1040-5488/15/9203-0324/0 VOL. 92, NO. 3, PP. 324Y328 OPTOMETRY AND VISION SCIENCE Copyright * 2015 American Academy of Optometry

ORIGINAL ARTICLE

Conjunctival Metaplasia after Pterygium Excision and Limbal Autograft Sang Beom Han*, Hee Kyung Yang*, Joon Young Hyon*, and Won Ryang Wee*

ABSTRACT Purpose. To investigate the changes of conjunctival epithelium in the pterygium and donor graft sites after pterygium excision and limbal conjunctival autograft. Methods. This study included 16 eyes of 15 patients who underwent pterygium excision and limbal conjunctival autograft. Epithelial impression cytology specimens of both the pterygium and donor graft were obtained preoperatively and at months 1, 3, and 6, at the pterygium and donor graft sites, respectively. In each specimen, changes in the conjunctival epithelium, including the nucleus-to-cytoplasm (N/C) ratio and goblet cell density (GCD), were evaluated. Morphologic changes in the cells and nuclei were also evaluated. Results. Preoperatively, both N/C ratio and GCD were significantly higher in specimens from the pterygium than in those from the donor site. At both sites, GCD decreased rapidly at 1 month after surgery and then gradually recovered. No significant difference in GCD was found between the two sites at 1, 3, and 6 months postoperatively. Although there was no significant difference in the N/C ratio at 1 and 3 months, the N/C ratio at the pterygium site was significantly greater than that at the donor site at 6 months. At 6 months postoperatively, changes suggesting squamous metaplasia, including elongation of the cells and pyknotic changes in the nuclei, were noted in five samples (31.3%) from the pterygium site but not in any of the samples obtained from the donor site. Conclusions. Conjunctival epithelial metaplasia may return after pterygium removal, which may be associated with the high rate of pterygium recurrence. (Optom Vis Sci 2015;92:324Y328) Key Words: conjunctival epithelium, impression cytology, metaplasia, pterygium

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terygia are pathologic changes to the conjunctiva and peripheral cornea that often result in unwanted cosmetic appearance or reduced best-corrected vision from induced irregular astigmatism. Surgical excision of pterygia is associated with a high rate of recurrence.1,2 In an effort to reduce the rate of pterygium recurrence, adaptations to the surgical technique have been developed. These include the use of postexcision conjunctival autografts, amniotic membrane transplantation, anterior lamellar keratoplasty, and the application of the antimetabolite mitomycin C.1Y6 Autologous limbal tissue grafts have also gained popularity as a method to facilitate the recovery of limbal barrier

*MD Department of Ophthalmology, Kangwon National University Hospital, Chuncheon, Korea (SBH); Department of Ophthalmology, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seongnam, Korea (HKY, JYH); and Department of Ophthalmology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Korea (WRW).

function.1,2,5Y9 In particular, limbal conjunctival autograft is conceivably an effective method of reconstructing both limbus and conjunctival epithelium, and several studies demonstrated its efficacy in reducing the recurrence rate.1,2,7 In addition to the recovery of limbal barrier function, changes in the conjunctival epithelium may also affect the postoperative clinical course of the pterygium. Therefore, investigation of the process of ocular surface healing at a microscopic level is necessary to elucidate the mechanism of ocular surface regeneration after pterygium surgery. Impression cytology is an easy, noninvasive method for obtaining ocular surface epithelial tissue using membrane filters and analyzing histologic features of the ocular surface. This technique is often used to examine the ocular surface epithelium in various ocular diseases including pterygium.10Y12 Previous researchers evaluated the histologic abnormalities of pterygia and conjunctival epithelial healing after pterygium excision using impression cytology10,12,13 and demonstrated that conjunctival epithelial cells on the pterygium showed signs of squamous metaplasia including elongated and enlarged cells with pyknotic

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Conjunctival Metaplasia after Pterygium ExcisionVHan et al.

changes in the nuclei, which lead to increased nucleus-to-cytoplasm (N/C) ratios.12 However, the comparison of preexcision and postexcision epithelial cells using impression cytology and evaluation of how level of metaplasia changed during the postoperative follow-up period have never been performed. Herein, we evaluated the changes in the conjunctival epithelium at the site of pterygium removal as well as the donor site by using impression cytology.

METHODS This study was approved by the Institutional Review Board of Seoul National University Bundang Hospital and adhered to the tenets of the Declaration of Helsinki. Informed consent was obtained from all study participants.

Study Participants This prospective study included 16 eyes of 15 consecutive patients who underwent limbal conjunctival autograft after pterygium excision at Seoul National University Bundang Hospital. All patients were followed up for 6 months or longer. The exclusion criteria included recurrent pterygium, connective tissue disorders, systemic vasculitis, glaucoma, diabetes mellitus, and prior history of vitreoretinal surgery. All surgeries were performed by two surgeons (SBH and JYH). In each case, the pterygium excision and limbal conjunctival autograft were performed under topical anesthesia with 0.5% proparacaine hydrochloride (Alcaine; Alcon Ltd, Fort Worth, TX), as previously described.7 Briefly, the body of the pterygium was dissected and excised using a Wescott scissor, and the head was dissected with a disposable Beaver surgical blade (Rudolph Beaver, Inc; Belmont, MA). Subconjunctival fibrovascular tissue was thoroughly removed. An autologous limbal-conjunctival graft harvested from the superior temporal bulbar conjunctiva was transferred onto the site where the pterygial tissue had been removed. The graft was then anchored to the adjacent conjunctiva with interrupted 8-0 Vicryl sutures, and the limbal margin of the graft was fixed at the limbus of the donor site with a long circumferential interrupted 10-0 nylon suture.

Impression Cytology In each case, the first impression cytology specimen was collected from the surface of the nasal pterygium and the superotemporal donor graft site. Additional samples were obtained from both surfaces at 1, 3, and 6 months postoperatively. In preparation, MF Millipore membrane filters (Millipore Corp, Billerica, MA) with a pore size of 0.22 Km were cut into strips of 5- by 7-mm pieces with a pointed tip in one corner to guarantee an optimal staining surface. By using an ophthalmodynamometer, gentle pressure (60 g) was applied to the filter membrane for 5 seconds. The membrane was then peeled off and placed into Falcon tubes (BD Biosciences, San Jose, CA) containing 95% ethanol.14,15 All specimens were subjected to periodic acid-Schiff staining and then dehydrated in graded sequences of ethanol (70, 80, 90, and 100%, in sequence) and, finally, xylene (80, 90, and 100%, in sequence).14 To evaluate the conjunctival surface, goblet cell densities (GCDs), N/C ratios, and features of conjunctival epithelial cell morphology were assessed using an Olympus BX 51 T-F light

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microscope (Olympus Optical Co Ltd, Tokyo, Japan). The N/C ratio of the nonYgoblet conjunctival epithelial cells was determined by measuring the long axis of the cytoplasm and nucleus under a light microscope at 200 magnification. Mean GCD was determined by averaging the number of goblet cells in five 1- by 1-mm areas defined by a calibrated microscope grid at 100 magnification.14 Regarding cell morphology, special attention was paid to changes suggestive of squamous metaplasia, such as cellular enlargement, elongation, or nuclear pyknosis.12 A single corneal specialist (SBH) masked to the site (superior donor graft site vs. nasal pterygium site) of the specimens evaluated all impression cytology samples.

Statistical Analysis Statistical analysis was performed using SPSS software version 18.0 (SPSS, Inc, Chicago, IL). The Mann-Whitney U test was used to compare N/C ratios and GCD between sites. P values less than 0.05 were considered statistically significant.

RESULTS The study populations comprised six men and nine women, and the mean (TSD) age was 53.6 (T11.1) years (range, 35 to 76 years). Preoperatively, the N/C ratio was significantly higher in specimens from the pterygium sites than in those from donor graft sites. At 1-month postoperatively, the N/C ratio of the specimens from the pterygium site had decreased. The N/C ratios for both sites were similar at 1 and 3 months postoperatively. At 6 months, the N/C ratio had increased among pterygium specimens, leading to a significant difference between specimens from the pterygium versus donor site (Table 1). The preoperative GCD was also significantly higher in specimens from the pterygium site than in those from the donor graft site. At 1 month postoperatively, GCD had decreased rapidly at both sites; GCD recovered at both sites. There was no significant difference in GCD between the sites at 1, 3, or 6 months postoperatively (Table 2). Cells from all 16 specimens obtained preoperatively from the surface of the pterygium exhibited morphologic features of squamous metaplasia, such as marked enlargement or elongation of the cells and pyknosis of the nucleus. In contrast, the cells from donor graft sites exhibited uniform, circular morphology, with no signs of squamous metaplasia. At 6 months after the surgery, changes in cell morphology suggesting squamous metaplasiaVstretching of the TABLE 1.

Comparison of the N/C ratios between the nasal pterygium site and superior donor graft site Nasal area (n = 16) Preoperative Postoperative 1 mo Postoperative 3 mo Postoperative 6 mo

1:4.75 T 1:2.50 T 1:1.63 T 1:1.81 T

0.52 0.37 0.39 0.36

Superior area (n = 16) 1:1.47 T 1:2.47 T 1:1.75 T 1:1.38 T

All data are expressed as mean T SD. *Student t test.

Optometry and Vision Science, Vol. 92, No. 3, March 2015

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0.34 0.34 0.41 0.34

p* G0.001 0.804 0.381 0.001

326 Conjunctival Metaplasia after Pterygium ExcisionVHan et al. TABLE 2.

Comparison of the GCDs between the nasal pterygium site and superior donor graft site Nasal area (n = 16) Preoperative Postoperative 1 mo Postoperative 3 mo Postoperative 6 mo

248.4 T 16.2 33.3 T 5.51 96.6 T 8.8 191.0 T 11.2

Superior area (n = 16) 195.9 33.8 97.1 187.4

T 16.5 T 7.2 T 8.2 T 6.9

p* G0.001 0.805 0.885 0.281

All data are expressed as mean T SD. *Student t test.

epithelial cells and pyknotic changes in the nucleiVwere found in five specimens (31.3%) from the pterygium site. No such changes were found in any specimens from the donor graft site (Fig. 1).

At 6 months postoperatively, no gross sign of corneal involvement or pterygial mass recurrence was visible at the site of excision. In all patients, the epithelial wound at the superotemporal donor graft site healed free of any complication such as limbal dysfunction, persistent conjunctival epithelial defects, or metaplastic changes.

DISCUSSION Previous studies have used impression cytology to show that conjunctival epithelial cells in pterygia exhibit signs of squamous metaplasia and increased GCD.10,12 Chan et al.12 showed that squamous metaplasia was found in the inferior and interpalpebral bulbar conjunctiva as well as the pterygial surface. Tseng et al.10 investigated histologic changes to the conjunctival epithelium of the pterygial excision site and showed that the ocular surface does not regenerate completely, even 1 year after surgery. However,

FIGURE 1. Representative photographs of conjunctival impression cytology. (A) Preoperative specimen from pterygial site. The N/C ratio of the conjunctival epithelial cells is 1:5 to 1:6. Enlargement and elongation of the cells and pyknotic changes in the nuclei are also observed. (B) Preoperative specimen from donor graft site. The N/C ratio is 1:1 to 1:1.5. Cell morphology is normal. (C) Specimen from pterygial site taken at 6 months postoperatively. The N/C ratio is 1:1 to 1:1.5. However, elongation of the cells and pyknotic change of the nucleus are observed. (D) Specimen from donor graft site taken at 6 months postoperatively. The N/C ratio is 1:1 to 1:1.5. There is no abnormality in cell morphology. Magnification: 200 for all photographs. A color version of this figure is available online at www.optvissci.com. Optometry and Vision Science, Vol. 92, No. 3, March 2015

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Conjunctival Metaplasia after Pterygium ExcisionVHan et al.

they did not evaluate the change of the squamous metaplasia after pterygium excision. In the present study, impression cytology revealed increased N/C ratios and GCD suggestive of squamous metaplasia and mucinous hyperplasia in pterygial tissue. These findings are consistent with those reported previously.10,12 Immediately after the surgery, few superficial epithelial cells were observed on the conjunctival graft, which is consistent with previous reports that most of these epithelial cells are devitalized by surgical insult or because of environmental modulation.10 At 6 months after surgery, an increase in N/C ratio, epithelial cell stretching, and nuclear pyknotic changes were observed at the pterygial excision site, which suggests that squamous metaplasia might return at the original pterygial site. Our results also showed a substantial decrease in GCD, followed by a gradual increase in cell number (Table 2). Notably, Tseng et al.10 demonstrated an increased density of nonmitotic epithelial cells in the wound area after pterygial excision. Epithelial regeneration appears to involve the migration of adjacent conjunctival epithelial cells onto the basement membrane of the conjunctival graft. These migrated epithelial cells enlarge and proliferate to form the stratified epithelium.10 Pterygium formation appears to be associated with chronic sunburn damage to the conjunctival epithelium, as pterygial epithelial cells show dyskeratotic changes similar to the histologic changes seen in chronically photodamaged skin cells.13 Therefore, graded dyskeratosis may be observed in the conjunctival epithelium adjacent to the pterygium; migration of the cells conceivably induces seeding of the dyskeratotic cells, potentially leading to regrowth of the original pterygial tissue.12 Chen et al.16 showed that vimentin-bearing epithelioid cells, presumed to be pterygial cells, were found more than 6 mm from the pterygial margin, on normal-appearing conjunctiva. Thus, complete removal of the dyskeratotic cells is conceivably often impossible, and migration of the pterygial cells onto the original pterygium site might not be uncommon. Our observations of squamous metaplasia at the original pterygium site support this assumption. The return of squamous metaplasia may, at least in part, account for the high rate of pterygium recurrence, particularly after simple excision. However, despite these metaplastic changes, invasion of the pterygium onto the cornea was not detected in any of the study participants. It is possibly because restoration of the intact limbal barrier function prevents regrown pterygial cells from invading the cornea. The efficacy of limbal autograft in lowering the rate of pterygium recurrence has been well established.1,2,5Y7 The results presented here also showed that conjunctival epithelium at the donor site regenerated successfully without any complication, suggesting that the risk of harvesting limbal conjunctival autograft tissue from superior conjunctiva might be low.7 The major limitation of this study is that we could collect only conjunctival epithelial cells, owing to the nature of impression cytology. It was not possible to evaluate the deeper conjunctival cells. Therefore, we only evaluated the epithelial changes during the healing process and did not fully describe the process of ocular surface healing. Although a biopsy can provide more information with regard to changes in deep-seated cells, it is impossible to perform such an invasive technique at each postoperative follow-up. Therefore, more advanced techniques, such as confocal microscopy, will be necessary for the evaluation of deeper conjunctival cells. In

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addition, the 6-month follow-up period might be considered short for an evaluation of the clinical course after pterygium excision. However, as 94% of recurrences are reported to occur within 6 months postoperatively, our findings on recurrence and the associated histologic changes remain valid.17 In conclusion, this study demonstrates that squamous metaplasia can recur even after noncomplicated pterygium excision and limbal conjunctival autograft, possibly caused by migration of the adjacent metaplastic conjunctival epithelial cells. Although this phenomenon may be associated with a high rate of pterygium recurrence, limbal reconstruction could potentially reduce the risk of recurrence by preventing metaplastic cells from invading the cornea.

ACKNOWLEDGMENTS The authors have no financial disclosure and declare no conflict of interest in the products or methods used in this study. Received September 11, 2014; accepted December 15, 2014.

REFERENCES 1. Ozer A, Yildirim N, Erol N, Yurdakul S. Long-term results of bare sclera, limbal-conjunctival autograft and amniotic membrane graft techniques in primary pterygium excisions. Ophthalmologica 2009; 223:269Y73. 2. Young AL, Leung GY, Wong AK, Cheng LL, Lam DS. A randomised trial comparing 0.02% mitomycin C and limbal conjunctival autograft after excision of primary pterygium. Br J Ophthalmol 2004; 88:995Y7. 3. Frau E, Labetoulle M, Lautier-Frau M, Hutchinson S, Offret H. Corneo-conjunctival autograft transplantation for pterygium surgery. Acta Ophthalmol Scand 2004;82:59Y63. 4. Al Fayez MF. Limbal versus conjunctival autograft transplantation for advanced and recurrent pterygium. Ophthalmology 2002; 109:1752Y5. 5. Oguz H, Kilitcioglu A, Yasar M. Limbal conjunctival miniautografting for preventing recurrence after pterygium surgery. Eur J Ophthalmol 2006;16:209Y13. 6. Miyai T, Hara R, Nejima R, Miyata K, Yonemura T, Amano S. Limbal allograft, amniotic membrane transplantation, and intraoperative mitomycin C for recurrent pterygium. Ophthalmology 2005;112:1263Y7. 7. Han SB, Hyon JY, Hwang JM, Wee WR. Efficacy and safety of limbal-conjunctival autografting with limbal fixation sutures after pterygium excision. Ophthalmologica 2012;227:210Y4. 8. Dushku N, Reid TW. P53 expression in altered limbal basal cells of pingueculae, pterygia, and limbal tumors. Curr Eye Res 1997; 16:1179Y92. 9. Dushku N, Reid TW. Immunohistochemical evidence that human pterygia originate from an invasion of vimentin-expressing altered limbal epithelial basal cells. Curr Eye Res 1994;13:473Y81. 10. Tseng SH, Chen YT, Cheng HC, Huang FC, Lee SC, Chen FK. Impression cytology study of conjunctival epithelial phenotypes on the healing ocular surface after pterygium excision. Cornea 2001; 20:244Y50. 11. Singh R, Joseph A, Umapathy T, Tint NL, Dua HS. Impression cytology of the ocular surface. Br J Ophthalmol 2005;89:1655Y9.

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328 Conjunctival Metaplasia after Pterygium ExcisionVHan et al. 12. Chan CM, Liu YP, Tan DT. Ocular surface changes in pterygium. Cornea 2002;21:38Y42. 13. Wang IJ, Lai WT, Liou SW, Chiu CZ, Hu FR, Kao WW, Hung PT. Impression cytology of pterygium. J Ocul Pharmacol Ther 2000; 16:519Y28. 14. Kim C, Shin YJ, Kim NJ, Khwarg SI, Hwang JM, Wee WR. Conjunctival epithelial changes induced by cilia in patients with epiblepharon or entropion. Am J Ophthalmol 2007;144:564Y9. 15. Martinez AJ, Mills MB, Jaceldo KB, Tio FO, Aigbivbalu IB, Hilsenbeck SB, Yee RW. Standardization of conjunctival impression cytology. Cornea 1995;14:515Y22. 16. Chen YT, Tseng SH, Tsai YY, Huang FC, Tseng SY. Distribution of vimentin-expressing cells in pterygium: an immunocytochemical study of impression cytology specimens. Cornea 2009;28:547Y52.

17. Chen PP, Ariyasu RG, Kaza V, LaBree LD, McDonnell PJ. A randomized trial comparing mitomycin C and conjunctival autograft after excision of primary pterygium. Am J Ophthalmol 1995; 120:151Y60.

Joon Young Hyon Department of Ophthalmology Seoul National University Bundang Hospital 300 Gumi-dong Bundang-gu Seongnam Gyeonggi 463-707 Korea e-mail: [email protected]

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Conjunctival metaplasia after pterygium excision and limbal autograft.

To investigate the changes of conjunctival epithelium in the pterygium and donor graft sites after pterygium excision and limbal conjunctival autograf...
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