RUBEOSIS IRIDIS GENERATED BY INSULIN HYPERSENSITIVITY* BY Alan
L. Shabo, MD (BY INVITATION), David S. Maxwell, PHD (BY INVITATION), Allan E. Kreiger, MD (BY INVITATION) AND Bradley R. Straatsma, MD INTRODUCTION
RUBEOSIS IRIDIS IS A SERIOUS OCULAR COMPLICATION OF DIABETES MELLITUS. IT occurs most frequently in eyes with proliferative diabetic retinopathy and
often leads to neovascular glaucoma and blindness. 1 The occurrence of rubeosis iridis was not generally appreciated until 1928 when Salus2 first described iris neovascularization in diabetics and named the condition "rubeosis iridis diabetica." Since that time, rubeosis iridis has been identified in a wide variety of ocular diseases.3 However, the rubeosis of diabetes is the most frequent cause of neovascular glaucoma about which our knowledge of pathogenesis and therapy is severely limited.4 Hoskins stated in a recent symposium on glaucoma that "a major obstacle to studying this process is lack of an experimental animal model."4 Despite numerous case reports in the literature, there have been few successful attempts to produce rubeosis iridis in the laboratory. Moreover, the methods employed have involved profound interruption of vasculature to the globe. Schulze3 reported that iris neovascularization could only be produced in rabbits by obliterating both of the long posterior ciliary arteries. Anderson and Morin5 produced rubeosis iridis in rabbits by a similar technique that resulted in anterior segment necrosis. We have conducted a series of experiments on monkeys sensitized to exogenous beef insulin. After injections of beef insulin into the vitreous, these monkeys develop a proliferative retinopathy with many features that resemble human diabetic retinopathy.6'7 Such features include the *From the Departments of Ophthalmology and Anatomy, Jules Stein Eye Institute, U.C.L.A. School of Medicine, Los Angeles, California. Supported in part by Grants from the American Diabetes Association and Fight-For-Sight, Inc. TR. AM. OPHTH. Soc., vol. LXXIV, 1976
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preretinal proliferation of fibrovascular tissue preferentially from the optic disc, vascular tortuosity with venous "beading," intraretinal hemorrhages and exudates, and retinal traction folds with detachment. This report describes the development of rubeosis iridis in this primate model which is also generated by insulin hypersensitivity.
MATERIALS AND METHODS
Eight healthy, adult Rhesus monkeys were sensitized to crystalline beef insulin (Lilly U-100) by 1 cc footpad injections of an insulin-Freund's complete adjuvant mixture (25 units insulin/l cc total volume) at weekly intervals over a three week period. The injections into the vitreous were made under Nembutal anesthesia. The pupil was dilated with 1% Cyclogyl and a topical antibiotic (Garamycin) was applied to the globe. From 2 to 5 units of crystalline beefinsulin in 0.05 ml total volume (without adjuvant) were injected into the posterior vitreous of each eye. The injections were made under direct observation by indirect ophthalmoscopy by inserting a 25 gauge needle (attached to a tuberculin syringe) through the temporal pars plana. The animals were followed closely by slit lamp biomicroscopy and iris fluorescein angiography for periods of up to seven months after the injection. At selected time intervals, the eyes were fixed by intracardiac perfusion or immersion fixation with 2% paraformaldehyde-2% glutaraldehyde in 0.1 M sodium cacodylate (pH 7.4) for study by light and electron microscopy. In some of the animals, the ipsilateral common carotid artery was isolated prior to enucleation, and 500 mg of horseradish peroxidase (Sigma Chemical Co.-Type II) in 5 cc sterile saline was slowly injected. Thirty minutes later, the eye was removed and fixed by immersion. The next day, the tissues from these peroxidase eyes were sectioned at 75-100l, with the Smith-Farquar tissue chopper and incubated in peroxidase substrate (3-3' diaminobenzidine) according to the method of Graham and Karnovsky.8 All of the tissues were then processed by standard methods for electron microscopy including postfixation in buffered osmium tetroxide, rapid alcohol dehydration, and embedment in Araldite. As controls, a sensitized monkey was given single (one eye) or multiple (the other eye) injection of 0.05 cc of sterile saline into the vitreous. In order to test the effects of crystalline beef insulin directly on ocular tissues, another monkey which was not sensitized to insulin was given three separate injections of 10 units crystalline beef insulin into the vitreous over a ten day period.
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FIGURE 1. Anterior segment nine days after insulin injection into the vitreous of a sensitized monkey. The iris is hyperemic and blood is present on the back of the cornea and is layered inferiorly in the anterior chamber. FIGURE 2. One month after insulin injection into the vitreous, dilated and tortuous blood vessels are present on the anterior iris surface adjacent to the minor arterial circle. The acute anterior segment inflammation has resolved. FIGURE 3. One month following injection of a large amount of insulin into the vitreous, this eye developed marked acute inflammation resulting in posterior synechiae that occluded the pupil. Prominent iris bombe is demonstrated in addition to a cataract and rubeosis iridis. FIGURE 4. Seven months after injection of a large amount ofinsulin, posterior synechiae join the partially atrophic iris to the anterior capsule of the cataractous lens. Numerous neovascular profiles are visible on the anterior iris surface. The resulting iris bombe has markedly shallowed the anterior chamber (See fluorescein angiogram - Fig. 10).
RESULTS
Within three days after a single injection of insulin into the vitreous, the sensitized monkeys develop inflammation in the anterior segment.
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Shabo et al Flare and cells are present in the aqueous humor and the iris exhibits hyperemia. Over the subsequent week, blood is present within the anterior chamber and may be layered inferiorly or adherent to the corneal endothelium (Fig. 1). There is marked vascular engorgement ofthe minor arterial circle. By two weeks after the injection, dilated, tortous vessels appear on the anterior iris surface adjacent to the minor arterial circle and extend toward the pupil and the iris root (Fig. 2). Histopathologic study demonstrates a large number of vascular profiles within the iris stroma and on the anterior iris surface (Fig. 5). These vessels are surrounded by acute and chronic inflammatory cells that closely invest the vascular endothelium even in regions where the vessels project into the anterior chamber (Fig. 5). The endothelium is irregular in outline, being very thin in some regions while large nuclei project into the lumina in other regions (Fig. 5). As the anterior chamber inflammatory reaction subsides, the iris vessels become more prominent. Iris fluorescein angiography demonstrates leakage of the dye into the anterior chamber. Studies with the ultrastructural tracer horseradish peroxidase confirms the abnormal permeability of the iris vasculature. Within 30 minutes following intracarotid peroxidase injection, the dense peroxidase reaction product is observed to have penetrated the vessel endothelium, stain the vessel basal lamina, and fill the extra vascular spaces of the iris stroma (Fig. 6). Peroxidase reaction product is present as well in the endothelial intercellular spaces suggesting a lack of the peroxidase-restrictive tight junctions (zonulae occludentes) that normally are present in iris vasculature (Fig. 7). Light microscopic examination of the iris two months following insulin injection demonstrates multiple large thin-walled blood vessels that permeate the iris stroma, extend to the anterior surface and even extend into the anterior chamber in some regions (Fig. 8). At this stage, scattered acute and chronic inflammatory cells including lymphocytes and plasma cells are present within the iris stroma but perivascular cuffing is less prominent (Fig. 8). Large dilated thin-walled vessels are found in the iris root in close proximity to the trabecular meshwork (Fig. 9). These vessels are also abnormally permeable as plasma components and the protein tracer peroxidase surround these vessels and fill the chamber angle including the intercellular spaces of the trabecular meshwork. Numerous inflammatory cells and erthrocytes are also found within the trabecular meshwork. In eyes with severe inflammatory reactions, posterior synechiae form and the lenses become cataractous. Within a few weeks after the injection, pupillary occlusion results in iris bombe (Fig. 3). Peripheral corneal
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Dark peroxidase reaction product is identified in the intercellular spaces (IS) of the vascular endothelium (E) suggesting intercellular passage of peroxidase and the absense of tight junctions in these new blood vessels. Lumen (L) x 32,000.
FIGURE 8
Light micrograph of the iris two months following insulin injection into the vitreous. Numerous, large thin-walled blood vessels permeate the iris stroma and are present on the anterior sufface. Scattered inflammatory cells are within the iris stroma but little perivascular cuffing is found. Anterior chamber (AC); posterior chamber (PC) X200.
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Shabo et al vascularization occurs in addition to rubeosis iridis. Figure 4 illustrates a monkey eye seven months following insulin injection into the vitreous. A dense cataract is present and posterior synechiae occlude the pupillary aperture. The iris, which is atrophic in some regions, demonstrates a complex network ofblood vessels on the anterior surface. There is bowing forward of the iris with marked shallowing of the anterior chamber. Iris angiography demonstrates the progressive leakage of fluorescein from these abnormal vessels with pooling in the shallow anterior chamber
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The control animal which was sensitized to insulin and received saline injections into the vitreous did not demonstrate any anterior or posterior segment inflammation. Similarly, no abnormalities developed in the animal who was given three separate insulin injections (10 units each) into the vitreous. DISCUSSION
The results of this study indicate that rubeosis iridis can be produced experimentally in monkeys sensitized to exogenous insulin. Following a single intraocular injection of insulin, the induced immunogenic inflammation also results in a variety of anterior segment changes ranging from acute hyphemas to posterior synechiae with cataract formation and iris bombe. This spectrum of clinical presentations is similar to that observed in diabetics with long-standing proliferative disease. The observation that immunogenic inflammation causes rubeosis iridis is consistent with several clinical inflammatory syndromes in which it can occur. Chronic uveitis,14 radiation induced uveitis,15 Eale's disease,16 and cranial arteritis17 are a few examples. Other clinical syndromes are characterized by ischemia and necrosis with associated inflammation such as anterior segment necrosis following retinal detachment surgery"8 and carotid-arterial occlusive19'20 diseases. In the laboratory, Schulze3 attempted over a dozen different methods to produce rubeosis iridis including ligation of the optic nerve, laceration and diathermy of central retinal vessels, diathermy ofvortex veins or directly to the iris surface, and injection of autogenous blood into the vitreous. The only successful technique for producing rubeosis iridis was obliteration of both long posterior ciliary arteries which resulted in anterior segment necrosis with associated inflammation. The classical histopathologic description of rubeosis iridis involves the development of a fibro-vascular membrane on the anterior iris surface which causes ectropion uvea and covers the angle structures.'6 Our
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experiments in the monkey result in profound neovascularization throughout the iris without preferential anterior surface lesions. The differences from the classical descriptions may be due in part to the fact that the monkeys are hyperimmune to insulin and that an accelerated inflammatory response occurs after intravitreal injections of relatively large amounts of antigen. In addition, the human pathologic studies are generally from patients who have had rubeosis iridis for long periods prior to enucleation.
The fact that intraocular insulin can produce rubeosis iridis and associated anterior segment changes in sensitized monkeys raises the question of whether insulin therapy initiates or potentiates the development of similar proliferative ocular lesions in diabetics. Commercial insulin, which is obtained principally from beef and pork pancreas, is an antigenic protein. 9 Its low molecular weight and chemical structure (cyclic aromatic acids) contribute to its antigenic activity.9 Most diabetics who receive exogenous insulin develop antibodies against it." The localized inflammatory reactions at the sites of injection as well as the development of insulin resistance reflect human immunity to insulin. Futhermore, fluorescent insulin-antibody binding has been demonstrated in the basement membranes of diabetic blood vessels in the eye. 12
Based upon these considerations, it is possible to speculate that leaky diabetic blood vessels (due to background retinopathy) may also admit small amounts of exogenous insulin into the eye. This slow, repetitive leakage could initiate or potentiate the development of proliferative retinal lesions and result in low grade anterior segment inflammation that ultimately produces rubeosis iridis. Multiple individual factors such as the status of the patient's immune system, the ability to respond to exogenous protein, the development of blocking antibodies, and the type or degree of background retinopathy could influence the potential for development of proliferative ocular disease including rubeosis iridis.
Another important consideration in regard to rubeosis iridis is the status of the blood-aqueous barrier. It is interesting to note that diabetics who undergo intraocular surgery are more likely to develop postoperative rubeosis iridis. This complication has been increasingly evident recently following vitrectomy in diabetics with proliferative retinopathy. Rubeosis iridis occurs in up to 25 % of such patients. 13 It is possible that the mechanical breakdown of the blood-aqueous barrier associated with surgery could also allow increased insulin leakage into the eye with subsequent immunogenic inflammation resulting in iris neovascularization.
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SUMMARY
There are important similarities between human and experimental monkey rubeosis iridis. We believe that we have developed a useful primate model to study iris neovascularization and that the possible role of immunity to insulin in the pathogenesis of human diabetic rubeosis iridis warrants further detailed consideration.
REFERENCES
1. Madsen PH: Rubeosis of the iris and hemorrhagic glaucoma in patients with proliferative diabetic retionopathy. Br J Ophthalmol 55:368, 1971. 2. Salus R: Rubeosis iridis diabetica, eine unbekaannte diabetische Irisveranderung. Med Klin 24:248, 1928. 3. Schulze RR: Rubeosis iridis. Am J Ophthalmol 63:487, 1967. 4. Hoskins HD Jr: Neovascular glaucoma: current concepts. Trans Am Acad Ophthalmol Otolaryngmol 78:330, 1974. 5. Anderson DM, JD Morin: Experimental anterior segment necrosis and rubeosis iridis. Can J Ophthalmol 6:196, 1971. 6. Shabo AL, Maxwell DS: Insulin-induced immunogenic retinopathy resembling the retinitis proliferans of diabetes. Trans Am Acad Ophthalmol Otolaryngol. In press. 7. Shabo AL, Maxwell DS, Straatsma BR: Experimental immunogenic retinopathy resembling proliferative diabetic retinopathy. III. Symposium on the Structure of the Eye, Tokyo. In press. 8. Graham RC Jr, Karnovsky MJ: The early absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistristry by a new technique. J Histochem Cytochem 14:291, 1966. 9. Kniker WT: Insulin and its immunologic reactions. In Vascular Complications of Diabetes Mellitus, Kimura and Caygill (eds), St Louis, CV Mosby, 1967, p 184. 10. Kinker WT: Immunogenic vasculitis: is it part of diabetes mellitus? In Vascular Complications of Diabetes Mellitus, Kimura and Caygill (eds), St Louis, CV Mosby, 1967, p 201. 11. Grodsky GM: Production of acute antibodies to insulin in man and rabbits. Diabetes 14:396, 1965. 12. Coleman SC, Becker B, Canaan S, Rosenbaum L: Fluorescent insulin staining of the diabetic eye. Diabetes 11:375, 1962. 13. Machemer R: Symposium on pars plana vitrectomy. Trans Am Acad Ophthalmol Otolaryngol. In press. 14. Marvas J: Biomicroscopic de la Chambre Anterieurre de L'iris et du Corps Ciliare, Paris, Masson, 1928, p 10. 15. Jones RF: Glaucoma following radiotherapy. Br J Ophthalmol 42:636, 1958. 16. Wise GN, Dollery CT, Henkind P: Anomalous vascular formations, neovascularization and microaneurysms. In The Retinal Circulation. New York, Harper and Row, 1971, p 276. 17. Wolter JR, Phillips LR: Secondary glaucoma in cranial arteritis. Am J Ophthalmol 59:625, 1965. 18. Wilson WA, Irvine SR: Pathological changes following disruption of blood supply to iris and ciliary body. Trans Am Acad Ophthalmol Otolaryngol 59:501, 1955. 19. Smith JL: Unilateral glaucoma in carotid occlusive disease. JAMA 182:683, 1962. 20. Swan KC, Raaf J: Changes in the eye and orbit following carotid ligation. Trans Am Ophthalmol Soc 49:435, 1952.
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DR HAROLD F. SPALTER. Mr President, Mr Secretary, members and guests. I am pleased to discuss this paper originating in the laboratories of our colleagues in Los Angeles just across the wide Pacific. The topic is timely and challenging. I am indebted to the authors for their well organized manuscript which, I received well in advence of the meeting and for this morning's lucid presentation. I am particularly honored by the Program Committee's invitation to open the formal discussion. Biologic experimental models such as developed by the authors may serve many useful purposes. Valid animal models illuminate pathogenetic mechanisms of human disease in a manner rarely afforded even by the most exhaustive clinical studies. With such models the investigator can employ his ingenuity to test our most important product as physicians, the chemotherapy and surgical approaches to disease prevention. To offer an immediate example, the experimental protocol presented today makes it possible to test methods of prophylaxis of rubeosis iridis in insulin dependent diabetics. Would the authors care to comment on the feasibility of programmed desensitization to insulin, surely a diffucult immunologic trick in man in order to prevent the complication of rubeosis? Does your experimental result lead us to conclude that the use of insulin and the management of clinical diabetes should be curtailed or, when possible, completely eliminated? Does your model not add additional support to the accumulating literature on the harmful potential of exogenous insulin in juvenile and even adult onset diabetics? As attractive as the investigators' hypothesis is, I would like to inquire of the speaker, how specific is this antigenic stimulus in the experimental production of rubeosis? Are control studies planned to evaluate the rubeosis induction potential of drugs other than insulin? Certainly rubeosis occurs in the diabetic receiving only oral hypoglycemic drugs and in the diabetic controlled by diet alone with no known exposure to exogenous insulin. Also, how do we explain the rubeosis encountered following retinal vein occlusion, anterior segment ischemia which you alluded to, and chronic uveitis where no known antigenic stimulus is operative? Another query, to be asked and hopefully to be answered by the authors' elegant model, is whether the rubeosis iridis of diabetis in man can be successfully treated and manipulated by extensive laser coagulation of the peripheral retina? One of the significant benefits of animal models is the availability of nonhuman subjects in whom new, often radical, therapeutic measures can be tested without the red tape and interminable committee delays and governmental restrictions for human clinical experimentation. Informed consent is not a problem with the Rhesus monkey! Several groups of laser enthusiasts have recently reported encouraging results with peripheral retinal ablation as a means of controlling rubeosis iridis in diabetics and retinal vein occlusions. Can we ask of the authors' model whether such results can be duplicated experimentally? The clinical challenge of a valid and reproducible animal disease model is sufficient to occupy the clinical and laboratory investigator full time. In order to accept such an exciting challenge
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we must be certain of the specificity and biologic uniqueness of that model. However, the mechanism of induction of experimental rubeosis is non-specific and can be provoked by antigens other than insulin. Bovine serum albumin alone can produce a similar sequence of events. We are in the authors' debt for providing this efficient animal workshop in which to test old, new, and yet unborn therapeutic measures in the still unsuccessful battle against one of ophthalmology's most challenging problems-rubeosis iridis. Thank you. DR GUNTER VON NOORDEN. I find the idea that insulin may contribute to the development of rubeosis iridis intriguing. To my knowledge rubeosis iridis was first reported by Salus of Prague in 1928, after the term rubeosis had been used earlier by my grandfather Carl von Noorden to describe the dilatation of facial capillaries in diabetics. It is not clear from the original publication by Salus whether two of the three patients in his report were treated with insulin. If rubeosis iridis occurs also in patients who are not on insulin, an etiological relationship to insulinrubeosis iridis would be more difficult to explain. DR WILLIAM H. SPENCER. The authors are to be congratulated on this most interesting paper. I would like to point out that as with many animal models this is truly not identical to what is seen histologically in the human iris in diabetic rubeosis or rubeosis due to any other cause. What the authors have shown appears to be a form of varicosity which occupies the stroma of the iris and protrudes toward its anterior surface. This differs from what is seen in the human iris where a fibrovascular membrane is seen which is pretty much limited to the anterior surface. In humans the difficulty that one encounters is usually with peripheral anterior synechia formation with proliferation of the connective tissue portion of the fibrovascular membrane into the chamber angle along with the formation of ectropion uvea. This apparently did not occur in Dr Shabo's animals. Posterior synechia formation is not a very common accompaniment of the usual form of rubeosis as we see it clinically and also histologically in the laboratory. I think that this is a very interesting form of vascular malformation induced by the mechanisms described by the authors but in my opinion it is not in fact a simile to the human condition. Thank you. DR WENDELL L. HUGHES. The report of this work by Dr Straatsma and associates represents a major step forward in the study of this serious condition which has resulted in the loss of so many eyes in the past. When opening of the anterior chamber becomes necessary for any reason, the prognosis has been so grave that surgery usually is put off until it is too late. The eye has been lost because of uncontrolled hemorrhage from the fragile dilated vessels on the iris that extend into the precapsular membrane to the lens. If there is an indication for opening the anterior chamber it would be advisable to be prepared for hemorrhage.
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In 1950 I reported at the International Congress of Ophthalmology in London on the use of thrombin as a means to prevent hemorrhage. The report described six eyes with rubeosis iridis, in which cataract extractions, combination procedure for cataract and glaucoma,and optical iridectomy were performed (XVI Concilium Ophthalmologicum 1950 [3ritain]). The anterior chamber was irrigated with the thrombin solution several times during surgery, with complete control in five and a minimal hemorrhage in one patient. No eyes were lost, and there were no reactions attributable to the use ofthe thrombin. As soon as the anterior chamber was entered, multiple bleeding points would appear but would be immediately stopped when the thrombin was introduced. Thrombin will convert one million times its own weight of fibrinogen to fibrin. The thrombin solution was prepared just prior to surgery, 500 units being diluted with 10 cc of saline. I was unable to find any reverence to the use of thrombin in the American literature but finally uncovered a report by Mary Savory of London (Trans Ophthalmol Soc Uk 67:323336, 1947). DR CLEMENT MCCULLOCH. Insulin was discovered for clinical use in 1922. I believe this was before the time of the paper that was quoted. The composition of commercial insulin is very well known. For the purpose of this discussion, it has two parts. One is the chemical insulin, and the other is the contaminants, such as animal proteins, which remain after purification from animal pancreas. I presume that you are using commercial insulin. If so, then your results relate either to the contaminant which is in commercial insulin or to the pure insulin itself. You have made an extremely interesting observation and there is no doubt that you need to check to decide if your findings are due to sensitivity to the insulin itself or to the contaminants in the preparation that you are using. DR FREDERICK C. BLODI. I would like to congratulate the essayists for this most important contribution to the understanding of rubeosis, and I would like to defend them against the implication that their animal model does not compare with what is going on in a patient. When Salus in Germany and von Noorden in Vienna described this condition, they emphatically called it rubeosis diabetica and differentiated it from the other rubeosis due to ischemia, central vein occlusion, etc. They made it crystal clear that what they described were dilatations and varicosities of the intrastromal vessels in the iris and not vascular overgrowth on the iris surface. This is a late stage of the disease. The essayists from Los Angeles have shown this to occur in their monkeys. This is an elegant and relevant animal model to diabetic rubeosis. DR ALAN L. SHABO. I would like to thank Doctor Spalter for his very careful and gracious review of our paper and also the other reviewers who made comments. I would like to answer some of the highlights because I know time is a factor. In terms of the question Doctor McCulloch raised "Are we sure this is a reaction to the insulin and not a contaminant?' Well, there is no ideal insulin. The insulin
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that we used was obtained from Lilly Pharmaceutical Company and is one which many diabetic patients are using on a daily basis. We were able to successfully obtain from Scandinavia (Novo Laboratories) a so-called monocomponent ("nonantigenic") insulin which is believed to be the purest available. We started a series of investigations with the monocomponent insulin and we soon became aware that this could induce the same type of pathological changes. In fact,one of the monkeys that we sensitized with the monocomponent insulin became diabetic as well. Therefore, we believe that it is the insulin and not the contaminants associated with it. In terms of Doctor Spencer's comments, I appreciate Doctor Blodi's supportive comments and I would just like to point out that when one deals with an animal model there are of course certain limitations and differences from human pathology. We have sensitized these animals and made them hyperimmune. We do not know enough at the present time to assess fully the state of immunity of human diabetic patients. We are, therefore, dealing with monkeys who are not diabetic, who are hyperimmune, who undergo an accelerated inflammatory reaction and, therefore, compress or telescope the kind of change that we have shown today into a very short period of time as opposed to the diabetic patient who suffers with his eye disease for many years. Therefore, I believe that in evaluating an animal model from an experimental pathologic viewpoint, one has to recognize the limitations but try to learn about the basic mechanisms which may apply to a clinical population. In response to Dr von Noorden's question, insulin was discovered in 1922. Whether or not the patients that Salus described in 1928 were using insulin is not known. It should be pointed out, however, that there are certainly other conditions unassociated with insulin usage where rubeosis iridis occurs. I think the basic common denominator in all of these conditions is anterior segment inflammation including the immunogenic inflammation associated with insulin. We believe that the first area to explore and the one in which we are presently involved is that related to patients undergoing vitrectomy. This form of intraocular surgery, as I mentioned, is commonly complicated by the development of rubeosis (up to 25%). Should insulin usage be curtailed or eliminated? No, of course not. Insulin is an extremely useful drug. The relative risks of stopping insulin therapy would far outweigh those of the possible complications induced by insulin. We do believe the patients who do not require insulin and who can be controlled with diet probably would be safest to be controlled with diet alone and not insulin, if at all possible. How specific is this antigenic stimulus? What about other antigens? We have done some studies on bovine serum albumin. We will soon report on proliferative retinal lesions in the posterior pole. We have not observed any rubeosis iridis with bovine serum albumin although I think it's a possibility in view of the underlying inflammatory nature of the reaction. Thank you very much for allowing us to share our experimental results with you today.
L. Shabo, MD (BY INVITATION), David S. Maxwell, PHD (BY INVITATION), Allan E. Kreiger, MD (BY INVITATION) AND Bradley R. Straatsma, MD INTRODUCTION
RUBEOSIS IRIDIS IS A SERIOUS OCULAR COMPLICATION OF DIABETES MELLITUS. IT occurs most frequently in eyes with proliferative diabetic retinopathy and
often leads to neovascular glaucoma and blindness. 1 The occurrence of rubeosis iridis was not generally appreciated until 1928 when Salus2 first described iris neovascularization in diabetics and named the condition "rubeosis iridis diabetica." Since that time, rubeosis iridis has been identified in a wide variety of ocular diseases.3 However, the rubeosis of diabetes is the most frequent cause of neovascular glaucoma about which our knowledge of pathogenesis and therapy is severely limited.4 Hoskins stated in a recent symposium on glaucoma that "a major obstacle to studying this process is lack of an experimental animal model."4 Despite numerous case reports in the literature, there have been few successful attempts to produce rubeosis iridis in the laboratory. Moreover, the methods employed have involved profound interruption of vasculature to the globe. Schulze3 reported that iris neovascularization could only be produced in rabbits by obliterating both of the long posterior ciliary arteries. Anderson and Morin5 produced rubeosis iridis in rabbits by a similar technique that resulted in anterior segment necrosis. We have conducted a series of experiments on monkeys sensitized to exogenous beef insulin. After injections of beef insulin into the vitreous, these monkeys develop a proliferative retinopathy with many features that resemble human diabetic retinopathy.6'7 Such features include the *From the Departments of Ophthalmology and Anatomy, Jules Stein Eye Institute, U.C.L.A. School of Medicine, Los Angeles, California. Supported in part by Grants from the American Diabetes Association and Fight-For-Sight, Inc. TR. AM. OPHTH. Soc., vol. LXXIV, 1976
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preretinal proliferation of fibrovascular tissue preferentially from the optic disc, vascular tortuosity with venous "beading," intraretinal hemorrhages and exudates, and retinal traction folds with detachment. This report describes the development of rubeosis iridis in this primate model which is also generated by insulin hypersensitivity.
MATERIALS AND METHODS
Eight healthy, adult Rhesus monkeys were sensitized to crystalline beef insulin (Lilly U-100) by 1 cc footpad injections of an insulin-Freund's complete adjuvant mixture (25 units insulin/l cc total volume) at weekly intervals over a three week period. The injections into the vitreous were made under Nembutal anesthesia. The pupil was dilated with 1% Cyclogyl and a topical antibiotic (Garamycin) was applied to the globe. From 2 to 5 units of crystalline beefinsulin in 0.05 ml total volume (without adjuvant) were injected into the posterior vitreous of each eye. The injections were made under direct observation by indirect ophthalmoscopy by inserting a 25 gauge needle (attached to a tuberculin syringe) through the temporal pars plana. The animals were followed closely by slit lamp biomicroscopy and iris fluorescein angiography for periods of up to seven months after the injection. At selected time intervals, the eyes were fixed by intracardiac perfusion or immersion fixation with 2% paraformaldehyde-2% glutaraldehyde in 0.1 M sodium cacodylate (pH 7.4) for study by light and electron microscopy. In some of the animals, the ipsilateral common carotid artery was isolated prior to enucleation, and 500 mg of horseradish peroxidase (Sigma Chemical Co.-Type II) in 5 cc sterile saline was slowly injected. Thirty minutes later, the eye was removed and fixed by immersion. The next day, the tissues from these peroxidase eyes were sectioned at 75-100l, with the Smith-Farquar tissue chopper and incubated in peroxidase substrate (3-3' diaminobenzidine) according to the method of Graham and Karnovsky.8 All of the tissues were then processed by standard methods for electron microscopy including postfixation in buffered osmium tetroxide, rapid alcohol dehydration, and embedment in Araldite. As controls, a sensitized monkey was given single (one eye) or multiple (the other eye) injection of 0.05 cc of sterile saline into the vitreous. In order to test the effects of crystalline beef insulin directly on ocular tissues, another monkey which was not sensitized to insulin was given three separate injections of 10 units crystalline beef insulin into the vitreous over a ten day period.
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FIGURE 1. Anterior segment nine days after insulin injection into the vitreous of a sensitized monkey. The iris is hyperemic and blood is present on the back of the cornea and is layered inferiorly in the anterior chamber. FIGURE 2. One month after insulin injection into the vitreous, dilated and tortuous blood vessels are present on the anterior iris surface adjacent to the minor arterial circle. The acute anterior segment inflammation has resolved. FIGURE 3. One month following injection of a large amount of insulin into the vitreous, this eye developed marked acute inflammation resulting in posterior synechiae that occluded the pupil. Prominent iris bombe is demonstrated in addition to a cataract and rubeosis iridis. FIGURE 4. Seven months after injection of a large amount ofinsulin, posterior synechiae join the partially atrophic iris to the anterior capsule of the cataractous lens. Numerous neovascular profiles are visible on the anterior iris surface. The resulting iris bombe has markedly shallowed the anterior chamber (See fluorescein angiogram - Fig. 10).
RESULTS
Within three days after a single injection of insulin into the vitreous, the sensitized monkeys develop inflammation in the anterior segment.
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Shabo et al Flare and cells are present in the aqueous humor and the iris exhibits hyperemia. Over the subsequent week, blood is present within the anterior chamber and may be layered inferiorly or adherent to the corneal endothelium (Fig. 1). There is marked vascular engorgement ofthe minor arterial circle. By two weeks after the injection, dilated, tortous vessels appear on the anterior iris surface adjacent to the minor arterial circle and extend toward the pupil and the iris root (Fig. 2). Histopathologic study demonstrates a large number of vascular profiles within the iris stroma and on the anterior iris surface (Fig. 5). These vessels are surrounded by acute and chronic inflammatory cells that closely invest the vascular endothelium even in regions where the vessels project into the anterior chamber (Fig. 5). The endothelium is irregular in outline, being very thin in some regions while large nuclei project into the lumina in other regions (Fig. 5). As the anterior chamber inflammatory reaction subsides, the iris vessels become more prominent. Iris fluorescein angiography demonstrates leakage of the dye into the anterior chamber. Studies with the ultrastructural tracer horseradish peroxidase confirms the abnormal permeability of the iris vasculature. Within 30 minutes following intracarotid peroxidase injection, the dense peroxidase reaction product is observed to have penetrated the vessel endothelium, stain the vessel basal lamina, and fill the extra vascular spaces of the iris stroma (Fig. 6). Peroxidase reaction product is present as well in the endothelial intercellular spaces suggesting a lack of the peroxidase-restrictive tight junctions (zonulae occludentes) that normally are present in iris vasculature (Fig. 7). Light microscopic examination of the iris two months following insulin injection demonstrates multiple large thin-walled blood vessels that permeate the iris stroma, extend to the anterior surface and even extend into the anterior chamber in some regions (Fig. 8). At this stage, scattered acute and chronic inflammatory cells including lymphocytes and plasma cells are present within the iris stroma but perivascular cuffing is less prominent (Fig. 8). Large dilated thin-walled vessels are found in the iris root in close proximity to the trabecular meshwork (Fig. 9). These vessels are also abnormally permeable as plasma components and the protein tracer peroxidase surround these vessels and fill the chamber angle including the intercellular spaces of the trabecular meshwork. Numerous inflammatory cells and erthrocytes are also found within the trabecular meshwork. In eyes with severe inflammatory reactions, posterior synechiae form and the lenses become cataractous. Within a few weeks after the injection, pupillary occlusion results in iris bombe (Fig. 3). Peripheral corneal
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Shabo et al vascularization occurs in addition to rubeosis iridis. Figure 4 illustrates a monkey eye seven months following insulin injection into the vitreous. A dense cataract is present and posterior synechiae occlude the pupillary aperture. The iris, which is atrophic in some regions, demonstrates a complex network ofblood vessels on the anterior surface. There is bowing forward of the iris with marked shallowing of the anterior chamber. Iris angiography demonstrates the progressive leakage of fluorescein from these abnormal vessels with pooling in the shallow anterior chamber
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The control animal which was sensitized to insulin and received saline injections into the vitreous did not demonstrate any anterior or posterior segment inflammation. Similarly, no abnormalities developed in the animal who was given three separate insulin injections (10 units each) into the vitreous. DISCUSSION
The results of this study indicate that rubeosis iridis can be produced experimentally in monkeys sensitized to exogenous insulin. Following a single intraocular injection of insulin, the induced immunogenic inflammation also results in a variety of anterior segment changes ranging from acute hyphemas to posterior synechiae with cataract formation and iris bombe. This spectrum of clinical presentations is similar to that observed in diabetics with long-standing proliferative disease. The observation that immunogenic inflammation causes rubeosis iridis is consistent with several clinical inflammatory syndromes in which it can occur. Chronic uveitis,14 radiation induced uveitis,15 Eale's disease,16 and cranial arteritis17 are a few examples. Other clinical syndromes are characterized by ischemia and necrosis with associated inflammation such as anterior segment necrosis following retinal detachment surgery"8 and carotid-arterial occlusive19'20 diseases. In the laboratory, Schulze3 attempted over a dozen different methods to produce rubeosis iridis including ligation of the optic nerve, laceration and diathermy of central retinal vessels, diathermy ofvortex veins or directly to the iris surface, and injection of autogenous blood into the vitreous. The only successful technique for producing rubeosis iridis was obliteration of both long posterior ciliary arteries which resulted in anterior segment necrosis with associated inflammation. The classical histopathologic description of rubeosis iridis involves the development of a fibro-vascular membrane on the anterior iris surface which causes ectropion uvea and covers the angle structures.'6 Our
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experiments in the monkey result in profound neovascularization throughout the iris without preferential anterior surface lesions. The differences from the classical descriptions may be due in part to the fact that the monkeys are hyperimmune to insulin and that an accelerated inflammatory response occurs after intravitreal injections of relatively large amounts of antigen. In addition, the human pathologic studies are generally from patients who have had rubeosis iridis for long periods prior to enucleation.
The fact that intraocular insulin can produce rubeosis iridis and associated anterior segment changes in sensitized monkeys raises the question of whether insulin therapy initiates or potentiates the development of similar proliferative ocular lesions in diabetics. Commercial insulin, which is obtained principally from beef and pork pancreas, is an antigenic protein. 9 Its low molecular weight and chemical structure (cyclic aromatic acids) contribute to its antigenic activity.9 Most diabetics who receive exogenous insulin develop antibodies against it." The localized inflammatory reactions at the sites of injection as well as the development of insulin resistance reflect human immunity to insulin. Futhermore, fluorescent insulin-antibody binding has been demonstrated in the basement membranes of diabetic blood vessels in the eye. 12
Based upon these considerations, it is possible to speculate that leaky diabetic blood vessels (due to background retinopathy) may also admit small amounts of exogenous insulin into the eye. This slow, repetitive leakage could initiate or potentiate the development of proliferative retinal lesions and result in low grade anterior segment inflammation that ultimately produces rubeosis iridis. Multiple individual factors such as the status of the patient's immune system, the ability to respond to exogenous protein, the development of blocking antibodies, and the type or degree of background retinopathy could influence the potential for development of proliferative ocular disease including rubeosis iridis.
Another important consideration in regard to rubeosis iridis is the status of the blood-aqueous barrier. It is interesting to note that diabetics who undergo intraocular surgery are more likely to develop postoperative rubeosis iridis. This complication has been increasingly evident recently following vitrectomy in diabetics with proliferative retinopathy. Rubeosis iridis occurs in up to 25 % of such patients. 13 It is possible that the mechanical breakdown of the blood-aqueous barrier associated with surgery could also allow increased insulin leakage into the eye with subsequent immunogenic inflammation resulting in iris neovascularization.
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SUMMARY
There are important similarities between human and experimental monkey rubeosis iridis. We believe that we have developed a useful primate model to study iris neovascularization and that the possible role of immunity to insulin in the pathogenesis of human diabetic rubeosis iridis warrants further detailed consideration.
REFERENCES
1. Madsen PH: Rubeosis of the iris and hemorrhagic glaucoma in patients with proliferative diabetic retionopathy. Br J Ophthalmol 55:368, 1971. 2. Salus R: Rubeosis iridis diabetica, eine unbekaannte diabetische Irisveranderung. Med Klin 24:248, 1928. 3. Schulze RR: Rubeosis iridis. Am J Ophthalmol 63:487, 1967. 4. Hoskins HD Jr: Neovascular glaucoma: current concepts. Trans Am Acad Ophthalmol Otolaryngmol 78:330, 1974. 5. Anderson DM, JD Morin: Experimental anterior segment necrosis and rubeosis iridis. Can J Ophthalmol 6:196, 1971. 6. Shabo AL, Maxwell DS: Insulin-induced immunogenic retinopathy resembling the retinitis proliferans of diabetes. Trans Am Acad Ophthalmol Otolaryngol. In press. 7. Shabo AL, Maxwell DS, Straatsma BR: Experimental immunogenic retinopathy resembling proliferative diabetic retinopathy. III. Symposium on the Structure of the Eye, Tokyo. In press. 8. Graham RC Jr, Karnovsky MJ: The early absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistristry by a new technique. J Histochem Cytochem 14:291, 1966. 9. Kniker WT: Insulin and its immunologic reactions. In Vascular Complications of Diabetes Mellitus, Kimura and Caygill (eds), St Louis, CV Mosby, 1967, p 184. 10. Kinker WT: Immunogenic vasculitis: is it part of diabetes mellitus? In Vascular Complications of Diabetes Mellitus, Kimura and Caygill (eds), St Louis, CV Mosby, 1967, p 201. 11. Grodsky GM: Production of acute antibodies to insulin in man and rabbits. Diabetes 14:396, 1965. 12. Coleman SC, Becker B, Canaan S, Rosenbaum L: Fluorescent insulin staining of the diabetic eye. Diabetes 11:375, 1962. 13. Machemer R: Symposium on pars plana vitrectomy. Trans Am Acad Ophthalmol Otolaryngol. In press. 14. Marvas J: Biomicroscopic de la Chambre Anterieurre de L'iris et du Corps Ciliare, Paris, Masson, 1928, p 10. 15. Jones RF: Glaucoma following radiotherapy. Br J Ophthalmol 42:636, 1958. 16. Wise GN, Dollery CT, Henkind P: Anomalous vascular formations, neovascularization and microaneurysms. In The Retinal Circulation. New York, Harper and Row, 1971, p 276. 17. Wolter JR, Phillips LR: Secondary glaucoma in cranial arteritis. Am J Ophthalmol 59:625, 1965. 18. Wilson WA, Irvine SR: Pathological changes following disruption of blood supply to iris and ciliary body. Trans Am Acad Ophthalmol Otolaryngol 59:501, 1955. 19. Smith JL: Unilateral glaucoma in carotid occlusive disease. JAMA 182:683, 1962. 20. Swan KC, Raaf J: Changes in the eye and orbit following carotid ligation. Trans Am Ophthalmol Soc 49:435, 1952.
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Shabo et al DISCUSSION
DR HAROLD F. SPALTER. Mr President, Mr Secretary, members and guests. I am pleased to discuss this paper originating in the laboratories of our colleagues in Los Angeles just across the wide Pacific. The topic is timely and challenging. I am indebted to the authors for their well organized manuscript which, I received well in advence of the meeting and for this morning's lucid presentation. I am particularly honored by the Program Committee's invitation to open the formal discussion. Biologic experimental models such as developed by the authors may serve many useful purposes. Valid animal models illuminate pathogenetic mechanisms of human disease in a manner rarely afforded even by the most exhaustive clinical studies. With such models the investigator can employ his ingenuity to test our most important product as physicians, the chemotherapy and surgical approaches to disease prevention. To offer an immediate example, the experimental protocol presented today makes it possible to test methods of prophylaxis of rubeosis iridis in insulin dependent diabetics. Would the authors care to comment on the feasibility of programmed desensitization to insulin, surely a diffucult immunologic trick in man in order to prevent the complication of rubeosis? Does your experimental result lead us to conclude that the use of insulin and the management of clinical diabetes should be curtailed or, when possible, completely eliminated? Does your model not add additional support to the accumulating literature on the harmful potential of exogenous insulin in juvenile and even adult onset diabetics? As attractive as the investigators' hypothesis is, I would like to inquire of the speaker, how specific is this antigenic stimulus in the experimental production of rubeosis? Are control studies planned to evaluate the rubeosis induction potential of drugs other than insulin? Certainly rubeosis occurs in the diabetic receiving only oral hypoglycemic drugs and in the diabetic controlled by diet alone with no known exposure to exogenous insulin. Also, how do we explain the rubeosis encountered following retinal vein occlusion, anterior segment ischemia which you alluded to, and chronic uveitis where no known antigenic stimulus is operative? Another query, to be asked and hopefully to be answered by the authors' elegant model, is whether the rubeosis iridis of diabetis in man can be successfully treated and manipulated by extensive laser coagulation of the peripheral retina? One of the significant benefits of animal models is the availability of nonhuman subjects in whom new, often radical, therapeutic measures can be tested without the red tape and interminable committee delays and governmental restrictions for human clinical experimentation. Informed consent is not a problem with the Rhesus monkey! Several groups of laser enthusiasts have recently reported encouraging results with peripheral retinal ablation as a means of controlling rubeosis iridis in diabetics and retinal vein occlusions. Can we ask of the authors' model whether such results can be duplicated experimentally? The clinical challenge of a valid and reproducible animal disease model is sufficient to occupy the clinical and laboratory investigator full time. In order to accept such an exciting challenge
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we must be certain of the specificity and biologic uniqueness of that model. However, the mechanism of induction of experimental rubeosis is non-specific and can be provoked by antigens other than insulin. Bovine serum albumin alone can produce a similar sequence of events. We are in the authors' debt for providing this efficient animal workshop in which to test old, new, and yet unborn therapeutic measures in the still unsuccessful battle against one of ophthalmology's most challenging problems-rubeosis iridis. Thank you. DR GUNTER VON NOORDEN. I find the idea that insulin may contribute to the development of rubeosis iridis intriguing. To my knowledge rubeosis iridis was first reported by Salus of Prague in 1928, after the term rubeosis had been used earlier by my grandfather Carl von Noorden to describe the dilatation of facial capillaries in diabetics. It is not clear from the original publication by Salus whether two of the three patients in his report were treated with insulin. If rubeosis iridis occurs also in patients who are not on insulin, an etiological relationship to insulinrubeosis iridis would be more difficult to explain. DR WILLIAM H. SPENCER. The authors are to be congratulated on this most interesting paper. I would like to point out that as with many animal models this is truly not identical to what is seen histologically in the human iris in diabetic rubeosis or rubeosis due to any other cause. What the authors have shown appears to be a form of varicosity which occupies the stroma of the iris and protrudes toward its anterior surface. This differs from what is seen in the human iris where a fibrovascular membrane is seen which is pretty much limited to the anterior surface. In humans the difficulty that one encounters is usually with peripheral anterior synechia formation with proliferation of the connective tissue portion of the fibrovascular membrane into the chamber angle along with the formation of ectropion uvea. This apparently did not occur in Dr Shabo's animals. Posterior synechia formation is not a very common accompaniment of the usual form of rubeosis as we see it clinically and also histologically in the laboratory. I think that this is a very interesting form of vascular malformation induced by the mechanisms described by the authors but in my opinion it is not in fact a simile to the human condition. Thank you. DR WENDELL L. HUGHES. The report of this work by Dr Straatsma and associates represents a major step forward in the study of this serious condition which has resulted in the loss of so many eyes in the past. When opening of the anterior chamber becomes necessary for any reason, the prognosis has been so grave that surgery usually is put off until it is too late. The eye has been lost because of uncontrolled hemorrhage from the fragile dilated vessels on the iris that extend into the precapsular membrane to the lens. If there is an indication for opening the anterior chamber it would be advisable to be prepared for hemorrhage.
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In 1950 I reported at the International Congress of Ophthalmology in London on the use of thrombin as a means to prevent hemorrhage. The report described six eyes with rubeosis iridis, in which cataract extractions, combination procedure for cataract and glaucoma,and optical iridectomy were performed (XVI Concilium Ophthalmologicum 1950 [3ritain]). The anterior chamber was irrigated with the thrombin solution several times during surgery, with complete control in five and a minimal hemorrhage in one patient. No eyes were lost, and there were no reactions attributable to the use ofthe thrombin. As soon as the anterior chamber was entered, multiple bleeding points would appear but would be immediately stopped when the thrombin was introduced. Thrombin will convert one million times its own weight of fibrinogen to fibrin. The thrombin solution was prepared just prior to surgery, 500 units being diluted with 10 cc of saline. I was unable to find any reverence to the use of thrombin in the American literature but finally uncovered a report by Mary Savory of London (Trans Ophthalmol Soc Uk 67:323336, 1947). DR CLEMENT MCCULLOCH. Insulin was discovered for clinical use in 1922. I believe this was before the time of the paper that was quoted. The composition of commercial insulin is very well known. For the purpose of this discussion, it has two parts. One is the chemical insulin, and the other is the contaminants, such as animal proteins, which remain after purification from animal pancreas. I presume that you are using commercial insulin. If so, then your results relate either to the contaminant which is in commercial insulin or to the pure insulin itself. You have made an extremely interesting observation and there is no doubt that you need to check to decide if your findings are due to sensitivity to the insulin itself or to the contaminants in the preparation that you are using. DR FREDERICK C. BLODI. I would like to congratulate the essayists for this most important contribution to the understanding of rubeosis, and I would like to defend them against the implication that their animal model does not compare with what is going on in a patient. When Salus in Germany and von Noorden in Vienna described this condition, they emphatically called it rubeosis diabetica and differentiated it from the other rubeosis due to ischemia, central vein occlusion, etc. They made it crystal clear that what they described were dilatations and varicosities of the intrastromal vessels in the iris and not vascular overgrowth on the iris surface. This is a late stage of the disease. The essayists from Los Angeles have shown this to occur in their monkeys. This is an elegant and relevant animal model to diabetic rubeosis. DR ALAN L. SHABO. I would like to thank Doctor Spalter for his very careful and gracious review of our paper and also the other reviewers who made comments. I would like to answer some of the highlights because I know time is a factor. In terms of the question Doctor McCulloch raised "Are we sure this is a reaction to the insulin and not a contaminant?' Well, there is no ideal insulin. The insulin
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that we used was obtained from Lilly Pharmaceutical Company and is one which many diabetic patients are using on a daily basis. We were able to successfully obtain from Scandinavia (Novo Laboratories) a so-called monocomponent ("nonantigenic") insulin which is believed to be the purest available. We started a series of investigations with the monocomponent insulin and we soon became aware that this could induce the same type of pathological changes. In fact,one of the monkeys that we sensitized with the monocomponent insulin became diabetic as well. Therefore, we believe that it is the insulin and not the contaminants associated with it. In terms of Doctor Spencer's comments, I appreciate Doctor Blodi's supportive comments and I would just like to point out that when one deals with an animal model there are of course certain limitations and differences from human pathology. We have sensitized these animals and made them hyperimmune. We do not know enough at the present time to assess fully the state of immunity of human diabetic patients. We are, therefore, dealing with monkeys who are not diabetic, who are hyperimmune, who undergo an accelerated inflammatory reaction and, therefore, compress or telescope the kind of change that we have shown today into a very short period of time as opposed to the diabetic patient who suffers with his eye disease for many years. Therefore, I believe that in evaluating an animal model from an experimental pathologic viewpoint, one has to recognize the limitations but try to learn about the basic mechanisms which may apply to a clinical population. In response to Dr von Noorden's question, insulin was discovered in 1922. Whether or not the patients that Salus described in 1928 were using insulin is not known. It should be pointed out, however, that there are certainly other conditions unassociated with insulin usage where rubeosis iridis occurs. I think the basic common denominator in all of these conditions is anterior segment inflammation including the immunogenic inflammation associated with insulin. We believe that the first area to explore and the one in which we are presently involved is that related to patients undergoing vitrectomy. This form of intraocular surgery, as I mentioned, is commonly complicated by the development of rubeosis (up to 25%). Should insulin usage be curtailed or eliminated? No, of course not. Insulin is an extremely useful drug. The relative risks of stopping insulin therapy would far outweigh those of the possible complications induced by insulin. We do believe the patients who do not require insulin and who can be controlled with diet probably would be safest to be controlled with diet alone and not insulin, if at all possible. How specific is this antigenic stimulus? What about other antigens? We have done some studies on bovine serum albumin. We will soon report on proliferative retinal lesions in the posterior pole. We have not observed any rubeosis iridis with bovine serum albumin although I think it's a possibility in view of the underlying inflammatory nature of the reaction. Thank you very much for allowing us to share our experimental results with you today.
