retraction of the eyelids secondary to thyroid ophthalmopathy-its

RETRACTION OF THE EYELIDSSECONDARY TO THYROID

OPHTHALMOPATHY-ITS SURGICALCORRECTION WITH SCLERA AND THE

FATE OF THE GRAFT

BY Joseph C. Flanagan, MD

INTRODUCTION

HISTORICAL CONSIDERATIONS

THE OCULAR MANIFESTATIONS OF THYROID DISEASE HAVE BEEN DOCUMENTED FORwell over a century and a half. In 1786 Parry' described the associationbetween enlargement of the thyroid gland and the development of exoph-thalmos. Graves2 redescribed this syndrome in 1835 and it has since beenknown as Graves' disease. Retraction ofthe eyelids is but one ofthe severaleye signs that are associated with Graves' disease. Severe lid retractionsecondary to Graves' disease may be corrected using scleral grafts.

Historically, human sclera has proven to be an excellent material forgrafts in ophthalmology because it is a homograft and is therefore verylikely to be accepted by the host. It is readily available from eye banks,amenable to sterilization and preservation, easy to work with and suf-ficiently strong.

Scleral grafts have been used to correct many abnormalities of the eyeand adnexa. In an attempt to treat degenerative myopia surgically, Borleyand Snyder3 used a seven millimeter donor scleral strip combined with ascleral resection. This was later modified by Miller and Borley4 by omittingthe scleral resection. Many related articles later appeared in the literatureincluding the work of Belyaev,5 Thompson6 and Snyder.7

Sclera was introduced in retinal detachment surgery by Paufique8'9 whoused it for local grafts. Rodrigues-Vasques'0 reported the use of sclera forencircling procedures in 1962. In 1966 Miller"l reported that he had beenusing a combination of a lamellar scleral resection and indentation withhuman preserved sclera in retinal detachment surgery since 1959. Sincethat time there have been many reports discussing different techniques forTR. AM. OPHTH. Soc. vol. LXXCVIII, 1980

using sclera to correct retinal detachments. 12-14 Wilson and Parker'5 usedscleral patches to cover exposed silicone buckles after retinal detachmentsurgery.

Scleral grafts have been used for segmental reconstruction of the exter-nal coats of the eye for a great many conditions including scleromalaciaperforans,16 malignant melanoma of the limbus,'7 implantation of akeratoprosthesis, 8 intercalary staphyloma in Marfan's syndrome, 19 recur-rent pterygium,20 anterior segment reconstruction,21 necrogranulomatousscleritis,22 sclero-chorio-retinal resection,Y' local excision of choroidalmalignant melanoma,24 iris prolapse,25 epithelial invasion of the anteriorchamber,2 and as a template for corneoscleral grafts.27

Scleral grafts have been utilized extensively in ophthalmic plasticsurgery. Helveston28 first reported the use ofa scleral patch for the repairof exposed or extruded orbital implants in 1969. His technique has beenmodified by many ophthalmic surgeons.29-32 In an attempt to preventextrusion, Soll3 used sclera as an integral part of the primary enucleationprocedure.The utilization of scleral strips to correct ptosis has been reported by

Bodian34 and Crawford.35 Other eyelid abnormalities in which sclera hasbeen useful are cicatricial entropion3W37 and eyelid retraction secondary tothyroid ophthalmopathy.37'38

ANATOMY

The retractors ofthe upper eyelid are the levator muscle and its aponeuro-sis and the sympathetic tarsal muscle of Muller. The upper eyelidaponeurosis is a continuation ofthe muscular portion ofthe levator muscleand it attaches to the anterior surface ofthe inferior one third ofthe tarsus.The levator muscle also has attachments to the midportion of the orbitalseptum and sends fibers into the skin of the upper eyelid, forming thesupratarsal fold. The sympathetic superior tarsal muscle of Muller arisesfrom the undersurface of the levator muscle at its junction with theaponeurosis and inserts onto the upper edge of the tarsus.The lower eyelid also has retractors which consist of the inferior tarsal

muscle and an aponeurosis which arises as part of the capsulopalpebralhead of the inferior rectus muscle. These retractors attach to the frontsurface of the tarsus and to the skin. The inferior tarsal muscle is primarilyconnective tissue and is more closely fused to the aponeurosis than thetarsal muscle of Muller in the upper lid. The inferior tarsal muscle arisesfrom the aponeurosis and inserts into the lower edge of the tarsus. Theposterior extension of the aponeurosis and the inferior tarsal muscle sur-

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Thyroid Ophthalmopathy

round the inferior oblique muscle to form its sheath. The retractors thenattach to the lower side of the inferior rectus muscle and help to form thesuspensory ligament of Lockwood. Both upper and lower eyelid retractorsystems have a third part extending to the conjunctiva as the suspensoryligament of the fornix.37

PATHOPHYSIOLOGY

Lid retraction in Graves' disease may be due to several factors. The first isexcessive contraction of Muller's muscle caused by the synergism betweenthyroxine and endogenous catecholamines (epinephrine and norepi-nephrine). Norepinephrine is a neurotransmitter which is stored in theterminal endings of sympathetic neurons and, when released, depolarizesthe adjacent effector cell at a specific receptor site to produce action ofthateffector cell.39When eyelid retraction is due to overaction of the sympathetically

innervated Muller's muscle alone, it can be relieved pharmacologically byusing adrenergic blocking agents such as guanethidine.40A41 Most of thefavorable reports utilizing guanethidine have been from Europe. Thistreatment has not been pursued in the United States because of the lack ofavailability and poor stability of guanethidine eyedrops. Guanethidine isused occasionally to aid in the diagnosis of eyelid retraction secondary toGraves' disease. For guanethidine to alleviate the lid retraction in Graves'disease it would have to be used early in the course of the disease beforefibrosis occurs, when there is primarily an overaction ofthe smooth muscleof Muller.

Eyelid retraction can occur in the euthyroid and hypothyroid states aswell as in the hyperthyroid state. Unilateral eyelid retraction is also com-mon. Because ofthese findings, other causes ofeyelid retraction in Graves'disease have to be considered. One such cause is the inflammation, thick-ening and ultimate fibrosis ofthe levator and Muller's muscle which occursin thyroid ophthalmopathy.Another factor is the overaction ofthe levator muscle which participates

much as a yolk muscle with the superior rectus muscle. The superior rectusmuscle overacts as it tries to overcome the resistance to upward gazecaused by fibrosis of the inferior rectus muscle. This mechanism is indi-cated if there is a definite upshoot of the upper lid as upward gaze passesthrough the primary straight ahead position and ifthere is no prominent lidlag on downward gaze.' Fibrosis of the lower lid aponeurosis and theinferior rectus muscle can transmit downward traction on the lower eyelid,causing lower lid retraction and scleral exposure inferiorly.

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TREATMENT OF THYROID EYEUD RETRACON

The ophthalmologist plays a significant role in the management ofpatientswith signs and symptoms of thyroid ophthalmopathy. The medical treat-ment ofthyroid.eyelid retraction may vary from the use oflubricating dropsand ointments, taping the eyelids closed during sleep, to the use ofsystem-ic steroids.

Ifthere is severe exposure keratitis secondary to proptosis or evidence ofoptic nerve involvement, an orbital decompression into the maxillary andethmoid sinuses may be indicated.4 If the exposure of the cornea is dueprimarily to eyelid retraction, recession of the retractors of the upper andlower eyelids with or without scleral grafts may be a safer procedure. Iftheeyelid retraction is corrected for cosmetic reasons, this safer procedurewould be indicated.

SURGICAL TREATMENT

Eyelid retraction secondary to Graves' disease has been treated surgicallyby a number ofprocedures on the levator aponeurosis and Muller's muscle.Blaskovics45 severed the levator (presumably Muller's muscle) from thesuperior tarsal border and retracted it with three sutures. For patients withmore than three millimeters of lid retraction, he dissected the levatormuscle from the skin and orbicularis and allowed it to retract freely into theorbit.

Cusick and SarrailW described an operation in which the levator, againprobably the tarsal muscle, was separated from the tarsal plate. Theycontrolled the amount of the recession with mattress sutures through thetarsal muscle. These sutures were tied loosely to the tarsus. Goldstein47utilized a levator recession in which an incision is made through the tarsusand its aponeurotic attachments one millimeter below its superior border.

Henderson8 devised a method of recessing conjunctiva and Muller'smuscle. This surgical procedure is usually performed under general anes-thesia, but in adults a frontal nerve block, as described by Hildreth andSilver,49 is adequate when operating on an upper eyelid. An infraorbitalnerve block works very well when operating on the lower eyelid. Theendocrine status of the patient should be stable for at least six monthsbefore surgical correction of the lid retraction is attempted.

SURGICAL CORRECTION OF MILD DEGREES OF LID RETRACTION

To correct mild degrees ofupper eyelid retraction (3mm or less), the upperlid is everted on a Desmarres lid retractor. Conjunctiva and Muller'smuscle are then recessed in the same manner as described by Hender-son.48 A small buttonhole incision is made through the conjunctiva and

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FIGURE 1A: Passage of the iris spatula in the natural plane between Muller's muscle and the levatoraponeurosis. B: Conjunctiva and Muller's muscle (black arrow) dissected from the upper

border of the tarsus (arrowhead) and the levator aponeurosis (white arrow).

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Muller's muscle at the mid portion of the lid just above the upper tarsalborder. An iris spatula is inserted into this buttonhole and is passed nasallyin the natural plane between Muller's muscle and the levator aponeurosis(Fig 1 A). When the proper plane is entered, the spatula passes very easilyand the gleam ofthe spatula should be visible to the surgeon. Ifthe spatulais passed too superficially it will produce buttonholes in the conjunctivaand, if it is placed too deeply, the passage is made with great difficulty.When the spatula reaches the nasal aspect ofthe lid, it is kept in place whileone blade of the Stevens scissors is placed in the same plane to dissect theconjunctiva and Muller's muscle from the upper border of the tarsus. Theiris spatula is then placed through the buttonhole into the same naturalplane to the temporal aspect of the lid. Again, one blade of a Stevensscissors is placed in the same plane, and the conjunctiva and Muller'smuscle are dissected free from the upper border of the tarsus to thetemporal aspect of the lid.

The conjunctiva and Muller's muscle may then be dissected from thelevator aponeurosis for an area of8 mm above the upper border ofthe tarsus(Fig 1 B). This dissection may be pefformed with cotton tipped applicators.The free edge ofthe recessed conjunctiva and Muller's muscle are suturedto the levator aponeurosis approximately 8 mm above the upper tarsalborder with a running 7-0 catgut suture. This gives insurance that the freeedge ofthe recessed tissues will not re-attach to the upper tarsal border andthereby negate the correction that should have been obtained. A tractionsuture is then inserted and the upper lid is taped to the cheek for one week.If an overcorrection is evident, the traction suture is removed on the firstpostoperative day.

The surgical procedure is similar when treating mild degrees of eyelidretraction (3 mm or less) of the lower lid. The lower lid is everted on aDesmarres lid retractor and held in place with forceps (Fig 2 A). A smallbuttonhole incision is then made in the conjunctiva and the musculofacialretractors at the midportion of the lower lid just below the inferior tarsalborder (Fig 2 B). One blade ofthe Stevens scissors is inserted into the planejust deep to the conjunctiva and the musculofacial tissue which extendsfrom the inferior rectus muscle to the inferior border of the tarsus. Theconjunctiva and retractors of the lower lid are then dissected from theinferior border of the tarsus. This dissection extends to the nasal andtemporal extremity ofthe tarsus (Fig 2 C). The conjunctiva and musculofa-cial tissue are then recessed for approximately 8 mm (Fig 2 D) and suturedto the orbital septum with a running 7-0 chromic catgut suture. A tractionsuture is then inserted and the lower lid is taped to the brow for one week.

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FIGURE 2A: Lower eyelid everted on a Desmarres lid retractor. B: Buttonhole incision through theconjunctiva and the musculofacial retractors of the lower eyelid just below the inferior tarsalborder. c: Dissection of the conjunctiva and the musculofacial retractors of the lower eyelidfrom the inferior border of the tarsus. D: Conjunctiva and musculofacial retractors (white

arrow) recessed from the inferior border of the tarsus (black arrow).

If an overcorrection is evident, the traction suture is removed on the firstpostoperative day.The previously described upper and lower eyelid surgery may be all that

is necessary to correct mild degrees of eyelid retraction (Fig 3 A and 3 B).

INDICATIONS FOR USE OF A SCLERAL GRAFT

In cases with upper eyelid retraction greater than 4 mm, I suture anelliptical strip of fresh sclera to the free edge of the recessed conjunctivaand Muller's muscle and to the upper border ofthe tarsus with a continuoussuture of 7-0 catgut. The fresh sclera is obtained from an eye bank and isused within four days after the death ofthe donor. Preserved sclera may beused which has been stored in glycerin in a vacuum tube or with amolecular sieve, or frozen in neosporin solution.34' The strip of donorsclera is obtained in the operating room from an eye bank eye using a Bard

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FIGURE 3A: Patient with approximately 2 mm ofeyelid retraction of all four eyelids secondary to thyroidophthalmopathy. B: Patient shown in Figure 3 A one year after a bilateral recession of theconjunctiva and Muller's muscle ofthe upper eyelids and a bilateral recession ofthe conjunc-

tiva and retractors of the lower eyelids.

Parker blade, forceps, scissors, and a muscle caliper. The scleral strip isapproximately 10 mm in width and is taken from the entire circumferenceof the globe. It is then immersed in a medicine glass filled with PolymixinB-Bacitracin-Neomycin (Neosporin) ophthalmic solution (Burrows Well-come Co, Research Triangle Park, NC) for one hour and then transferred totwo medicine glasses successively which contain sterile saline. At surgerythe scleral strip is tailored to the desired length and height (Fig 4 A) beforebeing sutured into the recipient bed.The rule ofthumb which has been followed is that for every millimeter.of

sclera showing above the limbus, the graft should be 2 mm in verticallength because of the tendency for the graft to contract.37 51 After the

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FIGURE 4A: Measuring the vertical height ofa scleral strip. B: Elliptical strip ofsclera sutured to the freeedge ofthe recessed conjunctiva and Miuller's muscle (white arrow) and to the upper border of

the tarsus (black arrow).

scleral graft is sutured in place (Fig 4 B), antibiotic ointment is instilled intothe eye and the lid is flipped back to its normal position. A traction suture isthen inserted and the lid is taped to the cheek. It is not necessary to coverthe grafted sclera with conjunctiva because it is rapidly covered withepithelium and does not irritate the cornea.51 The traction suture is left inplace for one week ifan adequate correction is obtained. If an overcorrec-tion is evident, the traction suture is removed on the first postoperativeday.

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FIGURE 5Elliptical strip of sclera sutured to the free edge of the recessed conjunctiva and the lower

eyelid retractors (white arrow) and to the lower border of the tarsus (black arrow).

In cases with lower eyelid retraction greater than 4 mm, I suture anelliptical strip of fresh sclera to the free edge of the recessed conjunctivaand musculofacial retractors and to the lower border of the tarsus with acontinuous suture of7-0 catgut (Fig 5). Following the rule ofthumb for theupper lid, 2 mm of scleral graft are used for every millimeter of sclerashowing below the limbus. The patient shown in Figs 6 A and 6 B had 6 mmof sclera showing above the limbus and 5 mm of sclera showing below thelimbus. Bilateral scleral grafts measuring 12 mm and 10 mm in verticallength were therefore utilized in the upper and lower lids respectively (Fig6 C).When surgery is performed on the upper and lower lids at the same time,

traction cannot be exerted on the upper and lower lid simultaneously. Thus,it is beneficial to have the patient tape the upper lid to the cheek for halfofthe day and the lower lid to the brow for the other halfofthe day for a periodofone week. An antibiotic ointment is instilled into the eye four times a dayfor two weeks.

PURPOSE

A question exists as to whether a scleral graft remains in situ, virtually

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FIGURE 6A: Patient with severe retraction of the upper and lower eyelids secondary to thyroid opthal-mopathy. B: Close up view of the patient in Fig 6 A demonstrating 6 mm of scleral exposureabove the limbus and 5 mm of scleral exposure below the limbus. c: The appearance of the

patient in Fig 6 A and 6 B one year postoperatively.

indistinguishable from the surrounding host tissue, or if the scleral graftserves merely as a template or matrix upon which the host sets down itsfibroblastic scar tissue. Previous studies have shown the fate ofscleral graftsin other surgical sites. 52-M4 There is disagreement in the literature concern-ing the absorption of grafted sclera. Some authors have reported no graftresorption.4 Several authors state, however, that there is partial or totalabsorption of the grafted sclera. 12"18, 3Bodian states that the scleral stripsare replaced by host fibrous tissue.34

Periodontal scleral grafts show an ingrowth of connective tissue whichappears to bind the graft to bone and gingiva.52 Nersasian and co-workers'described the fate oforal scleral grafts. They reported that there is minimalhost antigenic response with the exception of areas of granulation tissuesurrounding the graft, probably due to suture material. With time, vesselsand fibroblast-like cells were seen to migrate into the scleral graft suggest-ing that the sclera served as a template for the host tissue.

Sabates et al54 reported that rabbit scleral grafts showed an initial in-flammatory cell and exudative reaction which disappeared within twoweeks. In one month there was an increased infiltration offibroblasts at thesite ofthe graft. By the second month there was no difference between thescleral graft and the host tissue except for fewer fibrocytes in the graft.One report on histologic studies of a scleral graft removed during the

revision of an eyelid arch described the findings in a patient who had ascleral graft four months prior to the date of the biopsy.37 The specimenrevealed fibrous tissue showing vascularization, necrosis and moderate tomarked infiltration of inflammatory cells. Lymphocytes and plasma cellspredominated, suggesting a graft rejection phenomenon.The purpose of this study was to evaluate the fate of grafted scleral

material. The biologic reaction to grafted sclera was studied using rabbitsclera grafted into rabbit eyelids. Since it may be almost impossible tohistologically detect the difference between the scleral graft and fibroblas-tic scar template replacing the graft, it was decided to investigate othermeans to distinguish scleral grafts from host tissue. I thought that the bestmethod of distinguishing sclera grafted into the eyelid was by immunohis-tological techniques.

In order to accomplish this, it was necessary to obtain an antibody tosclera. The preparation of antibodies to sclera dates back many years. In1963, Perkins and Wood' prepared antibodies against various oculartissues from guinea pigs. They indicated that unabsorbed sera to sclerareacted with many tissues ofthe eye and other tissues ofthe body. Follow-ing absorption with normal guinea pig serum, the antiserum to guinea pigsclera lost all of its activity. Their report indicated that it was difficult to

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prepare antibodies against sclera-specific antigens, even though it waspossible to produce antibodies against the immunoglobulins contained inthe sclera. In later reports, Allensmith et all% quantitated the amounts ofimmunoglobulins and albumin in the sclera. A recent report described thefailure to produce antibodies to sclera or demonstrate autoimmunity inrabbits immunized with sclera and adjuvant even though cell-mediatedimmunity was present.57Van der Eerden and Broekhuyse58 showed that the structural glycopro-

teins of the scleral and corneal stroma were immunologically similar, butyet distinct from the structural glycoproteins of the lens capsule. Thesestructural glycoproteins were reported to be the major structural antigenicdeterminants of the ocular connective tissues. In later works, Van derEerden and Broekhuyse59 reported that the antiserum against the structur-al glycoproteins of sclera showed a wide cross reactivity with collagenousconnective tissue, Bowman's capsule of the kidney, synovial membraneand fibrous capsule. This antiserum also had a very strong response to theimmediate surrounding chondrocytes in cartilage, in addition to reactingwith reticular tissue of the pancreas and spleen. The antibodies to scleralstructural glycoproteins reacted with glomerular basement membrane andthe basement membrane of small blood vessels, but not the basementmembranes of the skin, cornea, conjunctival epithelium, lens capsule, orDescemet's membrane. Other studies indicated that after absorption ofantibodies produced against sclera with normal serum, only one antigencould be detected in human scleral homogenates, while two antigens couldbe detected with this antiserum in human corneal homogenates.60

Collagen is another possible antigen in sclera and is present in largeamounts. Recent studies with electron microscopy have shown that thenumber offibers in the sclera are fewer than those found in the cornea. 61 Inaddition, the fibers in the sclera are much larger in diameter (100 nm + 48nm) than they are in the cornea (30 nm + 3 nm). Biochemically, the sclerahas less proteoglycan which is of a different type than that found in thecornea. In 1977, Schmut62 biochemically detected both Type III collagenand Type I collagen in bovine sclera. It is assumed that the larger fibers inthe sclera are Type III collagen, while the smaller fibers in the cornea areType I collagen. Collagen found in scar tissue is believed to be larger thanType I collagen. Since none of the types of collagen described is a majorimmunogen, it is possible that none are involved in the immunohistochem-ical responses observed.

In this thesis I describe the preparation of an antibody to sclera and itsuse in the detection of scleral antigens in grafts of rabbit eyelids for periodsof up to one year.

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FIGURE 7A: Scleral graft biopsied at one week showing suture material (black arrows) and inflammatorycells (white arrow) (Hematoxylin and eosin, x 10). B: Scleral graft biopsied at two weeksshowing suture material (black arrow) and inflammatory cells (arrowheads) (Hematoxylin and

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METHODS AND MATERIALS

PREPARATION OF ANTISERUM

Rabbit sclera was obtained from a freshly sacrificed New Zealand whiterabbit. The eye was enucleated, eviscerated and stripped of episclera andchoroidal pigment. After soaking for three hours in Neosporin solution thesclera was homogenized in 20 volumes of phosphate buffered saline in a"VirTis" homogenizer in a cold room for three hours to insure total disrup-tion of the tissue. The homogenate was filtered through several layers ofsterile gauze and mixed with an equal volume of complete Freund's ad-juvant, forming a suspension.A Hartly strain guinea pig was sensitized with 0.05 ml ofthe suspension

in each hind foot pad and 0.4 ml in each thigh muscle. Intramuscularinjections were repeated with incomplete Freund's adjuvant at two weeksand four weeks. The animal was sacrificed by exsanguination two weeksafter the last challenge. After the serum clotted, it was centrifuged and heatinactivated at 56 C for 30 minutes, followed by absorption with lyophilizednormal rabbit serum (20 mg/ml sera) and rabbit liver acetone powder (20mg/ml sera, obtained from Sigma Chemical Company, St Louis, Mo). Theserum was absorbed at 4 C for four days and the insoluble material wasremoved by centrifugation. The absorbed guinea pig anti-rabbit scleraserum was stored in small aliquots at - 20 C until it was used.

IMMUNOPEROXIDASE TESTING

Rabbit sclera was grafted into the eyelids of anesthetized 2.5 kg NewZealand white rabbits. The data presented in this thesis represents theresults of 15 rabbits bilaterally grafted with rabbit sclera. Scleral biopsieswere obtained at various intervals up to one year and prepared for frozensections. These sections were air dried for one hour and then washed twicefor five minutes in phosphate buffered saline. They were then incubatedwith absorbed guinea pig anti-rabbit sclera serum diluted to 1:20 in phos-

FIGURE 8A: Scleral graft biopsied at one week showing the positively stained sclera (S) and peripherallyinfiltrating peroxidase positive inflammatory cells (arrowheads) at the edge of the graft.Presence of suture material (black arows) is visualized by methyl green counterstain (Im-munoperoxidase stain with absorbed guinea pig anti-rabbit sclera serum and peroxidaseconjugated rabbit anti-guinea pig IgG, heavy and light chain specific, x 10). B: Scleral graftbiopsied at one week showing the negatively stained control for Fig 8 A. Peroxidase positiveinflammatory cells (arrowheads) are seen infiltrating the periphery of the scleral graft (S)(Immunoperoxidase stain with normal guinea pig serum and peroxidase conjugated rabbit

anti-guinea pig IgG, heavy and light chain specific, x 10).

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FIGURE 9A: Scleral graft biopsies at two weeks showing the positively stained sclera (S). Suture material(black arrow) is still present and peroxidase positive inflammatory cells (arrowheads) arepresent throughout the graft (Immunoperoxidase stain with absorbed guinea pig anti-rabbitsciera serum and peroxidase conjugated rabbit anti-guinea pig IgG, heavy and light chainspecific, x 10). B: Scleral graft biopsied at two weeks showing the negatively stained control forFig 9 A. Peroxidase positive inflammatory cells (arrowheads) are present throughout thescieral graft (S) (Immunoperoxidase stain with normal guina pig serum and peroxidase

conjugated rabbit anti-guinea pig IgG, heavy and light chain specific, x 10).

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phate buffered saline for 20 minutes in a humidified chamber. After incuba-tion, the serum was removed and the slides were washed twice for fiveminutes each in phosphate buffered saline. The slides were then dried andincubated with 1:100 diluted horseradish peroxidase conjugated rabbitheavy and light chain specific anti-guinea pig IgG' (Cappel Laboratories,Downingtown, Pa). After 20 minutes, the slides were washed twice for fiveminutes and dipped into diaminobenzadine and H202.The production of the brown chromagen was observed visually and the

reaction was stopped by transferring the slides to phosphate buffered salinewhen the intensity of the positive control was sufficient. The slides werecounterstained with 0.1% methyl green for ten minutes and then washedtwice in phosphate buffered saline, dehydrated, mounted and graded in alight microscope. Photomicrographs were obtained with a Zeiss Photo-microscope III. Controls, consisting ofnormal guinea pig serum, were runwith each assay.

RESULTS

IMMUNOHISTOLOGIC DETECTION OF SCLERA

For orientation purposes, frozen sections of all biopsies were studiedfollowing hematoxylin and eosin staining. Biopsies obtained at varioustimes following grafting were examined for the presence ofscleral antigensusing the guinea pig anti-rabbit sclera serum prepared as described. Dur-ing week one (Fig 7 A) and week two (Fig 7 B) the major histologicobservation was inflammation due to the suture reaction. Immunoperox-idase testing during this time revealed a strong positive coloration in thescleral graft (Fig 8 A). In comparison, the negative control using normalguinea pig serum showed virtually no staining of the sclera (Fig 8 B). Themethyl green counterstain employed in this technique stained the suturematerial, showing the limits ofthe scleral graft. Note the presence ofdarklystained cells in the negative control (Fig 8 B). These infiltrating cells are

FIGURE 10A: Suture material in rabbit lid biopsied at one week showing suture material (black arrows)and inflammatory cells (arrowheads) (Hematoxylin and eosin, x 10). B: Suture in rabbit lidbiopsied at one week showing suture material (black arrows) and peroxidase positive in-flammatory cells (arrowheads). Little staining is observed in this specimen (Immunoperox-idase stain with absorbed guinea pig anti-rabbit sclera serum and peroxidase conjugated rabbitanti-guinea pig IgG, heavy and light chain specific, x 10). c: Suture in rabbit lid biopsied atone week. The suture material was removed by artifact from the space indicated by the blackarrow. Peroxidase positive inflammatory cells (arrowheads) are seen in this negatively stainedcontrol for Fig 10 B (Immunoperoxidase stain with normal guinea pig serum and peroxidase

conjugated rabbit anti-guinea pig IgG, heavy and light chain specific, x 10).

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peroxidase positive and are probably neutrophils known to have endoge-nous peroxidase.The immunohistologic response at two weeks is similar to the first week as

characterized in Figs 9 A and 9 B. The suture material remains, allowingdirect observation ofthe sharp border between the scleral grafts and the lidtissue. During the first week peroxidase positive inflammatory cells werefound only in the periphery of the scleral grafts (Fig 8 A and 8 B). Duringthe second week peroxidase positive inflammatory cells thoroughly infil-trated into the central portion of the scleral graft (Figs 9 A and 9 B).To examine the suture granuloma phenomenon by the immunoperox-

idase technique, a suture was placed in a rabbit lid and biopsied one weeklater. Hematoxylin and eosin staining ofthe suture biopsy (Fig 10 A) clearlyindicated the location of the suture material and demonstrated basophilicinflammatory cells between the suture material. Immunohistological stain-ing ofthis material did not show scleral antigens in this lid biopsy (Figs 10 Band 10 C). The presence of peroxidase positive inflammatory cells at thesite of the suture is readily demonstrated in both Figures 10 B and 10 C.Thus, the lack of a more intense staihi in the rabbit lid with the guinea piganti-rabbit sclera serum, indicated that there are no scleral antigens in thenormal rabbit lid, providing a suitable negative control.

In order to see if scar tissue would react with the guinea pig anti-rabbitsclera antibody, a rabbit lid was denuded with a scalpel blade and allowedto heal for one week. The biopsies which were obtained showed a focalinflammatory reaction with a thickening ofthe epithelium at the site ofthescar (Fig 11 A). Immunohistologic staining ofthe lid with the scar tissue didnot indicate any increased staining with the guinea pig anti-rabbit scleraantibody as compared to the normal guinea pig serum (Fig 11 B and 11 C).Again, peroxidase positive inflammatory cells could be visualized in thesesections.

Additional scleral grafts were biopsied at one month. The lamellar orfibrillar nature of the sclera could still be appreciated at low power mag-nification (Fig 12 A). Immunoperoxidase staining again indicated the pres-

FIGURE 11A: Lid scar biopsied at one week showing thickened epithelium (e) and a focal inflammatoryinfiltrate (white arrow) (Hematoxylin and eosin, x 16). B: Lid scar biopsied at one week. Thereis no difference in the stain intenstiy when compared to the negative control specimen in Fig11 C. Note the peroxidase positive inflammatory cells (arrowheads) (Immunoperoxidase stainwith absorbed guinea pig anti-rabbit sclera serum and peroxidase conjugated rabbit anti-guinea pig IgG, heavy and light chain specific, x 16). C: Lid scar biopsied at one week.Peroxidase positive inflammatory cells (arrowheads) are seen in this negatively stained controlfor Fig 11 B (Immunoperoxidase stain with normal guinea pig serum and peroxidase conju-

gated rabbit anti-guinea pig IgG, heavy and light chain specific, x 16).

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ence of scleral antigens in these preparations (positive response in Fig 12 Bversus the negative control response in Fig 12 C). At this time, peroxidasepositive inflammatory cells were still present in the scleral biopsy but inlower numbers than at two weeks. The suture material also could no longerbe demonstrated. Biopsies taken at two, four, six and eight months aftergrafting, no longer demonstrated suture material or areas of inflammation.Scleral antigens, however, were still present as detected with the guineapig anti-rabbit sclera serum when compared to negative controls (Table I).Ready localization of the scleral grafts was possible one year after graft-

ing, because the sclera was still visible on the inner surface of the lid.Histologically, the scleral grafts could still be distinguished from the lidtissues by the lamellar structure ofthe grafts (Fig 13 A). Even as late as oneyear after grafting, scleral antigens could be detected in this lamellar area(positive response using guinea pig anti-rabbit sclera serum in Fig 13 B ascompared to the normal control using guinea pig serum in Fig 13 C).The serial detection of scleral antigens by the indirect immunoperox-

idase technique showed the greatest amount ofantigen (most intense stain)during the first weeks after grafting. With time, the amount of scleralantigen decreased (less intense stain) but was still visible after one year(Table I).

DISCUSSION

The use of donor sclera in ophthalmic and periodontal surgery has in-creased greatly in the last decade. The major impetus for this increased useis the apparent lack of host response to the grafted sclera. Indeed, asidefrom plasma proteins,5 56 there appear to be only one or two antigens inthe structural glycoproteins of the sclera.58 Some authors suggest thatsclera may not induce antibodies or autoimmunity. ,57

There are many surgical procedures that currently rely on the donorsclera remaining in place and maintaining its integrity for extended periodsof time. Some authors support this contention4 while others contend thatthis sclera is partially or completely absorbed. 12 18,33

FIGURE 12A: Scleral graft biopsied at one month showing the lamellar nature ofthe sclera (S) (Hematoxy-lin and eosin, X 10). B: Scleral graft biopsied at one month showing the positively stained sclera(S) and peroxidase positive inflammatory cells (arrowheads) (Immunoperoxidase stain withabsorbed guinea pig anti-rabbit sclera serum and peroxidase conjugated rabbit anti-guinea pigIgG, heavy and light chain specific, x 10). c: Scleral graft biopsied at one month showing thenegatively stained control for Fig 12 B. Peroxidase positive inflammatory cells are seenthroughout the sclerl graft (S) (Immunoperoxidase stain with normal guinea pig serum andperoxidase conjugated rabbit anti-guinea pig IgG, heavy and light chain specific, x 10).

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In this study I have examined the fate of scleral grafts in the eyelid. Theexperiment was designed to parallel the clinical condition as closely aspossible. Rabbit sclera was grafted into the internal lamina ofrabbit eyelidsusing standardized surgical techniques. The grafted sclera was observedclinically as a white tissue on the surface of the everted lid for a period ofone year. The scleral graft was readily identified by light microscopybecause of its lamellar pattern.

In the first few weeks, inflammatory cells were observed approachingand infiltrating the graft. These cells were predominantly peroxidase posi-tive inflammatory cells. By one month, the number of inflammatory cellsbegan to decline, and after two months, areas of inflammation were notobserved. The probable cause ofthe inflammation was the suture material.The decrease in the inflammatory response paralleled the disappearance ofsuture material after one month.

Since there is no histological method for distinguishing scleral graftsfrom a fibroblastic scar that might replace such a graft, an immunohistologictechnique was utilized in order to demonstrate the presence of scleralantigens remaining at the graft site. Although sclera has been reported tobe a poor immunogen,5M57 antibodies have been made to one or two scleralantigens, exclusive ofthe serum proteins normally found in the sclera.59 Ithas been previously reported that the immunogenic material ofthe sclera isa structural glycoprotein, previously referred to as keratoglycosaminogly-can. ' Antibodies to scleral structural glycoproteins have been found tocross react with tissues of the eye and other parts of the body.59 Theantiserum prepared for these studies reacted in the rabbit sclera as demon-strated by indirect immunoperoxidase testing. Using these techniques,examination ofeyelid grafts of sclera has shown differences in stain intensi-ty between the sclera and the surrounding lid material. This difference canbe utilized for the demonstration of scleral antigens remaining at the graftsite. In the absence of a graft, no staining could be observed in the normallid biopsy, or in the area of the suture granuloma. Thus, even though theantibody to scleral structural glycoprotein is known to cross react with thestructural glycoproteins in other tissues, it was not an interference in thisassay.

FIGURE 13A: Scleral graft biopsied at one year showing the retention of the lamellar nature of sclera (S)(Hematoxylin and eosin, x 10). B: Scleral graft biopsied at one year showing the positivelystained sclera (S) (Immunoperoxidase stain with absorbed guinea pig anti-rabbit sclera serumand peroxidase conjugated rabbit anti-guinea pig IgG, heavy and light chain specific, x 10). C:Scleral graft biopsied at one year showing negatively stained sclera (S) control for Fig 13 B(Immunoperoxidase stain with normal guinea pig serum and peroxidase conjugated rabbit

anti-guinea pig IgG, heavy and light chain specific, x 10).

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TABLE I: IMMUNOHISTOCHEMICAL DETECTIONOF RABBIT SCLERA IN RABBIT EYELIDS

Immunoperox-idase staining

grade (0 to 4 +)Time af- (positive/nega-ter graft Figure nos. tive control)0* 10B & 10C +1.0/+0.51wk 8A&8B +3/+0.52 wks 9A & 9B +2.5/+0.51mo 12B & 12C +2.5/0.52 mos +2.5/+0.56 mos +2.0/+0.58 mos +2.0/+0.51 yr 13B & 13C +1.5/+

*Biopsy of control rabbit eyelid in which nosclera was grafted.

Utilizing the indirect immunoperoxidase technique, scleral antigenshave been found to persist at the site of the graft for at least 12 months.During this time, there was a gradual diminution in the amount of scleralantigens detected, but little histological change in the characteristic struc-ture of the scleral graft as dected by light microscopy.

Since the scar tissue on the denuded eyelid did not stain using thisanti-serum (Fig 11 B), the gradual replacement ofscleral antigens with hostscar tissue might explain the loss of staining intensity recorded over aperiod of one year (Table I). Alternatively, it is possible that the antigeniccomponents of the scleral graft were slowly leached out and not replacedwith similar antigenic components by the host.The observations described in this thesis indicate that allogenic scleral

grafts in the rabbit eyelid are not replaced for a period ofup to a year. Thegraft is not absorbed but becomes an integral part of the eyelid. The fate ofan allogenic scleral graft in the eyelid is in agreement with other reports ofscleral grafts at other sites. 11,52-54

It may not be possible to extrapolate the data to humans. However, thesuccessful results obtained with donor sclera in patients suggest that the lidgrafts retain their strength and structural integrity. This impression hasbeen reinforced by the clinical observation of scleral tissue on the innersurface of an eyelid one year after it had been grafted.

SUMMARY & CONCLUSIONS

The historical considerations, anatomy, pathophysiology and the surgicaltreatment of thyroid lid retraction have been presented. The fate of

Flanagan682


allogenic scleral grafts in rabbit eyelids has been studied by immunohistol-ogy. I have, therefore, been able to reach the following conclusions:

1. Mild thyroid eyelid retraction may be corrected surgically by reces-sing Muller's muscle and conjunctiva in the upper eyelid and by recessingthe conjunctiva and musculofacial retractors of the lower eyelid.

2. Moderate to severe thyroid eyelid retraction may be corrected byusing scleral grafts in combination with the above mentioned procedures.

3. Two millimeters of sclera should be used for each millimeter ofdesired correction because of a tendency toward contracture.

4. Recession of the retractors of the upper and lower eyelids combinedwith scleral grafts may provide long term correction of endocrine lidretraction.

5. Allogenic grafts of sclera in the eyelid of the rabbit were foundclinically to survive for periods of up to one year.

6. After an initial inflammatory cell infiltrate characterized by perox-idase positive inflammatory cells, little histologic change was observed inthe lamellar or fibrillar structure of the scleral graft.

7. Employing an antiserum prepared from a guinea pig against rabbitsclera, scleral antigens could be detected in the graft up to one year aftergrafting.

8. During the course of one year the amount of scleral antigens dimin-ished but at the end ofthe year they could still be detected immunohistolo-gically.

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retraction of the eyelids secondary to thyroid ophthalmopathy-its