Ocular Pathology of Diabetes Mellitus

MYASTHENIA GRAVIS

VOL. 67, NO. 1 REFERENCES

1. Osserman, K. E. and Teng, P.: Studies in myastlienia gravis—a rapid diagnostic test : Further progress with edrophonium (Tensilon) chloride. JA.MA 160:153, 1956. 2. Breinin, G. M. : Electromyograph—a tool in ocular and neurologic diagnosis: I. Myastlienia gravis. Arch Ophth. 57:161, 1957. 3. Glaser, J. S., Miller, G. R. and Gass, J. D. :

21

The edrophonium tonogram test in myasthenia gravis. Arch Ophth. 76:368, 1966. 4. Lancaster, W. B. : Detecting, measuring, plot­ ting and interpreting ocular deviations. Arch Ophth. 22:867, 1939. 5. Cronin, Mary L. : Practical application of the Lancaster red-green test. Am. Orthopt. J. 13:57, 1963. 6. Eaton, L. : Unpublished data.

OCULAR PATHOLOGY O F DIABETES M E L L I T U S MYRON YANOFF,

M.D.

Philadelphia, Pennsylvania This paper describes the histopathologic findings in eyes removed from patients with diabetes mellitus. The present concepts of diabetic retinopathy are reviewed with spe­ cial emphasis on retinal microangiopathy and the cystoid degeneration of the iris pigment epithelium.

right eyes obtained at autopsy from patients with diabetes mellitus.

MATERIAL AND METHODS

The histopathology of the retinal capillar­ ies, that is, diabetic retinal microangiopathy, consisted predominantly of the triad of (1) capillary microaneurysms, (2) a decreased ratio of capillary pericytes to capillary endothelial cells, and (3) capillary sheathing. Except for a rare fusiform type, the mi­ croaneurysms were saccular, appearing as small outpouchings from the side of appar­ ently viable, cellular capillaries (fig. 1). They were present predominantly in the pos­ terior segment of the retina but were found occasionally in the far periphery and in one case just posterior to the ora serrata. There was a random distribution in both the arteriolar and venular capillaries, with clustering around acellular, presumably occluded capil­ laries (fig. 2). Pericytes were strikingly ab­ sent from the sites of aneurysmal formation. Small, probably young, microaneurysms had thin walls made up of a proliferation of endothelinl cells. Older ancurysms had either thickened walls of PAS-positive material (basement membrane material) or totally ob­ literated lumens (fig. 1). Some of the larger

All whole eyes from patients with diabetes mellitus on file in the Laboratory of Oph­ thalmic Pathology at the Hospital of the University of Pennsylvania were reviewed, a total of 20, of which three were autopsy eyes. Hematoxylin- and eosin-stained sec­ tions were available through the axial por­ tions of the eye, including pupil and optic nerve, in all cases. Sections stained with both Best's carmine method for glycogen and the periodic acid-Schiff ( P A S ) reaction, with and without diastase, Masson's trichrome, Verhoeff-van Gieson elastic, toluidine blue, crystal violet, Alcian blue and col­ loidal iron, with and without hyaluronidase and/or diastase, oil red-O on frozen sections and Prussian blue were available in selected cases. Serial sections were performed on the entire axial portion of three eyes. In addi­ tion, trypsin-digested retinas were available for study from the posterior halves of 12 From the Department of Ophthalmology and Laboratory of Surgical Pathology of the Hospital of the University of Pennsylvania.

RESULTS

The main histopathologic findings are listed in Table 1. RETINA

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VOL. 67, NO. 1

OCULAR PATHOLOGY OF DIABETES

microaneurysms contained what appeared to be blood clots which were interpreted as thrombi (fig. 3). In areas where clusters of microaneurysms were noted, there often were seen abnormal loops or buds of capil­ laries showing fusiform thickening and ex­ tensive endothelial proliferation probably in­ dicating new vessel formation (neovascularization) (fig. 4 ) . The capillaries, whether or not they con­ tained microaneurysms, showed a wide­ spread loss, or death, of pericytes with a subsequent decrease in the normal one-toone pericyte to endothelial cell ratio (fig. 5). The greater the loss of pericytes per retina, the greater the number of microaneurysms per retina.1 The endothelial nuclei also showed changes consisting of mild pyknosis and irregularities and even an occasional loss or drop-out (fig. 5).

23

Fig. 1 (Yanoff). Retina trypsin-digested prep­ aration. Microaneurysms appear saccular, as outpouchings from side of capillary. Aneurysm on right, probably young, has thin wall with prolifer­ ated endothelial cells (tiny nuclei are leukocytes in lumen of aneurysm). Aneurysm on left, probably old, has its lumen obliterated by basement membrane material. (Periodic acid-Schiff and hematoxylin, X220.)

Fig. 2 (Yanoff). Retina trypsin-digested preparation. Microaneurysms clustered around acellular (presumably nonviable) capillaries (arrows). (Periodic acid-Schiff and hematoxylin, X4S.)

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AMERICAN JOURNAL OF OPHTHALMOLOGY

JANUARY, 1969

Fig. 3 (Yanoff). Retina trypsin-digested preparation. Large microaneurysms containing a thrombus (arrow). (Periodic acid-Schiff and hematoxylin, XllS.)

The capillary sheathing was caused by a thickening of the wall due to the deposition of PAS-positive material within the wall. So-called capillary arteriolar-venular shunts were found in those retinas showing exten­ sive diabetic microangiopathy (fig. 4 ) . These "shunts" were composed of abnormally di­ lated capillaries that ran an almost straightline course from arteriole to venule, apparent­ ly bypassing the rich capillary anastomotic bed. The walls of these dilated capillaries fre­ quently contained an increased number of endothelial cells. Small hemorrhages were commonly seen mainly in the middle retinal layers, especially in the inner nuclear layer and, to a lesser ex­ tent, the outer plexiform layer (fig. 6-A). Occasionally, nerve fiber layer hemorrhages or larger hemorrhages involving all layers were present (fig. 6, B and C).

Exudates or pools of eosinophilic homoge­ neous material, mainly in the outer plexi­ form layer of the retina, were encountered in all but three eyes (fig. 7). These collec­ tions had an oblique or tangential form sur­ rounding the fovea, contained within the nerve fiber layer of Henle, but elsewhere ap­ peared rounded or spherical in microscopic sections, though, as pointed out by Bloodworth,2 they actually have a widely rami­ fying, snakelike structure when looked at in three dimensions. The exudates stained viv­ idly with the oil red-O stain for fat, presum­ ably due to the presence of lipoproteins (fig. 8 ) . In many areas, macrophages (histiocytes) with foamy or clear cytoplasm were seen within and surrounding the exudates, most likely engulfing and removing the material. Cytoid bodies, the microscopic counterpart of clinically seen "cotton wool"

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25

Fig. 4 (Yanoff). A, Retina trypsin-digested preparation. Nondiabetic retina just posterior to equator. (Pediodic acid-Schiff and hematoxylin, X160.) B, Retina trypsin-digested preparation. Diabetic retina just posterior to equator. Abnormal loops or buds of capillaries with fusiform thickening and extensive endothelial proliferation, probably indicating intraretinal neovascularization. (Periodic acid-Schiff and hematoxylin, X160.) C, Retina trypsin-digested preparation. Endothelial proliferation, microaneurysms and neovascularization mainly centered around acellular (presumably nonviable) capillaries (narrows). The microaneurysms are randomly distributed in the capillary bed between arterioles (a) and venules (v). An anteriolar-venular "shunt" is shown (double arrow). (Periodic acid-Schiff and hematoxylin, x32.) (Reproduced from article by Yanoff1 in the New England Journal of Medicine.)

spots or exudates, although known to occur in diabetic retinas, 2 · 3 were not found in this study. Neovascularization of the retina appeared as endothelial proliferation under (external to) the internal limiting membrane of the retina with extension through this membrane onto the internal surface of the retina (fig. 9). Well-developed neovascularization, in­ cluding a fibrous component (retinitis proliferans), was invariably associated with a rupture of the internal limiting membrane and vitreous detachment. By means of serial sections, the fibrovascular membrane could

be traced from within the retina, through a break in the internal limiting membrane, into the potential space between this membrane and the posterior vitreous or onto the poste­ rior surface of the vitreous (fig. 10). A number of rather large vessels could be traced into and out of the retina. Contraction of these fibrovascular bands appeared related to retinal detachment in 10 of the eyes ex­ amined. When diabetic retinopathy was severe (exudates, hemorrhages and retinitis proliferans), the internal limiting membrane (basement membrane secreted by Miiller's

26

AMERICAN JOURNAL OF OPHTHALMOLOGY

JANUARY, 196')

Fig. 5 (Yanoff). A, Retina trypsin-digested preparation. Nondiabetic showing an approximate 1:1 relation of the smaller, darker, rounded pericytes (p) to the lighter, oval endothelial cells (e) (Periodic acid-Schiff and hematoxylin, χ325.) (Reproduced from article by Yanoff1 in the New England Journal of Medicine) B, Retina trypsin-digested preparation. Diabetic showing mild pyknosis and irregularities of endothelial cells, but mainly a loss of pericytes leaving an empty shell of basement membrane material (arrows) and resulting in a decreased pericyte-to-endothelial-cell ratio. The capillary walls are thickened by a periodic acid-Schiff-positive material (basement membrane). (Periodic acid-Schiff and hematoxylin, X32S.) (Reproduced from article by Yanoff1 in the New England Journal of Medicine.) cells) seemed thickened. Whether or not the membrane is indeed thickened will have to await proof by electron microscopy. The cuticular portion of Bruch's membrane (base­ ment membrane of retinal pigment epithe­ lium) appeared thickened ; sometimes drusen as well as large eosinophic, PAS-positive

masses within the outer retinal layers were formed. Most of the eyes, however, had ad­ vanced disease and this thickening of Bruch's membrane may be nonspecific. VITREOUS

In all cases of retinitis proliferans, and

Fig. 6 (Yanoff). A, "Dot" or "blot" hem­ orrhages seen as small hemorrhages within the retinal inner nuclear layer with some spread to outer plexiform layer. (Hematoxylin-eosin, X260.) B. Flame-shaped hemorrhage involves retinal nerve fiber layer. (Hematoxylin-eosm, χ260.) C, Large hemorrhage involving all retinal layers. (Hematoxylin-eosin, χ130.)

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OCULAR PATHOLOGY OF DIABETES

27

occasionally in its absence, the vitreous body was posteriorly detached. The proliferating fibrovascular tissue formed sheets and bands on the posterior surface of the detached vit­ reous. Frequently, collections of blood were present within the vitreous. CHOROID

Pathologic alterations in the choroidal vasculature due to diabetes are difficult to evaluate since these vessels so readily un­ dergo degenerative changes in nondiabetics. The impression was gained, however, that arteriosclerosis was more common in the 20 diabetic eyes than in nondiabetic eyes of comparable ages. In addition to the charac­ teristic lesions of arteriosclerosis (athero­ sclerotic plaques of large and medium arter­ ies and hylinazation and fibrosis of media of small arteries and arterioles), frequently PAS-positive material collected in the walls of arterioles, markedly narrowing and dis­ torting their lumens (fig. 11). PAS-positive material also thickened the walls of, and oc­ casionally obliterated, the choriocapillaris (fig. 12). Since the inner portions of the choriocapillaris contribute to the outer parts of Bruch's membrane and may be thickened in diabetics, and since, as pointed out above, the inner parts of Bruch's membrane, that is, the cuticular part or basement membrane of retinal pigment epithelium, may also be

Fig. 8 (Yanoff). Exudates in outer plexiform layer, similar to those shown in Figure 7, stain positively with fat stains, (oil red-O, X160.)

thickened, Bruch's membrane may appear quite prominent in histologie sections of dia­ betic eyes. CILIARY BODY

In all the eyes in which PAS-stained sec­ tions were available, there was a diffuse thickening of the basement membrane of the pigment epithelium of the ciliary body, par­ ticularly in the pars plicata (fig. 13). This thickening was so extensive in some of the eyes as to obliterate partially much of the vasculature in the ciliary processes (fig. 13). The thickening was diffuse and should not be confused with the patchy thickening found in many eyes of elderly nondiabetic patients. The basement membrane of the nonpigmented ciliary epithelium also seemed dif­ fusely thickened, even over the pars plana. This latter change, though, was more subtle and must await electron miscroscopic verifi­ cation. IRIS

Fig. 7 (Yanoff). Homogeneous, eosinophilic exudates (hard, waxy) present in retinal outer plexiform layer. (Hematoxylin-eosin, χΐοθ.)

Lacy vacuolization of the pigment epithe­ lium of the iris, especially the posterior layer, was present in eight (40%) of the eyes. In hematoxylin- and eosin-stained sec­ tions the vacuoles appeared either empty or filled with a very faint amphophilic, slightly granular material (fig. 14). Two of the three paraffin-embedded and serially sectioned

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VOL. 67, NO. 1

29

OCULAR PATHOLOGY OF DIABETES

Fig. 10 (Yanoff). Section of "tented" area of retina. A large vessel, cut tangentially, can be traced from the retina into the overlying fibrovascular membrane (retinitis proliferans). The internal limiting membrane of the retina dis­ appears at point where retina and fibrovascular membrane fuse. (Pe­ riodic acid-Schliff and hematoxylin, X44.)

eyes contained these vacuoles and various special stains were performed on multiple sections. T h e vacuoles were PAS-positive and stained vividly with Best's carmine method for glycogen; no staining occurred, however, if the sections were pretreated with diastase, thereby signifying the presence of glycogen in the vacuoles (fig. 1 5 ) . Nineteen of the eyes showed rubeosis iridis, accompanied by peripheral anterior synechias in 18. T h e rubeosis was well devel­ oped in most instances with subsequent ectropion uveae (fig. 16), but in a few eyes the fibrovascular membrane on the anterior sur­ face of the iris was quite thin and appeared only at the extreme periphery of the iris or the pupillary region, or both. Frequently, a n anterior chamber hemorrhage was associated with the filmy fibrovascular iris membrane.

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Fig. 11 (Yanoff). Choroidal arterioles (arrows) show collections of periodic acid-Schifï-positive ma­ terial in their walls, narrowing and distorting their lumens. (Periodic acid-Schiff and hematoxylin, X200.)

LENS

Sixteen of the eyes contained cataracts; the other four had had a cataract extraction

Fig. 12 (Yanoff). Choriocapillaris is largely ob­ literated by periodic acid-Schifï-positive material. (Periodic acid-Schiff and hematoxylin, X47S.)

Fig. 9 (Yanoff). A, The internal limiting membrane of the retina ends abruptly (arrows). Young, budding capillaries extend from the retina, through the gap in the internal limiting membrane, into the subvitreal space (v—posterior surface of vitreous). (Hematoxylin-eosin, Xl30.) B, Higher magnification of figure 9-A to show capillaries extending from within retina out into subvitreal space. (Hematoxylineosin, X200.)

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AMERICAN JOURNAL OF OPHTHALMOLOGY

JANUARY, 1969

Fig. 13 (Yanoff). A, Basement membrane of the ciliary body pigment epithelium is diffusely thickened. (Periodic acid-Schiff and hematoxylin, χ63.) Β, Marked thickening of basement membrane of ciliary process partially obliterating the subepithelial vasculature. The basement membrane of the nonpigmented ciliary epithelium (arroivs) also appears thickened. (Periodic acid-Schiff and hematoxylin, χ160.)

prior to enucleation. The cataracts were cor­ tical, nuclear and/or posterior subcapsular, except for one eye which contained a mature cataract (fig. 17). There was nothing specific about the cataracts; they appeared identical to the senile cataracts of the nondiabetic. DISCUSSION

A number of authors 2 ' 3 believe that the presence of retinal capillary microaneurysms has been overemphasized as the sine qua non of diabetic retinopathy. Microaneurysms have been found in association with venous thrombosis, macroglobulinemia, hyperten­ sion, uveitis, and other conditions.2'3 In one

Fig. 14 (Yanoff). Pigment epithelium of the iris shows lacy vacuolization involving both layers. The vacuoles appear empty in hematoxylin-and eosinstained sections (Hematoxylin-eosin, X375.)

study,1 retinal capillary microaneurysms were found in 10 of 11 diabetic patients but also were found in three of 35 nondiabetics. Nevertheless, though a single retinal capil­ lary microaneurysm is an inadequate basis for the diagnosis of diabetic retinpoathy, the retinal changes as a whole are fairly charac­ teristic of the condition. There have been many theories proposed to explain the histopathogenesis of retinal capillary microaneurysms.4 Those most pop­ ular in recent years include anoxia of the retina or anoxic swelling, or both, in the sur­ rounding retina,5 the U-shaped capillary kink theory6 and the "mural cell" disappear­ ance theory.4 The study of retinal vascula­ ture and of the proposed theories has been greatly facilitated by Friedenwald's 7 applica­ tion of the PAS technique to whole mounts of retinas, by the development of an injec­ tion technqiue, first by Michaelson and Steedman,8 and then by Ashton," and by the technique of retinal trypsin digestion of Kuwabara and Cogan.10 In light of the knowl­ edge derived from these advances, the cur­ rent theories of the histopathogenesis of reti­ nal capillary microaneurysms will be exa­ mined. The U-shaped capillary kink theory pro­ posed by Ashton" suggests that a small focal segment of the retinal capillary undergoes

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OCULAR PATHOLOGY OF DIABETES

31

Fig. 15 (Yanoff). A, Material in iris pig­ ment epithelial vacuoles is vividly periodic acid-Schiff-positive. (Periodic acid-Schiff, χ160.) Β, Material in iris pigment epi­ thelial vacuoles is vividly Best's carmine positive. (Best's carmine, X160.) C, After pre-treatment of the sections with diastase, no staining of the material within the vacu­ oles occurs with periodic acid-Schiff (Pe­ riodic acid-Schiff, χ63.)

degenerative changes leading to varicose di­ lations and the formation of a U-shaped kink; the limbs of the kink subsequently fuse, producing a saccular configuration or aneurysmal dilatation of one side of the reti­ nal capillary wall. However, in light of the studies employing the trypsin digestion tech­ nique,1·4 this theory is no longer tenable, for the evolution of a microaneurysm can be fol­ lowed from a tiny, thin-walled capillary outpouching to a large, thick-walled structure in whose genesis vessel kinking obviously does not play a major role. Indeed, Ashton 3 in a more recent paper indicated that the Ushaped capillary kink theory can only apply in some cases and that a diverticulum may arise on one side of the capillary without preceding loop formation. The "mural cell" disappearance theory proposed by Cogan, and co-workers4 is not so easily refuted. The small cell with a dark, round, or horseshoe-shaped nucleus and cytoplasmic processes which wrap around the retinal capillary wall external to the endothe-

lial cell, but completely embedded in base­ ment membrane, has been called by many names, for example, mural cell,10 pericyte,11 intramural pericyte,12 and perithelial cell,13 and has been the subject of much controversy.12·14'15 Ashton and Oliveira12 presented cogent arguments against using the term mural cell as proposed by Kuwabara and Cogan.10 They substitute the term extramural pericyte for pericytes found out­ side the vessel wall and intramural pericyte for "mural cell" or pericytes within the wall.12 Since Rouget11 originally described these cells and actually showed an excellent illustration of these cells in a retinal capil­ lary of a rabbit. I have adopted his original term of pericyte (even though the cells are not contractile as he thought). Any term, so long as the meaning is well understood, will suffice. The "mural cell" disappearance theory of retinal microaneurysmal formation4 stated that iiiicroaneurysms form where pericytes have disappeared. The loss of pericytes pre-

32

AMERICAN JOURNAL OF OPHTHALMOLOGY

JANUARY, 1969

pericyte disappearance is widespread, microaneurysms appear in clusters in scattered areas. One would expect a more random dis­ tribution if the pericyte loss factor was pri­ mary in their evolution. Furthermore, the microaneurysms are arranged about foci of acellular and presumably occluded capillar­ ies 3,4,10,18,19 Th e p r e s e n t study indicates that

Fig. 16 (Yanoff). A, Fibrovascular membrane present on anterior surface of iris (rubeosis iriclis) causing peripheral anterior synechia and secondary angle closure glaucoma (Hematoxylin-eosin, Xl2). B, Higher power of 16A to show rubeosis iridis and ectropion uveae. (Hematoxylin-eosin, XlOO.)

Fig. 17 (Yanoff). Mature cataract is present. The vitreous (arrows) is posteriorly detached, has a fibrovascular membrane on its posterior surface and contains blood. The retina (R) is partially detached. (Hematoxylin-eosin, χ6.)

sumably weakens the wall and a saccular outpouching or microaneurysm results. That pericytes disappear selectively in retinal cap­ illaries of diabetics has been confirmed by qualitative18 and quantitative1 observations in human retinas and in experimentally pro­ duced diabetes in dogs.17 The cause of this pericyte loss is unknown. Even though this

the clusters of microaneurysms are asso­ ciated with abnormal capillary loops or buds and capillaries with fusiform thickening and endothelial proliferation. The pericyte loss alone, therefore, cannot explain either the distribution of the microaneurysms or the associated vascular changes. Anoxia, or at least hypoxia, has been ac­ credited with playing an important role in the genesis of retinal capillary microaneu­ rysms.5 As stated above, microaneurysms are usually found in clusters about hypoxic areas. Perhaps the morphologic sequence is as follows : occluded capillaries followed by hypoxia and tissue necrosis and healing, ac­ companied by vascular dilatation and endo­ thelial proliferation in neighboring vessels. With the development of endothelial prolif­ eration there is a constant modeling and re­ modeling of the pattern of the newly formed endothelial tubes. Many of the first-formed capillaries are obliterated,20 but some retain an outpouching or aneurysm at the site of previous capillary formation. Or perhaps the microaneurysm is an abortive attempt at new vessel growth as suggested by the character­ istic endothelial proliferation. Wise 5 stated, "These globular and saccular buds are not ac­ tual aneurysms, but aborted attempts at neovascularization in response to the adjacent development of tissue factor X." A similar conclusion was reached by de Venecia (de Venecia, G.—personal communication) after studying several serially sectioned and trypsin-digested preparations of diabetic eyes and correlating them with serial colored and fluorescein fundus photos. He felt that mi­ croaneurysms with many endothelial cells, especially those that appear to be budding from vessels, represented abortive attempts

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OCULAR PATHOLOGY OF DIABETES

at neovascularization. Furthermore, as has been pointed out above, these areas of microaneurysmal formation are accompanied by new vessel formation. Interestingly, microaneurysms are also found in other hypoxic conditions such as venous thrombosis, macroglobulinemia and hypertensive retinopathy, all of which may be accompanied by new vessel growth. Bloodworth,2 however, did not think neo­ vascularization played a role in the develop­ ment of microaneurysms, but believed that simple dilatation or relaxation of the capil­ lary wall to form a thin-walled microaneurysm appeared to be the initial change in dia­ betic retinopathy. He further stated that this change is due to a degeneration of the sur­ rounding nervous elements. Unfortunately, there is no good evidence that the surround­ ing nervous tissue elements offer any sup­ port. Certainly vigorous shaking of the ret­ ina after removal of the perivascular neural elements with trypsin digestion does not alter the shape of the capillary tubes. Also, in both juvenile and senile retinoschisis,21 blood vessels practically devoid of all sur­ rounding support course in the inner wall of the schisis cavity without the formation of microaneurysms. In Pelizaeus-Merzbacher disease22 no microaneurysms are noted even though there is marked loss of the neuronal elements in the inner retinal layers. Ashton," who in 1951 proposed that microaneurysms are arrested new-vessel formations, in 1963 stated that this theory of new vessel growth can now be set aside.3 He further wrote, "the endothelial proliferation seen so fre­ quently within aneurysms is more likely to be a secondary manifestation and may arise in three ways: from circulatory stagnation within the aneurysm, or from increased traf­ fic of nutritional substances through the wall, or, more especially from endothelial migration into the aneurysm, for frequently the adjacent capillaries are denuded of en­ dothelial cells." The first two ways can prob­ ably be discounted since there is little proof to substantiate them. The last suggestion is

33

probably erroneous since the adjacent capil­ laries are denuded because, as Ashton9 him­ self has shown, they are occluded and pre­ sumed dead. More important is the fact that many capillaries that give rise to microaneu­ rysms have a markedly increased number of endothelial cells, most likely due to prolifera­ tion. Ashton 3 also discounted the "mural cell" disappearance theory and ended by saying, "One must seriously entertain the possibility that microaneurysms result from nothing more specific than stasis and engorgement of the capillaries, and it is in the cause of these factors that the explanation of their appear­ ance in any particular condition can be found." Perhaps, but the pericyte loss, capil­ lary basement membrane thickening, multi­ ple microwounds, and much greater fre­ quency of microaneurysms in diabetic reti­ nas than in other retinas with similar capil­ lary stasis and engorgement cannot be dis­ counted. This increased frequency of microaneu­ rysms in diabetics may very well be related to the generalized characteristic small blood vessel involvement in diabetes and also to the pericyte loss and basement membrane altera­ tion in the retinal capillaries. The role that the pericyte may play in modulating capillary wall integrity and in regulating the flow of nutrients to the surrounding retina still re­ mains to be determined. The development of capillary arteriolarvenular shunts that bypass whole segments of the capillary bed is a poorly understood phenomena. Cogan19 stated "The primary defect is loss of the mural cells that normally maintain retinal capillary tone and normally effect uniform distribution of blood through­ out the vascular bed. Loss of the mural cells and of the vascular tone results in channel­ ing of blood into a few distended capillaries that become the direct-line shunts between arterioles and venules." According to this in­ terpretation the bypassed capillaries undergo secondary occlusion and in turn account for microinfarcts. Cogan and Kuwabara 15 pointed out, however, that there is no evi-

34

AMERICAN JOURNAL OF OPHTHALMOLOGY

dence that mammalian pericytes are contrac­ tile; in fact, in vivo observations by Fried­ man, Smith and Kuwabara 23 failed to detect any change in retinal capillary caliber. Fur­ thermore, the diabetic capillaries have a thickened wall due to the increased thickness of the basement membrane.24"27 It seems un­ likely that the pericyte which has no myofibrils or collagen precursors in its cytoplasm could offer significant support. Also, as pointed out above, with trypsin digestion and vigorous shaking of the retina, the capil­ laries remain remarkably intact and ap­ pear to have rather rigid walls. It is more likely that rather than the shunts being pri­ mary and resulting in occlusion of surround­ ing vessels, they are secondary to the hypoxic stimulus of the surrounding occluded vessels, as suggested by Levene and associates.16 Dollery28 also pointed out that capillary arteriolar-venular shunts are un­ likely to be the cause of capillary closure, be­ cause they do not function as high-flow shunts. The influence of the shunt vessels in retinal circulation is unknown. Morphologi­ cally, since there is a direct arteriolar-venu­ lar connection bypassing the capillary bed, one would expect an accelerated retinal circulation time, whereas diabetics have in fact a delayed retinal circulation time.29·30 Hill and co-workers29 stated, "Despite their dilated appearance the flow rate of fluorescein does not seem increased but occasionally slowed, suggesting that the vessels do not act in any way as shunts, but are survivors of an obliterative process." Ferrer 30 showed de­ layed retinal circulation time in the patients in her Cases 15 and 16, long-standing diabet­ ics with advanced diabetic retinopathy, whereas her patients in Cases 11, 12, 13, and 14, long-standing diabetics without overt ret­ inopathy, had normal circulation times. Newly formed capillaries are very fragile and are more permeable to red blood cells and fluid than are mature vessels.20 This partially explains the high incidence of hem­ orrhages and exudates in diabetic retinas. Another factor is the tendency toward in­

JANUARY, 1969

competence of all diabetic retinal vessels, new or old, observed by Norton and Gutman,31 and also personally. The hemorrhages are located primarily in the inner nuclear and to a lesser extent the outer plexiform layers (forming "dot" and "blot" hemor­ rhages), probably due to the abundance of capillaries in the former area with spread to the latter; capillaries extend only into the outer plexiform layer to the level of the mid­ dle limiting membrane.32 Much less fre­ quently hemorrhages are present in the nerve fiber layer (flame-shaped hemor­ rhages). Exudates (waxy exudates seen clinically) are located predominantly in the outer plexi­ form layer. This location may be explained by the fact that with the constant leakage of proteins accompanied by fluid, the entire ret­ ina becomes edematous. The rich capillary bed of the inner retinal layers may be able to reabsorb much, if not all, of the fluid whereas the poorly vascularized outer retinal layers may have little reabsorptive capability with the resulting collections becoming the familiar "hard waxy exudates." Bloodworth,2 however, denies a causal re­ lationship between exudates and vascular disease, suggesting waxy exudates result from neuronal and glial cell degeneration "perhaps associated with some plasma pro­ teins." Were this true, one would anticipate a random distribution of exudates through­ out all retinal layers whereas exudates are found almost exclusively within the outer plexiform layer.33 It is more likely that both leaked proteins and glial-neuronal break­ down products are constituents of exudates which then are related histogenetically and morphologically to the surrounding blood vessels. The statement has been made,34 "True neovasculogenesis within the retina probably does not occur in the adult retina. What ap­ pears as new vessel formation with the oph­ thalmoscope is either a dilatation of pre­ formed channels to form shunt vessels or a proliferation of vessels on the surface of the

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OCULAR PATHOLOGY OF DIABETES

retina internal to the inner limiting mem­ brane." In another paper, Cogan and associates* said they found little evidence of intraretinal neovascularization. Histologically, however, the presence of intraretinal neovascularization has been demonstrated by Ballantyne,35 Ashton 3 ' 6 and others. Also, ToIentino, and co-workers36 have shown by biomicroscopic studies that new-formed glial-vascular tissue is first "intraretinal, in the level of the retinal vessels." They show that the proliferating tissue may break through the internal limiting membrane of the retina and grow along the posterior sur­ face of the vitreous in the potential space be­ tween vitreous and internal limiting mem­ brane. With vitreous contraction the glialvascular tissue may grow along the posterior surface of the detached vitreous. The pres­ ent study confirms these biomicroscopic ob­ servations. New vessel formation is initially intraretinal; subsequent growth results in extension through the internal limiting mem­ brane and further extension between this membrane and vitreous or along the poste­ rior surface of a detached vitreous. Ballan­ tyne35 noted that as intraretinal neovascular tissue approached the internal limiting mem­ brane, the latter seemed to melt away as if by lysis. Just as the altered glomerulus of a dia­ betic with its thickened basement membrane will leak protein and water, so may the al­ tered retinal capillaries. Fluorescein leakage has been demonstrated even in areas with­ out ophthalmoscopically visible anatomic changes, observed by Norton and Gutman,31 and also personally. Along with the in­ creased capillary permeability and resultant retinal edema, small vessel disease character­ istic of diabetes mellitus, lipemia and other as yet unknown factors may result in occlu­ sion of small vessels, causing tiny areas of necrosis. Hill and associates,29 utilizing arte­ rial fluorescein studies in the living diabetic, and Ashton9 with his injection technique on histologie specimens of diabetic retinas, have shown areas of capillary closure with micro-

35

aneurysms surrounding these areas. Further­ more, Hill and co-workers29 showed occa­ sional evidence of local delay in the arteriolar circulation; in areas where the capillary circulation was abnormal, some precapillary arterioles filled later than their neighbors, and the feeding branch arteriole was slow to empty. They felt that the extent of arteriolar change was interesting and cited Ashton's 3 observation on the possibility of gradual arteriolar obliteration with its relationship to much of the capillary closure which oc­ curs in the arteriolar side of the network. This capillary closure leads to tissue necro­ sis. With the formation of tissue necrosis and a microwound the stage is set for the hypoxic theory as stated above. Microaneurysms, hemorrhages, exudates and neovascu­ larization as well as abnormal arteriolar-venular "shunt" vessels then result. The rate and extent at which these changes take place in a given diabetic retina, though, varies greatly. Some retinas show very slowly progressive disease over a period of many years whereas others evidence greatly accelerated disease. This type of varied behavior is also character­ istic of other organ systems of diabetic pa­ tients, such as kidney and nervous. The rate and extent of the disease, then, could be pre­ determined largely by the genetic makeup of an individual. Lacy degeneration of the pigment epithe­ lium of the iris was first described and illus­ trated by Kamocki in 1887.37 Recently, Schüpbach38 published an exhaustive review of the subject. Except for one report,39 how­ ever, the exact nature of the material in the pigment epithelial vacuoles was unknown, al­ though suspected of being glycogen. In 1961 Yamashita and Becker,39 using methacrylate-embedded tissue showed that the vac­ uoles stained intensely with PAS. This PASpositive material could be removed by incu­ bation with saliva (diastase) prior to stain­ ing, thereby showing that glycogen or glycogenlike polysaccharides are major compo­ nents of the PAS-positive materials. The present study, utilizing paraffin-embedded

36

AMERICAN JOURNAL OF OPHTHALMOLOGY

JANUARY, 1969

ers. Glial-neuronal cell breakdown products are likely additional constituents of these deep exudates. Cotton wool exudates (cytoid bodies), although known to occur in diabetic retinas, were not found in this study. 6. Neovascularization of the retina occurs primarily within the inner retinal layers, with secondary extension through the inter­ nal limiting membrane to grow into the po­ tential space between this membrane and the posterior vitreous or along the posterior sur­ face of a detached vitreous. 7. Diffuse thickening of the basement membrane of the pigment epithelium of the CONCLUSIONS pars plicata of the ciliary body is a constant 1. Microaneurysms are not diagnostic of finding. The basement membranes of the diabetic retinal microangiopathy but the nonpigmented ciliary epithelium, of Müller's triad of (1) capillary microaneurysms, (2) a cells (internal limiting membrane of retina), decreased ratio of capillary pericytes to en- of retinal pigment epithelium (cuticular part dothelial cells, and (3) capillary sheathing is of Bruch's membrane) and of choriocapilhighly characteristic. The extent of the loss laris may also be thickened. of pericytes is directly related to microaneu8. The vacuoles within the lacy degenera­ rysmal formation, that is, the greater the loss tion of the pigment epithelium of the iris of pericytes, the greater the number of mi­ contain glycogen. The histopathogenesis of croaneurysms. this material is unknown but may be similar 2. Microaneurysms most likely represent to the accumulation of glycogen in the renal a response to a hypoxic environment in the tubular cells of diabetics. form of either abortive attempts at neovasSUMMARY cularization or regressed changes, or both, in a previously proliferating vessel. Twenty whole eyes and 12 trypsin-di3. The retinal capillary arteriolar-venular gested retinal preparations from diabetic pa­ "shunts" probably result as a secondary phe­ tients were examined histopathologically. nomena, that is, secondary to the surround­ Diabetic retinal microanagiopathy was char­ ing hypoxic stimulus. The functions of the acterized by microaneurysms, loss of capil­ shunts is unknown. lary pericytes, and capillary sheathing. The 4. Retinal hemorrhages are located mainly loss of pericytes correlated with an increased in the inner nuclear layer with spread to the number of microaneurysms. Microaneu­ outer plexiform layer (dot and blot hemor­ rysms seem to occur in hypoxic areas and rhages), seemingly because this is the area probably represent abortive attempts at neo­ where the incompetent capillary bed is most vascularization or regressed changes, or both, rich. Nerve fiber layer hemorrhages (flame- in a previously proliferating vessel. Neovas­ cularization is initially intraretinal and later shaped) are seen infrequently. 5. Retinal waxy exudates are located breaks through the internal limiting mem­ mainly in the outer plexiform layer probably brane to extend onto the internal surface of because in an edematous retina, edema fluid the retina. Diffuse basement membrane thick­ most likely pools in an avascular area where ening of the pigment epithelium of the pars it cannot be reabsorbed into the capillary bed plicata of the ciliary body is a constant find­ which is present only in the inner retinal lay­ ing. The vacuoles in the lacy degeneration of

eyes, confirms their findings. The material stained positively with PAS and Best's car­ mine stains and the absence of such staining after pretreatment with diastase is rather conclusive evidence for the material being glycogen. Also, the material gives a typical glycogen pattern in electron micrographs.40 Why this material accumulates in the iris pigment epithelium is unknown. Perhaps the iris pigment epithelium has the ability to ac­ cumulate and store this material from the aqueous much like the diabetics' renal tubu­ lar cells accumulate and store glycogen.

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OCULAR PATHOLOGY OF DIABETES

the pigment epithelium of the iris are shown to contain glycogen. 3400 Spruce Street (19104) ADDENDUM

Since this paper was submitted, an addi­ tional quantitative study has confirmed the selective loss of pericytes from the retinal capillaries of diabetics.41

ACKNOWLEDGMENT

I wish to thank Dr. G. Clayton Kyle for en­ couraging me to do this work and for reviewing the manuscript. REFERENCES

1. Yanoff, M. : Diabetic retinopathy. New Eng. J. Med. 274:1344, 1966. 2. Bloodworth, J. M. B., Jr. : Diabetic retinopa­ thy. Diabetes 11:1, 1962. 3. Ashton, N. : Studies of retinal capillaries in relation to diabetic and other retinopathies. Brit. J. Ophth. 47:521, 1963. 4. Cogan, D. G., Toussaint, D. and Kuwabara, T. : Retinal vascular patterns. IV. Diabetic retinop­ athy. Arch. Ophth. 66:366, 1961. 5. Wise, G. N. : Retinal neovascularization. Tr. Am. Ophth. Soc. 54:729, 1956. 6. Ashton, N. : In discussion of Diabetic retinopa­ thy. Proc. Roy. Soc. Med. 44:747, 1951. 7. Friedenwald, J. S. : A new approach to some problems of retinal vascular disease. Tr. Am. Acad. Ophth. Otolaryn. 53 :73, 1948. 8. Michaelson, I. C. and Steedman, H. F.: Injec­ tion of the retinal vascular system in enucleated eyes. Brit. J. Ophth. 33 :376, 1949. 9. Ashton, N. : Injection of the retinal vascular system in the enucleated eye in diabetic retinopathy. Brit. J. Ophth. 34:38, 1950. 10. Kuwabara, T. and Cogan, D. G. : Studies of retinal vascular patterns. I. Normal architecture. Arch. Ophth. 64:904, 1960. 11. Rouget, C. : Mémoire sur le Développement, la Stnicture et les Propriétés physiologiques des Capillaires sanguins et Lymphatiques. Arch. Physiol. Norm. Path. 5:603, 1873. 12. Ashton, N. and de Oliveira, F.: Nomencla­ ture of pericytes. Brit. J. Ophth. 50:119, 1966. 13. Fine, B. : Retinal Structure : Light- and Elec­ tron-Microscopic observations. In McPherson, A. (ed) : New and Controversial Aspects of Retinal Detachment. New York: Hoeber Med. Div., Har­ per and Row, 1968, p. 16. 14. de Oliveira, F. : Pericytes in diabetic retinop­ athy. Brit. J. Ophth. 50:134, 1966. 15. Cogan, D. G. and Kuwabara, T. : The mural

37

cell in pespective. Arch. Ophth. 78:133, 1967. 16. Levene, R., Horton, G. and Gorn, R. : Flatmount studies of human retinal vessels. Am. J. Ophth. 61:283, 1966. 17. Engerman, R. L. and Bloodworth, J. M. B , Jr. : Experimental diabetic retinopathy in dogs. Arch. Ophth. 73 :205, 1965. 18. Cogan, D. G. and Kuwabara, T.: Capillary shunts in the pathogenesis of diabetic retinopathy. Diabetes 12:293, 1963. 19. Cogan, D. G. : Diabetic retinopathy. New Eng. J. Med. 270:787, 1964. 20. Florey, Sir H. : General Pathology. Philadel­ phia, Saunders, 1962, ed. 3, p. 451. 21. Yanoff, M., Rahn, E. G. and Zimmerman, L. E. : Histopathology of juvenile retinoschisis. Arch. Ophth. 7 :49, 1968. 22. Rahn, E. K, Yanoff, M. and Tucker, S. : Neuro-ocular considerations in Pelizaeus-Merzbacher disease : A clinicopathologic study. Am. J. Ophth. 66:1143, 1968. 23. Friedman, E., Smith, T. R. and Kuwabara, T. : Retinal microcirculation in vivo. Invest. Ophth. 3:217, 1964. 24. Yamashita, T. and Rosen, D. A. : Electronmicroscopic study of diabetic capillary aneurysm. Arch. Ophth. 67:785, 1962. 25. Toussaint, D. and Dustin, P. : Electron mi­ croscopy of normal and diabetic retinal capillaries. Arch. Ophth. 70:96, 1963. 26. Bloodworth, J. M. B., Jr. : Diabetic microangiopathy. Diabetes 12 :99, 1963. 27. Bloodworth, J. M. B., Jr. and Molitor, D. L. : Ultrastructural aspects of human and canine dia­ betic retinopathy. Invest. Ophth. 4:1037, 1965. 28. Dollery, C. T. : Dynamic aspects of the reti­ nal circulation. Arch. Ophth. 79 :536, 1968. 29. Hill, D. W , Dollery, C. T., Mailer, C. M., Oakley, N. VV. and Ramalho, P. S. : Arterial fluorescein studies in diabetic retinopathy. Proc. Royal Soc. Med. 58:535, 1965. 30. Ferrer, O. : Retinal circulation times : Studies by means of fluorescein rapid sequence photogra­ phy. Tr. Am. Acad. Ophth. Otolaryn. 72 :50, 1968. 31. Norton, E. W. D. and Gutman, F. : Diabetic retinopathy studied by fluorescein angiography Ophthalmologica 150:5, 1965. 32. Fine, B. S. and Zimmerman, L. E. : Miiller's cells and the "middle limiting membrane" of the human retina. Invest. Ophth. 1:304, 1962. 33. Hogan, M. J. and Zimmerman, L. E. (eds) : Ophthalmic Pathology. Philadelphia, Saunders, 1962, ed. 2, p. 510. 34. Cogan, D. G. : Neurology of the Visual Sys­ tem. Springfield, Thomas, 1966, p. 53. 35. Ballantyne, A. J. : The state of the retina in diabetes mellitus. Tr. Ophth. Soc. U. K. 66:503, 1946. 36. Tolentino, F. I., Lee, P. F. and Schepens, C. L. : Biomicroscopic study of vitreous cavity in dia­ betic retinopathy. Arch. Ophth. 75:238, 1966. 37. Kamocki, V. : Pathologisch-anatomische Un­ tersuchungen von Augen diabetischer Individuen. Arch. f. Augenh. 17 :247, 1887.

38

AMERICAN JOURNAL OF OPHTHALMOLOGY

38. Schüpbach, von M. : Über die zystoide Degeneration des Pigmentepithels der Iris bei Diabetes mellitus. Klin. Mbl. Augenh. 150 :1, 1967. 39. Yamashita, T. and Becker, B. : The basement membrane in the human diabetic eye. Diabetes 10:167, 1961.

40. Yanoff, M., Fine, B. S. and Berleow, J. W. : Diabetic lacy vacuolization of iris pigment epitheHum. In preparation. 41. Speiser, P. Gittelsohn, A. M. and Patz, A.: Studies on diabetic retinopathy. Arch. Ophth. 80: 332, 1968.

CLINICAL EXPERIENCE W I T H T H E H E R B E R T E.

K A U F M A N , M.D.

JANUARY, 1969

EPIKERATOPROSTHESIS

A N D A N T O N I O R.

GASSET,

M.D.

Gainesville, Florida

In April 1968 we 1 described experiments in rabbits and monkeys in which a methacrylate covering was bonded to the anterior sur­ face of the cornea by octyl cyanoacrylate monomer. The excellent tolerance of these lenses by the animals suggested the possibil­ ity that they might be useful in man for the protection of the cornea, as well as for opti­ cal improvement: in some cases obviate the need for flush-fitting contact lenses ; in others, permit the optical correction of patients who require the optical benefit of a contact lens but do not have tolerance for such a lens; and in still another group, protect the globe in a variety of conditions in which no other sight-preserving technique could be expected to give satisfactory results. Although the technique of epikeratoprosthesis ( E K P ) is simple and easily adapted to office procedure, meticulous attention to detail is important. This communication outlines our present technique for the application of the epikerato­ prosthesis and summarizes our clinical results to date. MATERIAL AND METHODS PROSTHESIS

The prosthesis is basically an ordinary methyl methacrylate lens similar in many ways to the common corneal contact lens. From the Department of Ophthalmology, College of Medicine, University of Florida. This work was supported in part by Research and Training Grants from the National Institute of Neurological Dis­ eases and Blindness, National Institutes of Health, U. S. Public Health Service, Bethesda, Maryland.

Any diameter E K P can be employed but larger diameters seem most useful. Most pa­ tients require protection of the cornea and it is desirable for the E K P to be large enough that the adhesive can remain at the periphery of the lens with the central part of the E K P clear. For these reasons, a 9.5-mm pros­ thesis is most commonly used. With an E K P of this size, care must be taken to center the prosthesis on the cornea and not to overlap the corneoscleral limbus. The normal corneal contact lens has in­ termediate and secondary curves that lift the edge away from the cornea to facilitate tear flow. With the EKP, the epithelium of the cornea is removed and the lens must abut tightly against Bowman's membrane so that epithelium does not grow under it. We use EKP's with a single curve and no intermedi­ ate peripheral curves. This brings the edge of the lens tightly against Bowman's mem­ brane. The usual corneal contact lens has a some­ what rounded edge so that it does not abrade the epithelium as it moves. Since there is no movement, E K P lenses are made with a sharp edge angled down to the cornea at an angle of about 45° (fig. 1). Of course, any optical correction can be incorporated into the lens and either a full-cut or lenticular an­ terior surface can be used. It is relatively easy and inexpensive to purchase single curve lens blanks and edge them down at the time the prosthesis is being applied, cutting the desired diameter. Single-cut lenses seems