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| ARTICLES |
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| Year : 1972 | Volume
: 20
| Issue : 2 | Page : 70-76 |
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Experimental lens-induced uveitis. I. Systemic sensitization
OP Ahuja, MS Kothalkar, BR Shulka
Institute of Opthalmology J. L. N. Medical College, Aligarh Muslim University, Aligarh, India
Correspondence Address:
O P Ahuja
Institute of Opthalmology J. L. N. Medical College, Aligarh Muslim University, Aligarh
India
 Source of Support: None, Conflict of Interest: None  | Check |
PMID: 4668480 
How to cite this article:
Ahuja O P, Kothalkar M S, Shulka B R. Experimental lens-induced uveitis. I. Systemic sensitization. Indian J Ophthalmol 1972;20:70-6 |
It has long been recognised that under certain circumstances, lens proteins may induce inflammatory reactions in the eye. In clinical practice, it is encountered in somecases of extracapsular lens extractions or following trauma to the lens. Following the recognition of antigenic properties of lens (Uhlenhuth - 1903), a number of clinical and experimental investigations were made on the subject. Verhoef and Lemoine (1922) published their report which lent support to the original idea of Uhlenhuth that immunological properties are responsible for lens induced uveitis. The studies of Burky et al (1933) and Izumi Kano (19'64) revealed that alphacrystalline fraction was responsible for organspecific properties of lens proteins. Many other studies demonstrated five to eight soluble antigenic components in vertebrate and human lens (Rao et al - 1955: Maisel - 1963; Little and Langham - 1964 and others).
Based on these properties of lens. several investigators produced uveal inflammation by sensitizing the experimental animals with lens proteins (Burky et at - 1934; Lyda et al 1955; Luntz and Wright - 1962; Goodner - 1964: and Selzer et al - 1966). In thse experiments the antigen employed was derived from pooled, homologue or heterologous Lens material. At the time of undertaking the present study i.e. January 1966, the authors had found no report in literature regarding the effect of senstization against autologous lens proteins. The present investigation was therefore undertaken to explore this aspect of the problem by way of sensitizing the animals, by various routes, against their own lens proteins.
Material and Methods
The present study was undertaken on seventyone adult albino rabbits of 3-4 pounds body-weight. All the animals were examined with slit-lamp and ophthalmoscope to exclude any pre-existing ocular disease.
Right eye of these animals was enucleated under aseptic conditions and lens antigen was prepared, separately from each eye, after the technique of Burky and Woods (1931). The animals were then sensitized with the autogenous antigen through various routes. A pre-existing hypersensitivity to lens proteins was excluded by seeing the reaction to intradermal injection of 0.1 cc of the antigen. The present report deals with systemic sensitization, while the results of sensitization produced by other routes will be discussed in subsequent reports.
Systemic Sensitization
Twenty rabbits were sensitized by subcusaneous injections of 0.5 cc, of lens antigen combined with 0.5 cc of Freund's adjuvant. Six injections at weekly intervals were given on medial aspect of thigh at different sites. During the period of sensitization, daily records ocular conditions were made by examining the eyes with slit-lamp and ophthalmoscope. At the end of six weeks, skin test of each animal with autologous lens antigen was repeated. In addition, precipitation reactions between animals' serum and their respective lens antigen were studied by employing Immunological technique of Oudin (1955) and double diffusion agar technique. These sensitized animals were then challenged through various routes.
Method of Challenge
In group A (7 animals), 0.2 cc of antigen was injected into vitreous through the region of pars-plana (after withdrawing same amount of vitreous) with a 26 gauge hypodermic needle. In group B (7 animals), 0.5 cc of lens antigen was injected through marginal ear vein. while in group C (6 animals), the lens capsule was incised and the cortex distrubed with a sharp needle.
After the challenge, daily records of condition of all the eyes were maintained for a period of six weeks. At the end of six weeks, skin test and precipitation reactions were again studied. Histopathological examination was carried out in selected eyes from each group.
Control Group
Two groups of six animals each, were sensitized according to the above schedule but with lens antigen and Freund's adjuvant separately. The challenge consisited of an intravitreal injection of the serum antigen (3 animals) and trauma to the lens (3 animals) in each group. The clinical and laboratory studies were made as in other groups.
Results
(i) During Sensitization -
No clinical evidence of ocular inflammation of anterior or posterior segment was seen in any of the animals. However, on 8th to 10th day a nodular swelling appeared at the site of subcutaneous injection, vhich gradually increased to a size of an almond by 4th. week. The swelling burst in most cases forming an ulcer with yellowish cheesy discharge. The ulcer healed in about two weeks time. Immunological studies revealed a single precipitation band [Figure - 1] in five animals. Histological examination did not show any sign of inflammation.
(ii) After Challenge -
In group A, all the eyes revealed signs of inflammation on 2nd. day in the form of circumcorneal congestion and slight irregular and sluggish pupil. The peak reached on 4th. day when most of the eyes had irregular pupil, fine posterior synechiae, fibrinous exudates in anterior chamebr, strong aqueous flare and hazy vitreous. This reaction persisted for three to four days, thereafter fading gradually. By 26th day most of the eyes were quiet.
Cutaneous reaction in two animals was positive (erythema 10-12 mm. with induration). In one animal it was weakly positive while in remaining, it was negative. Precipitation reactions were positive in those animals showing a positive skin reaction.
Histopathological examination of any eye enucleated on 4th. day revealed fibrino-cellular exudates in anterior chamber and vitreous showing lymphocytes, plasma cells and eosinophils [Figure - 2]. Iris and ciliary body were moderately in filtrated with these cells but choroid was nomal.
In group B, no signs of ocular inflammation were observed in any of the animals. Similarly, results of skin test and immunological reactions were uniformly negative. Histopathological examination revealed no changes.
In Group C, on 2nd. day there was marked circumcornal injection with slight haziness of cornea and fine deposits on its posterior surface. For the next five days, severity of inflammation increased with strong aqueous flare, fibrinoid exudates and lens matter in anterior chamber [Figure - 3]. From 9th. day onwards inflammation started reducing in severity and by 30th. day all eyes were quiet. Skin test and precipitation test (Single band) was positive in all the animals.
Histological examination of an eye enucleated five days after the challenge, showed mild cellular infiltration while another eye enucleated on 10th. day showed signs of marked inflammation. Exudates were present in anterior and posterior chambers with infiltration by lymphocytes, neutrophils and plasma cells. In addition there were many eosinophils in posterior chamber and few polymorphs were noted around lens fibres. Choroid was normal.
Various results after sensitization and challenge have been summarised in the [Table - 1].
Results in Control Group
No clinical evidence of inflammation was noticed in any of the animals during sensitization. However, a similar type of ulceration as described above was noticed in those animals in which subcutaneous injection of Freund's adjuvant was made. Results of skin test and precipitation reactions were also negative. After challenging these animals by intravitreal injection of lens antigen and trauma to lens respectively, there was a mild clinical reaction but no convincing histopathological evidence of inflammation as seen in treated animals was observed.
| Discussion | |  |
The animals of this group were sensitized by repeated subcutaneous injections of a combination of autologous lens antigen and Freund's adjuvant as detailed earlier. Varying results were obtained in the demonstration of a generalized hypersensitivity by way of skin reactions and precipitation test. The skin reaction was weakly positive in the majority (12 out of 20) of rabbits, while a strongly positive reaction was seen in four animals. In the remaining four animals, the reaction was negative. Similarly antilens precipitins were demonstrated only in a small number of animals in which a single preciptin band was observed in contrast to the multiple lines reported by others (Rao et al - 1955, Francois et al - 1956 and Little and Langham - 1964). This single precipitin band in this study suggests that demonstrable antibodies were produced against only one of the soluble crystallins. Contrary to these observations, there was an indirect evidence of productions of hyperesensitivity in the ocular tissues. When a batch of these animals was given an intravitreal injection of lens antigen, a reaction was produced which was much more severe than that produced by a similar injection in non-sensitized animals (Ahuja, Kothalkar and Shukla - 1969). In addition, a classical allergic response was observed histologically in the form of infiltration of uveal tissues with lymphocytes, plasma cells and eosinophils. On the other hand, a neutrophilic infiltration of vitreous was seen in the non-sensitized animals following an intravitreal injection.
To exclude the possibility of these reactions being nonspecific due to Freund's adjuvant, two batches of animals were sensitized by the same route, but with Freund's adjuvant and lens antigen individually. Both the batches were then challenged by intravitreal injection of lens antigen. Not withstanding the clinical reaction produced, the difference in histological picture of the two groups was very significant. In animals sensitized with Freund's adjuvant, a cellular response in the form of mild mononuclear infiltration was seen, while in the other group, the cellular infiltration consisted of few neutrophils and lymphocytes with occassional plasma cells. This observation does not only exclude the possibility of ocular reaction being nonspecific, but also supports the contention that Freund's aduvant enhances the antigenic properties of lens proteins. As already stated no allergic reaction was seen against the injection of lens protein in rabbits sensitized with Freund's adjuvant, while the allergic reaction produced in the rabbits sensitized with lens antigen alone was much less severe than in those, sensitized by the combination of the two.
In the same manner as in these animals, the presence of ocular hypersensitivity was demonstrated in another batch of animals similarly sensitized, in which a sterile trauma to lens was made to liberate the lens proteins in aqueous. Clinically a severe inflammatory reaction was produced and histologically a picture suggestive of allergic reaction was seen. Three weeks following trauma to the lens, skin reaction was positive in all the rabbits and a single precipitin band was seen in three out of six animals (one died and two were sacrificed). A similar trauma to lens on the other hand in nonsensitized animals produced a mild inflammatory response and there was no histological evidence of allergic reaction (Ahuia and Kothalkar - 1969). Similar results were seen in rabbits sensitized separately with lens antigen and Freund's adjuvant. This observation further supports the view of enhancement of antiaenicity of lenticular proteins when they are combined with Freund's adjuvant. Similar conclusions were made by Luntz and Wright (1962) in the production of endophthalmitis phacoanaphylactica by repeated needling of the lens, with simultaneious intramuscular injections of Freund's adjuvant. In his study no such reaction was produced in the animals in which the injections of Freund's adjuvant were omitted.
By the foregoing discussion it is obvious that although an ocular hypersensitivity was demonstrated in all the, animals, the results of demonstration of generalized hypersensitivity were irregular. This inconsistency is rather difficult to explain. However, it is likely that the lenticular proteins having weak antigenic properties, were able to provoke only a weak antibody response which may not be possible to demonstrate by the methods adopted in the present investigation More sensitive technique could have probably elicited positive response. Secondly, as suggested by Burky et al (1933) precipitin titres may not be directly related with the ocular reaction and the ocular sensitivity may develop independent of precipitins.
Whether an active allergic reaction inside the eye produced by challenge dose in the sensitized animals affects the pricipitin titer or the skin reaction. cannot be asserted with certainty. In our croup of animals which were challenged by trauma to lens, skin reaction became positive and demonstrable precipitin titer was seen in the majority.
In another group of similarly sensitized animals an intravenous injection of the lens antigen failed to produce any uveal reaction as seen clinically and histologically. Though it is assumed that an ocular hypersensitivity must have been produced in these animals (as in other similarly treated animals, the non-production of the disease was probably due to a marked dilution of antigen in the general circulation. It may also be due to the fact that because of an intact blood aqueous barrier the injected antigen could not gain access inside the eye.
Summary and Conclusions
An attempt was made to produce lens-induced uveitis with autologous lens proteins and to correlate clinical picture with serological and histological findings.
Varying results were obtained in demonstration of generalized by persensitivity by way of skin reactions and precipitation tests in systemically sensitized animals.
A strong allergic response was seen clinically, immunologically and histopathologically in those animals where lens was traumatized after systemic sensitization. Similarly, an allergic response was seen histologically after intravitreal challenge injection of lens antigen. Intravenous injection on the other hand did not reveal any ocular reaction.
It was therefore concluded that autologous lens proteins under certain circumstances are capable of sensitizing the uveal tissue to produce an autoimmune inflammation.
The sensitizing properties of lens antigen are markedly enhanced when they are combined with Freund's adjuvant[16].
| References | | |
| 1. | Ahuja, O.P. Kothalkar, MS and Shukla, B.R.: Unpublished date.  |
| 2. | Burky, E.L. and Woods, A.C. (1931) A.M.A. Arch. Ophthal. 6 : 548. |
| 3. | Burky, E.L. Woods, A.C. and Woodhall, M.B. (1933) A.M.A. Arch. Ophthal. 9: 446. |
| 4. | Burky. E.L. (1934) A.M.A. Arch. Ophthal 12: 536 |
| 5. | Francois, J. Rabaey, M. Wieme, R.J. and Kaminski, M. (1956 Amer. J. Ophth. 42,577. |
| 6. | Goodner, E. K. (1964) - Immunopathology of Uveitis, Baltimore. The William and Wilkins Company P. 233. |
| 7. | Izumi, K and Kano M. (1964) Acta. Soc. Opthal lap. 68: 1121. |
| 8. | Little, J. and Langham, _J. (1964) Amer J. Ophthal. 72: 820. |
| 9. | Luntz M.H. and Wright, R. (1962) Exper. Eye Res. 1: 317. |
| 10. | Lyda, W. and Lippincott, SW, (1955) Amer. J. Ophthal. 40: 120. |
| 11. | Maisel, H. (1963) - Amer. J. Opthal. 55: 1208. |
| 12. | Oudin, J Cited by Rao, S. S. Kulkarni, M.E. and Cooper, S.N. (1955). Brit. J Ophthal. 39: 163. |
| 13. | Rao, SS. Kulkarni, M.E. and Cooper, S.N (1955) Brit. J. Ophthal. 39: 1963. |
| 14. | Selzer, G. Path., F.E. Luntz, M.H. Sacks. M.l. and Path.. M-M. (1966) Amer. J. Ophthal 61: 1396. |
| 15. | Uhlenhuth. Quoted by Woods. A.C. (1961) - Page 230. |
| 16. | Verhoeff, F.H. and Lemoine, A.H. (1922) Amer. J. Ophthal. 5: 700. |
[Figure - 1], [Figure - 2], [Figure - 3]
[Table - 1]
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