The University of Wisconsin colloid based preserving solution (UW solution) has had a significant impact on organ transplantation. After the first report of UW solution for successful preservation of the pancreas (72 hours) presented at the Meeting of the American Society of Transplant Surgeons 19871 and the subsequent report for preservation of the liver (48 hours) and kidney (72 hours) at the Pittsburgh Meeting 1987,2,3 UW solution was introduced into routine clinical practice.4,5 Unfortunately, lack of organ preserving solutions hindered further progress in organ transplantation in China.6 In 1992 Changzheng Hospital (Shanghai, China) began developing a new organ preserving solution. After 10 years of concerted efforts, a modified UW solution designated as No. 1 Changzheng Organ Preserving Solution (CZ-1 solution) has passed the phase of animal experiments and is now undergoing clinical trials.
Organ preserving solution
CZ-1 solution contains per litre lactobionate acid 100 mmol, KH2PO4 25 mmol, MgSO4 5 mmol, adenosine 5 mmol, glutathione 3 mmol, dexamethasone 8 mg, allopurinol 1 mmol, NaOH 0.1 mmol, sucrose 60 mmol, dextran-40 50 g, verapamil 20 mg (pH 7.45±0.10) and osmotic pressure (310±10) mOsm/L. UW solution was present from the Organ Transplantation Centre of Wisconsin State University, USA.
HC-A solution: a modified Ross solution developed by Changzheng Hospital and Shanghai Blood Centre Station in 1982.7
Eur-Collins solution (EC solution): glucose 194 mmol, KH2PO4 15 mmol, K2HPO4 42 mmol, NaHCO3 10 mmol, KCl 15 mmol, 355 mOsm/L.
Healthy New Zealand male rabbits weighing 2.0–2.5 kg and male SD rats weighing 200–300 g were provided by the Research Institute of the Second Military Medical University (Shanghai, China).
Eighteen rabbit kidneys were divided into 3 groups and flushed with CZ-1 solution at 2°C - 4°C, with UW solution and HC-A solution at 0°C - 4°C respectively for 0, 24, 36, 48, 60, 64, 72 and 80 hours. Morphological changes were observed by electron and visible microscopy. The kidney mitochondrial respiration control rate, kidney Na+-K+-ATPase activity, kidney cortex chondriosome Ca2+, kidney cortex glutathione content and ATP content were measured. The rat kidneys were stored in cold CZ-1 solution and UW solution for 48 hours and 72 hours. After transplantation, the recovery of kidney function was observed.
Twelve rabbit livers were divided into 2 groups and flushed with CZ-1 and UW solution respectively at 0°C - 4°C for 0, 8, 12, 18, 24 and 48 hours. Morphological changes were observed by electronic and visible microscopy.
Thirty-six male SD rat livers were divided into 2 groups. Control livers were attached to the isolated perfusion circuit immediately. They were collected after cold storage. The reperfusion medium was Krebs-Henseleit bicarbonate solution (600 ml containing NaCl 118 mmol/L, KCl 4.7 mmol/L, KH2PO4 1.2 mmol/L and HEPES 20 mmol/L). A membrane oxygenator (Sci Med, USA) purged with O2:CO2 (95%:5%) was used to maintain pH 7.4±0.05 and PO2 >450 mmHg. The perfusate was recirculated and maintained at 37°C. After being flushed with cold UW or CZ-1 for 18, 24 or 30 hours, ATP content was tested; sinusoidal lining cell mortality (SLCM), Krebs-Henseleit buffer perfusate ketone body ratio (PKBR) and hepatic tissue water content (HTWC) were measured.
The efficacy of CZ-1 solution was evaluated by restoration of the transplanted liver to normal functioning within 24 hours after storage at low temperature.
Twelve rabbit hearts were divided into 2 groups and flushed with CZ-1 or UW at 0°C - 4°C for 0, 18, 24, 36 and 48 hours. Changes in morphology were observed by electron and visible microscopy.
The rabbit hearts were preserved cold for 0, 6, 12 and 18 hours and the myocardial tissue ATP content and myocardium mitochondrial Ca2+ biochemical indices were assessed.
The SD rat hearts were preserved in cold CZ-1 solution for 18 hours and 24 hours and the survival rates after allogeneic transplantation were recorded.
The rabbit lungs were divided into 2 groups and preserved in CZ-1 or UW at 4°C - 10°C for 0, 8,12, 24, 36 and 48 hours. Changes in morphology were observed.
The SD rat lungs used for triple cannulation transplantation8 were preserved in CZ-1 and Euro-Collins solution and then their arterial blood oxygen partial pressure, arterial blood carbon dioxide partial pressure and wet/dry weight ratios were compared.
Small intestine preservation
Eighteen SD rat small intestines were divided into 3 groups, flushed with CZ-1, UW or 50 ml HC-A, preserved at 2°C - 4°C for 0, 10, 18 and 24 hours after bleaching and then observed by visible and electron microscopy for morphology and intestinal mucous energy metabolism biochemical study.
The SD rat pancreas tissues were divided into 2 groups, flushed with UW or CZ-1 at 2°C-4°C, preserved at 0°C-4°C for 0, 24, 48 and 72 hours, and tested for 6-keto-PG1a and TXB2.
The testicles of five deceased donors (aged 24 to 30 years) were quickly dissected, immersed in CZ-1 solution at 2°C-4°C and then preserved at 0°C-4°C for 1 hour. Then, they were flushed with 50–100 ml CZ-1 solution through the spermatic artery, preserved in CZ-1 solution at 0°C-4°C for 12, 24, 36, 48 and 60 hours then observed for HE stained chromatins.
The ethics committees of the three hospitals approved the protocol from experimental study to clinical application. Every patient gave signed agreements of medical informed consent.
Twelve kidneys from 6 deceased donors (all males, aged from 22 to 29 years) were flushed in CZ-1 solution, preserved for 24–31 hours and then transplanted to patients. After operation, the patients’ renal function recovery was observed. The blood types of the donors were as follows: O in 3 donors, A in 2 and B in 1. The hot ischaemic time of the donor kidneys was 9 minutes and 8 seconds to 12 minutes and 35 seconds; warm ischaemic time was 1 hour and 21 minutes to 2 hours and 15 minutes; cold ischaemic time was 22 hours and 18 minutes to 29 hours and 3 minutes; and total ischaemic time was 23 hours and 56 minutes to 30 hours and 51 minutes. All 12 donees (7 males and 5 females, aged from 33 to 59 years, mean 41 years) suffered from chronic nephritic uraemia and had undergone 10 to 58 courses of haemodialysis before operation. Blood compatibility was: O:O in 6 cases, A:A in 3 cases, B:B in 2 cases and A:AB in one case.
All the data were expressed mean ± standard deviation (SD) and analyzed by the t test. All the statistical analyses were performed using SPSS 10.0. P value less than 0.05 was considered statistically significant.
Electron and visible microscopy showed that there was no significant difference in morphology between the kidney tissue preserved in CZ-1 solution for less than 72 hours at cold storage and that preserved in UW solution. The degeneration of tissue cells and organelles of the kidneys preserved in CZ-1 solution was milder than those preserved in UW solution.9
The biochemical indices, including kidney chondriosome respiratory control rate, kidney Na+-K+-ATPase activity and kidney cortex ATP content of the kidneys preserved in CZ-1 solution, were better than those of the kidneys preserved in UW solution (P <0.05, Table 1).
Six SD rat donor kidneys were preserved in UW solution and another 6 in CZ-1 solution for 48 hours of cold storage. After transplantation, 5 animals in each group survived for more than 7 days. There was no significant difference between UW group and CZ-1 group for 48 hours preservation. Of 8 donor kidneys preserved in UW solution for 72 hours, 4 survived after transplantation. Of 8 donor kidneys preserved in CZ-1 solution, 5 survived after transplantation. The surviving kidneys almost recovered to their normal function within 14 days. There was no significant difference between UW and CZ-1 groups for 72 hours of preservation.
Mitochondria, endoplasmic reticulum and cell nucleus in the livers preserved in CZ-1 solution for 30 hours remained almost normal. Rupture of sinus interstitial endothelial cells, swelling of epithelial cells and disengagement of sinus interstitial on liver cell walls were observed during preservation both in CZ-1 and UW groups.
When the cold storage time prolonged, the ATP content and KHB liquid inner ketone bodies rate (PKBR) of the rat liver tissue declined. After cold storage for 18–30 hours, there was no significant difference between the two groups. After preservation for 30 hours, significant difference was noticed between the two groups (P <0.05, Table 2).
Six SD rats underwent orthotopic liver transplantation after their donor livers were preserved in CZ-1 solution at cold storage for 24 hours. They all revived immediately after operation and 6 survived over 24 hours, 1 died after 24 hours, 1 died after 72 hours, and 4 survived over 1 week. The survival probability in UW solution was 5/6. There was no significant difference between the two groups.
Under the light microscope, no significant pathological changes were observed in muscular tissues during 18 hours of cold storage. After 24-hour storage, the cardiac muscle cells were found to have cloudy swelling with misshapen or broken muscle fibres and widened diastems. After 48-hour storage, scattered necroses were seen in myocardial cells. No significant histological difference was found between CZ-1 and UW groups. Under the electron microscope, mild to moderate injury to myocardial cells was observed after 18-hour storage. Ballooning degeneration was seen in mitochondria with crests disappearing. After 24-hour storage, myofibrils were broken and lysed and mitochondria became swollen and ruptured with liquefactive degeneration and pyknosis. The morphological changes in CZ-1 solution group were slighter than those in UW solution group during 18 hours cold storage.10,11
No statistically significant difference was found in ATP level between CZ-1 and UW solution groups. CZ-1 solution was more effective in preventing mitochondria calcium accumulation than UW solution (Table 3).
The survival rate of SD rats hearts preserved in UW solution for 18 hours and 24 hours was 4/6 and 2/6 respectively, while in CZ-1 solution it was 5/6 and 2/6. There was no significant difference.
After the lungs were preserved for 12 hours, the tissue structure remained unimpaired. When they were preserved for 18–24 hours, it became moderately oedematous, with freckled bleeding, moderate congestion and focal infarction in capillaries. Endothelial cellular swelling, hump like spikes and a small number of hyaline membranes were formed. Obvious swelling alveolus pulmonis II cells with local necrosis or obvious vacuolization and destroyed alveolar septa fabric were observed under the electron microscope. There was no significant difference between the two groups.
Postoperation indices of CZ-1 group (arterial oxygen partial pressure, carbon dioxide partial pressure and wet/dry weight rate of the lungs) were better than those of EC group. The lungs preserved in cold CZ-1 for 8 hours had essentially normal function.8,12
The intestinal mucosa in HC-A solution was injured more severely than that in UW solution and CZ-1 solution. There was no significant difference between CZ-1 and UW groups. As the cold storage time was prolonged, ATP and TNA (ATP+ADP+AMP) content of the intestinal mucosa decreased gradually, while the content of ATP and TNA preserved in HC-A for 24 hours was significantly lower than that in UW and CZ-1 solutions. The ATP and TAN contents of the intestinal mucosa in CZ-1 group was higher than that in UW group (P <0.05).
After the pancreases were preserved for 48 hours, the tissue structure remains unimpaired. Only mild to moderate oedema was seen in acinus glandular epithelial cells. By electron microscopy, swollen mitochondria, thickened and fused ridges, changed vacuoles, expanding endoplasmic and Golgi compounds, rough endoplasmic shelling, polynucleotide depolymerization and chromation periphery were observed. After 72 hours preservation, swelling and denaturation were observed in pancreas acinus glandular epithelial cells and the acinus structure became indistinct and disappeared partly. Mitochondrial ridges could not be seen under the electron microscope. There was no significant difference between CZ-1 and UW groups. There was also no significant difference in 6-keto-PG1a values of pancreas tissue and preservation period between the two groups. With the preservation time prolonged, the TXB2 value of pancreas tissue increased dramatically. After 72 hours, the TXB2 value of pancreas tissue in CZ-1 was lower than that in UW (P <0.05).
The testicles were flushed with CZ-1 and preserved for 48 hours in cold storage. The testicles convolute seminiferous epithelia was lined up in order. Sperm was found with mesenchyme oedema. By 48–60 hours, the swollen testicles convoluted seminiferous tubule epithelium had disordered arrangement; thickened nuclear membrane and the formation of intranuclear vacuoles were clearly observed. After 60 hours, convoluted seminiferous tubule epithelial cells were disrupted and necrosed.
Renal function recovery of 12 kidneys preserved in cold CZ-1 solution for 24–31 hours after transplantation ranged from 2 days to13 days (mean 3.8 days). Mean urine time after blood supply was 1–3 minutes; donee's urine on the first day was 3.7–21.4 L (mean 11.2 L).
UW solution was developed to provide an appropriate environment for organs during simple cold storage. An appropriate physical environment is composed of lactobionate, raffinose, hydroxyethylstarch, electrolytes and a hydrogen ion buffer (phosphate) that facilitates flushing out of the organ and suppress hypothermia induced cellular swelling. The biochemical environment for facilitating rapid restoration of normal metabolic functions after preservation is obtained with adenosine and phosphate to stimulate ATP regeneration on reperfusion, glutathione as an antioxidant, allopurinol as an inhibitor of oxygen free radical generation from xanthine oxidase and steroids for membrane stability and inhibition of phospholipases.
CZ-1 preserving solution was developed based on UW formulation and the latest development of organ preservation.13,14 The modifications were replacing hydroxyethylstarch with dextran-40 (relative molecule mass of 40 000), replacing raffinose with sucrose, removing heparin and penicillin, adding Ca2+ blocker, verapamil, and regulating pH value to 7.45±0.10.
The purpose of these changes was to reduce the cost, enhance the preserving property and meet the needs of organ graft in China.
The results of our comparative study showed that there was no significant difference between CZ-1 and UW solutions in preserving the kidneys, liver and heart. Some researchers believed that the fatal damage in preservation of the liver was the damage to microcirculation.15 Reversible damage due to breaking of endothelial cells and swelling of epithelial cells was observed, but with prolonged storage the damage became irreversible. We also observed the same phenomenon as that observed by Lamesch et al,15 who found that change of the sinus space was less severe with CZ-1 than that with UW.
Biochemical changes are the key indications for judging the survival rate of a transplanted organ. This paper reports the biochemical changes of the kidneys, the liver and the heart after preservation in CZ-1 solution. Mitochondria plays an important role in maintaining the energy supply of the kidneys, liver and heart. Preservation decreases the capability of mitochondria in carrying out oxidative phosphorylation. Recent studies suggest that this decrease is partly due to the effect of Ca2+ on cellular and mitochondrial metabolism.16,17 In our experiments of preserving rabbit hearts in cold storage for 0, 6, 12 and 18 hours, we observed that there was no significant difference in ATP content in the cardiac muscle between the two groups (P >0.05). However, ATP content of CZ-1 group dropped more slowly and its myocardial Ca2+ overload was less than that of UW group. CZ-1 is superior to UW solution because the presence of Ca2+ blocker may improve the microcirculation of the kidneys. Organ preservation requires not only preservation of parenchymal tissue but also the vascular system. Continuous perfusion of the kidneys for three days has little effect on the vascular system, as shown in a series of scanning electron micrographs.18 The kidneys preserved for this period were fully viable. Perfusion for 7 days caused severe disruption of the vascular system and these kidneys were no longer viable. Simple cold storage, however, does not directly cause vascular damage. The state of the vascular system following transplantation is unknown. It is at this point that vascular damage may become apparent and limit the viability of the graft.
The integrity of the organelle structure, especially the mitochondria, is the base for oxidization and phosphorylation. Another reason why CZ-1 is superior is that we replaced hydroxyethylstarch with dextran-40. Serum albumin has been a common perfusate colloid but has been shown to cause endothelial injury.19 The Wisconsin group found that hydroxyethyl starch is a more effective colloid than serum albumin and better for kidney preservation.20 The presence of a colloid may also facilitate initial flushing out of the organ and removal of cellular components of blood as well as enhance the distribution of the preserving solution throughout the organ. Recently, Jacobsson et al21 reported significant differences in weight changes and tissue oedema in kidney preservation in a rat model, when UW solution with hydroxyethylstarch and without it were compared. They concluded that the presence of colloid improved the rheological properties of UW solution and contributed to a reduction in cellular swelling. This is in contrast to the results produced by Biguzas et al,22 who were able to obtain 48-hour cold storage kidney preservation in a rat model with UW solution; however, no beneficial effect of hydroxyethylstarch was seen in kidney preservation. Dextran-40 can improve microcirculation and reduce the accumulation of toxic products.
Under hypothermic conditions, the ionic pumps in the plasma membrane are suppressed, resulting in a loss of the membrane potential.23 This leads to cellular swelling. Some changes in membranes permeability are Ca2+ dependent and affect cellular ATP metabolism. Reperfusion injury is related to the production of oxygen free radicals and Ca2+ metabolism. An increased concentration of cellular Ca2+ has been associated with cell death.24 The reperfusion during transplantation could increase the Ca concentration. Fortunately, Ca2+ channel blockers (Verapamil) reduce organ damage during preservation.25
The antioxidant glutathione can prevent proteins containing hydrosulfuric group from being oxidized; however, cold storage cannot protect glutathione in renal cortex, so glutathione is added in both CZ-1 and UW solutions.
The ATP content, SLCM, PKBR and HTWC were assessed in the rat liver tissue. Liver cells have low membranous permeability and trypan blue test is negative. Therefore, the dye rate of the sinus hepaticus endothelioid can accurately reflect the SLCM. After transplantation and recovery of blood supply, complete oxidizing and phosphorylating function of the chondriosome, hepaticus is the precondition for survival. PKBR represents the AcAc and β-LHB ratio released into the KHB after oxygen supply, reflecting the NAD+/NADH ratio in the chondriosome of liver cells. The results of our study demonstrated that PKBR declined in all 18, 24 and 30 hours groups preserved by CZ-1 and UW, indicating that CZ-1 and UW solutions are similar in maintaining the oxidizing and phosphorylating functions. The ATP content, SLCM, PKBR and HTWC suggest that CZ-1 solution is more effective than UW solution. This is the reason why we add Ca2+ blocker.
Another important requirement for successful preservation is to prevent cellular acidosis. Tissue acidosis will kill cells and can induce lysosomal instability, activate lysosomal hydrolases and alter mitochondrial properties.26 Therefore, preserving solution always includes a pH buffer. Increasing the buffering capacity of a preserving solution could improve renal function after preservation. In addition, storage of the liver and pancreases has been improved by flushing with a solution that has an alkaline pH 7.9 to 8.3.25,26 Flushing an organ with a solution having a high pH may retard the rate at which the pH of tissue is decreased during cold storage. CZ-1 solution, whose pH value is adjusted to 7.45±0.10, would prove to be helpful for organ preservation.
The survival study on delayed preservation of rat kidneys, liver, heart and lungs showed that CZ-1 could safely preserve kidneys for 72 hours, livers for 24 hours, hearts for 18 hours and lungs for 8 hours.
Twelve kidneys preserved in CZ-1 solution under cold storage for 22–31 hours were transplanted successfully and the mean renal function recovery time was (3.83±1.68) days. It is reported that that the mean renal function recovery time is 6.7 days with HC-A solution. In clinical practice, it is not possible to transplant organs after purposely preserving them for 72 hours. It is reported in this study that the longest preservation time is 31 hours. It is enough to prove the preservation effect of CZ-1 on cadaveric kidneys.27
The results of our study confirmed that the effect of CZ-1 solution is similar to that of UW solution in terms of cold preservation of the kidneys, liver, heart, pancreas and small intestine, and in certain aspects, it is superior to UW solution. For lung preservation, it is better than EC solution. Moreover, the cost of CZ-1 solution is much lower than that of UW solution.
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