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Biomedical subjects

C J Hunt

Publications and source records attributed to C J Hunt.

36 records · Page 2Linked to original sources
PubMed1989

Hypothermic preservation of corneas in a hyperkalaemic solution (CPTES): I. Short-term storage in the absence of colloid osmotic agents.

Preservation solutions for short-term storage of isolated donor corneas for use in penetrating keratoplasty have all been based on tissue culture medium, on the assumption that media designed to maintain the viability of cells at physiological temperatures will also provide suitable conditions for preservation at reduced temperatures. But for hypothermic preservation of some other tissues and organs, when ionic pumps are inhibited, it is unnecessary to support metabolism, and beneficial control of ion and water distribution between intra- and extracellular compartments is achieved by storage in appropriately formulated 'intracellular-type' solutions. We have therefore designed a solution that will restrict ionic imbalances and minimise endothelial cell swelling in corneas during exposure at reduced temperatures. This potassium-rich solution contains the biological pH buffer TES as an impermeant anion and is designated CPTES (corneal-potassium-TES). The structural and functional integrity of rabbit corneas stored at 0 degrees C in CPTES, without the addition of colloid osmotic agents, is compared with that of corneas stored in glutathione bicarbonate Ringers' solution (GBR), an 'extracellular-type' medium formulated for the maintenance of endothelial integrity during in-vitro perfusion at 34 degrees C. Corneas swelled significantly less during storage in CPTES than in GBR and could be stored for five days before reaching the same degree of hydration as corneas stored for only three days in GBR. Gross structural integrity and endothelial ultrastructure were maintained during storage for three and five days in CPTES. The rate of thinning of corneas stored in CPTES was significantly greater than in comparable groups of corneas stored in GBR. However, the efficient dehydration of corneas stored in CPTES was always preceded during perfusion by a brief period of additional swelling which was shown to be an osmotic response during the elution of the buffer compound TES that had permeated the stroma during storage. The omission of calcium or the addition of adenosine and glutathione to the CPTES preservation medium had no detectable effect on the integrity of the endothelium, but the omission of bicarbonate was beneficial, producing significantly higher rates of stromal thinning during normothermic perfusion. Additional benefits for extending storage by including colloid osmotic agents are described in a companion paper.

Animals↗
PubMed1989

Hypothermic preservation of corneas in a hyperkalaemic solution (CPTES): II. Extended storage in the presence of chondroitin sulphate.

Periods of preservation for donor corneas, even for short times, are necessary to facilitate optimum conditions in penetrating keratoplasty. However, current techniques for corneal storage at low temperatures may not provide optimal conditions for maintaining tissue integrity. In particular, the ionic composition of the storage medium has received little attention since it has been assumed throughout that the normal complement of ions in tissue culture media will also be suitable for preservation at reduced temperatures. This study extends our previous investigations on the merits of using CPTES (corneal-potassium-TES), a potassium-rich balanced salt solution containing an impermeant anionic pH buffer (TES), as a storage solution specifically designed to prevent the loss of intracellular potassium and minimise endothelial cell swelling during the time that the normal regulatory processes are switched off. The effect of adding the natural polymer chondroitin sulphate (CS) as a colloid osmotic agent to the hyperkalaemic storage medium is now examined. Corneas stored in CPTES containing 2.5% chondroitin sulphate retained a very high level of structural and functional integrity after three, five, and seven days storage at 0 degrees C; furthermore, stromal swelling was restricted to only 21%. All corneas stored in CPTES + 2.5% CS showed active endothelial function by thinning efficiently at rates that were greater than those previously reported for rabbit corneas stored for similar lengths of time in either M-K medium or K-sol. The zwitterionic buffers TES and HEPES were interchangeable in the hyperkalaemic solution and were non-toxic to corneal endothelium at a concentration of 100 mM. These compounds offer excellent pH buffering in bicarbonate-free medium.

Animals↗
PubMed1989

Tolerance of corneas to multimolar dimethyl sulfoxide at 0 degrees C. Implications for cryopreservation.

Attempts to improve current methods of cryopreservation of corneas, whether by conventional freezing and thawing or by vitrification in the absence of ice, will require the use of high concentrations of cryoprotectants. In this study we extend our previous investigation of the tolerance of rabbit corneas to multimolar concentrations of the cryoprotectant dimethyl sulfoxide (Me2SO) added and removed at 0 degrees C in CPTES, a hyperkalaemic preservation solution containing the impermeant anionic buffer N-Tris(hydroxymethyl)methyl-2-aminoethane sulphonate (TES). Isolated corneas were exposed to 1, 2 or 3 mol/l Me2SO at 0 degrees C to minimize any effect due to temperature-dependent chemical toxicity and attention was given to the procedure for diluting Me2SO from the tissue in order to minimize osmotic stress to the endothelium. Endothelial integrity following these procedures was assessed both by the ability to control stromal hydration during perfusion on the specular microscope and by the structural integrity when examined by light and electron microscopy. The presence of an active endothelial pump and good morphology were demonstrated in corneas exposed to 1 and 2 mol/l Me2SO; serial dilution of the cryoprotectant was more beneficial than a single-step direct dilution. Corneas immersed directly into 3 mol/l Me2SO were irreparably damaged irrespective of the method of dilution. Sequential addition of 1 M, then 2 M and finally 3 M cryoprotectant followed by serial dilution was, however, tolerated by the endothelium and minor alterations to the structural integrity of the endothelial layer were rapidly repaired. The osmotic nature of these observations are analyzed and discussed.

Animals↗
PubMed1988

Introduction and removal of cryoprotective agents with rabbit kidneys: assessment by transplantation.

Rabbit kidneys were perfused with up to 4 M glycerol or propane-1,2-diol (propylene glycol, PG) in three vehicle solutions: one normokalemic and made hypertonic with mannitol (HP5), one hyperkalemic but without mannitol (HP6), and one hyperkalemic and with mannitol (HP7). Subsequent function was assessed by autotransplantation. Up to 3 M glycerol in HP5 was well tolerated but not in HP6 or HP7. Conversely, up to 3 M PG in HP7 was compatible with excellent post-transplant function, but the same concentration in HP5 was severely damaging. PG (4 M) in either solution was severely injurious and no kidneys survived perfusion with this concentration. Vascular resistance was well controlled by the vehicle solutions with mannitol, but it was generally higher during perfusion with the hyperkalemic HP7 compared with the normokalemic HP5. No kidneys perfused with 3 M solutions of either of the cryoprotective agents and cooled briefly to -6 degrees C without freezing had any post-transplant function, and neither did kidneys perfused with 3 M PG or 4 M glycerol tolerate slow cooling to -80 degrees C and warming. The need to optimize perfusate composition for the CPA being used is clear, and the dramatic increase in toxicity of PG when the concentration exceeds 3 M supports the suggestion that mixtures of PG and glycerol should be considered. The observation of damage at high subzero temperatures, before freezing has occurred, requires further detailed study.

Animals↗
PubMed1987

Osmotic properties of the rabbit corneal endothelium and their relevance to cryopreservation.

The process of cryopreservation subjects cells to gross changes in the composition of the solution that surrounds them, changes that cause the cells first to shrink and then to swell by an osmotic mechanism. Empirical methods have been developed that permit many cells to survive freezing and thawing, but the cornea, which is crucially dependent upon the function of its endothelial monolayer, has proved quite refractory. In this paper we explore the osmotic response of the corneal endothelium of the rabbit to solutions ranging in osmolality from 0.25 to 8.6 X isotonic. Boyle van't Hoff behavior was observed between 0.43 and 8.6 X isotonic, and there was an apparent nonosmotic volume of 33.6%. However, ultrastructural damage was observed at the limits of this range, and it appeared that the tolerated range was 0.64-4.4 X isotonic. We show the extent to which dimethyl sulfoxide (Me2SO) would be expected to moderate changes in volume during freezing and suggest that its initial concentration should be at least 2M to prevent excessive shrinkage. We also show that cell swelling during removal of Me2SO is especially likely to be hazardous.

Animals↗
PubMed1987

Cryopreservation of the rabbit cornea: freezing with dimethyl sulphoxide in air or in medium.

There have been considerable difficulties in developing a satisfactory method for the cryopreservation of corneas. In this paper we describe the effect of two variables that appear to influence the effectiveness of preservation, the concentration of cryoprotectant and the medium that surrounds the cornea during freezing. Rabbit corneas were exposed to the cryoprotectant dimethyl sulphoxide (Me2SO) in concentrations of 1 or 2 mol/l in a high-potassium well-buffered solution, and then cooled to -196 degrees C either in air or surrounded by 5 ml of the Me2SO solution. After storage at -196 degrees C, thawing and removal of the Me2SO, survival was assessed by electron microscopy and measurement of stromal thickness during perfusion on the specular microscope. The least degree of damage was observed when corneas were equilibrated with 1M Me2SO and frozen in air. The evidence suggests that 2M may be an excessive concentration of Me2SO in this system and that damage to the stroma may be reduced by freezing the cornea in air rather than surrounded by the Me2SO solution.

Air↗
PubMed1986

Cryopreservation of rabbit corneas: assessment by microscopy and transplantation.

Rabbit corneas were frozen and thawed by three methods and compared by full thickness transplantation as well as specular microscopy, histology, and transmission electron microscopy. Two of the methods used a recently described technique, in which the excised cornea was immersed in a potassium-rich buffered solution containing the cryoprotectant dimethyl sulphoxide (Me2SO, 2 mol/l). This solution was designed to restrict the loss of intracellular potassium and to prevent cell swelling at low temperatures. In one group the corneas were frozen and thawed surrounded by 5 ml of medium, while in the second group corneas were drained of excess fluid and frozen in air. The third group consisted of corneas cryopreserved by Capella and colleagues' method. All the cryopreserved corneas were damaged, but those that had been frozen in air after exposure to the new medium showed better structure and function than corneas frozen by either of the other two techniques.

Animals↗
PubMed1985

The effects of osmotic stress on human platelets.

The effect of osmotic stress on human platelets was investigated at 0, 25, and 37 degrees C. The osmolality of the suspending plasma was decreased by adding water or increased by adding sodium chloride or sucrose. After 5 min, isotonicity was restored by dilution with an excess of isotonic phosphate-buffered saline. After centrifugation, the platelets were resuspended in autologous plasma and then incubated for 1 hr at 37 degrees C before assaying the active transport of 5-hydroxytryptamine (5-HT) and the hypotonic stress response. Anisosmotic conditions had a greater effect on the extent of volume reversal in the hypotonic stress test than on 5-HT uptake. At 25 degrees C, only moderate degrees of hypotonicity (0.25 osmol/kg) or hypertonicity (0.59 osmol/kg) were sufficient to depress the hypotonic stress response. In general, platelets tolerated departures from isotonic conditions better at 0 degree C than at the higher temperatures. Furthermore, at 0 and 25 degrees C approximately equiosmolal concentrations of sucrose and sodium chloride depressed the hypotonic stress response to similar extents, but at 37 degrees C high osmolalities (greater than 2 osmol/kg) were tolerated better when the additive was sucrose than when it was sodium chloride. Platelets shrank when subjected to hyperosmotic conditions, but their discoid shape and the peripheral band of microtubules were maintained.

Biological Transport, Active↗
PubMed1985

A new preservation solution for storage of corneas at low temperatures.

Cryopreservation methods are only rarely used today for storing donor material for use in penetrating keratoplasty. There remains, however, a continuing need for a reliable technique for the long-term preservation of corneas. As a first step in developing an improved method of corneal cryopreservation we have studied the composition of the preservation medium. On the basis of experiments with other tissues a new solution (designated CPTES) has been formulated, containing concentrations of potassium, sodium and chloride similar to normal intracellular levels, in order to restrict the ionic imbalances that occur when temperature is reduced and ionic pumps switched off. An impermeant anion was included to reduce cellular swelling during low temperature storage. Integrity of the corneal endothelium was demonstrated following exposure to CPTES medium at 0 degree C: control of corneal hydration and the presence of an active bicarbonate - dependent pump were demonstrated by specular microscopy; normal ultrastructure of the endothelial layer was revealed by electron microscopy. The incorporation and removal of 1M dimethyl sulphoxide (Me2SO) is tolerated in corneas bathed in CPTES medium at 0 degree C, again with retention of endothelial structural and functional integrity. CPTES is proposed as a medium which will help to control osmotic and ionic disturbances during experimental procedures designed to improve methods of corneal preservation both above and below 0 degree C.

Animals↗
PubMed1984

The effect of cooling rate and warming rate on the packing effect in human erythrocytes frozen and thawed in the presence of 2 M glycerol.

The effect of hematocrit (2 versus 75%) has been studied on human red blood cells frozen and thawed in 2 M glycerol at a range of cooling rates (0.8-850 degrees C/min) and warming rates (0.1-200 degrees C/min). The data obtained at a hematocrit of 2% agree well with the data of R. H. Miller and P. Mazur (Cryobiology 13, 404-414, 1976). The results at a hematocrit of 75% show a decrease in recovery with increased cell packing, primarily dependent on warming rate at cooling rates less than 100 degrees C/min and on cooling rate at higher cooling rates. Rapid warming reduced the packing effect, whereas cooling faster than 100 degrees C/min accentuated it. It has been argued that these effects are unlikely to be due to modulation of the generally accepted mechanisms of freezing injury, that is, solution effects and intracellular freezing. It has been suggested that they may be explained by effects of cooling and warming rates on the dimensions of the liquid channels in which the cells are accommodated during freezing and thawing.

Blood Preservation↗
PubMed1984

Studies on cellular structure and ice location in frozen organs and tissues: the use of freeze-substitution and related techniques.

Recent studies have led to the conclusion that extracellular ice per se can damage whole organs and tissues. Thus information on the amount and distribution of ice is an important factor in the design of cooling regimens that avoid intracellular ice formation and attempt to localize the ice formed in areas of the tissue where its disruptive effects can be minimized. Furthermore, ultrastructural studies at subfreezing temperatures can enhance the interpretation of information gained from morphological and function studies conducted before cooling and after rewarming. Although many techniques exist for observing and recording structure in the frozen state, not all are applicable to tissues or organs. Freeze-substitution and isothermal freeze-fixation provide two flexible techniques to explore the frozen state. Isothermal freeze-fixation is most suitable for studies close to the melting point, while freeze-substitution can be used at lower temperatures, extending as far as -120 degrees C. A careful choice of technique can provide an accurate assessment of the amount and distribution of the ice phase and the structure of the tissue matrix.

Animals↗
PubMed1983

Transplantation and in vitro perifusion of rat islets of Langerhans after slow cooling and warming in the presence of either glycerol or dimethyl sulfoxide.

The cryoprotectants dimethyl sulfoxide (Me2SO) and glycerol have been used for the cryopreservation of fetal rat pancreases but only Me2SO has been reported for the cryopreservation of adult rat islets. Since glycerol may be preferred to Me2SO for clinical use, this study was undertaken to compare the effectiveness of these cryoprotectants during the slow cooling of isolated adult rat islets. Islets of Langerhans prepared from the pancreases of WAG rats by collagenase digestion were stored at -196 degrees C after slow cooling (0.3 degrees C/min) to -70 degrees C in the presence of multimolar concentrations of either Me2SO or glycerol. Samples were rewarmed slowly (approximately 10 degrees C/min) and dilution of the cryoprotectant was achieved using medium containing sucrose. Function was assessed by determination of the time course of the glucose-induced insulin release during in vitro perifusion at 37 degrees C and also by isograft transplantation. Transplants were carried out by intraportal injection of a minimum of 1700 frozen and thawed islets into streptozotocin-induced diabetic recipients and tissue function was assessed by monitoring blood glucose levels and body weight changes. Without exception the islets frozen and thawed in the presence of glycerol failed to reduce high serum glucose levels of recipient rats and in vitro dynamic release curves showed to demonstrate a glucose-sensitive insulin release pattern. Reversal of the diabetic conditions was achieved in two of five animals receiving islets which had been frozen and thawed with 2 M Me2SO; and in one of three animals receiving islets cryopreserved with 3 M Me2SO. Nevertheless, perifusion studies showed that the pattern of insulin secretion from groups of cryopreserved islets which did show an ability to secrete insulin was atypical compared with that of untreated controls, suggesting that the tissue was altered or damaged in some way.

Animals↗
PubMed1982

Freeze-substitution and isothermal freeze-fixation studies to elucidate the pattern of ice formation in smooth muscle at 252 K (-21 degrees C).

Taenia coli muscle was cooled to 252 K in the presence of the cryoprotectant dimethylsulphoxide, at cooling rates known to reduce viability by significantly different amounts. The reduction in viability was known to be related to ice formation. Freeze-substitution and isothermal freeze-fixation studies were carried out to determine the distribution of ice within the muscle at this temperature. Freeze-substitution using ethylene glycol was unsuccessful but a new method, using high concentrations of the cryoprotectant as the substituting solvent, was able to maintain ice configuration at this relatively high substitution temperature. The results of freeze-substitution in dimethylsulphoxide were confirmed by isothermal freeze-fixation when both techniques were conducted under identical cooling conditions. The results indicated that the functional differences produced by cooling muscle at either 0.3 K min-1 or 2 K min-1 were related to the distribution of the ice phase within the tissue.

Animals↗
PubMed1981

Dual staining of corneal endothelium with trypan blue and alizarin red S: importance of pH for the dye-lake reaction.

Evaluation of corneal endothelial integrity by combined staining with the vital stain trypan blue and the intercellular stain alizarin red S provides a simple, quick technique for visualisation of both damaged and normal cells, thereby permitting the quantification of endothelial cell damage. Adjustment of the pH of the alizarin red S reagent to 4.2 is important for optimum dye-laking at the intercellular borders, and brief fixation with glutaraldehyde maintains the staining effect of both dyes.

Animals↗