retinal vascular patterns

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Retinal vascular patternsVII. Acellular change

Toichiro Kuwabara and David G. Cogan

The trypsin digestion technique has revealed a preferential loss of endothelial cells (inconditions other than diabetes) and eventually a total acellularity of many retinal capillaries.The surprise is that this may occur toithout recognized functional abnormality or withoutappreciable histologic change in the rest of the retina. Perhaps the retina has a large reservecapacity normally or perhaps the choroidal circulation compensates for loss of the retinalcirculation. In any case the retina shows little reparative response to these capillary changes.Unlike what one expects in most other tissues, there is no formation of fibrous scar tissue,no infiltration w ith histiocytes, a nd no neovasculo genesis. Instead .Midler s cells fill up thetissue defect and their processes insinuate into what had been the capillary lumina.

-Lrevious papers have described the char-acteristic patterns and cytology of theretinal vessels, as revealed by the trypsindigestion technique, in normal and patho-logic conditions.la f Electron microscopicobservations1 2> 3 have supplemented thesedescriptions. The importance of the muralcells has been especially emphasized.ld> lf> 4These cells appear to be differentiatedpericytes encased within the wall of theretinal capillary and have an especial vul-nerability in diabetes. Only casual refer-ence has been made to the endothelialcells.Loss or attenuation of the endothelial

From the Howe Laboratory of Ophthalmology,Harvard University Medical School, Massachu-setts Eye and Ear Infirmary, Boston, Mass.This investigation was supported by a PublicHealth Service Research Grant No. HE-04051from the National Heart Institute and by aPublic Health Service Research Grant No.NB-02698 from the National Institute ofNeurological Diseases and Blindness, PublicHealth Service.

cells from the capillaries is a eommop -find-ing, perh aps the most common finding,, in-various pathologic conditions and in other-wise normal eyes. Widespread loss of cellu-larity is a constant occurrence in theperipheral capillaries of elderly people.It is often preliminary to loss of the muralcells with the result that a totally acellularand fibrous plexus finally remains. The ab-sorption of these acellular vessels is eitherextremely slow or nonexistent so that theremaining scaffolding preserves perma-nently the architectural pattern of theoriginal blood vessels.In the present paper we propose todemonstrate the early changes in theendothelial cells and the later changes inthe acellular vessels as seen in flat prepara-tions of the retinal vessels and in electronmicrographs. We wish further to point outa u niqu e reactio n of Miiller s cells of th eretina accompanying loss of cells from theretinal vessels but a surprising lack ofcorrelation between acellularity of retinalcapillaries and histologic abnormalities inthe retinal tissue.


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1 5 Kuwabara and Cogan Investigative phthalmologyDecember 1965

Materials and methodsRetinal vessels from approximately 500 eyeswere isolated by the trypsin digestion techniquewhich we have previously described.111 Aboutone-half of the eyes were obtained postmortemfrom persons not known to have ophthalmic dis-ease and one-half were obtained surgically witha variety of ocular disorders.Electron micrographic studies were carried outon the normal eyes of four elderly and threeyoung persons, on three eyes with absolute glau-coma, and on two eyes with injury.Correlative observations were made on twentycats eyes which had been damaged experimen-tally. The experiments included systemic poison-ing by iodoacetic acid, mechanical trauma,

ischemia, and photocoagulation.Results

Normal retinal capillaries show a re-markably uniform caliber of their luminaand constant distribution of cells in theirwalls. The ellipsoidal pale nuclei of theendothelial cells are regularly spaced asare also the round, dark nuclei of themural cells (Fig. IB). In the developingstage and during the first decade of lifethe nuclei of both endothelial and mural

cells are relatively abundant and frequentlyappear to overlap one another (Fig. 1A).With increasing age the nuclei in thecapillary wall become less numerous perunit length and eventually the ratio of theendothelial cells to the mural cells de-clines progressively.5 Although the endo-thelial nucleus becomes somewhat elon-gated with age (Fig. 1C), the shape andstainability of the individual nuclei remainrelatively constant.

The fine structure of normal retinalcapillaries is characterized in practicallyall species by: (1) a thick basement mem-brane which forms the outer limits of thecapillary and at the same time separatesthe endothelial and mural cells; (2) a thickendothelial lining of the relatively smalllumen; and (3) the presence of muralcells.Early changes in the endothelial cells.As the earliest pathological change, endo-thelial cells become irregular in shape,nuclear stainability, and distribution (Fig.2). This irregularity is seen either through-out the whole vasculature or in small lo-

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A . BFig. 1. Vessel preparations of normal human retinas. (P.A.S.-hematoxylin stain. x256.) A,Highly cellular vessels of 8-year-old boy. B, A 40-year-old man; endothelial cells e) an dmural cells m) are regularly spaced. C, An 80-year-old man; only two endothelial nucleiare seen in this field whereas mural cells are numerous. Capillaries are somewhat smaller.


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calized areas. The reason for these changesis not always clear but often acute or sub-acute pathological conditions, such as sud-den elevation of the intraocular pressureor injury, is found to be the possible cause.Although the destructive change is not ap-parent at this stage, acelliilarity seems tofollow it.The next noticeable, nonspecific changein the capillary wall is the disappearanceof the endothelial nucleus (Fig. 1C). De-pending on conditions of the retina andage, the sparseness of the endothelialnuclei varies considerably. And the severityof the vascular change is not necessarilycorrelated with histologic abnormalities inthe rest of the retina. It is not uncommonto find total absence of endothelial nucleiin a large area of the vasculature of a his-tologically normal retina.Capillaries which have lost endothelialnuclei appear to maintain their originalcaliber as long as mural cells are intact.Although red blood cells are not frequentlyfound in capillaries which have lost theirendothelium, electron microscopic studysuggests that some of these capillaries may

still be patent. Cross sections of retinalcapillaries in instances where vessel prepa-rations of the adjacent area have revealedextensive endothelial acelliilarity show thatmany are patent and have the normalendothelial lining (Fig. 3). The fact thatthe chances of hitting the nucleus of theendothelial cell in the ultrathin sections ofthis retina are extremely small, whereasthe demonstration of the mural nucleusis quite frequent, may well indicate thatthese cross sections are of endothelialacellular capillaries.

The cytoplasmic lining of these capil-laries presumably belongs to some surviv-ing endothelial cells from distant portionsof the same vessel, or from the adjacentcapillary. The fine structure of these cellsis not so different from the normal condi-tion, except that the cytoplasm is a littlethinner and the basement membrane be-longing to the endothelial cell shows arelatively high incidence of vacuole forma-tion, whereas the part which surroundsthe main body of the mural cell usuallyavoids severe vacuolization (Fig. 4).Endothelial and, occasionally, mural

Fig. 2. Vessel preparation from the retina of a glaucoma patient. Endothelial cells are ir-regular in shape and distribution. Mu ral cells are regularly distributed. (P.A.S.-hematoxylinstain. x256.)


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1052 Kmoabara and Cogan Investigative phthalmologyDecember 1965

Fig. 3. Electron micrograph of a retinal capillary from a young patient with ocular injury.Th e vessel prepa ration of this retina righ t) shows extensive endothelial acelhilarity. Theelectron micrograp h left) probably corresponds to an area similar to that indicated by theline crossing a mural cell. The capillary is probably patent. The mural cell Mnuc) is nor-mal. Endothelial lining E)may be somewhat thinner.

cells in the capillaries which are showinga decrease in the number of their endo-thelial nuclei, often show lipofuscin gran-ules in their paranuclear areas. Thesemasses of the lipofuscin are found in flatpreparations as well as by electron mi-croscopy Fig . 5). They are P.A.S. posi-tive and sudanophilic and easily distin-guishable from melanin or hematogeneouspigments. Aside from senescence, lipofuscingranules are most commonly seen in thevessels of chronically pathological eyes.They were especially conspicuous in retinalcapillaries of a few cases of chronic mildvenous occlusion.

Another type of electron-dense materialis seen within the vacuoles of the base-ment membrane. These are also of lipidic

nature and composed of fine amorphousgranules.Small nodulelike accumulations ofmitochondria in the endothelial and muralcells of this pathological stage may bemore than an incidental finding, thoughthe microorganelles appear to be withinnormal limits. This is also clearly observedin the vessels prepared from retinas whichhave been incubated in a nitro blue-tetrazolium solution containing succinicacid as the substrate, and followed by thetrypsin digestion Fig. 6) . The blue-stained, nodulelike material correspondsto the site of the accumulation of themitochondria observed electron micro-scopically.

Retinal tissue which is undergoing vas-


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Fig. 4. Capillary of a normal retina of an elderly person. Vacuoles (arrow) are seen in thebasement membrane at the enclothelial side. Basement membrane over the main body of themural cell has no vacuolization.cular changes often shows a conspicuousincrease of membranous structure aroundthe vessels. This structure seems to haveoriginated when the cell membranes ofMidler s cell processes became closer to-gether, as a result of the disappearanceof some neuronal cells. Basement mem-brane often extends into these membranes.Fine fibrous material clinging around thevessels in the pathological cases, as is seenin flat preparations, corresponds to theabove fibrous material (Fig. 7).Destructive changes, such as karyolysisor karyorrhexis, are also seen especiallyin eyes with acute ischemia or followingembolism. These changes seem to be fol-lowed by a localized or diffuse death ofthe capillary. In flat preparations they areseen as wrinkled acellular strands.le

Except in the case of diabetic retinop-athy, proliferation of endothelial cellsis rare. Proliferation of the mural cell hasnot yet been observed.Late stage of the acellular vesselsEventually the damaged capillaries losethe nuclei of mural cells as well as ofendothelial cells. Totally acellular capil-laries are not patent, and are no longerfunctioning as circulatory channels. Asretinal tissue seemingly has very littlecapacity for absorbing dead tissue, par-ticularly the basement membrane sub-stance, remnants of the capillaries re-main at the original site, preserving thenormal pattern of plexus for a longperiod of time. Damaged vessels in thenonspecific pathological condition appearto be simply abandoned (Fig. 8). Re-


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1054 Kuwabara and Cos.an Investigative phthalmologyDecember 1965

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Fig. 5. Lipofuscin particles Lf ) seen in the endothelial cell of an elderly person. Lipidicgranules Lg ) are also seen in the vacuole in the basement membrane. Vessel preparationof this retina left) shows P.A.S.-positive granules in the capillary wall.

A BFig, G. Dehy drogenase activity in the retinal capillaries of a glaucoma patien t. A, Succinicacid dehydrogenase activity. Large-sized granules are characteristically found in pathologicretinas. xl28,) B, DPNH diaphorase activity is demonstrated in the mural cell and in groupsof mitochondrias arro w) . *256.)


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Fig. 7. Increased membranous structure about a retinal capillary of an elderly person. Base-ment membrane substance is being deposited in some membranes (arrow). Note the accumu-lation of mitochondrias in the mural cell. Vessel preparation from the same retina (belo w)shows membranous structures attached to the vessel (arrow). (P.A.S.-liematoxylin stain.x256.)paratory process or neovasculogenesis toreplace the damaged vessels is either ab-sent or extremely rare. The reparatoryreaction of the surrounding retinal tissueis also minimal.Depending upon the nature and degreeof the damage, acellular strands are seeneither as clusters, or as a total acellularvascular bed. Even in cases of sharplycircumscribed retinal degeneration, dam-aged vessels stay at the original site with-

out appreciable change in the surroundingvascular beds.These damaged vessels are seen asP.A.S.-positive material in. the histopatho-logical specimen. Electron microscop-ically, they are demonstrated either as acollapsed structure of basement membranesurround ed by Mtiller s cells, or as a struc-ture which maintains the original caliberof the capillary but filled with prolonga-tion of Mullens cells (Figs. 8C, 9).


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1056 Kuioabara and Cogan Investigative phthalmologyDecember 1965

Fig . 8. A 57-year-old man with central artery occlusion or ^ weexs uuruuu n. s\, s\u u ip-laries have lost cellularity, but still maintain their original vascular pattern. (P.A.S.-hematoxy-lin stain. xl84.) B, Histolog)r shows good preservation of the outer half of the retina. Theinner layers have lost cells. One large collapsed vessel is seen in the center. No capillaryis recognizable. {Hematoxylin-eosin stain, x 184.) C, Electron micrograph of the nerve fiberlayer of the same retina shows totally acellular capillary. The lumen L) is filled with the ex-tension of the cytoplasm of Miiller s cell. The basem ent mem brane is swollen and vacuolated.


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Fig. 9. Capillaiy of the cat retina of which the large arteries were damaged by photo-coagulation two months previously. Capillary lumen and the space of the mural cell cyto-plasms M) are completely filled with Miiller s cell. Higher magnification (rig ht) showsthe identical structure in both outside and inside L) of the capillary. The basement mem-brane preserves its original structure quite well. All experimentally damaged, and clinico-pathological cases show similar findings.

In either case, the basement membraneappears to be swollen or looser than nor-mal, and shows occasional breaks. Thecytoplasm of th e Mliller s cell which hasinvaded into the capillary lumen is iden-tical to that which is in the surroundingretina. No collagenous tissue or fibrousthrombus is demonstrable in these lumina.

Peter Poulimenos and Barbara Bridgman gavetechnical assistance in the electron microscopicobservations.R E F E R E N C E S

la . Kuwabara, T., and Cogan, D . G.: Studies ofretinal vascular patterns. I. Normal architec-ture, Arch. Ophth. 64: 904, 1960.

l b . Toussaint, D., Ku wabara, T., and Cog an, D,C : Studies of retinal vascular patterns. II.Human retinal vessels studied in three dimen-sions, Arch. Ophth. 65: 575, 1961.lc . Kuw abara, T ., Carroll, J. M., and Cogan, D .C : Studies of retinal vascular patterns. III.Age, hypertension, absolute glaucoma, injury,Arch. Ophth. 65: 708, 1961.Id. Cogan, D. C , Toussaint, D., and Kuwabara,T.: Studies of retinal vascular patterns. IV.Diabetic retinopathy, Arch. Ophth. 66: 366,1961.l e .Reinecke, R. D., Kuwabara, T., Cogan, D . C ,and Weis, D. R.: Studies of retinal vascularpatterns. V. Experimental ischemia of thecat eye, Arch. Ophth. 67: 470, 1962.If. Kuwabara, T., and Cogan, D. C : Studies ofretinal vascular patterns. VI. Mural cells of


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1058 Kuioabara and ogan Investigative phthalmologyDecember 1965

the retinal capillaries Arch. Ophth . 69: 4921963.Kissen A. T. and Bloodworth J. M. B. Jr.:Ultrastructure of retinal capillaries of the ratExper. Eye Res. 1 :1 1961.Ishikawa T.: F ine structure of retinal vessels

4. Cogan D. G. and Kuwabara T.: Capillaryshunts in the pathogenesis of diabetic retinop-athy Diabetes 12: 293 1963.5. Cogan D. C : Doyne memorial lecture 1963.Development and senescence of the humanretinal vasculature Tr. Ophth. Soc. 83 : 4651963.

retinal vascular patterns

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