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Year : 1972  |  Volume : 20  |  Issue : 2  |  Page : 84-87

Corneal immune reactions

Aligarh Muslim University Institute of Ophthalmology, Aligarh, India

Correspondence Address:
B R Shukla
Aligarh Muslim University Institute of Ophthalmology, Aligarh
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Source of Support: None, Conflict of Interest: None

PMID: 4668482

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How to cite this article:
Shukla B R, Gupta N C, Ahuja O P. Corneal immune reactions. Indian J Ophthalmol 1972;20:84-7

How to cite this URL:
Shukla B R, Gupta N C, Ahuja O P. Corneal immune reactions. Indian J Ophthalmol [serial online] 1972 [cited 2023 Dec 10];20:84-7. Available from: https://journals.lww.com/ijo/pages/default.aspx/text.asp?1972/20/2/84/34663

In the science of keratoplasty, immunological rejection of the grafted cornea is a well recognised fact. To enhance the chance of a successful corneal transplant, at­tempts are being made to suppress this phenomenon. Several proce­dures like irradiation (Sterzl Mar­chioro and Woddel [8] ), Corticos­teriod therapy (Leibowitz and Elloitt [2] , Smolin and Keates [7] and Olson [3] ) and certain immuno sup­pressive drugs have been evaluated in this respect. In the present study, attempts were made to in­vestigate the effect of Purinethol,* on experimentally produced cor­neal hypersensitivity reactions. The drug is a known nucleic acid anti-metabolite and a suppressive of haemopoietic system.

  Materials and Methods Top

The present study was under­taken on 24 albino rabbits of either sex and of average weight of 3 pounds (varying between 2.5 to 4 pounds). They were labelled 1 through 24. These rabbits were an­aesthetised with 2% sodium seco­nal injected into the marginal vein of pinna. It was supplemented with topical instillation of 1 % anae­thaine and 1 cc of 2% novocain retrobuibarly.

Each eye was proptosed and fixed with fixation forceps. 0.06 cc of sterile bovine serum albumin solu­tion (100 mg./cc) was injected in­tralamellarly at the centre of the cornea with a 1 cc tuberculin syringe fitted with a 27 gauge needle. The injection was repeated on the 20th day.

The animals were then divided into three groups for observation

Group I :- This consisted of rabbits 1 through 9. No post in­oculation medication was given. They were carefully examined daily by oblique illumination and biomicroscopy. Blood was with­drawn by intracardiac puncture on 5th, 14th, 22nd and 30th day after inoculation for estimation of antibody titre and total white cell count. Double agar diffusion technique was employed for an­tibody estimation.

One eye at similar intervals was enuclated under general an­aesthesia. The histological exa­mination was made of serial sec­tions stained with haematoxyline - eosine and a methyl green-­pyronine stain.

Group II :- This consisted of rabbits 10 through 18. They were given a daily intramuscular in­jection of purinethol (2 mg./lb body weight) for 20 days starting from the day of inoculation. Observations, similar to group I were made but in addition a daily record of weight was kept for any evidence of loss of weight. The animals were watch­ed for any diarrhoea or loss of appetite.

Group III:- Consisted of rab­bits 19 through 24. A biweekly subconjunctival injection of 0.25 cc of 5% purinethol solution was injected in both eyes of each animal. The medication was in­stituted on the day of inocula­tion and continued for 30 days. This group was studied on the lines laid down for groups I and II.

  Results Top

Group 1 (Control)

Within 4-7 days following the inoculation, there appeared corneal opacification circum limbal con­gestion and corneal vascularization These signs persisted for about a week and then all the signs dis­appeared except for a mild injec­tion opacity in the centre of the cornea. The eyes remained so up­to the 20th. day of inocuatlon when a second inoculation was made. 24 hours after the second injection there appeared circum corneal con­gestion, corneal vascularization and a white ring of opacity in the cor­nea around the injection site.

Histological examination showed a mononuclear infiltration of lim­bus during the first phase of reac­tion but in the second phase there were also present many plasma cells - mature and immature.

The precipitin reactions for cir­culating antibodies gave uniformly negative results in all the samples of blood when tested by double agar diffusion technique.

Group II (I/M Purinethol)

Only 7 out of 19 eyes developed the first reaction 4-7 days after in­oculation. Only a mild central opacity or ciliary congestion was present in the other 11.

None of the 11 eyes reinoculated on 20th day developed any reac­tion in the form of corneal vascu­larisation or white immune ring around injection site. Only two eyes showed some limbal vascular dilatation.

Histological examination con­firmed the suppression of clinical response as only a few mononu­clear cells with insignificant num­ber of plasma cells were seen in the limbal infiltrates.

Immunological tests gave nega­tive results like the control group

Group III (SIC Purinethol)

There was no effect of therapy in this group. Clinical, histological and immunological results were similar to those in control group.

Drug Toxicity :­

No toxic side effects of the drug were seen when given by subcon­junctival route. However on syste­mic therapy, there was a signifi­cant leucopenia and diarrhoea in 3 of the animals. One of them died on 14th day of therapy.

  Discussion Top

The first reaction following ino­culation developed after 4-7 days. This was characterised by corneal opacification, circum corneal con­gestion and a lymphocytic infiltra­tration of limbus. The picture was suggestive of delayed hypersensi­tivity reaction. No circulating an­tibodies were demonstrable then. 24 hours following the second ino­culation, there developed the second reaction characterised by circum corneal congestion, a white opaque ring around the injection site and a mononuclear limbal in­filtration with many plasma cells. This was against the observations of Germuth [1] who observed this rea­ction on the 14th day following a single inoculation. This was the immediate type of hypersensitivity reaction. As we failed to get the reaction following a single inocu­lation, and could get a similar reac­tion only after a second inoculation it is suggested that although a reactive level of circulating anti­bodies was reached, the antigen depot in the cornea had exhausted. This antigen was then provided by a second injection. This early ex­haustion of antigen is difficult to explain. It may be that the breed or strain of animals that we em­ployed was different from those used by other workers. The limbal infiltrate of delayed hypersensiti­vity reaction consisted of mono­nuclear cells, predominantly the lymphocytes. During the immediate response, large number of mature and immature plasma cells were also seen among the mononuclear cells. The appearance of plasma cells at this stage is suggestive of their role in the formation of anti­bodies locally or extra ocularly. Though the antigenantibody reac­tion was observed clinically and confirmed histologically yet in our study no circulating antibodies could be demonstrated. As the standard double agar diffusion te­chnique using the standard mate­rial was employed, the negative immunological tests only suggest that although a reactive level of antibodies was present in the serum, the titre was probably not high enough to be demonstrated on agar plates.

Intramuscular purinethol thera­py markedly suppressed both the types of hypersensitivity reactions. Only 7 eyes out of 18 eyes develop­ed typical delayed type of reac­tion. Immediate reaction developed in none of those 11 eyes which were reinjected on 20th day.

Purinethol, a synthetic purine analogue interferes with nucleic acid biosynthesis. It suppresses the formation of lymphocytes and thereby of antibodies. It is inter­esting to note that although syste­mic purinethol suppressed imme­diate hypersensitivity reaction in almost all the 11 eyes leucopenia was observed in only three ani­mals. This suggests that the im­muno-suppressive effect of the drug is not directly related to its lymphocyte suppressing effect on the haemopoietic system. It may however be possible that the drug inhibits antibody formation even in the presence of normal lympho­cytic count probably by inhibiting the conversion of lymphocytes in­to antibody forming plasma cells.

Suppression of delayed hyper­sensitivity by systemic purinethol suggests that it has some inhibitory effect on the sensitization of tis­sues. Thus the drug has both cen­tral and peripheral action.

Subconjunctival route failed to suppress the hypersensitivity reac­tion. It is understandable that this route, which allows a comparative­ly small amount of the agent to be introduced, does not produce effec­tive levels of drug in general cir­culation. As very little antibody if at all is formed locally, this route of medication did not sup­press the reactions.

Systemic therapy is toxic as it produced diarrhoea and leucopenia in some of the animals.

  Summary Top

Both eyes of all the 24 animals were inoculated by an intra cor­neal injection of B.S.A. The injec­tion was repeated on 20th. day.

The control group which receiv­ed no medication showed a delay­ed hypersensitivity reaction 4-7 days after first injection and an im­mediate hypersensitivity reaction 24 hours after second inoculation.

Among the treated groups, intra muscular purinethol therapy markedly suppressed both the de­layed and immediate types of hy­persensitivity. The sub conjuncti­val route of purinethol therapy did not have a significant effect.

Systemic purinethol therapy pro­duced toxic side effects like diar­rohoea and leucopenia but the sub­conjunctival route was non-toxic.

  References Top

Germuth. F.G. et al (1962) J. Expt. Med.; 115, 919.  Back to cited text no. 1
Leibowitz, H.M. Elliott, J.H. (1965). Arch. Ophth.; 74, 835.  Back to cited text no. 2
Olson, C.L. (1966) Arch Ophthal; 75, 651.  Back to cited text no. 3
Parks, J.J.: Leibowitz, H.M. and Maumenee. A.E. (1962) J. Exp. Med.: 113, 867.  Back to cited text no. 4
Schwartz, R.; Stack, J. and Dameshek, W. (1958); Proc. Soc. Exper. Biol. and Med.: 99, 164.  Back to cited text no. 5
Sellyei, L.F.; and Ellis P.P. (1966) Amen. J. Ophth.; 61, 702.  Back to cited text no. 6
Smolin, G,; and Kaetes. R. H. (1967): Am. J. Ophth., 63, 335.  Back to cited text no. 7
Sterzl, T.E. Marchiors, T.L.; and Woddell, W.R. (1963) Surg. Gynae. and Obstet 117, 385.  Back to cited text no. 8


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