Ischemic preconditioning limits infarct size following regional ischemia reperfusion in in situ mouse hearts

Transcription

1 Crdiovsculr Reserch 42 (1999) locte/ crdiores locte/ crdiores Abstrct Ischemic preconditioning limits infrct size following regionl ischemi reperfusion in in situ mouse herts, b, * Dine L. Miller, Donn M. Vn Winkle Deprtment of Anesthesiology, Oregon Helth Sciences University, Portlnd, OR, USA b Reserch nd Anesthesiology Services, P-8-ANES VA Medicl Center 710 SW US Veterns Hospitl Rod, Portlnd, OR USA Received 0 June 1998; ccepted 29 October 1998 Objective: Ischemic preconditioning hs been demonstrted in wide vriety of nimls, from dogs to rts. Experimentlly-induced murine myocrdil ischemi reperfusion hs been described, but ischemic preconditioning hs not been reported in mouse herts. To test the hypothesis tht mouse herts exhibit preconditioning-induced protection, experiments were conducted in nesthetized open chest mice subjected to regionl myocrdil ischemi reperfusion. Methods: Following brbiturte nesthesi the FVB nd C57BL/ 6J mice underwent trcheostomy nd were mechniclly ventilted. The hert ws exposed vi left thorcotomy performed with the id of dissecting microscope. A 7-0 silk suture on curved tper needle ws pssed under the proximl left nterior descending coronry rtery to form snre. Mice were then rndomly ssigned to receive either no preconditioning or preconditioning. All mice were subjected to 60 min regionl myocrdil ischemi followed by 2.5 h of reperfusion. Ischemic preconditioning (IP) ws induced with two (FVB mice) or three (C57BL/ 6J mice) cycles of 5 min coronry occlusion nd 5 min reperfusion. Control nimls did not receive preconditioning ischemi. Are-t-risk ws ssessed with fluorescent prticles. Infrct size ws ssessed with triphenyl tetrzolium chloride, nd is expressed below s percentge of the re-t-risk. Results: In FVB mice preconditioning reduced infrct size 49%, from % to % (P,0.05). In C57BL/ 6J mice preconditioning reduced infrct size by 66%, from % to % (P,0.05). Conclusion: From these dt we conclude tht the infrct limiting effect of ischemic preconditioning is demonstrble in murine herts Elsevier Science B.V. All rights reserved. Keywords: Ischemic preconditioning; Myocrdil infrct size; Mice 1. Introduction significnt dvntge in the study of crdic (ptho)- physiology: the vilbility of trnsgenic constructs. Al- Ischemic preconditioning hs been demonstrted in though there re few geneticlly mnipulted rt models, wide vriety of niml models of cute myocrdil is- the vst mjority of geneticlly engineered nimls re chemi reperfusion, including lrge species such s swine mice. However, the smll size of the mouse hert mndtes nd smller rodents such s rts [1 6]. Protection resulting minituriztion, nd specilized equipment nd supplies. from ischemic preconditioning is exhibited s decrese in We wnted to determine if myocrdil ischemic precondiinfrct size, decresed incidence of mlignnt rrhythmis, tioning could be demonstrted in mouse herts. Studies nd improved post-ischemic contrctile function. Ex- were conducted in nesthetized open chest mice subjected perimentlly-induced murine myocrdil ischemi re- to regionl myocrdil ischemi reperfusion. perfusion hs been described [7 12], but ischemic preconditioning hs not been reported in mouse herts. Despite their smll size, mouse herts offer one very 2. Methods *Corresponding uthor. Tel.: , ex 57404; fx: E-mil ddress: vnwinkd@ohsu.edu (D.M. Vn Winkle) Animls used in these studies were llowed ccess to Time for primry review 22 dys / 99/ $ see front mtter 1999 Elsevier Science B.V. All rights reserved. PII: S (99)00005-X

2 D.L. Miller, D.M. Vn Winkle / Crdiovsculr Reserch 42 (1999) food nd wter d libitum until induction of nesthesi. zinc cdmium sulfide prticles (through n 8-mm syringe With locl Institutionl Animl Cre nd Use Committee filter) into the left ventriculr lumen vi the pex. The zinc pprovl, ll nimls received humne tretment in com- cdmium sulfide prticles fluoresce bright yellow under plince with the Guide for the Cre nd Use of Lbortory UV light nd demrcte the re-t-risk s negtive Animls published by the US Ntionl Institute for Helth imge. Herts were then weighed, sliced trnsversely into (NIH publiction No. 85-2, revised 1985). 5 slices of 1-mm thickness, nd incubted in triphenyl tetrzolium chloride (TTC, 1% w/ v in sodium phosphte buffer, ph 7.4, 78C) for 20 min. Contrst between stined 2.1. Surgicl preprtion nd unstined tissue ws enhnced by incubtion of the hert slices in 10% formlin for 10 min. Hert slices Two strins of mice were used: FVB nd C57BL/ 6J. rrnged cephld surfce fcing upwrd on microscope Mice were obtined from Hrln (Cincinnti, OH, USA) slide nd then compressed slightly to uniform thickness nd studied t pproximtely 1 16 weeks of ge (24 6 of 0.62 mm, using nother microscope slide with four g). Mice were nesthetized with 90 mg/ g i.p. sodium cover slips (one t ech corner) s shims. The slices were pentobrbitl, nd nesthesi ws mintined with one i.p. photogrphed under UV nd full spectrum light nd the supplement if needed. After induction of nesthesi the imges projected to fill n pper. The outlines of mice were plced in supine position, trcheostomy the risk re (zinc cdmium negtive) nd the infrct re performed, nd the mice mechniclly ventilted. Ventil- (TTC negtive) were trced for ech slice, nd then tion rte ws breths/ min with tidl volume of 1 digitized using computer-ssisted plnimetry. The resultnt ml. End-tidl pco 2 ws continuously monitored to guide res were multiplied by slice thickness to clculte ventiltory djustments. In FVB mice blood pressure ws volume per slice, then summed. The sme individul monitored using til cuff nd cutneous pulse oximeter trced ll imges without knowledge of the experimentl probe distl to the til cuff. Core body temperture ws group. Ech imge ws trced three times nd verged. mesured vi rectl temperture probe nd mintined t For ech hert, infrct size is normlized to re-t-risk C with miniture heting blnket. An ECG ws monitored throughout the surgery nd experimentl 2.4. Dt nlysis protocol. Using dissecting microscope (Leic MS5) with 6. objective, left thorcotomy ws performed in the ECG ws recorded continuously on strip chrt recorder fourth intercostl spce. The medil spect of the incision (Model 400, Gould Instruments, Vlley View, OH, USA). ws extended crnilly to form flp tht ws retrcted to Mesurements of systolic pressure ws mde t bseline, expose the hert. A smll opening ws mde in the immeditely prior to the long CAO, immeditely before pericrdium nd 7-0 silk suture on curved tper needle reperfusion, nd t the end of reperfusion. ws pssed under the proximl left nterior descending The primry dependent vrible nlyzed to ssess coronry rtery nd both ends of the suture pssed through presence or bsence of crdioprotection ws infrct size. segment of flred PE10 tubing to form snre. Mice Becuse the mount of left ventriculr myocrdium tht were then rndomly ssigned to receive either no precondi- progresses to infrction depends upon the size of the risk tioning or preconditioning. zone, infrct size ws normlized s percentge of the risk zone. Becuse the risk versus infrct reltionship 2.2. Induction of ischemi displys non-zero intercept, we lso exmined the risk versus infrct reltionship for ech experimentl group. Ischemi ws induced by gently snugging the snre Dt nlysis ws performed with PC-bsed sttisticl tight, nd clmping the PE10 tubing with vsculr softwre pckge (CRUNCH 4, Crunch Softwre, Oklnd, bulldog clmp which ws supported on sling. Ischemi CA, USA). Differences between groups nd within time ws verified by epicrdil cynosis followed by blnching, points re ssessed with nlysis of vrince. Post-hoc nd ECG chnges. Reperfusion ws elicited by loosening testing between groups ws performed using the Newmn the snre, nd ws verified by epicrdil hyperemi. All Keuls test for multiple comprisons. Post-hoc testing mice were subjected to 60 min regionl myocrdil within groups is performed with the Dunnett test for ischemi followed by 2.5 h of reperfusion. Ischemic multiple comprisons. preconditioning (IP) ws induced with two (FVB mice) or three (C57BL/6J mice) cycles of 5 min coronry occlusion nd 5 min reperfusion. Control (C) nimls did not receive. Results preconditioning ischemi. Twenty-eight FVB nd fourteen C57BL/ 6J mice were 2.. Mesurement of risk nd infrct sizes rndomized into the study; twenty (ten C nd ten IP) nd fourteen (seven C nd seven IP) contributed to the finl At the end of reperfusion, the coronry snre ws dtset, respectively. Premture deths resulting from retightened, nd the risk re ws delineted by infusing progressive nd irreversible brdycrdi nd hypotension

3 682 D.L. Miller, D.M. Vn Winkle / Crdiovsculr Reserch 42 (1999) Tble 1 Hemodynmic vlues Group Bseline Pre- End- Begin End occlusion occlusion reperfusion reperfusion FVB Hert rte (bets/ min) C IP Systolic rteril pressure (mmhg) C IP C57BL/6J Hert rte (bets/ min) C IP Systolic rteril pressure (mmhg) C IP Dt re shown s men6s.e.m. There were no significnt differences either between or within groups; C, control; IP, ischemic preconditioning. occurred in two FVB mice (one C nd one IP). Six 4. Discussion dditionl FVB herts were excluded from nlysis due to technicl filures in delineting the risk zone. Ventriculr The mjor finding of the present dt is tht in situ fibrilltion did not occur in ny mice. mouse herts disply infrct limittion s consequence of Hemodynmic dt for the mice re shown in Tble 1 myocrdil ischemic preconditioning. There hve been no There were no differences between groups in hert rte or previous studies exmining myocrdil ischemic precondirteril pressure t bseline or ny other timepoint. Tble 2 tioning in mice. Recently, mesurement of infrct size nd Figs. 1 nd 2 show infrct dt. Non-preconditioned following experimentlly-induced myocrdil ischemi nd mice displyed mrked infrction following 60 min cor- reperfusion hs been described in murine in situ nd onry occlusion nd 2.5 h reperfusion. In FVB mice isolted herts [7 1]. In the in situ studies, mice were preconditioning reduced infrct size 49%, from subjected to permnent or reversible coronry rtery % to % (P#0.01, Fig. 1). In C57BL/ 6J ligtion nd infrct size ws ssessed with triphenyl mice preconditioning reduced infrct size by 66%, from tetrzolium. Hemodynmics in these studies were not % to % (P#0.01, Fig. 2.). Becuse reported [8,10,12,1]. In the isolted hert studies, herts there ws positive reltionship between bsolute infrct were subjected to globl ischemi reperfusion, nd protecvolume nd bsolute risk volume, nd this reltionship tion ssessed by chnges in post-ischemic contrctile exhibited positive x-intercept, we lso performed AN- function nd/ or infrct limittion. COVA nlysis of bsolute infrct volume with bsolute As noted by Kss et l., mny murine experimentl risk volume s the covrite. This nlysis lso showed preprtions devite mrkedly from the norml conscious tht preconditioning reduced infrct size in both FVB nd physiologic vlues of hert rte nd men rteril pressure C57BL/ 6J mice (P,0.01 both strins). ( bets/ min nd mmhg, respectively) Tble 2 Weights nd volumes Group Mouse wt. Hert wt. Risk Infrct Risk:hert Infrct:risk (g) (mg) (mm ) (mm ) Rtio (%) Rtio (%) FVB C IP ** ** * ** C57Bl/6J C IP ** ** C, control; IP, ischemic preconditioning; dt re shown s men6s.e.m. *, P#0.05 vs. C; **, P#0.01 vs. C.

4 D.L. Miller, D.M. Vn Winkle / Crdiovsculr Reserch 42 (1999) Fig. 2. As ws seen for FVB mice, infrct size ws lso smller in preconditioned C57BL/ 6J mice, whether expressed s infrct s per- centge of re-t-risk (top pnel) or bsolute infrct volume (bottom pnel). Empty symbols represent dt from individul herts, nd filled symbols indicte the group men6s.e.m. The dt point indicted by the rrow corresponded to hert with lrge risk zone (41 mm [], verge risk zone size525 mm ). C, control; IP, preconditioned. Levels of sttisticl significnce re indicted s follows: *, P (ANOVA);, P (ANOVA);, P (ANCOVA, risk volume s co-vrite). Fig. 1. Infrct size ws smller in preconditioned FVB mice, whether expressed s infrct s percentge of re-t-risk (top pnel) or bsolute infrct volume (bottom pnel). Empty symbols represent dt from individul herts, nd filled symbols indicte the group men6s.e.m. C, control; IP, preconditioned. Levels of sttisticl significnce re indicted s follows: *, P (ANOVA);, P (ANOVA);, P (ANCOVA, risk volume s co-vrite). [14]. This is certinly true in isolted murine herts which re not pced, in which the intrinsic hert rte is reported to be pproximtely 00 bets/ min [7,11]. Only one of ATP-sensitive potssium chnnels). However, severl the isolted murine hert studies reported utilized pcing, studies hve exmined endogenous crdioprotection in with hert rte set t 60 bets/ min [11]. As noted bove, mice by using trngeniclly induced over-expression of the in situ studies of murine myocrdil infrct size hve specific substnces. Mtherne et l. hve shown tht not reported hemodynmic vlues. Although we used crdic specific over-expression of the A1 denosine nesthetized mice, our presently reported hemodynmic receptor results in incresed time to ischemic contrcture vlues suggest tht the experimentl preprtion we em- nd improved functionl recovery following 0 min of ployed pproximtes norml physiologic prmeters firly globl ischemi reperfusion in isolted buffer perfused well. Hert rte ws nd bets/ min in the mouse herts. These dt suggest tht denosine functions FVB nd C57BL/ 6J mice, respectively, nd rteril pres- s n endogenous protectnt during ischemi in mice [11]. sure ws 961 mmhg (FVB). Similrly, over-expression of extrcellulr superoxide dis- It is not currently known whether the cellulr triggers mutse (EC-SOD) results in improved crdic function nd meditors of the infrct limiting effect of precondition- following brief (7 min) globl ischemi in working isolted ing in the mice re the sme s those described for other buffer perfused mouse herts [7]. Limittion of infrct size species (e.g., denosine, brdykinin, protein kinse C, following 0 min globl myocrdil ischemi hs lso

5 684 D.L. Miller, D.M. Vn Winkle / Crdiovsculr Reserch 42 (1999) been reported in murine herts over-expressing het shock References protein 72 [8]. Therefore, severl endogenous substnces tht re known to confer crdioprotection in other species [1] Liu GS, Thornton J, Vn Winkle DM, Stnley AWH, Olsson RA, (e.g., rbbit, rt, dog) re lso crdioprotective in mouse Downey JM. Protection ginst infrction fforded by preconditioning is medited by A1 denosine receptors in rbbit hert. Circulherts. It is likely tht ischemic preconditioning in mice is tion 1991;84: medited in mnner similr to tht used by herts of [2] Schott RJ, Rohmnn S, Brun ER, Schper W. Ischemic precondiother species. tioning reduces infrct size in swine myocrdium. Circ Res Our initil studies utilized FVB mice becuse of their 1990;66: lrger size nd clmer temperment. Once we demoninfrction in rt hert. Am J Physiol 1992;26:H1107 H1112. [] Liu Y, Downey JM. Ischemic preconditioning protects ginst strted tht preconditioning did limit infrct size in this [4] Gomoll AW. Crdioprotection ssocited with preconditioning in the strin, we exmined preconditioning in the blck C57 nesthetized ferret. Bsic Res Crdiol 1996;91:4 44. strin; this strin ws chosen becuse it is commonly used [5] McKen T, Mendenhll W. Comprison of the responses to hypoxi, for genetic mnipultion. The experiments with FVB mice ischemi nd ischemic preconditioning in wild mrmot nd lbortory rbbit herts. J Exp Biol 1996;199: used two cycles of preconditioning prior to myocrdil [6] Murry CE, Jennings RB. Reimer KA. Preconditioning with isischemi nd showed tht infrct size could be reduced by chemi: dely of lethl cell injury in ischemic myocrdium. 50%. In the second set of experiments with C57BL/ 6J Circultion 1986;74: mice, we hoped increse the difference between control [7] Chen EP, Bittner HB, Dvis D, Folz RJ, Vn Trigt P. Extrcellulr infrct size nd preconditioned infrct size by incresing superoxide dismutse trnsgene overexpression preserves postis- chemic myocrdil function in isolted murine herts. Circultion the preconditioning stimulus to three cycles. Our dt 1996;94: confirmed n incresed myocrdil protection with three [8] Hutter JJ, Mestril R, Tm EKW, Sievers RE, Dillmnn WH, Wolfe cycles of preconditioning, with infrct size reduced by CL. Overexpression of het shock protein 72 in trnsgenic mice 66%. We feel tht the greter difference in infrct size decreses infrct size in vivo. Circultion 1996;94: found with three cycles of preconditioning increses the [9] Yoshid T, Mulik N, Engelmn RM, Ho Y-S, Mgnent J-L, Rousou JA et l. Glutthione peroxidse knockout mice re suscepusefulness of the model. We did note difference in tible to myocrdil ischemic reperfusion injury. Circultion control infrct size between the two strins employed, with 1997;96: control infrct size being lrger in the C57BL/6J mice. [10] Bilik S, Geenen DL, Ssson IE, Cheng R, Horner JW, Evns SM et The reson for this difference in not known. Inter-strin l. Myocyte poptosis during cute myocrdil infrction in the physiologic differences hve been reported, but none mouse loclizes to hypoxic regions but occurs independently of p5. J Clin Invest 1997;100: redily explin vrition in infrct susceptibility [15]. [11] Mtherne GP, Linden J, Byford AM, Guthier NS, Hedrick JP. In summry, the present dt demonstrte tht in situ Trnsgenic A1 denosine receptor overexpression increses myocrmouse herts cn be preconditioned. The bility to pre- dil resistnce to ischemi. Proc Ntl Acd Sci USA 1997;94:6541 condition mouse herts permits the ppliction of trns genic technology to studies exmining the cellulr medil. Myocrdil ischemi nd reperfusion: A murine model. Am J [12] Michel LH, Entmn ML, Hrtley CJ, Youker KA, Zhu J, Hll SR et tion of ischemic preconditioning. Physiol 1995;269:H2147 H2154. [1] Li Q, Li B, Wng X, Leri A, Jn KP, Liu Y et l. Overexpression of insulin-like growth fctor-1 in mice protects from myocyte deth fter infrction, ttenuting ventriculr diltion, wll stress, nd Acknowledgements crdic hypertrophy. J Clin Invest 1997;100: [14] Kss DA, Hre JM, Georgkopoulos D. Murine crdic function. A cutionry til. Circ Res 1998;82: This work ws supported by VA Merit Review grnt [15] Doevendns PA, Demen MJ, de Muinck ED, Smits JF. Cr- (D.V.W.). diovsculr phenotyping in mice. Crdiovsc Res 1998;9:4 49.