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The enhancements of chill tolerance of Artemia salina to single (glycerol, glucose or sucrose) and double (glucose-sucrose) component cryoprotectant solutions were determined. Zero, three, six, twelve and twenty-four hour post-hatch larvae were immersed in -1°C cryoprotectant solutions for periods up to 90 minutes. The ability of different larval stages to survive an instantaneous change from 28° to -1° versus a slow cooling regiment (1°C/hr) from 28° to 18° followed by instantaneous cooling to -1°C were compared. Most cryoprotectant solutions showed definite enhancement of survival with the multiple better than single component systems. Survival exceeded 200–500% over -1°C cryoprotectant free controls. The significance of these results to the development of cryogenic larval banking techniques is discussed.

Background The benefits of hypothermia for preventing ischemic injury are well known, but its application in surgery to protect the whole body during procedures requiring circulatory arrest is currently limited to Methods and Results With the use of extracorporeal cardiac bypass, 14 adult dogs were exsanguinated during cooling; 11 dogs were blood substituted using in combination the “purge” and “maintenance” solutions (group 1), and 3 dogs were perfused throughout with the “purge” solution alone as controls (group 2). After cardiac arrest, the solutions were continuously circulated for 3 \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\frac{1}{2}\) \end{document} hours by the extracorporeal pump (flow rate, 40 to 85 mL · kg −1 · min −1 ; mean arterial blood pressure, 25 to 40 mm Hg). The temperature was maintained at P P P =.03), and for CK-BB, the group 1 value was 108±22 versus 617±154 for group 2 ( P =.03). Neurological evaluation using deficit scores (NDS) was based on a modification of the Glasgow Coma Scale score: 0, normal; 1, minimal abnormality; 2, weakness; 3, paralysis; 4, coma; and 5, death. At days 1 and 2 after surgery, NDS (mean±SEM) were 0±0 for the experimental group versus 1.5±0.5 for the control group. At days 3 and 7 after surgery, NDS were 0±0 for group 1 versus 1.0±1.0 for group 2. Conclusions The faster neurological recovery of dogs treated with the “intracellular-type” maintenance solution supports the biochemical data showing the benefits of this type of blood substitute for extending the safe limits of hypothermic cardiac arrest procedures to >3 hours.

The requirement for more effective cryopreservation (CP) methodologies in support of the emerging fields of cell bioprocessing and cell therapy is now critical. Current CP strategies appropriately focus on minimizing the damaging actions of physicochemical stressors and membrane disruption associated with extra- and intracellular ice formation that occurs during the freeze-thaw process. CP protocols derived from this conceptual paradigm, however, yield suboptimal survival rates. We now provide the first report on the identification of delayed-onset cell death following CP and the significance of modulating molecular biological aspects of the cellular responses (apoptosis) to low temperature as an essential component to improve postthaw outcome. In this study we quantitatively examined the molecular basis of cell death associated with CP failure in a canine renal cell model. In addition, we report on the significant improvement in CP outcome through the modulation of these molecular mechanisms by the utilization of an organ preservation solution. HypoThermosol. Further, the utilization of HypoThermosol as the preservation medium and the modulation of molecular-based cell death have led to a paradigm shift in biologic preservation methodologies. The recognition of molecular mechanisms associated with CP-induced cell death offers the promise of improved CP of more complex and/or fragile biological systems such as stem cells, engineered tissues, and human organs.

A new concept in cryopreservation solution design was developed that focuses on the use of an intracellular-type, hypothermic maintenance medium coupled with additives that inhibit cryopreservation-induced apoptosis. HypoThermosol' (HTS), a hypothermic (4 degrees C) maintenance medium utilized in the long-term storage of cell, tissue, and organ systems, was tested for cryoprotective capability on a renal cell line (Madin-Darby Canine Kidney cells). HTS and HTS derivatives were tested against conventional cell culture medium (Dulbecco's Minimal Essential medium, DME) as the cryoprotectant carrier solution because (1) cells are exposed to an extended state of hypothermia during the freeze-thaw process, and (2) HTS is designed to protect cells exposed to a hypothermic state. Cells separately cryopreserved in either HTS or DME + 5% dimethyl sulfoxide (DMSO) yielded equivalent 24-h postthaw survival (approximately 30%) and 5-d recovery (approximately 90%). Cells cryopreserved in CryoStor CS 5, a HTS derivative containing 5% DMSO, yielded approximately 75% 24-h postthaw survival and recovery to 100% within 3 d. DNA gel electrophoresis was performed to determine the mechanisms of cell death contributing to cryopreservation failure. Cells preserved in DME (DMSO-free) died primarily through necrosis, whereas cells preserved in either DME + 5% DMSO, HTS, or CryoStor CS 5 died through a combination of apoptosis and necrosis. This observation led to the inclusion of an apoptotic inhibitor designed to improve cryopreservation outcome. MDCK cells cryopreserved in CryoStor CS 5 supplemented with an apoptotic inhibitor (Caspase I Inhibitor V), hereafter termed CryoStor CS 5N, resulted in a 24-h postthaw survival and recovery rate exceeding that of any other cryoprotective solution tested (85%). We conclude that: (1) the use of HTS (a dextran-based, intracellular-type solution) without DMSO can yield postthaw viability equivalent to that of standard DMSO-based cryopreservation methods, (2) postthaw viability can be significantly increased through the use of an intracellular-type solution in conjunction with DMSO, (3) the use of HTS allows for cryopreservation to be accomplished with reduced levels of cryoprotectants, and (4) the regulation of apoptosis is essential for the improvement of cryopreservation outcome.

Cryosurgical treatment of unresectable hepatic malignancies has proven beneficial in adults. Concerns regarding its use in children include the effect on growth and the risk of injury to adjacent structures. To test the effect of cryoablation on adjacent vascular structures in a growing animal, liquid nitrogen cryoablation was performed on a juvenile murine model. Sprague Dawley rats underwent double freeze-thaw cryoablation of the abdominal aorta with interposed liver tissue. Serial sacrifices were performed over 120 days. Comparisons were made with sham-operated controls. Overall, animal growth paralleled that of sham controls through all time points. Gross examination of aortic diameter also showed similar growth in vessel size between the groups. Histologic analysis demonstrated injury after cryoablation with smooth muscle cell vacuolization, followed by cell death. Aortic media layer collapse resulted from cellular loss, however, elastin fiber composition was maintained. Aortic patency was preserved despite evidence of cellular injury and aortic wall remodeling. An associated thermal sink effect on the opposing wall was identified. After cryoablation adjacent to the abdominal aorta in adolescent rats, vascular patency is maintained and animal growth and structural function is preserved, despite cellular injury and wall compression. These observations suggest that cryoablation may be a useful treatment adjunct in young subjects.

Aqueous solutions containing d-ribose demonstrate the ability to form more than one vitreous domain when exposed to low temperatures. Differential scanning calorimetry revealed two glass transitions (at Tgs of -63 and -43 degrees C) upon cooling and warming at a constant rate of 5 degrees C.min-1. The bulk water of the solution crystallizes at -18 degrees C (Tc). Heat capacity and enthalpy changes, and the derivatives for each thermal event, are calculated. Relaxation studies on the observed Tgs produced anticipated overshoots characteristic of the presence of glassy states.

Elevated fructose, glucose and trehalose levels were found inPolistes exclamans andP. annularis after exposure to cold. Glycerol was found only after extensive exposure to cold or in mid-winter. InP. exclamans mean supercooling points (SCP) decreased to a low of −12.9° C in February. InP. annularis SCP were never below −6.8° C in the field, but laboratory acclimation at +5° C resulted in SCP of −10.7° C. Freezing was lethal to adultPolistes under all circumstances. Workers and future queens were separated on the basis of the appearance of their fat bodies. ForP. exclamans, only 17 % of the future queens died during 15 days at +5° C (N=29) while 76 % of the workers died during identical cold exposure (N=45). The surviving future queens had elevated cryoprotectant levels consisting mainly of fructose in their hemolymph, while the few workers that survived did not. These experiments indicate that there are physiological differences between workers and future queens inPolistes collected in autumn and that fat body appearance determined using non-invasive techniques is a reliable indicator of caste. Males were similar to future queens in their response to cold indicating that autumn mating activity may extend into winter.

Understanding of insect dispersal-migration, including behavior, physiology, and diversity of the species involved, has developed slowly. Early theories focussed on concepts of passive dispersal and undirectional, nonsurvival related migration (Elton 1925; Felt 1928;Darlington 1938). More recent studies have been concerned with return-flight, survival-based migrations (Williams 1965; Johnson 1969).

The integrity of the peripheral heterothermic response was monitored in adult Sprague-Dawley rats during cold acclimation. Subcutaneous peripheral temperature gradients were simultaneously recorded in the hindlimbs. One limb was exposed to room temperature (22 +/- 2 degrees C) while the contralateral limb was gradually cooled to 0 +/- 1 degrees C. Noncontrols were acclimated at 5 +/- 1 degrees C for periods up to 35 days. Controls responded to the cooling regimen (25 to 0 degrees C at 0.5 degrees C . min-1) in a "poikilothermic" manner indicating local cold-induced vasoconstriction (CIVC). CIVC was not released until tissue temperatures reached 22,3 +/- 2.5 degrees C whereupon nonpatterned limb temperature fluctuations, Lewis' hunting response, were often initiated. The hunting response occurred synchronously in the contralateral warmed limb despite its elevated temperature. The experiments revealed a progressive decrease in the intensity of heterothermy indicative of an earlier onset of cold-induced vasodilation as well as increased resistance to tissue cooling with increasing acclimation time. Following 21 days at 5 degrees C, limb exposure to 0 degrees C resulted in a 2-4 degrees C drop in tissue temperature. The time course of the diminution in peripheral heterothermy is discussed. In addition, evidence supporting the hypothesis of a central component in the regulation of the hunting response is presented.

There is considerable interest in the transplantation of mammalian tissues for the treatment of disease states and trauma and the correction of tissue malfunction. The logistics of tissue procurement for transplantation are such that some method of tissue preservation is highly desirable. To date, the most common method of preservation has been hypothermic storage, either cold storage above freezing (usually 4 °C) or else cryopreservation and storage in the frozen state (usually — 70 °C to — 196 °C). The advantage of hypothermic storage in the unfrozen state is that ice damage is avoided, but the disadvantage is that there is a limited storage duration of about 1 week or less (11, 19). The advantage of cryopreservation is that truly long-term preservation ranging from months to years can be obtained, while the disadvantage is in the lack of a complete understanding of the mechanism of cryoinjury, so that cryopreservation produces significant damage as a result of ice formation during the freezing process. Nevertheless, cryopreservation has decided advantages over refrigerated storage provided that effective methodologies can be developed for the specific tissue under consideration.

An analysis of 80% ethanol-soluble carbohydrates and polyols (polyhydric alcohols) from the lichens Ramalina stenospora Miill. Arg. and R. complanata (Sw.) Ach., was made by means of a high-pressure liquid chro- matographic method. Two chromatographic columns, utBondapak carbohy- drate and Aminex Q-150S, were used to effect the analysis. Of the two columns used the yBondapak column was found to resolve the larger number of carbohydrates from synthetic mixtures; however, the Aminex Q-150S column was required for resolution of mannitol-glucose-sorbitol and arabitol-ribitol complexes. Aminex Q-150S was more efficient in the separa- tion of polyols than monosaccharides. Utilization of high-pressure liquid chromatography was found to be a rap- id, sensitive method for analysis of carbohydrates and polyols routinely found in lichen extracts. It has the desirable features of small sample size, recover- able fractions, rapidity and ease of quantification of lichen metabolites. The assessment of carbohydrate and polyhydric alcohol content in lichens, as well as the biotrophic transport of these products, have become important parameters in the analyses of physiological processes in lichen symbionts. Several methods of anal- yses of carbohydrates and polyhydric alcohols (polyols) from lichens and fungi are in current use (Lewis & Smith, 1967; Culberson, 1969, 1970). Development of automated assay methods to determine these products in lichens has focused upon gas chroma- tography (Komiya & Shibata, 1971; Culberson, et al. 1977). The method of high-pressure liquid chromatography (HPLC), which is reported in this paper, has features of rapidity, sensitivity, resolution and fraction recovery, and eliminates the need of sample derivatization required in gas chromatography (Brown, 1973). HPLC was first applied to lichen metabolites to elucidate the derivatives of the