Ischaemia preconditioning attenuates hypothermic ischaemia-reperfusion related early graft dysfunction following orthotopic lung transplantation in th
作者：Zhang Xingyi,Sun Mei,Wang Rongyou
单位：
关键词：
白求恩医科大学学报000221 〔提 要〕 目的：为了证明缺血预处理对移植肺功能的影响。方法：使用套接吻合技术行大鼠左肺同位肺移植，大鼠分为两组①对照组(n=16)：同位肺移植未行缺血预处理。②IP组(n=15)：同位肺移植前行缺血预处理。结果：IP组血气变化和受鼠生存期较对照组有明显改善。血管外水分含量亦较对照组明显减少。结论：缺血预处理不仅能减少移植肺损伤而且能改善移植肺功能不全。
Ischaemia preconditioning attenuates hypothermic ischaemia
-reperfusion related early graft dysfunction following
, 百拇医药
orthotopic lung transplantation in the rat
Zhang Xingyi Sun Mei Wang Rongyou
(Department of Thoracic Surgery1 and Pathology2,Second Clinical
College of the Bethune University of Medical Sciences,Changchun,130041,China)
〔Abstract〕 Objective:To investigate the effect of ischaemia preconditioning (IP) on function of the transplanted lungs.Methods:Rat orthotopic left lung transplantation (OLT) was performed using the cuff anastomosis technique.The rats were divided into two groups ① CONT (n=16):OLT without IP.② IP (n=15):OLT plus IP.Results:In IP group blood gas exchange and recipient survivals were significantly improved and extravascular water content decreased compared with CONT group.Conclusion:Ischaemia preconditioning not only ameliorates graft injury but inhibits graft dysfunction after OLT.
, 百拇医药
〔Key words〕 Orthotopic lung transplantation;ischaemia preconditioning
〔CLC number〕 R332 〔Document code〕 A
〔Article ID〕 0253-3707(2000)02-0162-04
Despite orthotopic lung transplantation (OLT) has become a successful therapeutic option for patients with end-stage lung disease,early graft dysfunction associated with ischaemia-reperfusion injury still remains unsolved and contributes to high morbidity and mortality rates in clinical OLT.Each graft inevitably sustains injury,namely,ischaemia and reperfusion injury during the time it is removed from the donor,stored in a preservation solution and transplanted into the recipient.The precise mechanism of the injury up to date is still poorly understood.Our study is to investigate the effect of ischaemia preconditioning on the early graft function after OLT in the rat.
, 百拇医药
1 Materials and methods
1.1 Animals and materials Male Sprague Dawley rats with an initial weight of 240～320 g were used.The animals were maintained on a standard rat diet with free access to tap water.All animals were not subject to fasting before surgery.The PA cuff (inside diameter:1.56 mm;length:2.0 mm) and the PV cuff (inside diameter:2.0 mm;lenght:2.0 mm) were made using polyethene catheters (Protex,Hythe,UK).
1.2 Anesthesia and surgical procedures With the aid of an operating microscope (×16 magnification),orthotopic left lung transplantation was performed based on the techniques described by Mizuta et al〔1〕 and us〔2〕 with some more important modifications.All surgical procedures were performed under anaesthesia with chloralhydrate (36 mg/kg body weight intraperitoneally) using a clean but non-sterile procedure.
, http://www.100md.com
1.3 Experimental groups The animals were randomly divided into two groups ①CONT (n=16):OLT absent from ischaemia preconditioning served as control. ② IP (n=15): OLT plus ischaemia preconditionging.
1.4 Orthotopic lung transplantation With the aid of transillumination of the neck allowing direct vision of the glottis,endotracheal intubation was performed with a 14- gauge angiocatheter following induction of anaesthesia.The lung was ventilated with room air using a rodent ventilator (tidal volume:5～10 ml;respiratory frequency:60～80 breaths per minute).The donor rat was placed in supine position. A bolus of 1000 U heparin was administered via penis vein.An entire anterior chest wall removal was performed via rib divisions on both sides of the spine,which ensures quick and adequate exposure of the mediastinum and the pulmonary hilus.In IP group,ischaemia preconditioning was performed by occluding the left main bronchus and the PA for 5 min followed by a period of 10 min reperfusion via occlusion releasing.Thereafter,organ was harvested following perfusion of the graft with lactate Ringer′s solution (4℃)。After attachment of cuffs to the PA and the PV was accomplished,the organ was then stored in a refrigerator at 4℃ for 4 hours.In the recipient operation,the left main bronchus was occluded using a 6-0 silk near to the carina and was transected proximally to the lung.The PA and the PV were cross-clamped together with an untraumatic Satinski clamp as near to the heart as possible.The native lung was removed.The donor lung was placed in the orthotopic position in the recipient thoracic cavity.The donor′s PV and PA attached to cuffs were inserted into their incised corresponding vessels of the recipient and were secured in place with 6-0 silk.Graft perfusion was restored by removal of the vessels clamping.Following the accomplishment of bronchial anastomosis using a 8-0 running suture (Prolene,Ethicon),the bronchial occluding sutures on the bronchus were removed and the graft was initially hyperinflated to eliminate atelectasis.
, 百拇医药
1.5 Ligation of the right pulmonary artery and bronchus Thirty minutes after the reestablishement of graft ventilation,ligation of the right PA and right bronchus in the recipient was performed,making the recipient rat totally dependent upon the pulmonary function of the transplanted lung.A single ligature was placed on the right hilar structures (PA and bronchus) by mediastinomy via the left thoractomy route,thus additional right thoractomy or sternotomy was not necessary.
, http://www.100md.com
1.6 Blood gas analysis In the recipient animals arterial blood samples were taken for the determinations of arterial oxygen tension (PO2) and arterial carbon dioxide tension (PCO2) at the time points of 0 min,10 min,20 min and 30 min after ligation of the right PA and the right bronchus.
Recipient survivals In the recipient animals,survivals were calculated at the time points of 0 min,10 min,20 min and 30 min after ligation of the right PA and the right bronchus.
, http://www.100md.com
1.7 Determination of extravascular water content At the end of the experiment,the pulmonary grafts were excised.A small section of the grafts was taken for histology.The remainings were weighted using an eletronic analysis balance to determine wet weight.The lungs were then placed in an heating oven at 80℃ for 24 hours and reweighed to determine dry weight.The extravascular water content of the lungs was then calculated by the formula:Extravascular water content=〔wet weight (mg)-dry weight (mg)/dry weight (mg)〕.
, http://www.100md.com
1.8 Satistics Results were expressed as x±s.Data were evaluated using the Mann-Whitney U test and Fisher′s exact test.P<0.05 was taken as showing statistical significance.
2 Results
2.1 In this study,31 rat OLT operations were accomplished using our modified techniques to the cuff anastomosis in the recipient operation.A 100% of operation successful rate was achieved.The average time for PA and PV cuff anastomosis was less than 8 min.
, 百拇医药
2.2 Blood gas exchange and recipient survivals The consequences of blood gas exchanges (PO2 and PCO2) and recipient survivals after ligation of right PA and right bronchus of the recipient were shown by table.A tendency of time-dependent changes of PO2 and PCO2 was presented in both CONT and IP group,indicating that functions of the transplanted lungs were to some extent improved.
2.3 Pulmonary extravascular water content Donor lung extravascular water content (7.6±0.5) in the CONT group was significantly higher compared with the IP group (8.5±0.4,P<0.05).This means that profound pulmonary edema occured in the transplanted lungs of the CONT group.
, http://www.100md.com
Table Time-course change of PO2,PCO2 (
±s,kPa) and survival rate (%) following ligation of the native right RA and bronchus on OLT recipients Time (min)
group
0
10
20
30
IP
CONT
, http://www.100md.com
IP
CONT
IP
CONT
IP
CONT
PO2
(x±s,kPa)
38.7±2.39
36.5±2.12
34.6±1.72
33.2±1.99
, 百拇医药
26.9±2.79
20.1±2.52
22.3±2.12
9.4±1.72
PCO2(x±s，kPa)
2.5±1.1
2.3±1.19
3.0±1.93
3.4±1.66
3.8±1.46
5.0±1.19
, http://www.100md.com
5.7±1.18
8.1±1.59
Survival(%)
100
100
100
96
86*
59
75*
33
*P<0.05 vs CONT
, 百拇医药
3 Discussion
There is a growing body of evidence to suggest that early graft dysfunction induced by ischaemia and reperfusion injury remains a major cause of morbidity and mortality in clinical lung transplantation.So far the precise mechanism underlying such injury during lung transplantation is unclear although numerous strategies have been explored to minimize such injury.Emerging evidence suggests that ischaemia preconditioning is protective against cardiac ischaemia-reperfusion injuries〔3～5〕.Moreover,the skin and liver graft injury may also be possibly attenuated by ischaemia preconditioning〔6,7〕.In this investigation,we found that the increase of extravascular water content was significantly blunted in the lung grafts exposed to ischaemia preconditioning,which are consistent with work of others〔8,9〕.In addition,we also found that ischaemia preconditioning significantly improved blood gas exchange and recipient survivals which are critical parameters for evaluation of graft function after OLT.It has been a well-established approach used in this study to fully evaluate funtion of the lung grafts by ligation of the contralateral PA and bronchus to mimic contralateral pneumonectomy,because it makes the recipient rat totally dependent upon the pulmonary function of the transplanted lung.However,this approach is not suitable for evaluation of long-term function of the transplanted lungs because majorities of the rectipient rats die within the first one or two hours following the contralateral pneumonectomy.In conclusions,the results of this study add to the growing body of evidence to suggest that ischaemia preconditioning affords to attenuate ischaemia-reperfusion related organ injury during transplantation.Moreover,the improved blood gas exchanges and recipient survivals by the phenomena of ischaemia preconditioning suggest that ischaemia preconditioning may enhance recovery from graft dysfunction following OLT.
, 百拇医药
〔Biography〕 Zhang Xingyi(1962-),male,Attending doctor,Medical doctor,Dunhua City,Cardiac and pulmonary transplantation.
〔References〕
〔1〕Mizuta T,Kawaguchi A,Nakahara K,et al.Simplified rat lung transplantation using a cuff technique〔J〕.Transplant Proc,1989,21:(1pt.3):2601～2602.
〔2〕Wang RY,Zhang XY,Li DF.Experimental study on application of Tripterygium wilfordii in orthotopic lung transplantation in rats〔J〕.J Bethune Univ Med Sci,1994,20(5):547～548.
, 百拇医药
〔3〕Mc′Nulty PH,Darling A,Whiting JM.Glycogen depletion contributes to ischemic preconditioning in the rat heart in vivo〔J〕.Am J Physiol,1996,271(6 Pt 2):2283～2289.
〔4〕Abd-Elfattah AS,Wechsler AS.Myocardial preconditioning:a model or a phenomenon〔J〕.J Card Surg,1995,10(4 suppl):381～388.
〔5〕Asimakis GK,Inners-McBride K,Medellin G,et al.Ischemic preconditioning attenuates acidosis and postischemic dysfunction in isolated rat heart〔J〕.Am J of Physiol,1992,263(3 Pt 2):887～894.
, http://www.100md.com
〔6〕Zahir KS,Syed SA,Zink JR,et al.Ischemic preconditioning improves the survival of skin and myocutaneous flaps in a rat model〔J〕.Plast Reconstr Surg,1998,102(1):140～150;discussion 151～152.
〔7〕Yin DP,Sankery HN,Chong AS,et al.Protective effect of ischemic preconditioning on liver preservation-reperfusion injury in rats〔J〕.Transplantation,1998,66(2):152～157.
〔8〕Du ZY,Hicks M,Winlaw D,et al.Ischemic preconditioning enhances donor lung preservation in the rat〔J〕.J Heart Lung Transplant,1996,15912):1258～1267.
〔9〕Li GH,Chen SX,Lou WH,et al.Protective effect of ischaemic preconditioning on donor lung in canine lung transplantation〔J〕.Chest,1998,113:1356～1359.
〔Received date〕 1999-01-04, 百拇医药
单位：
关键词：
白求恩医科大学学报000221 〔提 要〕 目的：为了证明缺血预处理对移植肺功能的影响。方法：使用套接吻合技术行大鼠左肺同位肺移植，大鼠分为两组①对照组(n=16)：同位肺移植未行缺血预处理。②IP组(n=15)：同位肺移植前行缺血预处理。结果：IP组血气变化和受鼠生存期较对照组有明显改善。血管外水分含量亦较对照组明显减少。结论：缺血预处理不仅能减少移植肺损伤而且能改善移植肺功能不全。
Ischaemia preconditioning attenuates hypothermic ischaemia
-reperfusion related early graft dysfunction following
, 百拇医药
orthotopic lung transplantation in the rat
Zhang Xingyi Sun Mei Wang Rongyou
(Department of Thoracic Surgery1 and Pathology2,Second Clinical
College of the Bethune University of Medical Sciences,Changchun,130041,China)
〔Abstract〕 Objective:To investigate the effect of ischaemia preconditioning (IP) on function of the transplanted lungs.Methods:Rat orthotopic left lung transplantation (OLT) was performed using the cuff anastomosis technique.The rats were divided into two groups ① CONT (n=16):OLT without IP.② IP (n=15):OLT plus IP.Results:In IP group blood gas exchange and recipient survivals were significantly improved and extravascular water content decreased compared with CONT group.Conclusion:Ischaemia preconditioning not only ameliorates graft injury but inhibits graft dysfunction after OLT.
, 百拇医药
〔Key words〕 Orthotopic lung transplantation;ischaemia preconditioning
〔CLC number〕 R332 〔Document code〕 A
〔Article ID〕 0253-3707(2000)02-0162-04
Despite orthotopic lung transplantation (OLT) has become a successful therapeutic option for patients with end-stage lung disease,early graft dysfunction associated with ischaemia-reperfusion injury still remains unsolved and contributes to high morbidity and mortality rates in clinical OLT.Each graft inevitably sustains injury,namely,ischaemia and reperfusion injury during the time it is removed from the donor,stored in a preservation solution and transplanted into the recipient.The precise mechanism of the injury up to date is still poorly understood.Our study is to investigate the effect of ischaemia preconditioning on the early graft function after OLT in the rat.
, 百拇医药
1 Materials and methods
1.1 Animals and materials Male Sprague Dawley rats with an initial weight of 240～320 g were used.The animals were maintained on a standard rat diet with free access to tap water.All animals were not subject to fasting before surgery.The PA cuff (inside diameter:1.56 mm;length:2.0 mm) and the PV cuff (inside diameter:2.0 mm;lenght:2.0 mm) were made using polyethene catheters (Protex,Hythe,UK).
1.2 Anesthesia and surgical procedures With the aid of an operating microscope (×16 magnification),orthotopic left lung transplantation was performed based on the techniques described by Mizuta et al〔1〕 and us〔2〕 with some more important modifications.All surgical procedures were performed under anaesthesia with chloralhydrate (36 mg/kg body weight intraperitoneally) using a clean but non-sterile procedure.
, http://www.100md.com
1.3 Experimental groups The animals were randomly divided into two groups ①CONT (n=16):OLT absent from ischaemia preconditioning served as control. ② IP (n=15): OLT plus ischaemia preconditionging.
1.4 Orthotopic lung transplantation With the aid of transillumination of the neck allowing direct vision of the glottis,endotracheal intubation was performed with a 14- gauge angiocatheter following induction of anaesthesia.The lung was ventilated with room air using a rodent ventilator (tidal volume:5～10 ml;respiratory frequency:60～80 breaths per minute).The donor rat was placed in supine position. A bolus of 1000 U heparin was administered via penis vein.An entire anterior chest wall removal was performed via rib divisions on both sides of the spine,which ensures quick and adequate exposure of the mediastinum and the pulmonary hilus.In IP group,ischaemia preconditioning was performed by occluding the left main bronchus and the PA for 5 min followed by a period of 10 min reperfusion via occlusion releasing.Thereafter,organ was harvested following perfusion of the graft with lactate Ringer′s solution (4℃)。After attachment of cuffs to the PA and the PV was accomplished,the organ was then stored in a refrigerator at 4℃ for 4 hours.In the recipient operation,the left main bronchus was occluded using a 6-0 silk near to the carina and was transected proximally to the lung.The PA and the PV were cross-clamped together with an untraumatic Satinski clamp as near to the heart as possible.The native lung was removed.The donor lung was placed in the orthotopic position in the recipient thoracic cavity.The donor′s PV and PA attached to cuffs were inserted into their incised corresponding vessels of the recipient and were secured in place with 6-0 silk.Graft perfusion was restored by removal of the vessels clamping.Following the accomplishment of bronchial anastomosis using a 8-0 running suture (Prolene,Ethicon),the bronchial occluding sutures on the bronchus were removed and the graft was initially hyperinflated to eliminate atelectasis.
, 百拇医药
1.5 Ligation of the right pulmonary artery and bronchus Thirty minutes after the reestablishement of graft ventilation,ligation of the right PA and right bronchus in the recipient was performed,making the recipient rat totally dependent upon the pulmonary function of the transplanted lung.A single ligature was placed on the right hilar structures (PA and bronchus) by mediastinomy via the left thoractomy route,thus additional right thoractomy or sternotomy was not necessary.
, http://www.100md.com
1.6 Blood gas analysis In the recipient animals arterial blood samples were taken for the determinations of arterial oxygen tension (PO2) and arterial carbon dioxide tension (PCO2) at the time points of 0 min,10 min,20 min and 30 min after ligation of the right PA and the right bronchus.
Recipient survivals In the recipient animals,survivals were calculated at the time points of 0 min,10 min,20 min and 30 min after ligation of the right PA and the right bronchus.
, http://www.100md.com
1.7 Determination of extravascular water content At the end of the experiment,the pulmonary grafts were excised.A small section of the grafts was taken for histology.The remainings were weighted using an eletronic analysis balance to determine wet weight.The lungs were then placed in an heating oven at 80℃ for 24 hours and reweighed to determine dry weight.The extravascular water content of the lungs was then calculated by the formula:Extravascular water content=〔wet weight (mg)-dry weight (mg)/dry weight (mg)〕.
, http://www.100md.com
1.8 Satistics Results were expressed as x±s.Data were evaluated using the Mann-Whitney U test and Fisher′s exact test.P<0.05 was taken as showing statistical significance.
2 Results
2.1 In this study,31 rat OLT operations were accomplished using our modified techniques to the cuff anastomosis in the recipient operation.A 100% of operation successful rate was achieved.The average time for PA and PV cuff anastomosis was less than 8 min.
, 百拇医药
2.2 Blood gas exchange and recipient survivals The consequences of blood gas exchanges (PO2 and PCO2) and recipient survivals after ligation of right PA and right bronchus of the recipient were shown by table.A tendency of time-dependent changes of PO2 and PCO2 was presented in both CONT and IP group,indicating that functions of the transplanted lungs were to some extent improved.
2.3 Pulmonary extravascular water content Donor lung extravascular water content (7.6±0.5) in the CONT group was significantly higher compared with the IP group (8.5±0.4,P<0.05).This means that profound pulmonary edema occured in the transplanted lungs of the CONT group.
, http://www.100md.com
Table Time-course change of PO2,PCO2 (
±s,kPa) and survival rate (%) following ligation of the native right RA and bronchus on OLT recipients Time (min)group
0
10
20
30
IP
CONT
, http://www.100md.com
IP
CONT
IP
CONT
IP
CONT
PO2
(x±s,kPa)
38.7±2.39
36.5±2.12
34.6±1.72
33.2±1.99
, 百拇医药
26.9±2.79
20.1±2.52
22.3±2.12
9.4±1.72
PCO2(x±s，kPa)
2.5±1.1
2.3±1.19
3.0±1.93
3.4±1.66
3.8±1.46
5.0±1.19
, http://www.100md.com
5.7±1.18
8.1±1.59
Survival(%)
100
100
100
96
86*
59
75*
33
*P<0.05 vs CONT
, 百拇医药
3 Discussion
There is a growing body of evidence to suggest that early graft dysfunction induced by ischaemia and reperfusion injury remains a major cause of morbidity and mortality in clinical lung transplantation.So far the precise mechanism underlying such injury during lung transplantation is unclear although numerous strategies have been explored to minimize such injury.Emerging evidence suggests that ischaemia preconditioning is protective against cardiac ischaemia-reperfusion injuries〔3～5〕.Moreover,the skin and liver graft injury may also be possibly attenuated by ischaemia preconditioning〔6,7〕.In this investigation,we found that the increase of extravascular water content was significantly blunted in the lung grafts exposed to ischaemia preconditioning,which are consistent with work of others〔8,9〕.In addition,we also found that ischaemia preconditioning significantly improved blood gas exchange and recipient survivals which are critical parameters for evaluation of graft function after OLT.It has been a well-established approach used in this study to fully evaluate funtion of the lung grafts by ligation of the contralateral PA and bronchus to mimic contralateral pneumonectomy,because it makes the recipient rat totally dependent upon the pulmonary function of the transplanted lung.However,this approach is not suitable for evaluation of long-term function of the transplanted lungs because majorities of the rectipient rats die within the first one or two hours following the contralateral pneumonectomy.In conclusions,the results of this study add to the growing body of evidence to suggest that ischaemia preconditioning affords to attenuate ischaemia-reperfusion related organ injury during transplantation.Moreover,the improved blood gas exchanges and recipient survivals by the phenomena of ischaemia preconditioning suggest that ischaemia preconditioning may enhance recovery from graft dysfunction following OLT.
, 百拇医药
〔Biography〕 Zhang Xingyi(1962-),male,Attending doctor,Medical doctor,Dunhua City,Cardiac and pulmonary transplantation.
〔References〕
〔1〕Mizuta T,Kawaguchi A,Nakahara K,et al.Simplified rat lung transplantation using a cuff technique〔J〕.Transplant Proc,1989,21:(1pt.3):2601～2602.
〔2〕Wang RY,Zhang XY,Li DF.Experimental study on application of Tripterygium wilfordii in orthotopic lung transplantation in rats〔J〕.J Bethune Univ Med Sci,1994,20(5):547～548.
, 百拇医药
〔3〕Mc′Nulty PH,Darling A,Whiting JM.Glycogen depletion contributes to ischemic preconditioning in the rat heart in vivo〔J〕.Am J Physiol,1996,271(6 Pt 2):2283～2289.
〔4〕Abd-Elfattah AS,Wechsler AS.Myocardial preconditioning:a model or a phenomenon〔J〕.J Card Surg,1995,10(4 suppl):381～388.
〔5〕Asimakis GK,Inners-McBride K,Medellin G,et al.Ischemic preconditioning attenuates acidosis and postischemic dysfunction in isolated rat heart〔J〕.Am J of Physiol,1992,263(3 Pt 2):887～894.
, http://www.100md.com
〔6〕Zahir KS,Syed SA,Zink JR,et al.Ischemic preconditioning improves the survival of skin and myocutaneous flaps in a rat model〔J〕.Plast Reconstr Surg,1998,102(1):140～150;discussion 151～152.
〔7〕Yin DP,Sankery HN,Chong AS,et al.Protective effect of ischemic preconditioning on liver preservation-reperfusion injury in rats〔J〕.Transplantation,1998,66(2):152～157.
〔8〕Du ZY,Hicks M,Winlaw D,et al.Ischemic preconditioning enhances donor lung preservation in the rat〔J〕.J Heart Lung Transplant,1996,15912):1258～1267.
〔9〕Li GH,Chen SX,Lou WH,et al.Protective effect of ischaemic preconditioning on donor lung in canine lung transplantation〔J〕.Chest,1998,113:1356～1359.
〔Received date〕 1999-01-04, 百拇医药