THE PATHOGENESIS OF HYALINE ARTERIOLOSCLEROSIS

BRIAN MCKINNEY * Department of Pathology, Royal Free Hospital, London

(PLATE CXXI)

THERE are two opposing schools of thought on the pathogenesis of hyaline arteriolosclerosis : one that i t is due to degeneration of arteriolar muscle, the other that its origin is hcematogenous.

Duguid and Anderson (1 952) studied the histology of hyaline arteriolosclerosis and came to the conclusionthat the hyaline material doesnot represent a degenera- tion of the vessel wall, as is commonly supposed, but is the product of a deposit of hzmatogenous origin from the lumen of the vessel. By readjustment of the endothelium, the deposit becomes incorporated in the wall and thus gives the familiar picture that has long been taken for a degeneration. Duguid’s views, as he himself pointed out, are in part a reversion to those held by Rokitansky and contrast sharply with current orthodox teaching, according to which hyaline arteriolosclerosis is the end result of a degeneration of the muscle of the arteriolar wall (Montgomery and Muirhead, 1954).

It would be hard to exaggerate the importance of a full understanding of the pathogenesis of hyaline arterioloselerosis, closely linked as it is with atheroma (Duguid, 1952), but, although it is some years since Duguid’s work appeared, there have been only two papers written in support of hie contentions. Still and Hill (1959) used methods similar to his own, based almost entirely on the study of sections stained with hzematoxylin and eosin, and one cannot say, except from morphological appearances, that the hyaline thickenings are of hmnatogenous origin or that they contain fibrin. Crawford and Woolf (1960) using suitably labelled human anti-fibrin sera have shown by fluorescent microscopy that hyalin contains a large amount of fibrin.

I n this study, emphasis has been laid on the histological identifica- tion of fibrin in the hyaline material of the arteriolar walls and an attempt has been made to show how hyalin is deposited as hsmato- genous material.

METHODS AND MATERIALS

The material consisted of (i) 50 spleens and kidneys obtained at routine necropsies on subjects of all age groups, but with a predominance of the middle- aged and elderly, (ii) the kidneys from the necropsies of 8 diabetic subjects and (iii) 19 spleens of normal males aged 20-30 yr obtained from the Department of Aviation Pathology, The R.A.F. Institute of Pathology and Tropical Medicine, Halton. It was fixed in 4 per cent. formaldehyde-saline as soon as possible after necropsy, dehydrated and embedded in paraffin wax. Sections were cut at 5 p.

Weigert’s fibrin stain, Mallory’s phosphotungstic acid-hzematoxylin, Lendrum’s (1 949) acid picro-Mallory, and

* Fresent address : The Department of Pathology, Makerere College, The University

J. PATE. BACT.-VOL. 83 (1962) 449 2 F

The following fibrin stains were used :

- College of Eart Africa, Kampala,, Uganda.

450 BRIAN McKIiVNE”

the fuchsin-miller method (Slidders, 1961). Hzmatoxylin and eosin, periodic acid-Schiff, haemalum-congo red, Gomori’s aldehyde-fuchsin elastin stain and Bielchowsky’s silver impregnation reticulin stain were also used.

To control the specificity of the fibrin stains a section of fibrinous pleurisy or fibrinous pericarditis, and a series of fibrin clots were put through with each batch of slides being stained to demonstrate fibrin. Fibrin clots were prepared according to the method of Gitlin and Craig (1957) except that the clots were made separately in 2-ml. containers and not on porcelain plates. Fibrin clots containing 5 , 2.5, 1.25, 0.625 and 0.312 per cent. of albumin were also prepared. Fibrinogen was prepared according to the method of Biggs and Macfarlane (1957) from out-dated transfusion blood ; and the thrombin used was obtained from Messrs Parke Davis ; the albumin was pure freeze-dried albumin obtained from the Lister Institute. London.

RESULTS Fibrin was identified by its characteristic staining reactions in

the hyalin in the walls of many arterioles showing hyaline arteriolo- sclerosis in both spleen and kidney. The fibrin deposits were found only in hyaline material and never in any other part of the vessel wall.

Phosphotungstic acid-haematoxylin, Weigert’s fibrin stain and the fuchsin-miller method all stained fibrin clots a uniform and constant colour, deep blue, blue and reddish purple respectively. The fibrinous exudates gave similar results. The picro-Mallory staining technique, however, gave very different staining reactions for different types of fibrin. Thus fibrin clots were blue, unless they contained 2 per cent. or more of albumin when they were red, a finding similar to that of Gitlin and Craig (1957). Fibrin in exudates was often stained a deep reddish-orange colour instead of the usually expected red.

Sections showing hyaline arteriolosclerosis and stained for fibrin with phosphotungstic acid-haematoxylin (figs. 1 and 2), Weigert’s fibrin stain (fig. 3) and by the fuchsin-miller technique (fig. 4) gave results for fibrin in hyalin similar to those for fibrin clots and fibrinous exudates.

With the picro-Mallory technique, however, fibrin in hyalin was stained either red or yellow. Fairly often, however, both types occurred together ; when this happened the red always surrounded the yellow and never vice versa.

Three types of fibrin deposits in hyalin were recognised, but there was no sharp distinction between them and they are separated merely for convenience of description.

(a ) Superficial deposits. In this type of lesion, hyalin consists entirely or almost entirely of fibrin of homogeneous appearance. The deposits are attached to the intima and may completely occlude the lumen (fig. 5). A lumen, however, usually remains and here the fibrin may be partly covered by endothelium (fig. 6).

Deposits of fibrin in these sites lie immediately below the intima and may constitute all the hyalin present. Often, however, there is below this layer a considerable amount of hyalin that contains no fibrin (fig. 7). Sometimes a thicker eccentric

( b ) Subendothelial deposits.

J. PATH. BACT.-VOI,. 83

HYALINE ARTERIOLOSCLEROSIS

FIG. 1 .-Phosphotungstic acid- hmnatoxylin. x 480.

FIG. 3.-Weigert’s fibrin stain, which does not give a clear differentiation of fibrin. ~ 4 8 0 .

PLATE CXXI

FIG. 2.-P.T.A.H. x245.

PIG. 4.-Fibrin stained a reddish purple colour. Fuchsin-miller method. x 245.

F I G . &-Upper arteriole completely occluded FIG. 6.--On left arteriole with small by hyalin. Centre stained yellow, periphery lumen partly lined by endothelium. red. Lower arteriole contains hyalin which The other arteriole shows fibrin stains blue, but few collagen fibres can be buried deeply within the hyalin of detected and no fibrin demonstrated. Acid the vessel wall. Acid picro-Mallory. picro-Mallory stain. x 480. x 245.

FIG. 7.-Hyalin containing fibrin sub- FIG. 8.-Thick eccentric deposit of intimally but not elsewhere. Acid hyalin in part, of vessel wall, picro-Mallory. x 245. displacing or pressing down into

the muscle. Acid picro-Mallory. x 245.

All figs. are of arterioles of spleen.

H YALINE ARTERIOLOSCLEROSIS 451

State of patients (no. in brackets)

Normotensive (44) . . . Hypertensive (25) . . .

No. of patients showing

5brin deposits

superficial subendothelial in %$: Of

no flbrin deposits

10 6 20 28

4 8 17 I 19

Frequency of deposits Fibrin deposits were found in the hyalin of arteriolar walls in 55

of the 69 spleens and 42 of the 58 kidneys examined. The table shows the frequency with which the above types of distribution occurred. These figures, however, have little significance beyond showing that the deposits can be demonstrated without difficulty or prolonged search. Only those deposits that gave the characteristic staining reactions for fibrin have been included as these staining reactions are rapidly lost as a deposit ages. This, and the fact that only relatively few arterioles were examined, show that these deposits are very common indeed.

Organisation of fibrin deposits During the examination of this material I was struck by some

peculiarities in the process by which the fibrin deposits become organised and was led to consider why the mechanism of organisation of fibrin in these small vessels should differ from the classical process of organisa- tion, as seen when a thrombus occludes a vein. On theoretical grounds two factors are possibly responsible : (1) the difference in pressure and (2) the difference in oxygen tension within the two types of thrombus.

In the case of an occluding thrombus the pressure in the interstices

H YALINE ARTERIOLOSCLEROSIS 451

deposit of fibrin can be seen, localised to one part of the vessel wall and displacing or pressing down into the muscle of the surrounding arteriolar wall (fig. 8). This type of deposit is seen particularly often near the division of the arteriole into small branches, or at bends in its course.

(c) .Fibrin in the depths of hyalin. Masses of fibrin situated deeply within the hyalin and surrounded by collagenous material were encountered in many of the vessels examined (fig. 6). They are composed of dense lumps or strands of fibrin and appear to be remnants of larger deposits such as are present in the lesions described in (a ) and (b).

Some arterioles have been seen that are hyalinised but contain no fibrin (fig. 5).

TABLE Incidence of fibrin in hyalin

Frequency of deposits Fibrin deposits were found in the hyalin of arteriolar walls in 55

of the 69 spleens and 42 of the 58 kidneys examined. The table shows the frequency with which the above types of distribution occurred. These figures, however, have little significance beyond showing that the deposits can be demonstrated without difficulty or prolonged search. Only those deposits that gave the characteristic staining reactions for fibrin have been included as these staining reactions are rapidly lost as a deposit ages. This, and the fact that only relatively few arterioles were examined, show that these deposits are very common indeed.

Organisation of fibrin deposits During the examination of this material I was struck by some

peculiarities in the process by which the fibrin deposits become organised and was led to consider why the mechanism of organisation of fibrin in these small vessels should differ from the classical process of organisa- tion, as seen when a thrombus occludes a vein. On theoretical grounds two factors are possibly responsible : (1) the difference in pressure and (2) the difference in oxygen tension within the two types of thrombus.

In the case of an occluding thrombus the pressure in the interstices

452 BRIAN McKJNNE Y

of the thrombus will differ little, if any, from interstitial tension else- where in the body and there will be little if any impediment to the organisation of the fibrin by the emigration of fibroblasts from the media into the substance of the fibrin deposits. In addition, the oxygen tension will be low in the centre of the thrombus in a vein, thus providing the physiological stimulus for organisation.

On the other hand, in the case of fibrin depositions on the intimal surfaces of arterioles, the effect of an appreciably higher pressure within the lumen of the arteriole has to be taken into account. In the super- ficial parts of the fibrin deposits the pressure will approximate to that of the arteriolar blood pressure and only in the deepest parts of the vascular wall will there be any appreciable fall below this level. In addition to the inhibitory effects of high interstitial tension there is the lack of the physiological stimulus of anoxia, for the fibrin deposit receives a rich supply of oxygen by diffusion from the arterial blood.

Histological investigations The description that follows is based on the study of a large number

of sections of blocks of spleen and kidney showing hyaline arteriolo- sclerosis stained to demonstrate fibrin. Stress has been laid on the fact that the picro-Mallory stain shows two distinct types of fibrin in hyaline material, whilst other fibrin stains show no such distinction ; the descriptions are based on the picro-Mallory stain.

Superjicial deposits. The hyaline material appears to be laid down at first on the surface of the intima and initially may even completely occlude the lumen of the arteriole. If this happens the lumen may become partially recanalised and the lining re-covered with endothelial cells. At an early stage the hyalin consists almost entirely of the yellow-stained variety, but often, even before i t has been endothelialised, it is stained red. This occurs at first around the edges of the yellow hyalin, but later the whole mass takes up the red stain of fibrin. Endothelial cells may begin to grow into the substance of the hyalin and cover the strands of fibrin for variable distances. As the fibrin consolidates many of the invading endothelial cells become flattened between the fibrin strands, but with the high oxygen tension they appear to remain viable, and it may be that some of these cells become converted into fibroblasts responsible for organisation of the superficial parts of the fibrin.

Organisation from the media. Fibrin deposits near the media may undergo organisation in a similar manner, but here fibroblasts can grow in directly from the media and the two processes of organisation may link up. Not infrequently, however, possibly because of high intravascular pressure and oxygen tension, the two processes fail to link up and the fibrin remains as an unorganised mass between the two zones of organisation. Such fibrin may long retain its typical staining properties, but may later lose its capacity to take up fibrin

H YALINE ARTERIOLOSCLEROSIS 453

stains, whilst showing no sign of organisation, although it takes up collagen stains weakly. This process may be repeated on several occasions so that successive layers of fibrin may be laid down on the vascular endothelium, and the amount of hyaline material increased.

Fibrin may also under suitable conditions penetrate the arteriolar endothelium and form a subendothelial layer which completely en- circles the circumference of the vessel. Or fibrin may be deposited in one section of the arteriolar wall where it may force the muscle outwards. These deposits appear to undergo a change similar to that in the material deposited directly on the surface of the endothelium.

One may postulate that if this process of fibrin deposition occurs repeatedly the media may eventually become atrophic and appear to have undergone " hyaline degeneration )', though in fact it has been displaced by fibrin of haematogenous origin.

DISCUSSION These results largely support Duguid's theory of the haematogenous

origin of hyalin. Hyalin appears to be laid down from the blood stream and to consist of fibrin initially, although it may contain, at least at first, some other haematogenous substances. It shows the characteristic amorphous appearance and staining reactions of fibrin. The organisa- tion of the fibrin usually takes place from both the endothelial and medial aspects, but very frequently this process does not extend very far into the fibrin, which may remain in situ for a considerable period and then break down into amorphous material which takes collagen stains weakly, but is not collagen.

Hyalin may also be laid down beneath the vascular endothelium, possibly as a result of the increased permeability of the endothelium to fibrin and plasma proteins. It is possible that a layer of hyaline material so formed may compress underlying muscle and subsequently cause atrophy. If fibrin is deposited below the intima of one part of the vessel wall a similar state of affairs may result, but the medial atrophy will then be localised to this area alone.

These findings provide support for Duguid in assuming the hamato- genous origin of hyalin, but do not agree completely with what would be expected from his views, in that they demonstrate (1) that the material may be laid down initially either within the vessel lumen or beneath the endothelium, ( 2 ) that i t is fibrin that is deposited initially and that (3) the material is partially organised, but largely remains as an amorphous material.

SUMNARY Sections of 69 spleens and 58 kidneys obtained at necropsy have

been stained by various methods to demonstrate fibrin in the hyalin of arteriolar walls. Hyalin consists initially of fibrin and plasma protein. The fibrin is gradually organised into collagen, or more usually into an amorphous collagen-like material. Hyalin appears to be laid down

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454 BRIAN McKINNE Y

from the blood stream, but though commonly deposited in a regular fashion on the intimal surface it may be laid down subintimally and in isolated sections of the arteriolar wall. Atrophy of the surrounding media and repeated deposits of hyalin may give the false impression that hyalin arises from degenerated muscle.

I would like to thank Professor Kenneth Hill for suggesting to me that I should investigate this subject ; without his help and advice this work would not have been carried out. Ny thanks are also due to the technical staff of the Histology Laboratory, Royal Free Hospital, for their work with many different staining techniques, and to the Photographic Departments of the Royal Free Hospital and Makerere College Medical School for assistance with the colour photography.

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GITLIN, D., AND CRAIG, J. M.. . LENDRUM, A. C. . . . . . MONTGOMERY, P. O’B., AND MUIR-

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