Phosphate-buffered Sucrose Research Articles

Ex vivo tissue preservation solutions for ultrasonic atomization

Tissue water content has been observed to influence ultrasonic atomization efficiency, which raises questions regarding ex vivo tissue storage time and viability. Here, we investigate the influence of tissue preservation solutions on acoustic properties and atomization efficiency. Samples from ten bovine livers were immediately submerged in phosphate-buffered saline (PBSaline), phosphate-buffered sucrose (PBSucrose), phosphate-buffered raffinose (PBRaffinose), or ViaSpan® clinical organ transplantation solution; no solution was also tested. Acoustic attenuation, sound speed, water content, and atomization efficiency were evaluated on either day 1 or day 2. For atomization efficiency, samples were partially submerged in water with the liver-air interface aligned with a 2-MHz focused transducer operating with 10-ms pulses at 1 Hz and peak pressures of 65/-16 MPa. Overall, no difference in atomization efficiency was observed. Water content in tissue was 66–78%, with PBSaline showing a significant increase between days (p = 0.0013). Only tissues in no solution showed a significant difference between days in acoustic attenuation (p = 0.0003). Sound speed measurements had more variation with only ViaSpan and PBSucrose showing no significant differences. As ViaSpan and PBSurcrose showed the most similarities in the tested acoustic properties, PBSurcose may be a low-cost alternative to the clinical ViaSpan solution for preserving ex vivo tissues. [Work supported by NIH DK043881 and EB017857.]Tissue water content has been observed to influence ultrasonic atomization efficiency, which raises questions regarding ex vivo tissue storage time and viability. Here, we investigate the influence of tissue preservation solutions on acoustic properties and atomization efficiency. Samples from ten bovine livers were immediately submerged in phosphate-buffered saline (PBSaline), phosphate-buffered sucrose (PBSucrose), phosphate-buffered raffinose (PBRaffinose), or ViaSpan® clinical organ transplantation solution; no solution was also tested. Acoustic attenuation, sound speed, water content, and atomization efficiency were evaluated on either day 1 or day 2. For atomization efficiency, samples were partially submerged in water with the liver-air interface aligned with a 2-MHz focused transducer operating with 10-ms pulses at 1 Hz and peak pressures of 65/-16 MPa. Overall, no difference in atomization efficiency was observed. Water content in tissue was 66–78%, with PBSaline showing a significant increase be...

Influence of phosphate-buffered sucrose solution on early graft function in feline renal autotransplantation

Graft perfusion with cold heparinized saline has known to induce ischemia and reperfusion injury in feline kidney transplantation. In this study, the effects of phosphate-buffered sucrose solution and heparinized saline solution on early kidney graft function were compared in feline kidney autotransplantation. Perfusion of grafts with or without hypothermic storage with chilled phosphate-buffered sucrose solution prevented ischemia and reperfusion injury despite a very short ischemic time. The results of our study suggest that phosphate-buffered sucrose perfusion and storage solution should be effective to reduce ischemia and reperfusion injury despite a very short ischemic time in feline kidney transplantation.

High-Molecular-Weight Polyethylene Glycol Enhances Hypothermic Storage of Feline Kidney Cells

ABSTRACTPhosphate-buffered sucrose (PBSc) solution is effective for short-term hypothermic preservation of tissue during feline kidney transplantation. A high-molecular-weight polyethylene glycol (35,000 Da, PEG35) reportedly enhanced the protective effects against cold-induced tubular injuries in animal kidney transplantation models. We investigated the ability of PBSc solution containing PEG35 to preserve cultured feline kidney cells using in vitro WST-8 cell proliferation assays. PEG35 significantly improved cell viability during 24 hr of cold preservation. PBSc containing 20 g/l PEG35 achieved an effect almost equal to that of University of Wisconsin (UW) solution, the gold standard preservation solution used in human clinical kidney transplantation, for up to 24 hr of preservation. Our results suggest that PBSc containing PEG35 provides an excellent medium for graft cold storage during feline kidney transplantation.

Neurons of the A5 region are required for the tachycardia evoked by electrical stimulation of the hypothalamic defence area in anaesthetized rats

In order to assess the possible interactions between the pontine A5 region and the hypothalamic defence area (HDA), we have examined the pattern of double staining for c-Fos protein immunoreactivity (c-Fos-ir) and tyrosine hydroxylase, throughout the rostrocaudal extent of the A5 region in spontaneously breathing anaesthetized male Sprague-Dawley rats during electrical stimulation of the HDA. Activation of the HDA elicited a selective increase in c-Fos-ir with an ipsilateral predominance in catecholaminergic and non-catecholaminergic A5 somata (P < 0.001 in both cases). A second group of experiments was done to examine the importance of the A5 region in modulating the cardiorespiratory response evoked from the HDA. Cardiorespiratory changes were analysed in response to electrical stimulation of the HDA before and after ipsilateral microinjection of muscimol within the A5 region. Stimulation of the HDA evoked an inspiratory facilitatory response, consisting of an increase in respiratory rate (P < 0.001) due to a decrease in expiratory time (P < 0.01). The respiratory response was accompanied by a pressor response (P < 0.001) and tachycardia (P < 0.001). After muscimol microinjection within the A5 region, pressor and heart rate responses to HDA stimulation were reduced (P < 0.01 and P < 0.001, respectively). The respiratory response persisted unchanged. Finally, to confirm functional interactions between the HDA and the A5 region, extracellular recordings of putative A5 neurones were obtained during HDA stimulation. Seventy-five A5 cells were recorded, 35 of which were affected by the HDA (47%). These results indicate that neurones of the A5 region participate in the cardiovascular response evoked from the HDA. The possible mechanisms involved in these interactions are discussed.

PBSH: A New Improved Cardiac Preservation Solution in Comparison With Three Clinically Proven Solutions

Introduction A solution under development at our institute, based on phosphate-buffered sucrose, has shown good preservation for kidneys and livers. This work used a refined version of this solution (PBSH—phosphate-buffered sucrose for the heart) in heart preservation, comparing it to solutions already widely used (University of Wisconsin, St. Thomas's, and Celsior solutions). Methods Following an initial washout phase and control working mode on a Langendorff system, hearts were flushed with preservation solution and after 6 hours at 4°C were then reperfused for 15 minutes followed by working heart mode for a further 30 minutes. Hemodynamic parameters were measured and compared with their preischemic values and expressed as percentage recovery. Enzyme measurement came from the collection of the initial 1.5 mL of the coronary effluent after the storage period. This was to test for creatine phosphokinase (CPK) and lactate dehydrogenase (LDH). Hearts were then placed immediately into liquid nitrogen for adenosine triphosphate (ATP), lactate, and creatine phosphate (CP) testing. Spectrophotometric analysis was used to assess both the release of CPK and LDH into the coronary effluent, and the level of ATP, lactate, and CP in the frozen heart tissue. Results These results show that hearts that are preserved in PBSH are hemodynamically as well preserved as the hearts preserved in other solutions tested and their enzyme and lactate content is lower while having higher levels of energy compounds in these hearts. Conclusion Overall these results show that PBSH is at least as effective in cardiac preservation in the rat model of 6 hours of cold ischemia as these other widely used solutions tested.

Influence of thePotato leafroll virusand virus-infected plants on the arrestment of the aphid, Myzus persicae

A series of experiments was conducted using membrane sachets containing MP148 diet or phosphate-buffered sucrose with and without purified Potato leafroll virus to determine if direct encounter with the virus would arrest the aphid, Myzus persicae (Sulzer) (Homoptera:Aphididae). In only two out of 36 tests were there significantly more aphids settled on sachets containing the virus. In all other tests, there were either significantly fewer aphids on sachets containing virus or there were no differences between virus treatments and control sachets without virus. In an experiment using excised Physalis floridana leaves, twice as many M. persicae settled on virus-infected leaves as on noninfected control leaves. Taken together, the results indicate that arrestment of M. persicae on potato leaf roll virus-infected plants may be due to enhanced nutritional qualities resulting from disease, but not from direct encounter with or detection of the virus.

Comparative efficacy of renal preservation solutions to limit functional impairment after warm ischemic injury

In kidney transplantation, cold storage is the dominant modality used to prolong organ viability ex vivo, but is inevitably followed by a period of warm ischemia. Preservation fluids limit tissue damage during the ischemic period, but there is little information on the influence of preservation fluids on the physiologic consequences of warm ischemia alone, or on the comparative ability of such preservation fluids to limit warm ischemic injury. In this study, warm ischemia was induced in rat kidneys by crossclamping the left renal pedicle for 45 min with contralateral nephrectomy. The ischemic kidneys were flushed with Euro-Collins (EC), hyper osmolar citrate (HOC), University of Wisconsin (UW), or phosphate buffered sucrose (PBS)140 solution. Over a period of 2 h after reperfusion, urine and blood samples were collected and physiological parameters related to the function of the postischemic kidneys were assessed. The data show that postischemic renal function can be influenced by the choice of preservation fluid. Essentially, the continued use of EC as a renal preservation solution finds little support in these data, and, while HOC and UW solutions were better able to limit the decline in renal function after warm ischemia than EC, the solution most able to limit functional impairment after warm ischemia was PBS140. This analysis compares the efficacies of the commonly used preservation solutions and could form the basis for future solid-organ transplant studies that may ultimately allow us to propose best-practice guidelines and an optimum platform for improved preservation solutions.

Reduced N-Acetylaspartate Levels in Mice Lacking Aralar, a Brain- and Muscle-type Mitochondrial Aspartate-glutamate Carrier

Aralar is a mitochondrial calcium-regulated aspartate-glutamate carrier mainly distributed in brain and skeletal muscle, involved in the transport of aspartate from mitochondria to cytosol, and in the transfer of cytosolic reducing equivalents into mitochondria as a member of the malate-aspartate NADH shuttle. In the present study, we describe the characteristics of aralar-deficient (Aralar-/-) mice, generated by a gene-trap method, showing no aralar mRNA and protein, and no detectable malate-aspartate shuttle activity in skeletal muscle and brain mitochondria. Aralar-/- mice were growth-retarded, exhibited generalized tremoring, and had pronounced motor coordination defects along with an impaired myelination in the central nervous system. Analysis of lipid components showed a marked decrease in the myelin lipid galactosyl cerebroside. The content of the myelin lipid precursor, N-acetylaspartate, and that of aspartate are drastically decreased in the brain of Aralar-/- mice. The defect in N-acetylaspartate production was also observed in cell extracts from primary neuronal cultures derived from Aralar-/- mouse embryos. These results show that aralar plays an important role in myelin formation by providing aspartate for the synthesis of N-acetylaspartate in neuronal cells.

Protective Effects of Polyethylene Glycol (20 mol/L) in Phosphate-Buffered Sucrose for Rat Liver Preservation

Protective Effects of Polyethylene Glycol (20 mol/L) in Phosphate-Buffered Sucrose for Rat Liver Preservation

Protective effects of lactobionate in modified phosphate-buffered sucrose

Protective effects of lactobionate in modified phosphate-buffered sucrose

Comparison of the protective effects of phosphate-buffered sucrose and University of Wisconsin solution in a non–heart-beating liver donor experiment

Comparison of the protective effects of phosphate-buffered sucrose and University of Wisconsin solution in a non–heart-beating liver donor experiment

Effect of various short-term storage methods on viability of cancellous bone fragments

Abstract Objective To determine effects of various storage methods on ex vivo viability of cancellous bone fragments. Sample Population Cancellous bone fragments obtained from 4 New Zealand White rabbits. Procedure Cancellous bone fragments were stored for 3 hours on ice in 1 of 5 preservation solutions or in 0.9% NaCI. Fragments were then reperfused (37 C) in oxygenated physiologic buffer solution for 1 hour. Cellular viability in fragments was assessed by ethidium monoazide labeling and fluorescence microscopy. Results Viability in fresh cancellous bone ranged from 85 to 100% (mean ± SEM, 88 ± 7%). Storage of fragments significantly reduced viability. Viability in bone fragments stored at 22 C in blood-soaked sponges or 0.9% NaCI solution was 63.8 ± 3 and 65.2 ± 7%, respectively. Use of cold 0.9% NaCI solution reduced viability to 53.6 ± 3%. Viability was significantly better for fragments stored in cold phosphate-buffered sucrose (70.4 ± 2%) and EuroCollins (71.4 ± 3%) solutions. After warm reperfusion, viability was best for fragments stored in cold phosphate-buffered sucrose (70.2 ± 4%), EuroCollins (72.6 ± 3%), or UW lactobionate solutions (69.6 ± 3%), compared with those stored in cold 0.9% NaCI (47.6 ± 3%) or hypertonic citrate (53.0 ± 3%) solutions or blood-soaked sponges (57.2 ± 3%). Conclusions Hypothermic storage in solutions designed to prevent temperature-dependent cell injury were best for maintaining cancellous bone fragment viability. Clinical Relevance Hypothermia may be advantageous for use in storing cancellous bone fragments during procedures that dictate a prolonged period between harvest and placement of graft fragments. (Am J Vet Res 1999;60:63–67)

Hypothermic storage of feline kidneys for transplantation: successful ex vivo storage up to 7 hours.

To describe the effect of hypothermic storage on transplanted feline kidneys. Kidneys were stored in University of Wisconsin (UW) sodium gluconate (n = 3) or phosphate-buffered sucrose (n = 5) solutions before transplantation. Eight cats with renal failure and seven normal cats as kidney donors. Kidneys were perfused through the renal artery with cold (10 degree C) storage solution and immersed in the solution on ice until transplantation. Mean ex vivo storage time was 4.8 +/- 0.36 hours (range, 3.5 to 7 hours). Seven recipient cats survived surgery. Five of the cats had decreased serum creatinine concentrations from a mean of 8.2 mg/dL (range, 4.0 to 15.8 mg/dL) preoperatively to 1.7 mg/dL (range 1.3 to 2.2 mg/dL) within 4 days of surgery. In one cat, serum creatinine concentration dropped from 15.1 to 3.7 mg/dL in 3 days, but the cat developed a ureteral stricture that required revision. One graft did not function, and the cat died on day 19. The mean postoperative survival time of cats that were discharged from the hospital (n = 6) was 254 days (range, 49 to 717 days) at the time of this report. Long-term renal function (> 60 days postoperatively; n = 5) was excellent with mean serum creatinine concentrations of 1.6 +/- 0.15 mg/dL. Hypothermic storage is feasible for short-term preservation of feline kidneys. The maximal length of feasible storage remains unknown. Hypothermia protects against ischemia-induced nephron loss during ex vivo manipulation of the allograft and allows longer safe vascular anastomosis times. Short-term hypothermic storage also provides time to accommodate modifications in scheduling or anesthetic management of the recipient operation.

Comparison of solutions for preservation of the rabbit liver as tested by isolated perfusion

Abstract The University of Wisconsin (UW) solution consists of a relatively complex mixture of agents. In this study we compared simpler preservation solutions, namely, histidine-tryptophan-ketoglutarate (HTK) and phosphate-buffered sucrose (PBS) with different compositions of UW solution in the isolated perfused rabbit liver model. Livers were stored cold for 24 and 48 h. After 24 h of preservation, the amount of bile produced in UW-preserved livers was significantly greater (P < 0.05) than that in HTK-preserved livers. Also, there was less LDH released into the perfusate in UW-preserved livers. There was more edema and lower K +/Na + rations in HTK-preserved livers than in UW-preserved livers (all data P < 0.05). After 48 h of preservation, the differences between livers preserved in UW or HTK solution were less noticeable than at 24 h and bile production was similar. LDH and AST release were greater in HTK-preserved livers than in UW livers, but these differences were not statistically significant. Preservation in PBS for 48 h was worse than in either UW or HTK solution. Substitution of polyethylene glycol (PEG) for hydroxyethyl starch (HES) in 48-h UW-preserved livers was not effective. We conclude that solutions simpler in composition than UW solution may be effective in kidney transplantation but do not appear suitable for successful liver preservation.

Comparison of Phosphate-Buffered Sucrose, Modified EuroCollins, and University of Wisconsin Solutions

Phosphate-buffered sucrose (PBS) has been shown to be highly effective for renal graft storage. It may, therefore, be useful for lung graft storage. Recent studies have suggested a possible role for University of Wisconsin (UW) solution in lung preservation. The object of this study was to evaluate these two solutions in comparison with EuroCollins (EC) solution for lung graft preservation in an isolated rat lung model. Lungs were stored for 6 hr at 4 degrees C after a single pulmonary artery flush with either PBS with prostacyclin (n = 10), EC with prostacyclin (n = 5), or UW (n = 5) solution. Reperfusion of the isolated lung was carried out for 1 hr using a venovenous extracorporeal circulation from a ventilated support rat. The support animals and isolated lungs were ventilated with room air. Control values were obtained from lungs reperfused immediately after harvesting (n = 5). At 1 hr, PBS provided a similar level of protection to EC: pO2, 45 +/- 10 mmHg and 54 +/- 6 mmHg; graft blood flow, 4.1 +/- 1.2 ml/min and 3.5 +/- 0.42 ml/min; peak airway pressure, 32 +/- 2.5 mmHg and 36 +/- 3.6 mmHg; weight gain, 4.1 +/- 0.6 g and 4.2 +/- 0.6 g, respectively (P = NS). However, the UW group provided superior function, which was similar to the control group: pO2, 128 +/- 2.7 mmHg and 126 +/- 5 mmHg; graft blood flow, 9.9 +/- 0.4 ml/min and 10.2 +/- 0.8 ml/min; peak airway pressure, 17.6 +/- 0.4 mmHg and 16.5 +/- 0.6 mmHg; weight gain, 0.12 +/- 0.1 g and 0.19 +/- 0.13 g, respectively (P = NS). UW was superior in all parameters to PBS and EC (P < 0.001). This suggests that the renal solutions PBS and EC are inappropriate for lung graft preservation, and that the requirements of the lung during hypothermic storage differ from those of the kidney.

Comparison of Phosphate-Buffered Sucrose, Modified EuroCollins, and University of Wisconsin Solutions

Comparison of Phosphate-Buffered Sucrose, Modified EuroCollins, and University of Wisconsin Solutions

Mucosal Glutaminase Activity and Histology as Parameters of Small Bowel Preservation Injury

The present study in Lewis rats was designed to assess the predictive value of the mucosal enzyme activities of glutaminase, maltase, and xanthine oxidase and of histology as parameters to delineate the degree of small bowel preservation injury. Small bowel grafts were flushed with saline or a modified phosphate-buffered sucrose (PBS) solution, stored at 8°C for 1, 6, or 12 hr, and transplanted heterotopically. Tissue samples for determination of mucosal enzyme activities were taken after the cold storage period, 20 min after reperfusion and 2 and 7 days postoperatively. Biopsies for light microscopic evaluations were obtained at the same time points, but not after cold storage. Glutaminase activity was well maintained after cold storage, regardless of the duration of preservation. Enzyme activities measured 20 min after reperfusion decreased with increasing duration of preservation (saline: R 2 = 32.8%; P < 0.01; PBS: R 2 = 52.3%; P ⩽ 0.001) and with increasing histologic preservation injury. Glutaminase activities were predictive for survival of grafts preserved with the PBS solution ( R 2 = 49.6%; P ⩽ 0.001; sensitivity 92%; specificity 100%), while the activities of maltase and of xanthine oxidase failed to do so. The degree of histologic preservation injury seen in graft specimens obtained 20 min after reperfusion was a good predictor of graft survival with a sensitivity of 90% for saline-preserved grafts and 92% for PBS-preserved grafts and a specificity of 88 and 67%, respectively. These data indicate that the determination of mucosal glutaminase activity in combination with standard histology serves well as monitor of small bowel preservation injury.

THE REFLUSH EFFECT—A PROSPECTIVE ANALYSIS OF LATE PERFUSION

This prospective randomized trial examines the effect of a "reflush" with preservation solution immediately prior to renal allograft implantation, using hyperosmolar citrate (HOC, n = 10) or phosphate-buffered sucrose (PBS140, n = 10) versus no reflush (n = 10). All kidneys had been stored in HOC. The HOC reflush did not alter the postpreservation intra- or extracellular electrolyte milieu, whereas the PBS140 reflush resulted in an apparent overall loss of both sodium and potassium from the kidney (P < 0.0005). A small amount of calcium was released into the venous effluent in both reflush groups. A similar amount of lactic acid was released into the venous effluent of the two reflush groups, reflected by a lower pH (P < 0.0005), and there was a similar loss of lactate dehydrogenase and creatine phosphokinase. An analysis of procoagulant activity in the first urine produced was performed as a marker of reperfusion injury. The median value was higher in the No Reflush group at 457.5 units, compared with 263.0 and 209.0 units for the PBS140 and HOC Reflush groups, respectively (P = 0.06). Reflushing the kidneys reduced the postoperative dialysis requirement (from 40% to 15%), but by the end of the first posttransplant week there was no significant difference between the renal functional analyses of the three groups, and there was no difference at one year. The proposed mechanism for the early renal functional improvement is a reduction in the calcium paradox and free radical formation, by release of calcium and ATP breakdown products into the venous effluent prior to implantation.

IMPROVED PORCINE RENAL PRESERVATION WITH A SIMPLE EXTRACELLULAR SOLUTION—PBS140

In this prospective randomized trial a porcine model of renal autotransplantation was used to compare quality of preservation, as reflected by detailed analysis of posttransplant renal function, following 24-hr cold storage in phosphate-buffered sucrose (PBS140), hyperosmolar citrate (HOC), and University of Wisconsin (UW) preservation solutions. There were 6 deaths with primary nonfunction: 3 of 5 HOC, 2 of 5 UW, but only 1 of 5 PBS140. Analysis of the whole group and separate analysis of the survivors demonstrated significantly better renal function following preservation with PBS140 when compared with both HOC and UW, with a lower peak serum creatinine (P = 0.02) and improved loop of Henle function (P = 0.02). The animals in the PBS140 group also demonstrated a more rapid return to normal creatinine, higher GFR, improved tubular function, and higher effective renal plasma flow, with figures approaching statistical significance (P = 0.06-0.07). The proposal of UW as a universal storage medium prompted this study, and its results suggest the need for a clinical comparison of renal preservation using UW and PBS140 in a prospective randomized trial.

RANOLAZINE—A NEW DRUG WITH BENEFICIAL EFFECTS ON RENAL PRESERVATION

Ranolazine is a new drug with a novel mode of action as a metabolic modulator and membrane stabilizer. In this prospective randomized double-blind trial, a porcine model of renal autotransplantation was used to assess the effects of this drug during preservation and reperfusion of kidneys cold-stored for 24 hr in phosphate-buffered sucrose (PBS140). Three groups of 10 animals were compared: a Placebo group (placebo given intravenously to the animal before nephrectomy, added to the preservation solution, and given again to the animal during reperfusion); a Storage group (Ranolazine before and during storage, placebo during reperfusion); and a Reperfusion group (placebo before and during storage, Ranolazine during reperfusion). Detailed analysis of posttransplant renal function was carried out over a 14-day follow-up period. There were 7 deaths with primary nonfunction: 2 Placebo, 1 Storage, 4 Reperfusion. Analysis of the whole group and separate analysis of the survivors demonstrated significantly improved glomerular (P less than 0.05), tubular (P less than 0.05), and loop of Henle (P less than 0.05) function in the Storage group. The results of this study clearly demonstrate the beneficial effects of Ranolazine during the storage phase of porcine renal preservation, and further investigation of this drug is warranted.