Biomedical subjects

Gregory M Fahy

Publications and source records attributed to Gregory M Fahy.

11 recordsLinked to original sources

PubMed2006

Cryopreservation of rat precision-cut liver and kidney slices by rapid freezing and vitrification.

Precision-cut tissue slices of both hepatic and extra-hepatic origin are extensively used as an in vitro model to predict in vivo drug metabolism and toxicity. Cryopreservation would greatly facilitate their use. In the present study, we aimed to improve (1) rapid freezing and warming (200 degrees C/min) using 18% Me(2)SO as cryoprotectant and (2) vitrification with high molarity mixtures of cryoprotectants, VM3 and VS4, as methods to cryopreserve precision-cut rat liver and kidney slices. Viability after cryopreservation and subsequent 3-4h of incubation at 37 degrees C was determined by measuring ATP content and by microscopical evaluation of histological integrity. Confirming earlier studies, viability of rat liver slices was maintained at high levels by rapid freezing and thawing with 18% Me(2)SO. However, vitrification of liver slices with VS4 resulted in cryopreservation damage despite the fact that cryoprotectant toxicity was low, no ice was formed during cooling and devitrification was prevented. Viability of liver slices was not improved by using VM3 for vitrification. Kidney slices were found not to survive cryopreservation by rapid freezing. In contrast, viability of renal medullary slices was almost completely maintained after vitrification with VS4, however vitrification of renal cortex slices with VS4 was not successful, partly due to cryoprotectant toxicity. Both kidney cortex and medullary slices were vitrified successfully with VM3 (maintaining viability at 50-80% of fresh slice levels), using an optimised pre-incubation protocol and cooling and warming rates that prevented both visible ice-formation and cracking of the formed glass. In conclusion, vitrification is a promising approach to cryopreserve precision-cut (kidney) slices.

Adenosine Triphosphate↗

PubMed2006

Cryopreservation of rat hippocampal slices by vitrification.

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 degrees C/min to -20 degrees 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 degree 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. 22-35, 2004). It was not mitigated by glycerol, aspirin, indomethacin, or mannitol addition to aCSF. When RPS-2 (Cryobiology 21, pp. 260-273, 1984) was used as a carrier solution for up to 50% w/v V(EG) solutes, 0 degree C was more protective than 10 degrees 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.

Animals↗

PubMed2006

Cryopreservation of complex systems: the missing link in the regenerative medicine supply chain.

Transplantation can be regarded as one form of "antiaging medicine" that is widely accepted as being effective in extending human life. The current number of organ transplants in the United States is on the order of 20,000 per year, but the need may be closer to 900,000 per year. Cadaveric and living-related donor sources are unlikely to be able to provide all of the transplants required, but the gap between supply and demand can be eliminated in principle by the field of regenerative medicine, including the present field of tissue engineering through which cell, tissue, and even organ replacements are being created in the laboratory. If so, it could allow over 30% of all deaths in the United States to be substantially postponed, raising the probability of living to the age of 80 by a factor of two and the odds of living to 90 by more than a factor of 10. This promise, however, depends on the ability to physically distribute the products of regenerative medicine to patients in need and to produce these products in a way that allows for adequate inventory control and quality assurance. For this purpose, the ability to cryogenically preserve (cryopreserve) cells, tissues, and even whole laboratory-produced organs may be indispensable. Until recently, the cryopreservation of organs has seemed a remote prospect to most observers, but developments over the past few years are rapidly changing the scientific basis for preserving even the most difficult and delicate organs for unlimited periods of time. Animal intestines and ovaries have been frozen, thawed, and shown to function after transplantation, but the preservation of vital organs will most likely require vitrification. With vitrification, all ice formation is prevented and the organ is preserved in the glassy state below the glass transition temperature (T(G)). Vitrification has been successful for many tissues such as veins, arteries, cartilage, and heart valves, and success has even been claimed for whole ovaries. For vital organs, a significant recent milestone for vitrification has been the ability to routinely recover rabbit kidneys after cooling to a mean intrarenal temperature of about -45 degrees C, as verified by life support function after transplantation. This temperature is not low enough for long-term banking, but research continues on preservation below -45 degrees C, and some encouraging preliminary evidence has been obtained indicating that kidneys can support life after vitrification. Full development of tissue engineering and organ generation from stem cells, when combined with the ability to bank these laboratory-produced products, in theory could dramatically increase median life expectancy even in the absence of any improvements in mitigating aging processes on a fundamental level.

Animals↗

PubMed2005

Identification of potential caloric restriction mimetics by microarray profiling.

To facilitate the development of assays for the discovery of pharmaceuticals capable of mimicking the effects of caloric restriction (CR) 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 approximately three-quarters of the genomic effects of LTCR in 8 wk (CR8). Eight weeks of metformin treatment was superior to CR8 at reproducing LTCR-like 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.

Animals↗

PubMed2004

Improved vitrification solutions based on the predictability of vitrification solution toxicity.

Long-term preservation of complex engineered tissues and organs at cryogenic temperatures in the absence of ice has been prevented to date by the difficulty of discovering combinations of cryoprotectants that are both sufficiently non-toxic and sufficiently stable to allow viability to be maintained and ice formation to be avoided during slow cooling to the glass transition temperature and subsequent slow rewarming. A new theory of the origin of non-specific cryoprotectant toxicity was shown to account, in a rabbit renal cortical slice model, for the toxicities of 20 vitrification solutions and to permit the design of new solutions that are dramatically less toxic than previously known solutions for diverse biological systems. Unfertilized mouse ova vitrified with one of the new solutions were successfully fertilized and regained 80% of the absolute control (untreated) rate of development to blastocysts, whereas ova vitrified in VSDP, the best previous solution, developed to blastocysts at a rate only 30% of that of controls. Whole rabbit kidneys perfused at -3 degrees C with another new solution at a concentration of cryoprotectant (8.4M) that was previously 100% lethal at this temperature exhibited no damage after transplantation and immediate contralateral nephrectomy. It appears that cryoprotectant solutions that are composed to be at the minimum concentrations needed for vitrification at moderate cooling rates are toxic in direct proportion to the average strength of water hydrogen bonding by the polar groups on the permeating cryoprotectants in the solution. Vitrification solutions that are based on minimal perturbation of intracellular water appear to be superior and provide new hope that the successful vitrification of natural organs as well as tissue engineered or clonally produced organ and tissue replacements can be achieved.

Animals↗

PubMed2004

Cryopreservation of organs by vitrification: perspectives and recent advances.

The cryopreservation of organs became an active area of research in the 1950s as a result of the rediscovery of the cryoprotective properties of glycerol by Polge, Smith, and Parkes in 1949. Over the ensuing four decades of research in this area, the advantages of vitrification, or ice-free cryopreservation, have become apparent. To date, experimental attempts to apply vitrification methods to vascularized whole organs have been confined almost entirely to the rabbit kidney. Using techniques available as of 1997, it was possible to vitrify blood vessels and smaller systems with reasonable success, but not whole organs. Beginning in 1998, a series of novel advances involving the control of cryoprotectant toxicity, nucleation, crystal growth, and chilling injury began to provide the tools needed to achieve success. Based on these new findings, we were first able to show that an 8.4M solution (VMP) designed to prevent chilling injury at -22 degrees C was entirely non-toxic to rabbit kidneys when perfused at -3 degrees C and permitted perfusion-cooling to -22 degrees C with only mild additional damage. We next investigated the ability of the kidney to tolerate a 9.3M solution known as M22, which does not devitrify when warmed from below -150 degrees C at 1 degrees C/min. When M22 was added and removed at -22 degrees C, it was sometimes [corrected] fatal, but when it was perfused for 25min at -22 degrees C and washed out simultaneously with warming, postoperative renal function recovered fully. When kidneys loaded with M22 at -22 degrees C were further cooled to an average intrarenal temperature of about -45 degrees C (about halfway through the putative temperature zone of increasing vulnerability to chilling injury), all kidneys supported life after transplantation and returned creatinine values to baseline, though after a higher transient creatinine peak. However, medullary, papillary, and pelvic biopsies taken from kidneys perfused with M22 for 25min at -22 degrees C were found to devitrify when vitrified and rewarmed at 20 degrees C/min in a differential scanning calorimeter. It remains to be determined whether this devitrification is seriously damaging and whether it can be suppressed by improving cryoprotectant distribution to more weakly perfused regions of the kidney or by rewarming at higher rates. In conclusion, although the goal of organ vitrification remains elusive, the prospects for success have never been more promising.

Animals↗

PubMed2003

Ultrarapid nonsuture mated cuff technique for renal transplantation in rabbits.

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.

Anastomosis, Surgical↗

PubMed2003

Physiological evaluation of a rabbit kidney perfused with VS41A.

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.

Absorption↗

PubMed2003

Apparent induction of partial thymic regeneration in a normal human subject: a case report.

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.

Dehydroepiandrosterone↗

PubMed2002

Normothermic blood perfusion of isolated rabbit kidneys. III. In vitro physiology of kidneys after perfusion with Euro-Collins solution or 7.5 M cryoprotectant (VS4).

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 mlxmin(-1)xg(-1)) relative to untreated controls (0.07 mlxmin(-1)xg(-1)) or EC-perfused kidneys (0.06 mlxmin(-1)xg(-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 micromolexmin(-1)xg(-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.

Animals↗

PubMed2002

Inhibition of bacterial ice nucleation by polyglycerol polymers.

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 microL volume. The mean freezing temperature was lowered from -6.8 degrees C (control) to -8.0,-9.4,-12.5, and -13.4 degrees C for 0.001, 0.01, 0.1, and 1% w/w PGL concentrations, respectively (SE < 0.2 degrees 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.

Bacterial Outer Membrane Proteins↗