placebo-controlled trial of preventionof jection · t. a. g. bell, d. l. easty, andk. g. mccullagh...

British Jouirnal of Ophthalmology, 1982. 66, 303-308

A placebo-controlled blind trial of cyclosporin-A inprevention of corneal graft rejection in rabbitsT. A. G. BELL'. D. L. EASTY'. AND K. G. McCULLAGH2

From the 'Bristol Eve Hospital and 2Searle Research and Development

SUMMARY Cvclosporin-A (CvA) administered to rabbits intramuscularlv in a dose of 25 mg/kg/davfor 14 davs following interlamellar corneal grafting had a significant effect in preventing rejection ofthe corneal graft (p<0)05), 'and the benefit was maintained. Rejection was attended bv an initialmild inflammatorv reaction and followed bv a protracted intense response involving a varietv of celltvpes, with subsequent loss of epithelium, endothelium and keratocvtes, and the development ofareas of stromal necrosis. CvA suppressed this rejection response.

Cvclosporin A, a cvclic polvpeptide produced bv avarietv of fungi, has been shown to have a markedimmunosuppressive action when given for shortperiods at the time of sensitisation in a varietv ofimmunological experiments in rats and mice.' Itappears that the drug impairs the earlv stage of anti-genic sensitisation of immunocompetent lvmphoidcells.' 2 The drug has been shown to prolong survivalof organ transplants in animals34 and man.5 The pur-pose of the present studv was to determine bv aplacebo-controlled blind trial the effect of CvA inpreventing comeal graft rejection in rabbits and todescribe the rejection process following interlamellarcorneal grafting and simultaneous skin grafting.

Materials and Methods

INTERLAMELLAR GRAFTINGOutbred New Zealand White rabbits (3-4 kg) wereused. The technique used was based on that describedbv Basu and Ormsby.6 A 5 mm penetrating donorbutton was placed in a half-depth pouch in therecipient cornea. This was fashioned by cutting apartial-depth corneal incision close to the superiorlimbus and dissecting downwards with a lamellardissector. Stitches were not used.

SKIN GRAFTINGA 5 cm square piece of shaved abdominal skin wasremoved from the donor rabbit, cut into 4 squares,and each inserted into small pockets fashioned under

Correspondence to Mr D. L. Eastv. Bristol Eve Hospital. LowerMaudlin Street. Bristol BSI 2LX.

the skin of the abdomen of the recipient rabbit. Thiswas performed at the time of comeal grafting in orderto ensure maximal rejection of the corneal graft.6Plastic sprav was applied to the skin incisions andpenicillin 100000 units administered intramuscularlvfor 4 davs.

TREATMENTA heat sterilised preparation of CvA in olive oil (50mg/ml) was prepared in 2 ml glass vials. Sterilisedolive oil was prepared in the same wav as a placebo.The preparations were coded and paired, and thevwere administered bv intramuscular injection to 13pairs of rabbits such that one of each pair receivedCvA 25 mg/kg/dav and the other placebo.

Results

Slit-lamp examination was performed throughout the14 davs following grafting and comparisons of theobservations made at 7 davs and at 14 davs. There-after 2 pairs were maintained without treatment for afurther 8 weeks. Histological examination of cornealtissue was performed at 2 weeks and at 10 weeks.

In both groups during the first 7 days blood vesselsgrew into the region of the incision, and inflammatorvcells were apparent in the recipient stroma. Thevessels were more apparent in untreated rabbits. At 7days 2 of the untreated rabbits had developed markedvascularisation and oedema of the recipient stroma(Table 1). Small opacities between the donor buttonand recipient cornea were present in both groups.At 14 days (Table 1) 9 untreated rabbits showed

marked rejection with opacification and vascularisa-303

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Fig. I Rabbit cornea. (a) Untreated rabbit at 25 dayvsshowing intense vascularisation and oedema extending intocentral donor button (a). Incision (b). (b) CvA treated rabbitat 25 davs showing clear recipient cornea and donor button.Interspace opacities outline the margin ofdonor tissue (a).(c) CvA treated rabbit at 10 weeks. The donor buitton hasbecome incorporated and remains clear (a).

Ic

tion of the proximal recipient stroma. Eleven treatedanimals showed no clinical evidence of rejection,and the corneae were completelv free of vessels andopacification.At 4 weeks the rejection process in the untreated

rabbits was maximal (Fig. la), while at 6 weeksresolution was noted with reduction in oedema,vascularisation, and the appearance of macrophagesinfiltrating the recipient stroma and graft button.Treated rabbits (Fig. Ib) remained free of inflamma-tion: at 6 weeks macrophages were again noted.At 10 weeks (Fig. Ic) in untreated rabbits the

proximal recipient stroma was no longer oedematousbut was diffuselv opaque. Within the deep stroma ofthe graft button opacities were noted. At the samestage in treated rabbits the graft edges were indistinctfollowing incorporation of the graft into the recipienttissue. Fig. 2 shows the sequence of events in (a)untreated and (b) treated rabbits.

Opacification and vascularisation of the recipientstroma, opacification of the graft button, and limbalinjection were regarded as potential criteria of graftrejection and were graded according to severitv intreated and untreated animals. Stromal opacification

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A placebo-controlled blind trial ofcyclosporin-A in prevention of corneal graft rejection in rabbits

Day I Day 3 Day 7

Day 14 Week 4 Week 6 Week 8

2a

Day 1 Day 3 Day 7

Day 14 Week 4 Week 8

2hFig. 2 Sequence ofevents following interlamellar corneal grafting and skin grafting. (a) Untreated: Vessels and cells developin the region ofthe incision and small interspace opacities (white) develop at the graft edge. The vessels and cell.s increasecoincidentallv with oedema orf the proximal recipient stroma involving the graft tissute. Resollution occurs with the appearanceofmacrophages in the recipient stroma and graft. Opacities (hatched) are noted deep in the graft, and the recipient stroma isslight1v opacified (white). (b) Treated: Vessels and cells develop in the region of the incision and cells reach the donor tissue.The vessels and cells regress. Interspace opacities (white) appear centrallv. Macrophages appear in the recipient stroma (ndgraft and the graft edges become indistinct.

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T. A. G. Bell, D. L. Easty, and K. G. McCullagh

Table I Rejection response in treated and untreated rabbttsat 7 davs and 14 davs

Marked Equivocal Norejection rejection rejction

Dav 76 pairs examined Treated 0 4 2

Untreated 2 4 0Dav 1413 pairs examined Treated I I 11

Untreated 9 1

Table 4 Distribution oftreatedand untreated rabbits withinlow and high rejection groupsfor each rejection criteria andtotal score

Rejection criteria No. in low No. in highrejection group rejection group

Treated Untreated Treated Untreated

Stromal opacification I1 4 2 8Stromal vascularisation 11 3 2 9Graft opacities 12 6 1 6Limbal injection to 4 2 7Total scores I1 3 2 9

Table 2 Grading ofopacification in recipient stroma

Appearance Grade

Absolutelv clear 0Diffuse opacification on slit-lamp examination ± small

occasional opacitiesDiffuse opacification just visible to naked eve ± occasionaldense opacities

Diffuse opacification easilv seen bv naked eve ± smalldense occasional opacities 3

Diffuse dense opacification 4

Table 3 Score ranges wherebv rabbits were allocated intohigh and low rejection groups

Rejection Value decided as Value decided ascriteria low rejection high rejection

Stromal opacification 0-2 3-4Stromal vascularisation (-1 2-3Graft opacities (-1 2-3Limbal injection (-1 2-3Total scores 0-4 5-14

was graded as shown in Table 2. Stromal vascularisa-tion, opacification of the graft button, and limbalinjection was graded according to a simple 0-3 scalewhich corresponded to absent, mild, moderate, andintense responses.At 2 weeks each rabbit was examined and a score

decided for each criterion. The total score for eachrabbit was also obtained. Rabbits were dividedaccording to their score into either a group displavingabsent/mild rejection or a group displayingmoderate/marked rejection for each criterion andtotal score (Table 3). The code was then broken, andTable 4 shows the distribution of treated and un-treated rabbits within these 2 rejection groups.

Statistical analvsis demonstrated a significantdifference in the distribution of treated and untreatedrabbits within the 2 rejection groups for stromalopacification, stromal vascularisation, and total scoreof rejection criteria (p<0-05,<0-05,<0-0l respect-ively by chi-square test; Table 5).

Table5 Probabilitv thatcvclosporin-A influenced rejectionfavourablv

Rejection criteria 2 P<

Stromal opacification 4-87 (005Stromal vascularisation 6-74 (-)IGraft opacities 3-64 NSLimbal injection .3-53 NSTotal score 6-74 0-(1

NS=not significant.

AVERAGE SCORE OF REJECTION CHANES IN TREATED AND UNTREATED RAUITS10 1

I

4

2

I

T

...I4..*..Io

STRML STMAL RAFT LIMAL TOTALOPACITIES VESELS OPACTES INCTIN SCORES

Fig. 3 A verage score ofrejection criteria and total score intreated and untreated rabbits. Standard errors are shown.Dark shade: untreated rabbits. Light shade: CvA treatedrabbits.

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A placebo-controlled blind trial ofcyclosporin-A in prevention ofcorneal graft rejection in rabbits

Fig. 4 Histology ofdonor tisslie.(x 260). (a) Untreated rabbit at 10weeks. The donor tissue is devoid ofkeratocvtes. Epithelilim andendothelilim are absent. (b) CvAtreated rabbit at 10 weeks.Keratocvtes are present and theepithelilum remains healthv. Shedepithelial cells form interspaceopacities (a).

4h

The average score for the rejection criteria in thetreated and untreated rabbits are shown withstandard errors (Fig. 3).

H ISTO LOGY

Histological examination at 14 davs in the lintreatedrabbits revealed an inflammatorv reaction comprisingpolvmorphonuclear leucocvtes, lvmphocvtes, fibro-blasts, and vascularisation in the proximal recipientstroma located in the plane of the original dissection,this reaction extending towards the graft button. Thecellular infiltrate was present in the tissue both

anterior and posterior to the graft button, and cellsinvaded the epithelium and endothelium of the donortissue. Cells were also noted in the graft stroma inreduced numbers. A dense lymphocytic reaction was

present in the ciliary body. Histological examinationin the treated rabbits revealed no inflammatory reac-

tion other than healing of the incision. Remnants ofendothelium were present, and the epithelium was

preserved.Histological examination at 10 weeks in the uin-

treated rabbits (Fig. 4a) revealed an absence of donorepithelium and a marked hvpocellularitv of the graft

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stroma with extensive areas of necrosis in the deeperlavers. Vessels extended into the graft, macrophageswere evident in recipient and donor tissue, andlvmphocvtes were noted in the ciliarv bodv. In thetreated rabbits (Fig. 4b) the graft was intact, with anormal population of keratocvtes and an intactepithelium. Macrophages were evident in the donorbutton and in the recipient stroma. A macrophageand mild lvmphocvtic reaction was present in theciliarv bodv.

Discussion

The results show that CvA significantlv reduced thedevelopment of stromal opacification and stromalvascularisation, which were the most obvious signs ofgraft rejection in this model.

Interspace opacities appeared to correspond withareas of degenerating epithelium. In untreatedrabbits they were associated with inflammatory cells.while in treated rabbits they resulted from shedepithelial cells. Whereas oedema of the graft buttonat 2-4 weeks was a reliable indicator of rejection, theinterspace opacitv was not.

In this model corneal grafting is attended bv theearlv presence of cells derived from the limbal vesselswhich develop in response to the incision. In theabsence ofCvA this cellular infiltrate is followed afteran interval of several davs bv a marked inflammatorvreaction, with intense vascularisation, increasedcellular infiltration, and oedema. This protractedreaction resulted in the destruction of epithelium,endothelium, and keratocvtes and would almostcertainly result in failure of penetrating keratoplasty.The recipient stroma remains vascularised andopacified.

In the presence of CvA earlv inflammatorv cellsappear in the proximal stroma. but there is no sub-sequent inflammatorv response. The observationssupport the suggestion that CvA impairs the earlvstage of antigenic sensitisation of immunocompetentcells.The prolonged survival of the graft following with-

drawal of treatment mav be due to an induction ofspecific tolerance to the antigen bv CvA as describedbv Green et al.7 Alternativelv it is possible that thecorneal graft regains immune privilege after healingof the incision. It is interesting that Shepherd et al.were unable to induce rejection of a corneal graft in a

control rabbit by sensitising 14 days later with a skingraft if the wound was not vascularised,8 suggestingthat the earlv inflammatorv response induced in thelocalitv of the incision constitutes a breakdown ofcomeal immune privilege.

Cvclosporin A has been shown to prolong survivalof penetrating corneal grafts in rabbits,8 and thepresent blind, placebo-controlled studv supports thisresult. The suggested mechanism of action appears tobe relevant to corneal grafting and provides arationale for short-term use of the drug until cornealimmune privilege is re-established.CvA has been used as the immunosuppressive

agent in renal transplants in man.5 Toxic side effectsincluding transient jaundice and changes in the liverand kidnev are reported,2 and there mav be a 10%incidence of lvmphoma in patients receiving the drugon a long-term basis.2 There mav be little indicationfor prolonged use in corneal grafting; consequentlv itwould seem unlikelv that side effects will prove to be ahazard. More information on the total dosage of CvAand the incidence of svstemic complications isrequired before short-term use of this drug can berecommended.

We gratefullv acknowledge assistance from Dr J. F. Borel of SandozLtd. Professor Grunsell. Department of Veterinarv Medicine. BristolUniversitv, and Miss Clare Carter, Miss Anne McSmvthurs. and MrsJanet Williams. Pharmacv Department. Bristol Eve Hospital forassistance -vith preparation and coding of the materials.

References

I Borel JF. Feurer C. Magnee C. St5ihelen H. Fffects of the newanti-lvmphocvtic peptide cvclosporin A in animals. Immunology1977;32: 1017-25.

2 Editorial. Lancet 1979; ii: 779-80.3 Green CJ. Allison AC. Extensive prolongation of rabbit kidnev

allograft survival after short term cvclosporin A treatment. Lancet1978:i: I182-3.

4 Caine RY. White DJG. Rolles K. Smith DF, Herhertson BM.Prolonged survival of pig orthoptic heart grafts treated with cvclo-sporin A. Lancet 1978: i: 1183-5.

5 Caine RY, White DJG. Thiru S. et al. Cvclosporin A in patientsreceiving renal allografts from cadaver donors. Lancet 1978; ii:1323-6.

6 Basu PK. Ormshv HL. Studies of immunitv with interlamellarcorneal homografts in rabbits. Am J Ophthalmol 1957: 44:598-602.

7 Green CJ. Allison AC. Precious S. Induction of specific tolerancein rabbits hv kidnev allografting and short periods of cvclosporin Atreatment. Lancet 1979: ii: 123-5.

8 Shepherd WFI. Coster DJ. Chin Fook. Rice NSC. Jones BR.Effect of cvclosporin A on the survival of cornecl grafts in rabbits.Br J Ophthalmol 1980; 64: 148-53.

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