- Email: [email protected]
In vivo pancreatic-specific hypothermia: retrograde ductal perfusion experimental studies
In Vivo Pancreatic-Specific Hypothermia: Retrograde Ductal Perfusion Experimental Studies C.P. Morrison, S.A. Wemyss-Holden, F.G. Court, B.D. Teague, A.R. Dennison, and G.J. Maddern
C
ELLULAR DAMAGE caused by warm ischaemia is a well-recognized problem in all areas of transplantation.1,2 These problems are particularly apparent in the living donor or autotransplantation situation, where conventional methods for cooling the organ in situ by vascular flushing with a cold preservation solution3 cannot be utilized. In the case of living kidney donors, the most common scenario for living donors, the consistent and relatively simple vascular anatomy allows the organ to be fully mobilized on a vascular pedicle, thereby minimizing warm ischaemic time.4 However, this is not always the situation for other organs (for example, total pancreatectomy for benign disease, usually chronic pancreatitis). The scarred and fibrotic nature of the diseased pancreas makes resection a lengthy procedure, and the complex arterial supply precludes the mobilization of the gland on a vascular pedicle. A combination of both these factors results in the resected pancreas being subjected to a considerable duration of warm ischaemia. This is obviously not important if the pancreas is simply being resected; however, if islet isolation and autotransplantation are being performed in conjunction with resection, the duration of warm ischaemia and the subsequent islet damage are vital factors in the success or failure of the islet autotransplant. This study investigated the feasibility of producing pancreatic-specific in vivo hypothermia by retrograde perfusion of the pancreatic duct with a cooled solution.
MATERIALS AND METHODS Eighteen specific pathogen-free female domestic white pigs were used for the study. Mean weight was 32 kg. The study consisted of six groups, with three animals in each group. All pigs were anaesthetised in the same manner. Sedation was achieved with an intramuscular injection of ketamine (20 mg/kg) and xylazine (1.5 mg/kg). Each animal was cleaned with a solution of chlorhexidine and taken into the operating theatre. A laryngeal mask airway was inserted and anaesthesia maintained with 1.5% halothane in oxygen. Oxygen saturation and heart rate were monitored continuously throughout the procedure. A midline laparotomy was performed and the pancreas was identified. The overlying loops of bowel were dissected free from the gland. The distal tip of the pancreas was mobilized and the distal 2 cm was resected to create an open duct system. A 5 cm duodenotomy was performed 20 cm distal to the pylorus. The
pancreatic duct was identified and then cannulated with an 18 gauge intravenous cannula. Three fine wire thermocouples (Dick Smith Electronics K-Type Thermocouple Q 1439) were then inserted into the pancreatic parenchyma at 5, 10, and 15 cm from the ampulla. A rectal thermometer was used to measure core temperature. Baseline readings were taken from the three thermocouples and rectal thermometer. The pancreas was then isolated from the surrounding bowel with gauze packs. An intraabdominal ice pack (1000 g) was placed around the pancreas. Temperature readings were taken from the three pancreatic thermocouples, along with core temperature every 5 minutes. The ice pack was replenished (500 g of ice) at 40 minute intervals. After 1 hour, retrograde perfusion of the pancreatic duct, via the ductal cannula, was commenced. The perfusing solution, Haemaccel (Hoechst Marion Roussel, Aust R 12401), was cooled to between 4°C and 6°C using an ice bath and a counter current perfusion apparatus. The perfusion rate (0, 1, 2, 3, 4, or 5 mL per minute) was randomly selected after the initial hour of ice pack cooling and was controlled by an intravenous infusion pump. Temperature readings were then recorded, at 5 minute intervals, for a further hour. At the end of the experiment the animal was killed with a lethal intracardiac injection of barbiturate. The data from both pancreatic and core temperature readings were analysed statistically using Scheffe multiple comparisons analysis of variance.
RESULTS
The results of the temperature readings for each given perfusion rate (0 to 5 mL/min) were analysed together (Fig 1). Lower pancreatic temperatures were achieved at the higher perfusion rates. A significant degree (P ⬍ .05) of pancreatic cooling was achieved at all the perfusion rates when compared to no perfusion (ice pack cooling alone). However, there was no significant (P ⬎ .05) difference in the extent of cooling achieved when comparing the lower From the University of Adelaide, Department of Surgery, The Queen Elizabeth Hospital, Adelaide, Australia (C.P.M., S.A.W.H., F.G.C., B.D.T., G.J.M.) and the Department of Surgery, Leicester General Hospital, Leicester, UK (A.R.D.). Address reprint requests to Professor G.J. Maddern, University of Adelaide, Department of Surgery, The Queen Elizabeth Hospital, Woodville Road, Woodville, SA 5011, Australia. E-mail: [email protected]
© 2002 by Elsevier Science Inc. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/02/$–see front matter PII S0041-1345(02)03604-7
Transplantation Proceedings, 34, 3351–3353 (2002)
3351
3352
MORRISON, WEMYSS-HOLDEN, COURT ET AL
Fig 1. Mean temperatures (pancreatic and core) achieved by pancreatic perductal cooling at a range of perfusion rates.
perfusion rates of 2 and 3 mL/min. At the higher perfusion rates of 4 and 5 mL/min the degree of pancreatic cooling was significantly (P ⬍ .05) greater than that achieved at the lower rates (Fig 1). The degree of pancreatic cooling attributable to ductal perfusion, rather than ice packing, was determined by calculating the difference between pancreatic temperatures at each perfusion rate (1 to 5 mL/min) and pancreatic temperatures achieved with ice pack cooling alone. This additional cooling effect was greatest at the higher perfusion rates (Fig 2). The relationship of mean rectal temperature and pancreatic temperature was also analysed statistically. The pancreatic temperature at each of the perfusion rates (including no perusion) was significantly (P ⬎ .05) lower than the mean core temperature (Fig 1). DISCUSSION
This study demonstrates that the in situ cooling of the porcine pancreas by ductal perfusion is feasible. The significantly increased cooling achieved with ductal perfusion when compared with ice packing alone suggests that the cooling effect is a direct result of ductal perfusion rather
than solely due to ice packing of the pancreas. Although the core temperature of the animal had decreased during the course of the experiments, to a low of 33°C at 2 hours, it was always significantly greater than the pancreatic temperature. Cellular energy requirement and oxygen utilization decreases with a reduction in temperature. Approximately 40% of cellular energy consumption is by the Na/K ATPase pump, the activity of which is temperature-dependent.5 Oxygen consumption has also been shown to be significantly reduced by moderate hypothermia, with the oxygen requirements of rat livers at 20°C being approximately one half of their requirements at 37°C.6 Pancreatic temperatures of 15°C to 16°C were achieved at the higher perfusion rates of 4 and 5 mL/min. The extent of the pancreaticspecific hypothermia produced by ductal perfusion in this study is of sufficient order to decrease oxygen requirements and Na/K pump activity. Also the already cooled, vascularised pancreas will been further cooled as its blood supply is ligated during pancreatic resection. During total pancreatectomy with islet autotransplantation the diseased pancreas is subjected to a period of warm ischaemia as it is resected, due to the scarred and fibrotic nature of the gland. Some authors think that this warm
IN VIVO PANCREATIC-SPECIFIC HYPOTHERMIA
3353
Fig 2. Mean (with standard deviation) additional cooling effect of pancreatic ductal perfusion when compared to ice pack cooling alone.
ischaemic insult may have a significant deleterious effect on islet yield,7 especially as the initially available islets in this group of patients are usually reduced. The technique used in this study of in situ pancreaticspecific hypothermia by retrograde ductal perfusion, although described here in a simplified experimental model, may be of potential use during total pancreatectomy in combination with islet autotransplantation. The ability to cool the pancreas in situ prior to and during resection may result in a reduction of the warm ischaemic damage to the pancreas. This in turn may have a beneficial effect on islet isolation in the areas of both islet yield and function.
REFERENCES 1. Bilde T, Dahlager JI, Asnaes S, et al: Scand J Urol Nephrol 11:165, 1977 2. Tellez-Yudilevich M, Tyhurst M, Howell SL, et al: Transplantation 23:217, 1977 3. Bjorken C, Lundgren G, Ringden O, et al: Br J Surg 63:517, 1976 4. Bettschart V, Schneider R, Halabi G, et al: Ann Urol (Paris) 35:5, 2001 5. Honig A, Oppermann H, Budweg C, et al: Am J Physiol 266:S10, 1994 6. Fujita S, Hamamoto I, Nakamura K, et al: Nippon Geka Hokan 62:58, 1993 7. Morrison CP, Wemyss-Holden SA, Dennison AR, et al: Arch Surg 137:80, 2002
C
ELLULAR DAMAGE caused by warm ischaemia is a well-recognized problem in all areas of transplantation.1,2 These problems are particularly apparent in the living donor or autotransplantation situation, where conventional methods for cooling the organ in situ by vascular flushing with a cold preservation solution3 cannot be utilized. In the case of living kidney donors, the most common scenario for living donors, the consistent and relatively simple vascular anatomy allows the organ to be fully mobilized on a vascular pedicle, thereby minimizing warm ischaemic time.4 However, this is not always the situation for other organs (for example, total pancreatectomy for benign disease, usually chronic pancreatitis). The scarred and fibrotic nature of the diseased pancreas makes resection a lengthy procedure, and the complex arterial supply precludes the mobilization of the gland on a vascular pedicle. A combination of both these factors results in the resected pancreas being subjected to a considerable duration of warm ischaemia. This is obviously not important if the pancreas is simply being resected; however, if islet isolation and autotransplantation are being performed in conjunction with resection, the duration of warm ischaemia and the subsequent islet damage are vital factors in the success or failure of the islet autotransplant. This study investigated the feasibility of producing pancreatic-specific in vivo hypothermia by retrograde perfusion of the pancreatic duct with a cooled solution.
MATERIALS AND METHODS Eighteen specific pathogen-free female domestic white pigs were used for the study. Mean weight was 32 kg. The study consisted of six groups, with three animals in each group. All pigs were anaesthetised in the same manner. Sedation was achieved with an intramuscular injection of ketamine (20 mg/kg) and xylazine (1.5 mg/kg). Each animal was cleaned with a solution of chlorhexidine and taken into the operating theatre. A laryngeal mask airway was inserted and anaesthesia maintained with 1.5% halothane in oxygen. Oxygen saturation and heart rate were monitored continuously throughout the procedure. A midline laparotomy was performed and the pancreas was identified. The overlying loops of bowel were dissected free from the gland. The distal tip of the pancreas was mobilized and the distal 2 cm was resected to create an open duct system. A 5 cm duodenotomy was performed 20 cm distal to the pylorus. The
pancreatic duct was identified and then cannulated with an 18 gauge intravenous cannula. Three fine wire thermocouples (Dick Smith Electronics K-Type Thermocouple Q 1439) were then inserted into the pancreatic parenchyma at 5, 10, and 15 cm from the ampulla. A rectal thermometer was used to measure core temperature. Baseline readings were taken from the three thermocouples and rectal thermometer. The pancreas was then isolated from the surrounding bowel with gauze packs. An intraabdominal ice pack (1000 g) was placed around the pancreas. Temperature readings were taken from the three pancreatic thermocouples, along with core temperature every 5 minutes. The ice pack was replenished (500 g of ice) at 40 minute intervals. After 1 hour, retrograde perfusion of the pancreatic duct, via the ductal cannula, was commenced. The perfusing solution, Haemaccel (Hoechst Marion Roussel, Aust R 12401), was cooled to between 4°C and 6°C using an ice bath and a counter current perfusion apparatus. The perfusion rate (0, 1, 2, 3, 4, or 5 mL per minute) was randomly selected after the initial hour of ice pack cooling and was controlled by an intravenous infusion pump. Temperature readings were then recorded, at 5 minute intervals, for a further hour. At the end of the experiment the animal was killed with a lethal intracardiac injection of barbiturate. The data from both pancreatic and core temperature readings were analysed statistically using Scheffe multiple comparisons analysis of variance.
RESULTS
The results of the temperature readings for each given perfusion rate (0 to 5 mL/min) were analysed together (Fig 1). Lower pancreatic temperatures were achieved at the higher perfusion rates. A significant degree (P ⬍ .05) of pancreatic cooling was achieved at all the perfusion rates when compared to no perfusion (ice pack cooling alone). However, there was no significant (P ⬎ .05) difference in the extent of cooling achieved when comparing the lower From the University of Adelaide, Department of Surgery, The Queen Elizabeth Hospital, Adelaide, Australia (C.P.M., S.A.W.H., F.G.C., B.D.T., G.J.M.) and the Department of Surgery, Leicester General Hospital, Leicester, UK (A.R.D.). Address reprint requests to Professor G.J. Maddern, University of Adelaide, Department of Surgery, The Queen Elizabeth Hospital, Woodville Road, Woodville, SA 5011, Australia. E-mail: [email protected]
© 2002 by Elsevier Science Inc. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/02/$–see front matter PII S0041-1345(02)03604-7
Transplantation Proceedings, 34, 3351–3353 (2002)
3351
3352
MORRISON, WEMYSS-HOLDEN, COURT ET AL
Fig 1. Mean temperatures (pancreatic and core) achieved by pancreatic perductal cooling at a range of perfusion rates.
perfusion rates of 2 and 3 mL/min. At the higher perfusion rates of 4 and 5 mL/min the degree of pancreatic cooling was significantly (P ⬍ .05) greater than that achieved at the lower rates (Fig 1). The degree of pancreatic cooling attributable to ductal perfusion, rather than ice packing, was determined by calculating the difference between pancreatic temperatures at each perfusion rate (1 to 5 mL/min) and pancreatic temperatures achieved with ice pack cooling alone. This additional cooling effect was greatest at the higher perfusion rates (Fig 2). The relationship of mean rectal temperature and pancreatic temperature was also analysed statistically. The pancreatic temperature at each of the perfusion rates (including no perusion) was significantly (P ⬎ .05) lower than the mean core temperature (Fig 1). DISCUSSION
This study demonstrates that the in situ cooling of the porcine pancreas by ductal perfusion is feasible. The significantly increased cooling achieved with ductal perfusion when compared with ice packing alone suggests that the cooling effect is a direct result of ductal perfusion rather
than solely due to ice packing of the pancreas. Although the core temperature of the animal had decreased during the course of the experiments, to a low of 33°C at 2 hours, it was always significantly greater than the pancreatic temperature. Cellular energy requirement and oxygen utilization decreases with a reduction in temperature. Approximately 40% of cellular energy consumption is by the Na/K ATPase pump, the activity of which is temperature-dependent.5 Oxygen consumption has also been shown to be significantly reduced by moderate hypothermia, with the oxygen requirements of rat livers at 20°C being approximately one half of their requirements at 37°C.6 Pancreatic temperatures of 15°C to 16°C were achieved at the higher perfusion rates of 4 and 5 mL/min. The extent of the pancreaticspecific hypothermia produced by ductal perfusion in this study is of sufficient order to decrease oxygen requirements and Na/K pump activity. Also the already cooled, vascularised pancreas will been further cooled as its blood supply is ligated during pancreatic resection. During total pancreatectomy with islet autotransplantation the diseased pancreas is subjected to a period of warm ischaemia as it is resected, due to the scarred and fibrotic nature of the gland. Some authors think that this warm
IN VIVO PANCREATIC-SPECIFIC HYPOTHERMIA
3353
Fig 2. Mean (with standard deviation) additional cooling effect of pancreatic ductal perfusion when compared to ice pack cooling alone.
ischaemic insult may have a significant deleterious effect on islet yield,7 especially as the initially available islets in this group of patients are usually reduced. The technique used in this study of in situ pancreaticspecific hypothermia by retrograde ductal perfusion, although described here in a simplified experimental model, may be of potential use during total pancreatectomy in combination with islet autotransplantation. The ability to cool the pancreas in situ prior to and during resection may result in a reduction of the warm ischaemic damage to the pancreas. This in turn may have a beneficial effect on islet isolation in the areas of both islet yield and function.
REFERENCES 1. Bilde T, Dahlager JI, Asnaes S, et al: Scand J Urol Nephrol 11:165, 1977 2. Tellez-Yudilevich M, Tyhurst M, Howell SL, et al: Transplantation 23:217, 1977 3. Bjorken C, Lundgren G, Ringden O, et al: Br J Surg 63:517, 1976 4. Bettschart V, Schneider R, Halabi G, et al: Ann Urol (Paris) 35:5, 2001 5. Honig A, Oppermann H, Budweg C, et al: Am J Physiol 266:S10, 1994 6. Fujita S, Hamamoto I, Nakamura K, et al: Nippon Geka Hokan 62:58, 1993 7. Morrison CP, Wemyss-Holden SA, Dennison AR, et al: Arch Surg 137:80, 2002