Delayed Corneal Wound Healing Following Radial Keratotomy

Delayed Corneal Wound Healing Following Radial Keratotomy JANET K. DEG, AA, EDWARD Y. ZAVALA, BS, PERRY S. BINDER, MD, FACS Abstract: Four human corneal specimens obtained 5 to 47 months following radial keratotomy were evaluated by correlative microscopy. Repeat radial keratotomies had been performed in two cases. We documented gaping keratotomy incisions, epithelial plugs, and epithelial-lined incisions. In all cases, Bowman's layer was malapposed with slight to moderate fibroblastic activity at the incision sites. Epithelial and endothelial radial ridges were seen in two cases. No endothelial damage was seen under the radial incisions. The morphological evaluation of these specimens show the potential for a poor wound-healing response when corneas with previous corneal surgery and/or pathologic states undergo radial keratotomy. [Key words: corneal stroma, corneal wound healing, epithelium, keratotomy, radial keratotomy, refractive corneal surgery.] Ophthalmology 92:734-740, 1985 The radial keratotomy procedure is designed to correct myopia by flattening the corneal curvature. The procedure was first developed by Sato l and was subsequently modified by Fyodorov.2 Since the introduction of radial keratotomy to the United States in 1978, the procedure has become increasingly popular. Early techniques used optical pachymetry of the central cornea to determine incision depth. Sixteen, 24, or 32 incisions combined with optical zones as small as 2.5 mm were performed using razor-blade fragments. Incisions were directed through the corneal limbus beginning in the paracentral cornea. 2,3 Laboratory and clinical studies have demonstrated that optical zones 3.0 mm or smaller and up to 16 deep radial incisions with sparing of the limbus can result in a signifcant decrease in myopia. 4,5 However, the possibility of endothelial damage occurs with deeper incisions. Postoperative glare occurs more frequently with smaller optical zones. 6 - 1O Micrometer handles for razor-blade From the Ophthalmology Research Laboratory, Sharp Cabrillo Hospital, San Diego, California. Supported in part by a grant from the San Diego Eye Foundation and National Eye Institute grant EY 04557-03 A 1. Presented at the Eighty·ninth Annual Meeting of the American Academy of Ophthalmology, Atlanta, Georgia, November 11-15, 1984. Reprint requests to Perry S. Binder, MD, 9834 Genesee Avenue, Suite 200, La Jolla, CA 93037. 734 knives and, more recently, for diamond knives have produced more predictable wounds and reduced stromal scarring. II ,12 The use of ultrasonic pachymetry has improved incision-depth predictability and the mean correction achieved. 4 With these new techniques, the visual and ocular complications (Table 1) following radial keratotomy have been reported to be decreasing. To date, one corneal button has been studied following radial keratotomyY One separate biopsy specimen documented an epithelial cyst in a radial keratotomy wound. 14 We now report a morphologic study of four specimens obtained following radial keratotomy. MATERIALS AND METHODS Three full-thickness corneal buttons obtained at keratoplasty and one lamellar corneal specimen were fixed in 1% glutaraldehyde/ 1% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4, 340 mOsm). The specimens were postfixed in 2% osmium tetroxide in 0.1 M phosphate buffer for one hour, then divided and prepared for correlative microscopy. In one case, an additional piece was taken for paraffin histology and fixed in 10% neutral buffered formalin. Specimens intended for scanning electron microscopy were dehydrated in a series of graded ethanol from 50 to 90% for five minutes each, then put through two DEG, et al • DELAYED CORNEAL WOUND HEALING Table 1. Visual and Ocular Complications Reported with Radial Keratotomy Visual Complications Undercorrection and overcorrection Astigmatism Rebound Glare + photophobia Nyctalopia Poor quality of vision Presbyopia Vision fluctuation Ocular Complications Decreased sensation Recurrent erosions Epithelial inclusion cysts Anterior synechiae Limbal "peaks" Peripheral pannus Intrastromal abscess Vascular ingrowth Corneal scarring Endothelial cell loss Cataract Endophthalmitis Fig 1. Clinical photograph of a 32-incision radial keratotomy procedure (case 3). The paired incisions are easily visualized. changes of 100% ethanol for ten minutes each and critical-point dried in a Tousimis Samdri-790 dryer using liquid CO 2 , sputter coated with gold/palladium, and examined using a Hitachi S-520 scanning electron microscope at 20 KV.15 Specimens intended for transmission electron microscopy and light microscopy were dehydrated in a graded series of ethanol followed by propylene oxide and embedded in polybed 812. Two-micron sections were stained with toluidine blue O. The paraffin-embedded specimen was stained with periodic acid Schiff. All light microscope sections were viewed arid photographed using an Olympus Vanox light microscope. A calibrated Olympus reticule at xl 0 magnification was used to measure corneal thickness and incision depth. Reported incision depth was measured as a percent of total corneal thickness. CASE REPORTS Case 1. A 42-year-old physician with a history of keratoconus had an 8-mm corneal transplant in 1973. Postoperatively, the cornea developed significant astigmatism. In January 1980, a radial keratotomy procedure with a 2.5-mm optical zone was performed using razor-blade knife incisions. Six standard radial incisions were combined with five parallel incisions in the steep meridian on both sides of the donor through the recipient cornea to the limbus. One incision was made perpendicular to each of these five incisions. The patient subsequently developed a decrease in best corrected vision, photophobia, and lacrimation. Five and a half months after the procedure, an 8.5-min corneal transplant was performed. This case has been previously published. l3 Case 2. A 46-year-old man with keratoconus, central apical scarring, and best corrected visual acuity of 20/70 underwent a radial keratotomy procedure in April 1983. The procedure was performed using a diamond knife to make eight radial incisions with a 3-mm optical zone. Postoperatively, the cornea remained clear, but the apical scarring resulted in best corrected vision of 20/60 + 2. Uncorrected visual acuity data was unavailable. In October 1983, six months following the radial keratotomy procedure, a penetrating keratoplasty was performed. At surgery, the keratotomy incisions in the recipient cornea were noted to separate easily, which necessitated interrupted 10-0 monofilament nylon sutures to close gapping peripheral host incisions during the procedure. The excised corneal button was placed in M-K medium and left at room temperature for 48 hours before fixation for correlative microscopy. Case 3. A 43-year-old man underwent a 16-incision radial keratotomy in December 1979 using a 3-mm optical zone and razor-blade fragment ihcisions. Four months later in April, 1980, a second 16-incision procedure was again performed on the same eye because of undercorrection (Fig 1). Following the second procedure, the patient complained of photophobia, glare, fluctuation in visual acuity, and loss of best corrected acuity from 20/20 to 20/50. On November 20, 1983, 43 months following the second l6-incision procedure, the patient underwent an 8-mm corneal transplant. At the time of surgery, the peripheral host keratotomy incisions separated easily but did not require suturing. The best corrected acuity in January 1985 was 20/30. Case 4. A 43-year-old man underwent an eight-incision radial keratotomy procedure in February 1982 for correction of myopia using razor-blade fragments. Two months later, in April 1982, eight additional incisions were performed due to an undercorrection. The eye remained myopic after the second procedure. The patient underwent a homoplastic myopic keratomileusis procedure in November 1983, 19 months following the second radial keratotomy procedure. The best corrected visual acuity prior to the keratomileusis procedure was 20/40. The keratomileusis specimen was removed from the patient and was initially placed in KM-26 cryoprotective solution and lathed. The lenticle fragmented along the gapping radial incisions from the previous surgeries. The corneal specimen was then placed in fixative, and a donor cornea was used in its place. The best acuity one year after this third procedure was 20/60. RESULTS Case 1: scanning electron microscopy of the cornea 5.5 months postoperatively demonstrated a rough and scarred epithelial surface. Some incisions were marked by a raised epithelial ridge, while others were indistinct. The epithelial cell borders were vague; surface microvilli 735 OPHTHALMOLOGY • JUNE 1985 • VOLUME 92 • NUMBER 6 Fig 3. Left. gaping radial incision six months postoperatively lined with epithelial cells (case 2). Note slight fibroblastic activity beneath incisions (light microscopy, Toluidine blue 0, X65). Right, an epithelial plug remaining in an incision. Note poor apposition of Bowman's layer and slight fibroblastic activity under the incision (light microscopy, Toluidine blue 0, X65). Fig 2. Scanning electron microscopy of an eight radial incision procedure (case 2). Note peripheral gaping wounds six months postoperatively (X35). were not seen. Posterior radial ridges under each incision were visible, but no endothelial cell loss or damage could be directly associated with these ridges. Centrally, the endothelial cells appeared swollen and pleomorphic with pitted borders. The peripheral endothelial cell junctions became increasingly difficult to visualize. Light microscopy revealed epithelial plugs as large as 15 cell layers deep and II cell layers wide. A thin epithelial cell layer with poor differentiation was found between incision sites. Bowman's layer was fractured and severely misaligned at each wound. Approximate depth of the incisions measured at the periphery was 16.5% of the total cornea thickness, 60% at mid-incision, and 77% paracentrally. Disrupted lamellae and moderate fibroblastic activity were seen directly below the epithelial plugs and along the length of each incision. The stromal keratocytes appeared normal. Superficial vascularization along the incision at the 5 o'clock and 11 o'clock meridians was noted clinically at the time of the penetrating keratoplasty, but the neovascularization was not visible in cross section. Transmission electron microscopy demonstrated a well-developed basement membrane underlying the ep- ithelial plugs. The epithelial cells at the bottom of the plugs had a normal complement of cytoplasmic organelles, desmosomes, and hemidesmosome attachments. Fibroblasts at the base of the plugs contained an increased number of mitochondria and extensive rough endoplasmic reticulum. Endothelial cells under the incisions exhibited cytoplasmic vacuoles and an increased number of mitochondria. Case 2: the corneal epithelial surface six months postoperatively had gaping wounds at the peripheral edge of the radial incisions (Fig 2). Centrally, the radial incisions appeared to be healed. The epithelial surface had normal-appearing microvilli with distinct cell borders. Histologically, there was a thin, poorly differentiated epithelial layer, possibly due to cell migration during storage in M-K medium at room temperature before fixation. Some radial incisions appeared as deep troughs lined with epithelium, while epithelial plugs remained in others (Fig 3). A discontinuous basement membrane underlying the epithelium was seen, suggesting the epithelial plugs were present prior to the penetrating keratoplasty (Fig 4). Disrupted lamellae identified the depth of the radial incision to be up to 90% of the corneal thickness at the periphery, 78% at mid-incision, and 77% paracentrally. Only slight fibroblastic activity was seen along the incisions (Figs 3,4). Posterior corneal ridges were not present under the incisions; however, isolated areas of damaged endothelial cells were seen. The endothelium, overall, appeared swollen and pleomorphic with overlapping intercellular borders, possibly an artifact of the extended M-K medium storage. Case 3: 47 months following the radial keratotomy, the epithelial surface was well healed with prominent, raised epithelial ridges delineating approximately half of the 32 radial incisions (Fig 5). Prominent epithelial nuclei, dense surface microvilli, and distinct cell borders DEG; et al • DELAYED CORNEAL WOUND HEALING Fig 4. Epithelial-stromal junction at the base of a gaping radial incision (case 2). A partial and irregular basement membrane is present (arrow) (transmission electron microscopy, uranyl acetate, Reynolds lead citrate, XSIOO). were also evident (Fig 5). Posterior radial ridges were present, but no endothelial damage, cell loss, or change in endothelial cell size was noted corresponding to these ridges or elsewhere (Fig 6). Histologically, the duplication of the radial incisions was clearly seen. One of the paired incisions commonly exhibited complete wound closure, while an epithelial plug of three to five cell layers deep and two to three cells wide still remained in the other, presumably more recent, incision (Fig 7). The close approximation of the paired incisions caused Bowman's layer to be grossly malapposed (Fig 7). In one case, a double epithelial plug was seen with a portion of Bowman's layer completely severed and appearing in a V-shape between the epithelial plugs (Fig 8). A normal-appearing epithelial cell layer was seen adjacent to and covering the incisions. The average depth of the incisions measured 71 % of the total corneal thickness at the periphery, 76.6% at mid-incision, and 57.7% paracentrally. Disrupted and randomly arranged lamellae indicated the site of the incisions. Only slight fibroblastic activity was seen in the area of the incision sites and epithelial plugs. Adjacent stromal keratocytes appeared normal. Neovascularization was absent. Fig 5. Raised epithelial ridges in case 3 (scanning electron microscopy, X40). Inset, high magnification of the epithelial cells demonstrating dense microvilli (SEM, X35(0). Case 4: this specimen was obtained by a lamellar keratectomy 19 months following a repeat radial keratotomy. By scanning electron microscopy, all incisions were easily visualized. A few basal epithelial cells remained on the surface of a fully exposed Bowman's layer. The incisions appeared to be spaced randomly. Two adjacent incisions were noted to intersect in one case (Fig 9). Some incisions exhibited complete wound closure and appeared intact, except for the fracture in Bowman's layer. Other radial incisions (presumably the most recent) were completely perforated and gaping from Bowman's layer through the thinly lathed stromal layer (Fig 9). Disrupted, randomly apposed lamellae, a fracture in Bowman's layer, and moderate fibroblastic activity were the only indications of the closed radial incisions by light microscopy. The surrounding stroma showed signs of freeze damage with occasional vacuolated keratocytes and loss of epithelium. IS The results of these four cases are summarized in Table 2. DISCUSSION The most common pathologic finding in these four cases of radial keratotomy was delayed wound healing 737 OPHTHALMOLOGY • JUNE 1985 • VOLUME 92 • NUMBER 6 Fig 7. Peripheral, paired radial incision (case 3). Bowman's layer between the incisions is grossly malapposed and an epithelial plug still remains in one incision (light microscopy, periodic acid-Schiff, X 100). Fig 6. Posterior corneal ridges present in case 3. Inset, endothelial cells overlying these radial ridges appear normal in structure (scanning electron microscopy, X22(0). Fig 9. Low power scanning electron micrograph of the cornea in case 4. Note gaping and closed fractures in Bowman's layer, intersecting adjacent incisions (arrow), and scattered basal epithelial cells remaining on the Bowman's membrane (scanning electron microscopy, X40). FigS. Repeat incision site (case 3). Bowman's layer is severed and appears in a V-shape within the double epithelial plug (light microscopy, Toluidine blue 0 XIOO). evidenced by epithelial plugs persisting up to 47 months postoperatively (Figs 7,8) and peripheral, unhealed wounds up to six months following surgery (Figs 2, 3 left). Three of the four cases contained epithelial plugs at 5.5, 6, and 47 months postoperatively with underlying 738 basement membranes (Figs 3 right, 7,8). A previously published study of a corneal biopsy six months postoperative noted poor wound healing that included epithelial inclusion cysts, epithelial degeneration, and basal lamina duplication following a 16-incision radial keratotomy.14 The normal corneal wound healing process includes regression of the epithelial plug within 6 to 14 days with the transformation of fibroblasts into fibrocytes with scar stabilization in three to six months. 16 The persistence of these plugs 6 to 47 months postoperatively suggests a reduction of expected wound healing. In most cases, DEG, et al • DELAYED CORNEAL WOUND HEALING Table 2. Clinical, Surgical, and Morphological Data on Four Radial Keratotomy Specimens Case No. Age (years) Sex 42/M Preoperative Clinical Observations Surgery Time of Postoperative Removal Penetrating keratoplasty for keratoconus performed August 1973 6 radial incisions, 5 parallel incisions in steep meridian both sides of cornea, 1 incision perpendicular to each of the 5 incisions January 1980 5V2 months penetrating keratoplasty Morphology Epithelial plugs in incisions, malapposed Bowman's layer, fibroblastic activity at wound site, epithelial and endothelial ridges 2 46/M Keratoconus 8 radial incisions April 1983 6 months penetrating keratoplasty Epithelial-lined incisions, peripheral-gaping incisions, malapposed Bowman's layer 3 43/M Healthy cornea 16 radial incisions December 1979, additional 16 incisions April 1980 47 months penetrating keratoplasty Epithelial plugs in some incisions, malapposed Bowman's layer at incision sites, epithelial and endothelial radial ridges 4 43/M Healthy cornea 8 radial incisions February 1982, additional 8 radial incisions April 1982 19 months from repeat radial keratotomy, lamellar keratoplasty Some gaping incisions, malapposed Bowman's layer M = male. slight to moderate fibroblastic activity was seen beneath the gaping wounds and epithelial plugs extending along the depth of the closed incision, but fibroblastic activity was greatly reduced in the area surrounding the unhealed wound extending to the epithelial surface (Figs 3,7,8). The epithelial plugs, edematous epithelium overlying the incisions, and the malapposition of Bowman's layer may account for the raised epithelial ridges (Figs 5,7,8). In case 3, clinical observations revealed raised epithelial ridges marking the sites of approximately 16 of the 32 incisions, possibly an artifact of the close approximation of the incisions which were intended to reopen and deepen the original 16 incisions. These factors may have contributed to the patient's photophobia and glare. The micrometer diamond blade knife was designed to improve incision depth predictability and thereby augment reproducibility. Studies comparing the micrometer razor blade knife to the micrometer diamond blade knife have shown no significant improvement of incision depth accuracy of one type over the other. However, the sharp diamond blade knife appeared to reduce stress, trauma, subsequent corneal scarring, and endothelial cell damage. ll ,12,17-19 The use of a micrometer diamond knife in case 2 did not show any differences in morphology or improved incision accuracy over cases 1 and 3. Inconsistency of incision depth was a common feature of cases 1, 2, and 3, with increased incision depth noted in the periphery in cases 2 and 3. Posterior corneal ridges have been described imme- diately after radial keratotomy. The actual ridges may be an artifact of fixation since they have not been seen when the anterior chamber was perfused with fixative. 20 Studies immediately following radial keratotomy, however, have demonstrated linear endothelial cell damage immediately under each radial incision.1O The cell damage in these cases was focal and was not present months following surgery (unpublished observations). Ridges were not seen in case 2, but it is uncertain whether their absence was a result of the use of a diamond-blade knife or due to gaping wounds and manipulation of the soft cornea. Posterior corneal ridges were seen in cases 1 and 3 (Fig 6). We saw no evidence of endothelial cell damage under the incisions. Repeat keratotomies are performed to achieve further correction by reopening the original incisions or by additional incisionsY Eight additional incisions one year postkeratotomy in monkeys have shown that additional (but unpredictable) correction can be achieved. 21 Repeat radial keratotomy has been reported to be associated with significant endothelial cellloss. 22 The repeat procedure (case 3) to reopen and deepen the incisions resulted in a repeated loss of visual acuity from 20/20 to 20/50 and reported photophobia and glare. Histological evaluation revealed that, instead of reopening the original incision, a second, closely approximated incision was made. There was no endothelial damage or cell changes apparent in this double radial keratotomy specimen. 739 OPHTHALMOLOGY • JUNE 1985 • Common factors which may have contributed to the delay or disruption in the wound-healing process were keratoconus (case 2), radial keratotomy performed on a postoperative corneal transplant case (case 1), and repeat radial keratotomy procedures with close approximation of incisions in cases 2 and 4 (Table 2). We were unable to determine if any of the cases received topical glucocorticoids following the keratotomy procedures, but all cases received topical steroids at some time. Based on this study, the radial keratotomy procedure may not be appropriate on corneas that are diseased or have had previous surgery. The two cases of repeat radial keratotomies raise the question of whether the sets of incisions performed months apart, number of incisions, or the close approximation of the incisions may have caused a poor wound-healing response. Based on our laboratory experience lO ,13 and the analysis of these four specimens, we have begun to formulate a theory of how radial keratotomy achieves a reduction of myopia. Previous research on the intrastromal implantation of hydrogel lenses has demonstrated little or no effect on the anterior corneal curvature when the lens was implanted within an intrastromal pocket and Bowman's membrane was left intact. 23 In contrast, significant changes in the anterior corneal curvature can be obtained when Bowman's membrane is severed 360 0 with a microkeratome and a hydrogel lens implanted within the stroma. 24 In the radial keratotomy procedure, Bowman's membrane is severed 360 0 , except for the central 3 to 4 mm which permits a steepening of the cornea in the incised area and a compensatory flattening in the uncut central cornea. Analysis of the four human specimens in the present study and one separately published case l4 demonstrated delayed wound healing, especially in the area of Bowman's membrane. We therefore theorize that this delayed healing response permits a permanent curvature change. Undercorrections may be due to more complete or more rapid wound healing whereas significant overcorrections would represent significantly delayed wound healing and anterior wound gaping filled with epithelium. Further analysis of clinical cases and specimens will help elucidate the mechanism of action of radial keratotomy. REFERENCES· 1. Sato T. Posterior incision of cornea; surgical treatment for conical cornea and astigmatism. Am J Ophthalmol 1950; 33:943-8. 740 VOLUME 92 • NUMBER 6 2. Fyodorov SN, Dumev VV. Operation of dosaged dissection of corneal circular ligament in cases of myopia of mild degree. Ann Ophthalmol1979; 11:1885-90. 3. Sato T, Akiyama K, Shibata H. A new surgical approach to myopia. Am J Ophthalmol 1953; 36:823-9. 4. Bores LD, Myers W, Cowden J. Radial keratotomy: an analysis of the American experience. Ann Ophthalmol1981; 13:941-8. 5. Sail J, Lee JS, Jester JV, et al. Radial keratotomy in fresh human cadaver eyes. Ophthalmology 1981; 88:742-6. 6. Miller D, Miller R. Glare sensitivity in simulated radial keratotomy. Arch Ophthalmol1981; 99:1961-2. 7. Rowsey JJ, Balyeat HD. Radial keratotomy: preliminary report of complications. Ophthalmic Surg 1982; 13:27-35. 8. Yamaguchi T, Polack FM, Valenti J, Kaufman HE. Endothelial damage after anterior radial keratotomy; an electron microscopic study of rabbit cornea. Arch Ophthalmol 1981; 99:2151-8. 9. Yamaguchi T, Kaufman HE, Fukushima A, et al. Histologic and electron microscopic assessment of endothelial damage produced by anterior radial keratotomy in the monkey cornea. Am J Ophthalmol 1981; 92:313-27. 10. Binder PS, Stainer GA, Zavala EY, et al. Acute morphologic features of radial keratotomy. Arch Ophthalmol1983; 101:1113-5. 11. Rowsey JJ, Balyeat HD, Rabinovitch B, et al. Predicting the results of radial keratotomy. Ophthalmology 1983; 90:642-54. 12. Galbavy EJ. Use of diamond knives in ocular surgery. Ophthalmic Surg 1984; 15:203-5. 13. Stainer GA, Shaw EL, Binder PS, et al. Histopathology of a case of radial keratotomy. Arch Ophthalmol 1982; 100:1473-7. 14. Jester JV, Villasenor RA, Miyashiro J. Epithelial inclusion cysts following radial keratotomy. Arch Ophthalmol1983; 101:611-5. 15. Binder PS, Zavala EY, Deg J, Akers PH. Refractive keratoplasty; tissue dyes and cryoprotective solutions. Arch Ophthalmol 1983; 101: 1591-6. 16. Binder PS, Wickham MG, Zavala EY, Akers PH. Corneal anatomy and wound healing. In: Symposium on Medical and Surgical Diseases of the Cornea; Transactions of the New Orleans Academy of Ophthalmology. St Louis: CV Mosby, 1980; 1-35. 17. Unterman SR, Rowsey JJ. Diamond-knife corneal incisions. Ophthalmic Surg 1984; 15:199-202. 18. Sail JJ, Lee T, Jester JV, et al. Analysis of incision depth following experimental radial keratotomy. Ophthalmology 1983; 90:655-9. 19. Yamaguchi T, Asbell PA, Ostrick M, et al. Endothelial damage in monkeys after radial keratotomy performed with a diamond blade. Arch Ophthalmol1984; 102:765-9. 20. Jester JV, Steel D, Sail J, et al. Radial keratotomy in non-human primate eyes. Am J Ophthalmol1981; 92:153-71. 21. Cowden JW, Weber B. Repeat radial keratotomy in monkeys. Ophthalmology 1983; 90:251-5. 22. Sail JJ. Progressive endothelial cell loss following repeat radial keratotomy-a case report. Ophthalmic Surg 1982; 13:997-9. 23. Sendele DD, Abelson MB, Kenyon KR, Hanninen LA. Intracomeal lens implantation. Arch Ophthalmol1983; 101:940-4. 24. Binder PS, Deg JK, Zavala EY, Grossman KR. Hydrogel keratophakia in non-human primates. Curr Eye Res 1981/82; 1:535-42.