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Experimental Retinal Detachment in the Owl Monkey
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todo personale di provocazione di distacco retinico sperimentale. Boll. Oculist. 43:87, 1964. 11. Landholm, W . M. and Watzke, R. C. : E x perimental retinal detachment with a sulfated polysaccharide. Invest. Ophth. 4:42, 1965. 12. Freilich, D. B., Lee, P. and Freeman, H . M. : Experimental retinal detachment. Arch. Ophth. 76 :432, 1966.
S E P T E M B E R , 1568
13. Fraunfelder, F. T. and Potts, A . M . : An experimental study of retinal detachments. Am. J. Ophth. 62 :561,1966. 14. Custodis, E. : Beobachtungen bei der diathermischen Behandlung der Netzhautablösung und ein Hinweis zur Therapie der Amotio retinae, Ber. Deutsch. Ophth. Ges. 57:227,1952.
E X P E R I M E N T A L R E T I N A L D E T A C H M E N T IN T H E O W L M O N K E Y HISTOLOGY
II.
OF RETINA
ROBERT
A N D PIGMENT
MACHEMER,
EPITHELIUM
M.D.*
Miami, Florida dase was injected into the vitreous, and under direct visual control the vitreous was repeatedly aspirated and then reinjected against the temporal equatorial retina, producing a retinal hole with surrounding detachment. The eyes were enucleated at 1 hour, 1 day, 3 days, and 1, 2, 4, 8, 12, 14 and 20 weeks after the production of the hole. Each group consisted of two to three eyes. They were fixed in a modified Kolmer fixative for 24 hours (one part acetic acid, four parts saturated watery solution of potassium dichromate, and four parts 4% formaldehyde) . T o keep the retina attached and to preMETHODS vent shrinkage of the eye, about 0.1 cc of the As previously described, retinal detach- fixative was injected into the anterior chamments were produced in the owl monkey ber. After one day of fixation a small calotte (Aotus trivirgatus) : One unit of hyaluroni- was cut off from the nasal side to avoid deformation of the globe during the dehydraFrom the Bascom Palmer Eye Institute, Departtion and to give the embedding material acment of Ophthalmology, University of Miami School of Medicine. This investigation was supcess to the inner eye. The eyes were slowly ported in part by U S P H S Research Grant M B dehydrated in alcohol, brought into methyl06841 from the National Institute of Neurological benzoate and, after only five minutes in chloDiseases and Blindness; in part by Research to Prevent Blindness, Inc.; in part by the Florida roform, embedded in paraffin.
The serial histologic changes in human retinal detachment are, as of the present time, still incompletely described because of the unavailability of pathologic tissue. Only a few cases of early human detachment are available. " A study of detached rabbit retina was made as early as 1909, but this retina differs from that of man in many respects. The purpose of this report is to describe the sequence of histologic changes in the experimentally detached owl monkey retina, a species whose eye is quite similar to that of man. 1
4
5
6
Lions' Eye Bank, Inc.; in part by the Florida Mid-Winter Seminar; in part by the National Council to Combat Blindness, Inc., Grant-in-Aid Special Fellowship No. F-211, and in part by Deutscher Akademischer Austauschdienst, Bonn, Germany. Read in part before the 17th annual session of the New Orleans Academy of Ophthalmology, Symposium on Retina and Retinal Surgery, New Orleans, February 15, 1968. * Visiting Research Fellow, Clinic, 34 Goettingen, Germany.
University
Eye
Vertical serial sections of 5-micron thickness were made in five areas of the eye : ( 1 ) through the retinal hole, ( 2 ) through the macular area, ( 3 ) through the disc, and (4 and 5) through nasal parts of the eye at distances from the disc corresponding to those of areas 1 and 2. The sections were stained with hematoxylin-eosin, P A S , and trichrome
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Fig. 1 (Machemer). Histology of normal retina in owl monkey very similar to that of human. Kolmer fixation preserves structure of retina well. ( P A S , X190.)
stain after Masson after they had been in a 5% solution of sodium bicarbonate for one hour. Controls included ( 1 ) seven untreated eyes, ( 2 ) two eyes injected intravitreously with one unit of lyophilized hyaluronidase (Wyeth Co.) dissolved in 0.1 cc of saline, and ( 3 ) four eyes injected with one unit of hyaluronidase in which the vitreous was then destroyed mechanically on the temporal side by repeated aspiration and reinjection. The latter six eyes were enucleated after 24 hours. RESULTS RETINA In the normal retina the histologic picture was very similar to that of the human eye (fig. 1 ) . However, there was no fovea, but a macular area with a large number of gan-
glion cells. Single photoreceptors, consisting of the inner and outer segments which were parallelly arranged and in good contact with the pigment epithelium, (fig. 2 ) could be seen. It should be emphasized that retinal capillaries never go deeper than the outside of the inner nuclear layer. After administration of hyaluronidase, or after vitreous destruction together with hyaluronidase, no histologic changes could be found in the retina. One hour after initial production of the retinal detachment, no additional retina detached spontaneously. The initial detachment was produced by fluid injected behind the retina, but only the retina near the retinal hole showed signs of mechanical destruction. The remainder of the detached retina looked normal. After one day the first signs of a loosen-
Fig. 2 (Machemer). Photoreceptors of normal retina consist mainly of parallelly arranged rods which can be easily distinguished. ( P A S , X750.)
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Fig. 3 (Machemer). Retina with 1-day-old detachment. Inner portions of retina, including inner nuclear layer, show diffuse loosening of structure, caused by edema. ( P A S , X 1 9 0 ) .
ing of the retinal structure in the inner layers appeared, with the outer layers of the detached retina becoming irregular (fig. 3 ) . Very few macrophages—most of them full of pigment granules—appeared at the outside of the outer segment layer. Most impressive after three days was a diffuse loosening of the inner layers of the detached retina (fig. 4 ) . Its severity was not uniform, so that all the stages between loosening of the structure and cystoid space formation could be found. Usually this structural loosening was most severe in the inner plexiform layer, but as soon as cystoid spaces were formed, they were found in the ganglion cell, inner plexiform, and inner nuclear layers. Horizontal structures tended
less to edema, so that the nerve fiber layer and a bandlike structure in the inner plexiform layer were often more compact than the environment. The outer nuclear layer usually appeared normal. A few nuclei were possibly pyknotic. With the formation of cysts in the inner layers, a few small empty spaces were sometimes seen in the outer nuclear layer. The photoreceptor layer showed very noticeable changes (fig. 5 ) . The inner segments appeared fairly normal with vertical arrangement, but the transition zone between the inner and outer segments, the area of the connecting ciliae, showed many small, empty spaces. The outer segments became irregularly arranged, could not be separated from
Fig. 4 (Machemer). Three-day-old detachment Cystoid spaces develop mainly in ganglion cell layer and in inner nuclear layer. Horizontal structures, such as nerve-fiber layer and part of inner plexiform layer, are less edematous. ( P A S , X190.)
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Fig. 5 (Machemer). Three-dayold detachment. Whereas inner segments of photoreceptor layer appear fairly normal, outer segments are irregularly arranged and cannot be distinguished from one another. Cystoid spaces appear. Macrophages are seen in this layer. ( P A S , X750.)
each other, and were of different lengths. Some pigment granules were seen at the outer margin and between the outer segments. Macrophages could be seen between outer segments. By the seventh day of detachment, it became more apparent that the edema in the retina varied significantly in different portions. In addition, there were areas with larger cystoid spaces (fig. 6 ) . When they developed, no edema could be found in the inner retinal layers, only a few small pseudocysts in the ganglion cell layer. These cystoid spaces gave the inner nuclear layer up to three times its normal thickness. They pushed the outer parts of the retina into folds, making the back of the retina wavy. They were vertically oriented
and could penetrate through the whole thickness of the layer. Usually the cells were found at the base and at the top of the cystoid spaces. The septa between the pseudocysts consisted of columns of cells, but could be so thin that only fine strands of acellular nerve fibers ran through them. There was still vascular supply of the whole inner nuclear layer, since capillaries were seen in the septa, and they reached the outer parts of this layer. A two-week-old detachment showed all the changes already described, but the cystoid space formation was more marked : the cavities could be so large that they extended into the outer nuclear layer (fig. 7 ) . The outer segments had degenerated further. This layer was thinned out and faded away
Fig. 6 (Machemer). One-week-old detachment. Edema as well as cystoid spaces may cause posterior side of retina to bulge. Clinically, this corresponds to shagreenlike pattern seen in highly detached retinas. Note that edema of innermost layers has vanished. Only a few cystoid spaces are found in the ganglion cell layer. ( P A S , X190.)
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Fig. 7 (Machemer). Two-week-old detachment. Large cystoid spaces in the inner nuclear layer have broken through inner plexiform layer into outer nuclear layer. Separating walls are very thin and in final stage consist only of nerve fibers. ( P A S , X190.)
somewhat toward its outer edge. It was impossible to distinguish any single outer segment. The layer was full of macrophages (fig. 8 ) . Three-, four-, five- and eight-week-old detachments showed no further changes, except that the cystoid spaces became larger. After 12 to 14 weeks of detachment, there were still portions of the retina that appeared normal, but most of the retina showed all stages of detachment, from diffuse loosening of structure to extensive cyst formation. Large pseudocysts had broken down the septa in many. Here, finally, the inner nuclear layer had almost disappeared, and the outer nuclear layer was reduced to one or two cells (fig. 9 ) . Heavy atrophy of the photoreceptors occurred, with short and
Fig. 8 (Machemer). Two-weekold detachment Outer segments are less numerous. The layer is thinned out and full of macrophages (see also fig. 7 ) . ( P A S , X7S0.)
thick inner segments, reduced in number; the outer segments had almost disappeared (fig. 10). It was noted that in flatly detached retinas the described changes always occurred to a much lesser degree. For example, a 14-day-old, flatly detached retina (no more than three times the thickness of a normal retina) showed no loosening of its structures in the inner parts. Thus it appeared normal in many portions ; but where the detachment was highest, the retina was thickened, as was apparent from the wavy appearance of the outer retina, although no loosening of the structure could be seen histologically. The photoreceptor layer was in much better condition than in highly detached retinas (fig. 11). Depending on the height of the detach-
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Fig. 9 (Machemer). Fourteen-week-old detachment with huge cystoid spaces and loss of cells in inner and outer nuclear layer. ( P A S , X190.)
ment, it was possible to identify even single outer segments ; they were sometimes still parallelly arranged. Not only flatly detached
areas of the retina could be well preserved, but sometimes also highly detached portions, Proliferation on the surface of the retina
Fig. 10 (Machemer). Higher magnification of retina shown in Figure 9. Inner segments are shorter and thicker; their number is reduced. Only small residues of the outer segments are left. ( P A S , X7S0.)
Fig. 11 (Machemer). Fourteenday-old, very flat detachment In comparison to changes in a highly detached retina (see Figure 8 ) , outer segments are much better preserved. ( P A S , X750.)
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Fig. 12 (Machemer). One-hour-old detachment. Edges of retinal hole are curled inwardly. (PAS,
was histologically observed three weeks after detachment, but there was clinical evidence —in one case with massive preretinal retraction after 14 days—that this can happen earlier, and may be very extensive. These changes will be described and discussed in a subsequent paper.
xSO.)
RETINAL HOLE
On the third day the edge of the retina at the hole was rounded, and it looked as though the nerve fiber layer had pulled the outer layer of the retina inward around the edge. No signs of proliferative activity were found. The outer nuclear layer contained many pyknotic nuclei, and wherever these were found the inner segments of the neuroepithelium were destroyed. This destruc-
One hour after and one day after the production of the detachment the edges of the retinal hole were found to be curled inwardly to a great extent (fig. 12). The edge of the hole—the retinal wound—was irregular and sometimes red blood cells covered it, depending on whether or not a vessel was hit during the production of the hole. The edge was not yet rounded, and the outer layers were not yet pulled inward. In the immediate vicinity of the hole the retina showed pyknotic nuclei in the outer nuclear layer and, to a lesser extent, in the inner nuclear layer, with destruction of the outer segments, with irregularity and shortening of the photoreceptors (fig. 13).
Fig. 13 (Machemer). High-power view of retina in Figure 12 shows fresh retinal wound. In immediate vicinity of hole many nuclei in inner and outer nuclear layers are pyknotic; outer segments missing. ( P A S , X7S0.)
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Fig. 14 (Machemer). One-week-old detachment. A t edge of hole outer layers of the retina are pulled inwardly. Inner limiting membrane is wrinkled. ( P A S , X195.)
tion was observed only in the immediate vicinity of the retinal hole. After one week the inner limiting membrane near the hole was wrinkled and the outer layers were pulled inward more than previously (fig. 14). Near the edge of the retinal wound there were a few cells with lightly staining, large, round nuclei and a prominent nucleolus. These were seen also in the outer nuclear layer (fig. 15), more evident after two weeks (fig. 16). There were more of these cells, and they were found in all layers. Some still resembled the above-described cells but there were gradual changes to cells with cigar-shaped nuclei. Mitoses were observed.
Older holes sometimes showed almost no proliferation, others had newly formed tissue, with the staining qualities of collagen, covering the wound. Some eyes showed progressive proliferation and strand formation along the retinal surface as early as four weeks after the beginning of the detachment. PIGMENT EPITHELIUM One hour after and one day after a detachment of the retina the pigment epithelium was normally flat and the pigment granules were still vertically oriented. A part of the pigment cell became visible that was hidden as long as the retina was attached and
Fig. 15 (Machemer). Highpower view of Figure 14. Cells with lightly basophilic, large, round nuclei appear near the edge of retinal wound. (PAS, x750.)
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Fig. 16 (Machemer). Two-weekold detachment. Many cells proliferating at retinal wound, covering it with "scar" tissue. ( P A S , X750.)
Fig. 17 (Machemer). Artificially induced retinal detachment occurring during fixation process. Pigment epithelium seems to consist of clear base with partly visible nuclei and pigmented top. ( P A S , Xl.900.)
which could not be seen in an artificial detachment, due to the histologic process (fig. 17). Overlying the pigment granules there was a faintly staining, nearly translucent area with a thickness one-third of the cell (fig. 18), irregularly limited to the subretinal fluid and with a fine vertical structure. The pigment granules protruded into this portion of the cell, which is never seen in an artificial detachment, due to the histologic process. On the third day the pigment epithelium cell tended to protrude. The pigment granules were no longer mainly vertically oriented, but often irregular. Only a few granules protruded into the translucent portion of the cell. During the first to the eighth weeks the translucent layer thickened from one-third to sometimes two-thirds of the thickness of the cell. The cells protruded further, some assuming the shape of a collar-button, with the
upper portion containing the pigment granules and the lower portion the nucleus (fig. 19). There were always flat cells, more numerous as the detachment became older. The pigment granules protruded more often into the translucent portion of the cell. There were cells with nuclei larger than normal and others that had two or more nuclei in one cell (figs. 20 and 2 1 ) . Finally, after 12 to 20 weeks, the pigment epithelium became flat. Only a few protruberant cells were seen. The translucent portion was about one-half the thickness of the cells. The pigment granules were located and oriented as in the normal eye; that is, they covered the inner part of the nuclei and were mainly vertically oriented and protruded like fingers into the translucent portion (fig. 2 2 ) . At that time, the nuclei were all about the same size. Cells with two nuclei were rarely found.
Fig. 18 (Macnemer). One-hour-old detachment Pigment epithelium has translucent portion which overlies pigmented part and represents cytoplasmatic processes of these cells. ( P A S , X1,900.)
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Fig. 19 (Machemer). Four-weekold detachment. Pigment epithelial cells protrude and may be collarbutton-shaped. Translucent layer becomes more prominent in older detachment. ( P A S , X 1,900.)
Figs. 20 and 21 (Machemer). Two-week-old detachment. Pigment epithelial cells often display large nuclei or have double nuclei ( P A S , X 1,900.)
PIGMENT EPITHELIUM UNDER THE RETINAL HOLE With the production of the retinal detachment, the pigment epithelium immediately underneath the retinal hole was destroyed by the Jetstream. Thus, after one hour and after one day no pigment cells were found in this area, but only a bare basement membrane (fig. 2 3 ) . Three days later no defect in the pigment epithelium could be seen. However, the cells in this area were flattened out, with thin nuclei and far-reaching cytoplasm (fig. 2 4 ) . Other cells seemed to have lost direct connection with the pigment epithelium and were lying on it rather loosely. These cells were round, with round nuclei, some very large. Most of them contained pigment granules.
After two weeks, heavy proliferative activity had often taken place. Not only was the defect filled with more or less pigmented cells, but newly formed strands covered the cells. These strands were from one to two layers thick ; sometimes there was a mass of proliferation with mitotic activity (fig. 2 5 ) . The cells no longer resembled ordinary pig-
Fig. 22 (Machemer). Fourteen-week-old detachment. In old detachments the pigment epithelial cells become flat again. ( P A S , Xl,500.)
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or three layers with irregularly arranged cells that contained less pigment than usual. In older detachments the pigment proliferated more, but usually did not develop strands. DISCUSSION RETINA Fig. 23 (Machemer). One-hour-old detachment. Directly underneath the retinal hole is a pigment epithelial defect caused by pressure of injected fluid. ( P A S , X750.)
ment epithelium cells. Their nuclei were often irregularly shaped and of various size, both smaller and larger than normal pigment epithelium cells. They were surrounded by tissue containing collagen fibers. Other cells were still scattered loosely. Older detachments produced similar findings. PIGMENT EPITHELIUM PROLIFERATION AT ORA When the retinal detachment extended only to the ora, no proliferation of pigment epithelium was found (fig. 2 6 ) . However, when a large portion of the nonpigmented layer of the pars plana detached, the pigment began to proliferate in the whole area of the detached pars plana epithelium (fig. 2 7 ) , the first case occurring after seven days. Here the pigment epithelium was thickened to two
From the first day of detachment the retinal structure of the inner layer becomes loose, probably due to edema. At first this is a diffuse edema of the layers between the inner limiting membrane and the outer plexiform layer. The retina becomes thicker. Horizontal structures such as the nerve-fiber layer and portions of the inner plexiform layer are less edematous. They may cause swelling, which pushes the retinal tissue backward, resulting in a buckling of the posterior retina in many places. The inner lay-
Fig. 24 (Machemer). Three-day-old detachment A few cells with flattened cytoplasm cover defect in pigment epithelium. ( P A S , X7S0.)
Fig. 25 (Machemer). Two-week-old detachment Heavy proliferation of pigment epithelial cells in area of original defect. ( P A S , X600.)
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Figs. 26 and 27 (Machemer). Two-week-old detachment. Spontaneous proliferation of pigment epithelium of pars plana occurs when unpigmented epithelium is detached. Figure 26 shows detachment ending at ora; Figure 27 represents detachment, in same eye, of unpigmented epithelium of pars plana. Spontaneous proliferation of pigment epithelium of pars plana occurs when unpigmented epithelium detaches.
ers are never pushed inwardly. These findings correspond to the clinical picture: The edema results in a decrease of translucency of the retina; the posterior buckling gives the retina a fine, wavy shagreenlike appearance, similar to sand on a beach, whereas the inner surface of the retina shows no wrinkles. The edema fluid accumulates later, mainly in the inner nuclear layer, and simultaneously vanishes from the other layers. Thus a cystoid space formation in the inner nuclear layer develops whereas the other layers appear normal. The process of fluid formation continues, and the spaces get larger, including the outer plexiform layer and the outer nuclear layer. However, at this time (up to the 12th week) no signs of cell death are found in the inner layers. By careful observation with a contact lens under high power, these larger cavities can be seen clinically. They resemble the Blessig-Iwanoff cysts found in human eyes at the ora. The edema shows there must be a metabolic change in the inner part of the retina, although this portion of the retina is still full of capillaries fed by the central retinal artery. Since mechanical destruction of the vitreous and hyaluronidase do not produce any
retinal change visible through the light microscope, this metabolic change must be due to detachment of the retina from the pigment epithelium. Thus the normal metabolism, even of the inner retinal parts, is not only dependent on the blood supply from the central artery, but also narrowly linked with the metabolic activity of the outer retinal layer and, respectively, of the pigment epithelium. Only in prolonged detachments (14 weeks) may the fluid-filled cavities become so large that cells die, perhaps by mechanical destruction due to overstretching of the cytoplasm in the thin walls or by compression on both sides of the cyst or by a final breakdown of an already unbalanced metabolism. The result is that the number of cells in the inner and outer nuclear layers is decreased. The early neuroepithelial changes occur in the outer segments. Although immediately after production of the detachment the outer segments are arranged parallelly as in the attached retina, they become irregular as early as the first day after detachment. Changes associated with dissolution occur. From the first day onward macrophages are lying between the outer segments. The inner segments change little. Their vertical and paral-
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lei arrangement can always be found unless there is destruction of the outer nuclear layer by pseudo-cyst formation. The normal metabolism between the pigment epithelium and the neuroepithelium is disturbed by the detachment of the retina. The outer segments are the most sensitive parts of these cells and degenerate with the beginning of detachment. The remainder of the cell is still functioning, as shown by its normal structure. This could be due to nourishment from the inner retina or to diffusion of metabolic products from the pigment epithelium through the subretinal fluid. Some evidence for this latter hypothesis is given by the observation that in a flat detachment the changes of the whole retina are much less prominent than in a high detachment. Obviously the importance of the pigment epithelium, not only for the outer segments but also for the metabolism of the whole retina, cannot be overstressed. RETINAL HOLE
Whenever a large hole of more than three disc diameters was produced or developed, the edges of the hole were rolled inward immediately. The fact that no vitreous strands were found at the edge of the hole to cause this inward rolling, and that after a few days the outer layers of the retina seemed to be pulled around the outermost edge of the hole, indicates factors within the retina. A possible explanation is the edema that causes the retina to swell around the edge of the hole, possibly in combination with some elastic forces in the horizontally oriented inner layers of the retina. The large number of pyknotic nuclei and the loss of the outer segments in the immediate vicinity of the retinal hole an hour after the production of the hole are due to mechanical destruction of this portion of the retina by the "Jetstream" of fluid injected against the retina to produce a hole with detachment. On close observation of the retina at the margin of the wound, proliferation of cells
S E P T E M B E R . 1968
can be found as early as the beginning of the second week. These cells differ clearly from macrophages—which are always found around the retinal wound—by their loose, round, large, nucleus and an easily visible nucleolus. After two weeks it becomes evident that these cells belong to the group of cells forming connective tissue, since all stages between these round cells and elongated cells with smaller and denser nuclei are found. They produce collagen, and thus the retinal wound is covered by "scar" tissue. The whole process can terminate at this stage, but in some cases further proliferation of cells on the retinal surface occurs. This will be reported in a subsequent publication. PIGMENT
EPITHELIUM
The first observation is that the pigment epithelium as a single layer of cells in the observed period of 20 weeks is always intact. It differs from normal pigment epithelium, in its close interconnection with the neuroepithelium of the retina by cytoplasmic interdigitations with the outer segments. This connection is not mechanically strong. Shrinkage of the retina during the histologic fixation process detaches the retina. The pigment epithelium then consists of two parts: a basal part with clear cytoplasm and part of the nucleus, and a top part full of pigment granules, often obscuring the upper portion of the nucleus. In an intra vitam detachment the pigment epithelium looks different. A thick, translucent inner portion of the cell is seen and becomes thicker with time. Its vertical structure suggests that this layer consists of cytoplasmic processes which in the normal retina interdigitate with the outer segments and are thus not visible. Another change in the pigment epithelium refers to the shape of the cell. Where the usual cell is rectangular and flat in cross section, the cell in detached areas protrudes. This is conspicuous after seven days. At the same time large nuclei and double-nucleated cells appear. Eventually, when the size of the nuclei normalizes, double-nucleated cells dis-
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appear and the translucent inner part of the
W h y some cells tend to accumulate in v e r y
cells thickens progressively, the pigment epi-
loose groups and whether they all originate
thelium cells flatten out again. T h e buckling
in the pigment epithelium is unknown.
of the cell m a y be due to ( 1 ) nuclei larger than normal, ( 2 ) m o r e than one nucleus, and (3)
the physical phenomenon that a single
cell in a fluid tends to be round. T h e pigment epithelium
is
firmly
attached o n one
PIGMENT
E P I T H E L I U M A T ORA
During
detachment
of
"Ringschwiele"
when fluid instead o f retina overlies it. U p o n
tachments o f h u m a n eyes.
of
the
translucent
unpigmented
cells at the pars plana reminds one of
side, but the other side can become rounded thickening
the
epithelium the proliferation of the pigmented the
seen in prolonged total de-
7
cytoplasmic SUMMARY
portion it becomes flat again. T h u s this p o r tion of the cell does not obstruct that o f the
T h e histologic
findings
of the retina, the
adjacent cell in the valley between t w o buck-
retinal hole, and the pigment epithelium in
led cells.
an eye with experimental retinal detachment
I n the period of observation the pigment epithelium showed n o signs of
are described.
degeneration
T h e most notable change in the inner lay-
during the retinal detachment. O n the con-
ers of the retina is an increasing edema that
trary, it appeared to have been highly active.
finally
T h e relationship between the preservation o f
spaces with degeneration of cells. T h e outer
leads to formation of large cystoid
the retinal structure and flat detachment ac-
segments o f the photoreceptor layers show a
counts not only for this finding, but the large
progressive
nuclei o r double-nucleated cells suggest the
the retinal change seems to depend not only
possibility of higher than normal metabolic
o n the length of the detachment period but
activity. A n o t h e r explanation must be con-
also on the height of the detachment. A t the hole,
degeneration.
early
T h e intensity
degeneration
of
of
sidered: T h e s e cells m a y eventually divide,
retinal
producing the macrophages. T h e s e are always
probably due to mechanical destruction,
cells,
pigmented, although there is no scattering of
found. Factors within the retina cause the
pigment granules after production of the de-
edge of the hole to roll inward, and the reti-
is
tachment, and the number of macrophages
nal w o u n d is closed b y "scar" tissue. U p o n
seems to be directly correlated with the n u m -
detachment, cytoplasmatic processes of
ber of double-nucleated cells.
pigment epithelium become visible; the cells
the
tend to protrude and are often binucleated. PIGMENT
EPITHELIUM
UNDER T H E RETINAL
T h e pigment epithelium defect under the ret-
HOLE
inal hole is closed b y encroaching prolifera-
A disturbance in the continuity of the pig-
tion. U p o n detachment of the unpigmented
ment epithelium layer is created only in the
epithelium o f the pars plana intensive prolif-
area where the pigment epithelium
eration of the underlying pigment epithelium
is
stroyed mechanically b y the Jetstream
dethat
produced the retinal hole. A f t e r three days the defect is closed, mainly b y
flattening
1638 N. W. Tenth Avenue ,(33136)
of
available cells. B u t proliferation is visible, too, since in some places cells begin to accumulate. T h i s proliferation can be so excessive that finally either a flat layer or a dense tongue of n e w cells overlie the repaired defect. Both m a y show proliferative
occurs.
activity
even after 1 4 days, as indicated b y mitoses.
ACKNOWLEDGMENT
I wish to acknowledge the skillful work of Mrs. M. Halley in the preparation of the histologic slides. REFERENCES
1. Kümmel, R. : Zur Entwicklung der Netzhautabhebung. Arch. f. Augenheilk. 95:214, 1925.
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2. Kümmel, R. : Zur Anatomie der frischen Netzhautablösung. Arch. f. Augenheilk. 314:100, 1936. 3. Kronfeld, P. C. : Histologie appearance of recent retinal tears. Arch. Ophth. 13:779, 1935. 4. Hervouet, F. : L'anatomie pathologique du décollement de la rétine. Bibl. Ophth. 3:1, 1965. 5. Birch-Hirschfeld, A . and Inouye, T. : Experi-
EXPERIMENTAL
S E P T E M B E R , 1968
mentelle und histologische Untersuchungen über Netzhautabhebung. Arch. f. Ophth. 70:486, 1909. 6. Machemer, R. and Norton, E. W . D. : Experimental retinal detachment in the owl monkey. I. Methods of production and clinical picture, Am. J. Ophth. 66:388,1968. 7. Hogan, M. J. and Zimmerman, L. E. : Ophthalmic Pathology. Philadelphia, Saunders, ed. 2, 1962.
RETINAL DETACHMENT
IN T H E O W L
MONKEY
I I I . ELECTRON MICROSCOPY OF RETINA AND PIGMENT EPITHELIUM ARNOLD J. KROLL, M . D . AND ROBERT MACHEMER, M . D . * Miami, Florida
It has been shown in the preceding paper that experimental retinal detachment in the owl monkey produced characteristic histologic changes. In the present paper, we describe the electron microscopic characteristics of the retina and pigment epithelium in retinal detachment. 1
MATERIALS AND METHODS In a series of 14 owl monkeys (Aotus trivirgatus), retinas were experimentally detached as previously described. Eyes were enucleated, without regard to conditions of light- or dark-adaptation, at intervals ranging from one hour to 14 weeks. Control eyes were subjected to intravitreous hyaluronidase injection alone, or injection plus vitreous aspiration and reinjection. Areas of attached retina in eyes with partial detachment also served as controls. 2
From the Bascom Palmer Eye Institute, Department of Ophthalmology University of Miami School of Medicine. This investigation was supported in part by U S P H S Research Grants No. NB05918 and NB06841 from the National Institute of Neurological Diseases and Blindness, by the National Council to Combat Blindness, Inc. Grantin-Aid Special Fellowship No. F-211; by funds from Research to Prevent Blindness, Inc. and by the Florida Lions Eye Bank, Inc. Read in part before the 17th annual session of the New Orleans Academy of Ophthalmology, Symposium on Retina and Retinal Surgery, New Orleans, February 15, 1968, and before the Club Jules Gonin, Cambridge, England, April 2, 1968. •Visiting research fellow University Eye Clinic, Göttingen, Germany.
After enculeation, each globe was opened near the ora serrata. A portion of midperipheral retina and pigment epithelium remote from the retinal breaks was excised with a sharp razor blade. The specimens were then fixed in 2 % phosphate buffered osmium tetroxide, dehydrated in a graded series of ethanol, embedded in epon, and sectioned and stained for both phase-contrast and electron microscopy as described elsewhere. 3
OBSERVATIONS The detailed observations are recorded as legends which accompany the figures. In essence, experimental retinal detachment produced the following changes: The horizontally layered discs in the photoreceptor outer segments underwent marked degeneration. This included loss of horizontal orientation, fragmentation, and eventual atrophy. Macrophages appeared and phagocytized fragments of outer segments. The pigment epithelial lamellar inclusion bodies " disappeared. The apical surface of the pigment epithelial cells became convexly protuberant, and the apical processes increased in number, and became thickened. Melanin granules withdrew from the apical processes. Cystoid extracellular spaces of varying sizes appeared in the middle and inner retina. These spaces consisted of enlargement of extracellular spaces. They were bordered by the cytoplasmic membranes of contiguous retinal 4
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todo personale di provocazione di distacco retinico sperimentale. Boll. Oculist. 43:87, 1964. 11. Landholm, W . M. and Watzke, R. C. : E x perimental retinal detachment with a sulfated polysaccharide. Invest. Ophth. 4:42, 1965. 12. Freilich, D. B., Lee, P. and Freeman, H . M. : Experimental retinal detachment. Arch. Ophth. 76 :432, 1966.
S E P T E M B E R , 1568
13. Fraunfelder, F. T. and Potts, A . M . : An experimental study of retinal detachments. Am. J. Ophth. 62 :561,1966. 14. Custodis, E. : Beobachtungen bei der diathermischen Behandlung der Netzhautablösung und ein Hinweis zur Therapie der Amotio retinae, Ber. Deutsch. Ophth. Ges. 57:227,1952.
E X P E R I M E N T A L R E T I N A L D E T A C H M E N T IN T H E O W L M O N K E Y HISTOLOGY
II.
OF RETINA
ROBERT
A N D PIGMENT
MACHEMER,
EPITHELIUM
M.D.*
Miami, Florida dase was injected into the vitreous, and under direct visual control the vitreous was repeatedly aspirated and then reinjected against the temporal equatorial retina, producing a retinal hole with surrounding detachment. The eyes were enucleated at 1 hour, 1 day, 3 days, and 1, 2, 4, 8, 12, 14 and 20 weeks after the production of the hole. Each group consisted of two to three eyes. They were fixed in a modified Kolmer fixative for 24 hours (one part acetic acid, four parts saturated watery solution of potassium dichromate, and four parts 4% formaldehyde) . T o keep the retina attached and to preMETHODS vent shrinkage of the eye, about 0.1 cc of the As previously described, retinal detach- fixative was injected into the anterior chamments were produced in the owl monkey ber. After one day of fixation a small calotte (Aotus trivirgatus) : One unit of hyaluroni- was cut off from the nasal side to avoid deformation of the globe during the dehydraFrom the Bascom Palmer Eye Institute, Departtion and to give the embedding material acment of Ophthalmology, University of Miami School of Medicine. This investigation was supcess to the inner eye. The eyes were slowly ported in part by U S P H S Research Grant M B dehydrated in alcohol, brought into methyl06841 from the National Institute of Neurological benzoate and, after only five minutes in chloDiseases and Blindness; in part by Research to Prevent Blindness, Inc.; in part by the Florida roform, embedded in paraffin.
The serial histologic changes in human retinal detachment are, as of the present time, still incompletely described because of the unavailability of pathologic tissue. Only a few cases of early human detachment are available. " A study of detached rabbit retina was made as early as 1909, but this retina differs from that of man in many respects. The purpose of this report is to describe the sequence of histologic changes in the experimentally detached owl monkey retina, a species whose eye is quite similar to that of man. 1
4
5
6
Lions' Eye Bank, Inc.; in part by the Florida Mid-Winter Seminar; in part by the National Council to Combat Blindness, Inc., Grant-in-Aid Special Fellowship No. F-211, and in part by Deutscher Akademischer Austauschdienst, Bonn, Germany. Read in part before the 17th annual session of the New Orleans Academy of Ophthalmology, Symposium on Retina and Retinal Surgery, New Orleans, February 15, 1968. * Visiting Research Fellow, Clinic, 34 Goettingen, Germany.
University
Eye
Vertical serial sections of 5-micron thickness were made in five areas of the eye : ( 1 ) through the retinal hole, ( 2 ) through the macular area, ( 3 ) through the disc, and (4 and 5) through nasal parts of the eye at distances from the disc corresponding to those of areas 1 and 2. The sections were stained with hematoxylin-eosin, P A S , and trichrome
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Fig. 1 (Machemer). Histology of normal retina in owl monkey very similar to that of human. Kolmer fixation preserves structure of retina well. ( P A S , X190.)
stain after Masson after they had been in a 5% solution of sodium bicarbonate for one hour. Controls included ( 1 ) seven untreated eyes, ( 2 ) two eyes injected intravitreously with one unit of lyophilized hyaluronidase (Wyeth Co.) dissolved in 0.1 cc of saline, and ( 3 ) four eyes injected with one unit of hyaluronidase in which the vitreous was then destroyed mechanically on the temporal side by repeated aspiration and reinjection. The latter six eyes were enucleated after 24 hours. RESULTS RETINA In the normal retina the histologic picture was very similar to that of the human eye (fig. 1 ) . However, there was no fovea, but a macular area with a large number of gan-
glion cells. Single photoreceptors, consisting of the inner and outer segments which were parallelly arranged and in good contact with the pigment epithelium, (fig. 2 ) could be seen. It should be emphasized that retinal capillaries never go deeper than the outside of the inner nuclear layer. After administration of hyaluronidase, or after vitreous destruction together with hyaluronidase, no histologic changes could be found in the retina. One hour after initial production of the retinal detachment, no additional retina detached spontaneously. The initial detachment was produced by fluid injected behind the retina, but only the retina near the retinal hole showed signs of mechanical destruction. The remainder of the detached retina looked normal. After one day the first signs of a loosen-
Fig. 2 (Machemer). Photoreceptors of normal retina consist mainly of parallelly arranged rods which can be easily distinguished. ( P A S , X750.)
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Fig. 3 (Machemer). Retina with 1-day-old detachment. Inner portions of retina, including inner nuclear layer, show diffuse loosening of structure, caused by edema. ( P A S , X 1 9 0 ) .
ing of the retinal structure in the inner layers appeared, with the outer layers of the detached retina becoming irregular (fig. 3 ) . Very few macrophages—most of them full of pigment granules—appeared at the outside of the outer segment layer. Most impressive after three days was a diffuse loosening of the inner layers of the detached retina (fig. 4 ) . Its severity was not uniform, so that all the stages between loosening of the structure and cystoid space formation could be found. Usually this structural loosening was most severe in the inner plexiform layer, but as soon as cystoid spaces were formed, they were found in the ganglion cell, inner plexiform, and inner nuclear layers. Horizontal structures tended
less to edema, so that the nerve fiber layer and a bandlike structure in the inner plexiform layer were often more compact than the environment. The outer nuclear layer usually appeared normal. A few nuclei were possibly pyknotic. With the formation of cysts in the inner layers, a few small empty spaces were sometimes seen in the outer nuclear layer. The photoreceptor layer showed very noticeable changes (fig. 5 ) . The inner segments appeared fairly normal with vertical arrangement, but the transition zone between the inner and outer segments, the area of the connecting ciliae, showed many small, empty spaces. The outer segments became irregularly arranged, could not be separated from
Fig. 4 (Machemer). Three-day-old detachment Cystoid spaces develop mainly in ganglion cell layer and in inner nuclear layer. Horizontal structures, such as nerve-fiber layer and part of inner plexiform layer, are less edematous. ( P A S , X190.)
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Fig. 5 (Machemer). Three-dayold detachment. Whereas inner segments of photoreceptor layer appear fairly normal, outer segments are irregularly arranged and cannot be distinguished from one another. Cystoid spaces appear. Macrophages are seen in this layer. ( P A S , X750.)
each other, and were of different lengths. Some pigment granules were seen at the outer margin and between the outer segments. Macrophages could be seen between outer segments. By the seventh day of detachment, it became more apparent that the edema in the retina varied significantly in different portions. In addition, there were areas with larger cystoid spaces (fig. 6 ) . When they developed, no edema could be found in the inner retinal layers, only a few small pseudocysts in the ganglion cell layer. These cystoid spaces gave the inner nuclear layer up to three times its normal thickness. They pushed the outer parts of the retina into folds, making the back of the retina wavy. They were vertically oriented
and could penetrate through the whole thickness of the layer. Usually the cells were found at the base and at the top of the cystoid spaces. The septa between the pseudocysts consisted of columns of cells, but could be so thin that only fine strands of acellular nerve fibers ran through them. There was still vascular supply of the whole inner nuclear layer, since capillaries were seen in the septa, and they reached the outer parts of this layer. A two-week-old detachment showed all the changes already described, but the cystoid space formation was more marked : the cavities could be so large that they extended into the outer nuclear layer (fig. 7 ) . The outer segments had degenerated further. This layer was thinned out and faded away
Fig. 6 (Machemer). One-week-old detachment. Edema as well as cystoid spaces may cause posterior side of retina to bulge. Clinically, this corresponds to shagreenlike pattern seen in highly detached retinas. Note that edema of innermost layers has vanished. Only a few cystoid spaces are found in the ganglion cell layer. ( P A S , X190.)
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Fig. 7 (Machemer). Two-week-old detachment. Large cystoid spaces in the inner nuclear layer have broken through inner plexiform layer into outer nuclear layer. Separating walls are very thin and in final stage consist only of nerve fibers. ( P A S , X190.)
somewhat toward its outer edge. It was impossible to distinguish any single outer segment. The layer was full of macrophages (fig. 8 ) . Three-, four-, five- and eight-week-old detachments showed no further changes, except that the cystoid spaces became larger. After 12 to 14 weeks of detachment, there were still portions of the retina that appeared normal, but most of the retina showed all stages of detachment, from diffuse loosening of structure to extensive cyst formation. Large pseudocysts had broken down the septa in many. Here, finally, the inner nuclear layer had almost disappeared, and the outer nuclear layer was reduced to one or two cells (fig. 9 ) . Heavy atrophy of the photoreceptors occurred, with short and
Fig. 8 (Machemer). Two-weekold detachment Outer segments are less numerous. The layer is thinned out and full of macrophages (see also fig. 7 ) . ( P A S , X7S0.)
thick inner segments, reduced in number; the outer segments had almost disappeared (fig. 10). It was noted that in flatly detached retinas the described changes always occurred to a much lesser degree. For example, a 14-day-old, flatly detached retina (no more than three times the thickness of a normal retina) showed no loosening of its structures in the inner parts. Thus it appeared normal in many portions ; but where the detachment was highest, the retina was thickened, as was apparent from the wavy appearance of the outer retina, although no loosening of the structure could be seen histologically. The photoreceptor layer was in much better condition than in highly detached retinas (fig. 11). Depending on the height of the detach-
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Fig. 9 (Machemer). Fourteen-week-old detachment with huge cystoid spaces and loss of cells in inner and outer nuclear layer. ( P A S , X190.)
ment, it was possible to identify even single outer segments ; they were sometimes still parallelly arranged. Not only flatly detached
areas of the retina could be well preserved, but sometimes also highly detached portions, Proliferation on the surface of the retina
Fig. 10 (Machemer). Higher magnification of retina shown in Figure 9. Inner segments are shorter and thicker; their number is reduced. Only small residues of the outer segments are left. ( P A S , X7S0.)
Fig. 11 (Machemer). Fourteenday-old, very flat detachment In comparison to changes in a highly detached retina (see Figure 8 ) , outer segments are much better preserved. ( P A S , X750.)
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Fig. 12 (Machemer). One-hour-old detachment. Edges of retinal hole are curled inwardly. (PAS,
was histologically observed three weeks after detachment, but there was clinical evidence —in one case with massive preretinal retraction after 14 days—that this can happen earlier, and may be very extensive. These changes will be described and discussed in a subsequent paper.
xSO.)
RETINAL HOLE
On the third day the edge of the retina at the hole was rounded, and it looked as though the nerve fiber layer had pulled the outer layer of the retina inward around the edge. No signs of proliferative activity were found. The outer nuclear layer contained many pyknotic nuclei, and wherever these were found the inner segments of the neuroepithelium were destroyed. This destruc-
One hour after and one day after the production of the detachment the edges of the retinal hole were found to be curled inwardly to a great extent (fig. 12). The edge of the hole—the retinal wound—was irregular and sometimes red blood cells covered it, depending on whether or not a vessel was hit during the production of the hole. The edge was not yet rounded, and the outer layers were not yet pulled inward. In the immediate vicinity of the hole the retina showed pyknotic nuclei in the outer nuclear layer and, to a lesser extent, in the inner nuclear layer, with destruction of the outer segments, with irregularity and shortening of the photoreceptors (fig. 13).
Fig. 13 (Machemer). High-power view of retina in Figure 12 shows fresh retinal wound. In immediate vicinity of hole many nuclei in inner and outer nuclear layers are pyknotic; outer segments missing. ( P A S , X7S0.)
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Fig. 14 (Machemer). One-week-old detachment. A t edge of hole outer layers of the retina are pulled inwardly. Inner limiting membrane is wrinkled. ( P A S , X195.)
tion was observed only in the immediate vicinity of the retinal hole. After one week the inner limiting membrane near the hole was wrinkled and the outer layers were pulled inward more than previously (fig. 14). Near the edge of the retinal wound there were a few cells with lightly staining, large, round nuclei and a prominent nucleolus. These were seen also in the outer nuclear layer (fig. 15), more evident after two weeks (fig. 16). There were more of these cells, and they were found in all layers. Some still resembled the above-described cells but there were gradual changes to cells with cigar-shaped nuclei. Mitoses were observed.
Older holes sometimes showed almost no proliferation, others had newly formed tissue, with the staining qualities of collagen, covering the wound. Some eyes showed progressive proliferation and strand formation along the retinal surface as early as four weeks after the beginning of the detachment. PIGMENT EPITHELIUM One hour after and one day after a detachment of the retina the pigment epithelium was normally flat and the pigment granules were still vertically oriented. A part of the pigment cell became visible that was hidden as long as the retina was attached and
Fig. 15 (Machemer). Highpower view of Figure 14. Cells with lightly basophilic, large, round nuclei appear near the edge of retinal wound. (PAS, x750.)
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Fig. 16 (Machemer). Two-weekold detachment. Many cells proliferating at retinal wound, covering it with "scar" tissue. ( P A S , X750.)
Fig. 17 (Machemer). Artificially induced retinal detachment occurring during fixation process. Pigment epithelium seems to consist of clear base with partly visible nuclei and pigmented top. ( P A S , Xl.900.)
which could not be seen in an artificial detachment, due to the histologic process (fig. 17). Overlying the pigment granules there was a faintly staining, nearly translucent area with a thickness one-third of the cell (fig. 18), irregularly limited to the subretinal fluid and with a fine vertical structure. The pigment granules protruded into this portion of the cell, which is never seen in an artificial detachment, due to the histologic process. On the third day the pigment epithelium cell tended to protrude. The pigment granules were no longer mainly vertically oriented, but often irregular. Only a few granules protruded into the translucent portion of the cell. During the first to the eighth weeks the translucent layer thickened from one-third to sometimes two-thirds of the thickness of the cell. The cells protruded further, some assuming the shape of a collar-button, with the
upper portion containing the pigment granules and the lower portion the nucleus (fig. 19). There were always flat cells, more numerous as the detachment became older. The pigment granules protruded more often into the translucent portion of the cell. There were cells with nuclei larger than normal and others that had two or more nuclei in one cell (figs. 20 and 2 1 ) . Finally, after 12 to 20 weeks, the pigment epithelium became flat. Only a few protruberant cells were seen. The translucent portion was about one-half the thickness of the cells. The pigment granules were located and oriented as in the normal eye; that is, they covered the inner part of the nuclei and were mainly vertically oriented and protruded like fingers into the translucent portion (fig. 2 2 ) . At that time, the nuclei were all about the same size. Cells with two nuclei were rarely found.
Fig. 18 (Macnemer). One-hour-old detachment Pigment epithelium has translucent portion which overlies pigmented part and represents cytoplasmatic processes of these cells. ( P A S , X1,900.)
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Fig. 19 (Machemer). Four-weekold detachment. Pigment epithelial cells protrude and may be collarbutton-shaped. Translucent layer becomes more prominent in older detachment. ( P A S , X 1,900.)
Figs. 20 and 21 (Machemer). Two-week-old detachment. Pigment epithelial cells often display large nuclei or have double nuclei ( P A S , X 1,900.)
PIGMENT EPITHELIUM UNDER THE RETINAL HOLE With the production of the retinal detachment, the pigment epithelium immediately underneath the retinal hole was destroyed by the Jetstream. Thus, after one hour and after one day no pigment cells were found in this area, but only a bare basement membrane (fig. 2 3 ) . Three days later no defect in the pigment epithelium could be seen. However, the cells in this area were flattened out, with thin nuclei and far-reaching cytoplasm (fig. 2 4 ) . Other cells seemed to have lost direct connection with the pigment epithelium and were lying on it rather loosely. These cells were round, with round nuclei, some very large. Most of them contained pigment granules.
After two weeks, heavy proliferative activity had often taken place. Not only was the defect filled with more or less pigmented cells, but newly formed strands covered the cells. These strands were from one to two layers thick ; sometimes there was a mass of proliferation with mitotic activity (fig. 2 5 ) . The cells no longer resembled ordinary pig-
Fig. 22 (Machemer). Fourteen-week-old detachment. In old detachments the pigment epithelial cells become flat again. ( P A S , Xl,500.)
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or three layers with irregularly arranged cells that contained less pigment than usual. In older detachments the pigment proliferated more, but usually did not develop strands. DISCUSSION RETINA Fig. 23 (Machemer). One-hour-old detachment. Directly underneath the retinal hole is a pigment epithelial defect caused by pressure of injected fluid. ( P A S , X750.)
ment epithelium cells. Their nuclei were often irregularly shaped and of various size, both smaller and larger than normal pigment epithelium cells. They were surrounded by tissue containing collagen fibers. Other cells were still scattered loosely. Older detachments produced similar findings. PIGMENT EPITHELIUM PROLIFERATION AT ORA When the retinal detachment extended only to the ora, no proliferation of pigment epithelium was found (fig. 2 6 ) . However, when a large portion of the nonpigmented layer of the pars plana detached, the pigment began to proliferate in the whole area of the detached pars plana epithelium (fig. 2 7 ) , the first case occurring after seven days. Here the pigment epithelium was thickened to two
From the first day of detachment the retinal structure of the inner layer becomes loose, probably due to edema. At first this is a diffuse edema of the layers between the inner limiting membrane and the outer plexiform layer. The retina becomes thicker. Horizontal structures such as the nerve-fiber layer and portions of the inner plexiform layer are less edematous. They may cause swelling, which pushes the retinal tissue backward, resulting in a buckling of the posterior retina in many places. The inner lay-
Fig. 24 (Machemer). Three-day-old detachment A few cells with flattened cytoplasm cover defect in pigment epithelium. ( P A S , X7S0.)
Fig. 25 (Machemer). Two-week-old detachment Heavy proliferation of pigment epithelial cells in area of original defect. ( P A S , X600.)
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Figs. 26 and 27 (Machemer). Two-week-old detachment. Spontaneous proliferation of pigment epithelium of pars plana occurs when unpigmented epithelium is detached. Figure 26 shows detachment ending at ora; Figure 27 represents detachment, in same eye, of unpigmented epithelium of pars plana. Spontaneous proliferation of pigment epithelium of pars plana occurs when unpigmented epithelium detaches.
ers are never pushed inwardly. These findings correspond to the clinical picture: The edema results in a decrease of translucency of the retina; the posterior buckling gives the retina a fine, wavy shagreenlike appearance, similar to sand on a beach, whereas the inner surface of the retina shows no wrinkles. The edema fluid accumulates later, mainly in the inner nuclear layer, and simultaneously vanishes from the other layers. Thus a cystoid space formation in the inner nuclear layer develops whereas the other layers appear normal. The process of fluid formation continues, and the spaces get larger, including the outer plexiform layer and the outer nuclear layer. However, at this time (up to the 12th week) no signs of cell death are found in the inner layers. By careful observation with a contact lens under high power, these larger cavities can be seen clinically. They resemble the Blessig-Iwanoff cysts found in human eyes at the ora. The edema shows there must be a metabolic change in the inner part of the retina, although this portion of the retina is still full of capillaries fed by the central retinal artery. Since mechanical destruction of the vitreous and hyaluronidase do not produce any
retinal change visible through the light microscope, this metabolic change must be due to detachment of the retina from the pigment epithelium. Thus the normal metabolism, even of the inner retinal parts, is not only dependent on the blood supply from the central artery, but also narrowly linked with the metabolic activity of the outer retinal layer and, respectively, of the pigment epithelium. Only in prolonged detachments (14 weeks) may the fluid-filled cavities become so large that cells die, perhaps by mechanical destruction due to overstretching of the cytoplasm in the thin walls or by compression on both sides of the cyst or by a final breakdown of an already unbalanced metabolism. The result is that the number of cells in the inner and outer nuclear layers is decreased. The early neuroepithelial changes occur in the outer segments. Although immediately after production of the detachment the outer segments are arranged parallelly as in the attached retina, they become irregular as early as the first day after detachment. Changes associated with dissolution occur. From the first day onward macrophages are lying between the outer segments. The inner segments change little. Their vertical and paral-
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lei arrangement can always be found unless there is destruction of the outer nuclear layer by pseudo-cyst formation. The normal metabolism between the pigment epithelium and the neuroepithelium is disturbed by the detachment of the retina. The outer segments are the most sensitive parts of these cells and degenerate with the beginning of detachment. The remainder of the cell is still functioning, as shown by its normal structure. This could be due to nourishment from the inner retina or to diffusion of metabolic products from the pigment epithelium through the subretinal fluid. Some evidence for this latter hypothesis is given by the observation that in a flat detachment the changes of the whole retina are much less prominent than in a high detachment. Obviously the importance of the pigment epithelium, not only for the outer segments but also for the metabolism of the whole retina, cannot be overstressed. RETINAL HOLE
Whenever a large hole of more than three disc diameters was produced or developed, the edges of the hole were rolled inward immediately. The fact that no vitreous strands were found at the edge of the hole to cause this inward rolling, and that after a few days the outer layers of the retina seemed to be pulled around the outermost edge of the hole, indicates factors within the retina. A possible explanation is the edema that causes the retina to swell around the edge of the hole, possibly in combination with some elastic forces in the horizontally oriented inner layers of the retina. The large number of pyknotic nuclei and the loss of the outer segments in the immediate vicinity of the retinal hole an hour after the production of the hole are due to mechanical destruction of this portion of the retina by the "Jetstream" of fluid injected against the retina to produce a hole with detachment. On close observation of the retina at the margin of the wound, proliferation of cells
S E P T E M B E R . 1968
can be found as early as the beginning of the second week. These cells differ clearly from macrophages—which are always found around the retinal wound—by their loose, round, large, nucleus and an easily visible nucleolus. After two weeks it becomes evident that these cells belong to the group of cells forming connective tissue, since all stages between these round cells and elongated cells with smaller and denser nuclei are found. They produce collagen, and thus the retinal wound is covered by "scar" tissue. The whole process can terminate at this stage, but in some cases further proliferation of cells on the retinal surface occurs. This will be reported in a subsequent publication. PIGMENT
EPITHELIUM
The first observation is that the pigment epithelium as a single layer of cells in the observed period of 20 weeks is always intact. It differs from normal pigment epithelium, in its close interconnection with the neuroepithelium of the retina by cytoplasmic interdigitations with the outer segments. This connection is not mechanically strong. Shrinkage of the retina during the histologic fixation process detaches the retina. The pigment epithelium then consists of two parts: a basal part with clear cytoplasm and part of the nucleus, and a top part full of pigment granules, often obscuring the upper portion of the nucleus. In an intra vitam detachment the pigment epithelium looks different. A thick, translucent inner portion of the cell is seen and becomes thicker with time. Its vertical structure suggests that this layer consists of cytoplasmic processes which in the normal retina interdigitate with the outer segments and are thus not visible. Another change in the pigment epithelium refers to the shape of the cell. Where the usual cell is rectangular and flat in cross section, the cell in detached areas protrudes. This is conspicuous after seven days. At the same time large nuclei and double-nucleated cells appear. Eventually, when the size of the nuclei normalizes, double-nucleated cells dis-
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appear and the translucent inner part of the
W h y some cells tend to accumulate in v e r y
cells thickens progressively, the pigment epi-
loose groups and whether they all originate
thelium cells flatten out again. T h e buckling
in the pigment epithelium is unknown.
of the cell m a y be due to ( 1 ) nuclei larger than normal, ( 2 ) m o r e than one nucleus, and (3)
the physical phenomenon that a single
cell in a fluid tends to be round. T h e pigment epithelium
is
firmly
attached o n one
PIGMENT
E P I T H E L I U M A T ORA
During
detachment
of
"Ringschwiele"
when fluid instead o f retina overlies it. U p o n
tachments o f h u m a n eyes.
of
the
translucent
unpigmented
cells at the pars plana reminds one of
side, but the other side can become rounded thickening
the
epithelium the proliferation of the pigmented the
seen in prolonged total de-
7
cytoplasmic SUMMARY
portion it becomes flat again. T h u s this p o r tion of the cell does not obstruct that o f the
T h e histologic
findings
of the retina, the
adjacent cell in the valley between t w o buck-
retinal hole, and the pigment epithelium in
led cells.
an eye with experimental retinal detachment
I n the period of observation the pigment epithelium showed n o signs of
are described.
degeneration
T h e most notable change in the inner lay-
during the retinal detachment. O n the con-
ers of the retina is an increasing edema that
trary, it appeared to have been highly active.
finally
T h e relationship between the preservation o f
spaces with degeneration of cells. T h e outer
leads to formation of large cystoid
the retinal structure and flat detachment ac-
segments o f the photoreceptor layers show a
counts not only for this finding, but the large
progressive
nuclei o r double-nucleated cells suggest the
the retinal change seems to depend not only
possibility of higher than normal metabolic
o n the length of the detachment period but
activity. A n o t h e r explanation must be con-
also on the height of the detachment. A t the hole,
degeneration.
early
T h e intensity
degeneration
of
of
sidered: T h e s e cells m a y eventually divide,
retinal
producing the macrophages. T h e s e are always
probably due to mechanical destruction,
cells,
pigmented, although there is no scattering of
found. Factors within the retina cause the
pigment granules after production of the de-
edge of the hole to roll inward, and the reti-
is
tachment, and the number of macrophages
nal w o u n d is closed b y "scar" tissue. U p o n
seems to be directly correlated with the n u m -
detachment, cytoplasmatic processes of
ber of double-nucleated cells.
pigment epithelium become visible; the cells
the
tend to protrude and are often binucleated. PIGMENT
EPITHELIUM
UNDER T H E RETINAL
T h e pigment epithelium defect under the ret-
HOLE
inal hole is closed b y encroaching prolifera-
A disturbance in the continuity of the pig-
tion. U p o n detachment of the unpigmented
ment epithelium layer is created only in the
epithelium o f the pars plana intensive prolif-
area where the pigment epithelium
eration of the underlying pigment epithelium
is
stroyed mechanically b y the Jetstream
dethat
produced the retinal hole. A f t e r three days the defect is closed, mainly b y
flattening
1638 N. W. Tenth Avenue ,(33136)
of
available cells. B u t proliferation is visible, too, since in some places cells begin to accumulate. T h i s proliferation can be so excessive that finally either a flat layer or a dense tongue of n e w cells overlie the repaired defect. Both m a y show proliferative
occurs.
activity
even after 1 4 days, as indicated b y mitoses.
ACKNOWLEDGMENT
I wish to acknowledge the skillful work of Mrs. M. Halley in the preparation of the histologic slides. REFERENCES
1. Kümmel, R. : Zur Entwicklung der Netzhautabhebung. Arch. f. Augenheilk. 95:214, 1925.
AMERICAN JOURNAL OF OPHTHALMOLOGY
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2. Kümmel, R. : Zur Anatomie der frischen Netzhautablösung. Arch. f. Augenheilk. 314:100, 1936. 3. Kronfeld, P. C. : Histologie appearance of recent retinal tears. Arch. Ophth. 13:779, 1935. 4. Hervouet, F. : L'anatomie pathologique du décollement de la rétine. Bibl. Ophth. 3:1, 1965. 5. Birch-Hirschfeld, A . and Inouye, T. : Experi-
EXPERIMENTAL
S E P T E M B E R , 1968
mentelle und histologische Untersuchungen über Netzhautabhebung. Arch. f. Ophth. 70:486, 1909. 6. Machemer, R. and Norton, E. W . D. : Experimental retinal detachment in the owl monkey. I. Methods of production and clinical picture, Am. J. Ophth. 66:388,1968. 7. Hogan, M. J. and Zimmerman, L. E. : Ophthalmic Pathology. Philadelphia, Saunders, ed. 2, 1962.
RETINAL DETACHMENT
IN T H E O W L
MONKEY
I I I . ELECTRON MICROSCOPY OF RETINA AND PIGMENT EPITHELIUM ARNOLD J. KROLL, M . D . AND ROBERT MACHEMER, M . D . * Miami, Florida
It has been shown in the preceding paper that experimental retinal detachment in the owl monkey produced characteristic histologic changes. In the present paper, we describe the electron microscopic characteristics of the retina and pigment epithelium in retinal detachment. 1
MATERIALS AND METHODS In a series of 14 owl monkeys (Aotus trivirgatus), retinas were experimentally detached as previously described. Eyes were enucleated, without regard to conditions of light- or dark-adaptation, at intervals ranging from one hour to 14 weeks. Control eyes were subjected to intravitreous hyaluronidase injection alone, or injection plus vitreous aspiration and reinjection. Areas of attached retina in eyes with partial detachment also served as controls. 2
From the Bascom Palmer Eye Institute, Department of Ophthalmology University of Miami School of Medicine. This investigation was supported in part by U S P H S Research Grants No. NB05918 and NB06841 from the National Institute of Neurological Diseases and Blindness, by the National Council to Combat Blindness, Inc. Grantin-Aid Special Fellowship No. F-211; by funds from Research to Prevent Blindness, Inc. and by the Florida Lions Eye Bank, Inc. Read in part before the 17th annual session of the New Orleans Academy of Ophthalmology, Symposium on Retina and Retinal Surgery, New Orleans, February 15, 1968, and before the Club Jules Gonin, Cambridge, England, April 2, 1968. •Visiting research fellow University Eye Clinic, Göttingen, Germany.
After enculeation, each globe was opened near the ora serrata. A portion of midperipheral retina and pigment epithelium remote from the retinal breaks was excised with a sharp razor blade. The specimens were then fixed in 2 % phosphate buffered osmium tetroxide, dehydrated in a graded series of ethanol, embedded in epon, and sectioned and stained for both phase-contrast and electron microscopy as described elsewhere. 3
OBSERVATIONS The detailed observations are recorded as legends which accompany the figures. In essence, experimental retinal detachment produced the following changes: The horizontally layered discs in the photoreceptor outer segments underwent marked degeneration. This included loss of horizontal orientation, fragmentation, and eventual atrophy. Macrophages appeared and phagocytized fragments of outer segments. The pigment epithelial lamellar inclusion bodies " disappeared. The apical surface of the pigment epithelial cells became convexly protuberant, and the apical processes increased in number, and became thickened. Melanin granules withdrew from the apical processes. Cystoid extracellular spaces of varying sizes appeared in the middle and inner retina. These spaces consisted of enlargement of extracellular spaces. They were bordered by the cytoplasmic membranes of contiguous retinal 4
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