Documenta Opthalmologica 77: 39-46, 1991. 9 1991 Kluwer Academic Publishers. Printed in the Netherlands.
The subretinal fluid in retinal detachment
A cytologic study PAOLO TOTI, 1 A U G U S T O MOROCUTTI, 2 C L A U D I A SFORZI, 2 M A R I A M A R G H E R I T A DE SANTI, 1 ANNA M A R I A CATELLA I & STEFANO BAIOCCHI 2 llstituto di Anatomia Patologica, Universitg~ degli Studi di Siena; 2Istituto di Scienze Ofialmologiche, Universit~ degli Studi di Siena Accepted 17 May 1991 Key words: Cytology, subretinal fluid, retinal detachment, transmission electron microscopy Abstract. Following retinal detachment, subretinal fluid (SRF) fills the neoformed space.
Subsequently subretinal and preretinal strands of proliferative tissue begin to form. We have collected the subretinal fluid withdrawn during retinal detachment surgery. We have studied subretinal fluid cytologically to evaluate the number and the type of cells present in the fluid, and by means of transmission electron microscopy. The first cell type to be present in the fluid represented degenerated aspects of pigmented epithelial cells (PECs). Successively, other cell types appeared in the fluid as nerve cells (rods, cones and glial cells), macrophages and well preserved pigmented epithelial cells. Abbreviations: PECs, pigmented epithelial cells; SRF, subrctinal fluid
Introduction Following retinal detachment which results from the separation of pigmented epithelium with neuropithelium, a subretinal fluid (SRF) fills the neoformed space. The SRF originates partly by the tiquifaction of vitreous humor [1] and partly by an increase in permeability of the choroidal vessels [2]. Subsequently, subretinal and preretinal strands of proliferative tissues begin to form [3]. Once proliferated, these strands generally lead to massive vitreous retraction. The presence of these subretinal strands has been associated with long standing retinal detachment [4]. In fact, subretinal strands tend to hinder the resettling of a detached retina. On the other hand, where reattachment does occur, most subretinal strands are found to lie flat beneath the reattached retina, as a permanent indicator of a previous detachment [5]. In both cases (detachment or reattachment), however, the strands may reach the posterior camera, through openings in the retina. The retraction of these strands distorts the retina thus impeding a new reattachment. Today, vitreous surgery techniques (Vitrectomy) allow treatment for the most severe degrees of retinal detachment and where indicated, subreti-
40 nal strands may be severed and removed. Such material has been extensively studied in order to identify their cellular composition; fundamental histological studies on retinal detachment have been performed on animal models as, for example, the owl monkeys of Machemer and Laqua who revealed a clinical picture and pathology similar to that of human beings. A lot of different cell types have been recognized, such as glial cells [6, 7], PEC cells [8-10] and fibroblasts. The retraction of such strands is due to the contraction of actin filaments contained in the cells and to their three dimensional arrangement described by Hamilton [11] and Yamamoto [12]. In the following study, we analysed SRF cytology in human subjects, in order to study the first steps in the membrane formation and to examine the cellular composition of the medium in which membranes are dipped [13]. Materials and methods
Subretinal fluid was obtained from the eyes of 28 patients (16 males and 12 females between 17 and 74 years of age) suffering from retinal detachment, and analysed. Out of the 28 patients, 14 had a myopia over 8 Diopters, 4 were aphakic (they were operated with the intra capsular technique), 3 revealed a previous trauma, 3 suffered from hemorrhages previous to detachment, 2 were diabetic, in 1 patient a total uveitis was observed while another myopic and aphakic patient revealed a detachment recurrence. All patients underwent an operation of equatorial buckling. A silicon sponge (4-5 mm in diameter) was used each time. During each operation, we performed an evacuating puncture or scleroctomy in order to drain SRF. The fluid was withdrawn with a cylinder needle attached to a sterilized syringe and introduced into the scleroctomic incision. In all cases we introduced sterilized air in the vitreous chamber to permit the evacuation of subretinal fluid and the reattachment of the detached retina. The cryocoagulation of retinal wounds was always used. The SRF removed, ranged from 0.3 to 1.2 cc. Part of the fluid collected was cytocentrifuged and the smears obtained were stained with either Papanicolau or May-Grunwald Giemsa. Another part of the fluid was immediately fixed in 2.5% glutaraldehyde, in 0.1M cacodylate buffer at 4 ~ for 3 hrs, postfixed in 1% buffered OsO4 for 2 hrs, dehydrated and embedded in epoxy resin. The resulting ultrathin sections were cut with LKB V Ultratome, counterstained with uranyl acetate and lead citrate, and finally observed through a Zeiss EM-109 electron microscope. Results
No cells were visible in the SRF withdrawn and observed 5 days after detachment. In the SRFs observed 7 and 8 days after detachment only few
41
Table I. Results data. The first column indicates the number of days between retinal detachment and the collection of subretinal fluid. Of course, this time has been estimated by patients inquiry. The number of cells found in the fluid has been evaluated in a semi-quantitative way from 0, which means absence of cells to + + +. Days 5 7 8 10 10 11 12 12 13 15 15 18
20 20 23 23 25 26 27 33 39 40 60 60 60 100 130
Macrophages
Pigmented preserved
Epithelium cells degenerated
Neuro-retinal cells
0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 + ++ + + + + + + ++ ++ ++ ++ + +
0 + + + + + + + + + + ++ ++ ++ ++ ++ ++ + + + ++ +++ ++ + + + ++
0 0 0 + + + + + + + + + 0 +
+
0 + ++ ++ ++ + + + ++ ++ + ++
+
+++ ++ +++ + 0 0 + +++ + + + ++
++
++
++
++
§ + + +
+ +
+ + + +
+ + +
cells w e r e p r e s e n t . In t h e f o l l o w i n g fluids e x a m i n e d a g r e a t n u m b e r o f cells o f d i f f e r e n t t y p e s w e r e o b s e r v e d . T h e s e cells i n c l u d e d p i g m e n t e p i t h e l i u m cells, m a c r o p h a g e s with a v a r i a b l e c o n t e n t o f m e l a n i c p i g m e n t an d n e r v e cells ( T a b l e 1).
Pigmented epithelium cells (PECs).
T h i s cell t y p e a p p e a r e d in t h e s u b r e t i n a l fluid e x a m i n e d t w o w e e k s a f t e r d e t a c h m e n t . T h e cells a p p e a r e d e i t h e r i s o l a t e d o r g r o u p e d ( 4 - 1 0 cells) as a ' m o r u l a ' . T h e y w e r e r e l a t i v e l y l ar g e a n d r o u n d , a n d t h e c y t o p l a s m was filled with b r o w n , o v a l an d e l o n g a t e d p i g m e n t g r a n u l e s . T h e c y t o p l a s m b o u n d a r y was well d e f i n e d b u t t h e n u c l e u s w as n o t p e r f e c t l y visible, b e i n g c o v e r e d by th e n u m e r o u s p i g m e n t g r an u l es. T h e s e cells w e r e s im i la r to t h e P E C s a d h e r i n g to c h o r o i d , e x c e p t f o r the r o u n d s h a p e t h e y a s s u m e d in t h e liquid. It is, h o w e v e r , a fact t h a t cells
42
Fig. 1. Papanicolau 50 x : 18 days after detachment. Numerous cell types are present in SRF. In this picture, well preserved 1' and degenerated ~ PECs and macrophages 9 are recognized,
Fig. 2. Papanicolau 125 •
23 days after detachment 'Morula' of PECs; oval, elongated pigment granules are well visible and partly mash the cell nuclei.
43
Fig. 3. May-Grunwald-Giemsa 400 • : 25 days after detachment. Macrophages with foamy, vacuolar cytoplasm and reniform nuclei.
Fig. 4. Transmission Electron Microscopy 12 000 • : 10 days after detachment. In the lower part of the picture, the cytoplasm of a macrophage with fagocytosis granules 9 contain cell debris and a pigment granule 1'; another pigment granule is free in the fluid, near the macrophage. In the upper part of the picture, the outer part of a neuropithelial cell shows a broken and disorganized disk pile.
44
Fig. 5. May-Grunwald-Giemsa400x: 10 days after detachment. A nerve cell that recalls the morphology of a bipolar cell: at the upper end of the cell, a bifurcating eytoplasmaticextension is visible. released in a fluid adopt a round shape because of the loss of polarity and the subsequent disorganization of cellular cytoskeleton. In the first withdrawals there was a lack of well defined pigmented epithelium cells. Instead, similar cells were depicted as having a partial loss of pigmented granules, an indistinct cytoplasm and a globoid nucleus rarefied by a clumped cromatin attached to the nuclear membrane.
Macrophages. This cell type was found in the subretinal fluid approx. 15-20 days after detachment. The cells revealed either a common reniform indented nucleus with a homogeneous, sharply marked cytoplasm, or a vacuolated cytoplasm with pigmented granules and an indistinct cell boundary. The latter morphology can be linked to a regressive change caused by a longer interval in the fluid. Neuro retinal cells. These cells showed a homogeneous, azurophilic cytoplasm; the nucleus was oval with homogeneous cromatin. Their shape varied according to the cell of origin and the amount of time they spent in the fluid. Some cells appeared oval-shaped with the nucleus slightly on one side, while on the other side, the cytoplasm revealed some shrinkage. The morphology of these cells clearly recalled the neuroepithelial cells. The electron microscopy examination confirmed light microscopy. It was possible to identify the cells through the presence of piles of disks, neurotubules and neurofilaments in the cytoplasm. Furthermore, changes were always present in the cells: swelling and lack of perfect order of the disk piles and untidy tubules and filaments dispersed in the cytoplasm. Other cells had a shape which recalled bipolar or ganglion cells. We found nerve cells in every withdrawal
45 examined. However, when retinal detachment occurred many days before medical care, many of the cells showed a round or oval shape and no extension. The presence of cytoplasm and nuclear vacuoles was sometimes observed.
Discussion
We found cells in SRF soon after detachment. The first cell type to be present in the fluid represented a degenerated aspect of pigmented epithelial cells. Since this type appeared very early on after detachment, we believe that degenerative changes of such cells are not only due to the period they spend in the fluid but could be the direct effect or the direct cause of the detachment. The pigmented epithelial cells are strictly linked to each other via junctional complexes (zonula occludens, zonula adherens and desmosomes); on the other hand, there is no real link between PEC and neuroepithelium, even if the outer part of rods and cones is completely surrounded by the villous projections of the PECs cytoplasm. However, the breaking down of such a structure is very rare when a previous metabolic damage of the retina is absent. In our opinion necrobiosis of PEC could be one of the reasons causing retinal detachment. After the weak link between rods and cones and PEC is broken these cells (PECs) start to proliferate. Machemer and Laqua found that the first PECs incorporated radiolabeled thymidin after 3 days, and the peak was reached after 7 days. This proliferating tissue clearly represented an obstacle for reattachment of the retina. The strands, once proliferated, crossed openings of the retina and reached the vitreous causing complete detachment and blindness by anterior PVR. Two weeks after the detachment, cells originating from the retina began to be present in the SRF. This event is well described in ancient literature though it seems to have been lost and forgotten today. We believe, however, that unsatisfactory anatomic and functional results which are observed in long term retinal detachments is not only caused by the presence of proliferative membranes in the sub-retinal space but also by the destruction of a variable number of neuropithelial cells.
References
1. Gonin J. Pathog~nieet anatomie pathologique des d6collementsr6tiniens. Bull Mem Soc Fr Ophtalmol 1920; 33: 1-18. 2. Tsuboi S, Taki-Noie J, Emi K, Manabe R. Fluid dynamicsin eyes with rhegmatogenous retinal detachments. Am J Ophthalmol 1985; 99: 673-76. 3. Laqua H, Machemer R. Clinical pathological correlation in massive periretinal proliferation, Am J Ophthalmol 1975; 80: 1-31.
46 4. Sternberg P Jr, Machemer R. Subretinal proliferation. Am J Ophthamol 1984, 98: 456-62. 5. Trese MT, Chandler DB, Machemer R. Subretinal strands: ultra-structural features. Graefe's Arch Clin Exp Ophthalmol 1985; 223: 35-40. 6. Horn DL, Aaberg TM, Machemer R, Fenzl R. Glial cell proliferation in human retinal detachment with massive preretinal proliferation. Am J Ophthalmol 1977; 84: 383-89. 7. Laqua H, Machemer R. Glial cells proliferation in retinal detachment (massive preretinal proliferation). Am J Ophthalmol 1975; 80: 602-18, 8. Machemer R. Pathogenesis and classification of massive periretinal proliferation. Br J Ophthalmol 1978; 62: 737-47. 9. Machemer R, Laqua H. Pigment epithelial proliferation in retinal detachment (massive periretinal proliferation). Am J Ophthalmol 1975; 80: 1-23. 10. Machemer R, van Horn DL, Aaberg TM. Pigment epithelial proliferation in retinal detachment with massive periretinal proliferation. Am J Ophthalmol 1978; 85: 181-91. 11. Hamilton CW, Chandler D, Klintorth GH, Machemer R. A transmission and scanning electron microscopic study of surgically excised preretinal membrane proliferations in diabetes mellitus. Am J Ophthalmol 1982, 94: 473-88. 12. Yamamoto T, Yamashita H, Hori S. Electron microscopic observation of preretinal membranes. Jpn J Ophthalmol 1989; 33: 151-65. 13. Bartoli F, Diversi A, Feira C, Liuzi L. Rama P. Indagini biochimiche sul liquido sottoretinico. Ed Minerva Medica, 1981; 35-42.
Address for correspondence: Dr. C. Sforzi, Clinica Oculistica, Universita degli studi di Siena, Viale Bracci, 1-53100 Siena, Italy.
The subretinal fluid in retinal detachment
A cytologic study PAOLO TOTI, 1 A U G U S T O MOROCUTTI, 2 C L A U D I A SFORZI, 2 M A R I A M A R G H E R I T A DE SANTI, 1 ANNA M A R I A CATELLA I & STEFANO BAIOCCHI 2 llstituto di Anatomia Patologica, Universitg~ degli Studi di Siena; 2Istituto di Scienze Ofialmologiche, Universit~ degli Studi di Siena Accepted 17 May 1991 Key words: Cytology, subretinal fluid, retinal detachment, transmission electron microscopy Abstract. Following retinal detachment, subretinal fluid (SRF) fills the neoformed space.
Subsequently subretinal and preretinal strands of proliferative tissue begin to form. We have collected the subretinal fluid withdrawn during retinal detachment surgery. We have studied subretinal fluid cytologically to evaluate the number and the type of cells present in the fluid, and by means of transmission electron microscopy. The first cell type to be present in the fluid represented degenerated aspects of pigmented epithelial cells (PECs). Successively, other cell types appeared in the fluid as nerve cells (rods, cones and glial cells), macrophages and well preserved pigmented epithelial cells. Abbreviations: PECs, pigmented epithelial cells; SRF, subrctinal fluid
Introduction Following retinal detachment which results from the separation of pigmented epithelium with neuropithelium, a subretinal fluid (SRF) fills the neoformed space. The SRF originates partly by the tiquifaction of vitreous humor [1] and partly by an increase in permeability of the choroidal vessels [2]. Subsequently, subretinal and preretinal strands of proliferative tissues begin to form [3]. Once proliferated, these strands generally lead to massive vitreous retraction. The presence of these subretinal strands has been associated with long standing retinal detachment [4]. In fact, subretinal strands tend to hinder the resettling of a detached retina. On the other hand, where reattachment does occur, most subretinal strands are found to lie flat beneath the reattached retina, as a permanent indicator of a previous detachment [5]. In both cases (detachment or reattachment), however, the strands may reach the posterior camera, through openings in the retina. The retraction of these strands distorts the retina thus impeding a new reattachment. Today, vitreous surgery techniques (Vitrectomy) allow treatment for the most severe degrees of retinal detachment and where indicated, subreti-
40 nal strands may be severed and removed. Such material has been extensively studied in order to identify their cellular composition; fundamental histological studies on retinal detachment have been performed on animal models as, for example, the owl monkeys of Machemer and Laqua who revealed a clinical picture and pathology similar to that of human beings. A lot of different cell types have been recognized, such as glial cells [6, 7], PEC cells [8-10] and fibroblasts. The retraction of such strands is due to the contraction of actin filaments contained in the cells and to their three dimensional arrangement described by Hamilton [11] and Yamamoto [12]. In the following study, we analysed SRF cytology in human subjects, in order to study the first steps in the membrane formation and to examine the cellular composition of the medium in which membranes are dipped [13]. Materials and methods
Subretinal fluid was obtained from the eyes of 28 patients (16 males and 12 females between 17 and 74 years of age) suffering from retinal detachment, and analysed. Out of the 28 patients, 14 had a myopia over 8 Diopters, 4 were aphakic (they were operated with the intra capsular technique), 3 revealed a previous trauma, 3 suffered from hemorrhages previous to detachment, 2 were diabetic, in 1 patient a total uveitis was observed while another myopic and aphakic patient revealed a detachment recurrence. All patients underwent an operation of equatorial buckling. A silicon sponge (4-5 mm in diameter) was used each time. During each operation, we performed an evacuating puncture or scleroctomy in order to drain SRF. The fluid was withdrawn with a cylinder needle attached to a sterilized syringe and introduced into the scleroctomic incision. In all cases we introduced sterilized air in the vitreous chamber to permit the evacuation of subretinal fluid and the reattachment of the detached retina. The cryocoagulation of retinal wounds was always used. The SRF removed, ranged from 0.3 to 1.2 cc. Part of the fluid collected was cytocentrifuged and the smears obtained were stained with either Papanicolau or May-Grunwald Giemsa. Another part of the fluid was immediately fixed in 2.5% glutaraldehyde, in 0.1M cacodylate buffer at 4 ~ for 3 hrs, postfixed in 1% buffered OsO4 for 2 hrs, dehydrated and embedded in epoxy resin. The resulting ultrathin sections were cut with LKB V Ultratome, counterstained with uranyl acetate and lead citrate, and finally observed through a Zeiss EM-109 electron microscope. Results
No cells were visible in the SRF withdrawn and observed 5 days after detachment. In the SRFs observed 7 and 8 days after detachment only few
41
Table I. Results data. The first column indicates the number of days between retinal detachment and the collection of subretinal fluid. Of course, this time has been estimated by patients inquiry. The number of cells found in the fluid has been evaluated in a semi-quantitative way from 0, which means absence of cells to + + +. Days 5 7 8 10 10 11 12 12 13 15 15 18
20 20 23 23 25 26 27 33 39 40 60 60 60 100 130
Macrophages
Pigmented preserved
Epithelium cells degenerated
Neuro-retinal cells
0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 + ++ + + + + + + ++ ++ ++ ++ + +
0 + + + + + + + + + + ++ ++ ++ ++ ++ ++ + + + ++ +++ ++ + + + ++
0 0 0 + + + + + + + + + 0 +
+
0 + ++ ++ ++ + + + ++ ++ + ++
+
+++ ++ +++ + 0 0 + +++ + + + ++
++
++
++
++
§ + + +
+ +
+ + + +
+ + +
cells w e r e p r e s e n t . In t h e f o l l o w i n g fluids e x a m i n e d a g r e a t n u m b e r o f cells o f d i f f e r e n t t y p e s w e r e o b s e r v e d . T h e s e cells i n c l u d e d p i g m e n t e p i t h e l i u m cells, m a c r o p h a g e s with a v a r i a b l e c o n t e n t o f m e l a n i c p i g m e n t an d n e r v e cells ( T a b l e 1).
Pigmented epithelium cells (PECs).
T h i s cell t y p e a p p e a r e d in t h e s u b r e t i n a l fluid e x a m i n e d t w o w e e k s a f t e r d e t a c h m e n t . T h e cells a p p e a r e d e i t h e r i s o l a t e d o r g r o u p e d ( 4 - 1 0 cells) as a ' m o r u l a ' . T h e y w e r e r e l a t i v e l y l ar g e a n d r o u n d , a n d t h e c y t o p l a s m was filled with b r o w n , o v a l an d e l o n g a t e d p i g m e n t g r a n u l e s . T h e c y t o p l a s m b o u n d a r y was well d e f i n e d b u t t h e n u c l e u s w as n o t p e r f e c t l y visible, b e i n g c o v e r e d by th e n u m e r o u s p i g m e n t g r an u l es. T h e s e cells w e r e s im i la r to t h e P E C s a d h e r i n g to c h o r o i d , e x c e p t f o r the r o u n d s h a p e t h e y a s s u m e d in t h e liquid. It is, h o w e v e r , a fact t h a t cells
42
Fig. 1. Papanicolau 50 x : 18 days after detachment. Numerous cell types are present in SRF. In this picture, well preserved 1' and degenerated ~ PECs and macrophages 9 are recognized,
Fig. 2. Papanicolau 125 •
23 days after detachment 'Morula' of PECs; oval, elongated pigment granules are well visible and partly mash the cell nuclei.
43
Fig. 3. May-Grunwald-Giemsa 400 • : 25 days after detachment. Macrophages with foamy, vacuolar cytoplasm and reniform nuclei.
Fig. 4. Transmission Electron Microscopy 12 000 • : 10 days after detachment. In the lower part of the picture, the cytoplasm of a macrophage with fagocytosis granules 9 contain cell debris and a pigment granule 1'; another pigment granule is free in the fluid, near the macrophage. In the upper part of the picture, the outer part of a neuropithelial cell shows a broken and disorganized disk pile.
44
Fig. 5. May-Grunwald-Giemsa400x: 10 days after detachment. A nerve cell that recalls the morphology of a bipolar cell: at the upper end of the cell, a bifurcating eytoplasmaticextension is visible. released in a fluid adopt a round shape because of the loss of polarity and the subsequent disorganization of cellular cytoskeleton. In the first withdrawals there was a lack of well defined pigmented epithelium cells. Instead, similar cells were depicted as having a partial loss of pigmented granules, an indistinct cytoplasm and a globoid nucleus rarefied by a clumped cromatin attached to the nuclear membrane.
Macrophages. This cell type was found in the subretinal fluid approx. 15-20 days after detachment. The cells revealed either a common reniform indented nucleus with a homogeneous, sharply marked cytoplasm, or a vacuolated cytoplasm with pigmented granules and an indistinct cell boundary. The latter morphology can be linked to a regressive change caused by a longer interval in the fluid. Neuro retinal cells. These cells showed a homogeneous, azurophilic cytoplasm; the nucleus was oval with homogeneous cromatin. Their shape varied according to the cell of origin and the amount of time they spent in the fluid. Some cells appeared oval-shaped with the nucleus slightly on one side, while on the other side, the cytoplasm revealed some shrinkage. The morphology of these cells clearly recalled the neuroepithelial cells. The electron microscopy examination confirmed light microscopy. It was possible to identify the cells through the presence of piles of disks, neurotubules and neurofilaments in the cytoplasm. Furthermore, changes were always present in the cells: swelling and lack of perfect order of the disk piles and untidy tubules and filaments dispersed in the cytoplasm. Other cells had a shape which recalled bipolar or ganglion cells. We found nerve cells in every withdrawal
45 examined. However, when retinal detachment occurred many days before medical care, many of the cells showed a round or oval shape and no extension. The presence of cytoplasm and nuclear vacuoles was sometimes observed.
Discussion
We found cells in SRF soon after detachment. The first cell type to be present in the fluid represented a degenerated aspect of pigmented epithelial cells. Since this type appeared very early on after detachment, we believe that degenerative changes of such cells are not only due to the period they spend in the fluid but could be the direct effect or the direct cause of the detachment. The pigmented epithelial cells are strictly linked to each other via junctional complexes (zonula occludens, zonula adherens and desmosomes); on the other hand, there is no real link between PEC and neuroepithelium, even if the outer part of rods and cones is completely surrounded by the villous projections of the PECs cytoplasm. However, the breaking down of such a structure is very rare when a previous metabolic damage of the retina is absent. In our opinion necrobiosis of PEC could be one of the reasons causing retinal detachment. After the weak link between rods and cones and PEC is broken these cells (PECs) start to proliferate. Machemer and Laqua found that the first PECs incorporated radiolabeled thymidin after 3 days, and the peak was reached after 7 days. This proliferating tissue clearly represented an obstacle for reattachment of the retina. The strands, once proliferated, crossed openings of the retina and reached the vitreous causing complete detachment and blindness by anterior PVR. Two weeks after the detachment, cells originating from the retina began to be present in the SRF. This event is well described in ancient literature though it seems to have been lost and forgotten today. We believe, however, that unsatisfactory anatomic and functional results which are observed in long term retinal detachments is not only caused by the presence of proliferative membranes in the sub-retinal space but also by the destruction of a variable number of neuropithelial cells.
References
1. Gonin J. Pathog~nieet anatomie pathologique des d6collementsr6tiniens. Bull Mem Soc Fr Ophtalmol 1920; 33: 1-18. 2. Tsuboi S, Taki-Noie J, Emi K, Manabe R. Fluid dynamicsin eyes with rhegmatogenous retinal detachments. Am J Ophthalmol 1985; 99: 673-76. 3. Laqua H, Machemer R. Clinical pathological correlation in massive periretinal proliferation, Am J Ophthalmol 1975; 80: 1-31.
46 4. Sternberg P Jr, Machemer R. Subretinal proliferation. Am J Ophthamol 1984, 98: 456-62. 5. Trese MT, Chandler DB, Machemer R. Subretinal strands: ultra-structural features. Graefe's Arch Clin Exp Ophthalmol 1985; 223: 35-40. 6. Horn DL, Aaberg TM, Machemer R, Fenzl R. Glial cell proliferation in human retinal detachment with massive preretinal proliferation. Am J Ophthalmol 1977; 84: 383-89. 7. Laqua H, Machemer R. Glial cells proliferation in retinal detachment (massive preretinal proliferation). Am J Ophthalmol 1975; 80: 602-18, 8. Machemer R. Pathogenesis and classification of massive periretinal proliferation. Br J Ophthalmol 1978; 62: 737-47. 9. Machemer R, Laqua H. Pigment epithelial proliferation in retinal detachment (massive periretinal proliferation). Am J Ophthalmol 1975; 80: 1-23. 10. Machemer R, van Horn DL, Aaberg TM. Pigment epithelial proliferation in retinal detachment with massive periretinal proliferation. Am J Ophthalmol 1978; 85: 181-91. 11. Hamilton CW, Chandler D, Klintorth GH, Machemer R. A transmission and scanning electron microscopic study of surgically excised preretinal membrane proliferations in diabetes mellitus. Am J Ophthalmol 1982, 94: 473-88. 12. Yamamoto T, Yamashita H, Hori S. Electron microscopic observation of preretinal membranes. Jpn J Ophthalmol 1989; 33: 151-65. 13. Bartoli F, Diversi A, Feira C, Liuzi L. Rama P. Indagini biochimiche sul liquido sottoretinico. Ed Minerva Medica, 1981; 35-42.
Address for correspondence: Dr. C. Sforzi, Clinica Oculistica, Universita degli studi di Siena, Viale Bracci, 1-53100 Siena, Italy.
