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Histopathologic Features of Neovascularization in Sickle Cell Retinopathy
Histopathologic Features of Neovascularization in Sickle Cell Retinopathy D. SCOTT McLEOD, CAROL MERGES, MAS, ASAKO FUKUSHIMA, MD, MORTON F. GOLDBERG, MD, AND GERARD A. LUTTY, PHD
• PURPOSE: To examine the histopathologic and morphometric features of neovascular lesions in human proliferative sickle cell retinopathy. • METHODS: Postmortem ocular tissue was ob tained from three subjects (aged 20, 28, and 40 years) with SS hemoglobinopathy and prepared for adenosine diphosphatase flat-embedding. Mor phometric analysis was performed before serial sectioning. • RESULTS: Numerous active and autoinfarcted lesions were found that represented virtually all stages in the life cycle of preretinal neovasculariza tion. These formations ranged from single small loops extending from arteries and veins along the retinal surface to the typical complex, elevated sea fan formations. Sea fans developed at hairpin loops and at arteriovenous crossings. There was an average of 5.6 connections between sea fans and retinal vessels; of these, 45% were arteriolar, 52.5% were venular, and 2.6% were at the capil lary level. Six of eight sea fans were located at arteriovenous crossings. Autoinfarction appeared
Accepted for publication June 24, 1997. From the Wilmer Ophthalmological Institute, Johns Hopkins Universi ty School of Medicine, Baltimore, Maryland. This work was funded by NIH grants HL45922 (Dr Lutty) and EY01765 (Wilmer Institute), and by an unrestricted research grant from Research to Prevent Blindness, Inc, New York, New York. Dr Lutty is an American Heart Association Established Investigator. Reprint requests to Gerard A. Lutty, PhD, 170 Woods Research Building, Johns Hopkins Hospital, 600 N Wolfe St, Baltimore, MD 21287-9115; fax: (410) 955-3447; e-mail: galutty@welchlink. welch.jhu.edu
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to occur initially within the sea fan capillaries. The average height of sea fans was 123 (Am above the retinal surface. • CONCLUSIONS: Preretinal neovascularization in sickle cell retinopathy can arise from both the arterial and venous sides of the retinal vasculature and can assume a variety of morphologic configu rations. Multiple feeding arterioles and draining venules are common, and autoinfarction appears to occur initially at the preretinal capillary level rather than at feeding arterioles. Arteriovenous crossings may be a preferential site for sea fan development.
S
ICKLE CELL DISEASE IS CAUSED BY A POINT MUTAtion in the hemoglobin gene. Deoxygenation or acidic conditions cause the hemoglobin mole cules to polymerize, making the sickle erythrocytes less pliable and more likely to assume unusual shapes. This increases blood viscosity and causes the individ ual cells to function as microemboli, becoming trapped within the microcirculation.1 Peripheral vas cular occlusion, presumably caused by sickle erythro cytes, is a hallmark of sickle cell retinopathy and is the initiating event that stimulates a cascade of events including neovascularization. Peripheral retinal nonperfusion and early forms of angiogenesis occur in approximately 40% (SS, sickle cell anemia) to 80% (SC) of sickle cell subjects.2 Proliferative sickle cell retinopathy is most often associated with hemoglobin SC disease and to a much lesser extent with other hemoglobinopathies (Sb-thalassemia and SS).1 Neo-
© AMERICAN JOURNAL OF OPHTHALMOLOGY 1997;124:455-472
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vascular formations, which usually occur in the equatorial plane, can eventually spread along the retinal surface and adopt a fan-shaped configuration, which morphologically resembles the marine inverte brate Gorgonia flabellum. With continued develop ment, sea fans may hemorrhage, be dragged off the retinal surface by adherent vitreous, or autoinfarct.3 Angiographic studies have shown that sea fans arise from arteriovenous anastomoses bordering avascular retina.14 These anastomoses form as a result of vascular remodeling after occlusive events.5 Their growth extends peripherally toward the ora serrata in an apparent attempt at revascularizing avascular and See also pp. 488-497, 506-515, and 516-520.
hypoxic retina. The most common sites for sea fan formations in the fundus are superotemporal and inferotemporal.1 Although proliferative sickle cell retinopathy has been studied extensively, clinically and angiographically, few histopathologic studies and no detailed morphometric analyses have been per formed on sea fans or other neovascular lesions in sickle cell subjects.6 In this study, we used adenosine diphosphatase (ADPase) flat-embedded retinas7 from sickle cell subjects to perform site-specific analysis of neovascular formations. The earliest forms of neovascularization, various types of active sea fans, and partially or totally autoinfarcted formations were all critically examined; they appeared to represent virtu ally all stages in the life cycle of preretinal neovascularization in sickle cell retinopathy.
METHODS
were aged 20 (Patient 1), 28 (Patient 2), and 40 (Patient 3) years. Medical histories showed each to have SS hemoglobinopathy. Eyes from each subject were opened 1 cm posterior to the limbus and examined macroscopically before processing. One eye of each subject was fixed, cryopreserved, and frozen for immunohistochemical analysis that will be report ed elsewhere. The retinas from the three fellow eyes were teased from the retinal pigment epithelium, fixed, washed, and processed for ADPase histochemical staining of the retinal vasculature, as previously described.7 The ADPase-incubated retinas were cut into piec es, flattened, postfixed, and embedded in JB-4 plastic, as described in detail previously.7 Using a Zeiss Photomicroscope II (Oberkochen, Germany) with darkfield illumination to visualize the lead phosphate reaction product, we photographically documented blocks of retina and obtained digitized images from a video camera attached to the microscope. Computerassisted morphometric analysis was performed using a Macintosh Ilci personal computer with image analysis software (National Institutes of Health Image soft ware, version 1.47). Serial cross sections (2.5 |xm thick) were cut on an ultramicrotome fitted with a video camera and macroscopic lens system, which were interfaced with the computer. This allowed us to visualize and document the vasculature en bloc during sectioning. Images of the blocks were obtained every 150 |xm of tissue sectioned and provided precise correlation of section location within the vasculature. For the purpose of being consistent, in this study we will refer to intraretinal parent vessels that give rise to neovascularization as arteries and veins, based on their vascular hierarchy. Vessels that traverse the internal limiting membrane to feed or drain preretinal neovascularization will be referred to as arterioles and venules, based on their luminal diameters (Table).
HUMAN POSTMORTEM EYES WERE OBTAINED FROM THE
Medical Eye Bank of Maryland, the Medical Eye Bank of Florida, and the Mid-America Eye and Tissue Bank of St Louis, Missouri. The Johns Hop kins University School of Medicine Human Subjects Board approved the use of human tissue for this study. The cause of death, time of death, death to enucleation time (elapsed time from time of death to enucleation and storage at 4 C), and brief medical history were provided for each donor. The donors 456
RESULTS • CASE l: A 20-year-old woman died from cardiac arrest. Examination of the retinal vasculature dis closed mild peripheral vaso-occlusive disease and early forms of neovascularization bordering a narrow field of avascular peripheral retina. These changes
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TABLE. Sea Fans From Patients 2 and 3
Sea Fan
Area (mm2)/ Maximum Height
No.
Above Retina (|xm)
1
1.01/70
2 3 4 5 6 7 8
3.43/100 0.96/150 4.62/75 0.28/115 1.43/160 0.38/200 3.05/113
Viable
Viable
Viable
Art Connections (luminal diameter)
Ven Connections (luminal diameter)
Cap Connections (luminal diameter)
Total Viable Connections
Location
0
5
A-V crossing
0
5
Hairpin loop
0
3
A-V crossing
0
9
A-V crossing
0
6
Hairpin loop
1 (6.4) 0 0
7
A-V crossing
0 4
A-V crossing A-V crossing
1 (11.9) 3 (17.1 ± 5.6)* 1 (13.3) 5 (8.5 ± 4.2)* 2 (10.3 ± 3.1)* 3 (13.0 ± 7.2)* 0 2 (16.2 ± 3.3)*
4 (16.5 ± 2 (21.7 ± 2 (19.2 ± 4 (17.1 ± 4 (8.8 ± 3 (12.8 ± 0 2 (29.3 ±
7.8)* 6.9)* 4.5)* 9.6)* 2.2)* 5.7)*
6.7)*
Art = arterial; Ven = venous; Cap = capillary; A-V == arteriovenous. *Mean luminal diameter ±SD.
were confined to the superotemporal quadrant. The neovascular lesions were primarily intraretinal and consisted of corkscrew-like configurations within fields of dilated capillaries. • CASE 2: A 28-year-old man died from cardiac arrest. He was septic at the time of death. The peripheral retinal vasculature demonstrated vascular obstructions, capillary dropout, and vascular remod eling secondary to vaso-occlusive disease. Angiopathic changes were mostly confined to the far periph ery in the superior and temporal quadrants. A sea fan formation was present at approximately the 11-o'clock position of the left eye. • CASE 3: Extensive occlusive and proliferative retinopathy was observed in a 40-year-old woman who died from respiratory arrest. The entire circumference of the retina demonstrated loss of vasculature, which was most pronounced in the superior quadrant. Neovascular formations were observed at the border of vascularized peripheral retina in the equatorial plane of the superotemporal and superonasal regions from the 10-o'clock to the 2-o'clock positions (Figure 1) of the right eye. Some formations were relatively small and consisted primarily of large-caliber vessels VOL.124, No. 4
forming loop-like structures (Figure 1, arrows 3), whereas others demonstrated a typical sea fan appear ance (Figure 1, arrows 4 through 6). Some sea fans appeared to be quite flat and spread out along the retinal surface. Others appeared to be more con densed and elevated (Figure 1, arrows 7A, 7C, and 7E), with ill-defined intercapillary spaces. One for mation appeared to be degenerative based on its weak ADPase staining intensity and morphologic appear ance (Figure 1, arrow 8). In some areas in Patient 1, vascular buds with unusually high ADPase activity were observed ex tending toward the vitreous from hairpin loop forma tions within recanalized segments of the terminal portions of occluded vessels (Figure 2, A). At low magnification, budding appeared to be associated with the venous side of the circulation; however, on closer examination, these buds appeared as loops, originating from both the arterial and venous compo nents of remodeled vasculature (Figure 2, B). Capil laries at the border of perfused-nonperfused retina appeared to be dilated, and morphometric measure ments disclosed capillary diameters to be twofold greater in this region than in more posterior locations (16.35 ± 2.3 |o,m vs 8.7 ± 0.7 |xm, respectively). Cross-sectional analysis of the loop-like formations
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FIGURE 1. Patient 3. Superior retina showing numerous neovascular formations bordering the avascular retina. Numbered arrows correspond to regions shown in subsequent figures (see Case 3 in Results) (brightfield illumination, X4.5).
(Figure 2, C through E) showed dilated vessels containing packed erythrocytes extending toward the vitreous cavity. Examination of the tips of these vessels disclosed endothelial cell hyperplasia. The unusually high ADPase activity observed in these formations in the flat perspective was indicative of endothelial cell proliferation in these vessel segments, as we have documented in intraretinal microvascular abnormality formations within diabetic retina.7 In some cases, single cells were observed projecting through small defects in the internal limiting mem brane (Figure 2, C). No abnormalities of the vitreous were evident at these sites. Analysis of the small loop-like formations in Pa tient 3, (Figure 1, arrows 3) disclosed simultaneous and independent arterial (a) and venous (v) prolifer ations (Figure 3). In the flat view (Figure 3, A), the formations appeared as loop-like structures on the internal surface of the retina that formed within a viable segment of the vein (Figure 3, A, arrows D and E) and at the terminal portion of an adjacent artery (Figure 3, A, arrows B and C). The arterial lesion bordered the avascular retina, and the venous lesion was associated with a relatively small area of capillary dropout. Sections taken through the arterial lesion
458
demonstrated that the neovascular formation was located on the retinal surface just proximal to a hairpin loop within the terminal portion of the arterial wall (Figure 3, B). The vessels in this forma tion had large lumina, thicker walls than the venous lesion had, and smooth muscle cells, mirroring the structure of the parent artery (Figure 3, C). The new vessels showed apparent adhesion to the cortical vitreous (Figure 3, B, C, and D, curved arrows). Sections collected through the venous loop formation showed the preretinal location of the vessels (Figure 3, D) and communications with the parent vein (not shown) through a discontinuous internal limiting membrane. The majority of preretinal vessels had large lumina with relatively thin walls and consisted of endothelial cell linings with scattered pericytes (Figure 3, D). These large, thin-walled vessels, which appeared to be an extruded segment of the parent vein, gave rise to capillaries (Figure 3, E) that had numerous bulky endothelial cell nuclei and relatively thin basement membranes. In this study, we critically examined eight sea fans from two subjects (seven from Patient 3, one from Patient 2). A summary of the histopathologic features of these formations is shown in the Table. Serial
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FIGURE 2. Patient 1. Border of vascularized retina as shown in flat view (top, A and B) and in histologic sections (bottom, C through E). (A) Neovascular buds (arrowheads) are present at the terminal ends of viable vascuiature in this field from the superotemporal retina. These buds appear to be related to the venous (v) and not the arterial (a) side of the circulation (darkfield illumination, X40). (B) At higher magnification, the arterial (a) and venular (v) components of the remodeled vascuiature terminate in hairpin loops (curved arrow). Neovascular buds (arrowheads) arise just posterior to the terminal ends of vascuiature. Regarding loop formation E, see the panel E legend below (darkfield illumination, X130). (C, D) Histologic sections from formations in A and B show vessels extending toward the vitreous. A single cell is shown projecting onto the inner retinal surface (arrow) (periodic acid-Schiff and hematoxylin, X650). (E) Section taken through the twisted loop formation (arrow E in B, top right) (periodic acid-Schiff and hematoxylin, X650).
sections were analyzed to determine the number, distribution, and luminal diameters of feeding arterioles and draining venules for each preretinal lesion (more than 8,000 serial sections were examined). The average area of the sea fans in this study was 2.1 ± 1.6 mm2, and the average height was 123 ± 44.5 (xm above the retinal surface. The number of viable connections between retinal vessels and sea fans averaged 5.6 ± 2.0 per formation. Of these connec tions, 52.4% were venular, 45.0% were arteriolar, and
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2.6% were at the capillary level. The average luminal diameters were 12.7 ± 5.0 |xm for the arteriolar connections and 17.9 ± 7.4 |xm for the venular connections. The largest feeding arteriole had a luminal diameter of 22.53 (xm, and the largest draining venule had a luminal diameter of 26.23 u,m. The distribution of feeders and drainers in sea fans varied; however, the largest were usually located in the central region of the neovascular formation. In some cases, the same intraretinal vessel gave rise to
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multiple connections with the fan. Smaller connec tions were located at the fringes of the sea fans and were generally connected to different intraretinal arteries and veins than were the centrally located connections. We observed typical sea fan formations at two types of anatomic locations within the peripheral vasculature: hairpin loops and arteriovenous crossings (Table). Figure 4 shows a sea fan from Patient 3 that has two connections that originated from a hairpin loop within a recanalized segment of an occluded artery (Figure 4, A and B, between the opposing arrows). These connections (Figure 4, C through F), which had luminal diameters of 22.42 \xm and 11.7 |JLm, were two of four feeders that originated from different segments of the same parent artery (Figure 4, A, straight arrows). This sea fan was 3.43 mm 2 in area and relatively flat, with a maximum height that was 100 |xm above the retinal surface (Table, sea fan 2). In addition to the four arteriolar connections, this formation also had two connections to the venous side of the circulation. All feeding and draining connections were considered patent because of their apparently viable endothelial cells and unobstructed lumina, which in some cases contained scattered erythrocytes and leukocytes. The majority of blood vessels within this neovascular formation were viable and consisted of arterioles, venules, and capillaries with walls of varying thickness. They were surround ed by a collagen-like matrix, and vitreous condensa tion was evident, as shown by darkfield microscopy. Generally, these preretinal vessels contained an en dothelial cell lining with smooth muscle cells associ ated with arterioles; pericytes were associated with
venules and capillaries. Some vessels appeared to be in various stages of degeneration (few apparently viable endothelial cell nuclei), whereas others were completely atrophic (no endothelial cell nuclei). No glial cells were apparent. A few inflammatory cells were observed in and around the vessels. In addition to originating from hairpin loops, most sea fans originated from sites at arteriovenous cross ings. In fact, five of the seven active fans and one autoinfarcted sea fan that we analyzed were located at arteriovenous crossings (two of the remaining three sea fans in Patient 3 also occurred at arteriovenous crossings). Figure 5 shows examples of sea fans from Patient 2 (A through C) and Patient 3 (D through F) that were located at arteriovenous crossings where the artery (Figure 5, A through C, arrow a) was situated internal to the vein (Figure 5, A through C, arrow v). The formation from Patient 2 (Figure 5, A through C) was 1.01 mm 2 in area, was virtually flat along the retinal surface, had one connection to an artery (Figure 5, B and C), and had four connections to a vein (Table, sea fan 1). No atrophic preretinal vessels or connections were observed in this sea fan. The feeding arteriole (Figure 5, B and C, curved arrow) broke through the internal membrane exactly at the arteriovenous crossing, where it formed a preretinal loop, and then extended peripherally into the fan (Figure 5, B and C). The feeding arteriole had a luminal diameter of 11.9 (xm. In this example, the underlying vein (Figure 5, A through C, arrow v) did not dip down or show any compression (Figure 5, C). Three of the venous connections in this formation occurred before the arteriovenous crossing. Again, preretinal vessels were found to have ample contrac-
FIGURE 3. Patient 3. (A) Preretinal loops (arrows D&E and B&C) originate from the terminal portion of an artery (a) and a straight portion of a vein (v) in this flat view. This region of retina is indicated by arrows 3 in Figure 1 (darkfield illumination, X38). (B) Section taken through the terminal portion of the artery (B&C in A, top) showing a hairpin loop formation within the hyalinized arterial wall (straight arrows). Note the apparent adherent vitreous (curved arrow) associated with the preretinal vessels (periodic acid-Schiff and hematoxylin, X450). (C) Section taken through the region indicated by arrow B&C in A showing an intraretinal segment of vessel projecting through a defect in the internal limiting membrane (arrowheads). The preretinal loops appear to have the same smooth-muscle cell component (straight arrows) as the parent artery has. Note the adherent vitreous (curved arrow) (periodic acid-Schiff and hematoxylin, X450). (D) Section taken through the region indicated by arrows D&E in A shows the preretinal disposition of the venous loop (straight arrow), adherent cortical vitreous (curved arrow), and a break in the internal limiting membrane (arrowheads) (periodic acid-Schiff and hematoxylin, X300). (E) Section taken through the region indicated by D&E in A showing capillaries (arrows) arising from the venous loop (periodic acid-Schiff and hematoxylin, X300).
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tile elements (pericytes and smooth muscle cells), and no glial cells or fibroblastic-like cells were observed. Darkfield microscopy showed vitreous condensation around the anterior portion of this sea fan, but no tractional fibers were evident. Many inflammatory cells were present in and around the preretinal vessels; this may have been related to the septic condition of this subject at the time of death. Another example of a sea fan located at this type of crossing (artery situated internal to the vein) is shown in Figure 5, D through F. This formation from Patient 3 was 4.62 mm 2 in area, relatively flat, had five connections to the arterial side of the circulation (four from different segments of the same artery), and four connections (three from the same vein) to the venous side (Table, sea fan 4). No atrophic feeders or drainers were observed in this formation; however, some atrophic preretinal vessels were found. The structure of preretinal vessels was similar to that described above. In contrast to the crossing shown in Figure 5, A through C, the underlying vein (Figure 5, D through F, arrow v), rather than the artery, broke through the internal limiting membrane and exited the retina just peripheral to this crossing point (Figure 5, F). Some compression of the vein was evident at the crossing. One half of the sea fans located at arteriovenous crossings had the vein internal to the artery. Figure 6 shows a sea fan that was 3.05 mm 2 in area and had two arterial and two venous connections (Table, sea fan 8). In this case, the major draining venule for the formation exited the retina at the crossing point (Figure 6, B), and the main feeder vessel exited the retina just after the crossing (Figure 6, C). In this sea fan, virtually all the morphologic stages in the
apparent life cycle of preretinal capillaries were repre sented (Figure 6, D through F). The preretinal vessels lay in close proximity to the cortical vitreous (Figure 6, B and C). Figure 6, D shows a section through a capillary at the anterior edge of the sea fan. The capillary, which is highly cellular, has unusually large and rounded endothelial cell nuclei and is poorly differentiated in terms of its structure. The lumen contains a leukocyte (Figure 6, D, arrow). In more posterior locations, capillaries were well differentiated and had a normal endothelial cell lining and pericyte component (Figure 6, E, arrow). Scattered through out the sea fan were capillaries that appeared to be degenerative, lacking endothelial cell and pericyte nuclei. These capillaries often contained aggregates of erythrocyte ghosts (Figure 6, F). We observed a subset of sea fans in Patient 3 that were remarkable because they appeared to be different morphologically from the typical flat fans that had peripherally directed growth patterns and welldefined intercapillary spaces (shown in Figures 4, 5, and 6). These atypical fans were smaller in area (Table, sea fans 3, 5, and 6), did not have a true fan-like configuration, appeared to be denser in terms of their vascularity, and had ill-defined intercapillary spaces (Figure 7, A, C, and E). Cross-sectional analysis of these formations disclosed that each had many infarcted preretinal channels embedded within a dense collagenous matrix (as determined with van Gieson stain) and a more mounded profile than did the typical flat sea fans (Figure 7, B, D, and F). The infarcted vessels were thick walled and acellular. They were intermingled with viable blood vessels, some of which demonstrated varying degrees of degeneration. Some of these vessels had lumina that were congested
FIGURE 4. Patient 3. Example of a sea fan (arrow 4 in Figure 1) that features connections to a hairpin loop in the terminal portion of a retinal artery. (A) Flat view of the sea fan showing the locations of all feeding arterioles (straight arrows) and draining venules (curved arrows) (darkfield illumination, X25). (B) Higher magnification view of the region between the opposing straight arrows in A, showing an arterial hairpin loop within the retina (arrows) (darkfield illumination, X63). (C) The same region as that shown in B, with the plane of focus on one of two connections between the hairpin loop and preretinal vessels (arrow) (darkfield illumination, XlOO). (D) Section showing the connection to the sea fan shown in C (arrow), a break in the internal limiting membrane (arrowheads), and the thick, hyalinized wall of the parent vessel where the hairpin loop formed (periodic acid-Schiff and hematoxylin, X380). (E) The same region as that shown in C except that the plane of focus is on a second connection between the sea fan and the hairpin loop (arrow) (darkfield illumination, X100). (F) Section showing the second connection, shown in E (arrow), a break in the internal limiting membrane (arrowheads), and the thick, hyalinized wall of the parent vessel. Note the close anatomic relationship between the new vessels and the cortical vitreous (periodic acid-Schiff and hematoxylin, X380).
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with erythrocytes, whereas others had collapsed lumina. Platelets, fibrin, and leukocytes were also observed within some vessels. All feeding arterioles and drain ing venules appeared to be viable and patent in each of these formations (not shown). Condensed, adher ent vitreous was present overlying these formations (Figure 7, D and F, arrowheads); however, no vitreous traction fibers were observed. In addition to active and partially infarcted sea fans, we analyzed an autoinfarcted formation from Patient 3 (Table, sea fan 7) that appeared to show recurrent growth of new vessels. Figure 8 shows a flat view of a neovascular formation (Figure 1, arrow 8) anterior to an arteriovenous crossing (vein internal to artery) that had little ADPase activity except at its base (Figure 8, arrowhead). Cross sections disclosed an elevated mass of atrophic vascular channels em bedded within a dense collagenous matrix (Figure 8, B). The extremely weak ADPase activity associated with this formation in the flat perspective was indica tive of degeneration of the cellular components within these vessels, which we have observed previ ously.7 A n atrophic connection to an atrophic oc cluded segment of a venous branch was identified (Figure 8, C, curved arrow). Just proximal to the autoinfarcted connection, new capillaries were ob served proliferating within the venous wall and along the retinal surface (Figure 8, C, arrowhead). This formation was interpreted as being new because it had formed at a location posterior to the atrophic connec
tion that originally drained the autoinfarcted sea fan and because it had unusually high ADPase activity, which is indicative of neovascularization.7
DISCUSSION IN THIS STUDY, WE USED THE ADPASE FLAT-EMBEDDING
technique to perform site-specific histopathologic analysis of retinal neovascularization in proliferative sickle cell retinopathy. The ADPase technique has been used previously to examine angiopathic changes in human retina. The technique uses ADPase enzyme histochemistry to visualize endothelium and smooth muscle cells of the vasculature in whole retinas.7 Because arteries have both endothelial and a smoothmuscle cell components, the arterial vessels are stained more intensely than the venular side of the circulation is. Ghost vessels, or vessels that lack these cellular components, are not labeled, whereas active neovascularization routinely demonstrates unusually high enzymatic activity, which is apparently due to endothelial cell hyperplasia. The differential staining patterns provided by this technique are therefore useful in the identification and analysis of normal and pathologic vasculature.7 Although histopathologic studies on the ocular manifestations of sickle cell disease have been performed by others, 6 those studies have dealt with a variety of lesions associated with this disease. In this study, we concentrated on one
FIGURE 5. Two examples of sea fans located at arteriovenous crossings in the retina, where the artery is situated internal to the vein. Patient 2. (A) Low-magnification view of a sea fan from the superotemporal region showing its parent artery (a) and vein (v). The arteriovenous crossing is indicated by the large curved arrow (darkfield illumination, X45). (B) A higher magnification flat view of the arteriovenous crossing shown in A. This field of view is rotated 90 degrees counterclockwise from that shown in A so that it has the same orientation as the section shown in C. In this case, the feeding arteriole appears to loop out of the retina (curved arrow) as the parent artery (a) crosses the vein (v) (darkfield illumination, X130). (C) Section taken through the crossing in B showing the connection between the intraretinal parent artery (a) and the preretinal feeding arteriole of the sea fan (curved arrow). This connection occurs at a break in the internal limiting membrane (arrowheads), where the artery crosses the vein (v) (periodic acid-Schiff and hematoxylin, X420). Patient 3. (D) Low-magnification view of a sea fan from the superior region (arrow 5 in Figure 1) showing its parent artery (a), draining vein (v), and the site at which they cross (large curved arrow) (darkfield illumination, X22). (E) Higher magnification flat view of the arteriovenous crossing in D showing the retinal vein (v), the venular connection with the sea fan (curved arrow), and the retinal artery (a). Compared with the lowmagnification view shown in D, this view has been rotated 90 degrees clockwise so that it has the same orientation as the section shown in F (darkfield illumination, X100). (F) A section through the crossing in E showing the connection between the parent vein (v) and the sea fan draining venule (curved arrow). The connection occurs at a break in the internal limiting membrane (arrowheads) near the site where the vein crosses the artery (a) (periodic acid-Schiff and hematoxylin, X300).
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aspect of sickle cell retinopathy, namely, neovascularization. Although the number of subjects used in the current study is small, our results not only confirm some of the well-recognized clinical and angiographic features of proliferative sickle cell retinopathy but also present features that have not been described previ ously: intraretinal evidence of neovascularization (Figure 2), the earliest stages of preretinal growth (Figures 3), and the earliest stages of autoinfarction (Figures 6 and 7). We have documented, therefore, examples of what we believe represent virtually all stages in the life cycle of preretinal neovascularization in sickle cell retinopathy. These include not only the earliest stages of new blood vessel growth from the retina into the vitreous and autoinfarction but also recurrence after autoinfarction (Figure 8). The fact that all stages of retinal neovascularization were seen within one eye suggests that these vascular growth and regression patterns are subject to local rather than to systemic factors. It is well recognized from angiographic data that sea fans form at arteriovenous anastomoses in the peripheral retina.1'4 These anastomotic channels form as a result of vaso-occlusions and subsequent vascular remodeling. In this study, we found sea fans at two other anatomic locations within the vasculature: hairpin loops and arteriovenous crossings. Hairpin loops within the retinal vessels of sickle cell subjects have been described.5,8 They are frequently found at the terminal ends of occluded arteries and veins. It is
thought that these structures represent recanalizations of occluded segments of vessels and that they may function to redirect blood flow posteriorly into a patent side arm channel. 8 These structures may form by neovascular processes, whereby the viable endothelium of the patent segment of vessel proliferates and parasitizes the residual basement membrane of the atrophic vessel's distal segment, as has been described by Archer. 9 The high levels of basic fibroblast growth factor that we have observed in these acellular basement membrane remnants may facilitate this process.10 Perhaps the formation of preretinal vessels from hairpin loops represents exuberant at tempts at recanalization, and their course from intra retinal to preretinal locations may be influenced by an anatomic path of least resistance. The internal limit ing membrane is extremely thin or even absent over superficial retinal vessels,11 and this anatomically weak boundary may permit this process to occur. The impressions gained from examining the form, structure, and course of vessels exiting the retina in early forms of neovascularization, as well as in more complex, typical sea fans, suggest an atypical mode of development for some of these preretinal neovascular formations. In a previous histopathologic study,8 we showed extruded or avulsed segments of large retinal veins forming preretinal venous loops in sickle cell disease. Avulsed retinal vessels have been described only occasionally in other proliferative retinopathies.12,13 The avulsed segments, which form loops
FIGURE 6. Patient 3. Example of a sea fan located at an arteriovenous crossing where the vein is located internal to the artery. (A) Low-magnification flat view of the sea fan and the arrangement of its parent artery (a) and vein (v). The major draining venule for the sea fan (solid curved arrow) makes its connection to the parent retinal vein at the arteriovenous crossing and the major feeding arteriole for the sea fan (open curved arrow) makes its connection to the parent retinal artery just beyond the crossing (darkfield illumination, X30). (B) Section taken through the region indicated by the solid curved arrow in A showing the connection between the parent vein (v) and the sea fan draining venule (solid curved arrow) at the point where the vein crosses the underlying artery (a). Arrowheads show a break in the internal limiting membrane (periodic acid-Schiff and hematoxylin, X380). (C) Section taken from the region indicated by the open curved arrow in A showing the connection between the parent artery (a) and the sea fan feeding arteriole (open curved arrow) through a break in the internal limiting membrane (arrowheads) (periodic acid-Schiff and hematoxylin, X380). (D) Section from the peripheral edge of the sea fan demonstrates numerous large rounded nuclei and poorly developed lumen associated with an immature capillary. A leukocyte (arrow) is contained within the lumen (periodic acid-Schiff and hematoxylin, X 1,150). (E) More posteriorly within the fan, capillaries exhibited a more mature structure with a well-developed lumen, endothelial cell lining (arrowhead), and a pericyte (arrow) (periodic acid-Schiff and hematoxylin, X 1,150). (F) Occasional atrophic capillaries, which had degenerative endothelial cells (arrowhead), were observed in many of these otherwise active formations. These vessels often had red blood cell ghosts within their lumina (curved arrow) (periodic acid-Schiff and hematoxylin, X 1,150).
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FIGURE 7. Patient 3. Examples of sea fans with an atypical morphology (arrows 7A, 7C, and 7E in Figure 1). (A, C, E) Flat views of sea fans that did not display either the peripherally directed growth patterns or the well-defined intercapillary spaces associated with typical sea fan formations. These formations were generally smaller in area and appeared to be more densely vascularized (darkfield illumination, X33). (B, D, F) Cross sections from each of these formations disclosed a collagenous matrix containing numerous atrophic vessels intermingled with viable blood vessels. These formations were more elevated than typical sea fans, which tended to lie flat along the retinal surface. Some condensed vitreous was found overlying and apparently adherent to these formations (arrowheads) (toluidine blue-basic fuchsin, B and D, X230; F, X370). 468
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FIGURE 8. Patient 3. (A) Flat view of a formation peripheral to an arteriovenous crossing at the border of vascularized retina (arrow 8 in Figure 1). The adenosine diphosphatase activity in this sea fan is extremely weak and diffuse (long arrows), except near its base, where activity is unusually high in blood vessels (arrowhead). At this crossing, the vein (v) is located internal to the artery (a) (darkfield illumination, X46). (B) Section taken through the region indicated by the long arrows in A shows an elevated mass of acellular vessel remnants embedded within a collagenous matrix (arrows) (toluidine blue-basic fuchsin, X 74). (C) Section taken from the region of high activity indicated by the arrowhead in A shows capillary-like vessels proliferating within the wall of an atrophic branch of vein and internally to the retinal surface (arrowhead). Additionally, the remnants of an atrophic connection with the infarcted sea fan are present in this section (curved arrow) (toluidine blue-basic fuchsin, X140). VOL.124, No. 4
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above the retinal surface, may be arteriolar or venular; however, the former have been described only in cases of retinal breaks.14 Cogan and associates15 described a case of a vascular loop in a diabetic eye that erupted through the internal limiting membrane, extended into the vitreous, and then reverted into the retina. They considered these loops to constitute benign forms of retinal vasoproliferation. Hersh and associates12 correlated the clinical and histopathologic features of diabetic retinal venous loops and suggested that vitreous traction played a role in their pathogenesis. Our results demonstrate that vascular loops form on the retinal surface in sickle cell retinopathy possibly as a result of avulsion or extrusion of vessel segments. The exact pathogenic mechanisms respon sible for preretinal loop formation are not clear; however, we did not observe vitreous traction associ ated with vascular loops, as has been described in diabetic retinopathy. Nonetheless, vitreous was noted to be adherent to several early preretinal neovascular formations. Subsequent traction later in the clinical course may be responsible for subsequent hemorrhaging into the vitreous. Interestingly, in the very earliest specimens we have of neovascular breakthrough of the internal limiting membrane (Figure 2), vitreous adherence was not yet seen. Because the venous loop that we show (Figure 3, A) gave rise to new capillaries (Figure 3, E), the possibility exists that new preretinal vessels can proliferate from avulsed vascular segments. In this study we observed a number of sea fan formations in the vicinity of arteriovenous crossings. In fact, six of eight sea fans analyzed in this study were located at or near arteriovenous crossings (Ta ble), in addition to an early form of preretinal neovascularization (data not shown). In most cases, we found marked thickening and hyalinization of the vessel walls at crossings that gave rise to neovascular ization. It is well recognized that pathologic events occur at major retinal arteriovenous crossings in systemic diseases such as hypertension and arterio sclerosis.16 Branch retinal vein occlusion commonly occurs at an arteriovenous crossing.17 Pathologic events at arteriovenous crossings occur more proximally in these other diseases, but sickle cell retinopa thy is a disease that affects primarily the peripheral retinal vasculature. Therefore, it is quite possible that the peripheral arteriovenous crossings, like other 470
vascular components of peripheral retina, may be more susceptible to pathologic changes in sickle cell disease. It has been suggested by Seitz16 that attenua tion of the venous wall, as we observed at some crossings (Figure 5, F), may lead to discontinuation of flow under certain conditions. In markedly constrict ed tubes, fluids may experience a loss of energy and eddy currents may form. The whirl-like movement of an eddy current, which theoretically occurs at a pathologic crossing, causes vertical acceleration of fluid and redirection of flow.16 It is quite possible that such forces induced by eddy currents could drive vessels or a segment of a vessel toward the retinal surface; any attenuation of the internal limiting membrane might facilitate the growth or partial avulsion of vessels into the vitreous. We did not observe luminal compression at all crossings where sea fans formed, and it is not clear whether all of the arteriovenous crossings at these sites were native or had formed as a result of vascular remodeling. Never theless, considering how blood flow can be affected at arteriovenous crossings, the possibility exists that hemodynamic factors in combination with altered compliance of the vessel wall (through thickening and hyalinization) contribute to the eruption of blood vessels through the internal limiting membrane at these sites. An anatomic survey of proliferative dia betic retinopathy performed by Taylor and Dobree18 showed a high degree of correlation between prereti nal neovascularization and arteriovenous crossings. The structure of preretinal vessels in sea fans is similar in some ways to that which has been described in neovascular formations in other proliferative retinopathies. We found three basic cell types associated with sea fans: endothelium, pericytes, and smoothmuscle cells. We did not observe glia, pigmented cells, or myofibroblasts in any of the formations we ana lyzed. Occasional inflammatory cells were observed. In most cases, they were within the lumina of blood vessels. The structure of capillaries in sea fans varied from highly cellular, with bulky endothelium and poorly developed lumina at their advancing tips, to well developed, with thin endothelium, substantial basal lamina, and pericytes associated with vessels located more posteriorly. The actively proliferating and poorly differentiated vessels at the leading edge of sea fans (Figure 6, D) may have inadequate barrier properties, which could contribute to the leakage of
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fluorescein dye, which is typically shown in angiographic studies.1,4 Alternatively or additionally, the endothelial cells of these vessels may have fenestrae, as has been demonstrated in preretinal vessels of diabetic subjects.19'20 In either event, large sea fans are characterized angiographically by profuse leakage of fluorescein at their peripheral, more immature borders (as in the capillary in Figure 6, D), whereas older, more mature neovascular capillaries near the parent arteries and veins of the sea fans leak dye far less or not at all. The lack of leakage from the latter capillaries, which may be comparable to the capillary shown in Figure 6, E, indicates physiologic or anatom ic development of barrier properties that are expected from the normal blood-retinal barrier. Spontaneous regression (autoinfarction) of neovascularization is well documented in proliferative sickle cell retinopathy.3,21 Some of the mechanisms pro posed for autoinfarction of sea fans include occlusion of feeder arterioles or vitreous traction causing hemodynamic alterations in the sea fan and its feeder vessels, resulting in sluggish blood flow and eventual occlusion.3 Our results suggest another possible mech anism for sea fan autoinfarction. Vaso-occlusions occur initially at the capillary (McLeod DS, unpub lished results, 1996) and precapillary level in the retina of sickle cell subjects. Eventually, arterioles, venules, arteries, and veins can become obstructed.8 In this study, we found evidence of preretinal capil lary occlusion and atrophy in neovascular formations, with patent feeding arterioles and draining venules. The increase in elevation and change in morphology we observed as more vessels became infarcted did not appear to be related to vitreous traction. In more advanced clinical cases, however, vitreous traction is considered to be the major cause of intravitreal hemorrhage from preretinal neovascularization. Hem orrhage was not observed in any of the sea fans in our study. The elevated position of sea fans as they autoinfarct may in some part be caused by retraction of the atrophic collagenous tubes and perivascular collagenous matrix in the relative absence of a framework of supportive tissue. In summary, this study has illustrated the life cycle of sea fans using site-specific, high-resolution histopathologic analysis. In addition to arteriovenous anas tomoses, preretinal neovascularization in sickle cell retinopathy can arise from hairpin loops in recanalVOL.124, No. 4
ized segments of peripheral retinal vessels, from preretinal vascular loops, and at arteriovenous cross ings. The initial formation appears to be loop-like instead of the fusiform buds described by Ausprunk and Folkman22 in other neovascularizations. Sea fans have multiple connections to intraretinal arteries and veins, suggesting that they arise from multiple, simul taneous angiogenic events. Autoinfarction of these formations appears to occur initially at the preretinal capillary level and may involve the same vasoocclusive processes that occur in retinal vessels. ACKNOWLEDGMENTS
The authors thank Drs W. R. Green and I. Fukushima for their critical evaluation of this manu script and Wilmer Photography for their excellent assistance.
REFERENCES 1. Goldberg MF. Retinal neovascularization in sickle cell reti nopathy. Trans Am Acad Ophthalmol Otolaryngol 1977;83: 409-431. 2. Kent D, Arya R, Aclimandos W, Bellingham A, Bird A. Screening for ophthalmic manifestations of sickle cell disease in the United Kingdom. Eye 1994;8:618-622. 3. Nagpal KC, Patrianakos D, Asdourian GK, Goldberg MF, Rabb M, Jampol LM. Spontaneous regression (autoinfarc tion) of proliferative sickle retinopathy. Am ] Ophthalmol 1975;80:885-892. 4. Raichand M, Goldberg MF, Nagpal KC, Goldbaum MH, Asdourian GK. Evolution of neovascularization in sickle cell retinopathy. Arch Ophthalmol 1977;95:1543-1552. 5. Galinos SO, Asdourian GK, Woolf MB, et al. Spontaneous remodeling of the peripheral retinal vasculature in sickling disorders. Am J Ophthalmol 1975;79:853-870. 6. Romayananda N, Goldberg MF, Green WR. Histopathology of sickle cell retinopathy. Trans Am Acad Ophthalmol Otolaryngol 1973;77:652-676. 7. Lutty GA, McLeod DS. A new technique for visualization of the human retinal vasculature. Arch Ophthalmol 1992; 110: 267-276. 8. McLeod D, Goldberg M, Lutty G. Dual perspective analysis of vascular formations in sickle cell retinopathy. Arch Ophthalmol 1993;111:1234-1245. 9. Archer DB. Retinal neovascularization. Trans Ophthal UK 1983;103:2-27. 10. Lutty G, Merges C, Crone S, McLeod D. Immunohistochemical insights into sickle cell retinopathy. Curr Eye Res 1994;13:125-138. 11. Foos RW. Vitreoretinal junction over retinal vessels. Albrecht v Graefes Arch Klin Exp Ophthal 1977;204:223-23412. Hersh PS, Green WR, Thomas JV. Tractional venous loops in diabetic retinopathy. Am J Ophthalmol 1981;92:661-671.
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13. Vine AK. Avulsed retinal veins without retinal breaks. Am ] Ophthalmol 1984;98:723-727. 14. Robertson DM, Curtin VT, Norton EW. Avulsed retinal vessels with retinal breaks: a cause of recurrent vitreous hemorrhage. Arch Ophthalmol 1971;85:669-672. 15. Cogan DG, Trussaint D, Kuwabara T. Retinal vascular patterns, IV: diabetic retinopathy. Arch Ophthalmol 1961; 66:366-378. 16. Seitz R. The retinal vessels. Saint Louis: Mosby, 1964:20-74. 17. Wise GN, Dollery CT, Henkind P. The retinal circulation. New York: Harper and Row, 1971:391. 18. Taylor E, Dobree JH. Proliferative diabetic retinopathy. Site and size of lesions. Br ] Ophthalmol 1970;54:11-18.
19. Taniguchi Y. Ultrastructure of newly formed blood vessels in diabetic retinopathy. Jap J Ophthalmol 1976;20:19-31. 20. Wallow IHL, Geldner PS. Endothelial fenestrae in prolifera tive diabetic retinopathy. Invest Ophthalmol Vis Sci 1980;19: 1176-1183. 21. Condon PI, Serjeant GR. Behaviour of untreated prolifera tive sickle retinopathy. Br ] Ophthalmol 1980;64:404-411. 22. Ausprunk DH, Folkman J. Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumor angiogenesis. Microvasc Res 1977;14: 53-65.
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472
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• PURPOSE: To examine the histopathologic and morphometric features of neovascular lesions in human proliferative sickle cell retinopathy. • METHODS: Postmortem ocular tissue was ob tained from three subjects (aged 20, 28, and 40 years) with SS hemoglobinopathy and prepared for adenosine diphosphatase flat-embedding. Mor phometric analysis was performed before serial sectioning. • RESULTS: Numerous active and autoinfarcted lesions were found that represented virtually all stages in the life cycle of preretinal neovasculariza tion. These formations ranged from single small loops extending from arteries and veins along the retinal surface to the typical complex, elevated sea fan formations. Sea fans developed at hairpin loops and at arteriovenous crossings. There was an average of 5.6 connections between sea fans and retinal vessels; of these, 45% were arteriolar, 52.5% were venular, and 2.6% were at the capil lary level. Six of eight sea fans were located at arteriovenous crossings. Autoinfarction appeared
Accepted for publication June 24, 1997. From the Wilmer Ophthalmological Institute, Johns Hopkins Universi ty School of Medicine, Baltimore, Maryland. This work was funded by NIH grants HL45922 (Dr Lutty) and EY01765 (Wilmer Institute), and by an unrestricted research grant from Research to Prevent Blindness, Inc, New York, New York. Dr Lutty is an American Heart Association Established Investigator. Reprint requests to Gerard A. Lutty, PhD, 170 Woods Research Building, Johns Hopkins Hospital, 600 N Wolfe St, Baltimore, MD 21287-9115; fax: (410) 955-3447; e-mail: galutty@welchlink. welch.jhu.edu
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to occur initially within the sea fan capillaries. The average height of sea fans was 123 (Am above the retinal surface. • CONCLUSIONS: Preretinal neovascularization in sickle cell retinopathy can arise from both the arterial and venous sides of the retinal vasculature and can assume a variety of morphologic configu rations. Multiple feeding arterioles and draining venules are common, and autoinfarction appears to occur initially at the preretinal capillary level rather than at feeding arterioles. Arteriovenous crossings may be a preferential site for sea fan development.
S
ICKLE CELL DISEASE IS CAUSED BY A POINT MUTAtion in the hemoglobin gene. Deoxygenation or acidic conditions cause the hemoglobin mole cules to polymerize, making the sickle erythrocytes less pliable and more likely to assume unusual shapes. This increases blood viscosity and causes the individ ual cells to function as microemboli, becoming trapped within the microcirculation.1 Peripheral vas cular occlusion, presumably caused by sickle erythro cytes, is a hallmark of sickle cell retinopathy and is the initiating event that stimulates a cascade of events including neovascularization. Peripheral retinal nonperfusion and early forms of angiogenesis occur in approximately 40% (SS, sickle cell anemia) to 80% (SC) of sickle cell subjects.2 Proliferative sickle cell retinopathy is most often associated with hemoglobin SC disease and to a much lesser extent with other hemoglobinopathies (Sb-thalassemia and SS).1 Neo-
© AMERICAN JOURNAL OF OPHTHALMOLOGY 1997;124:455-472
455
vascular formations, which usually occur in the equatorial plane, can eventually spread along the retinal surface and adopt a fan-shaped configuration, which morphologically resembles the marine inverte brate Gorgonia flabellum. With continued develop ment, sea fans may hemorrhage, be dragged off the retinal surface by adherent vitreous, or autoinfarct.3 Angiographic studies have shown that sea fans arise from arteriovenous anastomoses bordering avascular retina.14 These anastomoses form as a result of vascular remodeling after occlusive events.5 Their growth extends peripherally toward the ora serrata in an apparent attempt at revascularizing avascular and See also pp. 488-497, 506-515, and 516-520.
hypoxic retina. The most common sites for sea fan formations in the fundus are superotemporal and inferotemporal.1 Although proliferative sickle cell retinopathy has been studied extensively, clinically and angiographically, few histopathologic studies and no detailed morphometric analyses have been per formed on sea fans or other neovascular lesions in sickle cell subjects.6 In this study, we used adenosine diphosphatase (ADPase) flat-embedded retinas7 from sickle cell subjects to perform site-specific analysis of neovascular formations. The earliest forms of neovascularization, various types of active sea fans, and partially or totally autoinfarcted formations were all critically examined; they appeared to represent virtu ally all stages in the life cycle of preretinal neovascularization in sickle cell retinopathy.
METHODS
were aged 20 (Patient 1), 28 (Patient 2), and 40 (Patient 3) years. Medical histories showed each to have SS hemoglobinopathy. Eyes from each subject were opened 1 cm posterior to the limbus and examined macroscopically before processing. One eye of each subject was fixed, cryopreserved, and frozen for immunohistochemical analysis that will be report ed elsewhere. The retinas from the three fellow eyes were teased from the retinal pigment epithelium, fixed, washed, and processed for ADPase histochemical staining of the retinal vasculature, as previously described.7 The ADPase-incubated retinas were cut into piec es, flattened, postfixed, and embedded in JB-4 plastic, as described in detail previously.7 Using a Zeiss Photomicroscope II (Oberkochen, Germany) with darkfield illumination to visualize the lead phosphate reaction product, we photographically documented blocks of retina and obtained digitized images from a video camera attached to the microscope. Computerassisted morphometric analysis was performed using a Macintosh Ilci personal computer with image analysis software (National Institutes of Health Image soft ware, version 1.47). Serial cross sections (2.5 |xm thick) were cut on an ultramicrotome fitted with a video camera and macroscopic lens system, which were interfaced with the computer. This allowed us to visualize and document the vasculature en bloc during sectioning. Images of the blocks were obtained every 150 |xm of tissue sectioned and provided precise correlation of section location within the vasculature. For the purpose of being consistent, in this study we will refer to intraretinal parent vessels that give rise to neovascularization as arteries and veins, based on their vascular hierarchy. Vessels that traverse the internal limiting membrane to feed or drain preretinal neovascularization will be referred to as arterioles and venules, based on their luminal diameters (Table).
HUMAN POSTMORTEM EYES WERE OBTAINED FROM THE
Medical Eye Bank of Maryland, the Medical Eye Bank of Florida, and the Mid-America Eye and Tissue Bank of St Louis, Missouri. The Johns Hop kins University School of Medicine Human Subjects Board approved the use of human tissue for this study. The cause of death, time of death, death to enucleation time (elapsed time from time of death to enucleation and storage at 4 C), and brief medical history were provided for each donor. The donors 456
RESULTS • CASE l: A 20-year-old woman died from cardiac arrest. Examination of the retinal vasculature dis closed mild peripheral vaso-occlusive disease and early forms of neovascularization bordering a narrow field of avascular peripheral retina. These changes
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TABLE. Sea Fans From Patients 2 and 3
Sea Fan
Area (mm2)/ Maximum Height
No.
Above Retina (|xm)
1
1.01/70
2 3 4 5 6 7 8
3.43/100 0.96/150 4.62/75 0.28/115 1.43/160 0.38/200 3.05/113
Viable
Viable
Viable
Art Connections (luminal diameter)
Ven Connections (luminal diameter)
Cap Connections (luminal diameter)
Total Viable Connections
Location
0
5
A-V crossing
0
5
Hairpin loop
0
3
A-V crossing
0
9
A-V crossing
0
6
Hairpin loop
1 (6.4) 0 0
7
A-V crossing
0 4
A-V crossing A-V crossing
1 (11.9) 3 (17.1 ± 5.6)* 1 (13.3) 5 (8.5 ± 4.2)* 2 (10.3 ± 3.1)* 3 (13.0 ± 7.2)* 0 2 (16.2 ± 3.3)*
4 (16.5 ± 2 (21.7 ± 2 (19.2 ± 4 (17.1 ± 4 (8.8 ± 3 (12.8 ± 0 2 (29.3 ±
7.8)* 6.9)* 4.5)* 9.6)* 2.2)* 5.7)*
6.7)*
Art = arterial; Ven = venous; Cap = capillary; A-V == arteriovenous. *Mean luminal diameter ±SD.
were confined to the superotemporal quadrant. The neovascular lesions were primarily intraretinal and consisted of corkscrew-like configurations within fields of dilated capillaries. • CASE 2: A 28-year-old man died from cardiac arrest. He was septic at the time of death. The peripheral retinal vasculature demonstrated vascular obstructions, capillary dropout, and vascular remod eling secondary to vaso-occlusive disease. Angiopathic changes were mostly confined to the far periph ery in the superior and temporal quadrants. A sea fan formation was present at approximately the 11-o'clock position of the left eye. • CASE 3: Extensive occlusive and proliferative retinopathy was observed in a 40-year-old woman who died from respiratory arrest. The entire circumference of the retina demonstrated loss of vasculature, which was most pronounced in the superior quadrant. Neovascular formations were observed at the border of vascularized peripheral retina in the equatorial plane of the superotemporal and superonasal regions from the 10-o'clock to the 2-o'clock positions (Figure 1) of the right eye. Some formations were relatively small and consisted primarily of large-caliber vessels VOL.124, No. 4
forming loop-like structures (Figure 1, arrows 3), whereas others demonstrated a typical sea fan appear ance (Figure 1, arrows 4 through 6). Some sea fans appeared to be quite flat and spread out along the retinal surface. Others appeared to be more con densed and elevated (Figure 1, arrows 7A, 7C, and 7E), with ill-defined intercapillary spaces. One for mation appeared to be degenerative based on its weak ADPase staining intensity and morphologic appear ance (Figure 1, arrow 8). In some areas in Patient 1, vascular buds with unusually high ADPase activity were observed ex tending toward the vitreous from hairpin loop forma tions within recanalized segments of the terminal portions of occluded vessels (Figure 2, A). At low magnification, budding appeared to be associated with the venous side of the circulation; however, on closer examination, these buds appeared as loops, originating from both the arterial and venous compo nents of remodeled vasculature (Figure 2, B). Capil laries at the border of perfused-nonperfused retina appeared to be dilated, and morphometric measure ments disclosed capillary diameters to be twofold greater in this region than in more posterior locations (16.35 ± 2.3 |o,m vs 8.7 ± 0.7 |xm, respectively). Cross-sectional analysis of the loop-like formations
NEOVASCULARIZATION IN SICKLE CELL RETINOPATHY
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\
7C
7E
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FIGURE 1. Patient 3. Superior retina showing numerous neovascular formations bordering the avascular retina. Numbered arrows correspond to regions shown in subsequent figures (see Case 3 in Results) (brightfield illumination, X4.5).
(Figure 2, C through E) showed dilated vessels containing packed erythrocytes extending toward the vitreous cavity. Examination of the tips of these vessels disclosed endothelial cell hyperplasia. The unusually high ADPase activity observed in these formations in the flat perspective was indicative of endothelial cell proliferation in these vessel segments, as we have documented in intraretinal microvascular abnormality formations within diabetic retina.7 In some cases, single cells were observed projecting through small defects in the internal limiting mem brane (Figure 2, C). No abnormalities of the vitreous were evident at these sites. Analysis of the small loop-like formations in Pa tient 3, (Figure 1, arrows 3) disclosed simultaneous and independent arterial (a) and venous (v) prolifer ations (Figure 3). In the flat view (Figure 3, A), the formations appeared as loop-like structures on the internal surface of the retina that formed within a viable segment of the vein (Figure 3, A, arrows D and E) and at the terminal portion of an adjacent artery (Figure 3, A, arrows B and C). The arterial lesion bordered the avascular retina, and the venous lesion was associated with a relatively small area of capillary dropout. Sections taken through the arterial lesion
458
demonstrated that the neovascular formation was located on the retinal surface just proximal to a hairpin loop within the terminal portion of the arterial wall (Figure 3, B). The vessels in this forma tion had large lumina, thicker walls than the venous lesion had, and smooth muscle cells, mirroring the structure of the parent artery (Figure 3, C). The new vessels showed apparent adhesion to the cortical vitreous (Figure 3, B, C, and D, curved arrows). Sections collected through the venous loop formation showed the preretinal location of the vessels (Figure 3, D) and communications with the parent vein (not shown) through a discontinuous internal limiting membrane. The majority of preretinal vessels had large lumina with relatively thin walls and consisted of endothelial cell linings with scattered pericytes (Figure 3, D). These large, thin-walled vessels, which appeared to be an extruded segment of the parent vein, gave rise to capillaries (Figure 3, E) that had numerous bulky endothelial cell nuclei and relatively thin basement membranes. In this study, we critically examined eight sea fans from two subjects (seven from Patient 3, one from Patient 2). A summary of the histopathologic features of these formations is shown in the Table. Serial
AMERICAN JOURNAL OF OPHTHALMOLOGY
OCTOBER 1997
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FIGURE 2. Patient 1. Border of vascularized retina as shown in flat view (top, A and B) and in histologic sections (bottom, C through E). (A) Neovascular buds (arrowheads) are present at the terminal ends of viable vascuiature in this field from the superotemporal retina. These buds appear to be related to the venous (v) and not the arterial (a) side of the circulation (darkfield illumination, X40). (B) At higher magnification, the arterial (a) and venular (v) components of the remodeled vascuiature terminate in hairpin loops (curved arrow). Neovascular buds (arrowheads) arise just posterior to the terminal ends of vascuiature. Regarding loop formation E, see the panel E legend below (darkfield illumination, X130). (C, D) Histologic sections from formations in A and B show vessels extending toward the vitreous. A single cell is shown projecting onto the inner retinal surface (arrow) (periodic acid-Schiff and hematoxylin, X650). (E) Section taken through the twisted loop formation (arrow E in B, top right) (periodic acid-Schiff and hematoxylin, X650).
sections were analyzed to determine the number, distribution, and luminal diameters of feeding arterioles and draining venules for each preretinal lesion (more than 8,000 serial sections were examined). The average area of the sea fans in this study was 2.1 ± 1.6 mm2, and the average height was 123 ± 44.5 (xm above the retinal surface. The number of viable connections between retinal vessels and sea fans averaged 5.6 ± 2.0 per formation. Of these connec tions, 52.4% were venular, 45.0% were arteriolar, and
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2.6% were at the capillary level. The average luminal diameters were 12.7 ± 5.0 |xm for the arteriolar connections and 17.9 ± 7.4 |xm for the venular connections. The largest feeding arteriole had a luminal diameter of 22.53 (xm, and the largest draining venule had a luminal diameter of 26.23 u,m. The distribution of feeders and drainers in sea fans varied; however, the largest were usually located in the central region of the neovascular formation. In some cases, the same intraretinal vessel gave rise to
NEOVASCULARIZATION IN SICKLE CELL RETINOPATHY
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multiple connections with the fan. Smaller connec tions were located at the fringes of the sea fans and were generally connected to different intraretinal arteries and veins than were the centrally located connections. We observed typical sea fan formations at two types of anatomic locations within the peripheral vasculature: hairpin loops and arteriovenous crossings (Table). Figure 4 shows a sea fan from Patient 3 that has two connections that originated from a hairpin loop within a recanalized segment of an occluded artery (Figure 4, A and B, between the opposing arrows). These connections (Figure 4, C through F), which had luminal diameters of 22.42 \xm and 11.7 |JLm, were two of four feeders that originated from different segments of the same parent artery (Figure 4, A, straight arrows). This sea fan was 3.43 mm 2 in area and relatively flat, with a maximum height that was 100 |xm above the retinal surface (Table, sea fan 2). In addition to the four arteriolar connections, this formation also had two connections to the venous side of the circulation. All feeding and draining connections were considered patent because of their apparently viable endothelial cells and unobstructed lumina, which in some cases contained scattered erythrocytes and leukocytes. The majority of blood vessels within this neovascular formation were viable and consisted of arterioles, venules, and capillaries with walls of varying thickness. They were surround ed by a collagen-like matrix, and vitreous condensa tion was evident, as shown by darkfield microscopy. Generally, these preretinal vessels contained an en dothelial cell lining with smooth muscle cells associ ated with arterioles; pericytes were associated with
venules and capillaries. Some vessels appeared to be in various stages of degeneration (few apparently viable endothelial cell nuclei), whereas others were completely atrophic (no endothelial cell nuclei). No glial cells were apparent. A few inflammatory cells were observed in and around the vessels. In addition to originating from hairpin loops, most sea fans originated from sites at arteriovenous cross ings. In fact, five of the seven active fans and one autoinfarcted sea fan that we analyzed were located at arteriovenous crossings (two of the remaining three sea fans in Patient 3 also occurred at arteriovenous crossings). Figure 5 shows examples of sea fans from Patient 2 (A through C) and Patient 3 (D through F) that were located at arteriovenous crossings where the artery (Figure 5, A through C, arrow a) was situated internal to the vein (Figure 5, A through C, arrow v). The formation from Patient 2 (Figure 5, A through C) was 1.01 mm 2 in area, was virtually flat along the retinal surface, had one connection to an artery (Figure 5, B and C), and had four connections to a vein (Table, sea fan 1). No atrophic preretinal vessels or connections were observed in this sea fan. The feeding arteriole (Figure 5, B and C, curved arrow) broke through the internal membrane exactly at the arteriovenous crossing, where it formed a preretinal loop, and then extended peripherally into the fan (Figure 5, B and C). The feeding arteriole had a luminal diameter of 11.9 (xm. In this example, the underlying vein (Figure 5, A through C, arrow v) did not dip down or show any compression (Figure 5, C). Three of the venous connections in this formation occurred before the arteriovenous crossing. Again, preretinal vessels were found to have ample contrac-
FIGURE 3. Patient 3. (A) Preretinal loops (arrows D&E and B&C) originate from the terminal portion of an artery (a) and a straight portion of a vein (v) in this flat view. This region of retina is indicated by arrows 3 in Figure 1 (darkfield illumination, X38). (B) Section taken through the terminal portion of the artery (B&C in A, top) showing a hairpin loop formation within the hyalinized arterial wall (straight arrows). Note the apparent adherent vitreous (curved arrow) associated with the preretinal vessels (periodic acid-Schiff and hematoxylin, X450). (C) Section taken through the region indicated by arrow B&C in A showing an intraretinal segment of vessel projecting through a defect in the internal limiting membrane (arrowheads). The preretinal loops appear to have the same smooth-muscle cell component (straight arrows) as the parent artery has. Note the adherent vitreous (curved arrow) (periodic acid-Schiff and hematoxylin, X450). (D) Section taken through the region indicated by arrows D&E in A shows the preretinal disposition of the venous loop (straight arrow), adherent cortical vitreous (curved arrow), and a break in the internal limiting membrane (arrowheads) (periodic acid-Schiff and hematoxylin, X300). (E) Section taken through the region indicated by D&E in A showing capillaries (arrows) arising from the venous loop (periodic acid-Schiff and hematoxylin, X300).
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tile elements (pericytes and smooth muscle cells), and no glial cells or fibroblastic-like cells were observed. Darkfield microscopy showed vitreous condensation around the anterior portion of this sea fan, but no tractional fibers were evident. Many inflammatory cells were present in and around the preretinal vessels; this may have been related to the septic condition of this subject at the time of death. Another example of a sea fan located at this type of crossing (artery situated internal to the vein) is shown in Figure 5, D through F. This formation from Patient 3 was 4.62 mm 2 in area, relatively flat, had five connections to the arterial side of the circulation (four from different segments of the same artery), and four connections (three from the same vein) to the venous side (Table, sea fan 4). No atrophic feeders or drainers were observed in this formation; however, some atrophic preretinal vessels were found. The structure of preretinal vessels was similar to that described above. In contrast to the crossing shown in Figure 5, A through C, the underlying vein (Figure 5, D through F, arrow v), rather than the artery, broke through the internal limiting membrane and exited the retina just peripheral to this crossing point (Figure 5, F). Some compression of the vein was evident at the crossing. One half of the sea fans located at arteriovenous crossings had the vein internal to the artery. Figure 6 shows a sea fan that was 3.05 mm 2 in area and had two arterial and two venous connections (Table, sea fan 8). In this case, the major draining venule for the formation exited the retina at the crossing point (Figure 6, B), and the main feeder vessel exited the retina just after the crossing (Figure 6, C). In this sea fan, virtually all the morphologic stages in the
apparent life cycle of preretinal capillaries were repre sented (Figure 6, D through F). The preretinal vessels lay in close proximity to the cortical vitreous (Figure 6, B and C). Figure 6, D shows a section through a capillary at the anterior edge of the sea fan. The capillary, which is highly cellular, has unusually large and rounded endothelial cell nuclei and is poorly differentiated in terms of its structure. The lumen contains a leukocyte (Figure 6, D, arrow). In more posterior locations, capillaries were well differentiated and had a normal endothelial cell lining and pericyte component (Figure 6, E, arrow). Scattered through out the sea fan were capillaries that appeared to be degenerative, lacking endothelial cell and pericyte nuclei. These capillaries often contained aggregates of erythrocyte ghosts (Figure 6, F). We observed a subset of sea fans in Patient 3 that were remarkable because they appeared to be different morphologically from the typical flat fans that had peripherally directed growth patterns and welldefined intercapillary spaces (shown in Figures 4, 5, and 6). These atypical fans were smaller in area (Table, sea fans 3, 5, and 6), did not have a true fan-like configuration, appeared to be denser in terms of their vascularity, and had ill-defined intercapillary spaces (Figure 7, A, C, and E). Cross-sectional analysis of these formations disclosed that each had many infarcted preretinal channels embedded within a dense collagenous matrix (as determined with van Gieson stain) and a more mounded profile than did the typical flat sea fans (Figure 7, B, D, and F). The infarcted vessels were thick walled and acellular. They were intermingled with viable blood vessels, some of which demonstrated varying degrees of degeneration. Some of these vessels had lumina that were congested
FIGURE 4. Patient 3. Example of a sea fan (arrow 4 in Figure 1) that features connections to a hairpin loop in the terminal portion of a retinal artery. (A) Flat view of the sea fan showing the locations of all feeding arterioles (straight arrows) and draining venules (curved arrows) (darkfield illumination, X25). (B) Higher magnification view of the region between the opposing straight arrows in A, showing an arterial hairpin loop within the retina (arrows) (darkfield illumination, X63). (C) The same region as that shown in B, with the plane of focus on one of two connections between the hairpin loop and preretinal vessels (arrow) (darkfield illumination, XlOO). (D) Section showing the connection to the sea fan shown in C (arrow), a break in the internal limiting membrane (arrowheads), and the thick, hyalinized wall of the parent vessel where the hairpin loop formed (periodic acid-Schiff and hematoxylin, X380). (E) The same region as that shown in C except that the plane of focus is on a second connection between the sea fan and the hairpin loop (arrow) (darkfield illumination, X100). (F) Section showing the second connection, shown in E (arrow), a break in the internal limiting membrane (arrowheads), and the thick, hyalinized wall of the parent vessel. Note the close anatomic relationship between the new vessels and the cortical vitreous (periodic acid-Schiff and hematoxylin, X380).
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with erythrocytes, whereas others had collapsed lumina. Platelets, fibrin, and leukocytes were also observed within some vessels. All feeding arterioles and drain ing venules appeared to be viable and patent in each of these formations (not shown). Condensed, adher ent vitreous was present overlying these formations (Figure 7, D and F, arrowheads); however, no vitreous traction fibers were observed. In addition to active and partially infarcted sea fans, we analyzed an autoinfarcted formation from Patient 3 (Table, sea fan 7) that appeared to show recurrent growth of new vessels. Figure 8 shows a flat view of a neovascular formation (Figure 1, arrow 8) anterior to an arteriovenous crossing (vein internal to artery) that had little ADPase activity except at its base (Figure 8, arrowhead). Cross sections disclosed an elevated mass of atrophic vascular channels em bedded within a dense collagenous matrix (Figure 8, B). The extremely weak ADPase activity associated with this formation in the flat perspective was indica tive of degeneration of the cellular components within these vessels, which we have observed previ ously.7 A n atrophic connection to an atrophic oc cluded segment of a venous branch was identified (Figure 8, C, curved arrow). Just proximal to the autoinfarcted connection, new capillaries were ob served proliferating within the venous wall and along the retinal surface (Figure 8, C, arrowhead). This formation was interpreted as being new because it had formed at a location posterior to the atrophic connec
tion that originally drained the autoinfarcted sea fan and because it had unusually high ADPase activity, which is indicative of neovascularization.7
DISCUSSION IN THIS STUDY, WE USED THE ADPASE FLAT-EMBEDDING
technique to perform site-specific histopathologic analysis of retinal neovascularization in proliferative sickle cell retinopathy. The ADPase technique has been used previously to examine angiopathic changes in human retina. The technique uses ADPase enzyme histochemistry to visualize endothelium and smooth muscle cells of the vasculature in whole retinas.7 Because arteries have both endothelial and a smoothmuscle cell components, the arterial vessels are stained more intensely than the venular side of the circulation is. Ghost vessels, or vessels that lack these cellular components, are not labeled, whereas active neovascularization routinely demonstrates unusually high enzymatic activity, which is apparently due to endothelial cell hyperplasia. The differential staining patterns provided by this technique are therefore useful in the identification and analysis of normal and pathologic vasculature.7 Although histopathologic studies on the ocular manifestations of sickle cell disease have been performed by others, 6 those studies have dealt with a variety of lesions associated with this disease. In this study, we concentrated on one
FIGURE 5. Two examples of sea fans located at arteriovenous crossings in the retina, where the artery is situated internal to the vein. Patient 2. (A) Low-magnification view of a sea fan from the superotemporal region showing its parent artery (a) and vein (v). The arteriovenous crossing is indicated by the large curved arrow (darkfield illumination, X45). (B) A higher magnification flat view of the arteriovenous crossing shown in A. This field of view is rotated 90 degrees counterclockwise from that shown in A so that it has the same orientation as the section shown in C. In this case, the feeding arteriole appears to loop out of the retina (curved arrow) as the parent artery (a) crosses the vein (v) (darkfield illumination, X130). (C) Section taken through the crossing in B showing the connection between the intraretinal parent artery (a) and the preretinal feeding arteriole of the sea fan (curved arrow). This connection occurs at a break in the internal limiting membrane (arrowheads), where the artery crosses the vein (v) (periodic acid-Schiff and hematoxylin, X420). Patient 3. (D) Low-magnification view of a sea fan from the superior region (arrow 5 in Figure 1) showing its parent artery (a), draining vein (v), and the site at which they cross (large curved arrow) (darkfield illumination, X22). (E) Higher magnification flat view of the arteriovenous crossing in D showing the retinal vein (v), the venular connection with the sea fan (curved arrow), and the retinal artery (a). Compared with the lowmagnification view shown in D, this view has been rotated 90 degrees clockwise so that it has the same orientation as the section shown in F (darkfield illumination, X100). (F) A section through the crossing in E showing the connection between the parent vein (v) and the sea fan draining venule (curved arrow). The connection occurs at a break in the internal limiting membrane (arrowheads) near the site where the vein crosses the artery (a) (periodic acid-Schiff and hematoxylin, X300).
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aspect of sickle cell retinopathy, namely, neovascularization. Although the number of subjects used in the current study is small, our results not only confirm some of the well-recognized clinical and angiographic features of proliferative sickle cell retinopathy but also present features that have not been described previ ously: intraretinal evidence of neovascularization (Figure 2), the earliest stages of preretinal growth (Figures 3), and the earliest stages of autoinfarction (Figures 6 and 7). We have documented, therefore, examples of what we believe represent virtually all stages in the life cycle of preretinal neovascularization in sickle cell retinopathy. These include not only the earliest stages of new blood vessel growth from the retina into the vitreous and autoinfarction but also recurrence after autoinfarction (Figure 8). The fact that all stages of retinal neovascularization were seen within one eye suggests that these vascular growth and regression patterns are subject to local rather than to systemic factors. It is well recognized from angiographic data that sea fans form at arteriovenous anastomoses in the peripheral retina.1'4 These anastomotic channels form as a result of vaso-occlusions and subsequent vascular remodeling. In this study, we found sea fans at two other anatomic locations within the vasculature: hairpin loops and arteriovenous crossings. Hairpin loops within the retinal vessels of sickle cell subjects have been described.5,8 They are frequently found at the terminal ends of occluded arteries and veins. It is
thought that these structures represent recanalizations of occluded segments of vessels and that they may function to redirect blood flow posteriorly into a patent side arm channel. 8 These structures may form by neovascular processes, whereby the viable endothelium of the patent segment of vessel proliferates and parasitizes the residual basement membrane of the atrophic vessel's distal segment, as has been described by Archer. 9 The high levels of basic fibroblast growth factor that we have observed in these acellular basement membrane remnants may facilitate this process.10 Perhaps the formation of preretinal vessels from hairpin loops represents exuberant at tempts at recanalization, and their course from intra retinal to preretinal locations may be influenced by an anatomic path of least resistance. The internal limit ing membrane is extremely thin or even absent over superficial retinal vessels,11 and this anatomically weak boundary may permit this process to occur. The impressions gained from examining the form, structure, and course of vessels exiting the retina in early forms of neovascularization, as well as in more complex, typical sea fans, suggest an atypical mode of development for some of these preretinal neovascular formations. In a previous histopathologic study,8 we showed extruded or avulsed segments of large retinal veins forming preretinal venous loops in sickle cell disease. Avulsed retinal vessels have been described only occasionally in other proliferative retinopathies.12,13 The avulsed segments, which form loops
FIGURE 6. Patient 3. Example of a sea fan located at an arteriovenous crossing where the vein is located internal to the artery. (A) Low-magnification flat view of the sea fan and the arrangement of its parent artery (a) and vein (v). The major draining venule for the sea fan (solid curved arrow) makes its connection to the parent retinal vein at the arteriovenous crossing and the major feeding arteriole for the sea fan (open curved arrow) makes its connection to the parent retinal artery just beyond the crossing (darkfield illumination, X30). (B) Section taken through the region indicated by the solid curved arrow in A showing the connection between the parent vein (v) and the sea fan draining venule (solid curved arrow) at the point where the vein crosses the underlying artery (a). Arrowheads show a break in the internal limiting membrane (periodic acid-Schiff and hematoxylin, X380). (C) Section taken from the region indicated by the open curved arrow in A showing the connection between the parent artery (a) and the sea fan feeding arteriole (open curved arrow) through a break in the internal limiting membrane (arrowheads) (periodic acid-Schiff and hematoxylin, X380). (D) Section from the peripheral edge of the sea fan demonstrates numerous large rounded nuclei and poorly developed lumen associated with an immature capillary. A leukocyte (arrow) is contained within the lumen (periodic acid-Schiff and hematoxylin, X 1,150). (E) More posteriorly within the fan, capillaries exhibited a more mature structure with a well-developed lumen, endothelial cell lining (arrowhead), and a pericyte (arrow) (periodic acid-Schiff and hematoxylin, X 1,150). (F) Occasional atrophic capillaries, which had degenerative endothelial cells (arrowhead), were observed in many of these otherwise active formations. These vessels often had red blood cell ghosts within their lumina (curved arrow) (periodic acid-Schiff and hematoxylin, X 1,150).
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FIGURE 7. Patient 3. Examples of sea fans with an atypical morphology (arrows 7A, 7C, and 7E in Figure 1). (A, C, E) Flat views of sea fans that did not display either the peripherally directed growth patterns or the well-defined intercapillary spaces associated with typical sea fan formations. These formations were generally smaller in area and appeared to be more densely vascularized (darkfield illumination, X33). (B, D, F) Cross sections from each of these formations disclosed a collagenous matrix containing numerous atrophic vessels intermingled with viable blood vessels. These formations were more elevated than typical sea fans, which tended to lie flat along the retinal surface. Some condensed vitreous was found overlying and apparently adherent to these formations (arrowheads) (toluidine blue-basic fuchsin, B and D, X230; F, X370). 468
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FIGURE 8. Patient 3. (A) Flat view of a formation peripheral to an arteriovenous crossing at the border of vascularized retina (arrow 8 in Figure 1). The adenosine diphosphatase activity in this sea fan is extremely weak and diffuse (long arrows), except near its base, where activity is unusually high in blood vessels (arrowhead). At this crossing, the vein (v) is located internal to the artery (a) (darkfield illumination, X46). (B) Section taken through the region indicated by the long arrows in A shows an elevated mass of acellular vessel remnants embedded within a collagenous matrix (arrows) (toluidine blue-basic fuchsin, X 74). (C) Section taken from the region of high activity indicated by the arrowhead in A shows capillary-like vessels proliferating within the wall of an atrophic branch of vein and internally to the retinal surface (arrowhead). Additionally, the remnants of an atrophic connection with the infarcted sea fan are present in this section (curved arrow) (toluidine blue-basic fuchsin, X140). VOL.124, No. 4
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above the retinal surface, may be arteriolar or venular; however, the former have been described only in cases of retinal breaks.14 Cogan and associates15 described a case of a vascular loop in a diabetic eye that erupted through the internal limiting membrane, extended into the vitreous, and then reverted into the retina. They considered these loops to constitute benign forms of retinal vasoproliferation. Hersh and associates12 correlated the clinical and histopathologic features of diabetic retinal venous loops and suggested that vitreous traction played a role in their pathogenesis. Our results demonstrate that vascular loops form on the retinal surface in sickle cell retinopathy possibly as a result of avulsion or extrusion of vessel segments. The exact pathogenic mechanisms respon sible for preretinal loop formation are not clear; however, we did not observe vitreous traction associ ated with vascular loops, as has been described in diabetic retinopathy. Nonetheless, vitreous was noted to be adherent to several early preretinal neovascular formations. Subsequent traction later in the clinical course may be responsible for subsequent hemorrhaging into the vitreous. Interestingly, in the very earliest specimens we have of neovascular breakthrough of the internal limiting membrane (Figure 2), vitreous adherence was not yet seen. Because the venous loop that we show (Figure 3, A) gave rise to new capillaries (Figure 3, E), the possibility exists that new preretinal vessels can proliferate from avulsed vascular segments. In this study we observed a number of sea fan formations in the vicinity of arteriovenous crossings. In fact, six of eight sea fans analyzed in this study were located at or near arteriovenous crossings (Ta ble), in addition to an early form of preretinal neovascularization (data not shown). In most cases, we found marked thickening and hyalinization of the vessel walls at crossings that gave rise to neovascular ization. It is well recognized that pathologic events occur at major retinal arteriovenous crossings in systemic diseases such as hypertension and arterio sclerosis.16 Branch retinal vein occlusion commonly occurs at an arteriovenous crossing.17 Pathologic events at arteriovenous crossings occur more proximally in these other diseases, but sickle cell retinopa thy is a disease that affects primarily the peripheral retinal vasculature. Therefore, it is quite possible that the peripheral arteriovenous crossings, like other 470
vascular components of peripheral retina, may be more susceptible to pathologic changes in sickle cell disease. It has been suggested by Seitz16 that attenua tion of the venous wall, as we observed at some crossings (Figure 5, F), may lead to discontinuation of flow under certain conditions. In markedly constrict ed tubes, fluids may experience a loss of energy and eddy currents may form. The whirl-like movement of an eddy current, which theoretically occurs at a pathologic crossing, causes vertical acceleration of fluid and redirection of flow.16 It is quite possible that such forces induced by eddy currents could drive vessels or a segment of a vessel toward the retinal surface; any attenuation of the internal limiting membrane might facilitate the growth or partial avulsion of vessels into the vitreous. We did not observe luminal compression at all crossings where sea fans formed, and it is not clear whether all of the arteriovenous crossings at these sites were native or had formed as a result of vascular remodeling. Never theless, considering how blood flow can be affected at arteriovenous crossings, the possibility exists that hemodynamic factors in combination with altered compliance of the vessel wall (through thickening and hyalinization) contribute to the eruption of blood vessels through the internal limiting membrane at these sites. An anatomic survey of proliferative dia betic retinopathy performed by Taylor and Dobree18 showed a high degree of correlation between prereti nal neovascularization and arteriovenous crossings. The structure of preretinal vessels in sea fans is similar in some ways to that which has been described in neovascular formations in other proliferative retinopathies. We found three basic cell types associated with sea fans: endothelium, pericytes, and smoothmuscle cells. We did not observe glia, pigmented cells, or myofibroblasts in any of the formations we ana lyzed. Occasional inflammatory cells were observed. In most cases, they were within the lumina of blood vessels. The structure of capillaries in sea fans varied from highly cellular, with bulky endothelium and poorly developed lumina at their advancing tips, to well developed, with thin endothelium, substantial basal lamina, and pericytes associated with vessels located more posteriorly. The actively proliferating and poorly differentiated vessels at the leading edge of sea fans (Figure 6, D) may have inadequate barrier properties, which could contribute to the leakage of
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fluorescein dye, which is typically shown in angiographic studies.1,4 Alternatively or additionally, the endothelial cells of these vessels may have fenestrae, as has been demonstrated in preretinal vessels of diabetic subjects.19'20 In either event, large sea fans are characterized angiographically by profuse leakage of fluorescein at their peripheral, more immature borders (as in the capillary in Figure 6, D), whereas older, more mature neovascular capillaries near the parent arteries and veins of the sea fans leak dye far less or not at all. The lack of leakage from the latter capillaries, which may be comparable to the capillary shown in Figure 6, E, indicates physiologic or anatom ic development of barrier properties that are expected from the normal blood-retinal barrier. Spontaneous regression (autoinfarction) of neovascularization is well documented in proliferative sickle cell retinopathy.3,21 Some of the mechanisms pro posed for autoinfarction of sea fans include occlusion of feeder arterioles or vitreous traction causing hemodynamic alterations in the sea fan and its feeder vessels, resulting in sluggish blood flow and eventual occlusion.3 Our results suggest another possible mech anism for sea fan autoinfarction. Vaso-occlusions occur initially at the capillary (McLeod DS, unpub lished results, 1996) and precapillary level in the retina of sickle cell subjects. Eventually, arterioles, venules, arteries, and veins can become obstructed.8 In this study, we found evidence of preretinal capil lary occlusion and atrophy in neovascular formations, with patent feeding arterioles and draining venules. The increase in elevation and change in morphology we observed as more vessels became infarcted did not appear to be related to vitreous traction. In more advanced clinical cases, however, vitreous traction is considered to be the major cause of intravitreal hemorrhage from preretinal neovascularization. Hem orrhage was not observed in any of the sea fans in our study. The elevated position of sea fans as they autoinfarct may in some part be caused by retraction of the atrophic collagenous tubes and perivascular collagenous matrix in the relative absence of a framework of supportive tissue. In summary, this study has illustrated the life cycle of sea fans using site-specific, high-resolution histopathologic analysis. In addition to arteriovenous anas tomoses, preretinal neovascularization in sickle cell retinopathy can arise from hairpin loops in recanalVOL.124, No. 4
ized segments of peripheral retinal vessels, from preretinal vascular loops, and at arteriovenous cross ings. The initial formation appears to be loop-like instead of the fusiform buds described by Ausprunk and Folkman22 in other neovascularizations. Sea fans have multiple connections to intraretinal arteries and veins, suggesting that they arise from multiple, simul taneous angiogenic events. Autoinfarction of these formations appears to occur initially at the preretinal capillary level and may involve the same vasoocclusive processes that occur in retinal vessels. ACKNOWLEDGMENTS
The authors thank Drs W. R. Green and I. Fukushima for their critical evaluation of this manu script and Wilmer Photography for their excellent assistance.
REFERENCES 1. Goldberg MF. Retinal neovascularization in sickle cell reti nopathy. Trans Am Acad Ophthalmol Otolaryngol 1977;83: 409-431. 2. Kent D, Arya R, Aclimandos W, Bellingham A, Bird A. Screening for ophthalmic manifestations of sickle cell disease in the United Kingdom. Eye 1994;8:618-622. 3. Nagpal KC, Patrianakos D, Asdourian GK, Goldberg MF, Rabb M, Jampol LM. Spontaneous regression (autoinfarc tion) of proliferative sickle retinopathy. Am ] Ophthalmol 1975;80:885-892. 4. Raichand M, Goldberg MF, Nagpal KC, Goldbaum MH, Asdourian GK. Evolution of neovascularization in sickle cell retinopathy. Arch Ophthalmol 1977;95:1543-1552. 5. Galinos SO, Asdourian GK, Woolf MB, et al. Spontaneous remodeling of the peripheral retinal vasculature in sickling disorders. Am J Ophthalmol 1975;79:853-870. 6. Romayananda N, Goldberg MF, Green WR. Histopathology of sickle cell retinopathy. Trans Am Acad Ophthalmol Otolaryngol 1973;77:652-676. 7. Lutty GA, McLeod DS. A new technique for visualization of the human retinal vasculature. Arch Ophthalmol 1992; 110: 267-276. 8. McLeod D, Goldberg M, Lutty G. Dual perspective analysis of vascular formations in sickle cell retinopathy. Arch Ophthalmol 1993;111:1234-1245. 9. Archer DB. Retinal neovascularization. Trans Ophthal UK 1983;103:2-27. 10. Lutty G, Merges C, Crone S, McLeod D. Immunohistochemical insights into sickle cell retinopathy. Curr Eye Res 1994;13:125-138. 11. Foos RW. Vitreoretinal junction over retinal vessels. Albrecht v Graefes Arch Klin Exp Ophthal 1977;204:223-23412. Hersh PS, Green WR, Thomas JV. Tractional venous loops in diabetic retinopathy. Am J Ophthalmol 1981;92:661-671.
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13. Vine AK. Avulsed retinal veins without retinal breaks. Am ] Ophthalmol 1984;98:723-727. 14. Robertson DM, Curtin VT, Norton EW. Avulsed retinal vessels with retinal breaks: a cause of recurrent vitreous hemorrhage. Arch Ophthalmol 1971;85:669-672. 15. Cogan DG, Trussaint D, Kuwabara T. Retinal vascular patterns, IV: diabetic retinopathy. Arch Ophthalmol 1961; 66:366-378. 16. Seitz R. The retinal vessels. Saint Louis: Mosby, 1964:20-74. 17. Wise GN, Dollery CT, Henkind P. The retinal circulation. New York: Harper and Row, 1971:391. 18. Taylor E, Dobree JH. Proliferative diabetic retinopathy. Site and size of lesions. Br ] Ophthalmol 1970;54:11-18.
19. Taniguchi Y. Ultrastructure of newly formed blood vessels in diabetic retinopathy. Jap J Ophthalmol 1976;20:19-31. 20. Wallow IHL, Geldner PS. Endothelial fenestrae in prolifera tive diabetic retinopathy. Invest Ophthalmol Vis Sci 1980;19: 1176-1183. 21. Condon PI, Serjeant GR. Behaviour of untreated prolifera tive sickle retinopathy. Br ] Ophthalmol 1980;64:404-411. 22. Ausprunk DH, Folkman J. Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumor angiogenesis. Microvasc Res 1977;14: 53-65.
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