ELECTRON MICROSCOPIC STUDYOF EXPERIMENTAL ATHEROSCLEROSIS IN THE RAT

WJ.S. STruL, MD.,* AND ROBEIT M. ONKAL, M.D.

From the Department of Pathology, Washington University School of Medicine,St. Louis, Mo.

Compared with the rabbit, little use has been made of the rat in thestudy of experimental atherosclerosis until the last several years. NManyattempts were made in the past to induce the condition in rats since itwas realized that this animal had several advantages over the rabbit.In general, the lesions produced by various dietary means were unsatis-factory and bore little morphologic relation to the human disorder.More recently, however, Wissler, Eilert, Schroeder and Cohen' andFilhios, Andrus, Mann and Stare,2 by feeding diets containing choles-terol, sodium cholate and thiouracil to rats, have been able regularly toinduce arterial lesions which are comparable to the human condition.

It has been found possible in this laboratory to produce grossly visibleintimal lesions in the rat aorta by a diet containing large quantities ofsaturated fat in the form of butter, but without thiouracil.3 The presentcommunication presents an electron microscopic study of the aortic le-sions so produced. Ultrastructural methods were used in the hope thatthey would reveal the modes of formation and progression of these le-sions, since it seems likely that study of the experimental disease inanimals will throw little light on the human condition until the patho-genesis of the experimental lesions is known.

MATEIAL AND METHODSFour groups of male albino rats of the Wistar strain were used. Group I, consisting

of 6 rats, starting weight 200 gin., was fed the basal diet for periods ranging from4 to i6 weeks. This diet contained, in per cent by weight: butter, 40; cholesterol, 5;sodium cholate, 2; casein, 20; sucrose, 22; alphacel, 5; Wesson's salt mix,' 4; andNutritional Biochemicals "Diet Fortification Mixture," 2. The principal atherogenicconstituents were butter, cholesterol and sodium cholate. Group II, consisting of 4rats of the same starting weight, were fed the basal diet with the addition of 0.3per cent propylthiouracil.' GroupM consisted of 4 rats which had been fed the basaldiet for io to 12 months, and group IV (normal controls) consisted of 6 rats, starting

Supported by a grant from the Wesson Fund for Medical Research and Education, andResearch Grant H-4o39 from the National Heart Institute, National Institutes of Health,United States Public Health Service, Bethesda, Md.

Presented in part at the 45th Annual Meeting of the Federation of American Societiesfor Experimental Biology, Atlantic City, N. J., April I3, I96I.

Accepted for publication, July 14, ig6I.* Viting Asistant Professor from the Royal Free Hospital, London, England.

21

STILL AND O'NEAL

weight 200 gm., which were fed laboratory chow. In addition, several normal weanlingrats and rats weighing I50 gm. were used to study aortic changes due to age.At the end of i month, 2 rats from group I and 2 from group IV (normal controls)

were killed. After 8 weeks and at intervals of 2 weeks thereafter i rat from each ofgroups I. II and IV were killed. Rats in group III were killed when 2 had been onthe basal diet for io months and 2 for I2.

Animals were killed under ether anesthesia by the injection of i per cent osmiumtetroxide fixative' into the left ventricle. The aorta was removed entire and itsadventitia dissected away. The vessel was opened longitudinally along its anteriorsurface, pinned out on cardboard and immersed in a bath of buffered osmium fixative.Fixative was changed 3 times during fixation time of i hour at 40 C. Transverse seg-ments from the aorta just below the renal arteries were then taken. These weredehydrated and embedded in a mixture of methacrylate consisting of 6 parts of butylto i part methyl.

All blocks were rapidly screened by phase sections. From promising blocks thinsections were cut on a Porter-Blum microtome and viewed in an RCA ENMU-3Celectron microscope. Lesions which appeared representative were thoroughly exam-ined by multiple sections.

RESULTSNormal Intima

The ultramicroscopic structure of the normal rat aorta has been de-scribed by others in some detail.' 8 However, for the purposes of thisinvestigation certain features found in the intimas of the normal controlanimals need to be emphasized. For example, a connective tissue spacewas consistently present between the endothelium and internal elasticlamina. It contained multiple microfibrils, an occasional mononuclearcell (Fig. i) and more rarely a lymphocyte. Exmination of the weanlingrats and those of I50 gm. indicated that this space increased in prom-inence with age and growth.

In all normal aortas examined, the internal elastic lamina displayedregular and distinctive focal areas where a crescent of elastic tissue frag-ments split off from the lamina and bulged into the subendothelial space.Within bulges there were elements of one or more smooth muscle cells.In other areas there was more irregular fragmentation causing breachesin the elastic lamina where tentacles of smooth muscle cytoplasm andcytoplasmic processes from endothelial cells passed and intimal andmedial connective tissue mingled.

Atherosclerotic AortaThe changes to be described in the intima of the rats fed the high-fat

diets are a composite of the lesions seen to a greater or lesser degree inall aortas examined and are probably representative of the events takingplace. No essential differences were seen between rats given thiouraciland those given the basal diet only. Any minor differences found arementioned in the text. Only a rough correlation between the morphologicappearance of the lesions of various sizes and the length of time theanimals were subjected to their respective dietary regimens was at-

22 Vol. 4o, 1No. I

ATHEROSCLEROSIS IN THE RAT

tempted. For the purpose of this report therefore, minimal lesions areassumed to be "early," while lesions of large size are considered "late."

Endothelial Alterations. The most striking early change was vacuola-tion of the endothelial cells. It involved large areas of the intima and wasnot obviously related to other changes. The degree of vacuolation varied,with only one or two small vacuoles arranged near the nucleus (Fig. 2 )in some, while others were distorted into bizarre forms by large numbersof vacuoles (Fig. 3). Many vacuoles appeared empty, a number heldfoamy gray content, while a few contained electron-dense granules orwere lined by similar material. This vacuolation, although widespread,was not always continuous, and scattered among vacuolated cells wereothers which showed no such change.

Whether or not vacuoles were present in the endothelium, a numberof cytoplasmic processes or hooklets often projected from the lumensurface (Fig. 4). These hooklets varied in size and number, some wereshort and blunt, while others were long and fragile. The latter weresomewhat more frequently found in the endothelium overlying accumu-lations of foamy macrophages (see below), but this association was notconsistent. In normal endothelium the short blunt type of hooklet wasencountered, but rarely.

Subendothelial Alterations. Normal intima contains a subendothelialspace of variable width bounded on its external margin by the internalelastic lamina. An early cnge in all the butter-fed animals was the per-ceptible widening of this space, as compared to the normal, by the deposi-tion of a granular-fibrillary substance. This change was variable in itsextent and severity, and bore no apparent relation to vacuolation of en-dothelium. The granular-fibrillary substance showed no identifyingmorphologic features, although in general it bore a striking resemblanceto the blood plasma which had been incidentally fixed and sectioned withthe aortic wall. No extracellular lipid was seen in the subendothelialspace at this stage. In the deeper part of the space, next to the internalelastic lamina, the granular deposit could be seen merging with thenormal collagen there. At this stage changes were not observed in the in-ternal elastic lamina or the medial tissue adjacent to it, and lipid orvacuoles denoting lipid were not seen in the smooth muscle. Crescenticelastic tissue formations combined with smooth muscle cells were presentin this area as they were in the normal.

Accumulation of Macrophages. It was not possible from our studyto determine at what stage in the process macrophages began to appearbecause even after an interval on the dietary regimen as short as 4 weeks(the earliest examined) a few foam cells dotted the subendothelial space.From 8 weeks onward, however, in all aortas of the butter-fed group,

Jan., I962 23

STILL AND 0'NEAL

with or without thiouracil, accumulations of foam cells were conspicuous.These accumulations were separated from each other by areas of intima,normal but for the early changes already described.

In what was taken to be an early stage in this accumulating process,a single line of 3 or 4 vacuolated macrophages was found in the suben-dothelial zone, partially surrounded or separated by the granular-fibrillary material already described (Fig. 5). At times the subendothelialspace was only as wide as the foam cells it contained, but if it were widerthe foam cells were invariably situated just under the endothelium, therest of the space being occupied by a granular packing substance.The degree of vacuolation of the macrophages varied, but in most,

almost all the cytoplasm was occupied by vacuoles. The content of thesestructures was similar to that of the endothelium and only rarely was avacuole even partly filled by electron-dense material. It seemed, how-ever, that a greater amount of electron-dense material was present inthe macrophages of animals fed the basal diet than in the macrophagesof those with thiouracil added. The macrophages varied in size; nonehad more than one nucleus at this stage, and we were unable to discernany mitotic activity.The accumulations of macrophages were surmounted by intact but

attenuated endothelium, the main cell body of which was often trian-gular. Invariably, strands of endothelial cytoplasm were seen stretchinglike roots into the subendothelial space and often separating adjoiningmacrophages (Fig. 6). It was common also to see an entire endothelialcell forming a wedge between macrophages, extending from lumen tointernal elastica, so that the cell assumed a vertical position in relationto the surface. Many of the endothelial cells, in whatever position, stillshowed pronounced vacuolation of their cytoplasm, and some showedcleft formations resembling, but much smaller than, the cholesterolclefts in ordinary histologic preparations.The granular-fibrillary material, surrounding cells in the suben-

dothelial space, in some places had a more definite fibrillary structure.This appearance was particularly noticeable between macrophages andsurrounding structures where the material was somewhat compact. Nodefinitive structural markings such as banding could be observed in thesefibrils, but they were thinner than the collagen fibrils at the base of thelesion and resembled fibrin in general appearance.

Pathogenesis of the .Mlacrophage Lesions. WAe were particularlv inter-ested in how these accumulations of macrophages occurred. In the earlystages of the investigation foamy macrophages were frequently seenamong the blood elements incidentally fixed and sectioned with the aorticwall. Consequently, smears of tail blood were examined, and these

24 V'ol. 40, N'O. I

ATHEROSCLEROSIS IN THE RAT

showed that numbers of such cells were circulating in the blood stream.It became clear that some of the fat-filled macrophages could penetratethe endothelium since they were in various positions in relation to theendothelial lining (Figs. 7 and 8). The edge of the endothelial cytoplasmat its interruption by a macrophage was usually rounded and bulbous,with intact external cell membrane, and there could be little doubt thatthe appearance represented actual penetration rather than artifactualrupture of the endothelium. It could not be discerned, however, whetherthis penetration was taking place at cell boundaries or not. Of coursethere was no indication as to the direction in which the macrophageswere traveling, but we were surprised at the frequency with which wesaw the process occurring. For the most part the penetrating macro-phages already contained cytoplasmic fat vacuoles, but occasionallymononuclear cells with little or no fat were seen passing through the wall.At times macrophages containing fat and mononuclear cells without

fat were found deposited on or clinging to the surface of the endothelium,apparently in the process of being surrounded by arms of cytoplasmfrom the endothelium (Fig. 9). WN'hen this occurred, the cytoplasmicprocesses of the endothelial cells appeared to interdigitate with similarprocesses belonging to macrophages. Some of these morphologic appear-ances were reminiscent of those occurring when cells pass through thewalls of inflamed capillaries.Y

Other Subendothelial Cells. In addition to foamy macrophages, othercells were occasionally found in the subendothelial space. Most werenonvacuolated mononuclear cells, and they were found in both exper-imental and normal intimas but in fewer numbers in the latter. Similarly,a number of smooth muscle cells, combined with crescentic fragments ofelastic tissue, were found situated astride or peripheral to the internalelastic lamina, as they are in the normal. Occasionally, one of these cellscontained a single empty vacuole. Besides these two types of cells, anoccasional endothelial cell was found in early lesions in the suben-dothelial space, apparently out of contact with the surface.

Progression of the Lesions. The intimal lesions increased in size andextent by two processes; namely, further accumulation of macrophages,often now in layers, and a steady increase in the granular-fibrillary pack-ing material. In some lesions one type of change was more prominentthan the other. We saw no macrophages in the process of division, but itwas common to find evidence of macrophage rupture and with this theappearance of extracellular electron-dense lipid particles scatteredthroughout the granular-fibrillary network. At the same time, it becameincreasingly difficult to visualize boundaries of macrophages, the cellsseeming to merge and form small clusters.

fax., z962 25

STILL AND 0'NEAL

Later Lesions. The foam cell-intimal lesions in the animals fed thebasal high-fat diet for io to I2 months were, as one would expect, largerand their focal character more striking. The intervening areas of intimawere, however, not devoid of foam cells; one or two were nearly alwayspresent. The endothelium continued to show vacuolation, and there wasstill evidence of widening of the subendothelial space by the collection ofgranular-fibrillary material mixed now with extracellular lipid particles.Generally, in the later lesions a greater proportional increase of extra-cellular components rather than of cells was apparent. In the foam cellspresent, it was noticeable that many of the vacuoles now contained moreelectron-dense material (lipid) in contrast to the predominantly emptyones in earlier lesions.At times, in the larger lesions, endothelial cells were found in the

depths of plaques nearer to the internal elastic lamina than to the sur-face (Fig. io). These cells often retained wings of cytoplasm on eitherside. Superimposed on them were foamy macrophages and intercellularmaterial covered by intact endothelium. Occasionally, what appearedto be remnants of red cells and fibrin were found at the surface of thelesions, with a thin layer of compact substance (fibrin) separating themfrom the blood, an appearance frequently seen in conventional histologicsections.3The internal elastic lamina at this stage appeared more frequently

ruptured than in the normal, and through these breaks intimal contentsstreamed into the upper layers of the media. Here smooth muscle cellswere separated, and their normal configuration disrupted, by largeamounts of granular-fibrillary material, electron-dense granules of lipid,and occasionally cholesterol clefts. Macrophages were not seen penetrat-ing into the media, but an increased number of smooth muscle cells werevacuolated.

DISCUSSION

Detailed studies on the pathogenesis of experimental atherosclerosishave been largely confined to the disease produced in rabbits by feedingcholesterol. There is almost general agreement that the significant lesionis the accumulation of lipid-filled macrophages in the intima, but thereare conflicting views on the origin of these cells. Leary10 believed thatthey came from the blood stream and actively penetrated the endo-thelium. He further believed their source to be the reticuloendothelialcells of other organs such as the liver. Rannie and Duguid," while hold-ing that the macrophages circulated in the blood, sought to show thataggregations of these cells were deposited on the endothelial surface andincorporated into the intima by endothelial overgrowth. Poole and

26 Vol. 4o, No. I

ATHEROSCLEROSIS IN THE RAT

Florey 12 had no evidence to support the latter concept, but showed quiteclearly that macrophages could penetrate endothelium. Others came tothe conclusion that foamy macrophages were derived from pre-existingsubendothelial cells,'3 or that the endothelium itself and migratingsmooth muscle cells were the precursors of macrophages.'The present investigation has indicated that in the rat 3 processes

were involved in the production of the initial intimal lesions. They are(in probable chronologic order) vacuolation of endothelial cells, deposi-tion of granular-fibrillary material in the subendothelial space, and theaccumulation of macrophages. These 3 processes appeared singly or incombination, suggesting that they were not necessarily dependent one onthe other.

In the rat, as in the rabbit,"1"2 macrophages with or without lipidvacuoles can be found not only penetrating the endothelium but alsoclinging to the surface. Although we have no indication at present as tothe direction in which the penetration is taking place, it seems quiteclear that at some stage, macrophages can become attached to the endo-thelium by interdigitation of their cytoplasmic processes with those ofthe endothelium. When this event occurs, the appearance often suggeststhat the macrophages are gradually covered by endothelial overgrowthas first described by Rannie and Duguid." Whether this attachment isalso preliminary to the other mechanism by which macrophages gather-namely, penetration-we are unable to conclude with the availabledata.

In the early stage of the lesion any lipid present appeared to be intra-cellular (that is within macrophages or endothelium). No extraceliularparticles in any amount were seen until the lesion had progressed towhere a number of foam cells were present. The principal source of thisextraceliular lipid appeared to be from ruptured macrophages, althoughsome could have come from endothelial vacuoles discharging their con-tents into the subendothelial space.The accumulation of nonlipid extracellular material in the suben-

dothelial space was as prominent as the accumulation of macrophages inthe composition of the intimal lesion. In the early stages (4 weeks) thismaterial was granular and resembled elements of blood plasma. Later itbecame fibrillar in structure and at this stage more nearly resembledfibrin than any other fibrillar substance. More exact proof of its identityhas not yet been obtained, but in early human aortic lesions, fibrin orrelated substances have been shown to be present by various tech-niques.'5'7 Its presence in the rat lesion should therefore occasion nosurprise.The function of the scattered nonvacuolated mononuclear cells found

27Jan., I962

STILL AND O'NEAL

in the normal and thickened intimas is obscure. In the very early stagesof the atherosclerotic process, appearances suggested that they ingestedsome foreign substance, but they were often found unaltered in appear-ance even while surrounded by lipid and other particulate matter. Sim-ilarly, there was no good indication that any displaced endothelial cellbecame transformed into a foamy macrophage. Their fate in the depthsof the plaque was, however, also not clear. Smooth muscle cells did showsome vacuolation, but this change was only conspicuous in later stages,and in our rats was never severe. We saw no evidence of these cells mi-grating to the intima; nor was there any evidence that foamy macro-phages penetrated the internal elastic lamina. However, invariably, asthe intimal lesion progressed, an increased amount of extracellular lipidwas found in the upper portion of the media.The apparent changes in the osmiophilia of the lipid in the vacuoles

with the age of the lesion is of some interest. From the evidence availableconcerning osmic staining of various lipids,18 this phenomenon wouldseem to indicate the presence of an increasing amount of unsaturatedlipid. Although the reason for this change in composition is not clear,it parallels the alterations occurring with age in human atherosclerosis'9and the variety of forms the lipid assumes is similar in some ways to thatseen in electron microscopic studies of the human disease.20There are some observational differences between this investigation

and electron microscopic studies of cholesterol-induced aortic21 andcoronary atherosclerosis I in the rabbit and in human atherosclerosis.20For example, in the latter two investigations the lipid was described asbeing contained for the most part within smooth muscle cells. Moreover,in neither of the animal studies was conspicuous vacuolation of the endo-thelium or the attachment of macrophages to endothelial surfaces andtheir movement through endothelium reported. It must be assumed thatfailure to see the latter was a matter of chance since it has been estab-lished by conventional histologic means that they occur in experimentalatherosclerosis in the rabbit.""12

It seems evident that the role of the circulating macrophage in the ex-perimental atherosclerotic process requires further investigation. Ourfindings suggest that these cells play an active part in picking up lipidand depositing it in the aortic intima. Further, the actual numbers ofthese circulating cells may bear some relation to the speed of formationof the intimal lesions. For example, preliminary electron microscopicstudies in rats given a diet similar to group I except for the substitutionof 40 per cent corn oil for butter indicate that although the endothelialcells contain lipid vacuoles, and free lipid particles appear in the suben-dothelial space, foamy macrophages are relatively scanty in the periph-

28 Vol. 4o, No. I

JaX., 1962 ATHEROSCLEROSIS IN THE RAT 29

eral blood as compared with the butter-fed group, and do not accumulatein the intima for a period up to 9 months. We have also found that thenumbers of these circulating macrophages are related to the level of theblood cholesterol.23

SUMARY

Early intimal lesions produced in the rat's aorta by a high fat dietwere studied with the electron microscope. It was found that after 4weeks on the diet the endothelium showed considerable vacuolation, andthe subendothelial space was broadened by an accumulation of granular-fibrillary substance. Those animals on the diet for 8 weeks or longershowed steadily increasing accumulations of lipid-filled macrophages inthe subendothelial space. Macrophages similar in appearance were foundin the circulating blood of the rats, and were seen clinging to the endo-thelial surface and apparently penetrating the endothelium.

Later lesions showed proportionally more granular-fibrillary materialthan macrophages and an increasing amount of free lipid particles as thelatter ruptured. Although endothelial cells became buried in theselesions, there was no indication that they, or smooth muscle cells, wereconverted into macrophages, nor did the pre-existing mononuclear cellsfound in the intima of normal rats appear to play an important part inthe production of the foam cell lesions.

It is postulated that the circulating macrophages play an importantrole in the formation of these intimal lesions in experimental dietaryatherosclerosis in the rat.

REFERENCES

I. WISSLER, R. W.; ERET, M. L.; SCHROEDER, M. A., and COHEN, L. Produc-tion of lipomatous and atheromatous lesions in the albino rat. Arch. Path.,I954, 57, 333-35I-

2. FILLIos, L. C.; ANDRuS, S. B.; MANrN, G. V., and STARE, F. J. Experimentalproduction of gross atherosclerosis in the rat. I. Exper. Med., I956, 104, 539-554-

3. O'NFAL, R. M.; STILL, W. J. S., and HARTRoFT, W. S. Experimental athero-sclerosis in the rat. J. Path. & Bact. (In press)

4. WESSON, L. G. A modification of the Osborne-Mendel salt mixture containingonly inorganic constituents. Science, I932, 75, 339-340.

5. THOMAS, W. A., and HARTRoFT, W. S. Myocardial infarction in rats fed dietscontaining high fat, cholesterol, thiouracil and sodium cholate. Circulation,I959, 19, 65-72.

6. DALTON, A. V. A chrome-osmium fixative for electron microscopy. (Abstract)Anat. Rec., I955, 121, 28I.

7. BucK, R. C. The fine structure of endothelium of large arteries. J. Biophys. &Biochem. Cytol., I958, 4, I87-I9O.

30 STILL AND 0 NEAL Vol. 40, No. I

8. KEEcH, M. K Electron microscope study of the normal rat aorta. J. Biophys.& Biochem. Cytol., I960, 7, 533-538.

9. WILLIAuMsoN, J. R., and GRisHAm, J. W. Leucocytic emigration from inflamedcapillaries. Nature, London, I960, I88, 203.

io. LE4aB, T. The genesis of atherosclerosis. Arch. Path., I941, 32, 507-555.II. RANNI, I., and DUGTD, J. B. Pathogenesis of cholesterol arteriosclerosis in

the rabbit J. Path. & Bact., 1953, 66, 395-398.12. POOLE, J. C. F., and FLowmy, H. W. Changes in the endothelium of the aorta

and the behaviour of macrophages in experimental atheroma of rabbits. J.Path. & Bact., 1958, 75 245-251.

13. DuFr, G. L., and McMnLAN, G. C. The pathology of atherosclerosis. Am. I.Med., I951, II, 92-108.

14. ALIScHuI, R. Selected Studies on Arteriosclerosis. Charles C Thoma, Spring-field, Ill., I950, p. 56.

I5. DUGUD, J. B. Thrombosis as a factor in the pathogenesis of coronary athero-sclerosis. J. Path. & Bact., I946, 58, 207-2I2.

I6. MOVAT, H. Z.; HAUST, M. D., and MoRE, R H. The morphologic elements inthe early lesions of arteriosclerosis. Am. J. Path., 1959, 35, 93-I01.

I 7. WOOLF, N., and CRAWFoRm, T. Fatty streaks in the aortic intima studied byan immuno-histochemical technique. J. Path. & Bact., I960, 8o, 405-408.

i8. THOMAS, W. A., and O'NEAL, R M. Electron microscopy studies of butter andcorn oil in jejumal mucosa. Arch. Path., I960, 69, 12 I-129.

I9. BoTTCHER, C. J. F.; WooDFoRD, F. P.; TER HAR ROMENY-WACilTER, C. C. H.;BOELSMA VAN HouTE, E., and VAN GENT, C. M. Fatty-acid distribution inlipids of the aortic wall. Lancet, I960, I, 1378-I383.

20. GEER, J. C.; MCGILL, H. C., Jr., and STRONG, J. P. The fine structure of humanatherosclerotic lesions. Am. J. Path.,196I, 38, 263-287.

2I. BuCm, R C. The fine structure of the aortic endothelial lesions in experimentalatherosclerosis of rabbits. Am. J. Path., I958, 34, 897-909.

22. PARKER, F. An electron microscopic study of experimental atherosclerosis.Am. J. Path., I960, 36, I9-53.

23. SIMON, R.; STILL, W. J. S., and O'NLAL, R M. The circulating lipophage andexperimental atherosclerosis. (To be published)

The thin sections used in this study were cut by Miss Alice Atchison. We are gratefulto her.

LEGENDS FOR FIGURESFiG I. Normal rat aorta. An endothelial cell (E) surmounts the subendothelial

space (arrow). The latter contains a mononuclear cell (M) as well as multiplemicrofibrils. X 25,000.

FIG. 2. Early (fourth week) changes in the endothelium of a butter-fed rat. Theendothelial cell contains a singe large vacuole. Hook-like cytoplasmic processesare developing from its surface. The subendothelial space is widened and filledby granular materiaL X 17,000.

ATHEROSCLEROSIS IN THE RAT 31

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FIG. 3. More extensive vacuolation of a single endothelial cell. Note the varietv ofthe vacuolar contents. none of which are densely osmiophilic. The subendothelialspace (S) is of normal dimensions. x I 5-500.

FIG. 4. N'umerous hook-like cytoplasmic processes are seen on the surface of thisvacuolated endothelial cell. An intracytoplasmic .~myelin figure type of lipidform is present (F). X I5 500.

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FIG. 6. Several foamy macrophages are present. covered by an attenuated vacuo-lated endothelium. One endothelial cell stretches vertically (arrow) while otherssend processes deep into the subendothelial space. X 9.00C.

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FIG. 7. A foamy macrophage in the process of passing through the endothelium. Theedges of the latter are marked by arrows. X 12.000.

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34 l ol. 40, No. I

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FIG. IO. A larger lesion from a rat on the butter diet for i 2 months. Top right isthe aortic lumen (L >. Bottom center is a buried endothelial cell with its cvto-plasmic processes at either side (arrows '. A cholesterol-like cleft is present inone macrophage (upper left). The internal elastic lamina is at the bottom, left(E). X 6.ooo.

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