British J7ournal of Ophthalmology 1996;80:911-914

Temporary corneal stem cell dysfunction afterradiation therapy

Hiroshi Fujishima, Jun Shimazaki, Kazuo Tsubota

AbstractBackground-Radiation therapy cancause corneal and conjunctival abnor-malities that sometimes require surgicaltreatment. Corneal stem cell dysfunctionis described, which recovered after thecessation ofradiation.Methods-A 44-year-old man developed acorneal epithelial abnormality associatedwith conjunctival and corneal inflamma-tion following radiation therapy for maxil-lary cancer. He experienced ocular painand loss ofvision followed by conjunctivalepithelialisation of the upper and lowerparts ofthe cornea.Results-Examination of brush cytologysamples showed goblet cells in the upperand lower parts of the cornea, whichshowed increased fluorescein permeabil-ity, and intraepithelial lymphocytes. Im-pression cytology showed goblet cells inthe same part of the cornea. Specularmicroscopy revealed spindle type epithe-lial cells. Patient follow up included artifi-cial tears and an antibiotic ophthalmicointment. The corneal abnormalities re-solved after 4 months with improvedvisual acuity without any surgical inter-vention, but the disappearance ofthe pali-sades ofVogt did not recover at 1 year afterradiation.Conclusion-Radiation therapy in thispatient caused temporary stem cell dys-function which resulted in conjunctivali-sation in a part of the cornea. Althoughlimbal stem cell function did not fullyrecover, this rare case suggested thatmedical options should be consideredbefore surgery.(Br_J Ophthalmol 1996;80:911-914)

Radiation induced corneal epitheliopathy isone of the serious complications of radiationtherapy.' Manifestations of radiation inducedkeratopathy include superficial keratitis, stro-mal clouding, cell infiltration, and oedema ofthe cornea,' and in severe cases perforation ofthe cornea' can occur. Degeneration of thecorneal epithelium, probably due to an irre-versible inhibition of corneal mitosis afterradiation therapy, was reported in experimen-tal models.45

Corneal stem cells are essential to maintainthe epithelial organisation by undergoingcontinuous turnover throughout adult life.6 7The number of cells are maintained via theproliferation of a distinct subpopulation ofstem cells.8

When the stem cells are severely injured, theconjunctival epithelium extends across the cor-neal scleral limbus, creating a thin and irregu-lar surface over the cornea.9 Transdifferentia-tion of the conjunctiva to phenotypicallynormal corneal epithelium has been observedin some animal models and patients.1'° Limbaltransplantation (corneal stem cell transplanta-tion) should be considered"" when the stemcell deficiency is thought to be permanent. Wedescribe here a patient with radiation inducedcorneal epitheliopathy which resolved sponta-neously within 4 months. Clinical course andchange in tear function and ocular surfacecondition are reported.

Patient and methodsA 44-year-old Japanese man with advancedmaxillary cancer of the left parasinus wastreated with radiation from May to June 1993.A total dose of 61 Gy over 44 days (maximumdosage 3 Gy/day) was administrated via a rightangled pair of wedge filtered portals. Threedays after completion of the radiation therapyhe complained of blurred vision and ocularsurface pain in the left eye.The cornea and conjunctiva were evaluated

using slit-lamp examination, including fluores-cein and rose bengal staining. Schirmer's test,the cotton thread test,"5 and the clearance test'6were performed to evaluate the tear dynamics.Central corneal sensation was measured with aCochet and Bonnet aesthesiometer (LuneauOphtalmologie, France).'7 Conjunctival cellswere also evaluated by the brush cytologymethod.'8 Smears containing cellular materialwere spread on glass slides in the usualmanner, and then stained and fixed with aMay-Grunwald stain solution (eosin-methylene blue solution; Muto Pure Chemi-cals Ltd, Tokyo, Japan). Impression cytology

Figure 1 Corneal stem cell dysfunction 3 days aftercompletion of radiation therapy. Upper and lower cornealepithelial cells showed damage, and epithelial opacity wasapparent. The upper and lower parts of the cornea showedfluorescein staining.

Department ofOphthalmology, TokyoDental College, Chiba,JapanH FujishimnaJ ShimazakiK Tsubota

Department ofOphthalmology, KeioUniversity School ofMedicine, Tokyo, JapanH FujishimaK Tsubota

Correspondence to:Hiroshi Fujishima, MD,Department ofOphthalmology, TokyoDental College, 5-11-13Sugano, Ichikawa, Chiba,Japan 272.

Accepted for publication24 May 1996

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Table 1 Clinicalfindings

First day 4 weeks 8 weeks 4 months 6 months I _w

Visual acuity (left eye) 20/500 20/250 20/100 20/100 20/40 20/Pain +++ ++ + - - -Fluorescein stain +++ +++ ++ +Rose bengalstain +++ +++ ++ + ± +Epithelial opacity +++ +++ + +Corneal sensitivity (g/mm2) >17.68 17.68 12.Goblet cell +Palisade ofVogt -

Spindle type cell +

Figwre 2 Examination ofbrush cytology samples. GobletceJls were detected in the upper and lower areas of thecorneal surace.

was also performed using the method ofNelson et al (Schiffs Reagent; Muto PureChemicals Ltd, Tokyo, Japan)."9 Specularmicroscopy (Kowa; Tokyo, Japan) was per-

formed during the initial evaluation for blurredvision and again 12 months later.

ResultsThe previous examination, 6 months beforeinitiation, showed no ophthalmic abnormalitiesof radiation therapy. At the initial examination,the patient's visual acuity was 20/40 in his righteye and 20/500 in the left eye. A slit-lampexamination of the left eye showed corneal epi-thelial opacity, extending from the upper andlower limbus to the central cornea. Increasedfluorescein permeability was detected in thearea (Fig 1). Rose bengal stining was difffuselypositive on the cornea. The palisades of Vogt

* ..~~~~~~~. .o mm g . ... .Figre 3 Periodic acid Schiffsaining in pressn cytoloy at dt onset ofcomplicatioLThe mucin was stained red

(POV)`0 were not observed. Corneal sensationshowed 1.84 g/mm' in the right eye and greaterthan 17.68 g/mm2 in the left. Schirmer's test,the clearance test, and the tear function index(T1FI) were 9 mm, 8 x, and 72 (right eye) and17 mm, 1 x, and 17 (left eye). Results of thecotton thread test were 32 mm (right eye) and40 mm (left eye). The results indicated thattear flow was increased by the irritation but itsdrainage was poor in the left eye. Brush cytol-ogy samples in the upper and lower bulbarconjunctiva showed a few goblet cells includinglymphocytes from inflammatory cells (Fig 2).Impression cytology also revealed goblet cellson the entire cornea (Fig 3). Specular micros-copy in the central corneal epithelium revealedspindle-shaped and enlarged cells (Fig 4).The patient was treated with preservative-

free artificial tears (10 times a day) (SoftSantear eye drop; Santen Pharmaceutical Co,Osaka, Japan) and antibiotic ophthalmic oint-ment (twice a day) (Tarivid eye ointment;Santen Pharmaceutical Co, Osaka, Japan).After 4 weeks of follow up, epithelial opacityand vital stainings did not improve, and visualacuity remained at 20/250 (Fig 5). The patientstill had ocular pain at this time.

After 8 weeks of follow up, epithelial opacityas well as vital staining gradually decreased.The visual acuity of the left eye improved to20/100, and ocular pain was markedly de-creased. Four months after radiation therapy,scattered punctate epithelial staining was seenin the lower part of the cornea (Fig 6). Rosebengal staining was also decreased. Six monthsafter the therapy, the patient no longer hadocular pain and his visual acuity recovered to20/30 in the right eye and 20/40 in the left.Epithelial opacities and fluorescein staininghad completely disappeared; however, slightrose bengal staining in the lower part of thecornea remained. The results of tear functiontests performed 1 year after radiation therapywere as follows: Schirmer's test, clearance test,and TFI were 9 mm, 16 x, and 144 (right eye)and 29 mm, 1 x, and 29 (left eye), and cottonthread test was 35 mm (right eye) and 30 mm(left eye). Visual acuity recovered to 20/25 inthe left eye. Corneal sensation was 1.84 g/mm'(right eye) and it slightly recovered but still was12.84 g/mm' (left eye). No goblet cells weredetected by either brush cytology or impres-sion cytology. Specular microscopy showed nospindle-shaped cells and cell configuration wasnormal (Fig 7). There was no complaint ofocular pain; however, the disappearance of thepalisades of Vogt remained (Fig 8) (Table 1).

DiscussionX ray treatment for parasinus carcinoma deliv-ers a tumour lethal dose to the eyeball, whichmay lead to changes in the cornea andconjunctiva. Acute radiation reactions includetemporary corneal punctate epithelial erosions.Delayed corneal complications may resultdirectly from the effect of radiation or developin association with the dry eye syndrome as aresult ofreduced or absent lacrimal secretions.'A decrease in corneal sensitivity is a typicalearly sign of radiation keratopathy. Corneal

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Temporary corneal stem cell dysfunction after radiation therapy

.|s_.........~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~......Figure 4 Specular microscopy performed at the onset ofcomplicatons of radiation therapy.Spindle-type epitheial cells and large cells were seen.

"-'VJ~

Figure 5 Corneal stem cell dysfunction 60 days aftercomplication of radiation therapy. Slit-lamp examinationshowed a decrease in epithelial opacity. Fluorescein staininghad largely disappeared.

k......~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~....W~~~~~~~~~~~~~~~~~~~~~......_i

Figure 6 Slit-lamp examination 4 months aftercomplication of radiation therapy. Only cornealepithelialitis was seen in lower part of the cornea.

keratopathy, characterised by epithelial andstromal oedema,' has also been observed afterradiation therapy. Electron microscopy has

Figure 7 Specular microscopy performed 1 year after complication of radiation therapy.No specific changes were seen.

Figure 8 Slit-lamp examination 1 year after complicationof radiation therapy. Cornea and conjunctiva were normal.

shown degeneration of the corneal epitheliumprobably due to irreversible inhibition of mito-Sis."We have shown here a case of spontaneous

recovery of radiation induced corneal epithe-liopathy, with the presence of goblet cellswhose permeability to fluorescein remarkablyimproved within 6 months. Since the cornealsensation, tear function, and disappearance ofPOV did not fully recover during the follow up,the improvement of the corneal epithelium wasnot due to the recovery of the sensation or tearfunction. Thus, we present this case as a 'tem-porary corneal stem cell dysfunction', whichrecovered gradually after the cessation ofradiation.Stem cells are a distinct subpopulation of

basal cells located in various epithelial tissues.Corneal epithelial stem cells are believed to belocated in the basal cell layer of the peripheryof the cornea, the transition zone between thecorneal and conjunctival epithelium, and thecorneal limbus.212' Evaluation of POV'0 21 is ahallmark of the presence of stem cells. Undernormal conditions stem cells, conjunctival epi-thelial cells, and vessels do not penetrate thecornea and corneal clarity is maintained. Whenthe cornea is injured, active cellular renewaland differentiation can contribute to woundhealing.24 The concept that limbal stem celldysfunction includes conjunctivalisation wasfirst proved by Tseng et al in serial experimen-tal studies, and has been further confirmed byimpression cytology in a recent report." 11 24 25

Corneal stem cell dysfunction leads to cornealepithelial cell dysfunction. Conjunctival cellinvasion leads to a decrease in visual acuity. Inthe present case, the presence of goblet cells inthe cornea indicated that conjunctival cells hadinvaded the cornea and that epithelial cells hadbecome dysfunctional. The recovery wasthought to be due to the residual stem cells. Asthe temporal and nasal cornea were still clearand stem cells in these parts of the cornea stillremained and functioned, this keratopathycould recover spontaneously. Other conditions,such as thermal burns, alkaline burns, chemi-cal burns, contact lens injury, vernal conjuncti-vitis and radiation, may damage entire limbalstem cells so that the corneal epitheliumcannot be repopulated. Radiation may alsoaffect the mitotic rate of undifferentiated stemcells. The corneal abnormalities in the presentcase may have been related to temporary stem

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cell dysfunction, inhibition of epithelial mito-sis, goblet cell hyperplasia, or a combination ofthese factors.25

After 8 weeks to 6 months of follow up, cor-neal epithelial cells covered the entire cornea.No spindle-type cells were present, suggestingthat the rate of mitosis of corneal epithelialcells or stem cells may have recovered. A totaldaily dose of 3 Gy, with a standard fractiona-tion of 10 Gy per week in five fractions (2 Gyper treatment session), is considered safe forexternal beam radiation. For malignant tu-mours of the maxilla, a megavoltage dose of 60Gy or more is performed.22 However, thesedoses were determined using a low energy 220kV machine, which has still a higher incidenceof complications than contemporary radio-therapeutic machines.26 The onset of cornealchanges after radiation therapy varies from afew days to a year.'27

Initial treatment of radiation related cornealabnormalities aims to remove any noxious ele-ments that may cause further tissue damage.Specific treatment depends on the seriousnessof the condition. Preservative-free eyedropsmay be useful because tear dysfunction bringsabout these cases. Also, ointment and/or medi-cal use contact lenses may be appropriate for amild case. In a serious advanced case, limbalallo- or autograft transplantation must beconsidered.`2-4 While the limbal stem cellfunction did not fully recover at 1 year afterradiation, the present case showed clinicalrecovery of corneal and conjunctival abnor-malities within several months, suggesting thatmedical options should be explored before sur-gery is considered.

The authors thank Ms Yukiko Yagi for performing the brushcytology, Ms Saori Nishijima for taking slit photographs, andKe-Ping Xu, MD, for performing impression cytology.

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15 Sakamoto R, Bennet ES, Henry VA, Paragina S, Narumi T,Izumi Y, et al. The phenol red thread tear test: across-cultural study. Invest Ophthalmol Vis Sci 1993;34:3510-4.

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