|Year : 1972 | Volume
| Issue : 2 | Page : 63-69
Ocular response to uveal tissue
LP Agarwal, P Singh, PK Khosla
Department of Opthalmology, A.I.I.M.S., New Delhi, India
L P Agarwal
Department of Opthalmology, A.I.I.M.S., New Delhi
Source of Support: None, Conflict of Interest: None
|How to cite this article:|
Agarwal L P, Singh P, Khosla P K. Ocular response to uveal tissue. Indian J Ophthalmol 1972;20:63-9
Sympathetic ophthalmitis has been considered to be either of viral origin or a hypersenstive response to uveal tissue. Evidences in favour of each are equally balanced. Elschnig (1910a) first suggested that sympathetic ophthalmia is an allergic response to uveal tissue. In order that this could be accepted it had to be proved that uveal tissue has antigenic properties and is organ specific. Elschnig (1910 b) showed by animal experiments that it is the uveal tissue pigment which is the fraction responsible for such a reaction and that this fraction is organ specific. His findings were supported by Kummel (1912 and 1913) who in addition demonstrated the presence of antiuveal antibodies in 7 out of 13 cases `of sympathetic ophthalmia. Wissman, (1911) further strengthened this hypothesis by producing anaphylaxis by injecting the emulsion of the diseased eye (sympathetic ophthalmitis) of the individual in guinea pigs. passively sensitized by the serum of the same patient.
Fuchs and Meller (1914) however failed to demonstrate antiuveal antibodies in the serum of these patients. Rados (1913) and von Szily (1916) failed to demonstrate the production of antiuveal antibodies in animal experiments.
This led to considerable criticism of Elschnig's (1910) allergy theory of sympathetic ophthalmia. Woods (1916) confirmed Elschnig's (1910) work and he attributed von Szily's (1916) failure to faulty technique of heating the antigen which resulted in denaturation of proteins. Woods (1921) also demonstrated complement fixing antiuveal antibodies in serum of patients with sympathetic ophthalmia.
Vannas, Wedman and Tier (1935) produced a histopathological picture resembling sympathetic ophthalmia following the intraperitoneal implantation of the homologous eye. Collins (1949) and Aronson, Rogan and Zweigart (1963 a+b) produced in animals disease resembling sympathetic ophthalmia following repeated systemic injection of uveal tissue emulsion in combination with Freunds adjuvant (complete). But certain workers like Naquin (1955), failed to produce uveal hypersensitive reaction using a technique similar to that of Collins (1949).
A brief review of the sequence of events in this field indicates that nothing conclusive has been achieved so far. Allergic hypothesis needs more conclusive support to be finally accepted.
| Materials and Methods|| |
Healthy pigmented guinea pigs weighing between 400 to 500 gms were taken for the study. Animals were kept under observation for 15 days to rule out the presence of any concurrent ocular disease. Whole uveal tissue was used as antigen, so as to include all the antigenic components in their natural form.
The antigen was prepared from fresh enucleated guinea pig eyes. Uvea was dissected, pooled, weighed and homogenised in known amount of saline using a glass homogenizer (Poter Eleveghan). The final mixture contained 50 gm (wet weight) of uvea per cc. All this procedure was carried out under aspetic conditions. Sterility of the mixture was tested by inoculation on blood. agar. Fresh antigen was prepared for each batch of injections to guard against denaturation and alteration of antigenicity during storage.
Freunds adjuvant* (complete) was used throughout the study. The antigen adjuvant mixture was prepared by mixing the two in the ratio 7:3 (Aronson et al 1963 a). The mixture was prepared each time just before injecting.
Sensitization was carried out through intra-peritoneal and subcutaneous routes by four weekly injections of the angiten. Intraoocular, intramuscular or intracardiac challenge was given 2 weeks after the last immunization injection.
The study was divided into the following groups :
Intraocular injections:- to serve as data to determine the type of ocular response to primary injection of the antigen, as controls for comparison with experiments in group II & III.
a. 0.2 cc of uveal tissue antigen - 6 animals.
b. 0.2 cc of Freund's adjuvant (complete) mixed with saline in the ratio of 3:7 - 6 animals.
c. 0.2 cc. of adjuvant-antigen mixture in the ratio of 3:7 as in b. - 6 animals.
Intraperitoneal sensitization :
by 3 injections of the antigen given intraperitoneally at weekly intervals.
a. 1 cc of the uveal tissue antigen - 9 animals.
b. 1 cc of adjuvant-saline (3:7) mixture - 3 animals.
c. 1 cc of adjuvant-antigen (3:7) 3 animals.
... Subcutaneous sensitisation :- 9 animals, by three injections of 1 cc of uveal tissue antigen given subcutaneously at weekly intervals.
The animals in each group were challenged with the uveal antigen two weeks after the last sensitizing injection in the following ways :
i. intraocular challenge 0.02 cc -3 animals.
ii. intracardiac challenge 1.00 cc - 3 animals.
iii. intramuscular challenge 1.00 cc - 3 animals.
All intraocular injections were given in the anterior chamber of the right eye.
The animals were observed for a period of 22 days following the challenge injections.
Histopathological studies of the eyes were carried out on the days of sacrificing the animals i.e. 5th, 14th and 28th days. The eyes were embedded in paraffin and the sections stained with haematoxylin and eosin.
Immunological studies of the blood and aqueous were done on the days of challenge and of sacrificing the animals. Aqueous studies on the day of challenge were limited only to those animals getting an intraocular injection. This was to avoid unnecessary trauma to the eye which might interfere with the picture of ocular response. Double diffusion in agar by the technique of Agarwal, Gupta and Madan Mohan (1963) and immunoelectrophoresis by the technique of Schiedegger (1955) were the methods employed for the study of antibody response. The antigen for immune diffusion studies was centrifuged to remove the particulate matter. It was concentrated so that protein concentration was at least 10 mg./cc.
| Observations|| |
In all the groups, animals were observed clinically for a period of 28 days. Observations were recorded under two heads - I. Clinical and histopathological, II. Immunological.
I. Clinical and Histopathological Group I (a)
1st to 5th day : All injected eyes showed inflammation, characterized by ciliary congestion, miosis and corneal oedema with mild flare but no K. P.'s. Fundus was normal. Inflammation reached its peak on the 3rd day, and started regressing by the 5th day.
Histopathology of the eye enucleated on the 5 th day showed polymorphonuclear reaction in the cornea, iris and ciliary body with pigmented particles sticking on the back of cornea. The choroid showed areas of pigment proliferation.
6th-28th day : The mild signs of inflammation which were present si.arted disappearing progressively. On the 7th day there was no sign of inflammation. The eye remained normal till the last day of observation.
Histopathology of eyes enucleated on 14th day and 28th day did not reveal any sign of inflammation. The choroid showed areas of pigment proliferation.
Group I b
1st to 5th day : All the injected eyes showed signs of inflammation in the form of ciliary congestion, miosis and corneal oedema. As the anterior chamber contained the injected material it was, therefore, not possible to comment on the presence of K.P's and aqueous flare. Fundus could not be seen. Inflammation progressively increased. There was marked increase in ciliary congestion which formed a girdle around the limbus. Corneal haziness resembled a ground glass. The anterior chamber contained in addition to the injected material tats of exudates and in one animal it had formed hypopyon. The vitreous and fundus could not be visualized. Clinical picture did not show any abatement.
Histopathology on the 5th day showed diffuse polymorphonuclear cellular infiltration of the cornea, iris and ciliary body with exudation of cells into the anterior chamber. Stromal vessels of the iris and ciliary body showed dilatation. The choroid showed areas of pigment proliferation.
6th to 15th day : The clinical picture did not show signs of decline. The cornea showed increased opacificaton, a few limbal blood vessels were seen invading superficial layers of corneal stroma. Exudation showed signs of organization forming synechea between iris and back of cornea.
Histopathological picture was essentially the same as on the 5th day except that the cornea showed sub-epithelial vascularization in addition to infiltration. The choroid, iris and ciliary body showed pigment proliferation.
16th to 28th day : The Clinical picture remained unchanged. Histopathology showed increased irregularity of corneal lamellae, with subepithelial and stromal vascularization. In additon to diffuse poly-morphonuclear cellular reaction, some mononuclear and plasma cells could be seen in the region of the iris and ciliary body. Substance of the iris showed hyaline degeneration with pigment proliferation.
Group I c : ....
Clinical and histological studies were similar to those in group I b
Group II: (Intraperitoneal injection group).
Group II a (1)
Clinical, histopathological and immunological pictures were the same as in group Ia.
Group II a (ii & iii)
There was no clinical or histopathological evidence of uveitis in any of the animals in these groups.
Group 11 b (i)
The observations were the same as in group 1 a (i).
Group II b (ii & iii)
No clinical or histopathological evidence of uveitis was seen.
Group II c (i, ii & iii) -
Observations were comparable to those in II a (i, ii & iii) respectively.
Group III : (Subcutaneous Sensitization)
Group III a (i) - Clinical, histopathological and immunological pictures were similar to that in group I a (i).
Group III a (ii & iii) The eyes remained normal clinically as well as histopathologically during the period of observation.
No antiuveal antibodies could be demonstrated in either blood or aqueous of all the groups examined.
| Discussion|| |
Contradictory reports in literature are available on the immune nature of heterologous, homologous and autologus uvea. Elschnig (1910 a & b ) Kummel (1912 & 1913) and Woods (1916 and 1917) supported the allergic theory of sympathetic ophthalmic by showing that the homologous uvea is capable of an immune response in experimental animals. Von Szily (1916) and Naquin (1955) have, however, failed to demonstrate such a reaction.
Collins (1949) Aronson, Hogan and Zweigart (1963, a, b & c) have succeeded in producing immune uveitis but have either failed to or have not demonstrated the presence of antiuveal antibies in the serum of the animals.
Immunological studies in our series have failed to confirm the earlier works of ELschning (1910 b). Kummel (1912) and Woods (1916) who have shown that uveal tissue pigment is antigenic. We sensitised the animals with whole uveal tissue antigen, but have failed to find demonstratable antiuveal antibodies in either serum or aqueous. We believe that the failure to show demonstratable antibody titres is either due to the fact that homologous uveal tissue is not antigenic at all or is so weakly antigenic that it has failed to produce demonstrable antibody titres. It is, further surmised that the antibodies may not have been in sufficient concentration to produce an immune response which could be observed clinically or histologically.
Intraocular injection of uveal tissue antigen alone or in combination with Freund's adjuvant in the anterior chamber could not produce any clinical, histopathological or immunological evidence of ocular or general sensitization. This finding is however, contradictory to the earlier work `(Silverstein and Zimmerman (1959). We feel that difference could be due to two factors. The latter observers used bovine serum albumin which is a strong antigen as compared to uveal tissue and the injection was given into the vitreous which has the capacity to store and then slowly release the antigen.
Histological picture in which antigen and adjuvant or adjuvant alone was injected showed reaction in the early phase to which later mononuclear lymphocytes and plasma cells were added but no epithelioid cell. We attribute this granulomatous reaction to the presence of tubercle bacilli (dead) and the oils in Freund's adjuvant.
Our clinical and histological studies do not support the earlier works of Vannas et al (1935), Collins (1949) and Aronson and Hogan (1963 a, b, c.)
In our present study, the clinical and histopathological picture on intraocular challenge with uveal tissue antigen alone in animals sensitized either with uveal tissue antigen or with Freund's adjuvant was similar and it compared favourably with that in group I a. (without sensitization). This probably indicates that the reactions noticed have probably the same aetiological basis. This is further confirmed by the absence of immunolocial response in all these animals. The lack of antibody production in intraperitoneal sensitization group is also further supported by the absence of reaction following even intracardiac challenge and the absence of antiuveal antibodies in blood and aqueous in all the groups. The animals sensitized with a combination of uveal tissue antigen and adjuvant showed similar reaction which compares well with group Ia.
Presence of areas of pigment aggregation with lymphocytes which were considered diagnostic by Collins (1949) were, however, found in the animals in the control group and were considered to be of normal occurrence. Such nonspecific aggregations have already been reported by Naquin (1955). It was therefore. considered that an intraperitoneal injection of uveal tissue antigen alone or in combination with Freund's adjuvant (complete) can neither evoke an immune response nor can produce a clinical and histological picture of allergic uveitis.
Encouraged by the results of subcutaneous immunization with corneal antigen (Agarwal, Gupta and Mohan, 1963) we tried this route for immunization in group III. The final results were in no way different from those in group II.
We, therefore, feel that homologous uveal tissue antigen alone or in combination with Freund's adjuvant (complete) is neither capable of producing a detectable immune response nor can it produce a clinical picture of uveitis resembling sympathetic ophthalmia.
| Summary and Conclusions|| |
Response of ocular tissue to repeated injections of homologous uvea has been studied in guinea pigs. Animals were immunized to uveal tissue antigen alone and in combination with Freund's adjuvant (complete) through intraperitoneal and subcutaneous routes. They were observed clinically, histopathologically and immunologically. We draw the following conclusions :
(i) Intraocular injection into the anterior chamber of uveal tissue antigen alone produces a transitory reaction which completely subsides in 7 days. No ocular or general sensitization could be demonstrated.
(ii) Intraocular injection of Freund's adjuvant alone or in combination with uveal tissue antigen produces a very severe inflammatory reaction which does not show any sign of abatement during the period of observation. No ocular or general sensitization could be demonstrated.
(iii) Repeated intraperitoneal or subcutaneous injection of uveal tissue alone or in combination fails to produce any general or ocular sensitivity.
(iv) Picture resembling sym. pathetic ophthalmia could not be produced by repeated immunization with uveal tissue
(v) Pigment proliferation in choroid is of normal occurrence in guinea pig uveal tissue and, therefore, cannot be regarded as pathognomonic of sympathetic ophthalmia in these animals.
| References|| |
Agarwal, L.P.; Gupta, A.K. and Mohan M. (1963) Orient. A. Ophth.; 1. 217.
Aronson S.D., Hogan M.J. and Zweigart, P. (1963 a) A.M.A. Arch. of Ophth. 69, 105.
Aronson S.D., Hogan M.J. and Zweigart, P. (1963 b) ibid 69, 203.
Aronson S.D.. Hogan M.J. and Zweigart, P. (1963 c) ibid 69, 208.
Cellins, R.C. (1949) Am. J. Ophth. 32. 1687.
Eischnig, A. (1910a) Arch. Fur. Ophth. 75. 459.
Elschnig, A. (1910b) Arch. Fur. Ophth. 76, 499.
Fuchs, A. and Meller, J. (1914) Arch. F. Ophth. 88, 280.
Kummel, R. (1912) Arch. F. Ophth, 81. 486.
Kummel, R. (1913) Arch. F. Ophth. 84, 440.
Naquin G.A. (1955) Amer. J. Ophth. 39. 196.
Rados, A. (1913) Z. f. Immun Und. Exp. Therap. 19, 579.
Silver-Stein, A.M. and Zimmer-man L.E. (1959) Am. J. Ophth. 48, 447. (Nov. I & II)
Szily, A. Von (1916) Klin. Monatsbl. F. Augenh, 56, 79.
Schiedegger, J.J. (1955) Arch. Allergy. App. Immunol. 7, 103.
Vannas, S; Wedman, E; and Tier, N. (1935) Acta. Ophth. Kbh. 36, 618.
Woods, A.C. (1916) Arch. of Ophth. 45. 557.
Woods, A.C. (1917) Arch. of Ophthal. 46, 8.
Woods A.C. (1921) Tr. Sect. Ophthal. A M.A. 105.
Wissman R. (1911) Arch. of Ophth. 80, 399.