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The technology of split liver transplantation is becoming increasingly mature with rapid development. During ex vivo splitting, the depletion of intracellular energy sources [such as adenosine triphosphate (ATP)] and other metabolic disorders may lead to cell damage and dysfunction, which will be aggravated by reperfusion injury of liver transplantation, clinically manifested as postoperative complications and transplantation failure. To further improve the quality of donor liver in ex vivo split liver transplantation, research teams at home and abroad apply machine perfusion to enhance the quality of donor liver. In this article, the research progresses worldwide on machine perfusion of donor liver in ex vivo split liver transplantation were reviewed, and the application and prospect of dual hypothermic oxygenated machine perfusion (D-HOPE) in ex vivo split liver transplantation were elucidated, aiming to provide reference for increasing the source of donor liver for ex vivo split liver transplantation and further resolving the current status of donorliver shortage.
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@#Objective To investigate the effects of triptolide (T10) on biological activity of sciatic nerve in cold preservation and nerve regeneration after allogeneic transplantation. Methods Cell Counting Kit-8 (CCK-8) was used to test the proliferation of SCs in logarithmic phase in 1×10-6 mol/L, 1×10-7 mol/L, 1×10-8 mol/L and 1×10-9 mol/L of T10 solution. The sciatic nerves from Sprague-Dawley rats were pretreated in 0 mol/L, 1×10-6 mol/L, 1×10-7 mol/L, 1×10-8 mol/L and 1×10-9 mol/L of T10 solution at 4 ℃ or 37 ℃ for 24 hours (n = 6). The expression of nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) was detected with Western blotting. Other sciatic nerve fragments were randomly divided into fresh nerve group (group A, n = 30), DMEM preservation group (group B, n = 30), T10 preservation group (group C, n = 30), T10 pretreatment DMEM preservation group (group D, n = 30) and T10 pretreatment T10 preservation (group E, n = 30), and were stored under 4 ℃ for four weeks. Calcein-AM/PI double staining laser confocal microscope and flow cytometry were used to detect the living cells and dead cells. The expression of the major histocompatibility complex (MHC)-I, MHC-II and intercellular cell adhesion molecule-1 (ICAM-1) was detected with Western blotting. The corresponding sciatic nerves were used to repaire 10 mm defects in Wistar rats (named groups A', B', C', D' and E'), and fresh sciatic nerve from Wistar rats were also used to do it (group F'). Compound muscle action potential (CMAP) and motor nerve conduction velocity (MNCV) were tested 16 weeks after transplantation, and then the grafts were observed for the nerve regeneration. Results SCs proliferated as the controls in the T10 solution with a concentration of 1×10-9 to 1×10-7 mol/L (P > 0.05). The expression of all the neurotrophic factors was more under 37 ℃ than under 4 ℃ in all the concentrations of T10 solution, and it was the most in the concentration of 1×10-8 mol/L whenever under 37 ℃ or 4 ℃ (P < 0.05). After four weeks of cold preservation, compared with groups B, C and D, the living nerve cells were the most in group E, and the expression of MHC-I, MHC-II and ICAM-1 was the least (P < 0.05). CMAP, MNCV and the never regeneration were better in group E' than in groups A', B', C' and D' (P < 0.05). A large number of myelinated nerve fibers were observed in groups E' and F', uniformity in size, wide distribution, and with myelin sheath, compared with those in groups A', B', C' and D'. Conclusion A certain concentration of T10 can induce the sciatic nerve of rats to express neurotrophic factor in vitro, which can improve the biological activity of cold preservation nerves, reduce the immunogenicity, and promote the regeneration of recipient nerve after allogeneic transplantation. It is even better to be pretreated with T10 before cold preservation.
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INTRODUCCIÓN: La adecuada preservación del aloinjerto previo al trasplante renal es crucial para mantener buenos resultados luego del trasplante. En la actualidad existen dos métodos principales, la perfusión hipotérmica asistida por una máquina y la preservación en frío estático. El objetivo principal de este resumen es comparar ambos sistemas de preservación. MÉTODOS: Realizamos una búsqueda en Epistemonikos, la mayor base de datos de revisiones sistemáticas en salud, la cual es mantenida mediante el cribado de múltiples fuentes de información, incluyendo MEDLINE, EMBASE, Cochrane, entre otras. Extrajimos los datos desde las revisiones identificadas, analizamos los datos de los estudios primarios, realizamos un metanálisis y preparamos una tabla de resumen de los resultados utilizando el método GRADE. RESULTADOS Y CONCLUSIONES: Identificamos 10 revisiones sistemáticas que en conjunto incluyeron 34 estudios primarios, de los cuales 13 corresponden a ensayos aleatorizados. Concluimos que la preservación mediante perfusión hipotérmica de máquina probablemente disminuye el riesgo de retraso en el funcionamiento del injerto y podría llevar a un leve aumento en la sobrevida del injerto. Sin embargo, no existen diferencias en la sobrevida del paciente entre ambos métodos.
INTRODUCTION: The adequate preservation of the allograft prior to kidney transplant is key for a good outcome after transplantation. Currently, there are two main methods: hypothermic machine perfusion and static cold preservation. The main objective of this summary is to compare both preservation systems. METHODS: We searched in Epistemonikos, the largest database of systematic reviews in health, which is maintained by screening multiple information sources, including MEDLINE, EMBASE, Cochrane, among others. We extracted data from the systematic reviews, reanalyzed data of primary studies, conducted a meta-analysis and generated a summary of findings table using the GRADE approach. RESULTS AND CONCLUSIONS: We identified 10 systematic reviews including 34 primary studies, of which 13 were randomized trials. We concluded preservation by hypothermic machine perfusion probably decreases the risk of delayed graft function and could lead to a slight increase in graft survival. However, there are no differences in patient survival between the two methods.
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Humans , Organ Preservation/methods , Kidney Transplantation/methods , Delayed Graft Function/prevention & control , Perfusion/methods , Randomized Controlled Trials as Topic , Databases, Factual , Cold Temperature , Graft SurvivalABSTRACT
Objective: To investigate the feasibility of repairing nerve defect of sciatic nerve in rats by the pretreatment of cold preservation with tetramethylpyrazine. Methods: Forty Wistar rats and 40 SD rats were selected as donors and recipients, respectively [3 months, SPF male rat (200 ± 20) g], the sciatic nerve segments of 15 mm were harvested from the donors, then randomly preserved in 4℃ tetramethylpyrazine solution (0, 50, 100, and 200 mg/L, in A, B, C, and D groups, n = 20) for 6 weeks, the ultrastructure of sciatic nerve was observed by transmission electron microscope (TEM). Calcein-AM staining was used to observe the change of fluorescence intensity with laser scan confocal microscope (LSCM). The recipients were randomly divided into A′, B′, C′, and D′ groups (n = 10, corresponding to A, B, C, and D groups), 10 mm defect of sciatic nerve in recipient was repaired by the nerve preserved for 6 weeks. The gross appearance and sciatic nerve function index (SFI) were detected at different period after the surgery. The electrophysiological changes, regenerated nerve image analysis, and TEM were performed in 20 weeks after transplantation. Results: The nerve preserved for 6 weeks, there were severe demyelination and neuraxis denaturation in group A, but these changes in groups B, C, and D were more gentle than those in the group A. The fluorescence intensity in the groups B, C, and D was higher than that in group A, and in group B higher than that in groups C and D (P 0.05). All the rats survived to the end of the experiment. There were significant differences (P 0.05) in different times of SFI after transplantation, electrophysiological detection, the number of myelinated nerve fibers, and thickness of myelin at 20 weeks, The observation of TEM in 20 weeks showed that myelinated nerve fibers and thickness of myelin sheath in group A′ was thinner than those in groups B′, C′, and D′, and that the hyperplasia of connective tissues in group A′ was more serious than that in groups B′, C′, and D′. Conclusion: Tetramethylpyrazine plays a protective role on the cold preservation of sciatic nerve, and could promote the nerve regeneration after allogeneic transplantation.
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Objective To discuss the protective effect of creatine phosphate (CP)on isolated rat liver against cold preservation.Methods Isolated perfused rat liver model under simple cold preservation was established.The liver of the control group was perfused with pure University of Wisconsin (UW)solution. With UW solution as the base fluid,the liver of the low-dose group was perfused with 1 g/100 ml CP in UW solution;the liver of the middle-dose group was perfused with 2 g/100 ml CP in UW solution;the liver of the high-dose group was perfused with 3 g/100 ml CP in UW solution.The livers of each group were cold preserved in the corresponding perfusion fluid at 4 ℃.The content of alanine aminotransferase (ALT)and lactate dehydrogenase (LDH)in preservation solution in infrahepatic vena cava were determined.The content of malondialdehyde (MDA)and activity of myeloperoxidase (MPO)in liver tissues were detected.The apoptosis index (AI)of liver cells in liver tissues and positive expression rate of NF-κB in liver tissues were observed. Pathologic changes of liver tissues were observed under optical microscope.Results At 12 h after the cold preservation,the content of ALT and LDH in the rat livers of low-,middle-and high-dose groups were lower than those of the control group (all in P <0.05).At 18 h after the cold preservation,the content of MDA and MPO in the liver tissues of low-,middle-and high-dose groups were lower than those of the control group (all in P <0.05).At 12 h and 18 h after the cold preservation,AI and positive expression rate of NF-κB in liver cells in the rat livers of low-,middle-and high-dose groups were lower than those of the control group (all in P<0.05).At 24 h after the cold preservation,the content of ALT and MDA in preservation solution of the high-dose group was obviously higher than that of the control group as well as the low-and middle-dose groups (all in P <0.05).The results of pathological examination indicated that the injuries to the livers of the high-,middle-and low-dose groups were obviously lighter than that of the control group.There was no obvious difference among each dose group.Conclusions CP in UW solution may well protect the isolated rat liver against cold preservation,which is better than pure UW solution.
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Objective To investigate the effects of cold preservation on the expression of GATA in intrahepatic bile duct.Methods The intrahepatic bile duct tissues of SD rats were obtained by collagenase perfusion combined with mechanical separation.After being cut into fragments,the intrahepatic bile duct tissues were cultured in rat tail collagen gel for 48 hours before experiment.All the rats were divided into the control group,cold preservation 1 hour (CP1 h) group and cold preservation 12 hours (CP12 h) group.There were 5 rats in each group.The mRNA and protein expressions of GATA were detected by Real-Time polymerase chain reaction and Western blot.Measurement data with normal distribution were presented as (x) ± s.Comparison among 3 groups was done by ANOVA and pairwise comparisons were done by LSD test.Results The mRNA expressions of GATA3,GATA4,GATA6 were detected,while the mRNA expressions of GATA1,GATA2 and GATA5 were undetectable in intrahepatic bile duct tissue of the control group.The mRNA expressions of GATA4 in the CP1 h group,CP12 h group and the control group were 0.72 ± 0.08,0.56 ± 0.07 and 0.96 ± 0.06,with significant difference among the 3 groups (F =38.981,P ＜0.05).The mRNA expression of GATA4 in the CP12 h group was significantly lower than that in the CP1 h group and the control group,and the mRNA expression of GATA4 in the CP1 h group was significantly lower than that in the control group (P ＜ 0.05).The mRNA expression of GATA6 in the CP1 h group,CP12 h group and the control group were 0.83 ± 0.07,0.68 ± 0.12 and 0.98 ± 0.12,with significant difference among the 3 groups (F =10.175,P ＜ 0.05).The mRNA expression of GATA6 in the CP12 group was significantly lower than that in the CP1 h group and the control group,and the mRNA expression of GATA6 in the CP1 h group was significantly lower than that in the control group (P ＜ 0.05).The mRNA expressions of GATA3 in the CP1 h group,CP12 h group and the control group were 0.92 ± 0.06,0.89 ± 0.05 and 0.98 ± 0.11,with no significant difference among the 3 groups (F =1.674,P ＞ 0.05).The protein expressions of GATA4 in the CP1 h group,CP12 h group and the control group were 0.78 ± 0.07,0.64 ± 0.06 and 0.99 ± 0.10,with significant difference among the 3 groups (F =24.211,P ＜ 0.05).The protein expression of GATA4 in the CP12 h group was significantly lower than that in the CP1 h group and the control group,and the protein expression of GATA4 in the CP1 h group was significantly lower than that in the control group (P ＜ 0.05).The protein expressions of GATA6 in the CP1 h group,CP12 h group and the control group were 0.90 ± 0.04,0.75 ±0.06 and 0.98 ±0.11,with significant difference among the 3 groups (F=11.651,P＜0.05).The protein expression of GATA6 in the CP12 h group was significantly lower than that in the CP1 h group and the control group (P ＜ 0.05).Conclusion The expressions of GATA4 and GATA6 in the intrahepatic bile duct tissues are decreased significantly after cold preservation,which indicate that GATA4 and GATA6 might be involved in the pathophysiological process of the bile duct after cold preservation.
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Objective To develop rat models of graft cholangiopathies and evaluated their values.Methods Four groups were constructed.The long-term cold preservation group(n=24) contained homogeneic inbred rat liver orthotopic transplantations (ROLT) performed in a rat combination of ♂ Wistar→ ♂ Wistar with the donor liver preserved in 4℃ UW for 12 h,the vessels reconstructed by the two-cuff method,and the hepatic artery and extrahepatic bile duct rebuilt by a stent.The chronic rejection group (n=24)(CsA 1 mg · kg-1 · d-1,cold preserved for 1 h) was allogeneic inbred ♂ DA→♂ Lewis rats induced for ROLT,and the revascularization methods were the same as the longterm cold preservation group.The control group (n=24) (cold preserved for 1 h) was homogeneic inbred ♂ Wistar→♂ Wistar rats with ROLT techniques the same as above.The sham group (n=24)was ♂ Wistar rats that had an exploratory laparotomy.The animals were followed for 16 weeks,complications were compared,and liver tissues were harvested.Histopathological and morphometric techniques were used to construct a time course of histological changes after liver transplantation.Results In both the long-term cold preservation group and chronic rejection group,the rats recovered slowly,the incidence of complications and mortality were higher than those of other groups,and the intrahepatic bile duct proliferation and immune cell infiltration were noticeable after the operation.In 16 weeks,the hepatic lobules were separated by the proliferating bile ducts,the normal structure of hepatic lobules disappeared,many biliary epithelial cells necrosed with disappearing cytoplasms,and there was immune cell infiltration and obliterative arteriopathy.Conclusions The rat models of graft cholangiopathies induced by long-term cold preservation or chronic rejection donor livers are stable and easily standardized.This model is ideal for studying the pathogenesis and prevention of graft cholangiopathies.
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Objective: To observe the morphological changes of cold-stored rat kidneys flushed with multi-organ preservation solution containing ketamine. Methods: The kidneys of male SD rats were retrogradely perfused with 0-4°C HC-A (50-100 ml) only or together with ketamine (10-5 μmol/L), and were then isolated, cold stored for 24, 48, and 72 h. The morphological changes of the kidneys in both groups were observed under light microscope and transmission electron microscope. Results: Light microscopy and transmission electron microscopy showed that there were evident morphological changes in the ketamine group after 72 h, with necrotic cells and organelles, and severe renal interstitial edema. The above changes in the HC-2 group were slighter. Conclusion: Ketamine in the organ preservation solution is harmful for the cold storage of rat kidneys.
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Objective To evaluate the efficacy of liver grafts with warm ischemia and with different cold preservation time in liver transplantation.Methods The clinical data of 154 patients who received liver transplantation at the First Affiliated Hospital of Sun Yat-sen University from January 2006 to December 2007 were retrospectively analyzed.The warm ischemia time of the liver grafts obtained from the non-heart-beating donors was within 10 minutes.According to cold perservation time of the liver grafts,patients were divided into 3 groups:the cold preservation time of the liver grafts was within 8 hours,8-12 hours and above 12 hours in group I(n=58),group Ⅱ(n=62)and group Ⅲ(n=34),respectively.The peak level of alanine aminotransferase(ALT),primary graft dysfunction(PGD)after liver transplantation,acute rejection response,biliary complications,vessel complications,perioperative infections and the survival of liver grafts and recipients among the 3 groups were analyzed via chi-square test,t test and variance analysis.Results No PGD was detected in the 3 groups after liver transplantation.All patients were followed up for 8-32 months.The peak level of ALT,incidence of infection and biliary complication,survival of liver grafts and recipients were(482±357)U/L,12%(7/58),12%(7/58),86%(50/58)and 88%(51/58)in group Ⅰ,and were(1274±608)U/L,29%(10/34),26%(9/34),68%(23/34)and 71%(24/34)in group Ⅲ,with significant difference between the 2 groups(t=5.23,X~2=4.28,6.77,4.51,4.28,P<0.05).The peak level of ALT in group Ⅱ was(953±424)U/L,which was significant higher than(482±357)U/L in group Ⅰ(t=4.76,P＜0.05).Conclusions Liver grafts with a warm ischemia time shorter than 10 minutes could tolerate the injury caused by cold preservation with the maximum time of 12 hours.The incidences of biliary complications and postoperative infections are significantly increased and the survivals of liver grafts and recipients are decreased when the cold preservation time exceeds 12 hours.
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Objective To investigate the expression of Muc1,Muc3A and Muc4 in cultured intrahepatic bile duct(IBD)tissues from different hepatic segments after cold preservation.Methods The IBD tissues of SD rats were obtained by collagen perfusion combining mechanical separation and then were divided into large and small IBD.The 2 parts of IBD were seeded in rat tail collagen gel and were cultured for 48 hours,then the IBD tissues from 10 rats were stored in UW solution at 4℃ for 1 hour(group I,n=5)and 12 hours(group Ⅱ,n=5),respectively,and the IBD tissues from the rest 5 rats were cultured in incubator at 37℃ for 24 hours (control group,n=5).The expressions of Muc1,Muc3A and Muc4 were detected by RT-PCR and Western blot.All data were analysed via ANOVA or LSD test.Results The expressions of Muc1,Muc3 A and Muc4 were detected both in large and small IBD tissues.The mRNA expressions of Muc1,Muc3A and Muc4 were decreased in large IBD as time passed by,which were 0.95±0.14,0.26±0.04 and 0.24±0.06 in group Ⅰ,0.18±0.03,0.14±0.04 and 0.22±0.07 in group Ⅱ,1.00±0.20,1.00±0.09 and 1.00±0.21 in control group,with significant difference among the 3 groups(F=8.8,57.1,10.8,P＜0.05).The mRNA expressions of Muc1 and Muc3A in group Ⅱ were significantly lower than in group Ⅰ(P＜0.05).The protein expressions of Muc1 and Mue3A in large IBD were also decreased as time passed by,which were 0.82±0.13,0.73±0.10 in group Ⅰ,0.56±0.11,0.33±0.04 in group Ⅱ,1.05±0.41,1.06±0.38 in control group,with significant difference among the 3 groups(F=3.9,12.6,P＜0.05).The protein expression of Muc1 of group Ⅱ was significantly lower than in control group(P＜0.05),and the protein expression of Muc4 in group Ⅱ was significantly lower than in group Ⅰ(P＜0.05).The mRNA expressions of Muc3A in small IBD were increased as time passed by,which were 0.15±0.04 in group Ⅰ,0.19±0.05 in group Ⅱ and 0.06±0.03 in control group.Conclusion Decreases of Muc1,Muc3A and Muc4 in IBD after long time cold preservation may weaken the selfprotection of biliary epithelium and case sever injury to bile duct.
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Introdução: A preservação a frio geralmente é utilizada para minimizar a injúria renal durante a preservação. Testamos se a adição de sinvastatina, captopril, e ramipril à solução de Euro-Collins (EC) ou o pré-tratamento de rins de ratos doadores com ramipril e/ou sinvastatina poderia reduzir a injúria da preservação renal. Métodos: Inicialmente, adicionamos ramipril e sinvastatina à solução de EC, contendo fragmentos de rins de ratos que foram preservados a frio por 24 horas (n=6). O dano tecidual foi avaliado pela liberação de desidrogenase lática (DHL). In vivo, ramipril, por gavagem, e/ou sinvastatina, intraperitonealmente, foram administrados aos ratos doadores 18h antes da nefrectomia.Fragmentos renais foram estocados a frio em EC durante 24h ou 48h. O dano tecidual foi avaliado, utilizando a liberação de DHL, substâncias reativas ao ácido tiobarbitúrico (TBARS), teste do MTT e pelo exame morfológico após 0, 24, e 48h (n=10). Concomitantemente, captopril foi acrescentado à solução de EC e fragmentos adicionais dos rins de ratos controles foram estocados a frio durante 24h e 48h, com dano tecidual avaliado por liberação de DHL e exame histológico. Resultados: A adição de ramipril e sinvastatina à solução de EC não afetou a viabilidade dos fragmentos renais (p>0,05). A adução de captopril na solução de EC também não melhorou a viabilidade durante a preservação a frio, avaliada pela liberação de desidrogenase lática e pelo escore histológico (p>0,05). O pré-tratamento dos doadores renais com ramipril e/ou sinvastatina não alterou significativamente o MTT, MDA, DHL ou escores histológicos. Discussão: Inibidores da ECA e estatinas protegem contra a injúria da isquemia-reperfusão (I/R) após pré-tratamento por alguns dias e reduzem o estresse oxidativo e marcadores inflamatórios e poderiam melhorar a capacidade antioxidante da solução de EC...
Background: Cold storage is generally used to minimize kidney injury during preservation. We tested whether or not addition of simvastatin, captopril, and ramipril in the Euro-Collins solution (EC) or pre-treatment of rat kidney donors with ramipril and/or simvastatin reduced preserved kidney injury.Methods: We first added ramipril and simvastatin to EC with rat kidney fragments that were cold-stored for 24 hours (n=6). Tissue damage was assessed by lactate dehydrogenase (LDH). In vivo, ramipril, by gavage, and/or intraperitoneal simvastatin , were administered to donor rats 18h before nephrectomy. Kidney fragments were cold-stored in EC for 24h or 48h. Tissue damage was assessed by LDH release, TBARS, MTT-assay, and by morphologic assessment at 0, 24, and 48h (n=10). Concomitantly, captopril was added to EC and additional fragments of control rat kidneys were coldstored for 24 and 48h, with damage assessed by LDH release and histology. Results: The addition of ramipril and simvastatin to EC solution did not change the viability of rat kidney fragments (p>0.05). The addition of captopril in the EC solution also did not improve cold-storage viability, as assessed by LDH release levels and histological scores (p>0.05). Pre-treatment of kidney donors with ramipril and/or simvastatin did not significantly change MTT, MDA, LDH levels or histological scores.Discussion: ACEIs and statins protect against organ I/R injury after donor pre-treatment for a few days and reduce oxidant stress and inflammatory markers, and could improve the antioxidant capacity of EC solution. In the tested concentrations, inclusion of captopril, ramipril, and simvastatin in the EC solution did not improve the preservation quality of cold-stored rat kidney fragments...
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Animals , Antihypertensive Agents/analysis , Ischemia/therapy , Rats , SimvastatinABSTRACT
Objective To establish a model of rat orthotopic liver transplantation and investigate the relationship among cold preservation time, activation of nuclear transcription factor-κB (NF-κB) and donor preservation injury after liver transplantation. Methods Orthotopic liver transplantation was performed in Wistar rats which were randomly divided into the following groups according to the different duration of liver cold storage in UW solution: group A (sham operation, n=10), group B NF-κB in liver before and after transplantation was measured by electrophoretic mobility shift assays; protein expression of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) in the liver was measured by immunohistochemistry; the serum TNFα and IL-1β, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and hepatic cell apoptosis were examined. Results With extended cold storage duration, the activity of NF-κB in the donor liver increased (P<0.05, group D vs. groups A, B and C). TNF-α and IL-1β levels also increased (P<0.05, group D vs. groups A, B and C). Donor liver reperfusion injury was gradually aggravated with the prolonging of graft cold preservation. Both the serum TNF-α and IL-1β levels increased highly (P<0.05 group A vs. groups B, C and D),NF-κB in the liver significantly increased (P<0.05, group A vs. groups D, B and C) with gradual prolonging of graft cold preservation time. The serum ALT and AST level and apoptosis index level elevated greatly (P<0.05, group A vs. groups D, B and C). Conclusion NF-κB of donor liver was activated inductively in cold preservation phase and activated explosively in reperfusion phase. The longer cold preservation time was, the higher NF-κB level in donor liver became. NF-κB led to the expression of TNF-α and IL-1β in donor liver. Inflammatory factors are one of the most important mechanisms for the donor liver injury after liver transplantation.
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PURPOSE: The effect of intra-portal infusion of glucose-insulin-potassium (GIK) solution on the energy metabolism during cold preservation was investigated using a small-animal liver transplantation model. METHODS: Fifteen white rats were divided into 3 groups: the group A (feeding group) were fed normally before experiment. The group B (fasting group) and group C (GIK group) were fasted from 3 days before experiment, by which acute nutritional deficiency state was induced. In group A and B, the whole liver was procured after intra-portal perfusion of HTK solution and serial liver biopsies were performed during the cold preservation period with 4degrees C HTK solution. In group C, intra-portal GIK solution infusion for 1 hour preceded liver graft harvest. From the liver tissues, the relative intracellular glycogen contents and the ATP concentration were measured. RESULTS: Relative glycogen contents in group A were 100% at 0 h, 64.6% at 2 h, 54.9% at 4 h, and 16.2% at 8 h; 10.3%, 8.3%, 4.9% and 0%, respectively in group B; 109.2%, 96.9%, 54.2% and 9.7%, respectively in group C. There was a temporary supercharge of ATP level in group C only at 0 h. Apoptosis was less expressed in group C comparing with group A and B. CONCLUSION: Rapid intra- portal infusion of GIK solution could make intrahepatic glycogen content fully restored to the normal level. Considering that intracellular glycogen is the main energy source during immediate post-transplant period, its restoration may contribute to improvement of post-transplant graft function.
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Animals , Humans , Rats , Adenosine Triphosphate , Apoptosis , Biopsy , Cold Temperature , Energy Metabolism , Glucose , Glycogen , Insulin , Liver , Liver Transplantation , Malnutrition , Mannitol , Perfusion , Potassium , Potassium Chloride , Procaine , TransplantsABSTRACT
The purpose of this study was to examine the viability of PDL cells in rat molars by using in vivo MTT assay, which was used to compare fast cryopreservation group by liquid nitrogen (-196degrees C) with 4degrees C cold preservation group. A total of 74 Sprague-Dawley white female rats of 4 week-old with a body weight of 100 grams were used. The maxillary left and right, first and second molars were extracted as atraumatically as possible under ketamine anesthesia. Ten teeth of each group were divided as six experimental groups depending upon the preservation. Cryopreservation groups were Group 1 (5% DMSO 6% HES in F medium), Group 2 (10% DMSO in F medium), Group 3 (5% DMSO 6% HES in Viaspan(R)), Group 4 (10% DMSO in Viaspan(R)) which were cryopreserved for 1 week and cold preservation groups were Group 5 (F medium), Group 6 (Viaspan(R)) at 4degrees C for 1 week. Immediate extraction group was used as a control. After preservation and thawing, the in vivo MTT assay was processed. Two way ANOVA and Duncan's Multiple Range Test was performed at the 95% level of confidence. Another 2 teeth of each group were treated as the same manner and frozen sections 10 microm thick for microscopic observation. The value of optical density obtained after in vivo MTT analysis was divided by the value of eosin staining for tissue volume standardization. Group 1, 2 had significantly higher optical density than Group 3 and 4 which had the lowest OD value. Group 6 had higher OD value than in Group 5 (P < 0.05). Histological findings of periodontal ligament cell, after being stained with MTT solution were consistent with the in vivo MTT assay results. In this study, the groups which were frozen with DMSO as a cryoprotectant and the groups with F medium showed the best results.
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Animals , Female , Humans , Rats , Anesthesia , Body Weight , Cell Survival , Cryopreservation , Dimethyl Sulfoxide , Eosine Yellowish-(YS) , Frozen Sections , Ketamine , Molar , Nitrogen , Periodontal Ligament , Rats, Sprague-Dawley , ToothABSTRACT
Objective To investigate the effects and mechanism of Panax Notoginseng saponins (PNGS) on rat liver during cold preservation. Methods Using isolated perfusion of rat liver model (IPRL), Fura 2 method was used to measur the concentration of calcium ion in hepatic cells which had been preserved in DMEM solution with different concentration of PNGS added and cold preserved for 2 hours. Liver function, metabolic products of oxygen free radicals, energy substance and aucount of biliary flow as well as morphological study were measured from liver tissue which had been cold preserved in lactate riuges’s solution containing different dosages of PNGS for 24 hours and 30 minutes reperfusion.Results The contents of intracellur calcium of the rat hepatocytes,MDA, AST, ALT, LDH were lower than those in the control group,but SOD, ATP, TAN, EC and bile production were higher than those in the control group( P 0.05). Conclusion PNGS relieve the injury of the rat liver during cold preservation. The mechanism might through inhibition calcium overload, improve the energy metabolism, play a role against free radical injury realize.
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Hepatocyte transplantation is a potential treatment modality for liver diseased patients. Purified hepatocytes stimulates allospecific cytotoxicity by expressing the MHC class I antigen. Also, during cold preservation, hepatocytes are damaged by lipid peroxidation with oxygen free radicals, which may induce apoptosis on cold preserved hepatocyte. For measuring the degree of antigenicity on cold- preserved mice hepatocytes with UW solution, we studied the expression of MHC class I antigen in various time period by FACS and RT-PCR. For analysis of apoptotic hepatocyte death, we studied morphological changes and DNA fragmentation. We used flow cytometry techniques with rhodamine 123,3,3'-dihexiloxadicarbocyanine (DiOC6 (3)) and propidium iodide (PI). DiOC6 (3) is mitochondrial probe to measure the mitochondrial transmembrane potential that drops early in apoptosis. The percentage of cells undergoing chromatinolysis (subdiploid cells) was determined by ethanol fixation followed by RNA digestion and PI staining. The cold preserved hepatocytes expressed MHC class I constitutively, but revealed no significant differences among various preservation period. However, apoptosis of hepatocytes occured progressively during cold preservation. These results provides that the cold preservation of mice hepatocyte induces apoptosis with involvement of an oxidative process, but does not stimulate over expression of MHC class I antigen.