- Email: [email protected]
Correlation of Fluorescein Angiogram and Retinal Digest in Diabetic Retinopathy
VOL. 69, NO. 3
DEGENERATION OF PERIPHERAL RETINA
L. E. : Histopathology of juvenile retinoschisis. Arch. Ophth. 79:49, 1968. 15. Rutnin, U., and Schepens, C. L. : Fundus ap pearance in normal eyes: III. Peripheral degenera tions. Am. J. Ophth. 64:1040, 1967. 16. Zimmerman, L. E. : Applications of histo-
403
chemical methods for the demonstration of acid mucopolysaccharides to ophthalmic pathology. Tr. Am. Acad. Ophth. 62 :697, 1958. 17. Streeten, B. W. : Development of the human retinal pigment epithelium and the posterior seg ment. Arch. Ophth. 81:383, 1969.
CORRELATION OF FLUORESCEIN ANGIOGRAM AND RETINAL IN DIABETIC R E T I N O P A T H Y E V A M.
KOHNER,
M.D.,
AND P A U L
HENKIND,
DIGEST
M.D.
New York, New York Improved techniques have recently ex panded our knowledge of diabetic retinop-athy. Such techniques include vascular injections, 1 , a shake preparations, 3 and trypsinl digestion 4 ' 5 for histologie specimens, and1 fluorescence angiograms* for observation off the retinal lesions in life. Ashton 2 ' 7 · 8 used neoprene and India inkc injections to demonstrate the connections ofE microaneurysms to capillaries, abnormal cap illary loops, and areas of capillary nonperfusion. I n severe retinopathy, he noted in volvement of arterioles and shunt vessels3 crossing otherwise nonperfused areas. Cogani and his co-workers 4 · 5 used retinal digests to) observe cellular detail. T h e y found the selective loss of pericytes from capillaries corre sponding to the nonperfused vessels of Ash ton, hypercellular microaneurysms and hypercellular shunt vessels surrounding areass of capillary closure. 9 I n 1961, Novotny and Alvis 8 reported on1 fluorescence angiographie studies in diabeticC retinopathy. Since then, fluorescence photog raphy has become widely used in investigation1 of diabetic retinopathy during life. 10 " 14 From the Department of Ophthalmology, Newv York University, New York City. Dr. Kohner wass a Medical Research Council Alexander Pigottt Wernher Memorial Trust Fellow from the Royal1 Postgraduate Medical School Hammersmith Hospi tal, London. This work was supported by NIHI Grant NB-05-059-06 and the Scientific Section off the British Diabetes Association. Reprint requests to Paul Henkind, M.D., Newv York University, 550 First Avenue, New York,:, New York 10016.
H o w accurately fluorescence angiograms mirror the retinal vascular pathology is not really known. T h e r e has been only one report comparing a digest preparation with a fluorescence angiogram in the same patient. 15 I n that study, only microaneurysms were ob served in detail. It was found that those packed with red cells did not show u p on fluorescence photographs, while those with a partially hyalinized wall leaked and ones with a hypercellular wall retained fluorescein. This paper reports on the findings in a patient who died 39 months following yttrium-90 ( 9 0 Y ) implantation of her pituitary gland. T h e last fluorescence angiograms were performed five months prior to death. CASE
HISTORY
A 30-year-old woman was seen initially at the Hammersmith Hospital in March, 1965. She was referred for pituitary ablation because of diabetic retinopathy. Her diabetes was diagnosed when she was two years of age, and she was treated twice daily with regular insulin and a 160-gram carbohydrate diet. Diabetic control was fair. In 1955, she noted tran sient blurring of vision in her right eye. This cleared rapidly with improvement of the diabetic control. Late in 1964, the vision in her left eye became blurred and gradually deteriorated, so that at the time of her first visit, it had light perception only. Visual acuity in her right eye was 20/40. Her blood pressure was 145/80, with slight posrural hypotension. The peripheral pulses were normal. The knee jerks were sluggish and the ankle jerks were absent. Pin prick and light touch sensations were markedly reduced over the feet and lower legs. The right fundus had numerous microaneurysms,
404
AMERICAN JOURNAL OF OPHTHALMOLOGY
hemorrhages, dilated capillaries and possibly new vessels (fig. 1). The left retina showed massive retinitis proliferans and preretinal hemorrhage. Laboratory studies revealed a hemoglobin of 8.3 g with microcytic film and a hematocrit of 29%. Serum cholesterol, 290 mg%; serum pro teins, 6.8 g/100 ml; albumin 3.8 g and globulins 3.0 g. Renal involvement was indicated by a blood urea nigrogen (BUN) of 62 mg/100 ml (normal 20-40 mg%) and proteinuria of 2.9 g in 24 hours. It was felt that the patient would benefit from pi tuitary ablation, and "Ύ implantation was per formed in April, 1965. The postoperative period was complicated by transient hyponatremia and postural hypotension. At the three-month assessment, she was found to have only partial pituitary ablation, with normal pe riods and thyroid function, but a reduced adrenal reserve with symptomatic Addison's disease seven days after steroid withdrawal. Growth hormone levels after insulin-induced hypoglycemia reached only six nanograms at 120 minutes.1*·" The visual acuity remained unchanged, but the retinal appear ance improved in the right eye. Over the next three years the appearance of the right fundus remained improved (fig. 2), but visual acuity deteriorated to 20/60. Fluorescence photo graphs were taken before pituitary implantation, and at six and 14 months postoperatively. Unfortu nately, these were of poor quality and difficult to compare with the last two series taken at 24 and 34 months. During the first postoperative year the patient was well, but thereafter she had a gradually rising BUN and increasing hypertension. She was admit ted to hospital on several occasions because of ure mia and finally died in June, 1968. The right eye was obtained for histologie exami nation. F L U O R E S C E N C E ANGIOGRAMS
The fluorescence angiograms were done by the intra-arterial technique.12 The area studied was a 30° field extending from the disc to about one disc diameter lateral to the macula and bounded by the temporal vessels (fig. 3). Capillaries—Multiple areas of capillary nonperfusion were evident. The largest of these was in the perimacular region, and was about three times the diameter of the normal perimacular capillary-free zone. Large areas of capillary closure were also present in the superior temporal area while smaller ones were seen elsewhere. Microaneurysms were scattered through out the field. Almost all were adjacent to areas of capillary nonperfusion. On fluores
MARCH, 1970
cence angiograms they could not always be differentiated from capillary loops, since these also surrounded areas of capillary clo sure. Some of the microaneurysms retained dye after most of the capillary bed had emp tied. A few leaked fluorescein, but this was difficult to ascertain because of the back ground choroidal fluorescence (figs. 4 and 5). Considering the severe capillary abnor malities, the number of microaneurysms was small and the leakage through their walls slight. Abnormal capillary configurations were widespread. Some jutted into areas of capil lary closure, suggesting attempts at revascularization, while others had a haphazard ar rangement. A number of the vessels were di lated and it is impossible to tell whether they were capillaries or abnormal arteries and veins, or even new vessels. Arteries and veins—Some arterial branches passing through areas of capillary closure showed local dilatation, retention of dye and leakage (fig. 5). This was also seen in some venous branches, but was less marked. Small vessels showed occasional loops and increased tortuosity. Flow mea surements were not performed in this pa tient, but the marked lamination of fluores cein in the temporal arteries may indicate slow flow. The large veins showed no obvious lesions while many of the smaller ones had unusual loops, were very tortuous, and retained dye. Arteriovenous and veno-venous communi cations—Vesels larger than capillaries were seen to connect arteries and veins especially across areas of capillary closure. This was most marked in the macular area. Arteriove nous and veno-venous communications were seen between the branches of the superior and inferior temporal vessels lateral to the macula. Some of these vessels showed local ized dilatations, but none showed the charac teristic of shunt vessels, i.e., early appear ance of dye lamina in the vein indicating a low pressure-high flow channel. New vessels—Some of the abnormal ve-
VOL. 69, NO. 3
DIABETIC RETINOPATHY
405
nous side branches might be new vessels, but true new vessel formation was only obvious in the superior temporal region at the edge of the picture. DIGEST PREPARATION
Capillaries—Almost no normal capillaries were seen in any portion of the retinal digest from the disc border to the retinal periphery. Most were either pale, acellular basement membrane tubes or strands, or tortuous, hy percellular channels with proliferated endothelium. Intramural pericytes were rare, and where present, usually had either pale nuclei or ghost remnants. No capillaries were seen with normal-appearing intramural pericytes and absent endothelial cells. The cellular cap illaries seemed to be dilated when compared to normal retinal digests, and to have an un even increase in PAS-staining of the base ment membrane. Numerous aneurysmal dilatations of the capillaries were seen. They ranged from large, round, hypercellular lesions to small, hyalinized, or even disintegrated ones (fig. 6). The latter were mainly on acellular capil laries. The aneurysms were scattered throughout the field from posterior pole to
Fig. 1 (Kohner and Hcnkind). Fundus photograph of the right eye taken in March, 1965, showing nu merous microaneurysms, hemorrhages, dilated cap illaries and possibly neovascular tufts.
Fig. 2 (Kohner and Hcnkind). Fundus photograph of the right eye taken in 1968. Note the diminished number of hemorrhages and microaneurysms.
periphery. Some were closely related to precapillary arterioles or postcapillary venules, but most were within the midst of the capil lary bed itself. No aneurysms were evident in the distribution of the radial peripapillary capillaries. Arteries and veins—The larger retinal ar teries seemed to have a normal cellular popu lation and were without evident dilatation or tortuosity. Many of the precapillary arter ioles were abnormal, having intense PASpositive walls, increased numbers of endo thelial cells and occasional microaneurysms. The larger retinal veins were slightly ir regular in caliber but had a normal cellular configuration. The smaller venous branches were often abnormal with a marked hypercellularity of their walls. A number of tor tuous, hypercellular arteriovenous communi cations—larger than capillaries—were seen crossing areas of obliterated acellular capil laries. Neovascularization—Numerous markedly irregular, hypercellular and dilated vessels appeared to overlie the normal level of the retinal capillary bed. In areas of capillary obliteration, hypercellular channels linked adjacent arteries and veins (these arterio venous communications are the shunts de-
406
AMERICAN JOURNAL OF OPHTHALMOLOGY
, ^ * «wsa^
MARCH, 1970
v
.'vliSMii-
Fig. 3 (Kohner and Henkind). Arterial-capillary phase fluorescein angiogram of the right eye five months prior to the patient's death. The area covered is a 30° field including the macula and most of the disc. The black boxes, A and B, outline where fluorescein angiograms are compared with the retinal digest. Note the areas of capillary nonperfusion and microaneurysms. scribed by Cogan and Kuwabara. 9 No such channels were seen in areas of cellular capil laries. Detailed comparison between the digest preparation and fluorescence angiograms were done in the two regions illustrated in Figures 7-10. In general, the features dem onstrated are listed in Table 1. Other features not consistently seen in
clude leakage of fluorescein from some hypercellular vessels and hypercellular loops, and retention of dye in some of the vessels, which, on digest, look no more abnormal than other vessels in which fluorescein is neither retained nor leaked. Some apparent local thickening of arteries observed on the angio grams was seen on the digest preparation to represent cellular (sometimes hypercellular)
VOL. 69, NO. 3
DIABETIC RETINOPATHY
407
Fig. 4 (Kohner and Henkind). Capillary-venous phase fluorescein angiogram taken several seconds after Figure 3. Microaneurysms and capillary loop are visible as well as irregular dilated vessels.
arteriolar remnants with attached acellular capillaries. Figures 7-A and B show the capillary
and Figure 7-C the late venous phase of area A in Figure 3. In the diagram, Figure 7-D, the features discussed are represented. Fig-
Fig. 5 (Kohner and Henkind). Late venous phase fluorescein angio gram taken several seconds after Figure 4. Note microaneurysms with persisting fluorescence and fluores cein leakage ; also, some abnormally leaking new vessels.
408
AMERICAN JOURNAL OF OPHTHALMOLOGY
M A R C H , 1970
■0*
^&Λ^.
Fig. 6 (Kohner and Henkind). Retinal digest preparation showing a variety of aneurysms and dilata tions of the capillaries. Most of the aneurysms are acellular or only slightly cellular, while the dilated vessels appear hypercellular ( P A S and hematoxylin, χ 3 2 0 ) .
ure 8 is the digest preparation of the same area. The fluorescence angiogram shows that there are large areas of capillary nonperfvi sion and on the digest preparation these are shown to be either completely acellular ves sels, or vessels with ghost pericytes only, but no endothelial cells. The very close correla tion between perfusion and cellularity is demonstrated by the vessels indicated by arrow 1 in Figure 7-D. The loops arising from this vessel jutting into the nonperfused area may be attempts at neovascularization. It is not possible to decide whether the ter minal portions are vascular loops or microaneurysms. The irregular dilated and densely staining vessel (arrow 2, fig. 7-D) is seen on the digest as a hypercellular channel with many hypercellular buds, not all of which are clearly seen on the fluorescence angio gram. Arrow 3 (fig. 7-D) shows another ir regular, dilated vessel, and this appears to retain dye for a long period (fig. 7-C). On the digest it is hypercellular, but does not
really differ from other hypercellular vessels. Note that all the hypercellular vessels appear to lie in front of the acellular ones in the histologie preparation. Of the microaneurysms, one (arrow 4, fig. 7-D) has a highly cellular wall, and does not leak fluorescein, while the one marked by arrow 5 (fig. 8) lies on an acellular capillary and is not perfused. The cluster of micro aneurysms (arrow 6, fig. 7-D) shows some leakage. We are not certain which of these microaneurysms leaks. Arrow 7 (fig. 7-D), which indicates an aneurysm on the angiograms, looks like an abnormal bend in the capillary (fig. 8). The sluggish filling of some vessels is demonstrated in Figures 7 A-C (arrow 8, fig. 7-D). On fluorescence angiograms this vessel is perfused from capillaries, most of which are abnormal, and on the digest prepa ration they appear as possible intraretinal neovascular channels (arrow 9, fig. 8). The long retention of the dye and intense fluorés-
VOL. 69, NO. 3
DIABETIC RETINOPATHY
409
Fig. 7 (Kohner and Henkind). Capillary phase (A and B) and venous phase (C) fluorescein angiograms of area A in Figure 3. A diagram of the area is shown in D, bottom left. The significant features outlined are discussed in the text. cence on late pictures probably indicates se vere endothelial cell damage, because five months later, at the time of the digest prepa ration, a short segment of this vessel appears completely acellular ( a r r o w 10, fig. 8 ) . Figure 9 A - C is the area adjacent and su
perior to the macula (area B in fig. 3 ) . Fig ure 9-D is a diagramatic representation of the features discussed and Figure 10 shows the digest preparation of the same area. Sev eral arteriovenous communications are seen to cross the area of capillary closure (arrow
410
AMERICAN JOURNAL OF OPHTHALMOLOGY
Hufe-
MARCH, 1970
,V 4 'V Pr
:io
V*·
-Mt0F4
élXOb. mu.'*
<*"
I t^T-lf
\ ·*|
*fcj
a-
i
s $i
Fig. 8 (Kohner and Henkind). Digest preparation of area A in Figures 3 and 7. (PAS and hematoxylin, xl2S).
VOL. 69, NO. 3
DIABETIC RETINOPATHY
411
4 (fig. 9-D), while not one that is perfused (arrow 5, fig. 10) lies on an acellular capil lary. The localized thickening on the artery (arrow 6, fig. 9-D) indicates the origin of the acellular vessel on which microaneurysm 5 (fig. 10) is found. DISCUSSION
Since fluorescence angiograms are now routinely performed for the investigation of diabetic retinopathy, it is important to know what the angiographie lesions represent in Ψ! « J€l terms of pathologic changes. This report M r^L • V •\ II / ·» suggests the degree of correlation that can be achieved. Complete agreement was not ex pected for two reasons : the last fluorescence angiogram was taken five months before the patient's death, and alterations of the vascu lar architecture during the digesting and mounting procedures invariably result in Fig. 9 (Kohner and Henkind). Capillary phase some distortion. Furthermore, diabetic reti (A and B) and late venous phase (C) fluorescein nas, with their abnormal vessels, are easily angiograms of area B in Figure 3. A diagram of damaged. Despite these difficulties, close thefieldis shown in D. bottom, with significant fea correlation between the fluorescence angio tures outlined and discussed in the text. gram and the digest preparation was found. 1, fig. 9-D) ; these are irregular, hypercellu The fluorescence and digest preparation lar channels. On the fluorescence angiograms appearance of microaneurysms in this study there are several vascular segments which were similar to those obtained by de Venecia show increased density and leakage in the and Davis.15 In our preparation the nonperlate phase (fig. 9-C). These appear hypercel fused microaneurysms were on acellular cap lular on the digest preparation. They may illaries, and occasionally appeared to be dis represent intraretinal neovascularization. integrating. It was difficult to distinguish Concerning microaneurysms, some are per capillary loops from microaneurysms. Com fused, but do not leak (arrow 3, fig. 9-D). parison of the fluorescence angiogram with Histologically they are hypercellular. Two the digest was useful, but occasionally even partially hyalinized ones are probably re this did not give the answer. sponsible for the leakage indicated by arrows It has been assumed by many that areas ^
f
J
M"
^^^■^^
3vf
Λ A
^3
Fig. 10 (Kohner and Henkind). Digest preparation of area seen in Figures 3-B and 9. ( F A S and hematoxylin, χ 1 2 5 ) .
of capillary nonperfusion correspond to acel lular capillaries. This was confirmed in the present study. It seems that for perfusion an endothelial cell lining is essential. The hypercellular dilated vessels were ex amined. Some were obvious arteriovenous communications, and others dilated capillar ies surrounded by many acellular vessels. These vessels appeared both dilated and ir regular on fluorescence angiograms. This is of importance, since Oosterhuis and Vink13 postulated that the easy visibility of diabetic capillaries and their apparent dilatation on fluorescence angiograms indicates leakage of the dye into the lining endothelium. Though this may occur in some vessels, the majority we studied were truly dilated capillaries. Retention of the dye in, and leakage through, the vessel wall indicates a more se vere abnormality. It is not evident from the digest preparation what this abnormality is. The vessels leaking and retaining fluorescein usually appeared no more abnormal than others which filled and emptied normally.
TABLE 1 COMPARISON OF DIGEST PREPARATION AND FLUORESCEIN ANGIOGRAM
Digest Preparation Hypocellular or acellular capillaries: Endothelial cells present, intramural pericytes ab sent or abnormal Intramural pericytes pres ent but abnormal, endo thelial cells absent Completely acellular Microaneurysms: Cellular wall Partially hyalinized Acellular or on acellular capillaries Hypercellular vessels (larger than capillaries) : Hypercellular arteriovenous communications:
Fluorescein Study
Perfused capillaries Capillaries not per fused Capillaries fused
not
per
Perfused, not leaking Perfused, leaking Not perfused Dilated vessels (larger occasionally re taining dye) Dilated channels con necting arteries to veins; flow rate not increased, thus not true shunts
VOL. 69, NO. 3
DIABETIC RETINOPATHY
Some of the hypercellular vessels, espe cially those forming buds, appear to be at tempts at revascularization of nonperfused areas, and probably represent intraretinal neovascularization. It is of interest that they arise from both arteries and veins. Since the fluorescein and digest studies are two-dimen sional, the level of these vessels could not be definitely determined. Concerning the widespread capillary ab normalities, there were relatively few microaneurysms seen by angiography. This is partly because many were on acellular capil laries and therefore not perfused. Many of the vessels present may be newly formed, and these rarely form microaneurysms. The other possibility is the beneficial effect of pi tuitary ablation on microaneurysm forma tion. It has been shown previously 19 that fol lowing pituitary implant of 9 0 Y the number of new microaneurysms forming is greatly reduced, and the disappearance of existing ones accelerated. N o conclusion about the efficacy of pitui tary ablation can be drawn from this patient. This is not only because she is the sole ablated patient in whom a retinal digest preparation has been studied, but because her pituitary ablation was only partial. Also her terminal illness was associated with hy pertension (though there was no obvious hy pertensive retinopathy p r e s e n t ) . Clearly, whatever the benefits of the oper ation were, they did not prevent the closure of large areas of the capillary bed and wide spread abnormalities throughout the retina. It was rather surprising that this patient re tained relatively good vision. Whether slow occlusion of the retinal vascular bed per mits alternate metabolic pathways to develop, or allows choroidal nourishment to supply the entire retinal thickness is unknown. SUMMARY
A 30-year-old diabetic woman had a par tial pituitary ablation for the treatment of her diabetic retinopathy. Fluorescein angiograms of her right eye were performed pre-
413
and posthypophysectomy. At her death, 39 months after 0 0 Y implantation, her right eye was removed. T h e retina was digested and a comparison made between the fluorescein angiograms and the histologie preparation. A good correlation was found to exist between the angiographie appearances and the microcirculatory pathology. T h e major points are : ( 1 ) that perfused vessels have an intact endothelium, nonperfused ones lack endothelium ; ( 2 ) that perfused microaneurysms have a cellular wall, while acellular ones do not show up on the angiogram but can be found in the retinal digest preparation; ( 3 ) that the dilated vessels seen by angiography usually were hypercellular; ( 4 ) that arteriovenous communications were hypercellular channels lying in an otherwise atrophie cap illary bed. Angiography revealed that they were not highflow channels. ACKNOWLEDGMENTS
We thank Dr. Richards, Ashford Hospital, Mid dlesex, for obtaining the eye, postmortem, and Professor Russell Fraser for permitting us to study his patient. The line drawings were prepared by Mr. Philip C. Johnson, the photographs by Mr. Henrick Malpica, and the text typed by Mrs. Doris Ortega. Mr. Cybald Cole provided technical assistance. REFERENCES
1. Michaelson, I. C, and Stecdman, H. F. : Injec tion of the retinal vascular system in enucleated eyes. Brit. J. Ophth. 33:376, 1949. 2. Ashton, N. : Injection of the retinal vascular system in the enucleated eye in diabetic retinopathy. Brit. J. Ophth. 34:38, 1950. 3. Ashton, N. : Vascular changes in diabetes with particular reference to the retinal vessels. Brit. T. Ophth. 33 :407, 1949. 4. Kuwabara, T., and Cogan, D. G. : Studies of retinal vascular patterns. 1. Normal architecture. Arch. Ophth. 64:904, 1960. 5. Cogan, D. G., Toussaint, D., and Kuwabara, T. : Retinal vascular patterns. IV. Diabetic retinop athy. Arch. Ophth. 66:100, 1961. 6. Novotny, H. R., and Alvis, D. L. : Method of photographing fluorescence in circulating blood in the human retina. Circulation 24 :82, 1961. 7. Ashton, N. : The pathology of retinal micro aneurysms, Acta XVI. Concilium Ophthalmologicum (Britannia) 1:411, 1951. 8. Ashton, N. : Arteriolar involvement in diabetic retinopathy Brit. J. Ophth. 37:282, 1953. 9. Cogan, D. G., and Kuwabara, T. : Capillary
414
AMERICAN JOURNAL OF OPHTHALMOLOGY
shunts in the pathogenesis of diabetic retinopathy. Diabetes 12:293, 1963. 10. Scott, D. J., Dollery, C. T., Hill, D. W , Hodge, J. V., and Fraser, T. R. : Fluorescein stud ies in diabetic retinopathy. Brit. Med. J. 1:811, 1964. 11. Norton, E. W. D., and Gutman, F.: Diabetic retinopatby studied by fluorescein angiography. Ophthalmologica 150:5, 1965. 12. Kohner, E. M., Dollery, C. T., Paterson, J. W., and Oakley, N. W. : Arterial fluorescein stud ies in diabetic retinopathy. Diabetes 16:1, 1967. 13. Oosterhuis, J. A., and Vink, R. : Fluorescein photography in diabetic retinopathy, in Perspectives in Ophthalmology, Henkes, H. (ed.), Exerpta Medica Foundation, 1968. p. 1. 14. Wessing, A. : Fluorescenz Angiographie der
Retina. Stuttgart, Georg Thieme Verlag, 1968. 15. De Venecia, G., and Davis, M. D. : Histology and fluorescein angiography of microaneurysms in diabetes mellitus. Invest. Ophth. 6:555, 1967. 16. Fraser, T. R., and Wright, A. D. : Standard procedures for assessing hypersécrétion and secre tory capacity for human growth hormone using the radioimmunoassay. Postgrad. Med. 44:53, 1968. 17. Wright, A. D , Kohner, E. M., Oakley, N. W., Fraser, T. R., Joplin, G. F., and Hartog, M. : Serum growth hormone levels and the response of diabetic retinopathy to pituitary ablation. Brit. Med. J. 2 :346, 1969. 18. Kohner, E. M., Dollery, C. T., Fraser, T. R, and Bulpitt, C. J. : The effect of pituitary ablation in diabetic retinopathy studied by fluorescence pho tography. In preparation.
FINE STRUCTURE O F RABBIT SCLERAL M A N F R E D S P I T Z N A S , M . D . , * L I L I A N A L U C I A N O , M.D.,f
Collagen fibrils have a random distribution in sections from any portion of the sciera. In the equatorial region, the collagen fibrils a r e oriented both in oblique and meridional planes. I n addition to collagen, the sciera of this region contains some elastic fibers, fibroblasts and melanocytes. Unlike the cornea, a mucopolysaccharide matrix around the scierai fibers is entirely absent. 1 3 Electron microscopic studies of the mechanically fragmented sciera have been made by Schwarz, 1 · 2 ' 4 François and associates, 5 and Grignolo, 6 employing specimens which were shadowed. These techniques were satisfac tory to demonstrate surface anatomy but were not satisfactory to demonstrate the through-and-through structure of the sciera. Standard techniques were used in this study to demonstrate the scierai structure. * From the Universitäts-Augenklinik, 43 Essen, Germany. t Anatomisches Institut, Abteilung Elektronmi kroskopie, Medizinische Hochschule, 3 Hanover, Germany. Reprint requests to Manfred Spitznas, M.D., Uni versitäts-Augenklinik, 43 Essen, Hufelandstr. 55, Germany.
MARCH, 1970
COLLAGEN
AND ENRICO R E A L E , ! M.D.
MATERIALS AND METHODS
Rabbits weighing 3 to 4 k g were anesthe tized with pentobarbital sodium (Nembutal) and the common carotid on one side was perfused with a solution of 1% formalde hyde and 1.25% glutaraldehyde buffered with sodium cacodylate 0.1 M at p H 7.4 and containing 25 m g / 1 0 0 ml of calcium chloride. After enucleation of the homolateral eye the postequatorial sciera was cut into small pieces about 0.5 mm 3 in size and fixation was continued for about one hour. T h e tis sue was then fixed for an hour in a phos phate-buffered 1% O s 0 4 solution without glucose. T h e specimens were embedded in Epon 812, cut with an L K B Ultrotome I I I , placed on carbon-coated F o r m v a r membranes (Dowell 7 ) and stained with uranyl acetate or lead acetate, or both. A Siemens Elmiskop I A was used for these studies. RESULTS
The collagen fibrils among bundles of scierai collagen have varying diameters ( Figs. 1-A, B ) . In the outer sciera the average di-
DEGENERATION OF PERIPHERAL RETINA
L. E. : Histopathology of juvenile retinoschisis. Arch. Ophth. 79:49, 1968. 15. Rutnin, U., and Schepens, C. L. : Fundus ap pearance in normal eyes: III. Peripheral degenera tions. Am. J. Ophth. 64:1040, 1967. 16. Zimmerman, L. E. : Applications of histo-
403
chemical methods for the demonstration of acid mucopolysaccharides to ophthalmic pathology. Tr. Am. Acad. Ophth. 62 :697, 1958. 17. Streeten, B. W. : Development of the human retinal pigment epithelium and the posterior seg ment. Arch. Ophth. 81:383, 1969.
CORRELATION OF FLUORESCEIN ANGIOGRAM AND RETINAL IN DIABETIC R E T I N O P A T H Y E V A M.
KOHNER,
M.D.,
AND P A U L
HENKIND,
DIGEST
M.D.
New York, New York Improved techniques have recently ex panded our knowledge of diabetic retinop-athy. Such techniques include vascular injections, 1 , a shake preparations, 3 and trypsinl digestion 4 ' 5 for histologie specimens, and1 fluorescence angiograms* for observation off the retinal lesions in life. Ashton 2 ' 7 · 8 used neoprene and India inkc injections to demonstrate the connections ofE microaneurysms to capillaries, abnormal cap illary loops, and areas of capillary nonperfusion. I n severe retinopathy, he noted in volvement of arterioles and shunt vessels3 crossing otherwise nonperfused areas. Cogani and his co-workers 4 · 5 used retinal digests to) observe cellular detail. T h e y found the selective loss of pericytes from capillaries corre sponding to the nonperfused vessels of Ash ton, hypercellular microaneurysms and hypercellular shunt vessels surrounding areass of capillary closure. 9 I n 1961, Novotny and Alvis 8 reported on1 fluorescence angiographie studies in diabeticC retinopathy. Since then, fluorescence photog raphy has become widely used in investigation1 of diabetic retinopathy during life. 10 " 14 From the Department of Ophthalmology, Newv York University, New York City. Dr. Kohner wass a Medical Research Council Alexander Pigottt Wernher Memorial Trust Fellow from the Royal1 Postgraduate Medical School Hammersmith Hospi tal, London. This work was supported by NIHI Grant NB-05-059-06 and the Scientific Section off the British Diabetes Association. Reprint requests to Paul Henkind, M.D., Newv York University, 550 First Avenue, New York,:, New York 10016.
H o w accurately fluorescence angiograms mirror the retinal vascular pathology is not really known. T h e r e has been only one report comparing a digest preparation with a fluorescence angiogram in the same patient. 15 I n that study, only microaneurysms were ob served in detail. It was found that those packed with red cells did not show u p on fluorescence photographs, while those with a partially hyalinized wall leaked and ones with a hypercellular wall retained fluorescein. This paper reports on the findings in a patient who died 39 months following yttrium-90 ( 9 0 Y ) implantation of her pituitary gland. T h e last fluorescence angiograms were performed five months prior to death. CASE
HISTORY
A 30-year-old woman was seen initially at the Hammersmith Hospital in March, 1965. She was referred for pituitary ablation because of diabetic retinopathy. Her diabetes was diagnosed when she was two years of age, and she was treated twice daily with regular insulin and a 160-gram carbohydrate diet. Diabetic control was fair. In 1955, she noted tran sient blurring of vision in her right eye. This cleared rapidly with improvement of the diabetic control. Late in 1964, the vision in her left eye became blurred and gradually deteriorated, so that at the time of her first visit, it had light perception only. Visual acuity in her right eye was 20/40. Her blood pressure was 145/80, with slight posrural hypotension. The peripheral pulses were normal. The knee jerks were sluggish and the ankle jerks were absent. Pin prick and light touch sensations were markedly reduced over the feet and lower legs. The right fundus had numerous microaneurysms,
404
AMERICAN JOURNAL OF OPHTHALMOLOGY
hemorrhages, dilated capillaries and possibly new vessels (fig. 1). The left retina showed massive retinitis proliferans and preretinal hemorrhage. Laboratory studies revealed a hemoglobin of 8.3 g with microcytic film and a hematocrit of 29%. Serum cholesterol, 290 mg%; serum pro teins, 6.8 g/100 ml; albumin 3.8 g and globulins 3.0 g. Renal involvement was indicated by a blood urea nigrogen (BUN) of 62 mg/100 ml (normal 20-40 mg%) and proteinuria of 2.9 g in 24 hours. It was felt that the patient would benefit from pi tuitary ablation, and "Ύ implantation was per formed in April, 1965. The postoperative period was complicated by transient hyponatremia and postural hypotension. At the three-month assessment, she was found to have only partial pituitary ablation, with normal pe riods and thyroid function, but a reduced adrenal reserve with symptomatic Addison's disease seven days after steroid withdrawal. Growth hormone levels after insulin-induced hypoglycemia reached only six nanograms at 120 minutes.1*·" The visual acuity remained unchanged, but the retinal appear ance improved in the right eye. Over the next three years the appearance of the right fundus remained improved (fig. 2), but visual acuity deteriorated to 20/60. Fluorescence photo graphs were taken before pituitary implantation, and at six and 14 months postoperatively. Unfortu nately, these were of poor quality and difficult to compare with the last two series taken at 24 and 34 months. During the first postoperative year the patient was well, but thereafter she had a gradually rising BUN and increasing hypertension. She was admit ted to hospital on several occasions because of ure mia and finally died in June, 1968. The right eye was obtained for histologie exami nation. F L U O R E S C E N C E ANGIOGRAMS
The fluorescence angiograms were done by the intra-arterial technique.12 The area studied was a 30° field extending from the disc to about one disc diameter lateral to the macula and bounded by the temporal vessels (fig. 3). Capillaries—Multiple areas of capillary nonperfusion were evident. The largest of these was in the perimacular region, and was about three times the diameter of the normal perimacular capillary-free zone. Large areas of capillary closure were also present in the superior temporal area while smaller ones were seen elsewhere. Microaneurysms were scattered through out the field. Almost all were adjacent to areas of capillary nonperfusion. On fluores
MARCH, 1970
cence angiograms they could not always be differentiated from capillary loops, since these also surrounded areas of capillary clo sure. Some of the microaneurysms retained dye after most of the capillary bed had emp tied. A few leaked fluorescein, but this was difficult to ascertain because of the back ground choroidal fluorescence (figs. 4 and 5). Considering the severe capillary abnor malities, the number of microaneurysms was small and the leakage through their walls slight. Abnormal capillary configurations were widespread. Some jutted into areas of capil lary closure, suggesting attempts at revascularization, while others had a haphazard ar rangement. A number of the vessels were di lated and it is impossible to tell whether they were capillaries or abnormal arteries and veins, or even new vessels. Arteries and veins—Some arterial branches passing through areas of capillary closure showed local dilatation, retention of dye and leakage (fig. 5). This was also seen in some venous branches, but was less marked. Small vessels showed occasional loops and increased tortuosity. Flow mea surements were not performed in this pa tient, but the marked lamination of fluores cein in the temporal arteries may indicate slow flow. The large veins showed no obvious lesions while many of the smaller ones had unusual loops, were very tortuous, and retained dye. Arteriovenous and veno-venous communi cations—Vesels larger than capillaries were seen to connect arteries and veins especially across areas of capillary closure. This was most marked in the macular area. Arteriove nous and veno-venous communications were seen between the branches of the superior and inferior temporal vessels lateral to the macula. Some of these vessels showed local ized dilatations, but none showed the charac teristic of shunt vessels, i.e., early appear ance of dye lamina in the vein indicating a low pressure-high flow channel. New vessels—Some of the abnormal ve-
VOL. 69, NO. 3
DIABETIC RETINOPATHY
405
nous side branches might be new vessels, but true new vessel formation was only obvious in the superior temporal region at the edge of the picture. DIGEST PREPARATION
Capillaries—Almost no normal capillaries were seen in any portion of the retinal digest from the disc border to the retinal periphery. Most were either pale, acellular basement membrane tubes or strands, or tortuous, hy percellular channels with proliferated endothelium. Intramural pericytes were rare, and where present, usually had either pale nuclei or ghost remnants. No capillaries were seen with normal-appearing intramural pericytes and absent endothelial cells. The cellular cap illaries seemed to be dilated when compared to normal retinal digests, and to have an un even increase in PAS-staining of the base ment membrane. Numerous aneurysmal dilatations of the capillaries were seen. They ranged from large, round, hypercellular lesions to small, hyalinized, or even disintegrated ones (fig. 6). The latter were mainly on acellular capil laries. The aneurysms were scattered throughout the field from posterior pole to
Fig. 1 (Kohner and Hcnkind). Fundus photograph of the right eye taken in March, 1965, showing nu merous microaneurysms, hemorrhages, dilated cap illaries and possibly neovascular tufts.
Fig. 2 (Kohner and Hcnkind). Fundus photograph of the right eye taken in 1968. Note the diminished number of hemorrhages and microaneurysms.
periphery. Some were closely related to precapillary arterioles or postcapillary venules, but most were within the midst of the capil lary bed itself. No aneurysms were evident in the distribution of the radial peripapillary capillaries. Arteries and veins—The larger retinal ar teries seemed to have a normal cellular popu lation and were without evident dilatation or tortuosity. Many of the precapillary arter ioles were abnormal, having intense PASpositive walls, increased numbers of endo thelial cells and occasional microaneurysms. The larger retinal veins were slightly ir regular in caliber but had a normal cellular configuration. The smaller venous branches were often abnormal with a marked hypercellularity of their walls. A number of tor tuous, hypercellular arteriovenous communi cations—larger than capillaries—were seen crossing areas of obliterated acellular capil laries. Neovascularization—Numerous markedly irregular, hypercellular and dilated vessels appeared to overlie the normal level of the retinal capillary bed. In areas of capillary obliteration, hypercellular channels linked adjacent arteries and veins (these arterio venous communications are the shunts de-
406
AMERICAN JOURNAL OF OPHTHALMOLOGY
, ^ * «wsa^
MARCH, 1970
v
.'vliSMii-
Fig. 3 (Kohner and Henkind). Arterial-capillary phase fluorescein angiogram of the right eye five months prior to the patient's death. The area covered is a 30° field including the macula and most of the disc. The black boxes, A and B, outline where fluorescein angiograms are compared with the retinal digest. Note the areas of capillary nonperfusion and microaneurysms. scribed by Cogan and Kuwabara. 9 No such channels were seen in areas of cellular capil laries. Detailed comparison between the digest preparation and fluorescence angiograms were done in the two regions illustrated in Figures 7-10. In general, the features dem onstrated are listed in Table 1. Other features not consistently seen in
clude leakage of fluorescein from some hypercellular vessels and hypercellular loops, and retention of dye in some of the vessels, which, on digest, look no more abnormal than other vessels in which fluorescein is neither retained nor leaked. Some apparent local thickening of arteries observed on the angio grams was seen on the digest preparation to represent cellular (sometimes hypercellular)
VOL. 69, NO. 3
DIABETIC RETINOPATHY
407
Fig. 4 (Kohner and Henkind). Capillary-venous phase fluorescein angiogram taken several seconds after Figure 3. Microaneurysms and capillary loop are visible as well as irregular dilated vessels.
arteriolar remnants with attached acellular capillaries. Figures 7-A and B show the capillary
and Figure 7-C the late venous phase of area A in Figure 3. In the diagram, Figure 7-D, the features discussed are represented. Fig-
Fig. 5 (Kohner and Henkind). Late venous phase fluorescein angio gram taken several seconds after Figure 4. Note microaneurysms with persisting fluorescence and fluores cein leakage ; also, some abnormally leaking new vessels.
408
AMERICAN JOURNAL OF OPHTHALMOLOGY
M A R C H , 1970
■0*
^&Λ^.
Fig. 6 (Kohner and Henkind). Retinal digest preparation showing a variety of aneurysms and dilata tions of the capillaries. Most of the aneurysms are acellular or only slightly cellular, while the dilated vessels appear hypercellular ( P A S and hematoxylin, χ 3 2 0 ) .
ure 8 is the digest preparation of the same area. The fluorescence angiogram shows that there are large areas of capillary nonperfvi sion and on the digest preparation these are shown to be either completely acellular ves sels, or vessels with ghost pericytes only, but no endothelial cells. The very close correla tion between perfusion and cellularity is demonstrated by the vessels indicated by arrow 1 in Figure 7-D. The loops arising from this vessel jutting into the nonperfused area may be attempts at neovascularization. It is not possible to decide whether the ter minal portions are vascular loops or microaneurysms. The irregular dilated and densely staining vessel (arrow 2, fig. 7-D) is seen on the digest as a hypercellular channel with many hypercellular buds, not all of which are clearly seen on the fluorescence angio gram. Arrow 3 (fig. 7-D) shows another ir regular, dilated vessel, and this appears to retain dye for a long period (fig. 7-C). On the digest it is hypercellular, but does not
really differ from other hypercellular vessels. Note that all the hypercellular vessels appear to lie in front of the acellular ones in the histologie preparation. Of the microaneurysms, one (arrow 4, fig. 7-D) has a highly cellular wall, and does not leak fluorescein, while the one marked by arrow 5 (fig. 8) lies on an acellular capillary and is not perfused. The cluster of micro aneurysms (arrow 6, fig. 7-D) shows some leakage. We are not certain which of these microaneurysms leaks. Arrow 7 (fig. 7-D), which indicates an aneurysm on the angiograms, looks like an abnormal bend in the capillary (fig. 8). The sluggish filling of some vessels is demonstrated in Figures 7 A-C (arrow 8, fig. 7-D). On fluorescence angiograms this vessel is perfused from capillaries, most of which are abnormal, and on the digest prepa ration they appear as possible intraretinal neovascular channels (arrow 9, fig. 8). The long retention of the dye and intense fluorés-
VOL. 69, NO. 3
DIABETIC RETINOPATHY
409
Fig. 7 (Kohner and Henkind). Capillary phase (A and B) and venous phase (C) fluorescein angiograms of area A in Figure 3. A diagram of the area is shown in D, bottom left. The significant features outlined are discussed in the text. cence on late pictures probably indicates se vere endothelial cell damage, because five months later, at the time of the digest prepa ration, a short segment of this vessel appears completely acellular ( a r r o w 10, fig. 8 ) . Figure 9 A - C is the area adjacent and su
perior to the macula (area B in fig. 3 ) . Fig ure 9-D is a diagramatic representation of the features discussed and Figure 10 shows the digest preparation of the same area. Sev eral arteriovenous communications are seen to cross the area of capillary closure (arrow
410
AMERICAN JOURNAL OF OPHTHALMOLOGY
Hufe-
MARCH, 1970
,V 4 'V Pr
:io
V*·
-Mt0F4
élXOb. mu.'*
<*"
I t^T-lf
\ ·*|
*fcj
a-
i
s $i
Fig. 8 (Kohner and Henkind). Digest preparation of area A in Figures 3 and 7. (PAS and hematoxylin, xl2S).
VOL. 69, NO. 3
DIABETIC RETINOPATHY
411
4 (fig. 9-D), while not one that is perfused (arrow 5, fig. 10) lies on an acellular capil lary. The localized thickening on the artery (arrow 6, fig. 9-D) indicates the origin of the acellular vessel on which microaneurysm 5 (fig. 10) is found. DISCUSSION
Since fluorescence angiograms are now routinely performed for the investigation of diabetic retinopathy, it is important to know what the angiographie lesions represent in Ψ! « J€l terms of pathologic changes. This report M r^L • V •\ II / ·» suggests the degree of correlation that can be achieved. Complete agreement was not ex pected for two reasons : the last fluorescence angiogram was taken five months before the patient's death, and alterations of the vascu lar architecture during the digesting and mounting procedures invariably result in Fig. 9 (Kohner and Henkind). Capillary phase some distortion. Furthermore, diabetic reti (A and B) and late venous phase (C) fluorescein nas, with their abnormal vessels, are easily angiograms of area B in Figure 3. A diagram of damaged. Despite these difficulties, close thefieldis shown in D. bottom, with significant fea correlation between the fluorescence angio tures outlined and discussed in the text. gram and the digest preparation was found. 1, fig. 9-D) ; these are irregular, hypercellu The fluorescence and digest preparation lar channels. On the fluorescence angiograms appearance of microaneurysms in this study there are several vascular segments which were similar to those obtained by de Venecia show increased density and leakage in the and Davis.15 In our preparation the nonperlate phase (fig. 9-C). These appear hypercel fused microaneurysms were on acellular cap lular on the digest preparation. They may illaries, and occasionally appeared to be dis represent intraretinal neovascularization. integrating. It was difficult to distinguish Concerning microaneurysms, some are per capillary loops from microaneurysms. Com fused, but do not leak (arrow 3, fig. 9-D). parison of the fluorescence angiogram with Histologically they are hypercellular. Two the digest was useful, but occasionally even partially hyalinized ones are probably re this did not give the answer. sponsible for the leakage indicated by arrows It has been assumed by many that areas ^
f
J
M"
^^^■^^
3vf
Λ A
^3
Fig. 10 (Kohner and Henkind). Digest preparation of area seen in Figures 3-B and 9. ( F A S and hematoxylin, χ 1 2 5 ) .
of capillary nonperfusion correspond to acel lular capillaries. This was confirmed in the present study. It seems that for perfusion an endothelial cell lining is essential. The hypercellular dilated vessels were ex amined. Some were obvious arteriovenous communications, and others dilated capillar ies surrounded by many acellular vessels. These vessels appeared both dilated and ir regular on fluorescence angiograms. This is of importance, since Oosterhuis and Vink13 postulated that the easy visibility of diabetic capillaries and their apparent dilatation on fluorescence angiograms indicates leakage of the dye into the lining endothelium. Though this may occur in some vessels, the majority we studied were truly dilated capillaries. Retention of the dye in, and leakage through, the vessel wall indicates a more se vere abnormality. It is not evident from the digest preparation what this abnormality is. The vessels leaking and retaining fluorescein usually appeared no more abnormal than others which filled and emptied normally.
TABLE 1 COMPARISON OF DIGEST PREPARATION AND FLUORESCEIN ANGIOGRAM
Digest Preparation Hypocellular or acellular capillaries: Endothelial cells present, intramural pericytes ab sent or abnormal Intramural pericytes pres ent but abnormal, endo thelial cells absent Completely acellular Microaneurysms: Cellular wall Partially hyalinized Acellular or on acellular capillaries Hypercellular vessels (larger than capillaries) : Hypercellular arteriovenous communications:
Fluorescein Study
Perfused capillaries Capillaries not per fused Capillaries fused
not
per
Perfused, not leaking Perfused, leaking Not perfused Dilated vessels (larger occasionally re taining dye) Dilated channels con necting arteries to veins; flow rate not increased, thus not true shunts
VOL. 69, NO. 3
DIABETIC RETINOPATHY
Some of the hypercellular vessels, espe cially those forming buds, appear to be at tempts at revascularization of nonperfused areas, and probably represent intraretinal neovascularization. It is of interest that they arise from both arteries and veins. Since the fluorescein and digest studies are two-dimen sional, the level of these vessels could not be definitely determined. Concerning the widespread capillary ab normalities, there were relatively few microaneurysms seen by angiography. This is partly because many were on acellular capil laries and therefore not perfused. Many of the vessels present may be newly formed, and these rarely form microaneurysms. The other possibility is the beneficial effect of pi tuitary ablation on microaneurysm forma tion. It has been shown previously 19 that fol lowing pituitary implant of 9 0 Y the number of new microaneurysms forming is greatly reduced, and the disappearance of existing ones accelerated. N o conclusion about the efficacy of pitui tary ablation can be drawn from this patient. This is not only because she is the sole ablated patient in whom a retinal digest preparation has been studied, but because her pituitary ablation was only partial. Also her terminal illness was associated with hy pertension (though there was no obvious hy pertensive retinopathy p r e s e n t ) . Clearly, whatever the benefits of the oper ation were, they did not prevent the closure of large areas of the capillary bed and wide spread abnormalities throughout the retina. It was rather surprising that this patient re tained relatively good vision. Whether slow occlusion of the retinal vascular bed per mits alternate metabolic pathways to develop, or allows choroidal nourishment to supply the entire retinal thickness is unknown. SUMMARY
A 30-year-old diabetic woman had a par tial pituitary ablation for the treatment of her diabetic retinopathy. Fluorescein angiograms of her right eye were performed pre-
413
and posthypophysectomy. At her death, 39 months after 0 0 Y implantation, her right eye was removed. T h e retina was digested and a comparison made between the fluorescein angiograms and the histologie preparation. A good correlation was found to exist between the angiographie appearances and the microcirculatory pathology. T h e major points are : ( 1 ) that perfused vessels have an intact endothelium, nonperfused ones lack endothelium ; ( 2 ) that perfused microaneurysms have a cellular wall, while acellular ones do not show up on the angiogram but can be found in the retinal digest preparation; ( 3 ) that the dilated vessels seen by angiography usually were hypercellular; ( 4 ) that arteriovenous communications were hypercellular channels lying in an otherwise atrophie cap illary bed. Angiography revealed that they were not highflow channels. ACKNOWLEDGMENTS
We thank Dr. Richards, Ashford Hospital, Mid dlesex, for obtaining the eye, postmortem, and Professor Russell Fraser for permitting us to study his patient. The line drawings were prepared by Mr. Philip C. Johnson, the photographs by Mr. Henrick Malpica, and the text typed by Mrs. Doris Ortega. Mr. Cybald Cole provided technical assistance. REFERENCES
1. Michaelson, I. C, and Stecdman, H. F. : Injec tion of the retinal vascular system in enucleated eyes. Brit. J. Ophth. 33:376, 1949. 2. Ashton, N. : Injection of the retinal vascular system in the enucleated eye in diabetic retinopathy. Brit. J. Ophth. 34:38, 1950. 3. Ashton, N. : Vascular changes in diabetes with particular reference to the retinal vessels. Brit. T. Ophth. 33 :407, 1949. 4. Kuwabara, T., and Cogan, D. G. : Studies of retinal vascular patterns. 1. Normal architecture. Arch. Ophth. 64:904, 1960. 5. Cogan, D. G., Toussaint, D., and Kuwabara, T. : Retinal vascular patterns. IV. Diabetic retinop athy. Arch. Ophth. 66:100, 1961. 6. Novotny, H. R., and Alvis, D. L. : Method of photographing fluorescence in circulating blood in the human retina. Circulation 24 :82, 1961. 7. Ashton, N. : The pathology of retinal micro aneurysms, Acta XVI. Concilium Ophthalmologicum (Britannia) 1:411, 1951. 8. Ashton, N. : Arteriolar involvement in diabetic retinopathy Brit. J. Ophth. 37:282, 1953. 9. Cogan, D. G., and Kuwabara, T. : Capillary
414
AMERICAN JOURNAL OF OPHTHALMOLOGY
shunts in the pathogenesis of diabetic retinopathy. Diabetes 12:293, 1963. 10. Scott, D. J., Dollery, C. T., Hill, D. W , Hodge, J. V., and Fraser, T. R. : Fluorescein stud ies in diabetic retinopathy. Brit. Med. J. 1:811, 1964. 11. Norton, E. W. D., and Gutman, F.: Diabetic retinopatby studied by fluorescein angiography. Ophthalmologica 150:5, 1965. 12. Kohner, E. M., Dollery, C. T., Paterson, J. W., and Oakley, N. W. : Arterial fluorescein stud ies in diabetic retinopathy. Diabetes 16:1, 1967. 13. Oosterhuis, J. A., and Vink, R. : Fluorescein photography in diabetic retinopathy, in Perspectives in Ophthalmology, Henkes, H. (ed.), Exerpta Medica Foundation, 1968. p. 1. 14. Wessing, A. : Fluorescenz Angiographie der
Retina. Stuttgart, Georg Thieme Verlag, 1968. 15. De Venecia, G., and Davis, M. D. : Histology and fluorescein angiography of microaneurysms in diabetes mellitus. Invest. Ophth. 6:555, 1967. 16. Fraser, T. R., and Wright, A. D. : Standard procedures for assessing hypersécrétion and secre tory capacity for human growth hormone using the radioimmunoassay. Postgrad. Med. 44:53, 1968. 17. Wright, A. D , Kohner, E. M., Oakley, N. W., Fraser, T. R., Joplin, G. F., and Hartog, M. : Serum growth hormone levels and the response of diabetic retinopathy to pituitary ablation. Brit. Med. J. 2 :346, 1969. 18. Kohner, E. M., Dollery, C. T., Fraser, T. R, and Bulpitt, C. J. : The effect of pituitary ablation in diabetic retinopathy studied by fluorescence pho tography. In preparation.
FINE STRUCTURE O F RABBIT SCLERAL M A N F R E D S P I T Z N A S , M . D . , * L I L I A N A L U C I A N O , M.D.,f
Collagen fibrils have a random distribution in sections from any portion of the sciera. In the equatorial region, the collagen fibrils a r e oriented both in oblique and meridional planes. I n addition to collagen, the sciera of this region contains some elastic fibers, fibroblasts and melanocytes. Unlike the cornea, a mucopolysaccharide matrix around the scierai fibers is entirely absent. 1 3 Electron microscopic studies of the mechanically fragmented sciera have been made by Schwarz, 1 · 2 ' 4 François and associates, 5 and Grignolo, 6 employing specimens which were shadowed. These techniques were satisfac tory to demonstrate surface anatomy but were not satisfactory to demonstrate the through-and-through structure of the sciera. Standard techniques were used in this study to demonstrate the scierai structure. * From the Universitäts-Augenklinik, 43 Essen, Germany. t Anatomisches Institut, Abteilung Elektronmi kroskopie, Medizinische Hochschule, 3 Hanover, Germany. Reprint requests to Manfred Spitznas, M.D., Uni versitäts-Augenklinik, 43 Essen, Hufelandstr. 55, Germany.
MARCH, 1970
COLLAGEN
AND ENRICO R E A L E , ! M.D.
MATERIALS AND METHODS
Rabbits weighing 3 to 4 k g were anesthe tized with pentobarbital sodium (Nembutal) and the common carotid on one side was perfused with a solution of 1% formalde hyde and 1.25% glutaraldehyde buffered with sodium cacodylate 0.1 M at p H 7.4 and containing 25 m g / 1 0 0 ml of calcium chloride. After enucleation of the homolateral eye the postequatorial sciera was cut into small pieces about 0.5 mm 3 in size and fixation was continued for about one hour. T h e tis sue was then fixed for an hour in a phos phate-buffered 1% O s 0 4 solution without glucose. T h e specimens were embedded in Epon 812, cut with an L K B Ultrotome I I I , placed on carbon-coated F o r m v a r membranes (Dowell 7 ) and stained with uranyl acetate or lead acetate, or both. A Siemens Elmiskop I A was used for these studies. RESULTS
The collagen fibrils among bundles of scierai collagen have varying diameters ( Figs. 1-A, B ) . In the outer sciera the average di-