〔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。

Ischaemiapreconditioning attenuates hypothermic ischaemia
-reperfusion related early graft dysfunction following
orthotopic lung transplantation in the rat

Zhang Xingyi Sun Mei Wang Rongyou
( Department ofThoracic 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) onfunction of the transplanted lungs.Methods:Rat orthotopic left lung transplantation (OLT)was performed using the cuff anastomosis technique.The rats were divided into two groups① CONT (n=16):OLT without IP.② IP (n=15):OLT plus IP.Results:In IP group blood gasexchange and recipient survivals were significantly improved and extravascular watercontent decreased compared with CONT group.Conclusion:Ischaemia preconditioning not onlyameliorates graft injury but inhibits graft dysfunction after OLT.
〔 Key words 〕 Orthotopiclung transplantation;ischaemia preconditioning
〔 CLC number 〕 R332 〔 Document code 〕 A
〔 Article ID 〕 0253-3707(2000)02-0162-04

Despite orthotopic lung transplantation (OLT) has become asuccessful therapeutic option for patients with end-stage lung disease,early graftdysfunction associated with ischaemia-reperfusion injury still remains unsolved andcontributes to high morbidity and mortality rates in clinical OLT.Each graft inevitablysustains injury,namely,ischaemia and reperfusion injury during the time it is removed fromthe donor,stored in a preservation solution and transplanted into the recipient.Theprecise mechanism of the injury up to date is still poorly understood.Our study is toinvestigate the effect of ischaemia preconditioning on the early graft function after OLTin the rat.

1 Materials and methods

1.1 Animals and materials Male Sprague Dawleyrats with an initial weight of 240～320 g were used.The animals were maintained on astandard rat diet with free access to tap water.All animals were not subject to fastingbefore surgery.The PA cuff (inside diameter:1.56 mm;length:2.0 mm) and the PV cuff (insidediameter: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 anoperating microscope (×16 magnification),orthotopic left lung transplantation wasperformed based on the techniques described by Mizuta et al^〔1〕 and us^〔2〕 with some more important modifications.All surgical procedures were performed underanaesthesia with chloralhydrate (36 mg/kg body weight intraperitoneally) using a clean butnon-sterile procedure.
1.3 Experimental groups The animals were randomly divided intotwo groups ①CONT (n=16):OLT absent from ischaemia preconditioning served as control. ②IP (n=15): OLT plus ischaemia preconditionging.
1.4 Orthotopic lung transplantation With the aid oftransillumination of the neck allowing direct vision of the glottis,endotrachealintubation was performed with a 14- gauge angiocatheter following induction ofanaesthesia.The lung was ventilated with room air using a rodent ventilator (tidalvolume:5～10 ml;respiratory frequency:60～80 breaths per minute).The donor rat wasplaced in supine position. A bolus of 1000 U heparin was administered via penis vein.Anentire anterior chest wall removal was performed via rib divisions on both sides of thespine,which ensures quick and adequate exposure of the mediastinum and the pulmonaryhilus.In IP group,ischaemia preconditioning was performed by occluding the left mainbronchus and the PA for 5 min followed by a period of 10 min reperfusion via occlusionreleasing.Thereafter,organ was harvested following perfusion of the graft with lactateRinger′s solution (4℃)。After attachment of cuffs to the PA and the PV wasaccomplished,the organ was then stored in a refrigerator at 4℃ for 4 hours.In therecipient operation,the left main bronchus was occluded using a 6-0 silk near to thecarina and was transected proximally to the lung.The PA and the PV were cross-clampedtogether with an untraumatic Satinski clamp as near to the heart as possible.The nativelung was removed.The donor lung was placed in the orthotopic position in the recipientthoracic cavity.The donor′s PV and PA attached to cuffs were inserted into their incisedcorresponding vessels of the recipient and were secured in place with 6-0 silk.Graftperfusion was restored by removal of the vessels clamping.Following the accomplishment ofbronchial anastomosis using a 8-0 running suture (Prolene,Ethicon),the bronchial occludingsutures on the bronchus were removed and the graft was initially hyperinflated toeliminate atelectasis.
1.5 Ligation of the right pulmonary artery and bronchus Thirtyminutes after the reestablishement of graft ventilation,ligation of the right PA and rightbronchus in the recipient was performed,making the recipient rat totally dependent uponthe pulmonary function of the transplanted lung.A single ligature was placed on the righthilar structures (PA and bronchus) by mediastinomy via the left thoractomy route,thusadditional right thoractomy or sternotomy was not necessary.
1.6 Blood gas analysis In the recipient animals arterial bloodsamples were taken for the determinations of arterial oxygen tension (PO₂ ) andarterial carbon dioxide tension (PCO₂ ) at the time points of 0 min,10 min,20min and 30 min after ligation of the right PA and the right bronchus.
Recipient survivals In the recipient animals,survivals were calculated at the timepoints of 0 min,10 min,20 min and 30 min after ligation of the right PA and the rightbronchus.
1.7 Determination of extravascular water content At the end ofthe experiment,the pulmonary grafts were excised.A small section of the grafts was takenfor histology.The remainings were weighted using an eletronic analysis balance todetermine wet weight.The lungs were then placed in an heating oven at 80℃ for 24 hoursand reweighed to determine dry weight.The extravascular water content of the lungs wasthen calculated by the formula:Extravascular water content=〔wet weight (mg)-dry weight(mg)/dry weight (mg)〕.
1.8 Satistics Results were expressed as x±s.Data wereevaluated using the Mann-Whitney U test and Fisher′s exact test.P<0.05 was taken asshowing statistical significance.

2 Results

2.1 In this study,31 rat OLT operations wereaccomplished using our modified techniques to the cuff anastomosis in the recipientoperation.A 100% of operation successful rate was achieved.The average time for PA and PVcuff anastomosis was less than 8 min.
2.2 Blood gas exchange and recipient survivals The consequencesof blood gas exchanges (PO₂ and PCO₂ ) and recipient survivals afterligation of right PA and right bronchus of the recipient were shown by table.A tendency oftime-dependent changes of PO₂ and PCO₂ was presented in both CONTand IP group,indicating that functions of the transplanted lungs were to some extentimproved.
2.3 Pulmonary extravascular water content Donor lungextravascular water content (7.6±0.5) in the CONT group was significantly higher comparedwith the IP group (8.5±0.4,P<0.05).This means that profound pulmonary edema occured inthe transplanted lungs of the CONT group.

Table Time-course change of PO₂ ,PCO₂ (±s,kPa) andsurvival rate (%) following ligation of the native right RA and bronchus on OLT recipients

There is a growing body of evidence to suggestthat early graft dysfunction induced by ischaemia and reperfusion injury remains a majorcause of morbidity and mortality in clinical lung transplantation.So far the precisemechanism underlying such injury during lung transplantation is unclear although numerousstrategies have been explored to minimize such injury.Emerging evidence suggests thatischaemia preconditioning is protective against cardiac ischaemia-reperfusion injuries^〔3～5〕 .Moreover,theskin and liver graft injury may also be possibly attenuated by ischaemia preconditioning^〔6,7〕 .Inthis investigation,we found that the increase of extravascular water content wassignificantly blunted in the lung grafts exposed to ischaemia preconditioning,which areconsistent with work of others^〔8,9〕 .In addition,we also found thatischaemia preconditioning significantly improved blood gas exchange and recipientsurvivals which are critical parameters for evaluation of graft function after OLT.It hasbeen a well-established approach used in this study to fully evaluate funtion of the lunggrafts by ligation of the contralateral PA and bronchus to mimic contralateralpneumonectomy,because it makes the recipient rat totally dependent upon the pulmonaryfunction of the transplanted lung.However,this approach is not suitable for evaluation oflong-term function of the transplanted lungs because majorities of the rectipient rats diewithin the first one or two hours following the contralateral pneumonectomy.Inconclusions,the results of this study add to the growing body of evidence to suggest thatischaemia preconditioning affords to attenuate ischaemia-reperfusion related organ injuryduring transplantation.Moreover,the improved blood gas exchanges and recipient survivalsby the phenomena of ischaemia preconditioning suggest that ischaemia preconditioning mayenhance recovery from graft dysfunction following OLT.

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〔Received date〕 1999-01-04 (Zhang Xingyi,Sun Mei,Wang Rongyou)