Histidine-tryptophan-ketoglutarate Research Articles

Superior myocardial preservation with HTK solution over Celsior in rat hearts with prolonged cold ischemia

Increasing allograft ischemic time is a significant risk factor for mortality following heart transplantation (HTx). The purpose of this study was to evaluate the protective effects of histidine-tryptophan-ketoglutarate (HTK) and Celsior (CEL) using a rat HTx model with prolonged cold storage. The hearts were excised from donor rats, stored in cold preservation solution for either 6 or 18 hours, and heterotopically transplanted into syngeneic recipients. Serum creatine phosphokinase (CPK), serum troponin I, graft-infiltrating cells, graft mRNA levels for inflammatory mediators, and tissue adenosine triphosphate (ATP) levels were analyzed, as markers of graft injury. The recipients of grafts stored in HTK for 18 hours of prolonged cold ischemia had lower levels of serum CPK and tissue malondialdehyde, less upregulation of the mRNAs for IL-6 and inducible nitric oxide synthase, less apoptosis, and higher ATP levels than those receiving grafts stored in CEL and Saline. Cardiac contraction 3 hours after reperfusion was observed in 43% of the cardiac grafts stored in HTK for 18 hours, while no cardiac wall movement was seen in grafts stored in either saline or CEL. Cold storage in HTK exhibited superior protective effects against prolonged cold ischemia in a syngeneic rat transplantation model.

Prolonged cold storage using a new histidine-tryptophan-ketoglutarate-based preservation solution in isogeneic cardiac mouse grafts

The aim of this study was to evaluate a new histidine-tryptophan-ketoglutarate (HTK)-based cold preservation solution in comparison with traditional HTK solution in a mouse cardiac transplant model and to assess the impact of chloride ions and of iron chelators. After 24 h cold ischaemia, traditional HTK-preserved hearts survived up to 13 days (4.4 ± 1.7 days; n = 8)). Hearts stored in the new solution without iron chelators (N46) showed significantly prolonged survival up to 2 months (N46: 11.9 ± 8.7 days; P < 0.01; n = 7) and with iron chelators (DesfLK) up to 3 months (N46 DesfLK: 12.7 ± 13.7 days; n = 6). Re-beating time was significantly shorter with the new solution (HTK: 14.5 ± 5.9 min, N46: 9.2 ± 2.7 min, N46 DesfLK: 7.1 ± 3.7 min; P < 0.01). The new solution showed significantly decreased release of creatine kinase (HTK: 25998 ± 8471 U/L, N46: 13829 ± 7679 U/L, N46 DesfLK: 3093 ± 597 U/L; P < 0.01 and n = 7 each) and lactate dehydrogenase (HTK: 5391 ± 1062 U/L, N46: 3428 ± 1890 U/L, P < 0.05; N46 DesfLK: 682 ± 344 U/L, P < 0.01) and decreased histological evidence of injury. A chloride-poor variant of the new solution showed inferior graft survival. The new solution markedly attenuates myocardial injury and yields better graft survival than traditional HTK solution. The presence of chloride ions is crucial for heart preservation. Some protective effects are obviously caused by iron chelators.

A Comparison of Hypothermic Machine Perfusion Versus Static Cold Storage in an Experimental Model of Renal Ischemia Reperfusion Injury

There is increasing support for the use of hypothermic machine perfusion (HMP) in an attempt to reduce preservation injury. However, experimental evidence is needed to further examine the effects of HMP on renal ischemia reperfusion injury. Porcine kidneys were subjected to 10 min of warm ischemia followed by 18 hr of static cold storage with hyperosomolar citrate (HOC), histidine-tryptophan-ketoglutarate (HTK), or University of Wisconsin (UW) solutions or 18 hr HMP with Kidney Perfusion Solution using the Lifeport perfusion system. Renal function, oxidative damage, and morphology were assessed during 3 hr of reperfusion with autologous blood using an isolated organ perfusion system. During reperfusion, intrarenal resistance was significantly lower in the HMP group compared with HOC and UW (area under the curve; HMP 3.8+/-1.7, HOC 9.1+/-4.3, UW 7.7+/-2.2, HTK 5.6+/-1.9 mm Hg/min; P=0.006), and creatinine clearance was significantly higher compared with the UW group (area under the curve creatinine clearance; HMP 9.8+/-7.3, HOC 2.2+/-1.7, UW 1.8+/-1.0, HTK 2.1+/-1.8 mL/min/100 g; P=0.004). Tubular function was significantly improved in the HMP group (P<0.05); however, levels of lipid peroxidation were significantly higher (P=0.005). HMP demonstrated a reduced level of preservation injury compared with the static techniques resulting in improved renal and tubular function and less tubular cell inflammation during reperfusion.

Prolonged donor heart preservation with pinacidil: The role of mitochondria and the mitochondrial adenosine triphosphate–sensitive potassium channel

Prolonged donor heart preservation is important in cardiovascular surgery. This study examined the effect of pinacidil, a nonselective adenosine triphosphate-sensitive potassium channel opener, on donor heart preservation when added to histidine tryptophan ketoglutarate solution and the role of mitochondria in this protection. Sprague-Dawley rat hearts received one of 5 preservation solutions in the Langendorff perfusion apparatus (24 per group): (1) histidine tryptophan ketoglutarate solution; (2) histidine tryptophan ketoglutarate solution containing pinacidil; (3) histidine tryptophan ketoglutarate solution containing pinacidil and 5-hydroxydecanote, a mitochondrial adenosine triphosphate-sensitive potassium channel blocker; (4) histidine tryptophan ketoglutarate solution containing pinacidil and Hoechst-Marion-Roussel 1098, a sarcolemmal adenosine triphosphate-sensitive potassium channel blocker; and (5) histidine tryptophan ketoglutarate solution containing pinacidil, 5-hydroxydecanote, and Hoechst-Marion-Roussel 1098. After a 10-minute equilibration period, all the hearts in the different preservation solutions were placed in cold storage for 8 hours, followed by 60 minutes of reperfusion. Hemodynamics, mitochondrial respiratory function, adenosine triphosphate level, cardiac troponin I release, and ultrastructure were examined. Histidine tryptophan ketoglutarate solution containing 0.5 mmol/L pinicidal significantly improved heart function, coronary flow, myocardial ultrastructure, and cardiac troponin I release after reperfusion (P < .01 or P < .05). In the pinacidil group at the end of storage and the end of reperfusion, mitochondrial respiratory function and myocardial adenosine triphosphate levels were superior when compared with other groups (P < .01 or P < .05). These beneficial effects of pinacidil were blocked by 100 mumol/L 5-hydroxydecanote. Histidine tryptophan ketoglutarate solution containing pinacidil provides better cardioprotection with preservation of mitochondrial energy. This effect of pinacidil appears to depend on both mitochondrial and sarcolemmal adenosine triphosphate sensitive potassium channel.

Impact of Normothermic Perfusion and Protein Supplementation on Human Endothelial Cell Function During Organ Preservation

Hypothermia-induced changes in endothelial cell (EC) morphology and function after organ storage may influence the initial outcome and development of transplant-associated coronary artery disease. Human saphenous vein ECs were incubated with saline (NaCl), University of Wisconsin (UW), and histidine-tryptophan-ketoglutarate (HTK) solution, with and without protein additives, at 4 degrees C and 37 degrees C. After 6 hours, ECs were recultivated for 24 and 48 hours with culture medium (reperfusion). Mitochondrial activity, adenosine triphosphate concentration, cell count, and inflammatory responses were analyzed. Cold preservation did not affect the mitochondrial activity of ECs and allowed a complete regeneration of the metabolic turnover after reperfusion. However, under normothermic conditions the metabolism of the cells was influenced by time and type of preservation solution. While both the mitochondrial activity and cell count did not change after treatment with NaCl and culture medium, the metabolic turnover of cells treated with HTK and UW solution significantly increased (twofold) and decreased (twofold, p < 0.05), respectively, after reperfusion. The endothelial reactivity remained unchanged after treatment with NaCl and HTK. The addition of serum proteins significantly improved mitochondrial activity of cells treated with warm NaCl and HTK (p < 0.05). The UW-treated cells burned out through a significant up-regulation of the ATP concentration resulting in a complete metabolic regression after reperfusion and induction of apoptosis. Normothermic preservation in UW prevented regeneration of ECs, while treatment with HKT solution did not irreversibly affect mitochondrial activity of ECs and allowed complete regeneration of metabolism and function. Serum proteins improved the preservation effect of HTK and NaCl.

Pulsatile perfusion preservation of warm ischaemia-damaged experimental kidney grafts

Cold storage using histidine-tryptophan-ketoglutarate (HTK) solution is used widely in clinical practice for the preservation of warm ischaemia-damaged kidney grafts. This study assessed the efficacy of pulsatile machine perfusion in combination with Polysol for the preservation of warm ischaemia-damaged kidney grafts. After induction of warm ischaemia by clamping of the left renal pedicle for 30 min, pigs were subjected to left nephrectomy. Thereafter, grafts were preserved for 20 h by cold storage with HTK (CS-HTK) or Polysol (CS-PS), or machine preservation with Polysol (MP-PS). Subsequently, contralateral kidneys were removed and preserved kidneys were transplanted. Control pigs underwent unilateral nephrectomy. Renal function was assessed daily for 1 week. Kidney biopsies were analysed for morphology and proliferative response. Renal function of warm ischaemia-damaged grafts preserved using MP-PS was comparable to that of non-ischaemic controls. MP-PS and CS-PS groups showed improved renal function compared with the CS-HTK group, with more favourable results for MP-PS than for CS-PS. The proliferative response of tubular cells in the CS-HTK group was higher than in all other groups. This study demonstrated that the function of warm ischaemia-damaged kidney grafts after pulsatile perfusion preservation was comparable to that of non-ischaemic controls.

Survival of partial liver grafts is increased after cold storage in Custodiol-N (HTK-N)

Background: Ischemia/Reperfusion injury (IRI) remains a major issue in partial liver transplantation. Improved cold storage solutions may lead to reduced IRI and thus increase both graft survival and its function. Custodiol-N (HTK-N), a modified variant of Bretschneider’s Histidine-Tryptophan-Ketoglutarate (HTK) solution protects livers from IRI in various transplantation models. Since its effect in living donor liver transplantation has not been examined, this study was designed to compare its effect with HTK and University of Wisconsin (UW) solution after partial liver transplantation in rats.

Comparison of Preservation Solutions for Washout of Kidney Grafts: An Experimental Study

Objective The impact of different preservation solutions for washout of kidney grafts was evaluated regarding temperature, kidney weight, remaining red blood cells (RBCs) and histological evaluation after ex vivo washout using 500 mL cold preservation solution at 4°C followed by 24 hours cold storage (CS). Methods Kidneys retrieved from Landrace pigs (20–30 kg) were immediately washed (warm ischemic time 0 min [WIT 0]), using 500 mL cold University of Wisconsin solution (UW), histidine-tryptophan-ketoglutarate (HTK), or Polysol (PS) followed by 24 hours, CS. Also, kidneys were retrieved after a WIT of 30 minutes followed by washout using HTK or PS. Results After washout, the weight of kidneys washed out with HTK had increased, whereas that of organs in the UW or PS group had decreased. After washout with UW, the core temperature of WIT 0 kidneys was lower than that with HTK. The time needed for washout using 500 mL solution was shorter using PS compared with HTK for both WIT 0 and WIT 30 groups. The amount of remaining RBCs was similar between all WIT 0 groups; whereas in the WIT 30 groups the amount was higher in kidneys washed out using HTK compared with PS. Histological evaluation showed less tissue injury among PS-washed kidneys compared with UW or HTK. Conclusion Overall, kidneys washed-out with PS showed better preservation of structural integrity after 24 hours, CS compared with either UW or HTK. Washout of warm ischemically damaged kidneys was more effective using PS compared with HTK.

Influence on Energy Kinetics and Histology of Different Preservation Solutions Seen During Cold Ischemia in the Liver

Background and Purpose Cold flush preservation prolongs tissue viability during ischemia. However, there is little understanding of the effects of various preservation fluids on events during this period. A study of cold ischemia in rat livers was undertaken to compare biochemical and histological changes over time, using three preservation solutions: University of Wisconsin (UW), histidine-tryptophan-ketoglutarate (HTK), and Leeds solution (LS) under development at our institution. Leeds solution is a phosphate-based sucrose solution that like UW contains the impermeant lactobionate and the metabolite allopurinol (1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one) which acts as a competitive inhibitor of xanthine oxidase, stopping the breakdown of hypoxanthine to xanthine by oxidizing it to alloxanthine, inhibiting both the conversion of hypoxanthine to xanthine and the conversion of xanthine to uric acid. Materials and Methods At various time points, samples were analyzed for adenosine triphospate (ATP) and metabolites by high-performance liquid chromatography as well as for histological changes. Results In all livers, ATP, ADP, and AMP degraded over 4 hours. In UW and LS groups, degradation beyond hypoxanthine was halted, and it continued in the HTK group. This blockade led to a significant reduction in the accumulation of xanthine and uric acid. Histological analysis showed protected architecture and maintenance of reticulin scaffolds in the UW and LS groups, whereas tissue breakdown was seen from earlier time points in the HTK group. Additionally, throughout ischemia, signs of pathological injury were more pronounced with UW- than with LS-preserved tissue. Conclusions These results implied that cold ischemia in the liver is characterized by dynamic biochemical changes coincident with pathological injury which are initiated from the time of organ perfusion and influenced by the choice of the perfusion fluid. Allopurinol in UW and LS appears to be critical. We hypothesized that it may also affect the degree of subsequent reperfusion injury. The data supported the assertion that LS offerred improved preservation over UW, adding to the impetus to shorten ischemic times in clinical transplantation.

Myocardial Protective Effect of Urethane on Isolated Rat Hearts in Prolonged Hypothermic Preservation

One of the most important factors restricting heart transplantation is the limited myocardial ischemia time. This study investigated the effects of urethane on the hypothermic preservation of donor rat hearts. Hearts isolated from rats were divided into 2 groups (n = 8), a control group with histidine-tryptophan-ketoglutarate (HTK) solution alone and an experimental group with HTK solution plus 30 mM urethane. Hearts were mounted on a Langendorff apparatus to estimate the baseline cardiac function; the hearts were then arrested and stored in one of the 2 solutions for 6 hours and 18 hours at 4 degrees C. After preservation, the hearts were reperfused, and cardiac function was evaluated. Lactate dehydrogenase (LDH) release, adenosine triphosphate (ATP) content, cardiomyocyte apoptosis, and myocardial ultrastructure were examined. Compared with the control group, the experimental group showed a significantly higher recovery of cardiac function for both 6 hours and 18 hours of preservation and demonstrated a lower rate of cardiomyocyte apoptosis (8.5% + or - 1.2% versus 12.2% + or - 1.8% for 6 hours; 14.1% + or - 2.1% versus 31.4% + or - 2.7% for 18 hours). ATP content was significantly higher in the experimental group than in the control group after 18 hours of preservation (229.4 + or - 29.7 microg/g versus 153.2 + or - 21.1 microg/g). The experimental group also showed lower levels of LDH release after 18 hours of preservation. Electron microscopy studies demonstrated better cardiomyocyte structure in the experimental group for both 6 hours and 18 hours of preservation. Use of urethane improved cardiac functional recovery and led to significant protective effects on rat hearts placed in a hypothermic preservation solution for a prolonged period.

Risk Factors for Developing Ischemic-Type Biliary Lesions After Liver Transplantation

Ischemic-type biliary lesions (ITBL) account for a major part of patients' morbidity and mortality after orthotopic liver transplantation (OLT). The exact origin of this type of biliary complication remains unknown. This study retrospectively evaluated 1843 patients. Patients with primary sclerosing cholangitis were excluded from this study. The diagnosis of ITBL was established only when all other causes of destruction of the biliary tree were ruled out. Donor age (P = 0.028) and cold ischemic time (CIT) (P = 0.002) were found to be significant risk factors for the development of ITBL. Organs that were perfused with University of Wisconsin (UW) solution developed ITBL significantly more often than Histidine-Tryptophan-Ketoglutarate (HTK)-perfused organs (P = 0.036). The same applied to organs harvested externally and shipped to our center versus those that were procured locally by our harvest teams (P < 0.001). Pressure perfusion via the hepatic artery significantly reduced the risk of ITBL (P = 0.001). The only recipient factor that showed a significant influence was Child-Pugh score status C (P = 0.021). Immunologic factors had no significant impact on ITBL. The clinical consequences of this study for our institution have been the strict limitation of CIT to < 10 h and the exclusive use of HTK solution. We further advocate that all organ procurement teams perform pressure perfusion on harvested organs.

Mechanoenergetic function and troponin T release following cardioplegic arrest induced by St Thomas' and histidine-tryptophan-ketoglutarate cardioplegia – an experimental comparative study in pigs☆☆☆

The study compares the single dose histidine-tryptophan-ketoglutarate (HTK) cardioplegia to the repeatedly delivered St Thomas' Hospital Solution (STHS) with respect to preservation of left ventricular mechanoenergetics and leakage of troponin T in a porcine experimental model. Fourteen pigs were randomized to a single infusion of 30 ml/kg HTK cardioplegia (n=7) or 500 ml STHS (n=7) followed by 200 ml after 20 and 40 min. After 1 h of aortic cross-clamping on cardiopulmonary bypass (CPB), the pigs were weaned and the hearts reperfused for 4 h. Stroke work (SW) was determined by a conductance catheter in the left ventricle. Myocardial oxygen consumption (MvO(2)) was measured as a function of coronary blood flow and arterial-to-coronary sinus oxygen saturation difference. Troponin T was sampled from the coronary sinus. The slope of the SW-MvO(2) relationship increased by 1.09 (+/-0.53) in the HTK group compared with 0.33 (+/-0.70) in the STHS group following ischemia and 4 h of reperfusion (P=0.04). Troponin T was significantly higher in the HTK group compared with the STHS group (P=0.04). Repeatedly delivered STHS gives better preservation of postischemic mechanoenergetic function and lower troponin T release compared with single dose HTK cardioplegia, indicating improved cardioprotection with STHS.

P-Selectin Expression in Cold Preserved Kidneys in University of Wisconsin and Histidine-Tryptophan-Ketoglutarate Solutions

The differences and efficacy of standard preservation solutions in kidney transplantation, University of Wisconsin (UW) and histidine-tryptophan-ketoglutarate (HTK), remain a topic of debate in recent clinical studies. P-selectins represent glycoproteins expressed on endothelial cells and platelets responsible for the earliest events in ischemia/reperfusion injury in kidney transplantation. This study aimed to compare the levels of P-selectin expression between cold preserved kidney tissues in UW and HTK solutions. Thirty kidneys were procured from male Lewis rats and stored in cold (4°C) solutions for periods of 4, 12, 16, 20, and 24h. Group 1 (n=15) kidneys were stored in UW solutions, and group 2 (n=15) kidneys were submerged in HTK solutions. At the end of each time point, the kidneys underwent preparation and levels of P-selectin expression in the tissues were measured using Immunoblot analyses and adjusted volumetric quantification of Western blot signals. For all periods of cold preservation, P-selectin expression was significantly down-regulated in kidney tissues stored in UW compared with HTK solutions (P<0.001). In summary, UW demonstrated a significant benefit over HTK solution in down-regulating P-selectin expression in cold preserved kidney grafts.

Incidence of and risk factors for ischemic-type biliary lesions following orthotopic liver transplantation

Ischemic-type biliary lesions (ITBL) account for a major part of patients' morbidity and mortality after orthotopic liver transplantation (OLT). The exact origin of this type of biliary complication remains unknown. This study retrospectively evaluated 1843 patients. Patients with primary sclerosing cholangitis were excluded from this study. The diagnosis of ITBL was established only when all other causes of destruction of the biliary tree were ruled out. Donor age (P = 0.028) and cold ischemic time (CIT) (P = 0.002) were found to be significant risk factors for the development of ITBL. Organs that were perfused with University of Wisconsin (UW) solution developed ITBL significantly more often than Histidine-Tryptophan-Ketoglutarate (HTK)-perfused organs (P = 0.036). The same applied to organs harvested externally and shipped to our center versus those that were procured locally by our harvest teams (P < 0.001). Pressure perfusion via the hepatic artery significantly reduced the risk of ITBL (P = 0.001). The only recipient factor that showed a significant influence was Child-Pugh score status C (P = 0.021). Immunologic factors had no significant impact on ITBL. The clinical consequences of this study for our institution have been the strict limitation of CIT to <10 h and the exclusive use of HTK solution. We further advocate that all organ procurement teams perform pressure perfusion on harvested organs.

Improved vessel preservation after 4 days of cold storage: Experimental study in rat arteries

Cold storage of arteries for reconstructive and bypass surgery may result in injury of endothelial cells which may promote low perfusion and graft vasculopathy. A recently developed N-acetyl histidine-buffered, potassium-chloride enriched, and amino acid-fortified vascular storage solution augmented with iron chelators deferoxamine (100 micromol/L) and LK 614 (20 micromol/L) was studied in the rat superior mesenteric artery and aorta with respect to: (1) potassium-induced vessel tone, (2) endothelium-dependent and -independent relaxation, and (3) endothelial nitric oxide synthase (eNOS) protein expression over 4-days cold storage (4 degrees C).This solution was compared with traditional storage solutions, histidine-tryptophan-ketoglutarate (HTK) and physiological saline solution (PSS). Vessels stored for 4 days in the new solution were significantly better protected than those stored in traditional HTK or PSS. The protective effects comprised: (1) vessel tone development after stimulation with potassium-chloride solution, (2) endothelium-dependent and -independent vessel relaxation, and (3) eNOS expression. With iron chelators (deferoxamine 100 microM, LK 614 20 microM) present in the storage solution, endothelium-dependent relaxations (eNOS-dependent and K(Ca)-channel-dependent) were fully maintained after 96 hours of cold storage. Endothelial cell structure was significantly better maintained after 96 hours in the new solution than in HTK or PSS solutions. Already, 2 hours of cold storage in HTK resulted in a significant loss of structurally intact endothelium. The structural changes correlated significantly with the diminished vessel relaxation capacity. Furthermore, tissue reductive capacity was only preserved after 96 hours storage if the new solution was used. The new storage solution is superior to traditional HTK and PSS cold storage with respect to: (1) preservation of vessel structure and function; (2) the presence of iron chelators significantly improved protection of endothelial function; and (3) the new solution permits cold vessel storage for a minimum of 4 days with full maintenance of endothelial function and its coupling to smooth muscle.

Myocardial Protection With the Use of L-arginine and N-α-acetyl-histidine

Effective myocardial preservation is an important condition for cardiac surgery, especially in heart transplantation with long ischemia times. During ischemia and reperfusion, myocardial function is altered by cold-induced ischemic injury. Cold-induced ischemic injury is triggered by cold storage and the amino acid histidine, a main component of the storage solution histidine-tryptophan-ketoglutarate (HTK). Cold-induced ischemic injury generates free oxygen radicals in an iron-dependent way. We investigated the efficacy of new modifications with the addition of L-arginine and N-alpha-acetyl-histidine to the well-established HTK solution (Custodiol) using a rat heart transplant model. Heterotopic transplantation was performed in Lewis rats (n = 20). After 1 hour of ischemic preservation and 1 hour of reperfusion, we assessed myocardial function and energy charge potential. The modifications of HTK solution included the addition of L-arginine, partial replacement of histidine with acetyl-histidine, and reduction of chloride concentration (HTK-1). In a second group, Custodiol served as the control. After 1 hour of reperfusion, left ventricular systolic pressure (106 +/- 33 vs 69 +/- 9 mm Hg; P < .05) and minimum rate of pressure development (dP/dt) (-1388 +/- 627 vs -735 +/- 219 mm Hg/s; P < .05) were significantly higher among the HTK-1 group compared with the control group. Energy charge potential did not differ significantly between the groups. This study showed that the novel modified HTK-1 solution improved myocardial contractility and relaxation after heart transplantation.

Pretreatment With Calcitonin Gene-Related Peptide Attenuates Hepatic Ischemia/Reperfusion Injury in Rats

Objective Oxygen free radicals and apoptosis play important roles in liver ischemia/reperfusion (I/R) injury. We sought to investigate the protective effect of calcitonin gene-related peptide (CGRP) to attenuate liver I/R injury due to oxygen free radicals and apoptosis. Materials and Methods Harvested rat livers were perfused via the portal vein with 60 mL of 4°C histidine-tryptophan-ketoglutarate (HTK) solution alone in the control group, or with the same solution containing CGRP (3 μg/10 g body weight) in the experimental group. After 24 hours of cold storage, hepatic enzyme leakage, portal venous pressure, oxygen consumption, total adenine nucleotides (TAN), bile production, lipoperoxide (LPO) release, apoptosis, and histochemical changes were evaluated upon 45 minutes of isolated reperfusion. Results Compared with control livers, CGRP-treated organs showed significantly decreased alanine aminotransferase (ALT) and glutamate-lactate dehydrogenase (GLDH) leakage and portal venous pressure (2.0 ± 0.3 vs 4.0 ± 0.4 mm Hg; P < .01), with significantly increased bile production (8.56 ± 0.76 vs 3.34 ± 0.68 μL/g/45 min; P < .01), oxygen consumption (5.14 ± 0.4 vs 2.57 ± 0.2 μL/g/min; P < .01), and total adenine nucleotides (TAN) (11.1 ± 0.71 vs 7.02 ± 0.53 μmol/g; P < .01) upon reperfusion as signs of recovered viability. We observed infrequent positive terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling (TUNEL) staining, especially in sinusoidal lining cells (SLC). The percentage of TUNEL-positive cells in the CGRP group was significantly decreased compared with the control group: (4.1 ± 0.67 vs 8.0 ± 1.27; P < .05). Perfusate levels of low molecular weight (LMW) histone-associated DNA fragments (0.36 ± 0.04 vs 0.53 ± 0.06 AU; P < .05) were also decreased, coupled with strong 5′-nucleotidase (5′-NT) and LDH activity staining concentrated on the endothelial cells. LPO release in the perfusate was largely decreased: (0.12 ± 0.02 vs 0.36 ± 0.04 nmoL/g, P < .01). Conclusion CGRP ameliorated liver I/R injury due to reactive oxygen species and apoptosis.

Clinical experience with histidine‐tryptophan‐ketoglutarate solution in abdominal organ preservation: a review of recent literature

Histidine-tryptophan-ketoglutarate (HTK) solution was introduced by Bretschneider as a cardioplegia solution in the early 1980s and has subsequently been applied to organ preservation for transplantation in Europe during the 1980s and in North America during the last six yr. With the increasing use of HTK for primary preservation, it is important that the transplant community be aware of the properties of this solution and the advantages and disadvantages of its use. Even if a center uses an alternative preservation solution, it is likely that import offers will be made for allografts that have been preserved in HTK. In this review article, recent literature describing the clinical use of HTK in abdominal transplantation will be summarized with references to earlier reports when indicated.

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristic, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we have developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004, and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37°C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4°C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristic, organ procurement and preservation affect the isolation outcome. At University of Illinois at Chicago (UIC) we have developed a successful isolation protocol with an improved purification gradient. The program started in January 2004, and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37 degrees C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4 degrees C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.