Cryopreservation of solid organs might be possible using a mixture of cell-permeable agents, cryo... more Cryopreservation of solid organs might be possible using a mixture of cell-permeable agents, cryoprotectants (CPA), which are designed to completely preclude ice crystal formation during cooling to cryogenic temperatures. The effects of a specific prototype solution (VS4) were evaluated by normothermic blood perfusion in vitro. Rabbit kidneys were divided into three groups: untreated controls (n = 7), Euro-Collins (EC)-perfused controls (n = 6) and VS4 (4970, w/v) CPA-perfused kidneys (n = 7). After a 2-h blood perfusion, five of the seven CPA-perfused kidneys developed polyuria (0.21 mlxmin-'xg-') relative to untreated controls (0.07 mlxminp'xg-') or EC-perfused kidneys (0.06 mlxmin-'xgp'), owing to the lower reabsorption of water (34.3%), Naf (34.2%) and glucose (35.6%). Furthermore, two kidneys were non-functional with virtually no urine production. Reduced tubular function was associated with reduced oxygen consumption (3.6 versus 2.3 versus 2.0 pmolexmin-'xgp' for controls, EC-and CPA-perfused kidneys, respectively) and increased weight gain (17% versus 20% versus 30%, respectively) after blood perfusion. Therefore, the current results provide insight into both the physiological effects of VS4 and the limits of reversibility of renal pathophysiological states. Our results also indicate that in vitro monitoring of oxygen consumption and weight gain of perfused organs could be used as predictors of renal function.
Although much interest has attended the cryopreservation of immature neurons for subsequent thera... more Although much interest has attended the cryopreservation of immature neurons for subsequent therapeutic intracerebral transplantation, there are no reports on the cryopreservation of organized adult cerebral tissue slices of potential interest for pharmaceutical drug development. We report here the first experiments on cryopreservation of mature rat transverse hippocampal slices. Freezing at 1.2°C/min to À20°C or below using 10 or 30% v/v glycerol or 20% v/v dimethyl sulfoxide yielded extremely poor results. Hippocampal slices were also rapidly inactivated by simple exposure to a temperature of 0°C in artificial cerebrospinal fluid (aCSF). This effect was mitigated somewhat by 0.8 mM vitamin C, the use of a more ''intracellular'' version of aCSF having reduced sodium and calcium levels and higher potassium levels, and the presence of a 25% w/v mixture of dimethyl sulfoxide, formamide, and ethylene glycol (''V EG solutes''; Cryobiology 48, pp. 2004). It was not mitigated by glycerol, aspirin, indomethacin, or mannitol addition to aCSF. When RPS-2 (Cryobiology 21, pp. [260][261][262][263][264][265][266][267][268][269][270][271][272][273] 1984) was used as a carrier solution for up to 50% w/v V EG solutes, 0°C was more protective than 10°C. Raising V EG concentration to 53% w/v allowed slice vitrification without injury from vitrification and rewarming per se, but was much more damaging than exposure to 50% w/v V EG . This problem was overcome by using the analogous 61% w/v VM3 vitrification solution (Cryobiology 48, pp. 157-178, 2004) containing polyvinylpyrrolidone and two extracellular ''ice blockers.'' With VM3, it was possible to attain a tissue K + /Na + ratio after vitrification ranging from 91 to 108% of that obtained with untreated control slices. Microscopic examination showed severe damage in frozen-thawed slices, but generally good to excellent ultrastructural and histological preservation after vitrification. Our results provide the first demonstration that both the viability and the structure of mature organized, complex neural networks can be well preserved by vitrification. These results may assist neuropsychiatric drug evaluation and development and the transplantation of integrated brain regions to correct brain disease or injury.
The simple linear polymer polyglycerol (PGL) was found to apparently bind and inhibit the ice nuc... more The simple linear polymer polyglycerol (PGL) was found to apparently bind and inhibit the ice nucleating activity of proteins from the ice nucleating bacterium Pseudomonas syringae. PGL of molecular mass 750 Da was added to a solution consisting of 1 ppm freeze-dried P. syringae 31A in water. Differential ice nucleator spectra were determined by measuring the distribution of freezing temperatures in a population of 98 drops of 1 lL volume. The mean freezing temperature was lowered from )6.8°C (control) to À8:0; À9:4; À12:5, and )13.4°C for 0.001, 0.01, 0.1, and 1% w/w PGL concentrations, respectively (SE < 0.2°C). PGL was found to be an ineffective inhibitor of seven defined organic ice nucleating agents, whereas the general ice nucleation inhibitor polyvinyl alcohol (PVA) was found to be effective against five of the seven. The activity of PGL therefore seems to be specific against bacterial ice nucleating protein. PGL alone was an ineffective inhibitor of ice nucleation in small volumes of environmental or laboratory water samples, suggesting that the numerical majority of ice nucleating contaminants in nature may be of nonbacterial origin. However, PGL was more effective than PVA at suppressing initial ice nucleation events in large volumes, suggesting a ubiquitous sparse background of bacterial ice nucleating proteins with high nucleation efficiency. The combination of PGL and PVA was particularly effective for reducing ice formation in solutions used for cryopreservation by vitrification. Ó
Cryopreservation of solid organs might be possible using a mixture of cell-permeable agents, cryo... more Cryopreservation of solid organs might be possible using a mixture of cell-permeable agents, cryoprotectants (CPA), which are designed to completely preclude ice crystal formation during cooling to cryogenic temperatures. The effects of a specific prototype solution (VS4) were evaluated by normothermic blood perfusion in vitro. Rabbit kidneys were divided into three groups: untreated controls (n = 7), Euro-Collins (EC)-perfused controls (n = 6) and VS4 (4970, w/v) CPA-perfused kidneys (n = 7). After a 2-h blood perfusion, five of the seven CPA-perfused kidneys developed polyuria (0.21 mlxmin-'xg-') relative to untreated controls (0.07 mlxminp'xg-') or EC-perfused kidneys (0.06 mlxmin-'xgp'), owing to the lower reabsorption of water (34.3%), Naf (34.2%) and glucose (35.6%). Furthermore, two kidneys were non-functional with virtually no urine production. Reduced tubular function was associated with reduced oxygen consumption (3.6 versus 2.3 versus 2.0 pmolexmin-'xgp' for controls, EC-and CPA-perfused kidneys, respectively) and increased weight gain (17% versus 20% versus 30%, respectively) after blood perfusion. Therefore, the current results provide insight into both the physiological effects of VS4 and the limits of reversibility of renal pathophysiological states. Our results also indicate that in vitro monitoring of oxygen consumption and weight gain of perfused organs could be used as predictors of renal function.
The simple linear polymer polyglycerol (PGL) was found to apparently bind and inhibit the ice nuc... more The simple linear polymer polyglycerol (PGL) was found to apparently bind and inhibit the ice nucleating activity of proteins from the ice nucleating bacterium Pseudomonas syringae. PGL of molecular mass 750 Da was added to a solution consisting of 1 ppm freeze-dried P. syringae 31A in water. Differential ice nucleator spectra were determined by measuring the distribution of freezing temperatures in a population of 98 drops of 1 lL volume. The mean freezing temperature was lowered from )6.8°C (control) to À8:0; À9:4; À12:5, and )13.4°C for 0.001, 0.01, 0.1, and 1% w/w PGL concentrations, respectively (SE < 0.2°C). PGL was found to be an ineffective inhibitor of seven defined organic ice nucleating agents, whereas the general ice nucleation inhibitor polyvinyl alcohol (PVA) was found to be effective against five of the seven. The activity of PGL therefore seems to be specific against bacterial ice nucleating protein. PGL alone was an ineffective inhibitor of ice nucleation in small volumes of environmental or laboratory water samples, suggesting that the numerical majority of ice nucleating contaminants in nature may be of nonbacterial origin. However, PGL was more effective than PVA at suppressing initial ice nucleation events in large volumes, suggesting a ubiquitous sparse background of bacterial ice nucleating proteins with high nucleation efficiency. The combination of PGL and PVA was particularly effective for reducing ice formation in solutions used for cryopreservation by vitrification. Ó
Wire-length resonant coils, which are based on equating a wavelength of RF with the wire length u... more Wire-length resonant coils, which are based on equating a wavelength of RF with the wire length used in winding a coil, were previously developed for use in hyperthermia treatment for cancer therapy. Design modifications, coupled with a series of experiments, established their potential use for rapidly and uniformly rewarming canines following hypothermic cardiac surgery. The authors detail a new design which incorporates a single-end coaxial input and RF shielding, allowing it to be used outside of a screen room environment. Data from early experiments to investigate this system's usefulness in rewarming cryogenically preserved donor organs are presented and discussed.>
To facilitate the development of assays for the discovery of pharmaceuticals capable of mimicking... more To facilitate the development of assays for the discovery of pharmaceuticals capable of mimicking the effects of caloric restriction on life-and healthspan (CR mimetics), we evaluated the effectiveness of glucoregulatory and putative cancer chemopreventatives in reproducing the hepatic gene-expression profile produced by long-term CR (LTCR) using Affymetrix microarrays. We have shown that CR initiated late in life begins to extend lifespan, reduce cancer as a cause of death, and reproduce ~three quarters of the genomic effects of LTCR in 8 weeks (CR8). Eight weeks of metformin treatment was superior to CR8 at reproducing LTCRlike gene expression changes, maintaining a superior number of such changes over a broad range of statistical stringencies, and producing more gene ontology terms overlapping those produced by LTCR. Consistent with these results, metformin has been shown to reduce cancer incidence in mice and humans. Phenformin, a chemical cousin of metformin, extends lifespan and reduces tumor incidence in mice. Taken together, these results indicate that gene-expression biomarkers can be used to identify promising candidate CR mimetics. of the glucoregulatory compounds metformin (MET), glipizide (GLIP), GLIP plus MET (GM), and rosiglitazone (ROS) for their ability to reproduce the effects of LTCR on hepatic gene PG-00069-2005.R1
Magnetic resonance imaging was used to detect signs of regeneration of the thymus after approxima... more Magnetic resonance imaging was used to detect signs of regeneration of the thymus after approximately one month of human growth hormone administration. A 46-year-old human volunteer was placed on a regimen of recombinant human growth hormone and pharmaceutical grade dehydroepiandrosterone for one month. Mediastinal magnetic resonance images were collected at baseline and after the study period. Thymic cross sections were analyzed for total area and for the total gray area, which was taken to represent functional mass. Baseline and post-treatment blood samples were taken to follow changes in IGF-1 levels and related metabolites. The setting was an informal, non-institutional trial supervised by a physician will full informed consent of the volunteer. Visual inspection and image analysis demonstrated limited but distinct enlargement of the thymus after treatment, and an increase in the percent of thymic cross section represented by gray-appearing (functional) mass. Estimated total thymic functional volume was within the normal range at baseline, but after treatment was more than three standard deviations above the expected mean for a subject of this age, thus meeting a proposed definition of thymic hyperplasia for individuals. IGF-1 levels were confined to the upper range of normal for young adults. The present observations apparently provide the first demonstration of growth hormone induced partial reversal of established thymic involution in a normal human subject, and are consistent with previous measurements of restored immune function after the administration of human growth hormone to elderly individuals.
The objective of the present study was to determine whether rabbit kidneys could be perfused with... more The objective of the present study was to determine whether rabbit kidneys could be perfused with a 7.5 M vitrification solution (VS4, which vitrifies under applied pressure) without loss of function. To answer this question, kidneys were perfused with VS4 using a computer-based machine to gradually raise and lower concentration and then attached to the aorta and vena cava of a perfusor rabbit using an apparatus that permitted renal blood flow and renal function to be measured. About half (6/13) of the kidneys so evaluated resumed substantial immediate function after a transient period of severely reduced blood flow. Loss of function did not occur if cryoprotectant concentration was limited to 3.8 M. The loss of function produced by VS4 could be partially reproduced by artificially limiting blood reflow in control kidneys to simulate the transiently low flows caused by VS4 exposure. These results provide the first evidence that both the parenchyma and the vascular system of a sensitive mammalian organ can survive exposure to a vitrifiable concentration of cryoprotectant. ᭧ 1997 Academic Press
Cryoprotectant toxicity is a fundamental limiting factor for the successful cryopreservation of l... more Cryoprotectant toxicity is a fundamental limiting factor for the successful cryopreservation of living systems by both freezing and vitrification, and the ability to negate it would be attractive. Past attempts to demonstrate ''cryoprotectant toxicity neutralization" (CTN) have had many ups and downs. First convincingly introduced by Baxter and Lathe in 1971, the concept that certain amides can block toxic effects of dimethyl sulfoxide (Me 2 SO) was contradicted by direct experiments in 1990. But in 1995, the opposite mode of CTN, in which Me 2 SO blocked the damaging effects of formamide, was robustly demonstrated. Recent experiments have verified the original 1995 results and extended them to urea and acetamide, but no CTN was detected for N-methylamides (N-methylformamide, N,N-dimethylformamide, and N-methylacetamide). On the theory that the latter amides and acetamide might serve as low-toxicity structural analogs of formamide, urea, or Me 2 SO, competition experiments were carried out between them and formamide or urea, but CTN was not observed for these amide-amide systems. The idea that the N-methylamides might have non-specific rather than specific toxicity was supported by the fact that the concentrations of these amides that cause toxicity are similar to the concentrations that denature model proteins. Clear examples of neutralization of the toxicity of glycerol, propylene glycol, ethylene glycol, or Me 2 SO are presently lacking, but effects of the latter that depend on sulfhydryl oxidation have been reversed with reducing agents. In summary, CTN is a useful phenomenon with significant theoretical and practical implications.
24. Investigating hydroxyl radicals (OH) and 8-hydroxy-2 0 -deoxyguanosine as generic biomarkers ... more 24. Investigating hydroxyl radicals (OH) and 8-hydroxy-2 0 -deoxyguanosine as generic biomarkers of cryoinjury and cold storage stability.
Cryopreservation of solid organs might be possible using a mixture of cell-permeable agents, cryo... more Cryopreservation of solid organs might be possible using a mixture of cell-permeable agents, cryoprotectants (CPA), which are designed to completely preclude ice crystal formation during cooling to cryogenic temperatures. The effects of a specific prototype solution (VS4) were evaluated by normothermic blood perfusion in vitro. Rabbit kidneys were divided into three groups: untreated controls (n=7), Euro-Collins (EC)-perfused controls (n=6) and VS4 (49%, w/v) CPA-perfused kidneys (n=7). After a 2-h blood perfusion, five of the seven CPA-perfused kidneys developed polyuria (0.21 ml×min–1×g–1) relative to untreated controls (0.07 ml×min–1×g–1) or EC-perfused kidneys (0.06 ml×min–1×g–1), owing to the lower reabsorption of water (34.3%), Na+ (34.2%) and glucose (35.6%). Furthermore, two kidneys were non-functional with virtually no urine production. Reduced tubular function was associated with reduced oxygen consumption (3.6 versus 2.3 versus 2.0 μmole×min–1×g–1 for controls, EC- and CPA-perfused kidneys, respectively) and increased weight gain (17% versus 20% versus 30%, respectively) after blood perfusion. Therefore, the current results provide insight into both the physiological effects of VS4 and the limits of reversibility of renal pathophysiological states. Our results also indicate that in vitro monitoring of oxygen consumption and weight gain of perfused organs could be used as predictors of renal function.
Donor Schwann cells, perineurial cells, and vasculature are known to survive in grafts of periphe... more Donor Schwann cells, perineurial cells, and vasculature are known to survive in grafts of peripheral nerve. In the present study, we attempted to cryopreserve nerve to determine whether these cellular components of nerve would survive after transplantation and support host axonal regeneration through the graft. Four-centimeter lengths of peroneal nerves were removed from inbred adult American Cancer Institute (ACI) rats and placed into vials that contained a cryoprotective mixture of dimethyl sulfoxide and formamide (DF) at room temperature. Each vial with nerves in DF was cooled at a rate of 1–1.5°C/minute down to –40°C at which point the vials were plunged into liquid nitrogen at –196°C. After 5 weeks of storage, the nerves were thawed and DF removed. Some of the cryopreserved-thawed ACI nerves were transplanted as isografts into the legs of ACI rats. Other ACI nerves were used as allografts and inserted into immunologically normal Fischer (FR) rats that were untreated or were immunosuppressed with the drug Cyclosporin A (Cy-A). At surgery, only one end of the nerve graft was joined to the cut proximal end of the peroneal nerve of the host. The cellular elements of ACI grafts were present at 5 weeks in grafts removed from ACI rats and FR rats treated with Cy-A. Non-immunosuppressed FR rats rejected ACI nerves as did FR rats in whom Cy-A was stopped after 5 weeks of treatment. All surviving ACI grafts underwent Wallerian degeneration and consisted of columns of Schwann cells, which in their proximal portion were associated with regenerating host axons. The donor perineurial sheath and vasculature were also present in surviving grafts. ACI isografts only were examined 20 weeks postoperatively. All normal tissue components survived in these older grafts and contained regenerated and myelinated host axons throughout their 4 cm lengths. These results demonstrated that the cellular elements of nerve can be cryopreserved, and after transplantation, survive and function. Because nerves survived after prolonged cryopreservation, it seems feasible to establish a nerve bank from which grafts can be withdrawn to repair gaps in injured nerves. However, cryopreserved nerves used as allografts remain immunogenic and require immunosuppression for their survival. Published in 1993 by Wiley-Liss, Inc.
An effective and ultrarapid technique for kidney transplantation in the rabbit is introduced. Vas... more An effective and ultrarapid technique for kidney transplantation in the rabbit is introduced. Vascular anastomosis was completed using a novel cuff technique in which mating cuffs were used to join the delicate renal vein. The ureter was reconstructed by spatulated end-to-end anastomosis, with special attention to the rabbit's unique ureteral vascular anatomy. The total vascular anastomosis time was 3.4 ± 1.3 min, and there were no episodes of bleeding or thrombosis. The ureter complication rate was 7.3%. Kidneys transplanted after 5 h of cold storage using the new technique yielded better postoperative creatinine results than similar preserved kidneys transplanted using previously described methods. We suggest this technique for studies of long-and short-term kidney preservation and transplantation in the rabbit, as well as for veterinary transplantation in which donor kidneys must be stored for only a short time before use.
The current report compares the renal physiological impact of a standard vitrification solution, ... more The current report compares the renal physiological impact of a standard vitrification solution, VS41A, as measured by normothermic blood perfusion, to the physiological effects of VS4, a related but more dilute vitrification solution previously shown to be consistently compatible with life support function of transplanted rabbit kidneys. VS41A, which allows survival of only about half of the kidneys perfused with it, also appeared to be more damaging than VS4 based on in vitro functional indices and histology in one rabbit kidney so evaluated.
In contemplating the future of aging, it is helpful to recognize that aging postponement, arrest,... more In contemplating the future of aging, it is helpful to recognize that aging postponement, arrest, prevention, and reversal are commonplace but still under-appreciated natural and experimental phenomena. Aging is apparently completely preventable for prolonged periods in many whole animals by nutritional cues that are not confined to calorie restriction. Aging can be substantially postponed by interfering with active life-shortening processes that are triggered by sexual maturation and reproduction in most species, perhaps including humans, but, contrary to the idea that active life shortening is necessitated by a tradeoff against reproduction, aging can also be dramatically postponed by sexual maturation and reproduction (“negative reproductive costs”) in many species, including mammals. Many fundamental age-related changes are actually reversible in adults or adult systems by the application of simple physiological interventions (“segmental aging reversal”). These reversible elemental aspects of aging include, for example, reduced transcription, reduced translation, altered gene expression, reduced DNA repair, reduced mitochondrial function, reduced regenerative capacity, replicative senescence, lipofuscin accumulation, reduced immune function, thymic involution, loss of reproductive cycling in females, age-related organ atrophy, and hair graying and balding. In addition, many aging phenotypes appear to be interlinked, such that correction of one or a few central aging pathways leads to the correction of multiple downstream pathways without the need for specific intervention in any of these dependent pathways. Collectively, such observations reveal aging on multiple levels and in diverse phyla to be driven by biological mechanisms that are considerably more accessible than is classically expected. This body of observations implies that the future of human aging can be substantially different than its past and supports recent excitement over the possibilities of human intervention.
Cryopreservation of solid organs might be possible using a mixture of cell-permeable agents, cryo... more Cryopreservation of solid organs might be possible using a mixture of cell-permeable agents, cryoprotectants (CPA), which are designed to completely preclude ice crystal formation during cooling to cryogenic temperatures. The effects of a specific prototype solution (VS4) were evaluated by normothermic blood perfusion in vitro. Rabbit kidneys were divided into three groups: untreated controls (n = 7), Euro-Collins (EC)-perfused controls (n = 6) and VS4 (4970, w/v) CPA-perfused kidneys (n = 7). After a 2-h blood perfusion, five of the seven CPA-perfused kidneys developed polyuria (0.21 mlxmin-'xg-') relative to untreated controls (0.07 mlxminp'xg-') or EC-perfused kidneys (0.06 mlxmin-'xgp'), owing to the lower reabsorption of water (34.3%), Naf (34.2%) and glucose (35.6%). Furthermore, two kidneys were non-functional with virtually no urine production. Reduced tubular function was associated with reduced oxygen consumption (3.6 versus 2.3 versus 2.0 pmolexmin-'xgp' for controls, EC-and CPA-perfused kidneys, respectively) and increased weight gain (17% versus 20% versus 30%, respectively) after blood perfusion. Therefore, the current results provide insight into both the physiological effects of VS4 and the limits of reversibility of renal pathophysiological states. Our results also indicate that in vitro monitoring of oxygen consumption and weight gain of perfused organs could be used as predictors of renal function.
Although much interest has attended the cryopreservation of immature neurons for subsequent thera... more Although much interest has attended the cryopreservation of immature neurons for subsequent therapeutic intracerebral transplantation, there are no reports on the cryopreservation of organized adult cerebral tissue slices of potential interest for pharmaceutical drug development. We report here the first experiments on cryopreservation of mature rat transverse hippocampal slices. Freezing at 1.2°C/min to À20°C or below using 10 or 30% v/v glycerol or 20% v/v dimethyl sulfoxide yielded extremely poor results. Hippocampal slices were also rapidly inactivated by simple exposure to a temperature of 0°C in artificial cerebrospinal fluid (aCSF). This effect was mitigated somewhat by 0.8 mM vitamin C, the use of a more ''intracellular'' version of aCSF having reduced sodium and calcium levels and higher potassium levels, and the presence of a 25% w/v mixture of dimethyl sulfoxide, formamide, and ethylene glycol (''V EG solutes''; Cryobiology 48, pp. 2004). It was not mitigated by glycerol, aspirin, indomethacin, or mannitol addition to aCSF. When RPS-2 (Cryobiology 21, pp. [260][261][262][263][264][265][266][267][268][269][270][271][272][273] 1984) was used as a carrier solution for up to 50% w/v V EG solutes, 0°C was more protective than 10°C. Raising V EG concentration to 53% w/v allowed slice vitrification without injury from vitrification and rewarming per se, but was much more damaging than exposure to 50% w/v V EG . This problem was overcome by using the analogous 61% w/v VM3 vitrification solution (Cryobiology 48, pp. 157-178, 2004) containing polyvinylpyrrolidone and two extracellular ''ice blockers.'' With VM3, it was possible to attain a tissue K + /Na + ratio after vitrification ranging from 91 to 108% of that obtained with untreated control slices. Microscopic examination showed severe damage in frozen-thawed slices, but generally good to excellent ultrastructural and histological preservation after vitrification. Our results provide the first demonstration that both the viability and the structure of mature organized, complex neural networks can be well preserved by vitrification. These results may assist neuropsychiatric drug evaluation and development and the transplantation of integrated brain regions to correct brain disease or injury.
The simple linear polymer polyglycerol (PGL) was found to apparently bind and inhibit the ice nuc... more The simple linear polymer polyglycerol (PGL) was found to apparently bind and inhibit the ice nucleating activity of proteins from the ice nucleating bacterium Pseudomonas syringae. PGL of molecular mass 750 Da was added to a solution consisting of 1 ppm freeze-dried P. syringae 31A in water. Differential ice nucleator spectra were determined by measuring the distribution of freezing temperatures in a population of 98 drops of 1 lL volume. The mean freezing temperature was lowered from )6.8°C (control) to À8:0; À9:4; À12:5, and )13.4°C for 0.001, 0.01, 0.1, and 1% w/w PGL concentrations, respectively (SE < 0.2°C). PGL was found to be an ineffective inhibitor of seven defined organic ice nucleating agents, whereas the general ice nucleation inhibitor polyvinyl alcohol (PVA) was found to be effective against five of the seven. The activity of PGL therefore seems to be specific against bacterial ice nucleating protein. PGL alone was an ineffective inhibitor of ice nucleation in small volumes of environmental or laboratory water samples, suggesting that the numerical majority of ice nucleating contaminants in nature may be of nonbacterial origin. However, PGL was more effective than PVA at suppressing initial ice nucleation events in large volumes, suggesting a ubiquitous sparse background of bacterial ice nucleating proteins with high nucleation efficiency. The combination of PGL and PVA was particularly effective for reducing ice formation in solutions used for cryopreservation by vitrification. Ó
Cryopreservation of solid organs might be possible using a mixture of cell-permeable agents, cryo... more Cryopreservation of solid organs might be possible using a mixture of cell-permeable agents, cryoprotectants (CPA), which are designed to completely preclude ice crystal formation during cooling to cryogenic temperatures. The effects of a specific prototype solution (VS4) were evaluated by normothermic blood perfusion in vitro. Rabbit kidneys were divided into three groups: untreated controls (n = 7), Euro-Collins (EC)-perfused controls (n = 6) and VS4 (4970, w/v) CPA-perfused kidneys (n = 7). After a 2-h blood perfusion, five of the seven CPA-perfused kidneys developed polyuria (0.21 mlxmin-'xg-') relative to untreated controls (0.07 mlxminp'xg-') or EC-perfused kidneys (0.06 mlxmin-'xgp'), owing to the lower reabsorption of water (34.3%), Naf (34.2%) and glucose (35.6%). Furthermore, two kidneys were non-functional with virtually no urine production. Reduced tubular function was associated with reduced oxygen consumption (3.6 versus 2.3 versus 2.0 pmolexmin-'xgp' for controls, EC-and CPA-perfused kidneys, respectively) and increased weight gain (17% versus 20% versus 30%, respectively) after blood perfusion. Therefore, the current results provide insight into both the physiological effects of VS4 and the limits of reversibility of renal pathophysiological states. Our results also indicate that in vitro monitoring of oxygen consumption and weight gain of perfused organs could be used as predictors of renal function.
The simple linear polymer polyglycerol (PGL) was found to apparently bind and inhibit the ice nuc... more The simple linear polymer polyglycerol (PGL) was found to apparently bind and inhibit the ice nucleating activity of proteins from the ice nucleating bacterium Pseudomonas syringae. PGL of molecular mass 750 Da was added to a solution consisting of 1 ppm freeze-dried P. syringae 31A in water. Differential ice nucleator spectra were determined by measuring the distribution of freezing temperatures in a population of 98 drops of 1 lL volume. The mean freezing temperature was lowered from )6.8°C (control) to À8:0; À9:4; À12:5, and )13.4°C for 0.001, 0.01, 0.1, and 1% w/w PGL concentrations, respectively (SE < 0.2°C). PGL was found to be an ineffective inhibitor of seven defined organic ice nucleating agents, whereas the general ice nucleation inhibitor polyvinyl alcohol (PVA) was found to be effective against five of the seven. The activity of PGL therefore seems to be specific against bacterial ice nucleating protein. PGL alone was an ineffective inhibitor of ice nucleation in small volumes of environmental or laboratory water samples, suggesting that the numerical majority of ice nucleating contaminants in nature may be of nonbacterial origin. However, PGL was more effective than PVA at suppressing initial ice nucleation events in large volumes, suggesting a ubiquitous sparse background of bacterial ice nucleating proteins with high nucleation efficiency. The combination of PGL and PVA was particularly effective for reducing ice formation in solutions used for cryopreservation by vitrification. Ó
Wire-length resonant coils, which are based on equating a wavelength of RF with the wire length u... more Wire-length resonant coils, which are based on equating a wavelength of RF with the wire length used in winding a coil, were previously developed for use in hyperthermia treatment for cancer therapy. Design modifications, coupled with a series of experiments, established their potential use for rapidly and uniformly rewarming canines following hypothermic cardiac surgery. The authors detail a new design which incorporates a single-end coaxial input and RF shielding, allowing it to be used outside of a screen room environment. Data from early experiments to investigate this system's usefulness in rewarming cryogenically preserved donor organs are presented and discussed.>
To facilitate the development of assays for the discovery of pharmaceuticals capable of mimicking... more To facilitate the development of assays for the discovery of pharmaceuticals capable of mimicking the effects of caloric restriction on life-and healthspan (CR mimetics), we evaluated the effectiveness of glucoregulatory and putative cancer chemopreventatives in reproducing the hepatic gene-expression profile produced by long-term CR (LTCR) using Affymetrix microarrays. We have shown that CR initiated late in life begins to extend lifespan, reduce cancer as a cause of death, and reproduce ~three quarters of the genomic effects of LTCR in 8 weeks (CR8). Eight weeks of metformin treatment was superior to CR8 at reproducing LTCRlike gene expression changes, maintaining a superior number of such changes over a broad range of statistical stringencies, and producing more gene ontology terms overlapping those produced by LTCR. Consistent with these results, metformin has been shown to reduce cancer incidence in mice and humans. Phenformin, a chemical cousin of metformin, extends lifespan and reduces tumor incidence in mice. Taken together, these results indicate that gene-expression biomarkers can be used to identify promising candidate CR mimetics. of the glucoregulatory compounds metformin (MET), glipizide (GLIP), GLIP plus MET (GM), and rosiglitazone (ROS) for their ability to reproduce the effects of LTCR on hepatic gene PG-00069-2005.R1
Magnetic resonance imaging was used to detect signs of regeneration of the thymus after approxima... more Magnetic resonance imaging was used to detect signs of regeneration of the thymus after approximately one month of human growth hormone administration. A 46-year-old human volunteer was placed on a regimen of recombinant human growth hormone and pharmaceutical grade dehydroepiandrosterone for one month. Mediastinal magnetic resonance images were collected at baseline and after the study period. Thymic cross sections were analyzed for total area and for the total gray area, which was taken to represent functional mass. Baseline and post-treatment blood samples were taken to follow changes in IGF-1 levels and related metabolites. The setting was an informal, non-institutional trial supervised by a physician will full informed consent of the volunteer. Visual inspection and image analysis demonstrated limited but distinct enlargement of the thymus after treatment, and an increase in the percent of thymic cross section represented by gray-appearing (functional) mass. Estimated total thymic functional volume was within the normal range at baseline, but after treatment was more than three standard deviations above the expected mean for a subject of this age, thus meeting a proposed definition of thymic hyperplasia for individuals. IGF-1 levels were confined to the upper range of normal for young adults. The present observations apparently provide the first demonstration of growth hormone induced partial reversal of established thymic involution in a normal human subject, and are consistent with previous measurements of restored immune function after the administration of human growth hormone to elderly individuals.
The objective of the present study was to determine whether rabbit kidneys could be perfused with... more The objective of the present study was to determine whether rabbit kidneys could be perfused with a 7.5 M vitrification solution (VS4, which vitrifies under applied pressure) without loss of function. To answer this question, kidneys were perfused with VS4 using a computer-based machine to gradually raise and lower concentration and then attached to the aorta and vena cava of a perfusor rabbit using an apparatus that permitted renal blood flow and renal function to be measured. About half (6/13) of the kidneys so evaluated resumed substantial immediate function after a transient period of severely reduced blood flow. Loss of function did not occur if cryoprotectant concentration was limited to 3.8 M. The loss of function produced by VS4 could be partially reproduced by artificially limiting blood reflow in control kidneys to simulate the transiently low flows caused by VS4 exposure. These results provide the first evidence that both the parenchyma and the vascular system of a sensitive mammalian organ can survive exposure to a vitrifiable concentration of cryoprotectant. ᭧ 1997 Academic Press
Cryoprotectant toxicity is a fundamental limiting factor for the successful cryopreservation of l... more Cryoprotectant toxicity is a fundamental limiting factor for the successful cryopreservation of living systems by both freezing and vitrification, and the ability to negate it would be attractive. Past attempts to demonstrate ''cryoprotectant toxicity neutralization" (CTN) have had many ups and downs. First convincingly introduced by Baxter and Lathe in 1971, the concept that certain amides can block toxic effects of dimethyl sulfoxide (Me 2 SO) was contradicted by direct experiments in 1990. But in 1995, the opposite mode of CTN, in which Me 2 SO blocked the damaging effects of formamide, was robustly demonstrated. Recent experiments have verified the original 1995 results and extended them to urea and acetamide, but no CTN was detected for N-methylamides (N-methylformamide, N,N-dimethylformamide, and N-methylacetamide). On the theory that the latter amides and acetamide might serve as low-toxicity structural analogs of formamide, urea, or Me 2 SO, competition experiments were carried out between them and formamide or urea, but CTN was not observed for these amide-amide systems. The idea that the N-methylamides might have non-specific rather than specific toxicity was supported by the fact that the concentrations of these amides that cause toxicity are similar to the concentrations that denature model proteins. Clear examples of neutralization of the toxicity of glycerol, propylene glycol, ethylene glycol, or Me 2 SO are presently lacking, but effects of the latter that depend on sulfhydryl oxidation have been reversed with reducing agents. In summary, CTN is a useful phenomenon with significant theoretical and practical implications.
24. Investigating hydroxyl radicals (OH) and 8-hydroxy-2 0 -deoxyguanosine as generic biomarkers ... more 24. Investigating hydroxyl radicals (OH) and 8-hydroxy-2 0 -deoxyguanosine as generic biomarkers of cryoinjury and cold storage stability.
Cryopreservation of solid organs might be possible using a mixture of cell-permeable agents, cryo... more Cryopreservation of solid organs might be possible using a mixture of cell-permeable agents, cryoprotectants (CPA), which are designed to completely preclude ice crystal formation during cooling to cryogenic temperatures. The effects of a specific prototype solution (VS4) were evaluated by normothermic blood perfusion in vitro. Rabbit kidneys were divided into three groups: untreated controls (n=7), Euro-Collins (EC)-perfused controls (n=6) and VS4 (49%, w/v) CPA-perfused kidneys (n=7). After a 2-h blood perfusion, five of the seven CPA-perfused kidneys developed polyuria (0.21 ml×min–1×g–1) relative to untreated controls (0.07 ml×min–1×g–1) or EC-perfused kidneys (0.06 ml×min–1×g–1), owing to the lower reabsorption of water (34.3%), Na+ (34.2%) and glucose (35.6%). Furthermore, two kidneys were non-functional with virtually no urine production. Reduced tubular function was associated with reduced oxygen consumption (3.6 versus 2.3 versus 2.0 μmole×min–1×g–1 for controls, EC- and CPA-perfused kidneys, respectively) and increased weight gain (17% versus 20% versus 30%, respectively) after blood perfusion. Therefore, the current results provide insight into both the physiological effects of VS4 and the limits of reversibility of renal pathophysiological states. Our results also indicate that in vitro monitoring of oxygen consumption and weight gain of perfused organs could be used as predictors of renal function.
Donor Schwann cells, perineurial cells, and vasculature are known to survive in grafts of periphe... more Donor Schwann cells, perineurial cells, and vasculature are known to survive in grafts of peripheral nerve. In the present study, we attempted to cryopreserve nerve to determine whether these cellular components of nerve would survive after transplantation and support host axonal regeneration through the graft. Four-centimeter lengths of peroneal nerves were removed from inbred adult American Cancer Institute (ACI) rats and placed into vials that contained a cryoprotective mixture of dimethyl sulfoxide and formamide (DF) at room temperature. Each vial with nerves in DF was cooled at a rate of 1–1.5°C/minute down to –40°C at which point the vials were plunged into liquid nitrogen at –196°C. After 5 weeks of storage, the nerves were thawed and DF removed. Some of the cryopreserved-thawed ACI nerves were transplanted as isografts into the legs of ACI rats. Other ACI nerves were used as allografts and inserted into immunologically normal Fischer (FR) rats that were untreated or were immunosuppressed with the drug Cyclosporin A (Cy-A). At surgery, only one end of the nerve graft was joined to the cut proximal end of the peroneal nerve of the host. The cellular elements of ACI grafts were present at 5 weeks in grafts removed from ACI rats and FR rats treated with Cy-A. Non-immunosuppressed FR rats rejected ACI nerves as did FR rats in whom Cy-A was stopped after 5 weeks of treatment. All surviving ACI grafts underwent Wallerian degeneration and consisted of columns of Schwann cells, which in their proximal portion were associated with regenerating host axons. The donor perineurial sheath and vasculature were also present in surviving grafts. ACI isografts only were examined 20 weeks postoperatively. All normal tissue components survived in these older grafts and contained regenerated and myelinated host axons throughout their 4 cm lengths. These results demonstrated that the cellular elements of nerve can be cryopreserved, and after transplantation, survive and function. Because nerves survived after prolonged cryopreservation, it seems feasible to establish a nerve bank from which grafts can be withdrawn to repair gaps in injured nerves. However, cryopreserved nerves used as allografts remain immunogenic and require immunosuppression for their survival. Published in 1993 by Wiley-Liss, Inc.
An effective and ultrarapid technique for kidney transplantation in the rabbit is introduced. Vas... more An effective and ultrarapid technique for kidney transplantation in the rabbit is introduced. Vascular anastomosis was completed using a novel cuff technique in which mating cuffs were used to join the delicate renal vein. The ureter was reconstructed by spatulated end-to-end anastomosis, with special attention to the rabbit's unique ureteral vascular anatomy. The total vascular anastomosis time was 3.4 ± 1.3 min, and there were no episodes of bleeding or thrombosis. The ureter complication rate was 7.3%. Kidneys transplanted after 5 h of cold storage using the new technique yielded better postoperative creatinine results than similar preserved kidneys transplanted using previously described methods. We suggest this technique for studies of long-and short-term kidney preservation and transplantation in the rabbit, as well as for veterinary transplantation in which donor kidneys must be stored for only a short time before use.
The current report compares the renal physiological impact of a standard vitrification solution, ... more The current report compares the renal physiological impact of a standard vitrification solution, VS41A, as measured by normothermic blood perfusion, to the physiological effects of VS4, a related but more dilute vitrification solution previously shown to be consistently compatible with life support function of transplanted rabbit kidneys. VS41A, which allows survival of only about half of the kidneys perfused with it, also appeared to be more damaging than VS4 based on in vitro functional indices and histology in one rabbit kidney so evaluated.
In contemplating the future of aging, it is helpful to recognize that aging postponement, arrest,... more In contemplating the future of aging, it is helpful to recognize that aging postponement, arrest, prevention, and reversal are commonplace but still under-appreciated natural and experimental phenomena. Aging is apparently completely preventable for prolonged periods in many whole animals by nutritional cues that are not confined to calorie restriction. Aging can be substantially postponed by interfering with active life-shortening processes that are triggered by sexual maturation and reproduction in most species, perhaps including humans, but, contrary to the idea that active life shortening is necessitated by a tradeoff against reproduction, aging can also be dramatically postponed by sexual maturation and reproduction (“negative reproductive costs”) in many species, including mammals. Many fundamental age-related changes are actually reversible in adults or adult systems by the application of simple physiological interventions (“segmental aging reversal”). These reversible elemental aspects of aging include, for example, reduced transcription, reduced translation, altered gene expression, reduced DNA repair, reduced mitochondrial function, reduced regenerative capacity, replicative senescence, lipofuscin accumulation, reduced immune function, thymic involution, loss of reproductive cycling in females, age-related organ atrophy, and hair graying and balding. In addition, many aging phenotypes appear to be interlinked, such that correction of one or a few central aging pathways leads to the correction of multiple downstream pathways without the need for specific intervention in any of these dependent pathways. Collectively, such observations reveal aging on multiple levels and in diverse phyla to be driven by biological mechanisms that are considerably more accessible than is classically expected. This body of observations implies that the future of human aging can be substantially different than its past and supports recent excitement over the possibilities of human intervention.
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Papers by Gregory Fahy