Liver preservation by cold storage with hyperosmolar solutions for twenty-four hours.

Canine livers were successfully preserved in an ischemic state for 24 hr under hypothermic storage with hyperosmolar colloid or crystalloid solutions. Livers preserved with a colloid hyperosmolar solution (MSGF) showed slightly better survival results than the ones preserved with a crystalloid hyperosmolar solution. It is possible that hyperosmolarity associated with hyperkalemia is an important factor for liver preservation for transplantation.     

Pancreas preservation with TP-IV: a hyperosmolar colloid solution

This study compares the efficacy of a new hyperosmolar colloid solution (TP-IV) with Euro-Collins solution for long-term (72 hr) hypothermic storage of canine pancreas autografts. Four experimental recipient groups and their survival (30-day study period) results were as follows: Gr. I (n = 6) pancreatectomized controls, without autotransplant (X +/- SD = 5.83 +/- 3.06 days); Gr. II (n = 6) fresh nonpreserved autografts (X +/- SD = 23.83 +/- 10.12 days, 5 of 6 greater than 30 days); and Gr. III (n = 7) and Gr. IV (n = 5) receiving pancreas autografts stored at 4 degrees C for 72 hr in either Euro-Collins or TP-IV, respectively (Gr. III, 13.85 +/- 9.04 days; Gr. IV, 21.2 +/- 12.37 days). The results appear to indicate that TP-IV is superior to Euro-Collins solution for 72-hr hypothermic storage of pancreas grafts. In fact, survival in the TP-IV-presented group was comparable to that of fresh, non-preserved autografts.     

Lung transplantation protection after warm ischemia and 1 day preservation

This study attemps to determine the role of colloid hyperosmolar solutions in the preservation of nonischemic and ischemic lungs. Four groups of animals were studied: Control fresh lung allografts and lungs stored under hypothermic storage (4–7 °C) in a modified silica gel fraction (MSGF) for 24 hr with or without warm ischemia (0, 30, 60 min) prior to storage. Fresh lungs lived an average of 14.5 days after transplantation. Stored nonischemic lungs survived an average of 11.2 days, and ischemic (30, 60 min) stored lungs remained alive for an average of 10.5 and 9.2 days. There were no significant differences in survival between the fresh and preserved allografts. Pneumonia and/or rejection occurred in 71% of all groups. MSGF appears to be a good solution for 24-hr hypothermic storage of nonischemic and ischemic lungs.     

Cold storage preservation of islet and pancreas grafts as assessed by in vivo function after transplantation to diabetic hosts

The major goal of hypothermic (4–8 °C) preservation of intact pancreases or isolated islets will be to provide sufficient time for HLA typing, cross matching, selection, and preparation of recipients—logistical efforts requiring 12–72 hr for clinical kidney transplantation, usually <48 hours. Some investigators have studied in vitro function of islets after cold storage, but the critical test of viability—permanent restoration of normoglycemia after transplantation to diabetic recipients—has been tested in only a few experiments. Reversal of hyperglycemia by syngeneic or autogenic transplants in diabetic animals has been achieved after CS of dispersed pancreatic tissue from neonatal rats in GIB media for ? 146 hr, adult dogs in TCM 199 for ?24 hr, and adult DL-ethionine-treated rats in RPMI 1640 for ?72 hr. In the neonatal rat donor model, intravenous glucose tolerance test (IVGTT) results were similar in recipients of fresh or stored islets; in the dog model, IVGTT test results were variable, but generally inferior in recipients of stored as compared to fresh islets; in the adult rat donor model, recipients of ?24-hr coldstorage islets had insulin and IVGTT K values similar to those of recipients of fresh islets, but the success rate progressively declined for CS times >24 hr. Various agents were added to the media, but the need or the optimal concentrations were not critically determined by using different recipes for different groups of recipients. Cold storage of intact pancreas autografts has been tested in dogs; simple electrolyte solutions are satisfactory for 24 hr, but only a silica gel-filtered plasma-based solution has been reliable for 48 hr. Pulsatile machine perfusion (PMP) of canine pancreas grafts for 24 hr has had a success rate similar to CS in some experiments and lower in others. PMP has been almost totally unreliable for >24 hr. Further refinements are needed if preservation of islets for >24 hr and pancreases for >48 hr are to be consistently successful. If current experimental techniques are effective for human islets or pancreases, however, these times are sufficient to complete the logistical maneuvers required before transplantation.     

Development of a cold storage solution for pancreas preservation

Canine pancreas tissue slices were incubated at 5 degrees C for 24 hr in solutions containing different saccharides (raffinose, sucrose, mannitol, or glucose). At the end of incubation tissue water (TW expressed as kg H2O/kg dry wt) was determined as a measure of tissue edema. Tissue edema was greatest in slices stored in Eurocollins (EC) solution (TW = 4.96 +/- 0.14) which contains glucose for osmotic pressure. The degree of edema was decreased by saccharides in proportion to their molecular mass: mannitol (MW = 180, TW = 3.84 +/- 0.08), sucrose (MW = 348, TW = 3.54 +/- 0.08), and raffinose (MW = 594, TW = 3.30 +/- 0.07). Tissue edema was also greatest in slices incubated in solutions containing the smallest molecular mass anions: Cl- (TW = 4.02 +/- 0.16), gluconate (TW = 3.69 +/- 0.10), and lactobionate (TW = 3.28 +/- 0.13). Cold storage of the intact pancreas in EC solution for 24 hr did not induce as much edema as in slices (TW = 2.88 +/- 0.10). However, on isolated reperfusion at normothermia (37 degrees C) the pancreas became edematous (TW = 3.33 +/- 0.12). Storage of the pancreas in a lactobionate-raffinose solution did not induce edema after 90 min of normothermic reperfusion. The suppression of tissue edema in the pancreas may be essential to obtaining long-term preservation (24-72 hr) of this organ which is currently limited to about 6-8 hr in EC solution. The newly developed lactobionate-raffinose solution appears to control tissue edema in both tissue slices and the intact-flushed out organ.     

Renal preservation by hypothermic perfusion. IV. The use of gelatin polypeptides as the sole colloid

Rabbit kidneys were perfused with a solution of extracellular electrolyte composition, rendered hypertonic with glucose and containing 1.75% Haemaccel (Hoechst) as the sole colloid. Perfusions were carried out at 10 °C for 24 and 48 hr using perfusion pressures of 20 or 40 mm Hg. Function, was tested by autografting with immediate contralateral nephrectomy. All the kidneys perfused at 20 mm Hg for 24 hr showed excellent life-sustaining function and 7 of 10 survived in the 40 mm Hg group; the difference was not statistically significant. However, all the kidneys perfused for 48 hr failed to sustain life, and histological examination revealed extensive breakdown of the microcirculation. The 24 hr results were similar to those previously obtained with an albumin-based perfusate, but the 48-hr results were inferior: However, the obvious advantages of a well-standardised, cheap, and easily stored perfusate are such as to justify further study of gelatin-derived colloids for organ preservation.     

Effect of dehydratation and blood substitute infusion on the hemostasis system in a 21-hour bed rest experiment

The time course of the hemostasis parameters (activated partial thromboplastin time, prothrombin time, thrombin time, fibrinogen, plasminogen, D-dimer, soluble fibrin-monomer complexes (SFMC), as well as antithrombin III (AT III) and protein C (antiplasmin) activities) were estimated in eight male volunteers aged from 20 to 40 years during 21-h bed rest after dehydratation induced by diuretics, which was followed by intravenous infusion of colloid and crystalloid solutions. It was shown that noncompensated dehydratation during bed rest did not cause significant alterations in the procoagulant component of hemostasis. The intravenous infusion of the blood substitute sterofundin (crystalloid) and venofundin (colloid) to the bed-resting subjects resulted in an increase in coagulation within the physiological norm. A decrease in the AT III activity and plasminogen level resulting in an increase in SFMC were observed. The fibrinogen concentration remained stable, which indicated the absence of acute reaction of the body to the experimental effects.     

Alkaline phosphatase modulation by osmolality changes during the growth cycle of KB cell cultures.

Growing KB cells in hyperosmolar medium causes in reduction in total alkaline phosphatase activity associated with a decrease in the proportion of the heat-labile and an increase of the heat-stable enzyme components. In standard medium enzyme activity increases progressively during a 6-day growth cycle and the proportion of heat-stable activity remains constant. In hyperosmolar medium, activity increases only during the initial 24 hr after the change in osmolality and then levels off, but the heat-stable alkaline phosphatase activity increases 7-fold within 48 hr. The transmission in thermostability is discernible 24 hr after increasing the osmolality of the medium and toward the end of a growth cycle most of the activity is heat-stable.     

The role of quality control in a skin bank: tissue viability determination.

New surgical procedures requiring viable skin have increased rapidly over the last few years. The cell viability assessment in allograft skin is a major step forward in burn treatment, since it is well-known that taking is correlated with grafted tissue viability. Various methods, both qualitative and quantitative, are currently used. Although qualitative assays (histomorphology, immunocytochemistry) are routinely performed in our laboratory, there arose a need to set up a standardised quantitative assay in an attempt to obtain a cut-off value so that the skin sample could be determined valid or not for grafting. Therefore, two different tetrazolium salt compounds MTT and WST-1, were compared in order to determine their efficacy in the evaluation of tissue viability. Several experimental conditions were analysed: 1- cellular cultures of keratinocytes and fibroblasts, 2- fresh skin tissue samples, 3- the same specimen tested daily for at least 2 weeks, 4- after cryopreservation and thawing. Viable cells were analysed by the cleavage of tetrazolium salts to formazan by cellular enzymes. The formazan dye produced by metabolically active cells was then quantified by measuring the absorbance of the dye solution at the appropriate wavelength. It was seen that WST-1 is easier to handle, more stable, has a wider line arrange, accelerated colour development and is more sensitive than MTT on fresh specimens and cell suspension. However, after 72 hours of storage at 4°C, most of the WST-1 tested specimens no longer gave any absorbance signal, whilst MTT specimens were seen to give a signal for more than two weeks. Moreover, after thawing WST-1 tested samples were almost negative,whilst MTT samples continued to give strong signals. In conclusion, WST-1 assay offers rapid and precise results as to the cell viability of fresh allografts and cell cultures, whilst the MTT method is much more useful in establishing viability after long conservation and cryopreservation. In our clinical experience, allografts transplanted at 72 hr post-harvesting or after cryopreservation showed a mean of take more than of 80%, demonstrating that the MTT system is more reliable for the determination of allograft viability. Studies are ongoing with larger clinical cohorts to establish the precise cut-off value for skin graft validation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of cryopreservation on the immunogenicity of porcine arterial allografts in early stages of transplant vasculopathy

BACKGROUND: The number of revascularization procedures including coronary and lower extremity bypass, have increased greatly in the last decade. It suggests a growing need for vascular grafts. Cryopreserved allografts could represent a viable alternative but their immunologic reactivity remains controversial. METHODS: 71 pigs (40 recipients and 31 donors) were used. Two femoral grafts per recipient animal were implanted for 3, 7, and 30 days. Types of grafts: fresh autograft as a control graft (n=19), fresh allograft (n=31) and cryopreserved allograft (n=30). Histological and immunohistochemical studies were performed. RESULTS: Fresh allografts compared to autografts showed intimal inflammatory infiltration at 3 days (328 vs. 0 macrophages/mm2; P<0.05) and 7 days (962 vs. 139 T lymphocytes/mm2; P<0.05) post-transplantation. At 30 days, there was a loss of endothelial cells, presence of luminal thrombus and aneurismal lesions (total area=15.8 vs. 8.4 mm2; P<0.05). Cryopreservation did not reduce these lesions nor modify endothelial nitric oxide synthase (eNOS) expression nor modify the number of animals that developed anti-SLA antibodies. Moreover, at 7 days, cryopreserved allografts compared to fresh allografts showed a higher expression of P-selectin (5 out of 5 vs. 1 out of 5; P<0.05) and, at 30 days, a greater inflammatory reactivity (2692 vs. 1107 T lymphocytes/mm2 in media; P<0.05) with a trend towards a higher presence of multinucleated giant cells than in the fresh ones. CONCLUSIONS: The cryopreservation method used maintained immunogenicity of allografts and increased the inflammatory reactivity found in fresh allografts up to 30 days of vascular transplantation.     

Functional challenge affects aquaporin mRNA abundance in mouse blastocysts

The aquaporins (AQPs) are a family of channel proteins that facilitate diffusion of water across cell membranes. Three members of the AQP family have been detected in the mouse blastocyst: AQP 3 and 8 are located in the basolateral domain and AQP 9 predominantly in the apical domain of the trophoblast cells. These are believed to be involved in facilitating the accumulation of fluid into the blastocyst cavity. We have investigated the ability of mouse embryos to regulate AQP gene expression in response to different treatments expected to affect the passage of water across the trophoblast cells using real-time PCR. In the first experiment 8-cell embryos were allowed to develop to blastocysts in media from 300 to 400 mOsm. Blastocyst formation was unaffected by media made hyperosmolar by glycerol, whereas blastocyst formation was significantly reduced in sucrose-based 350 and 400 mOsm media. AQP 8 mRNA levels were reduced when embryos were cultured in glycerol-based hyperosmolar media. The mRNA levels of AQP 3, 7, 9, and 11 were not significantly affected by hyperosmolar media. In the second experiment blastocysts were punctured (0 hr) and allowed to re-expand. AQP mRNA levels were examined after 2, 6, and 10 hr. Compared to control embryos, the expression of AQP 3, 7, and 9 were upregulated after 2 hr. Upregulation was sustained only for AQP 9 and this was sustained up to 6 and 10 hr after puncture. In the third experiment we compared expression of AQPs between in vitro cultured and in vivo developed blastocysts. We found that in vitro culture resulted in lower levels of AQP 8, 9, and 11 compared to in vivo development. These experiments show that mouse embryos are capable of regulating AQP mRNA abundances in response to environmental alterations.     

Radioprotection provides functional mechanics but delays healing of irradiated tendon allografts after ACL reconstruction in sheep

Successful protection of tissue properties against ionizing radiation effects could allow its use for terminal sterilization of musculoskeletal allografts. In this study we functionally evaluate Achilles tendon allografts processed with a previously developed radioprotective treatment based on (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) crosslinking and free radical scavenging using ascorbate and riboflavin, for ovine anterior cruciate ligament reconstruction. Arthroscopic anterior cruciate ligament (ACL) reconstruction was performed using double looped allografts, while comparing radioprotected irradiated and fresh frozen allografts after 12 and 24 weeks post-implantation, and to control irradiated grafts after 12 weeks. Radioprotection was successful at preserving early subfailure mechanical properties comparable to fresh frozen allografts. Twelve week graft stiffness and anterior-tibial (A-T) translation for radioprotected and fresh frozen allografts were comparable at 30 % of native stiffness, and 4.6 and 5 times native A-T translation, respectively. Fresh frozen allograft possessed the greatest 24 week peak load at 840 N and stiffness at 177 N/mm. Histological evidence suggested a delay in tendon to bone healing for radioprotected allografts, which was reflected in mechanical properties. There was no evidence that radioprotective treatment inhibited intra-articular graft healing. This specific radioprotective method cannot be recommended for ACL reconstruction allografts, and data suggest that future efforts to improve allograft sterilization procedures should focus on modifying or eliminating the pre-crosslinking procedure.     

Forty-eight hours hypothermic pulsatile perfusion of canine hearts before transplantation.

Canine hearts preserved for 24 hr under hypothermic pulsatile perfusion had a good function after transplantation. The perfusate consisted of cryoprecipitated plasma that was modified by the addition of salt poor albumin, potassium chloride and glucose; the final osmolarity was 340 mOsm/L. Fresh allografts without perfusion survived for an average of two weeks after transplantation, and the 24 hr perfused hearts survived for more than 19 days after transplantation. When the perfusion was extended to 48 hr, the survival was decreased to 11 days. These data indicate that hypothermic pulsatile perfusion is completely safe and feasible for 24 hr without significant functional or histological impairment. The survival response of the hearts perfused for 48 hr was significantly decreased when compared to the hearts perfused for 24 hr.     

Differentiation of Cells in the Hypophysis and in Pancreatic Islets

Deparaffinized, 3-5μ, sections are brought to water, oxidized 3.5 min in an equal-parts mixture of 0.3% H₂SO₄ and 0.3% KMnO₄, and decolorized with 4% K₂S₂O₅. Nuclei are stained with Gomori's (1939) chromium-hematoxylin, and cell granules with Cason's (1950) mixture. The eosinophilic cells of the hypophysis and the alpha cells of pancreatic islets (of Langerhans) stain carmine red; basophilic and beta cells stain dark blue. Heidenhain's _susa is the most suitable fixative for hypophysis, Bouin's fluid for pancreas; but a satisfactory result is obtainable after formalin-sublimate or plain formalin. Besides studying the ratio of the cell types in the hypophysis or in pancreatic islets, it is possible to estimate the granule content of the cells. The method works on human autopsy material provided fixation of hypophysis occurs within 24 hr, and. pancreas, 12 hr post mortem, and it is suitable also for quite fresh organs.     

Saponification of Methylated Mucosubstances at Low Temperatures

A fresh 1% solution of KOH in 70% ethanol in 2 hr at 2-4 C restores basophilia to methylated acid mucosubstances satisfactorily without detaching or damaging tissue sections. A 0.5% solution of Ba(OH)₂ under the same conditions gives results nearly as good, but NaOH and KMnO₄ are unsatisfactory.     

Morphological aspects of the rat kidney preserved by cold storage. I. Glomerular morphometric changes

The absolute density of glomeruli in the microscopic field was determined in the rat kidney preserved by cold storage for 24, 48, 72 and 96 hrs in two different media: Sacks (hyperosmolar electrolytic solution of intracellular type) and Plasmagel (gelatin solution 4%). Progressive, statistically significant (p less than 0.01) decrease of glomerular density at 24 and 48 hrs was followed by return to initial values at 96 hrs. Decrease of the glomerular density was greater with Plasmagel.     

Toluidine Blue-Alizarin Red S Staining of Cartilage and Bone in Whole-Mount Skeletons in Vitro

Cartilage and bone of the developing skeleton can be reliably differentiated in whole-mount preparations with toluidine blue-alizarin red S staining after FAA fixation. The recommended staining procedure is based chiefly on the use of newborn white and Swiss-Webster mice, 4-9 days postnatal, but was tested also on mice and rats 3-8 wk of age. Procedure: Sacrifice, skin, eviscerate, remove body fat, and place specimens in FAA (formalin, 1; acetic acid, 1; 70% alcohol, 8) for approximately 40 min. Stain in 0.06% toluidine blue made in 70% ethyl alcohol for 48 hr at room temperature. Use 20 volumes of stain solution to the estimated volume of the specimen. Destain soft tissues in 35% ethyl alcohol, 20 hr; 50%, 28 hr; and 70%, 8 hr. Counterstain in a freshly prepared 1% aqueous solution of KOH to which is added 2-3 drops of 0.1% alizarin red S per 100 ml of solution. Each day for 3 days, transfer the specimen to a fresh 1% KOH-alizarin mixture, or until the bones have reached the desired intensity of red and soft tissues have cleared. Rinse in water, and place in a 1:1 mixture of glycerol and ethyl alcohol for 1-2 hr, then transfer the specimen to fresh glycerol-alcohol for final clearing and storage. Older mice and rats require procedural modifications: (1) fixation for 2 hr, (2) 0.12% toluidine blue, (3) maceration for 4 days in 3% KOH-alizarin, and (4) preliminary clearing for 24 hr in a mixture of glycerol, 2; 70% ethyl alcohol, 2; and benzyl alcohol, 1 (v/v) before placing in a 1:1 alcohol-glycerol mixture.     

Prolongation of canine pancreas allograft survival with cyclosporin A: preliminary report.

Studies were conducted on dogs to test the efficacy of cyclosporin A (CyA) in prolonging normoglycaemia and graft survival after whole-organ pancreas allograft transplantation. Five dogs subjected to pancreatectomy alone served as controls. Withholding immunosuppression after transplantation (five animals) resulted in the same median duration of survival as occurred in the controls (13 days). Azathioprine and steroids (seven animals) produced median durations of normoglycaemia and survival of 9 and 23 days respectively. Animals given CyA 18 mg/kg/day (five) and 25 mg/kg/day (10), however, showed median durations of normoglycaemia of 18 and 55 days (p less than 0.05 and p less than 0.02) respectively and median survival times of 36 and 85 days (NS and p less than 0.02). If CyA proved effective in controlling rejection of pancreas allografts in man it would offer unstable diabetics in renal failure a more hopeful outlook than conventional immunosuppression.     

Effects of crystalloid and colloid fluids on extravascular lung water in hypoproteinemic dogs

We compared the effect of crystalloid to colloid fluid infusion on extravascular lung water (EVLW) in hypoproteinemic dogs. Plasmapheresis was used to decrease plasma colloid osmotic pressure (COP) to less than 40% of its base-line level. Five animals were then infused with 0.9% sodium chloride (saline), five with 5% human serum albumin (albumin), and five with 6% hydroxyethyl starch (hetastarch) to increase the pulmonary arterial occlusive pressure by 10 Torr in comparison to the postplasmapheresis level for a 5-h study interval. On completion of the procedure, the lungs were harvested and EVLW measured by the blood-free gravimetric technique. Three to six times the volume of saline compared with albumin or hetastarch (P less than 0.001) was infused. In the saline animals, COP was decreased to 3.3 +/- 1.3 Torr, whereas COP was increased to 18.1 +/- 1.4 Torr in albumin animals (P less than 0.001) and 20.1 +/- 1.6 Torr in the hetastarch group (P less than 0.001). The saline-treated dogs developed gross signs of systemic edema. The EVLW was 8.1 +/- 0.9 ml/kg in saline animals compared with 5.3 +/- 2.1 ml/kg in the albumin (P less than 0.05) and 4.1 +/- 1.4 ml/kg in the hetastarch (P less than 0.01) groups. These data indicate that crystalloid fluid infusion during hypoproteinemia is associated with the development of both systemic and pulmonary edema.     

Unilateral exposure of Shaker B potassium channels to hyperosmolar solutions.

This study tests the hypothesis that ion channels will be affected differently by external (extracellular) versus internal (cytoplasmic) exposure to hyperosmolar media. We looked first for effects on inactivation kinetics in wild-type Shaker B potassium channels. Although external hyperosmolar exposure did not alter the inactivation rate, internal exposure slowed both onset and recovery from fast inactivation. Differential effects on activation kinetics were then characterized by using a noninactivating Shaker B mutant. External hyperosmolar exposure slowed the late rising phase of macroscopic current without affecting the initial delay or early rising phase kinetics. By contrast, internal exposure slowed the initial steps in channel activation with only minimal changes in the later part of the rising phase. Neither external nor internal hyperosmolar exposure affected tail current rates in these noninactivating channels. Additionally, suppression of peak macroscopic current was approximately twofold smaller during external, as compared with internal, hyperosmolar exposure. Single-channel currents, observed under identical experimental conditions, showed a differential suppression equivalent to that seen in macroscopic currents. Apparently, during unilateral hyperosmolar exposure, changes in macroscopic peak current arise primarily from changes in single-channel conductance rather than from changes in equilibrium channel gating. We conclude that unilateral hyperosmolar exposure can provide information concerning the potential structural localization of functional components within ion-channel molecules.