Journal of Refractive Surgery

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Light Microscopic and Ultrastructural Findings in Failed Epikeratoplasty

Hans E Grossniklaus, MD; Jonathan H Lass, MD; Gretta Jacobs, MD; Curtis E Margo, MD; Kevin M McAuliffe, MD

Abstract

ABSTRACT: Corneal buttons from two patients with failed epi keratoplasties requiring penetrating keratoplasty were examined by light and transmission electron microscopy. Penetrating keratoplasty was performed for lenticule scarring In the first case (adult aphakia) and inadequate best corrected visual acuity in the second case (keratoconus). Findings included decreased lenticule keratocytes and electron-dense, fibrillogranular material associated with either lenticule or recipient keratocytes in both corneas and defects in Bowman's layer in one cornea. In addition, light and electron microscopic evidence consistent with recipient keratocyte migration into the lenticule was demonstrated in one case. These findings were compared with three previous cases. [Refractive and Corneal Surgery. 1989; 5:296-301.]

Abstract

ABSTRACT: Corneal buttons from two patients with failed epi keratoplasties requiring penetrating keratoplasty were examined by light and transmission electron microscopy. Penetrating keratoplasty was performed for lenticule scarring In the first case (adult aphakia) and inadequate best corrected visual acuity in the second case (keratoconus). Findings included decreased lenticule keratocytes and electron-dense, fibrillogranular material associated with either lenticule or recipient keratocytes in both corneas and defects in Bowman's layer in one cornea. In addition, light and electron microscopic evidence consistent with recipient keratocyte migration into the lenticule was demonstrated in one case. These findings were compared with three previous cases. [Refractive and Corneal Surgery. 1989; 5:296-301.]

The histologic findings of the lenticule following epikeratoplasty and its relationship to the recipient bed has received little attention.13 Defects in recipient Bowman's layer,1-3 decreased lenticule keratocytes,13 electron-dense material at the lenticulerecipient interface,13 and variable electron-dense material associated with lenticule or recipient keratocytes have been reported.

Our report provides light and electron microscopic findings in two additional corneal buttons with epikeratoplasty lenticules. Our findings show that defects in recipient Bowman's layer are present prior to epikeratoplasty and confirm the observation of electron-dense material of unknown significance associated with recipient keratocytes, donor keratocytes, or both. In addition, histopathologic evidence consistent with recipient keratocyte migration into the lenticule following human epikeratoplasty is demonstrated, confirming previous evidence of lenticule repopulation by host keratocytes.4

CASE REPORTS

Case One

Clinical History

A 49-year-old man underwent epikeratoplasty of the left eye with a lyophilized, cryolathed lenticule (American Medical Optics, Irvine, Calif) for monocular aphakia and contact lens intolerance. His past history included proliferative diabetic retinopathy, bilateral scatter laser photocoagulation, and an extracapsular cataract extraction 2 years previously. His best corrected visual acuities were 20/30 in the right eye and 20/100 in the left eye. Postoperatively, a persistent epithelial defect was present over the inferior central cornea that failed to heal despite artificial tears, topical epidermal growth factor solution (Chiron Ophthalmics, Irvine, Calif) 1 mg/ml two drops four times a day for 1 month, a bandage soft contact lens, and a lateral tarsorraphy. The defect finally healed 4 months postoperatively after a 1 month course of fibronectin solution (Chiron) 3.5 mg/ ml two drops four times a day. A residual central scar in the lenticule and what was believed to be in the recipient stroma limited best corrected visual acuity to count fingers at 6 feet in the left eye (Figure 1). A penetrating keratoplasty was performed 7 months after the epikeratoplasty.

Pathologic Findings - Light Microscopy

Examination of the corneal button disclosed a recipient cornea with peripheral trephination wounds and an overlying lenticule. The center of the recipient cornea measured 0.56 mm and had a 0.36 mm overlying lenticule (the lenticule comprising approximately 40% of total thickness) (Figure 2). The peripheral lenticule trephination wounds were 45% and 60% of the total corneal thickness. There were subepithelial bullae, basal epithelial edema, absence of Bowman's layer, and stromal scarring in the lenticule in the area corresponding with the scar seen clinically. Keratocytes were concentrated in the anterior, peripheral, and most posterior portion of the lenticule with a relative absence of keratocytes centrally. Bowman's layer was intact in the recipient, except at trephination sites. Descemet's membrane was intact and there were 12 endothelial cells per high power field.

Figure 1: Patient one. Clinical appearance 4 months after surgery with scarring of the epikeratoplasty lenticule.Figure 2: Patient one. A) Corneal button (Masson's trichrome, original magnification ? 4). B) Corresponding diagram.

Figure 1: Patient one. Clinical appearance 4 months after surgery with scarring of the epikeratoplasty lenticule.

Figure 2: Patient one. A) Corneal button (Masson's trichrome, original magnification ? 4). B) Corresponding diagram.

Pathologic Findings - Electron Microscopy

Transmission electron microscopy disclosed features consistent with those seen by light microscopy with the additional features of rare areas of fibrillogranular material near lenticule keratocytes and foci of wide-spaced collagen at the lenticulerecipient trephination interface. At the trephination site adjacent collagen lamellae from the recipient and lenticule were perpendicular and in one area recipient keratocytes appeared to be migrating into the lenticule (Figures 3 & 4). Keratocytes in the center of the lenticule contained more degenerative changes than in the recipient.

Figure 3: Patient one. A) Keratocytes (arrow) are present anterior to cut margin of recipient Bowman's layer (arrowhead). Lenticule stroma contains relatively more keratocytes than recipient in this particular field; however, there was an overall decrease in the number of keratocytes in the lenticule, as compared to the recipient stroma (PAS, original magnification > 700). B] Electron micrograph of cut edge of recipient Bowman's layer (asterisk) corresponding with (A). Lenticule keratocytes (arrow) are draped over the edge of Bowman's layer. Electron-dense material is associated with a lenticule keratocyte (bracketed area) (original magnification ? H 700). C) Higher magnification of bracketed area shown in (B) demonstrates fibrillogranular material (arrowhead) associated with a lenticule keratocyte (asterisk) in surrounding collagen matrix, (original magnification ? 35 000).

Figure 3: Patient one. A) Keratocytes (arrow) are present anterior to cut margin of recipient Bowman's layer (arrowhead). Lenticule stroma contains relatively more keratocytes than recipient in this particular field; however, there was an overall decrease in the number of keratocytes in the lenticule, as compared to the recipient stroma (PAS, original magnification > 700). B] Electron micrograph of cut edge of recipient Bowman's layer (asterisk) corresponding with (A). Lenticule keratocytes (arrow) are draped over the edge of Bowman's layer. Electron-dense material is associated with a lenticule keratocyte (bracketed area) (original magnification ? H 700). C) Higher magnification of bracketed area shown in (B) demonstrates fibrillogranular material (arrowhead) associated with a lenticule keratocyte (asterisk) in surrounding collagen matrix, (original magnification ? 35 000).

Figure 4. Patient one. A) Trephination wound (arrowheads) Interface of recipient stroma on left and donor stroma on right. Note apparent migration of plump keratocytes (arrow) between recipient and lenticule (Masson's trichrome, original magnification ? 700). B) Electron micrograph of recipient lenticule interface (arrowheads corresponding to area shown In (A). Note pfump keratocyte (arrow) and apparent migration between recipient keratocyte on left and lenticule keratocyte on right (bracketed area) (original magnification ? 7800). C) Higher magnification of bracketed area shown in (B) shows keratocyte in recipient (arrowhead) is In contact with a keratocyte in the lenticule (arrow) via an opening in the collagen lamellae, (original magnification ? 24 000).

Figure 4. Patient one. A) Trephination wound (arrowheads) Interface of recipient stroma on left and donor stroma on right. Note apparent migration of plump keratocytes (arrow) between recipient and lenticule (Masson's trichrome, original magnification ? 700). B) Electron micrograph of recipient lenticule interface (arrowheads corresponding to area shown In (A). Note pfump keratocyte (arrow) and apparent migration between recipient keratocyte on left and lenticule keratocyte on right (bracketed area) (original magnification ? 7800). C) Higher magnification of bracketed area shown in (B) shows keratocyte in recipient (arrowhead) is In contact with a keratocyte in the lenticule (arrow) via an opening in the collagen lamellae, (original magnification ? 24 000).

Case Two

Clinical history

A 36-year-old man underwent epikeratoplasty of the right eye with a cryolathed, lyophilized lenticule (American Medical Optics) for keratoconus. His past history included obesity, blepharophimosis, and bilateral keratoconus with progression of the cones, scarring, and contact lens intolerance (Figure 5). His preoperative best corrected visual acuities were 20/50 in the right eye and 20/30 in the left eye. Postoperatively, the corneal scar noted preoperatively extended into the visual axis. The patients best corrected postoperative visual acuity was 20/50 in the right eye. A penetrating keratoplasty was performed 1 year later.

Pathologic findings - Light microscopy

Examination of the corneal button disclosed a recipient cornea with the edges corresponding with previous trephination wounds. The center of the recipient cornea measured 0.12 mm and had a 0.36 mm overlying lenticule (comprising 75% of the total corneal thickness) (Figure 6). The peripheral edges of the corneal button apparently did not contain the deepest portion of the epikeratoplasty trephination wounds. Keratocytes were concentrated in the anterior and peripheral lenticule with absence of keratocytes centrally. There were multiple breaks in Bowman's layer in the recipient with associated scarring (Figure 7) and central thinning of the recipient stroma. Twelve endothelial cells per high power field were present.

Pathologic findings - Electron microscopy

Transmission electron microscopy disclosed features consistent with those seen by light microscopy with the additional features of electron-dense granular material near recipient keratocytes and focally disoriented collagen lamellae in the host stroma. Descemet's membrane and endothelium were normal.

Table

TABLEClinicopathologic Studies of Failed Epi keratoplasty

TABLE

Clinicopathologic Studies of Failed Epi keratoplasty

Figure 5: Patient two. Preoperative appearance of recipient cornea with keratoconus. Note the apical scar.

Figure 5: Patient two. Preoperative appearance of recipient cornea with keratoconus. Note the apical scar.

DISCUSSION

Histopathologic and experimental studies have suggested lenticule repopulation by donor keratocytes in animal models and human corneal buttons following epikeratoplasty.46 Our study has demonstrated light microscopic and ultrastructural evidence consistent with keratocyte migration from the recipient bed into the lenticule (case 1, Figure 4). This was evidenced by plump keratocytes at the trephination interface that appeared to be migrating from the recipient to the donor.

An overall decrease in the number of keratocytes in the lenticules in both cases was not unexpected considering the length of time following surgery (Figure 8). Cryopreservation has been shown to kill keratocytes7; lenticules in place as long as 3.5 years have been noted to not be fully repopulated with keratocytes.3 Our two cases, however, differed in one major respect. Epidermal growth factor may have exerted a trophic effect in the first case, since there appeared to be enhanced repopulation of the central lenticule (Figure 8); however, other factors, such as viability of host keratocytes, may play a role.810

Figure 6: Patient two. A) Corneal button (Masson's trichrome, original magnification ? 4). B) Corresponding diagram.

Figure 6: Patient two. A) Corneal button (Masson's trichrome, original magnification ? 4). B) Corresponding diagram.

Figure 7: Patient two. A) Epikeratoplasty lenticule stroma overlies recipient Bowman's layer with defect (arrow) and stroma scar (asterisk). Recipient stroma, Descemet's membrane, and endothelium are present posterior to Bowman's layer (pediatric acidSchift original magnification ? 350). B) Irregular collagenous scar in recipient stroma corresponds with scar shown in (A) (original magnification ? 15 600).Figure 8: Patient one A) Corneal button (hematoxylin & eosin, original magnification ? 150) and B) corresponding diagram. Patient two C) Corneal button (hematoxylin & eosln, original magnification ? 150). D) Corresponding diagram. Relatively more keratocytes are noted In the anterior stroma and lentlcule-recipient interface In patient one than patient two.

Figure 7: Patient two. A) Epikeratoplasty lenticule stroma overlies recipient Bowman's layer with defect (arrow) and stroma scar (asterisk). Recipient stroma, Descemet's membrane, and endothelium are present posterior to Bowman's layer (pediatric acidSchift original magnification ? 350). B) Irregular collagenous scar in recipient stroma corresponds with scar shown in (A) (original magnification ? 15 600).

Figure 8: Patient one A) Corneal button (hematoxylin & eosin, original magnification ? 150) and B) corresponding diagram. Patient two C) Corneal button (hematoxylin & eosln, original magnification ? 150). D) Corresponding diagram. Relatively more keratocytes are noted In the anterior stroma and lentlcule-recipient interface In patient one than patient two.

We compared other histopathologic findings of our cases with three previously reported cases (Table).1'3 There were no defects in recipient Bowman's layer in our case 1; however, they were present in case two and the three previously reported cases. Two of the previously reported cases and our case 2 were epikeratoplasties over corneas with keratoconus and the remaining previously reported case may have had trauma and a defect in recipient Bowman's layer prior to surgery.13 A "frayed" appearance of recipient Bowman's layer is typical in keratoconus11 and was present in our case 2. Therefore, it is likely that the three previously reported cases and our case 2 had defects in recipient Bowman's layer prior to epikeratoplasty secondary to keratoconus or trauma. One study suggested that defects in recipient Bowman's layer may enhance keratocyte migration into lenticules.3 Our findings suggest that defects in recipient Bowman's layer do not necessarily enhance migration, since there were more lenticule keratocytes in our case 1 (with an intact recipient Bowman's) than case 2 (with defects in recipient Bowman's).

Electron-dense material anterior to recipient Bowman's layer, present in two of the three previous cases was absent in our cases.1,3 Perhaps basement membrane-like material from denuded recipient epithelium contributed to the appearance of the electron-dense material. Electron-dense material was also reported to be present around lenticule and recipient keratocytes.1-3 Electron-dense material was associated with keratocytes in the lenticule in our case 1 and recipient keratocytes in case 2. This material may be a product of abnormal keratocyte metabolism; however, its significance is unknown and has no known clinical relevance.

Our two cases along with previous studies provide evidence of lenticule repopulation with keratocytes from the donor bed. This process is variable and may take years for complete repopulation to occur. Further studies may elucidate the nature of the electrondense material associated with keratocytes and the mechanism of population of the lenticule with viable keratocytes.

REFERENCES

1. Binder PS, Baumgartner SD, Fogle JA. Histopathology of a case of epikeratophakia (aphakic epikeratoplasty). Arch Ophthalmol. 1985; 103:1357-1363.

2. Goodman GL, Peiffer RL, Werblin TP. Failed epikeratoplasty for keratoconus. Cornea. 1986; 5:29-34.

3. Jaeger MJ, Berson P, Kaufman HE, Green, WR. Epikeratoplasty for keratoconus. A clinicopathologic case report. Cornea. 1987; 6:131-139.

4. Baumgartner SD, Binder PS. Refractive keratoplasty. Histopathology of clinical specimens. Ophthalmology. 1985; 92:1606-1615.

5. Yamaguchi T, Koenig SB, Kimura T, Werblin TP, McDonald MB, Kaufman HE. Histological study of epikeratophakia in primates. Ophthalmic Surg. 1984; 15:230-235.

6. Samples JR, Binder PS, Zavala EY, Deg JK, Baumgartner SD. Epikeratophakia: clinical evaluation and histopathology of a non-human primate model. Cornea. 1984; 3:51-60.

7. Zavala EY, Binder PS, Deg J, Baumgartner SD. Refractive keratoplasty. Lathing and cryopreservation. CLAO J. 1985; 11:155-162.

8. Binder PS, Wickham MG, Zavala EY, Akers PH. Corneal anatomy and wound healing. In Transaction - New Orleans Academy of Ophthalmology: Symposium on medical and surgical diseases of the cornea. St Louis, Mo: CV Mosby Co; 1980:1-35.

9. Suda T, Nishida T, Oshashi Y, et al: Fibronectin appears at the site of corneal stromal wounds in rabbits. Curr Eye Res. 1981; 1:553-556.

10. Brightwell JR, Riddle SL, Eiferman RA, et al. Biosynthetic human EGF accelerates healing of neodecadron-treated primate cornea. Invest Ophthalmol Vis Sci. 1985; 26:105-110.

11. Spencer WH: Cornea. In: Spencer WH ed. Ophthalmic Pathology. An Atlas and Textbook. Philadelphia, Pa: WB Saunders Co; 1985:229-388.

TABLE

Clinicopathologic Studies of Failed Epi keratoplasty

10.3928/1081-597X-19890901-06

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