laboratory and animal investigationsProtective Effects of IschemicPreconditioning on Donor Lung inCanine Lung Transplantation*Guohii Li, MD; Shengxi Chen, MD; Wanzhong Lou, MD; and ErXiong Lu, MD

Background: Myocardial ischemic preconditioning has been found to protect the myocardium.We hypothesized that lung ischemic preconditioning might enhance canine lung preservationand reduce allograft lung dysfunction after transplantation.Methods: Ten pairs of adult canines underwent left lung allotransplantation. Five donors weretreated with ischemic preconditioning (their left hilus clamped for 10 min and released for 15 min[group IP]), and five donors were not treated with ischemic preconditioning (group C). The donorlungs were flushed with 4°C Euro-Collins solution (ECS) and stored in the same solution for 2lAh, then transplanted to the recipient canines. The animals were observed for 1 to 2 h aftertransplantation. The lung venous blood of the recipient and donor lung tissue was collected justafter thoractomy and 1 h after reperfusion of the transplanted lung in both groups.Results: The numbers of polymorphonuclear leukocytes (PMNs) in the pulmonary venous bloodafter reperfusion were significantly higher in group IP than in group C (p<0.05). However, thenumbers of PMNs in lung interstitium under microscopy were less in group IP than in group C.The thromboxane B2, malondialdehyde, and mean pulmonary artery pressure contents were

significantly lower in group IP than in group C (p<0.05), and the superoxide dismutase and mixedvenous oxygen tension values were significantly higher in group IP than in group C (p<0.05).Histologic findings show less damage in group IP than in group C.Conclusions: The protective effects of ischemic preconditioning in conjunction with ECS flushand storage were superior to using ECS alone. The possible mechanisms were that ischemicpreconditioning inhibited the accumulation and activation of PMNs in lung tissue and reducedthe production of oxygen-free radicals. (CHEST 1998; 113:1356-59)

Key words: canine; ischemic preconditioning; lung protection; lung transplantation; polymorphonuclearAbbreviations: ECS= Euro-Collins solution; MDA=malondialdehyde; MPAP=mean pulmonary7 arteiy pressure;PMN=polymoiphonuclear leukocyte; Pv02=mixed venous oxygen tension; SOD= superoxide dismutase;TXB2=thromboxane B2

T ung transplantation has become an effective-*-* treatment for a variety of patients with end-stagelung disease.1 However, early allograft dysfunctioncaused by ischemia-reperfusion remains an impor¬tant problem to be solved.23

Preconditioning, a phenomenon first reported byMurry and colleagues,4 has been demonstrated to bethe most powerful endogenous protection in myo-

*From the Department of Cardiothoracic Surgery7, Xiangya Hos¬pital, Hunan Medical University, Hunan, PR China.Manuscript received June 16, 1997; revision accepted October 9,1997.

cardium. Our previous studies have shown thatpreconditioning improves the myocardial preserva¬tion even when combined with cold crystalloid car¬

dioplegia in animals and humans.56 We also foundthat preconditioning protected the lungs from severe

ischemia-reperfusion injury in a normothermicallyexperimental model in rabbits.7 Donor lung flushingand core cooling are the two main methods used forlung preservation.3 In the present study, we testedthe hypothesis that ischemic preconditioning mightalso enhance the donor lung preservation in conjunc¬tion with the hypothermic Euro-Collins solution(ECS) flush and storage.

1356 Laboratory and Animal Investigations

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Materials and Methods

Ten pairs of adult canines were divided randomly into group IPand group C. In each group, five canines served as donors, andfive canines were used as recipients. Ischemic preconditioningwas used to treat group IP, while group C received no ischemicpreconditioning. All animals received humane care in compliancewith the "Principles of Laboratory Animal Care" formulated bythe National Society for Medical Research and the "Guide for theCare and Use of Laboratory Animals," prepared by the NationalInstitutes of Health (NIH publication 85, 23, revised 1985).

(Space Lab Inc; 90303B). Lung tissue was prepared for lightmicroscopic and electron microscopic (Hitachi 600; Japan) ob¬servation. According to the histologic examination methods ofMurata et al,9 we recorded the numbers of alveoli injury andPMNs under a light microscope in a blinded manner.

Statistical AnalysisAll data were presented as mean±SEM. Comparisons between

groups and within groups were made by Tukey's test. Differenceswere considered significant when the value was <0.05.

Donor Procedure

Donor animals were anesthetized with IV thiopental (10mg/kg) followed by atropine (0.3 mg) and intubated with an

endotracheal tube. The lungs were ventilated with 100% oxygen(Excel 210; Ohmeda) at a tidal volume of 250 mL, at a rate of 16breaths/min, and 5 cm H20 positive end-expiratory pressure.After a median sternotomy, the superior and interior venae cavae,the ascending aorta, the trunks of the pulmonary arteiy, and thetrachea were isolated. Animals were heparinized (30 mg/kg)before insertion of a curved metal-tipped cannula (Sarns, Inc;Ann Arbor, Mich) through a purse string suture in the main

pulmonary arteiy, and ischemic preconditioning then was per¬formed on the left lung (clamped left hilus for 10 min, releasedfor 15 min by ligature). The control group did not have ischemicpreconditioning. Cardiac inflow was occluded by ligation of thesuperior and inferior venae. The proximal inferior vena was cut,and the left atrial appendage was amputated for decompressionofthe pulmonary flush. The lungs were perfused immediately ata pressure of 30 mm Hg with cold (4°C) ECS (60 mL/kg). At thesame time, the lungs were ventilated with 100% oxygen. Whenthe flushing was completed, the trachea was clamped at endinspiration, and the heart lung block was excised. The harvestedorgans were stored in 4°C ECS for 2lA h before implantation.

Recipient Procedure

The recipient canines were anesthetized in the same manner as

the donor canines. A femoral arterial line and a femoral venous

Swan-Ganz catheter were placed and continuously transduced.The donor left lung was separated from the heart lung block, andleft lung single allotransplantion was performed with standardtechniques.8 The lung allograft was topically cooled with ice slushduring implantation. After reperfusion of the allograft, the BPwas maintained at a normal level. The lungs were ventilated with100% oxygen. The canines wrere then observed for 1 to 2 h. Twoblood samples were taken separately, one just after the recipientleft thoractomy and the other after 1 h of transplanted lungreperfusion. The blood samples were collected from the leftpulmonary veins and were measured respectively for polymor¬phonuclear leukocytes (PMNs), blood gas analysis (CIBA Corn¬ing 288, Blood Gas system), superoxide dismutase/malondialde-hyde (SOD/MDA) (Jiang Zheng Biological Engine Institute;China), and thromboxane B2 (TXB2) (RIA East Asia ImmuneTechnological Institute; China). Mean pulmonary artery pressure(MPAP) was measured by a pulmonary 5F Swan-Ganz catheter

Results

There were no significant differences between thegroups with respect to donor weight, recipientweight, flushing time, storage time, and transplantedtime (Table 1).The number of PMNs was significantly higher in

group IP than in group C. The TXB2, MDA, andMPAP values were significantly lower in group IPthan in group C, and the SOD and mixed venous

oxygen tension (Pv02) values were significantlyhigher in group IP than in group C (Table 2).

Light Microscopic Observation

We found lung edema and alveoli injury and more

PMNs in the lung interstitium after reperfusion for1 h in group C but less lung edema and alveoli injuryand fewer PMNs in the lung interstitium in group IP(Table 3).

Electron Microscopic Observation

We found that RBCs and plasma had entered thealveoli. Most of the mitochondria in the type II cellwere swollen and vesicular in group C, but very fewmitochondria were vesicular in group IP (Fig 1).

Discussion

The key factor in successful lung transplantation isthe best protection of the donor lung. Currently, themethods used to protect the donor lung are stillunsatisfactory. We carried out lung ischemic precon¬ditioning with 4°C ECS flushing of the donor lung.The results show that ischemic preconditioning im¬

proved lung gas exchange, reduced pulmonary arterypressure, and reduced lung tissue injury after lung

Group

Table 1.Characteristics of Experimental GroupDonor Weight, kg Recipient Weight, kg Flush Time, s Storage Time, min Transplanted Time, min

C (n=5)IP (n=5)

10.6±1.710.8±1.9

10.9±1.211.0±1.5

120±9122±8

148±21150±23

64±768±8

CHEST/113/5/MAY, 1998 1357

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Table 2.The Values ofPMN, SOD, MDA, TXB2,Pv02, and PvC02 in Lung Venous Blood and MPAP*

Preischemia Reperfusion 1 h

PMN, X109/LGroup C

Group IPMDA, nM/LGroup C

Group IPSOD, NU/mLGroup C

Group IPTXB2, pg/mLGroup C

Group IPPv02, mm HgGroup CGroup IP

PvC02, mm HgGroup CGroup IP

MPAP, mm HgGroup C

Group IP

2.07:12.08:!

1.96:11.89:1

89.9:!87.8-1

854.2-1868.2-1

465.7-1460.2-1

39.0-141.2-1

18.0-118.7-1

0.390.42

0.410.52

:4.75.6

:69.4:70.2

:29.227.0

8.1:7.3

:2.2:2.3

1.52±0.081.84±0.lV

4.35±0.343.20±0.46f

121.5±14.9142.7±17.3f

1435.1±63.3955.4±72.8f

243.5±27.7417.7±30.8f

46.1 + 7.143.1±6.8

31.5+3.722.5±3.9*

*Data were presented as mean± SEM. PvC02=mixed venous carbondioxide tension.

fp<0.05 vs control (n=5).

transplantation. The protective effects of ischemicpreconditioning together with ECS flush and storageofthe donor lung were superior to using ECS alone.

Despite a large number of studies, the specificpathophysiologic mechanism of lung ischemic reper¬fusion injury remains unclear. However, it has beendemonstrated that endothelial dysfunction,10 neutro¬

phil activation,11 oxygen-free radicals,12 and plateletactivation are involved.13 The neutrophil appears to

play a main role in lung ischemia reperfusion inju-11,14

In our study, the numbers of PMNs in lunginterstitium under light microscopy were less ingroup IP then in group C. Ischemic preconditioningreduced the number of PMNs in lung tissue afterreperfusion for 1 h. Neutrophils can produce reac¬

tive oxygen metabolites, such as the superoxideanion, hydroxyl radical, and hydrogen peroxide, andpromote their adherence to the endothelium, whichcan damage the pulmonary endothelium.1516 Isch¬emic preconditioning also reduces the production of

Table 3.Quantification ofAlveoli Injury and PMNs inLung Interstitium

Group C Group IP Valu

Alveoli injury, %PMN (n/HP)

32.0±4.19.2±2.1

9.1±2.33.2±0.8

<0.05<0.05

Figure 1. Electron microscopic view of left lower lobe in bothgroups. Top, a: group C; RBCs and plasma entered the alveoli;the mitochondria in the type II cell were swollen and vesicular.Bottom, h: group IP; the mitochondria in the type I cell wrere

vesicular (stained with lead citrate and uranium acetate, originalmagnification X 6,000).

TXB2 after lung reperfusion for 1 h. The lungsappear to be a major site of thromboxane synthesis.Neutrophils may play a direct role in causing thegeneration of thromboxane in the ischemic setting.17The vasoconstrictive effects of thromboxane appearto be responsible for the pulmonary hypertensionfollowing ischemia.18 This was supported by our

experiment of a temporal relationship between the

1358 Laboratory and Animal Investigations

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rise in lung venous blood TXB2 and increase inMPAP. Observation through an electron microscopeindicates that TXB2 can indirectly affect lung trans-vascular fluid filtration through an already damagedmicrovascular barrier. We found that RBCs andplasma had entered alveoli and found lung edemaand alveoli injury in group C. Lung ischemic precon¬ditioning decreased exhaustion of energy, whichshowed that very few mitochondria were vesicularand swollen in group IP. Lung ischemic precondi¬tioning decreased oxidant damage after transplantedlung reperfusion for 1 h. The content of MDA ingroup IP was lower than that in group C, but thevalues for SOD in group IP were higher than thosein group C. Du et al19 reported that lung ischemicpreconditioning decreased thiobarbituric acid reac¬

tive substances formation in rat lung preservation,and Chen et al7 reported that lung ischemic precon¬ditioning increased production of SOD in vivo inrabbits. Their experimental results could account forour findings.The present findings may allow us to hypothesize

that lung ischemic preconditioning inhibits the accu¬

mulation and activation of PMNs in lung tissue, thusreducing the production of oxygen-free radicals andTXB2, and decreasing the consumption of SOD andlung tissue injury.2021 Wang and Lou22 reported thatheat shock preconditioning could protect oleic acidlung in rats and found that heat shock proteins were

produced after heat shock preconditioning for 24 h.Whether lung ischemic preconditioning can produceheat shock proteins is unclear; therefore we shallcontinue to carry out the research.

In summary, the protective effects of ischemicpreconditioning in conjunction with ECS flush andstorage of the donor lung are superior to using ECSalone. The possible mechanisms are that ischemicpreconditioning inhibits the accumulation and acti¬vation of PMNs in lung tissue and reduces theproduction of oxygen-free radicals.

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