Ex vivo normothermic perfusion of isolated segmental porcine bowel: a novel functional model of the small intestine.

BACKGROUND: There is an unmet need for suitable ex vivo large animal models in experimental gastroenterology and intestinal transplantation. This study details a reliable and effective technique for ex vivo normothermic perfusion (EVNP) of segmental porcine small intestine. METHODS: Segments of small intestine, 1.5-3.0 m in length, were retrieved from terminally anaesthetized pigs. After a period of cold ischaemia, EVNP was performed for 2 h at 37°C with a mean pressure of 80 mmHg using oxygenated autologous blood diluted with Ringer's solution. The duration of EVNP was extended to 4 h for a second set of experiments in which two segments of proximal to mid-ileum (1.5-3.0 m) were retrieved from each animal and reperfused with whole blood (control) or leucocyte-depleted blood to examine the impact of leucocyte depletion on reperfusion injury. RESULTS: After a mean cold ischaemia time of 5 h and 20 min, EVNP was performed in an initial group of four pigs. In the second set of experiments, five pigs were used in each group. In all experiments bowel segments were well perfused and exhibited peristalsis during EVNP. Venous glucose levels significantly increased following luminal glucose stimulation (mean(s.e.m.) basal level 1.8(0.6) mmol/l versus peak 15.5(5.8) mmol/l; P < 0.001) and glucagon-like peptide 1 (GLP-1) levels increased in all experiments, demonstrating intact absorptive and secretory intestinal functions. There were no significant differences between control and leucocyte-depleted animals regarding blood flow, venous glucose, GLP-1 levels or histopathology at the end of 4 h of EVNP. CONCLUSIONS: This novel model is suitable for the investigation of gastrointestinal physiology, pathology and ischaemia reperfusion injury, along with evaluation of potential therapeutic interventions.

The use of ex-vivo normothermic perfusion for the resuscitation and assessment of human kidneys discarded because of inadequate in situ perfusion.

BACKGROUND: Many kidneys are rejected for transplantation due to inadequate in situ perfusion during organ retrieval because of the risk of additional ischaemic injury and microvasculature thrombosis. This study describes the use of ex vivo normothermic perfusion (EVNP) for the resuscitation and assessment of human kidneys that were discarded after inadequate in situ perfusion. METHODS: Twenty-two human kidneys were retrieved but then deemed unsuitable for transplantation, primarily due to inadequate in situ perfusion. After a period of static cold storage, kidneys were perfused for 60 min with an oxygenated red cell based solution at 36 °C. RESULTS: Nineteen out of 22 kidneys (86 %) were from DCD donors. During EVNP, kidneys were assessed and scored based on their macroscopic appearance, measures of renal blood flow and urine production. Kidneys were scored from 1 indicating the least injury to 5, indicating the worst. Twelve kidneys had an EVNP score of 1-2, 7 scored 3-4 and 3 kidneys scored 5. The EVNP score 5 kidneys had a low level of tubular function compared to the score 1-4 kidneys. Their perfusion parameters did not improve during EVNP and they were considered non-transplantable. There was no association between the histological evaluation and EVNP parameters. CONCLUSION: EVNP restores function ex vivo and enables an assessment of kidneys that have been declined for transplantation due to inadequate in situ perfusion.

Ex vivo normothermic perfusion for quality assessment of marginal donor kidney transplants.

BACKGROUND: A significant proportion of kidneys procured for transplantation are discarded because of concerns about their suitability. In this study ex vivo normothermic perfusion (EVNP) was used as a quality assessment device before renal transplantation. METHODS: Seventy-four human kidneys deemed unsuitable for transplantation following retrieval underwent 60 min of EVNP with an oxygenated red cell-based solution at 36°C. Receiver operating characteristic (ROC) curves were used to identify thresholds of renal blood flow and urine output. These thresholds and a grading of macroscopic appearance were incorporated into an EVNP assessment score (highest quality, 1; lowest, 5). This was applied to a series of 36 kidneys transplanted after EVNP. RESULTS: In the discarded kidney series, 60 (81 per cent) scored 1-4 and 14 (19 per cent) scored 5. Although none of these kidneys was transplanted, those with a score from 1 to 4 were considered suitable for transplantation. In the 36 transplanted kidneys, the score ranged between 1 and 3 (score 1, 17; score 2, 11; score 3, 8). All of these kidneys were transplanted without any complications or primary non-function. The delayed graft function rate was 6 per cent (1 of 17) in kidneys scoring 1, 0 per cent (0 of 11) in those scoring 2 and 38 per cent (3 of 8) in those scoring 3 (P = 0·024). The mean(s.d.) estimated glomerular filtration rate at 12 months was 51(16), 63(15) and 38(21) ml in kidneys scoring 1, 2 and 3 respectively (P = 0·015). CONCLUSION: EVNP combined with a simple scoring system is an innovative technology for pretransplant assessment of kidney quality and acceptability for transplantation. This study suggests that a high percentage of retrieved kidneys are being discarded unnecessarily.

Ischaemic conditioning reduces kidney injury in an experimental large-animal model of warm renal ischaemia.

BACKGROUND: Ischaemic conditioning, using short repeated sequences of intermittent ischaemia, is a strategy that may ameliorate ischaemia-reperfusion injury. The aim of the study was to assess the effects of direct and remote ischaemic conditioning in a porcine model of renal warm ischaemia-reperfusion injury. METHODS: Pigs (50 kg) underwent laparotomy and 60-min occlusion of the left renal pedicle followed by right nephrectomy. Animals were divided into three groups: untreated controls (n = 8); direct postconditioning involving six 15-s cycles of clamping then releasing of the left renal artery (n = 7); or remote periconditioning involving four 5-min cycles of clamping then releasing of the left common iliac artery (n = 8). After 7 days kidney tissue was harvested, and blood and urine samples were collected on postoperative days 1, 3 and 7. RESULTS: The direct postconditioning group had a lower area under the serum creatinine curve (mean(s.d.) 1378(157) versus 2001(1022) µmol/l · day respectively; P = 0.036) and peak creatinine level (316(46) versus 501(253) µmol/l respectively; P = 0.033) compared with values in control animals. There was a significant increase in serum levels of tumour necrosis factor α on day 1 in control animals but not in the conditioning groups (P = 0.013). Urinary levels of neutrophil gelatinase-associated lipocalin increased over the study period in both the control and remote groups (P = 0.001 for both), but not in the direct group (P = 0.176). There was no mortality and no complications related to either conditioning technique. CONCLUSION: In this in vivo large-animal model, direct renal artery ischaemic postconditioning protected kidneys against warm ischaemia injury. This straightforward technique could readily be translated into clinical practice. Surgical relevance Ischaemic conditioning has been shown to improve outcomes in both experimental studies and clinical trials in cardiac surgery. Evidence from small-animal and human studies assessing ischaemic conditioning techniques in renal transplantation have not yet established the optimal technique and timing of conditioning. In this study, a large-animal model of renal warm ischaemia was used to compare different conditioning techniques. Postconditioning applied directly to the renal artery was shown to reduce renal injury. Furthermore, new evidence is provided that shorter cycles of ischaemic postconditioning than previously described can protect against renal injury. Evidence from a large-animal model is provided for different conditioning techniques. The beneficial postconditioning technique described is straightforward to perform and provides an alternative method of conditioning following renal transplantation, with potential for application in clinical practice.

Use of Ex Vivo Normothermic Perfusion for Quality Assessment of Discarded Human Donor Pancreases.

A significant number of pancreases procured for transplantation are deemed unsuitable due to concerns about graft quality and the associated risk of complications. However, this decision is subjective and some declined grafts may be suitable for transplantation. Ex vivo normothermic perfusion (EVNP) prior to transplantation may allow a more objective assessment of graft quality and reduce discard rates. We report ex vivo normothermic perfusion of human pancreases procured but declined for transplantation, with ABO-compatible warm oxygenated packed red blood cells for 1-2 h. Five declined human pancreases were assessed using this technique after a median cold ischemia time of 13 h 19 min. One pancreas, with cold ischemia over 30 h, did not appear viable and was excluded. In the remaining pancreases, blood flow and pH were maintained throughout perfusion. Insulin secretion was observed in all four pancreases, but was lowest in an older donation after cardiac death pancreas. Amylase levels were highest in a gland with significant fat infiltration. This is the first study to assess the perfusion, injury, as measured by amylase, and exocrine function of human pancreases using EVNP and demonstrates the feasibility of the approach, although further refinements are required.

The role of magnetic resonance cholangiopancreatography in the management of acute gallstone pancreatitis.

INTRODUCTION: The aim of this study was to identify whether magnetic resonance cholangiopancreatography (MRCP) can be used selectively in patients with acute gallstone pancreatitis to detect choledocholithiasis, based on liver function tests (LFTs) and ultrasonography appearance. METHODS: All patients admitted between January 2008 and January 2011 with gallstone pancreatitis (amylase >300u/l) who underwent MRCP were included in the study. LFTs and radiology reports were obtained from the respective computer systems. RESULTS: Overall, 173 patients with acute gallstone pancreatitis underwent MRCP and 30% (52/173) showed choledocholithiasis. The mean bilirubin level was significantly higher in those with choledocholithiasis (46 ±5µmol/l vs 36 ±3µmol/l, p=0.0388) although there was no significant difference in alkaline phosphatase (276 ±25iu/l vs 229 ±16iu/l, p=0.1154). However, sensitivity of abnormal bilirubin (>21µmol/l) for choledocholithiasis was only 62% and specificity was 41%. Sensitivity of abnormal alkaline phosphatase (>140iu/l) for choledocholithiasis was only 75% and specificity was 37%. There was a significant association between biliary dilatation on ultrasonography and choledocholithiasis on MRCP (p=0.0099) although the sensitivity of biliary dilatation for choledocholithiasis was only 44% and the specificity was 79%. Furthermore, there was no difference in the incidence of choledocholithiasis on MRCP for those patients with persistently deranged LFTs versus those whose LFTs returned to normal (relative risk: 1.07, 95% confidence interval: 0.61-1.89, p=1.00). Overall, 10% of patients with choledocholithiasis on MRCP had entirely normal LFTs on admission and no biliary dilatation or choledocholithiasis on ultrasonography. CONCLUSIONS: All patients with acute gallstone pancreatitis should undergo specific imaging, preferably MRCP, to exclude choledocholithiasis as LFTs and ultrasonography are inaccurate in predicting common bile duct stones.

Rapamycin toxicity in MIN6 cells and rat and human islets is mediated by the inhibition of mTOR complex 2 (mTORC2).

AIMS/HYPOTHESIS: Rapamycin (sirolimus) is one of the primary immunosuppressants for islet transplantation. Yet there is evidence that the long-term treatment of islet-transplant patients with rapamycin may be responsible for subsequent loss of islet graft function and viability. Therefore, the primary objective of this study was to elucidate the molecular mechanism of rapamycin toxicity in beta cells. METHODS: Experiments were performed on isolated rat and human islets of Langerhans and MIN6 cells. The effects of rapamycin and the roles of mammalian target of rapamycin complex 2 (mTORC2)/protein kinase B (PKB) on beta cell signalling, function and viability were investigated using cell viability assays, insulin ELISA assays, kinase assays, western blotting, pharmacological inhibitors, small interfering (si)RNA and through the overproduction of a constitutively active mutant of PKB. RESULTS: Rapamycin treatment of MIN6 cells and islets of Langerhans resulted in a loss of cell function and viability. Although rapamycin acutely inhibited mTOR complex 1 (mTORC1), the toxic effects of rapamycin were more closely correlated to the dissociation and inactivation of mTORC2 and the inhibition of PKB. Indeed, the overproduction of constitutively active PKB protected islets from rapamycin toxicity whereas the inhibition of PKB led to a loss of cell viability. Moreover, the selective inactivation of mTORC2 using siRNA directed towards rapamycin-insensitive companion of target of rapamycin (RICTOR), mimicked the toxic effects of chronic rapamycin treatment. CONCLUSIONS/INTERPRETATION: This report provides evidence that rapamycin toxicity is mediated by the inactivation of mTORC2 and the inhibition of PKB and thus reveals the molecular basis of rapamycin toxicity and the essential role of mTORC2 in maintaining beta cell function and survival.

Case-matched comparison of long-term results of non-heart beating and heart-beating donor renal transplants.

BACKGROUND: Function and survival of non-heart-beating donor (NHBD) renal transplants have been shown to be comparable to those from heart-beating donors (HBDs) up to 10 years after transplantation. However, there are few data on outcome after 10 years, particularly from uncontrolled NHBD donors. METHODS: All NHBD renal transplants (predominantly uncontrolled) performed between April 1992 and January 2002 were retrospectively matched with HBD renal transplants performed over the same period. RESULTS: Some 112 NHBD renal transplants were compared with 164 HBD renal transplants. Delayed graft function was significantly higher in the NHBD group (83.9 versus 22.0 per cent respectively; P < 0.001). Primary non-function rates were similar (5.4 versus 1.8 per cent respectively; P = 0.164). Overall serum creatinine was significantly higher in NHBDs (P < 0.001). Median graft and patient survival was 126 months for NHBD and 159 months for HBD kidneys. Death-censored graft survival at 1, 5, 10 and 15 years was respectively 91.8, 77.5, 61.0 and 44.2 per cent for NHBD, and 91.1, 86.3, 71.7 and 58.5 per cent for HBD kidneys (P = 0.108). CONCLUSION: Despite increased delayed graft function rates and serum creatinine levels, the long-term survival of NHBD renal transplants was similar to those from HBDs. However, there was a trend to poorer function and survival from 10 years after transplant.

Surgical treatment of combined hepatic and pulmonary colorectal cancer metastases.

AIMS: Surgical resection of combined hepatic and pulmonary metastases remains controversial in light of limited supportive evidence. This study aimed to audit our initial experience with this aggressive surgical strategy. METHODS: Between 1997 and 2006 we assessed 19 patients with colorectal cancer metastases for combined liver and lung metastasectomy, of whom 16 patients underwent surgery. We retrospectively reviewed perioperative and survival data. RESULTS: Synchronous liver metastases were present in three out of 16 patients at time of diagnosis of the primary tumour, and one out of 16 patients had synchronous lung and liver metastases with the primary tumour. Of those 12 patients who developed metachronous metastases five patients developed liver metastases first, one patient developed pulmonary metastases first, and six patients developed synchronous liver and lung metastases. Thirty nine operations were performed on 16 patients. The median hospital stay was 5.5 (2-10) days for the pulmonary and 7 (1-23) days for the hepatic resections. There were no in-hospital deaths. Chemotherapy was given to five patients prior to metastasectomy and nine received adjuvant chemotherapy following metastasectomy. Median survival from diagnosis of metastatic disease was 44 months (8-87 months). Estimated 1-year survival from diagnosis of metastatic disease was 94%, estimated 5-year survival was 20%. CONCLUSION: We believe an aggressive but selective surgical approach to combined hepatic and pulmonary colorectal metastases is justified by limited resource requirements and encouraging survival.

Case-control comparison of laparoscopic and open washout for peritoneal dialysis-associated peritonitis.

BACKGROUND: Peritonitis is the major complication of peritoneal dialysis (PD). Some 25-40 per cent of patients require surgical intervention, traditionally laparotomy, washout and removal of the PD catheter. The aim was to compare this open procedure with laparoscopic washout and catheter removal. METHODS: In a case-control comparison, 20 patients who had laparoscopic washout for PD-associated peritonitis were matched by age and causative organism with 20 patients who had open washout. RESULTS: The groups were well matched for age, sex, causative organism, preoperative C-reactive protein level, white cell count and presence of bowel wall sclerosis. Laparoscopic surgery was quicker than open operation (mean(s.d.) 49(13) versus 73(30) min; P = 0.006) and postoperative morphine requirements were significantly lower (median 0 versus 27 mg; P < 0.001). Bowel function recovered more quickly in the laparoscopic group, as measured by time to first passage of flatus (mean(s.d.) 2(1) versus 5(4) days; P = 0.004) and resumption of free fluids (median 2 versus 4 days; P = 0.044). Reoperation rates and 30-day mortality were identical in the two groups. CONCLUSION: This study suggests that laparoscopic washout for PD peritonitis is as effective as open washout, but is quicker and less painful with earlier return of bowel function.

A successful scheme for reducing waiting times for arteriovenous fistula formation.

PURPOSE: In a bid to reduce waiting times for arteriovenous fistula (AVF) formation we introduced a scheme whereby nephrologists were able to place patients directly on the waiting list for surgery. This study evaluated the quality of these direct referrals and assessed the reduction in waiting times. METHODS: Fifty consecutive patients referred directly to the waiting list were compared with 50 patients placed on the waiting list after being assessed in our vascular access clinic. RESULTS: Forty-nine patients from the direct group and 47 patients from the clinic group underwent surgery. In the direct group 39 patients (80%) underwent the same procedure as they had been originally listed for, compared with 39 patients (83%) in the clinic group (p=0.80). A fistula suitable for needling was created in 37 patients (76%) of the direct group and 38 patients (81%) of the clinic group (p=0.62). The median wait from referral to surgery in the direct group was significantly lower than in the clinic group (62 vs.141 days; p<0.0001). CONCLUSION: This study demonstrates that nephrologists are able to effectively assess select patients for AVF formation. This significantly reduces waiting times for surgery, without an increase in cancelled operations, or a reduction in technical success.