Early ischemic ultrastructural and histochemical alterations in the myocardium of the rat following coronary artery occlusion

ESPERIiWWTAL

Early

AXI)

~LOLECULAH

PATHOLOGY

30,

12%11:3

(l!)‘i!))

lschemic Ultrastructural and Histochemical in the Myocardium of the Rat following Coronary Artery Occlusion ROBERT

A.

KLONER,

MICHAEL ASD

Departments Peter

of Medicine Bent

Hrigham

PETER

C. R.

I~ISHREIN,

CAROL

Alterations

31.

HARE,

MAROKO

f’athoiogy, Harvard dredical Hospital, Boston, Jlassachusrtts

and

School

and

0211.5

The early ult.rastrrtctural and hiskt~hemkal changes in t.he myocardium following coronary artery occlusion in the rat, were studied. Heat+ from rats in which the left main coronary artery had been occlrtded for 3, 10, 20, 30, 40, 60, and 180 min were examined. Evidence of damage to myorardial cells and the microvasculature was present at 5 min of ischemia and berame progressively more severe with t.ime. Bot,h by electron microscopic and histochemiral technicpIes glycogen deplet,ion was observed within 10 min of the onset, of ischemia; nentral fat accumulated at 60 to 180 min of ischemia, predominately in apparently viable cells srtrrottnding the cells with more severe ischemic injnry. Loss of oxidative enzyme acativity, as determined histochemically, lagged behind evidence of nltrastructural evidence of tnitorhondrial damage. That s morphologic ischemic myocardial datnage occurs earlier in the rut ihan in dogs. Wavy fihers were identified in the ischetnir zone, but showed less trlt rastrttctrtral damage than other cells in the same microscopic fields. By his1 ochetttical i ec*httiqrtes, wavy fibers in the ischemic zone showed glycogett loss but still had oxidative enzyme activity 3 hr after the oc~c*lttsion.

IKTRODUCTION Coronary occlusion iI the rat recently was used as a model for testing the efficiency of interventions designed t.o protect. the ischemic myocardium (Maclean et d., 1976; Deloche et al., 1977; Jlaclean et al., 1977a and b; JIaclean et aE., 1973; Kloner et al., 197s). Accordingly, t)he goal of t’his invest)igation was to describe in detail the ult,rast,ructural and histochemical changes occurring during the early st,ages of myocardial ischemia in t’his model. Three features were of special int,erest, : (1) correlation between ultraskuctural and histochemical findings; (2) ultrastructural and histochemical features of wavy fibers which recently have been described as an early sign of myocardial infarction (Bouchardy and Majno, 1974) ; and (3) ultrastructural and histochemical characterization of cells which accumu1at.e lipid in order to verify whet,her this metabolic abnormalit,?orcurs in reversibly or irreversibly injured cells. Supported in part by Contract No. NOl-HV-33000 under the Cardiac Diseases Branch, Division of Heart and Vascular Diseases, National Heart, Lung, and Blood Inst,itute, NIH, NIH Grants No. HL-06370-16 and No. HL-20199-01, and a grant from the John A. Hartford Formdation, Hartford, Connect kilt. 129 0014-4800/79/020129-15$02.00/O Copyright 0 1979 by Academic All

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Press. Inc. reserved.

FIG. 1. Panel A: Nonischemir rat myocardium. Sarcomeres are in regibter and mitochondria (m) show tightly packed cristae. Glycogen is ablmdant. ii)intercalated disc; (t)-t tubule iX 10,000). Panel B: Ischemic myocardium after 5 min of coronary occlusion. Mitochondria are swollen as manifest by 10~3 of matrix density and separation of cristae. An amorphous intramitochondrial dense body (arrow) is present and glycogen is sparse. The capillary endothelial cell

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Experiments were carried out in albino-Sprague-Dawley male rats (Charles River Breeding Laborat,ory), weighing 250 t,o 300 gm. E’ollowing ether anest’hesia, the chest was opened in the 5th intercost,al space, the left main coronary art’ery occluded 1 to 2 mm from its origin, and t’he chest closed with a purse-string sut,ure as described in detail elsewhere (Rlaclean et al., 1978) based on previously used techniques (Johns and Olson, 1954 ; Deloche et al., 1972). Icor elect)ron microscopic studies, the occlusions were maintained for the following t’ime int,ervals: 5 min (four rats); 10 min (four rats); 20 min (t)hree rats) ; 40 min (two rats) ; 60 nun (four rat.s) ; 180 nun (14 rats). Two sham-operat.ed rat.s wit,hout. coronary occlusion were also st.udied 3 hr after thoracotomy. Following the allot.ed time interval, t,he rats were reanesthetized and carbon black was inject’ed intravenously to det,ermine whether a successful occlusion had occurred and to distinguish perfused Gssue (black areas) from tissue to which coronary flow was reduced (pink areas). Immediat8ely thereafter the hearts were excised. Transmural sections for electron microscopy were obtained from bot)h t,he nonischemic midventricular sept)um (control) and from t,hree sites in the ischemic anterior vent,ricular free wall. I~ollowing trimming of t,he endocardium and epicardium, Gssue from each site was cut into six or seven transmural slices in cold 1% osmic acid. The thickness of each slice was 1 to 2 mm. Slices were then dehydrated in graded alcohols, passedthrough propylene oxide and a 1: 1 mixture of propylene oxide plus Epon S12 overnight,. Two slices were chosen randomly for final embedding in Epon Sl 2. Sections approximately 1 p-thick were st,ained with toluidine blue for light~microscopy. Thin sections were mount,ed on plain copper grids and stained with aqueous many1 acet)ate and lead cit,rat,e and examined on a Philips 201 electron microscope. Histochemical studies were carried out in 51 rats. live, 10, 20, 30, 60, 120, and 180 min following coronary artery occlusion the rats were sacrificed, t,heir hearts excised, cut int,o four transverse slices, and frozen rapidly in 2-methylbutane cooled in dry ice to -60°C. Ten micron t,hick serial sections were cut) from slices from t,he midventricular level (4 t#o 4.5 mm from the apex) and stained by the following hist,ologic and histochemical t,echniques as previously described (Pishhein et al., 1977) : hemat.oxylin and eosin fur standard histologic examinat,ion ; oil red 0 stain for neut,ral lipids; periodic-acid Schiff (PAS)-diat,ase mebhod for glycogen; and succinic dehydrogenase (SDH), lactic dehydrogenase (LDH), and reduced nicotinamide adenine dinucleot)idase-diaphorase (NADH-diaphorase) reactions for the activit)y of oxidative mitochondrial enzymes (l’earse, 1968). RESULTS He&on

drlicroscopic

Findings

Nonischemic myocardium. Rlyocardial tissue from sham-operated rats and the nonischemic midventricular sept,um (which was always perfused with carbon black) of rats wit,h coronary occlusions showed normal myocardial cells and a normal microvasculature (ttig. 1A). RIyocardial cells had sarcomeres which were in register and the mitochondria contained tightly packed crist’ae. lschemic zone. By 5 min of coronary artery occlusion several morphologic changes were present in many ~11s from the isrhemic zone (,pink areas aft,er

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carbon black injection), however, these changes were patchy (l:ig. 1B). Mtochondria demo&rated early swelling with a decrease in matrix densit’y and separation of cristae. Occasional intramitochondrial amorphous densit,ies were present. The sarcoplasmic reticulum was sometimes swollen and the presence of wide I bands suggested myocardial relasat,ion or stretching. C:lycogen granules appeared less abundant than in control rats and t,herc was early nuclear c.lJromatiIr showed loss Of clumping and margination. The capillary endothelial cell.< OftrIl pinocytotic vesicles and occasionally there were small lOcalized arf’as of swelling as manifested by intraluminal, mcm1)ranc-1)ouI~d Ijlrl~s ( l’ig. 113). Stasis of ergt,hrocyt,es was common. By 10 or 20 min of ischemia the alJove changes were more uniform. Intramitochondrial amorphous dense boclicr were numerous (I:ig. 1C). In addition, the sarcolemmal membrane often was separated from the myofilaments 1Jy localized spaces presumably repre,qenting areas IJf intracellular edema ; in some areas the sarcolemmal membrane was discontinuous. Glycogen was sparse (,I’ig. 1C). The damage to t.he endot,helium also was more marked ; endothelial blehs were more prominent, than at 5 min and plate1et.s often appeared to be plugging gaps in the endot.helium (E’ig. 1D). Extravasation of erythrocytes was observed. IJsually, the same areas that had vascular damage also had myocardial damage. However, less frequentI! were noted areas where myocardial damage was present wit,hlout adjacent capillary damage and ~YJIlVeIWly areas whnc capillary damage was present, without, adjacent IllyO(!ardial damage. By 40 or 60 min of isrhemia intermyofibrillar edema and su1Jsar~ollemmal 1Jlebs were frequent, (I:ig. “A). ~Iitoc*hondrial swelling was prominent and intramitochondrial amorphous densities now were more numerous. III addit)ion, there was a second type of dense bcjdy whic*h was linear in nature and often appeared to be present bet,wern the menihranes of cristae (I;ig. 21~). By IS0 min the changes described above were more severe. JIitochondrial cristae were disrupted and mgofilaments often showed disarray (E’ig. 32). Amorphous dense bodies were less numerous in the very swollen mitochondria : intercalat)ed discs remained intact,. Wavy fibers were observed both by light and electron microscopy between 5 and 1SO min after occlusion. In the 1 p-thick toluidine-blue stained sections, these fibers appeared more intact and less swollen than many nonwav!’ fibers in the same field (Ipig. 8). Way. fibers often were most abundant, at the periphery of t,he area of ischemic damage. E’itJers which were wavy 1Jy light microscopy between 10 and IS0 min of ischemia showed glycogen loss and occasional I 1Jands when studied by electron microscopy (Icig. 3). In general, however, these cells had fewer abnormal mitorhondria, less int’racellular edema and less membrane disruption compared to ot(her ischemic cells in the same field. llyocardial cells which appeared only minimally injured between GO and IS0 min of ischemia, and which appeared to surround more severely damaged cells, often contained large numbers of osrniophilic droplets (which were not, tJound by a membrane) characteristic of neutral fat,. The lipid droplets wrare often in rlose association with mitoc~lJontlria ( I;ig. 1). Histochermkal .Vonischemic

ventricular

Fiwlinys

m~ocarrli~~~r~. In sllanl-opcratcld rats and in the nonischemic midseptum of rats in whic*h coronayv occlusion had 1)een carried out)

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FIG. 2. Panel A : Ischemk myocardilnn after 60 min of c~m~nnry occlusion. Il-ide I hands are present and mitoc*hondria are markedly swollen and umtaitr amorphous dense bodies. The SWWlemma1 membrane (s) of the caellon the left k lifted off of the myofilaments and the snrcolemmal membrane of the cell on the right i* largely degenerated. Glyc*ogen is sparse. (X l-I,700). Panel B : Isrhemic myocardiurn after 60 min of u)ronary orchGon. At this time, mitochondria developed linear densities (arrows) which appeared between cristae. (X41,100). Panel C: Ischemic myocardium after 3 hr of coronary c~c*clnsion.1Iitocshondria show massive swelling and my&laments (m) are in disarray. (~20,000).

glycogen and oxidat,ive enzymes were distributed homogeneously throughout8 t,he myocardium and t)here was no intracellular accumulation of neutral lipid.

(;lycogen. There was rapid depletion of glycogen from the ischemic left ventricular free wall, a finding which correlated well with the ultrastructural appearance. By 5 min there was mild glycogen deplet’ion which became more pronounced at, 10 and 20 min (Fig. 5A). Wavy fibers, which were usually located toward the periphery of the ischemic zone, showed glyrogen depletion as early as 10 min after coronary occlusion (Icig. GA). Neutral fats. By 60 to IS0 min Of ischemia, neutral fat was found to accumulate in the cells at the margin of the ischemic zone but not in the central area, which showed glycogen depletion (E’ig. :iB). This correlated with the ultra&r-uctural finding of lipid accumulation in thosr cells which appeared cithcr normal or only minimally injured at, this time. reduced nicotinanlidc Oxidati~~e P~~Z~~IIIP.S. I,ac.tic del~yclrogt~nasc (LIIH), (SDH). ()nly ntlenine dinuclrotidc (Snl)H)-tliapllc,ras~~, saccinic dt’~1~drOgcIlaSP

FIG. 3. Patlel ,4: Tschemic myocardium after 3 hr of coronary orclusioll. \Vavy filers (arrows) are c‘umpact, ~ulswollen a:ld show general lar architecttue. In contrast, there are several non-wavy cells which are swollen and architectlnxlly disrupted Cc) within the same field. CX2000). Panel B : Electron micrograph of a wavy fiber from isc*hemk myoc*arditun after 1 hr of u,rc,nary oc~lluion. Other than glyrogen sional I bands (arrows) the cell appears intact. C X9300).

preservation Toluidine depletion

of cellublue stain. and occaq-

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after 2 hr of ischemia did loss of cwzynw adivity first hwanw apparent,. There was further loss of act.ivity of all t,hr.w osidativc t~nzynws I)>, 3 hr iE’ig. SC). Many wavy fibers still contained enzynw wtivity after 3 hr of iwlicniia (Fig. (iIS).

FIG. -5. Panel A : Ten mirron section of the left ventricle stained for glycogen fullowing 20 mill of c~m~nary occlusion. The anterior left vent rkrd:tr free wall fright side) hhows glyogen depletion as manifest 1)~ paleness. ( X10: stained with periodic-acid BchitT-dinsta
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In t,lw wllx wit,11 lipid accumulat,ion at the margin of tlie ischemic zone, t,lle staining pattern was mow granular tlrun normal ljut not dwrcawd in intewity.

Tlic n~orpliologi~ alterations which otair in niyocardial cells during the first few hours of acute myot~ardial infarction cannot be appreciat,ed lq routine l~istologic techniques, hut can 1x1 appreciated 1)~’ electron microscopy (Caulfield and Iilionsky, 1959; Herdson et al., 19&S; .Jennings et al., 19.X; ,Jennings et al., 1960 ; .Jennings et al., 1969 ; Ik la Iglesia and Immh, 1972 ; Iilouer et al., 1974a). ,Jennings et al. (1965) dew1ilwd in detail t,hr changes wllkll occur in the posterior papillary muscle of the dog following occlusion of the left circumflex corcjnar> artery. Witmllin 15 nlin of isclwrnia I hands widen suggesting rnyofibrillar rclauation or stretching, glycogen granules are reduced in numkr, nuclear chrtmatin begins to marginate and there is occasional clearing of the mitoc*hoIidrial matrix density. By 30 nlin of (sol-onary occlusion, the I hands become even wider, the nuclear cliromatin margiiiation and clunipirig lwonie prominent, there is a further decrease in glycogeu and nlitochoudria stlo~~ furtlrer clearing of their matrix density. In addit.ion, i~ltt~l~nr~tjfil)rillar rldtma l~won~rs apparent. By 40 ruin of occlusiou, rnitoctlondrial swelling with disruption of txristac hecomes prominent,. In additiou, intramitocl~ondrial anlorphous mat,ris dense bodies appear and glycogen is absent. By 60 nlin, evidence of intrawllular edema is inauifest~ by lifting of the sarctjk~ninia~ nienibrant off Of the inyofi~anients, and in some cells sarcolemmal mcmhranw a1e disruptt>d. II:ndot,hrlial damage does not l)ecrjme prornitiriit until 60 to 90 min of iwlieniia (Iiloncr ef nl., 19741)). It wa, whereas the rodent collateral circulat,ion, which is lessw-t>11 developed, results in more uniform and nearly always

KLONER

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FIG. 6. Panel A: Ten mkron section of the heart stained for glycogen following 3 hr of coronary occlusion. \Vavy fibers (arrows) within the ischemir region show glycnogen depletion. Nonwavy fibers from the nonischemic zone (lower left) which stain for glycogen, are shown for comparison (X800, stained with periodic--acid Schiff-diastase method). Panel B : Ten mixon section of the heart stained for NAI)H-diaphorase artivit’y after 3 hr of occlusion. Shown is an area from ischemic myocardinm well within the zone of glycogen depletion. Ischemic nonwavy fibers are present in the upper half of the pklrrre and show enzyme depletion. Ischemic wavy fibers are present in the lower half of the picture and show oxidative enzyme ac+ivity. (X600; N.41)H diaphorase stain).

transmural infarctions. Since the rat model of ischemic injury is being used to test interventions which can alter myocardial infarct size (Deloche et al., 1977; JIaclean et al., 1976; Allaclean et al., 1!)78), and sinre ischcmic changes evolve

more: rapidly in this nIod~1, tlttl ititt~rvcntiot~s uttdcr stud?- sltould IJC ntltititiistt~rc~tl t3rlior aftt>r tltc ~orottaq~ o(~(~lusifbtt tliatt itt tlrtb dog tnod~~l. Hi~toCll~~tnic~al st.udics sllo\v(~l tltni loss of osidativcb (‘IM>‘III( acotivit !. IJWQIII~~ nljpartattt otil~ aftclr 120 to IS0 tiiitr of isc*li~ttli:b. Sitic*o ultrastruc~tural tlaniaffc to some mitoCliotidriu oc~c~urrt~tl ~ottsitlt~t~al~l~~ IZWI!~ (,i t IJ 10 tniti), t~lt~Ctrott micfirosottz~mes as copy apJJrars to 1W n1orc sctttsitivc tlrntt Itistl~c~lJ~~t~~istt~?- of osidnt.ivc an indicator of tkarly ntitoc~l~ot~drial damage. Thus, t4>. cltattgt~:: in n~itoC1totidrial structure arc identifitd prior to Coml~lrtc~ a1Jolition of ettz~matitr aCt,ivit,y. Tlte Correlation of ultrastructural and Itistoc~l~rtnic~al findings in this study slloived t.hat glycogcn depletion would Ixt demonstrated vclr?- taarl\- 1)~ Ijotlt tcc~llniqurs. N&ral fat was found to ltavc ac~cumulatcd after 60 to IS0 mitt of isc,llttnia 1~) 1JcJtll techniques in Cells \vlAClt ot,lltxrwise apparrd Itcarlnorntal 1)~ eleCtron microsCop?- and wltiCl1 surrounded mart’ st~riousl~ damaged c*clls. l’revious ltistochetnical and ult.rastructural stud& (Wart.tnart et nl., 1%X ; I\auftusn et al., 19% ; Suja et al., 1Wi; l’age and l’olimeni, 1977) al so leave noted a Collec6on of lipid in a ZOIW surrounding m~ocardial infar& Hrttc~t~, during tlir early phase of ischemia, lipid accumulates in mc~tat)oliCall~~ aljttormnl but pro1~at~l~ revc>rsiMq injured cells un the ~tc~ripltrr~ of tltc isCltc~ntic~ zotw. Use of the liistocl~t~mic~al staining trcliniqut~s on frozen src~tiotis does liavc limitations. ~Ientl~rant~ c~ltati~t~s may 0cCtir during tlic frt>rzitig and thawing: proc’css for ltist.olo~ic~ stCti0ti.s. Altlrou~lt sul)stratrl and t~nz~n~c~s may ltA< from tlte ~lld, t)lie gc~rit7al dis;tri1Jutiott of t~tiz~~tiic arid gl~~c~~p~n los.3 \vas always witliiti the distribution clef tltr oCc*ludrd corottar~ artcry. WC lravtl found that liistocltemiCa1 studies performc~d otl uttfiscd frozctt ~ec~tiotts result in less loss of glyCogtln from normal tissue than wIttan tlttb tissue is first fist,d and tltcn staifitxl for glycogen. Wavy fibers bavc heett iwtd as a ~~a~hOlOgi~~ niarlit~r of nlyocardial itifarc~tion (Boucltarcl~ and Alajtto, 1974). I’rc~sutual~l~~ t1tt.y art’ a rtxtlt of PtrctCl~ing of isChemic m\r.ofibers 1)~ adjaccttt CotttraCting c&ells. Dttailcd studies on their u1trastruCturc during t1rC first ft,\\- ~ICJWS of ischctnit\ art’ 1aCkittg. In titc prrsrnt study, wavy filers ~erc present as early as 5 to 10 min of ischcmia. hlthougll they were present in tltc isc~llrmic~ zone and did demonstrattl glyc~~gcrl loss and oc~ca,sional I t)arids, ultrastruCturnl1~ t,ltth>. appt~arrd ottl~ tninimall~ injured Compared to otlic3r Cells in t1tc sati~c field. ‘T1rcx wavy. fiixlrs \vtxrth less swoktt and ltad fewer altered init~Jc~lOtidria. Ht~nc~r, althougll the appraranc*e of \vavy fi1)rrs duriti~ tlie very tlarlq’ pltnse of (~orottar~ oc~ClIIsion may indiCate t)llah isellrmia has occurred, at. the time tllvse Cells \v(hte studi lvavincss \vas not! indic~ativc of irreversible damage as asstassed 1,~ ultraSstruCtural alterations. Even in autops; specimen.; from patients lvith myocardial infarctions \vav?- fi1)eri; were not al\vag,s necrotir (Bouchard~ and AIajno, 1974). Histocllemical data ott wavy fillers Confirmed t#he ultrast,ructural ol)servatiott tllat, their gl~cogt~n was depleted 1)ut also sho\ved t,llat many Ivav?; fi1~~~ still COIlt~aiIlNl osidativc enzymes at 3 hr of oc~Clusion indicating that, tltey \vt’re less severely damaged than otltclr Cells I\-lriClt had lost the,ie enzyrtws by 3 hr.

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P.\cq E., and PUIJMWI, P. I. (1977). IJtrastrIIctIIr:d chatlges in the khelnic zone bordering experimental infarcts in rat left ventricles. dLtu. J. Palhol. 87, 81-101. PKMSE, A. G. E. (1968). Histochemistry theoretical and ;2pplird. 3rd etlilioll. Vol. 1 and 2, Churchill Livingston. New York. W.~KTM.~N, MT. B., JENNINGS, It. B., YOKOY.IM.\, H. I)., and CI,\II \I.GH, C:. 1~. (19%). Fatty changes of the mytteardi~ml in early experirncnt al irlf:m.t ion. rZi&. f’