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Corneal epithelium following intraepikeratophakia
Corneal epithelium following intraepikeratophakia Kazuo Tsubota, M.D.
Epithelialization of the cornea is the key to a successful epikeratophakia procedure. Recent developments in the intraepikeratophakia technique may facilitate epithelialization,l but the results are not known. In this report I evaluate the procedure using electron and specular microscopy.
MATERIALS AND METHODS Three pediatric aphakic patients, two adult aphakic patients, and three keratoconus patients were included in the study. In adult patients the corneal epithelium was examined by specular microscopy preoperatively and postoperatively. Part of the removed epithelium was examined by electron microscopy. Supported by a grant from the japanese Ministry Corneal Transplant. Presented in part at the Symposium
Oll
of Health
The modified intraepikeratophakia technique developed by Martel and Martel 1 was used in this study. The patients were given retrobulbar anesthesia using 4 ml of 2% xylocaine. A small amount of 100% ethyl alcohol was applied to the patient's corneal epithelium with the medical quick absorber. Thirty minutes after this application, a sheet of corneal epithelium was removed from Bowman's layer, using a blunt spatula, and cultured in TC199 medium during surgery (Figure 1). By avoiding unnecessary pulling, it was possible to preserve the epithelial sheet. The ordinary epikeratophakia procedure was performed using the commercially prepared keratolens (Allergan Medical Optics, Irvine, CA). At the conclusion of surgery, the epithelial
and Welfares Study Group for Developing Standard Requirements for
Cataract, IOL and Refractive Surgery, Washillgton, D.C., April 1989.
Reprint reqllests to Kawo TSllbota, M.D., Department of Ophthalmology, Tokyo Dental College, 7-1 Sugano 6 chome, Ichikawa, Chiba, 272, japan. 460
J CATARACT REFRACT SURG-VOL 17, JULY 1991
A
B
Fig. 1.
(Tsubota) The modified intraepikeratophakia teehnique used in this study. The epithelium was removed with a special blunt spatula (A, B) and preserved in TC199 medium (C). Arrow indieates the epithelial sheet floating in the medium.
C
sheet was placed on a keratolens and apressure patch was applied to it. Part of the epithelium removed from one patient was used for electron microscopic examination. This piece of epithelium was fixed with 2% glutaraldehyde and processed for scanning and transmission electron microscopic observation as described previously.2 Following surgery, the corneal epithelium was examined by biomicroscopy with daily ßuorescein staining until the staining ceased. A special contact lens was used to take epithelial pictures. 3 ,4 The pictures were taken before surgery and one week, one month, three months, and six months after surgery. The pictures were evaluated for the presence of abnormal cells and abnormalities in the cellular pattern.
opacification developed between the recipient and the donor cornea. Although the visual acuity of pediatric patients could not be obtained, adult patients increased their uncorrected acuity in all Table 1. Clinical results of intraepikeratophakia procedure.
Case 1. Adult aphakia
Preop Visual Aeuity
Pos top Visual Acuity
20/200 (20/30)*
20/40 (20/25)
Clear
20/200 (20/30)
Clear
2. Adult aphakia 20/800 (20/20)
Cornea
3. Keratoeonus
20/200 (20/20)
20/30 (20/30)
Clear
4. Keratoeonus
20/200 (20/25)
20/40 (20/25)
Clear
5. Keratoconus
20/100 (20/25)
20/40 (20/25)
Clear
6. Pediatrie aphakia
nlct
nie
Peripheral opacity
RESULTS
7. Pediatrie aphakia
nie
nie
Clear
The clinical results of the intraepikeratophakia procedure are presented in Table 1. The cornea was clear in all cases at six months except in one case of pediatric aphakia (Case 6) in which a peripheral
8. Pediatrie aphakia
n/e
nie
Clear
* Correeted visual aeuity in parentheses = not eheeked
t nie
J CATARACT REFRACT SURG-VOL 17, JULY 1991
461
Fig. 2.
(Tsubota) Two scanning electron microscopic observations of the superficiallayer of corneal epithelium removed during the intraepikeratophakia procedure. The superficiallayer is substantially damaged. Bar is 10 J.tm.
cases. In one case of adult aphakia (Case 2) and in one case of keratoconus (Case 3), the corrected visual acuity was decreased two lines although the patients did not complain of this reduction. Figure 2 shows scanning electron microscopic photographs ofthe corneal epithelium. The superficiallayer of the epithelium was damaged and many cracks in the cell border were observed (arrow). Figure 3 shows transmission electron microscopic photographs. The superficial cells (S) were damaged and the cellular organism and cell borderlines were destroyed. The basal cell layer (B) was also disturbed, but the wing celllayer (W) seemed to remain
intact with normal configurations of organs such as mitochondria. Table 2 summarizes the results of autografted corneal epithelium. In six of eight cases, the epithelium attached itself to the Bowman's layer 48 hours after surgery when the pressure patch was loosened (Figure 4, Zeft). The epithelium covered more than 80% of the keratolens, and the whole cornea was covered within 72 hours in seven cases. Nevertheless, the autografted epithelium was stained with fluorescein for one week (Figure 4, right). The epithelium did not show any fluorescein staining after a one-week observation period.
Fig. 3.
462
(Tsubota) Two transmission electron microscopic observations of corneal epithelium removed during the intraepikeratophakia procedure. The superficiallayer (S) was damaged but the wing celilayer (W) is intact with slight alteration of the basal celilayer (B). Bar is 2 J.t111.
J CATARACT REFRACT SURG-VOL 17, JULY 1991
Fig. 4.
(Tsubota) Biomicroscopic observation of corneal epithelium following surgery (Case 2). Left: 48 hours after surgery. The autografted epithelium is attached to the keratolens stained with ßuorescein (arrow). There is also an epithelial defect observed in the inferior portion (double arrows). Right: Seven days after surgery. The autografted epithelium is no longer stained with ßuorescein.
Specular microscopic observation of aphakia and keratoconus patients showed abnormal patterns even before surgery (Figure 5). Figure 6 shows the corneal epithelium one week, one month, three months, and six months postoperatively. In the early postoperative period, abnormal cells, including spindle-shaped cells, large cells, and cells with nuclei, were observed. These abnormal cells became less apparent over time, but the irregular cell pattern remained after six months of observation. Table 3 and Figure 7 summarize the results of specular microscopy.
DISCUSSION The epithelial healing process is attracting researchers' interest because the success rate of the
Table 2. Epithelialization of corneal epithelium following epikeratophakia.
Case
No Age (Years)1 Fluorescein Epithelialization Stain Sex
l. Adult aphakia
621M
72 ho urs
6 days
2. Adult aphakia
59/M
72 hours
7 days
3. Keratoconus
181M
72 hours
7 days
4. Keratoconus
191M
72 hours
6 days
5. Keratoconus
191M
7 days
7 davs (autograft off)
6. Pediatric aphakia
11M
48 ho urs
5 days
7. Pediatric aphakia
21M
72 hours
7 days
8. Pediatric aphakia
21M
7 days
7 davs (autograf"t off)
Fig. 5.
(Tsubota) Specular microscopic observation of corneal epithelium before surgery. Bar is 100 J.tm. Top: Aphakic patient (Case 1): The epithelium displays an irregular pattern and cells with nudei. Bottom: Keratoconus patient (Case 7): The epithelium shows a spindle-shaped cell pattern.
J CATARACT REFRACT SURG-VOL 17, JULY 1991
463
A
B
D
c Fig. 6.
(Tsubota) Specular microscopic observation of corneal epithelium following surgery (Case 5). Bar is 100 /-Lm. A: One week after surgery: The epithelium shows an abnormal cell pattern along with spindle-shaped cells, large cells, and cells with nudeL B: One month after surgery: The epithelium shows a more normal pattern than at the one-week observation, aIthough spindle-shaped cells exist. C: Three months after surgery: Large cells still exist. D: Six months after surgery: No spindleshaped cells, large cells, or cells with nudeL
epikeratophakia procedure depends on the re-epithelialization of the cornea. The idea of autografting the corneal epithelium to facilitate re-epithelialization is promising,l but a morphological evaluation of the autografting has not been performed. Examination showed that the superficiallayer and the basal celllayer were damaged by the procedure, but the wing cell layer remained intact. Since the most essentiallayer for epithelial healing is thought to be the basal cell layer, the ideal procedure should preserve it. Regardless of the practicality of the current method, a new method for intraepikeratophakia should be devised. Although the basal celllayer of the corneas in this study was damaged, the autografted epithelium adhered to Bowman's layer of the keratolens, sug-
gesting the viability of this grafting wh ich apparently facilitates epithelialization. However, it is not known whether superficially stained epithelium has a protective effect or not. Six of our eight cases showed earlier epithelialization, which is compatible with previous reports on intraepikeratophakia. The development of a special contact lens for specular microscopic observation facilitates epithelial pictures. 3 ,4 This is a good way to observe the epithelium at the cellular level. This study showed persistent abnormalities in epikeratophakia patients
5 4
Table 3. Cellular abnormalities observed under the specular microscope.
Interval
SpindleShaped Cells
Large Cells
Cells with Nudei
Irregular Cell Pattern
Preoperatively
3,4,5*
1,2
All
All
1 week
All
All
All
All
1 month
2,3,4,5
1,2,4,5
All
All
3 months
3,4,5
1,2,4
All
All
6 months
3,5
1,2,4
All
All
*Numbers indicate the case number shown in Table 1 464
3 2
0 Pre - Op
1 Week
1 Month 3 Months 6 Months
Time Fig. 7.
(Tsubota) Corneal epithelial changes after intraepikeratophakia. Spindle-shaped cells and large cells appeared in the first week after the surgery in all cases. 0 = spindle-shaped cell; 6 = large cell;. = cell with nudeus; ... = irregular cell pattern.
J CATARACT REFRACT SURG-VOL 17, JULY 1991
more than six months after the surgery, as reported by Rao and coauthors. 5 The epithelium appeared abnormal preoperatively in adult aphakic and keratoconus patients, suggesting possible abnormalities in pediatric aphakic patients as weil. These findings indicate that careful evaluation ofthe corneal epithelium in the epikeratophakia procedure is necessary. If we can correlate the abnormal specular observation and the result of epithelialization after the intraepikeratophakia procedure, we may be able to eliminate patients with a risk of poor wound healing of the corneal epithelium.
REFERENCES 1. Martel J, Martel J. Intraepikeratophakia. Ann Ophthalmol 1987; 19:287-292 2. Tsubota K, Kenyon KR, Cheng HM. Hard contact lensinduced metabolie changes in rabbit corneas. Exp Eye Res 1989; 49:769-775 3. Tsubota K. A contact lens for specular microseopie observation. Am J Ophthalmol 1988; 106:627-628 4. Tsubota K, Mashima Y, Naoi S. A new contact lens for specular microseopie photography. Jpn J Clin Ophthalmol 1989; 43:997-999 5. Rao CN, Canti S, Aquavella Jv. Specular microscopy of corneal epithelium after epikeratophakia. Am J Ophthalmol 1987; 103:392-396
J CATARACT REFRACT SURG-VOL 17, JULY 1991
465
Epithelialization of the cornea is the key to a successful epikeratophakia procedure. Recent developments in the intraepikeratophakia technique may facilitate epithelialization,l but the results are not known. In this report I evaluate the procedure using electron and specular microscopy.
MATERIALS AND METHODS Three pediatric aphakic patients, two adult aphakic patients, and three keratoconus patients were included in the study. In adult patients the corneal epithelium was examined by specular microscopy preoperatively and postoperatively. Part of the removed epithelium was examined by electron microscopy. Supported by a grant from the japanese Ministry Corneal Transplant. Presented in part at the Symposium
Oll
of Health
The modified intraepikeratophakia technique developed by Martel and Martel 1 was used in this study. The patients were given retrobulbar anesthesia using 4 ml of 2% xylocaine. A small amount of 100% ethyl alcohol was applied to the patient's corneal epithelium with the medical quick absorber. Thirty minutes after this application, a sheet of corneal epithelium was removed from Bowman's layer, using a blunt spatula, and cultured in TC199 medium during surgery (Figure 1). By avoiding unnecessary pulling, it was possible to preserve the epithelial sheet. The ordinary epikeratophakia procedure was performed using the commercially prepared keratolens (Allergan Medical Optics, Irvine, CA). At the conclusion of surgery, the epithelial
and Welfares Study Group for Developing Standard Requirements for
Cataract, IOL and Refractive Surgery, Washillgton, D.C., April 1989.
Reprint reqllests to Kawo TSllbota, M.D., Department of Ophthalmology, Tokyo Dental College, 7-1 Sugano 6 chome, Ichikawa, Chiba, 272, japan. 460
J CATARACT REFRACT SURG-VOL 17, JULY 1991
A
B
Fig. 1.
(Tsubota) The modified intraepikeratophakia teehnique used in this study. The epithelium was removed with a special blunt spatula (A, B) and preserved in TC199 medium (C). Arrow indieates the epithelial sheet floating in the medium.
C
sheet was placed on a keratolens and apressure patch was applied to it. Part of the epithelium removed from one patient was used for electron microscopic examination. This piece of epithelium was fixed with 2% glutaraldehyde and processed for scanning and transmission electron microscopic observation as described previously.2 Following surgery, the corneal epithelium was examined by biomicroscopy with daily ßuorescein staining until the staining ceased. A special contact lens was used to take epithelial pictures. 3 ,4 The pictures were taken before surgery and one week, one month, three months, and six months after surgery. The pictures were evaluated for the presence of abnormal cells and abnormalities in the cellular pattern.
opacification developed between the recipient and the donor cornea. Although the visual acuity of pediatric patients could not be obtained, adult patients increased their uncorrected acuity in all Table 1. Clinical results of intraepikeratophakia procedure.
Case 1. Adult aphakia
Preop Visual Aeuity
Pos top Visual Acuity
20/200 (20/30)*
20/40 (20/25)
Clear
20/200 (20/30)
Clear
2. Adult aphakia 20/800 (20/20)
Cornea
3. Keratoeonus
20/200 (20/20)
20/30 (20/30)
Clear
4. Keratoeonus
20/200 (20/25)
20/40 (20/25)
Clear
5. Keratoconus
20/100 (20/25)
20/40 (20/25)
Clear
6. Pediatrie aphakia
nlct
nie
Peripheral opacity
RESULTS
7. Pediatrie aphakia
nie
nie
Clear
The clinical results of the intraepikeratophakia procedure are presented in Table 1. The cornea was clear in all cases at six months except in one case of pediatric aphakia (Case 6) in which a peripheral
8. Pediatrie aphakia
n/e
nie
Clear
* Correeted visual aeuity in parentheses = not eheeked
t nie
J CATARACT REFRACT SURG-VOL 17, JULY 1991
461
Fig. 2.
(Tsubota) Two scanning electron microscopic observations of the superficiallayer of corneal epithelium removed during the intraepikeratophakia procedure. The superficiallayer is substantially damaged. Bar is 10 J.tm.
cases. In one case of adult aphakia (Case 2) and in one case of keratoconus (Case 3), the corrected visual acuity was decreased two lines although the patients did not complain of this reduction. Figure 2 shows scanning electron microscopic photographs ofthe corneal epithelium. The superficiallayer of the epithelium was damaged and many cracks in the cell border were observed (arrow). Figure 3 shows transmission electron microscopic photographs. The superficial cells (S) were damaged and the cellular organism and cell borderlines were destroyed. The basal cell layer (B) was also disturbed, but the wing celllayer (W) seemed to remain
intact with normal configurations of organs such as mitochondria. Table 2 summarizes the results of autografted corneal epithelium. In six of eight cases, the epithelium attached itself to the Bowman's layer 48 hours after surgery when the pressure patch was loosened (Figure 4, Zeft). The epithelium covered more than 80% of the keratolens, and the whole cornea was covered within 72 hours in seven cases. Nevertheless, the autografted epithelium was stained with fluorescein for one week (Figure 4, right). The epithelium did not show any fluorescein staining after a one-week observation period.
Fig. 3.
462
(Tsubota) Two transmission electron microscopic observations of corneal epithelium removed during the intraepikeratophakia procedure. The superficiallayer (S) was damaged but the wing celilayer (W) is intact with slight alteration of the basal celilayer (B). Bar is 2 J.t111.
J CATARACT REFRACT SURG-VOL 17, JULY 1991
Fig. 4.
(Tsubota) Biomicroscopic observation of corneal epithelium following surgery (Case 2). Left: 48 hours after surgery. The autografted epithelium is attached to the keratolens stained with ßuorescein (arrow). There is also an epithelial defect observed in the inferior portion (double arrows). Right: Seven days after surgery. The autografted epithelium is no longer stained with ßuorescein.
Specular microscopic observation of aphakia and keratoconus patients showed abnormal patterns even before surgery (Figure 5). Figure 6 shows the corneal epithelium one week, one month, three months, and six months postoperatively. In the early postoperative period, abnormal cells, including spindle-shaped cells, large cells, and cells with nuclei, were observed. These abnormal cells became less apparent over time, but the irregular cell pattern remained after six months of observation. Table 3 and Figure 7 summarize the results of specular microscopy.
DISCUSSION The epithelial healing process is attracting researchers' interest because the success rate of the
Table 2. Epithelialization of corneal epithelium following epikeratophakia.
Case
No Age (Years)1 Fluorescein Epithelialization Stain Sex
l. Adult aphakia
621M
72 ho urs
6 days
2. Adult aphakia
59/M
72 hours
7 days
3. Keratoconus
181M
72 hours
7 days
4. Keratoconus
191M
72 hours
6 days
5. Keratoconus
191M
7 days
7 davs (autograft off)
6. Pediatric aphakia
11M
48 ho urs
5 days
7. Pediatric aphakia
21M
72 hours
7 days
8. Pediatric aphakia
21M
7 days
7 davs (autograf"t off)
Fig. 5.
(Tsubota) Specular microscopic observation of corneal epithelium before surgery. Bar is 100 J.tm. Top: Aphakic patient (Case 1): The epithelium displays an irregular pattern and cells with nudei. Bottom: Keratoconus patient (Case 7): The epithelium shows a spindle-shaped cell pattern.
J CATARACT REFRACT SURG-VOL 17, JULY 1991
463
A
B
D
c Fig. 6.
(Tsubota) Specular microscopic observation of corneal epithelium following surgery (Case 5). Bar is 100 /-Lm. A: One week after surgery: The epithelium shows an abnormal cell pattern along with spindle-shaped cells, large cells, and cells with nudeL B: One month after surgery: The epithelium shows a more normal pattern than at the one-week observation, aIthough spindle-shaped cells exist. C: Three months after surgery: Large cells still exist. D: Six months after surgery: No spindleshaped cells, large cells, or cells with nudeL
epikeratophakia procedure depends on the re-epithelialization of the cornea. The idea of autografting the corneal epithelium to facilitate re-epithelialization is promising,l but a morphological evaluation of the autografting has not been performed. Examination showed that the superficiallayer and the basal celllayer were damaged by the procedure, but the wing cell layer remained intact. Since the most essentiallayer for epithelial healing is thought to be the basal cell layer, the ideal procedure should preserve it. Regardless of the practicality of the current method, a new method for intraepikeratophakia should be devised. Although the basal celllayer of the corneas in this study was damaged, the autografted epithelium adhered to Bowman's layer of the keratolens, sug-
gesting the viability of this grafting wh ich apparently facilitates epithelialization. However, it is not known whether superficially stained epithelium has a protective effect or not. Six of our eight cases showed earlier epithelialization, which is compatible with previous reports on intraepikeratophakia. The development of a special contact lens for specular microscopic observation facilitates epithelial pictures. 3 ,4 This is a good way to observe the epithelium at the cellular level. This study showed persistent abnormalities in epikeratophakia patients
5 4
Table 3. Cellular abnormalities observed under the specular microscope.
Interval
SpindleShaped Cells
Large Cells
Cells with Nudei
Irregular Cell Pattern
Preoperatively
3,4,5*
1,2
All
All
1 week
All
All
All
All
1 month
2,3,4,5
1,2,4,5
All
All
3 months
3,4,5
1,2,4
All
All
6 months
3,5
1,2,4
All
All
*Numbers indicate the case number shown in Table 1 464
3 2
0 Pre - Op
1 Week
1 Month 3 Months 6 Months
Time Fig. 7.
(Tsubota) Corneal epithelial changes after intraepikeratophakia. Spindle-shaped cells and large cells appeared in the first week after the surgery in all cases. 0 = spindle-shaped cell; 6 = large cell;. = cell with nudeus; ... = irregular cell pattern.
J CATARACT REFRACT SURG-VOL 17, JULY 1991
more than six months after the surgery, as reported by Rao and coauthors. 5 The epithelium appeared abnormal preoperatively in adult aphakic and keratoconus patients, suggesting possible abnormalities in pediatric aphakic patients as weil. These findings indicate that careful evaluation ofthe corneal epithelium in the epikeratophakia procedure is necessary. If we can correlate the abnormal specular observation and the result of epithelialization after the intraepikeratophakia procedure, we may be able to eliminate patients with a risk of poor wound healing of the corneal epithelium.
REFERENCES 1. Martel J, Martel J. Intraepikeratophakia. Ann Ophthalmol 1987; 19:287-292 2. Tsubota K, Kenyon KR, Cheng HM. Hard contact lensinduced metabolie changes in rabbit corneas. Exp Eye Res 1989; 49:769-775 3. Tsubota K. A contact lens for specular microseopie observation. Am J Ophthalmol 1988; 106:627-628 4. Tsubota K, Mashima Y, Naoi S. A new contact lens for specular microseopie photography. Jpn J Clin Ophthalmol 1989; 43:997-999 5. Rao CN, Canti S, Aquavella Jv. Specular microscopy of corneal epithelium after epikeratophakia. Am J Ophthalmol 1987; 103:392-396
J CATARACT REFRACT SURG-VOL 17, JULY 1991
465