530g) were given either 0.2ml saline (C) or 100 ug/kg physostigmine salicylate in 0.2 ml saline via tail vein 15 min prior to the start of running to exhaustion at 10, 15, 26, or 30 degrees C. In both C- and PH-treated groups, endurance decreased and HR increased with increasing Ta from 15 to 30 degrees C. At 15 and 26 degrees C the C rats ran significantly (p < .05) longer and had significantly lower HR than the PH rats: C15 = 90 +/- 8 min, 0.022 +/- 0.006 degrees C/min; C26 = 67 +/- 6, 0.051 +/- 0.007; PH15 = 57 +/- 5, 0.052 +/- 0.008; and PH26 = 43 +/- 6, 0.092 +/- 0.007. At 10 and 30 degrees C there were no significant differences between C and PH-treated rats. A Ta of 30 degrees C was too high for effective cooling in either group, and at 10 degrees C both groups were able to dissipate heat despite the increased metabolic rate of the PH-treated rats. The PH-treated rat model of cholinergic drug effect is useful at a Ta of 15 and 26 degrees C.     

Changes in biogenic amines and microbiological analysis in albacore (Thunnus alalunga) muscle during frozen storage

Albacore specimens of extra quality were analyzed for their biogenic amine contents after 1, 3, 6, and 9 months of frozen storage at -18 degrees C or -25 degrees C. A high-performance liquid chromatography method involving a linear elution gradient was optimized for the identification and determination of putrescine, cadaverine, histamine, spermidine, and spermine in albacore tuna. Putrescine was the biogenic amine that showed the highest increase, reaching concentrations of 59.04 ppm (815% of the initial level) and 68.26 ppm (942% of the initial level) in the white muscle of albacore after 9 months of frozen storage at -18 and -25 degrees C, respectively. Cadaverine, histamine, and spermidine concentrations were below 3, 5, and 11 ppm, respectively, after 9 months of frozen storage, while spermidine underwent a significant decrease at both storage temperatures. Microbiological analysis confirmed the absence of species of Enterobacteriaceae in 75% of the albacore specimens after 9 months of frozen storage; coliforms were always below 3 CFU/g. The survival rate of the psychrotrophic microorganisms after 9 months of frozen storage at -25 degrees C was 4.6%, while 38.9 and 92.1% of the aerobic mesophiles present in the white muscle of albacore before freezing survived 9 months of storage at -18 and -25 degrees C, respectively.     

MPP+ induced substantia nigra degeneration is attenuated in nNOS knockout mice

Differential scanning calorimetry was used to characterize thermal events associated with freezing and melting of suspensions and extracts of Panagrolaimus davidi, an Antarctic nematode which can survive intracellular freezing. Nematode suspensions produced a single freezing exotherm with a shoulder on the peak representing the freezing of the nematodes. A shoulder on the peak of melting endotherms indicates the melting of the nematodes and of the water surrounding them. Exotherms were also detected from individual nematodes mounted in liquid paraffin. The freezing of nematodes was very rapid and in marked contrast to that of freezing-tolerant insects and vertebrates, which take hours or days to freeze. Eighty-two percent of the nematodes' body water froze. High levels of survival were obtained in nematodes exposed to temperatures down to -40 degrees C. No additional thermal events were observed after the freezing event and before the melting of samples cooled to -40 degrees C, indicating no changes in the proportion of body water frozen. Ice nucleating activity is present in nematode suspensions but not in supernatants from nematode extracts. No thermal hysteresis activity was detected in nematode extracts.     

Thermoregulatory vasoconstriction impairs active core cooling

BACKGROUND: Many clinicians now consider hypothermia indicated during neurosurgery. Active cooling often will be required to reach target temperatures < 34 degrees C sufficiently rapidly and nearly always will be required if the target temperature is 32 degrees C. However, the efficacy even of active cooling might be impaired by thermoregulatory vasoconstriction, which reduces cutaneous heat loss and constrains metabolic heat to the core thermal compartment. The authors therefore tested the hypothesis that the efficacy of active cooling is reduced by thermoregulatory vasoconstriction. METHODS: Patients undergoing neurosurgical procedures with hypothermia were anesthetized with either isoflurane/nitrous oxide (n = 13) or propofol/fentanyl (n = 13) anesthesia. All were cooled using a prototype forced-air cooling device until core temperature reached 32 degrees C. Core temperature was measured in the distal esophagus. Vasoconstriction was evaluated using forearm minus fingertip skin-temperature gradients. The core temperature triggering a gradient of 0 degree C identified the vasoconstriction threshold. RESULTS: In 6 of the 13 patients given isoflurane, vasoconstriction (skin-temperature gradient = 0 degrees C) occurred at a core temperature of 34.4 +/- 0.9 degree C, 1.7 +/- 0.58 h after induction of anesthesia. Similarly, in 7 of the 13 patients given propofol, vasoconstriction occurred at a core temperature of 34.5 +/- 0.9 degree C, 1.6 +/- 0.6 h after induction of anesthesia. In the remaining patients, vasodilation continued even at core temperatures of 32 degrees C. Core cooling rates were comparable in each anesthetic group. However, patients in whom vasodilation was maintained cooled fastest. Patients in whom vasoconstriction occurred required nearly an hour longer to reach core temperatures of 33 degrees C and 32 degrees C than did those in whom vasodilation was maintained (P < 0.01). CONCLUSIONS: Vasoconstriction did not produce a full core temperature "plateau," because of the extreme microenvironment provided by forced-air cooling. However, it markedly decreased the rate at which hypothermia developed. The approximately 1-h delay in reaching core temperatures of 33 degrees C and 32 degrees C could be clinically important, depending on the target temperature and the time required to reach critical portions of the operation.     

Cryopreservation of mouse spermatozoa from inbred and F1 hybrid strains

Mouse epididymal spermatozoa from inbred(BALB/c, C3H/He, C57BL/6N, CBA/JN and DBA/2N) and F1 hybrid (B6C3F1, BDF1 and CDF1) strains suspended in cryopreservation solution (18% raffinose and 3% skim milk in distilled water) were frozen and stored at -196 degrees C. After thawing at room temperature, sperm motility and fertilizing ability were examined. Spermatozoa from all of the strains were successfully frozen, although the motility and the fertilization rates of frozen-thawed spermatozoa (the proportions of the fresh oocytes from Jcl:ICR strain which developed to pronuclear oocytes and 2-cell embryos after insemination by frozen-thawed spermatozoa) varied among strains (motility: 23% for C57BL/6N to 62% for DBA/2N; fertilization rates: 26% for C57BL/6N to 89% for DBA/2N). Nearly all 2-cell embryos fertilized by frozen-thawed spermatozoa were transferred to the oviducts of pseudopregnant recipients and 35-62% of 2-cell embryos developed into normal young.     

Freezing temperature of finger skin

In 45 subjects, 154 frostnips of the finger were induced by cooling in air at -15 degrees C with various wind speeds. The mean supercooled skin temperature at which frostnip appeared was -9.4 degrees C. The mean skin temperature rise due to heat of fusion at ice crystallization was 5.3 degrees C. The skin temperature rose to what was termed the apparent freezing point. The relation of this point to the supercooled skin temperature was analyzed for the three wind speeds used. An apparent freezing point for a condition of no supercooling was calculated, estimating the highest temperature at which skin freezes at a given wind speed. The validity of the obtained differences in apparent freezing point was tested by an analysis of covariance. Although not statistically significant, the data suggest that the apparent freezing point with no supercooling decreases with increasing wind velocity. The highest calculated apparent freezing point at -15 degrees C and 6.8 m/s was 1.2 degrees C lower than the true freezing point for skin previously determined in brine, which is a statistically significant difference.     

A simplified method for cryopreservation of hematopoietic stem cells with -80 degrees C mechanical freezer with dimethyl sulfoxide as the sole cryoprotectant

A simplified method for cryopreservation was developed with 10% dimethylsulfoxide (DMSO) as the sole cryoprotectant without rate-controlled freezing. This method produced high recovery rate for mononucleated cells (87%) and elevated trypan blue viability (90%). Autologous peripheral blood stem cells (PBSCs) and bone marrow cells with plasma and 10% DMSO were frozen and stored in a -80 degrees C mechanical freezer. Eleven patients with solid and hematological malignancies were transplanted with autologous bone marrow or PBSCs. The median number of infused mononuclear cells (MNC) and CD34+ cells were 3.63 x 10(8)/Kg and 4.80 x 10(6)/Kg, respectively. The median number of infused post-thawing CFU-GM was 20 x 10(4)/Kg. All patients showed a rapid and sustained engraftment. The mean times to reach a neutrophil count of 0.5 x 10(9)/L and a platelet count of 50 x 10(9)/L were 11 and 13 days, respectively. All patients are alive and 10 in unmaintained complete remission for 3-9 months after transplantation. These results show the efficacy of this simplified cryopreservation technique that will be useful for institutions without rate-controlled freezing facilities.