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Histopathology of Experimental Photocoagulation in the Dog Eye
HISTOPATHOLOGY O F E X P E R I M E N T A L PHOTOCOAGULATION IN T H E DOG EYE* PART III.
MICROANEURYSMLIKE FORMATIONS FOLLOWING BRANCH VEIN OCCLUSION EDWARD O K U N ,
M.D.
Saint Louis, Missouri AND ELEANOR M.
COLLINS
Bethesda, Maryland In 1951, Becker and Post 1 reported find ing venous capillary aneurysms in flat mounts of each of 39 cases of central retinal vein occlusion. They also described a technique for producing retinal vein occlusion in the cat eye by introducing a wire electrode into the eye and cauterizing in the region of the optic disc. Flat mounts of the retinal capil laries in these cases several months after oc clusion of the veins revealed microaneurysms similar to those seen in human eyes. Today photocoagulation affords a much easier and less traumatic method for occluding veins experimentally. Recently, Kuwabara and Cogan2 described a method of trypsin digestion to isolate the retinal vessels from the remainder of the retina and vitreous, thus allowing good vis ualization of the retinal vasculature with routine and special stains. Utilizing the pho tocoagulation technique for the production of venous occlusion, and the trypsin diges tion technique for the preparation of flat mounts of the retina, we proposed to investi gate the effect of branch vein occlusion on the retinal vasculature of the dog eye. MATERIALS AND METHODS
In the right eye of eight dogs, one of the two major horizontal veins which enter the disc was occluded by photocoagulation. This ♦From the Ophthalmology Branch, National In stitute of Neurological Diseases and Blindness, Na tional Institutes of Health, U. S. Public Health Service, Department of Health, Education and Welfare. This material was presented in part in exhibit form at the 110th annual meeting of the American Medical Association, New York, June 26-30, 1961, and at the 66th annual session of the American Academy of Ophthalmology and Otolaryngology, October 8-13, 1961, Chicago.
was done by first slowing the blood flow with lesions placed both proximal and distal to the planned site of occlusion. The vein was then occluded with a slightly more intense burn placed between the previous two. The changes in the coagulated vessels were fol lowed by repeated ophthalmoscopic examina tions, and in selected cases, by fundus draw ings and photographs.3 The left eye of each animal received coagulation over several of the arteries and arterioles with attempts to occlude at least one major branch. Eyes were enucleated at one hour, one day, one week, one month, four months, and eight months after attempted vein occlusion. Each eye was opened by removal of a verti cal calotte, which cut across the occluded vessel. The eyes were serially sectioned and every tenth section was stained with hematoxylin and eosin. Flat mounts were pre pared from the calottes which contained the previously occluded vessels. Calottes from the fellow eyes as well as those from 10 additional senile dog eyes which were not subjected to photocoagulation served as con trols. The trypsin digestion technique of Kuwabara and Cogan2 was used, and the flat mounts were stained with PAS and hematoxylin. RESULTS OPHTHALMOSCOPIC FINDINGS
At the spot of photocoagulation there was an immediate narrowing of the visible blood column within the vein, and marked stagna tion of blood flow within the segment of vein isolated for occlusion. Within several min utes, deep and superficial hemorrhages ap peared near the point of occlusion. Twenty40
HISTOPATHOLOGY OF PHOTOCOAGULATION four areas were next
hours postcoagulation sausage-shaped of dilation were still present, and there many more hemorrhages. Within the week the vessel became entirely
41
sheathed. One month after coagulation the occluded vessel appeared as a grayish line. Four and eight months later the vessel be came even more indistinct. Very small hem-
Fig. 1 (Okun and Collins). (A) Occluded vein one hour after coagulation. Note marked dilatation of vessel with aggregation and margination of leukocytes within fibrin clot. (Hematoxylin-eosin, X87.) (B) High-power view of vessel in (A). (Hematoxylin-eosin, χ350.)
42
EDWARD OKUN AND ELEANOR M. COLLINS
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Fig. 2 (Okun and Collins). (A) Occluded vein near photocoagulation site three months after coagula tion. (Hematoxylin-eosin, χ87.) (Β) Markedly thickened vein away from photocoagulation site three months after coagulation. (Hematoxylin-eosin, χ87.)
orrhages and/or microaneurysms could still be seen in the vicinity of the occluded ves sels. The fundus four and eight months fol lowing coagulations showed mottling of the pigmentary pattern in the region of the occluded vein.
In the fellow eyes which received coagu lation over the arteries, no immediate vascu lar change was noted. At subsequent ophthalmoscopic examinations, it was confirmed that the attempts at arterial occlusion, with but one exception, were uniformly unsuccessful.
HISTOPATHOLOGY OF PHOTOCOAGULATION
43
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'J$B**&1 "·'"""..""*"- ' ***"''* '"" ' '"- ' Fig. 3 (Okun and Collins). Occluded vein six months after coagulation. (Masson trichrome stain, X87.)
Fig. 4 (Okun and Collins). Trypsin-digested flat mount showing venous branches distal to occlusion site (proximal area of same vessel shown in cross section Figure 2-A and B). (X88.)
44
EDWARD OKUN AND ELEANOR M. COLLINS
Fig. S (Okun and Collins). Microaneurysmlike formation at site where atrophie capillary joins venule. (PAS-hematoxylin, X3S.)
Fig. 6 (Okun and Collins). Microaneurysmlike formation at site where atrophie capillary joins venule. (PAS-hematoxylin, X430.)
The course of the artery or arteriole was in some instances affected by the adjacent scar, but the vessels remained patent. Sections of the occluded vein one hour after occlusion revealed a coagulation clot, containing coagulated red blood cells, a moderate number of polymorphonuclear leu kocytes and fibrin (fig. 1-A and B ) . Several months following occlusion, the veins were markedly thickened and hypercellular, even in areas away from the photocoagulation site (fig. 2-A and B). Although complete occlu sion could be demonstrated in some eyes (fig. 3), most of the sections revealed a very small central lumen. Three to six months postphotocoagulation, both flat mounts and cross sections of the previously occluded vessels continued to show hypercellularity of the vessel wall. The flat mounts at this time contained budlike proliferations of cells arising either from nearby capillaries or directly from the hyper cellular venules (fig. 4 ) . These buds had the appearance of microaneurysms.* Extending from the domes of each of these aneurysmlike formations were atrophie capillaries (figs. 5, 6 and 7). Similar microaneurysmlike formations
were seen in the flat mounts of two of the control eyes. In each instance these were single, isolated formations, as compared to the finding of from four to 12 buds about each occluded vessel in the experimental eyes. The photocoagulated arteries and arterioles did not appear hyperplastic, as did the veins.
* Since red blood cells could not be demonstrated in any of these buds, there is the possibility that they represent solid cords of proliferating cells. Until cross section studies definitely clarify their nature, they shall be referred to as "microaneurysm like formations."
DISCUSSION
In attempting to reconstruct the mechanism of microaneurysmlike formations following branch vein occlusion, two previously un recognized findings appear to be of impor tance: 1. The hypercellularity of both the oc cluded vein and the microaneurysmlike for mations (figs. 4, 5 and 6).
Fig. 7 (Okun and Collins). Microaneurysmlike formation and fingerlike proliferating bud. (PAShematoxylin, X475).
HISTOPATHOLOGY OF PHOTOCOAGULATION 2. The location at a site where either a venule or capillary gives rise to an atrophie capillary. Since only the previously occluded veins appear hypercellular, it is reasonable to as sume that this hyperplastic response is re lated to the coagulation clot and resultant stagnation of blood flow. Whether the clot itself, or the subsequent nutritional impair ment, stimulate this response cannot be an swered from the present data. However, the proliferative reaction appears to continue long after the clot is dissolved and circula tion is re-established. The microaneurysmlike formations which are produced under these circumstances are most likely the re sult of the same process which is occurring in the vessel wall. It seems plausible that the lumen of a previously patent vessel could serve as a natural lining along which cells could proliferate. Whether the capillary atrophy occurs before or after the prolifera tive process is unknown. However, there certainly are many more atrophie capillaries than there are microaneurysmlike forma tions. It is of interest to compare these aneurysmlike formations to the aneurysms dem onstrated by this same technique in human diabetic retinopathy.5 The main difference appears to be the location of the experi mentally produced lesions exclusively on the venous side of the retinal capillary system, while the diabetic lesions are present throughout the capillary network. The more recent diabetic aneurysms are cellular, but not as cellular as those appearing with ve nous oclusion. The vessels from which dia betic aneurysms arise are also hypercellular, and many are located at the site where an atrophie capillary joins a viable one. Cogan
45
and Kuwabara note that these aneurysms point toward an acellular zone, and that there is a loss of mural cells in those capil laries which give rise to aneurysms. In the present study the mural cells are largely ob scured by the hyperplastic endothelial cells. However, when they can be identified in the vessels which give rise to aneurysmlike for mations, they appear to be diminished in number. The proliferative response to venous oc clusion could conceivably give rise to either microaneurysms or neovascular tufts depend ing upon subsequent changes. If these forma tions fail to proliferate any further and be come canalized, they would be microaneu rysms; if they continue to proliferate and become canalized, they would become neo vascular tufts. These preliminary studies are compatible with the concept that the micro aneurysms of experimental vein occlusion represent abortive attempts at neovascularization.4 They also support the concept that the aneurysms seen in vein occlusion are fundamentally similar to those seen in dia betic retinopathy, the main difference being that the former are located almost exclu sively at the venous side of the capillary net work. SUMMARY
Branch vein occlusions were produced in the dog eye by means of the Zeiss photocoagulator. Flat mounts of the trypsin-digested retinas three to eight months follow ing venous occlusion showed microaneurysm like formations. These hypercellular buds were located at the point where an atrophie capillary joined either a hyperplastic venule or capillary. 640 South Kingshighway (10). Ophthalmology Branch.
REFERENCES
1. Becker, B., and Post, L. T., Jr.: Retinal vein occlusion: Clinical and experimental observations. Am. J. Ophth., 34:677, 1951. 2. Kuwabara, T., and Cogan, D. G. : Studies on retinal vascular patterns, I. Normal architecture. AMA Arch. Ophth., 64:904, 1960. 3. Okun, E., and Collins, E. M.: Histopathology of experimental photocoagulation in the dog eye: Part I. Graded lesions, vitreous effect, and complications. Am. J. Ophth., 54:3, 1962. 4. Wise, G. N.: Retinal microaneurysms. AMA Arch. Ophth., 57:151, 1957. 5. Cogan, D. G., Toussaint, D., and Kuwabara, T.: Retinal vascular patterns, IV. Diabetic retinopathy. AMA Arch. Ophth., 67:366, 1961.
MICROANEURYSMLIKE FORMATIONS FOLLOWING BRANCH VEIN OCCLUSION EDWARD O K U N ,
M.D.
Saint Louis, Missouri AND ELEANOR M.
COLLINS
Bethesda, Maryland In 1951, Becker and Post 1 reported find ing venous capillary aneurysms in flat mounts of each of 39 cases of central retinal vein occlusion. They also described a technique for producing retinal vein occlusion in the cat eye by introducing a wire electrode into the eye and cauterizing in the region of the optic disc. Flat mounts of the retinal capil laries in these cases several months after oc clusion of the veins revealed microaneurysms similar to those seen in human eyes. Today photocoagulation affords a much easier and less traumatic method for occluding veins experimentally. Recently, Kuwabara and Cogan2 described a method of trypsin digestion to isolate the retinal vessels from the remainder of the retina and vitreous, thus allowing good vis ualization of the retinal vasculature with routine and special stains. Utilizing the pho tocoagulation technique for the production of venous occlusion, and the trypsin diges tion technique for the preparation of flat mounts of the retina, we proposed to investi gate the effect of branch vein occlusion on the retinal vasculature of the dog eye. MATERIALS AND METHODS
In the right eye of eight dogs, one of the two major horizontal veins which enter the disc was occluded by photocoagulation. This ♦From the Ophthalmology Branch, National In stitute of Neurological Diseases and Blindness, Na tional Institutes of Health, U. S. Public Health Service, Department of Health, Education and Welfare. This material was presented in part in exhibit form at the 110th annual meeting of the American Medical Association, New York, June 26-30, 1961, and at the 66th annual session of the American Academy of Ophthalmology and Otolaryngology, October 8-13, 1961, Chicago.
was done by first slowing the blood flow with lesions placed both proximal and distal to the planned site of occlusion. The vein was then occluded with a slightly more intense burn placed between the previous two. The changes in the coagulated vessels were fol lowed by repeated ophthalmoscopic examina tions, and in selected cases, by fundus draw ings and photographs.3 The left eye of each animal received coagulation over several of the arteries and arterioles with attempts to occlude at least one major branch. Eyes were enucleated at one hour, one day, one week, one month, four months, and eight months after attempted vein occlusion. Each eye was opened by removal of a verti cal calotte, which cut across the occluded vessel. The eyes were serially sectioned and every tenth section was stained with hematoxylin and eosin. Flat mounts were pre pared from the calottes which contained the previously occluded vessels. Calottes from the fellow eyes as well as those from 10 additional senile dog eyes which were not subjected to photocoagulation served as con trols. The trypsin digestion technique of Kuwabara and Cogan2 was used, and the flat mounts were stained with PAS and hematoxylin. RESULTS OPHTHALMOSCOPIC FINDINGS
At the spot of photocoagulation there was an immediate narrowing of the visible blood column within the vein, and marked stagna tion of blood flow within the segment of vein isolated for occlusion. Within several min utes, deep and superficial hemorrhages ap peared near the point of occlusion. Twenty40
HISTOPATHOLOGY OF PHOTOCOAGULATION four areas were next
hours postcoagulation sausage-shaped of dilation were still present, and there many more hemorrhages. Within the week the vessel became entirely
41
sheathed. One month after coagulation the occluded vessel appeared as a grayish line. Four and eight months later the vessel be came even more indistinct. Very small hem-
Fig. 1 (Okun and Collins). (A) Occluded vein one hour after coagulation. Note marked dilatation of vessel with aggregation and margination of leukocytes within fibrin clot. (Hematoxylin-eosin, X87.) (B) High-power view of vessel in (A). (Hematoxylin-eosin, χ350.)
42
EDWARD OKUN AND ELEANOR M. COLLINS
als*" -'?»,;;·»·· ;>
'
■
^ . . , , ^ ^ ί , ' - · -
^;·"?·
^JP^^*«
Fig. 2 (Okun and Collins). (A) Occluded vein near photocoagulation site three months after coagula tion. (Hematoxylin-eosin, χ87.) (Β) Markedly thickened vein away from photocoagulation site three months after coagulation. (Hematoxylin-eosin, χ87.)
orrhages and/or microaneurysms could still be seen in the vicinity of the occluded ves sels. The fundus four and eight months fol lowing coagulations showed mottling of the pigmentary pattern in the region of the occluded vein.
In the fellow eyes which received coagu lation over the arteries, no immediate vascu lar change was noted. At subsequent ophthalmoscopic examinations, it was confirmed that the attempts at arterial occlusion, with but one exception, were uniformly unsuccessful.
HISTOPATHOLOGY OF PHOTOCOAGULATION
43
Jg^
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ίί-*Ι
;
r
w ..; .&*"
'J$B**&1 "·'"""..""*"- ' ***"''* '"" ' '"- ' Fig. 3 (Okun and Collins). Occluded vein six months after coagulation. (Masson trichrome stain, X87.)
Fig. 4 (Okun and Collins). Trypsin-digested flat mount showing venous branches distal to occlusion site (proximal area of same vessel shown in cross section Figure 2-A and B). (X88.)
44
EDWARD OKUN AND ELEANOR M. COLLINS
Fig. S (Okun and Collins). Microaneurysmlike formation at site where atrophie capillary joins venule. (PAS-hematoxylin, X3S.)
Fig. 6 (Okun and Collins). Microaneurysmlike formation at site where atrophie capillary joins venule. (PAS-hematoxylin, X430.)
The course of the artery or arteriole was in some instances affected by the adjacent scar, but the vessels remained patent. Sections of the occluded vein one hour after occlusion revealed a coagulation clot, containing coagulated red blood cells, a moderate number of polymorphonuclear leu kocytes and fibrin (fig. 1-A and B ) . Several months following occlusion, the veins were markedly thickened and hypercellular, even in areas away from the photocoagulation site (fig. 2-A and B). Although complete occlu sion could be demonstrated in some eyes (fig. 3), most of the sections revealed a very small central lumen. Three to six months postphotocoagulation, both flat mounts and cross sections of the previously occluded vessels continued to show hypercellularity of the vessel wall. The flat mounts at this time contained budlike proliferations of cells arising either from nearby capillaries or directly from the hyper cellular venules (fig. 4 ) . These buds had the appearance of microaneurysms.* Extending from the domes of each of these aneurysmlike formations were atrophie capillaries (figs. 5, 6 and 7). Similar microaneurysmlike formations
were seen in the flat mounts of two of the control eyes. In each instance these were single, isolated formations, as compared to the finding of from four to 12 buds about each occluded vessel in the experimental eyes. The photocoagulated arteries and arterioles did not appear hyperplastic, as did the veins.
* Since red blood cells could not be demonstrated in any of these buds, there is the possibility that they represent solid cords of proliferating cells. Until cross section studies definitely clarify their nature, they shall be referred to as "microaneurysm like formations."
DISCUSSION
In attempting to reconstruct the mechanism of microaneurysmlike formations following branch vein occlusion, two previously un recognized findings appear to be of impor tance: 1. The hypercellularity of both the oc cluded vein and the microaneurysmlike for mations (figs. 4, 5 and 6).
Fig. 7 (Okun and Collins). Microaneurysmlike formation and fingerlike proliferating bud. (PAShematoxylin, X475).
HISTOPATHOLOGY OF PHOTOCOAGULATION 2. The location at a site where either a venule or capillary gives rise to an atrophie capillary. Since only the previously occluded veins appear hypercellular, it is reasonable to as sume that this hyperplastic response is re lated to the coagulation clot and resultant stagnation of blood flow. Whether the clot itself, or the subsequent nutritional impair ment, stimulate this response cannot be an swered from the present data. However, the proliferative reaction appears to continue long after the clot is dissolved and circula tion is re-established. The microaneurysmlike formations which are produced under these circumstances are most likely the re sult of the same process which is occurring in the vessel wall. It seems plausible that the lumen of a previously patent vessel could serve as a natural lining along which cells could proliferate. Whether the capillary atrophy occurs before or after the prolifera tive process is unknown. However, there certainly are many more atrophie capillaries than there are microaneurysmlike forma tions. It is of interest to compare these aneurysmlike formations to the aneurysms dem onstrated by this same technique in human diabetic retinopathy.5 The main difference appears to be the location of the experi mentally produced lesions exclusively on the venous side of the retinal capillary system, while the diabetic lesions are present throughout the capillary network. The more recent diabetic aneurysms are cellular, but not as cellular as those appearing with ve nous oclusion. The vessels from which dia betic aneurysms arise are also hypercellular, and many are located at the site where an atrophie capillary joins a viable one. Cogan
45
and Kuwabara note that these aneurysms point toward an acellular zone, and that there is a loss of mural cells in those capil laries which give rise to aneurysms. In the present study the mural cells are largely ob scured by the hyperplastic endothelial cells. However, when they can be identified in the vessels which give rise to aneurysmlike for mations, they appear to be diminished in number. The proliferative response to venous oc clusion could conceivably give rise to either microaneurysms or neovascular tufts depend ing upon subsequent changes. If these forma tions fail to proliferate any further and be come canalized, they would be microaneu rysms; if they continue to proliferate and become canalized, they would become neo vascular tufts. These preliminary studies are compatible with the concept that the micro aneurysms of experimental vein occlusion represent abortive attempts at neovascularization.4 They also support the concept that the aneurysms seen in vein occlusion are fundamentally similar to those seen in dia betic retinopathy, the main difference being that the former are located almost exclu sively at the venous side of the capillary net work. SUMMARY
Branch vein occlusions were produced in the dog eye by means of the Zeiss photocoagulator. Flat mounts of the trypsin-digested retinas three to eight months follow ing venous occlusion showed microaneurysm like formations. These hypercellular buds were located at the point where an atrophie capillary joined either a hyperplastic venule or capillary. 640 South Kingshighway (10). Ophthalmology Branch.
REFERENCES
1. Becker, B., and Post, L. T., Jr.: Retinal vein occlusion: Clinical and experimental observations. Am. J. Ophth., 34:677, 1951. 2. Kuwabara, T., and Cogan, D. G. : Studies on retinal vascular patterns, I. Normal architecture. AMA Arch. Ophth., 64:904, 1960. 3. Okun, E., and Collins, E. M.: Histopathology of experimental photocoagulation in the dog eye: Part I. Graded lesions, vitreous effect, and complications. Am. J. Ophth., 54:3, 1962. 4. Wise, G. N.: Retinal microaneurysms. AMA Arch. Ophth., 57:151, 1957. 5. Cogan, D. G., Toussaint, D., and Kuwabara, T.: Retinal vascular patterns, IV. Diabetic retinopathy. AMA Arch. Ophth., 67:366, 1961.