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Histopathological findings after intracorneal ring segment implantation in keratoconic human corneas
J CATARACT REFRACT SURG - VOL 33, FEBRUARY 2007
Histopathological findings after intracorneal ring segment implantation in keratoconic human corneas Se´pideh Samimi, MD, Franc¸ois Leger, MD, PhD, David Touboul, MD, Joseph Colin, MD
PURPOSE: To evaluate histopathological changes induced in keratoconic corneas after implantation of Intacs intracorneal ring segments (Addition Technology, Inc.). SETTING: Departments of Ophthalmology and Pathology, Hospital Pellegrin, Bordeaux, France. METHODS: This retrospective study included 8 keratoconic, contact-lens-intolerant eyes of 8 patients who had penetrating keratoplasty (PKP) after removal of Intacs inserts because of a poor refractive outcome or insert extrusion. Light microscopy was performed on all specimens after conventional staining. Immunohistochemistry was performed to identify cell types located next to the tunnel using AE1/AE3 cytokeratins, CD34, vimentin, collagen IV, and a-smooth muscle actin monoclonal antibodies. RESULTS: Conventional histology showed hypoplasia of the epithelium immediately surrounding the channel. There was no evidence of an inflammatory response or foreign-body granuloma. Keratocyte density was decreased above and below the tunnel, and collagen IV synthesis was seen in the scar area. All samples stained negatively with a-smooth muscle actin, indicating that myofibroblasts were not present. These changes were no longer visible when PKP was performed more than 6 months after Intacs explantation. CONCLUSIONS: Intacs induced keratocyte apoptosis, probably through a switch to a collagenous synthetic phenotype. Although histological changes seem to be entirely reversible after implant removal, longer follow-up is necessary to determine whether they accelerate corneal thinning and keratoconus progression via apoptosis and release of metalloprotease. J Cataract Refract Surg 2007; 33:247–253 Q 2007 ASCRS and ESCRS
Keratoconus is a noninflammatory, progressive, bilateral thinning disease of the cornea characterized by development of myopia and irregular astigmatism with resulting loss of vision. Intacs (Addition Technology, Inc.) are poly(methyl methacrylate) corneal inserts. They were approved by the U.S. Food and Drugs Administration in 1999 for the correction of low myopia and in 2004 for the treatment of myopia and astigmatism associated with keratoconus. They are also indicated for the treatment of corneal ectasia after laser in situ keratomileusis. Intacs inserts have a crescent-shaped arc length of 150 degrees, with an inner diameter and outer diameter of 6.8 mm and 8.1 mm, respectively. Inserts of varying thickness are available, ranging from 0.25 to 0.45 mm. Intracorneal rings may be an alternative to penetrating keratoplasty (PKP) for the treatment of keratoconus in patients who are contact lens intolerant and have a clear central cornea (stages II and III in Krumeich et al.’s classification1). Intracorneal rings confer the advantage of reshaping the Q 2007 ASCRS and ESCRS Published by Elsevier Inc.
abnormal cornea without removing tissue or touching the central cornea.2,3 Moreover, studies show that Intacs inserts can be easily removed with reversal of visual, refractive, and topographic changes.4–6 Although the clinical effects of Intacs implantation have been extensively studied since Colin and Velou2 performed the first Intacs placement for keratoconus in 1997, few data are available on the histopathological changes after ring implantation.7–11 Our current study investigated corneal button histology in patients having PKP after Intacs explantation to determine the morphological effects of Intacs inserts and their potential impact on keratoconus progression. PATIENTS AND METHODS This retrospective study included 8 keratoconic, contactlens-intolerant eyes of 8 patients who had PKP after removal of Intacs inserts because of a poor refractive outcome or insert extrusion (Table 1). Explantation of the inserts was performed several months before PKP (range 5 to 27 months) or during the same 0886-3350/07/$-see front matter doi:10.1016/j.jcrs.2006.08.059
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INTACS IN KERATOCONUS: HISTOPATHOLOGICAL FINDINGS
Table 1. Characteristics of study patients and treatment details.
Treated Eye
Intacs Thickness (mm)
Case
Sex
Age (Y)
Reason for Intacs Explant
1 2 3 4 5 6 7
M M F M M F M
53 19 37 42 39 22 23
Left Left Left Right Right Left Right
0.45 0.35 0.45 0.45 0.45 0.45 0.45
IR IR IR Extrusion of 1 segment Extrusion of both segments IR ER
8
M
32
Left
0.45
IR
Time from Intacs Implant to Explant (Mo)
Segment
Time from Intacs Explant to PKP (Mo)
10 15 13 3 4 13 10 37 24
d d d d d d Superior Inferior d
0 0 0 15 5 0 27 0 0
Implant Z implantation; Explant Z explantation; ER Z excessive refractive result; IR Z insufficient refractive result, PKP Z penetrating keratoplasty
surgical session as PKP. No intraoperative or postoperative complications with removal were noted in any case. Conventional histology and immunohistochemistry were performed on tissue removed from the 8 corneal buttons immediately posterior to PKP to identify any morphological changes induced by Intacs implantation. Conventional Histology Histological study was performed using 4 mm sections of excised corneal button tissue. Tissue sections were placed on formalin-fixed paraffin-embedded blocks stained with hemateineosin-safran, trichrome Masson, and periodic acid-Schiff solutions and examined by light microscopy.
RESULTS
Immunohistochemistry Immunohistochemistry was performed on cells located next to the Intacs tunnel. Tissue sections were dewaxed in xylene, rehydrated through a graded ethanol series, and washed with phosphate-buffered saline. Antigen retrieval was achieved by heat treatment in a water bath (20 to 40 minutes at 98 C) using a pH 6 target retrieval solution. Samples were stained using a sensitive polymer-based detection system (En Vision Plus,
Accepted for publication August 25, 2006. From the Ophthalmology and Pathology Departments, Hospital Pellegrin, Bordeaux, France. Joseph Colin is a consultant to Addition Technology, Inc. No other author has a financial or proprietary interest in any material or method mentioned. Presented at the Winter Refractive Surgery Meeting of the European Society of Cataract & Refractive Surgeons, Monte Carlo, Monaco, February 2006, and the ASCRS Symposium on Cataract, IOL and Refractive Surgery, San Francisco, California, USA, March 2006. Corresponding author: Dr. Joseph Colin, Service Ophtalmologie, CHU Pellegrin Place, Ame´lie Raba-Le´on 33 076, Bordeaux Cedex, France. E-mail: [email protected].
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DakoCytomation automated immunostainer). The reference, and dilution factor of primary antibodies were as follows: AE1/ AE3 cytokeratin (Dako M3515, 1/100), vimentin (Dako M0725, 1/800), and CD34 to detect keratocytes (Dako M7165, QBEnd10, 1/200); collagen IV (Immunotech 1115, 1/100); and a-smooth muscle actin to detect myofibroblasts (Dako M851, 1/100). The CD34 antigen is a 105-120 kDa transmembrane glycoprotein that is expressed on the surface of lymphohematopoietic stem and progenitor cells, small-vessel endothelial cells, embryonic fibroblasts, and some cells in fetal and adult nervous tissues. Its expression is highest in the most primitive stem cells and decreases gradually as lineage-committed progenitors differentiate. The CD34 antigen is also present on capillary endothelial cells and on bone marrow stromal cells.
Conventional Histology
Table 2 shows the results of the conventional histological evaluation. Focal epithelial hypoplasia immediately above the Intacs tunnel was present in 5 eyes. However, epithelial thickness appeared to be normal toward the center of the cornea (Figures 1 and 2). Six eyes had a break in Bowman’s layer, characteristic of advanced keratoconus. Channel haze was observed in the stroma in all eyes except case 4; the haze was at a depth ranging between 50% and 75% (Figure 3). There was no evidence of inflammatory cells, foreign-body granulomas, or neovascularization even though the Intacs inserts had been inserted next to the limbus. All corneas appeared to have a structurally sound stroma with parallel collagen lamellar fibers, except at the site next to insertion of the Intacs segments, where the fibers deviated to give way to the tunnel. As expected, no lipid droplets were observed in the sections because lipids are dissolved during preparation of the tissue for light microscopy. However, acidophilic densification suggestive of collagen synthesis was seen in
J CATARACT REFRACT SURG - VOL 33, FEBRUARY 2007
INTACS IN KERATOCONUS: HISTOPATHOLOGICAL FINDINGS
Table 2. Conventional histology findings.
Case
Epithelial Hypoplasia
Bowman’s Disruption
Channel Localization (%)
Granuloma
1 2 3 4 5 6 7 8
Yes Yes Yes No No Yes Yes Yes
Yes Yes Yes Yes No Yes No Yes
50 75 66 None observed 50–66 66 75 75
No No No No No No No No
2 eyes (cases 3 and 8) adjacent to the tunnel segments (Figure 4). A heterogeneous cellular group was found in 3 eyes (cases 6, 7, and 8) around the channel section (Figure 5). Immunohistochemistry
Immunochemistry of cells inside the tunnel and around the inner edge of the tunnel stained positively for collagen IV in 4 eyes (cases 1, 3, 6, and 8), suggesting that new collagen IV synthesis had occurred (Figure 6). Positive AE1/AE3 cytokeratin staining was limited to the corneal epithelium in all cases. Of particular note, the cellular formations next to the tunnel in samples from cases 6, 7, and 8 were cytokeratin AE1/AE3 negative. All samples stained negatively with a-smooth muscle actin, indicating myofibroblasts were not present. Results for the CD34 stain showed that fewer keratocytes were present above and below the Intacs insertion site than the lateral sides of the insertion site or the center of the cornea (Figures 7 and 8), except in case 4 in which the corneal scar from the insert was no longer visible. All keratocytes and endothelial cells stained positively with vimentin. However, in the deposit areas, a few cells that stained positive with vimentin stained negative with CD34.
Figure 1. Focal hypoplasia of the epithelium overlying the Intacs channel. Note that the epithelial thickness toward the center of the cornea is normal (case 6).
DISCUSSION
Most publications focus on clinical and refractive outcomes after intracorneal ring segment placement without considering the pathology of the corneal wound-healing response. When this issue has been addressed, the histopathological changes have usually been investigated using confocal microscopy,12 optic coherence tomography,13 ultrasound,14 or specular microscopy.15 Moreover, the largest study of the histological effects of intracorneal ring segment implantation conducted to date was performed in 5 New Zealand rabbits.7 Further histological studies in humans are needed because the healing response after Intacs implantation could have profound consequences on both keratoconus progression and the prognosis of future PKP. This study evaluated the histopathological effects of Intacs implantation in 8 keratoconic human corneas. Our findings of epithelial hypoplasia overlying the intracorneal ring segments and maintained stromal structure are in agreement with those in previous studies.7,8 However, normal epithelial thickness above the channel scar was found in cases in which PKP was performed several months
Figure 2. Focal hypoplasia of the epithelium (case 6).
J CATARACT REFRACT SURG - VOL 33, FEBRUARY 2007
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INTACS IN KERATOCONUS: HISTOPATHOLOGICAL FINDINGS
Figure 3. Channel haze in 50% depth of the stroma (case 1).
(5 to 27) after Intacs explantation. This suggests that hypoplasia of the epithelium over the segments initially regulates the corneal surface but is reversible after insert removal. In addition, the channel fibrous scar eventually heals after insert explantation; evidence in cases 4 and 5 suggest that the healing process requires between 5 months and 15 months. Studies have established that corneal implants evoke the intracellular and extracellular production of lipids, and this is thought to be a nonspecific response to the stress of degenerating cells.7,8 We were unable to identify lipid deposits in our study as we did not perform cryopreservation. However, we identified weak extracellular spaces adjacent to the scar tissue, suggesting an artifact of lipid removal. Our keratocyte density findings are particularly noteworthy. Results of CD34 staining showed that keratocyte density was lower in areas above and below the Intacs insertion site than in the center of the cornea and comparatively lower in the corneal region immediately next to the channel, albeit to a lesser degree. The CD34 antigen is a transmembrane glycophosphoprotein expressed on fibroblast-like cells in connective
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Figure 4. Acidophilic densification adjacent to the segments tunnel suggestive of collagen synthesis (case 3).
tissue.16 It has been known since 2002 that CD34 is expressed on human keratocytes but not by the epithelium or endothelium.17 In 2003, Joseph et al.16 suggested CD34 plays a role in cytoadhesion and signaling related to differentiation and proliferation. To our knowledge, our study is the first to report a decrease in keratocyte density. Twa et al.,7 who evaluated the histology of the corneal stroma after intrastromal corneal ring implantation in 5 New Zealand rabbits, found increased cellular bodies along the inner radius of the ring segments that were shown on conventional histological examination to be keratocytes. As this finding was associated with an elevated calcium level in the 6-month period after surgery, the authors considered it to be an initial response to surgical trauma. However, the study did not report whether keratocytes were present in the stromal tissue above and below the rings. Increased keratocyte density has also been reported in humans. Dawson et al.,11 who used electron microscopy to evaluate a unique corneal button from a patient with a history of previous corneal ring segments, found that activated keratocytes seemed to persist indefinitely.
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INTACS IN KERATOCONUS: HISTOPATHOLOGICAL FINDINGS
Figure 5. Nonhomogeneous cellular formation around the channel section (case 8).
However, neither the duration of corneal ring implantation nor the interval before PKP was reported. In a case report of a patient who had photorefractive keratectomy, Ferrara corneal rings implantation, and PKP with trephination of the corneal tissue containing the implant 2.5 years later, Twa et al.8 identified an increased density of vimentin-positive cells in the deposit region. They considered that the cellular response associated with the deposit was caused by keratocytes, most likely with a myofibroblastic phenotype. Moreover, they found a greater density of cells in the area central to the corneal implant. Spirn et al.9 report similar findings in a patient with progressive corneal keratectasia under the intracorneal ring segment. Ruckhofer,12 who performed confocal microscopy in
Figure 6. Collagen IV synthesis in the channel scar (case 1).
Figure 7. Decrease of CD34 positive cells superiorly and inferiorly to the rings (patient 1).
18 eyes of 12 patients, found that the stroma adjacent to the intracorneal segments appeared to have seemingly compressed layers with many keratocytes, especially underneath the segments.
Figure 8. Decrease of CD34 positive cells superiorly and inferiorly to the rings (patient 6).
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Although these studies show increased keratocyte density in the deposit region, little is known about keratocyte activity in other regions of the cornea. Moreover, there are no objective data comparing cell density in the area next to the rings with that in more distal sites. A decrease in CD34-positive cells could be related to a reduction in keratocytes or to a change in their phenotype via the cell life cycle and apoptosis.18 In primitive fibroblasts, such as dendritic cells in tumors and healing wounds, the CD34 disappears as the cells attain a more differentiated a-smooth muscle actin–positive myofibroblastic or collagenous synthetic phenotype.19 It was based on this understanding of keratocyte phenotypology that we decided to include vimentin staining in our study because the technique enables identification of nonspecific mesenchymatous cells. Although the results for vimentin and CD34 stains were reasonably comparable, some cells, especially in the deposit region, were CD34 negative and vimentin positive. This finding is probably a result of a reduction in keratocytes and a change in keratocyte phenotype. Because all samples stained negatively for a-smooth muscle actin, keratocyte differentiation does not appear to be of a myofibroblastic phenotype and is probably of a collagenous synthetic form. Indeed, our study identified collagen synthesis in the deposit areas. This has been reported,7 but to our knowledge we are the first to characterize the collagen as type IV. Kim et al.20 detected apoptotic keratocytes in the anterior stroma, particularly in the areas with Bowman’s disruption, in 60% of keratoconic corneas and 35% of stromal dystrophy corneas. Fabre et al.21 showed that chronic epithelial injury induces apoptosis via interleukin 1 and that keratoconic eyes may be more susceptible to apoptosis. The authors hypothesize that apoptosis, with normal release of degradative enzymes and other components, would damage surrounding cells. Thus, if Intacs inserts cause keratocyte apoptosis in the area next to the tunnel, it is possible the rings accelerate the progression of corneal ectasia through metalloproteinase release. This hypothesis could explain the cases of progressive keratolysis after Intacs implantation reported by Bourges et al.22 In cases in our study in which PKP was performed several months after Intacs explantation (case 4, 5, and 7), the cornea had become repopulated with keratocytes, probably from neighboring cells. In case 4, the scar was no longer visible. Several reports show that the refractive and topographic changes induced by Intacs inserts are reversible after explantation of the segments. Our study demonstrates that the histological effects of Intacs implantation are also reversible after insert removal. Nevertheless, further studies are needed to establish whether there is an association between Intacs placement and progression of keratoconus
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or graft rejection. Further study is also required evaluating the long-term consequences of reduced peripheral keratocytes associated with Intacs implantation, particularly with regard to axial stromal transparency.
REFERENCES 1. Krumeich JH, Daniel J, Knu¨lle A. Live-epikeratophakia for keratoconus. J Cataract Refract Surg 1998; 24:456–463 2. Colin J, Velou S. Current surgical options for keratoconus. J Cataract Refract Surg 2003; 29:379–386 3. Alio´ JL, Artola AL, Hassanein A, et al. One or 2 Intacs segments for the correction of keratoconus. J Cataract Refract Surg 2005; 31:943–953 4. Alio´ JL, Artola A, Ruiz-Moreno JM, et al. Changes in keratoconic corneas after intracorneal ring segment explantation and reimplantation. Ophthalmology 2004; 111:747–751 5. Siganos CS, Kymionis GD, Kartakis N, et al. Management of keratoconus with Intacs. Am J Ophthalmol 2003; 135:64–70 6. Asbell PA, Uc¸akhan O¨O¨, Odrich M. Photorefractive keratectomy after intrastromal corneal ring segment explantation. Am J Ophthalmol 1999; 128:755–756 7. Twa MD, Ruckhofer J, Kash RL, et al. Histologic evaluation of corneal stroma in rabbits after intrastromal corneal ring implantation. Cornea 2003; 22:146–152 8. Twa MD, Kash RL, Costello M, Schanzlin DJ. Morphologic characteristics of lamellar channel deposits in the human eye; a case report. Cornea 2004; 23:412–420 9. Spirn MJ, Dawson DG, Rubinfeld RS, et al. Histopathological analysis of post-laser-assisted in situ keratomileusis corneal ectasia with intrastromal corneal ring segment. Arch Ophthalmol 2005; 123:1604–1607 10. Quantock AJ, Kincaid MC, Schanzlin DJ. Stromal healing following explantation of an ICR (intrastromal corneal ring) from a nonfunctional human eye. Arch Ophthalmol 1995; 113:208–209 11. Dawson DG, Edelhauser HF, Grossniklaus HE. Long-term histopathologic findings in human corneal wounds after refractive surgical procedures. Am J Ophthalmol 2005; 139:168–178 12. Ruckhofer J, Bo¨hnke M, Alzner E, Grabner G. Confocal microscopy after implantation of intrastromal corneal ring segments. Ophthalmology 2000; 107:2144–2151 13. Wirbelauer C, Winkler J, Scholz C, et al. Experimental imaging of intracorneal ring segments with optical coherence tomography. J Refract Surg 2003; 19:367–371 14. Reinstein DZ, Srivannaboon S, Holland SP. Epithelial and stromal changes induced by Intacs examined by three-dimensional very high-frequency digital ultrasound. J Refract Surg 2001; 17:310–318 15. Azar RG, Holdbrook MJ, Lemp M, Edelhauser HF. Two-year corneal endothelial cell assessment following INTACS implantation; KeraVision study group. J Refract Surg 2001; 17:542–548 16. Joseph A, Hossain P, Jham S, et al. Expression of CD34 and L-selectin on human corneal keratocytes. Invest Ophthalmol Vis Sci 2003; 44: 4689–4692 17. Toti P, Tosi GM, Traversi C, et al. CD-34 stromal expression pattern in normal and altered human corneas. Ophthalmology 2002; 109: 1167–1171 18. Espana EM, Kawakita T, Liu C-Y, Tseng SCG. CD-34 expression by cultured human keratocytes is downregulated during myofibroblast differentiation induced by TGF-b1. Invest Ophthalmol Vis Sci 2004; 45:2985–2991 19. Silverman JS, Tamsen A. Fibrohistiocytic differentiation in subcutaneous fatty tumors; study of spindle cell, pleomorphic, myxoid, and atypical lipoma and dedifferentiated liposarcoma cases composed
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in part of CD34C fibroblasts and FXIIIaC histiocytes. J Cutan Pathol 1997; 24:484–493 20. Kim W-J, Rabinowitz YS, Meisler DM, Wilson SE. Keratocyte apoptosis associated with keratoconus. Exp Eye Res 1999; 69:475–481 21. Fabre EJ, Bureau J, Pouliquen Y, Lorans G. Binding sites for human interleukin 1a, gamma interferon and tumor necrosis factor on cultured
fibroblasts of normal cornea and keratoconus. Curr Eye Res 1991; 10:585–592 22. Bourges J-L, Trong TT, Ellies P, et al. Intrastromal corneal ring segments and corneal anterior stromal necrosis. J Cataract Refract Surg 2003; 29:1228–1230
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Histopathological findings after intracorneal ring segment implantation in keratoconic human corneas Se´pideh Samimi, MD, Franc¸ois Leger, MD, PhD, David Touboul, MD, Joseph Colin, MD
PURPOSE: To evaluate histopathological changes induced in keratoconic corneas after implantation of Intacs intracorneal ring segments (Addition Technology, Inc.). SETTING: Departments of Ophthalmology and Pathology, Hospital Pellegrin, Bordeaux, France. METHODS: This retrospective study included 8 keratoconic, contact-lens-intolerant eyes of 8 patients who had penetrating keratoplasty (PKP) after removal of Intacs inserts because of a poor refractive outcome or insert extrusion. Light microscopy was performed on all specimens after conventional staining. Immunohistochemistry was performed to identify cell types located next to the tunnel using AE1/AE3 cytokeratins, CD34, vimentin, collagen IV, and a-smooth muscle actin monoclonal antibodies. RESULTS: Conventional histology showed hypoplasia of the epithelium immediately surrounding the channel. There was no evidence of an inflammatory response or foreign-body granuloma. Keratocyte density was decreased above and below the tunnel, and collagen IV synthesis was seen in the scar area. All samples stained negatively with a-smooth muscle actin, indicating that myofibroblasts were not present. These changes were no longer visible when PKP was performed more than 6 months after Intacs explantation. CONCLUSIONS: Intacs induced keratocyte apoptosis, probably through a switch to a collagenous synthetic phenotype. Although histological changes seem to be entirely reversible after implant removal, longer follow-up is necessary to determine whether they accelerate corneal thinning and keratoconus progression via apoptosis and release of metalloprotease. J Cataract Refract Surg 2007; 33:247–253 Q 2007 ASCRS and ESCRS
Keratoconus is a noninflammatory, progressive, bilateral thinning disease of the cornea characterized by development of myopia and irregular astigmatism with resulting loss of vision. Intacs (Addition Technology, Inc.) are poly(methyl methacrylate) corneal inserts. They were approved by the U.S. Food and Drugs Administration in 1999 for the correction of low myopia and in 2004 for the treatment of myopia and astigmatism associated with keratoconus. They are also indicated for the treatment of corneal ectasia after laser in situ keratomileusis. Intacs inserts have a crescent-shaped arc length of 150 degrees, with an inner diameter and outer diameter of 6.8 mm and 8.1 mm, respectively. Inserts of varying thickness are available, ranging from 0.25 to 0.45 mm. Intracorneal rings may be an alternative to penetrating keratoplasty (PKP) for the treatment of keratoconus in patients who are contact lens intolerant and have a clear central cornea (stages II and III in Krumeich et al.’s classification1). Intracorneal rings confer the advantage of reshaping the Q 2007 ASCRS and ESCRS Published by Elsevier Inc.
abnormal cornea without removing tissue or touching the central cornea.2,3 Moreover, studies show that Intacs inserts can be easily removed with reversal of visual, refractive, and topographic changes.4–6 Although the clinical effects of Intacs implantation have been extensively studied since Colin and Velou2 performed the first Intacs placement for keratoconus in 1997, few data are available on the histopathological changes after ring implantation.7–11 Our current study investigated corneal button histology in patients having PKP after Intacs explantation to determine the morphological effects of Intacs inserts and their potential impact on keratoconus progression. PATIENTS AND METHODS This retrospective study included 8 keratoconic, contactlens-intolerant eyes of 8 patients who had PKP after removal of Intacs inserts because of a poor refractive outcome or insert extrusion (Table 1). Explantation of the inserts was performed several months before PKP (range 5 to 27 months) or during the same 0886-3350/07/$-see front matter doi:10.1016/j.jcrs.2006.08.059
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INTACS IN KERATOCONUS: HISTOPATHOLOGICAL FINDINGS
Table 1. Characteristics of study patients and treatment details.
Treated Eye
Intacs Thickness (mm)
Case
Sex
Age (Y)
Reason for Intacs Explant
1 2 3 4 5 6 7
M M F M M F M
53 19 37 42 39 22 23
Left Left Left Right Right Left Right
0.45 0.35 0.45 0.45 0.45 0.45 0.45
IR IR IR Extrusion of 1 segment Extrusion of both segments IR ER
8
M
32
Left
0.45
IR
Time from Intacs Implant to Explant (Mo)
Segment
Time from Intacs Explant to PKP (Mo)
10 15 13 3 4 13 10 37 24
d d d d d d Superior Inferior d
0 0 0 15 5 0 27 0 0
Implant Z implantation; Explant Z explantation; ER Z excessive refractive result; IR Z insufficient refractive result, PKP Z penetrating keratoplasty
surgical session as PKP. No intraoperative or postoperative complications with removal were noted in any case. Conventional histology and immunohistochemistry were performed on tissue removed from the 8 corneal buttons immediately posterior to PKP to identify any morphological changes induced by Intacs implantation. Conventional Histology Histological study was performed using 4 mm sections of excised corneal button tissue. Tissue sections were placed on formalin-fixed paraffin-embedded blocks stained with hemateineosin-safran, trichrome Masson, and periodic acid-Schiff solutions and examined by light microscopy.
RESULTS
Immunohistochemistry Immunohistochemistry was performed on cells located next to the Intacs tunnel. Tissue sections were dewaxed in xylene, rehydrated through a graded ethanol series, and washed with phosphate-buffered saline. Antigen retrieval was achieved by heat treatment in a water bath (20 to 40 minutes at 98 C) using a pH 6 target retrieval solution. Samples were stained using a sensitive polymer-based detection system (En Vision Plus,
Accepted for publication August 25, 2006. From the Ophthalmology and Pathology Departments, Hospital Pellegrin, Bordeaux, France. Joseph Colin is a consultant to Addition Technology, Inc. No other author has a financial or proprietary interest in any material or method mentioned. Presented at the Winter Refractive Surgery Meeting of the European Society of Cataract & Refractive Surgeons, Monte Carlo, Monaco, February 2006, and the ASCRS Symposium on Cataract, IOL and Refractive Surgery, San Francisco, California, USA, March 2006. Corresponding author: Dr. Joseph Colin, Service Ophtalmologie, CHU Pellegrin Place, Ame´lie Raba-Le´on 33 076, Bordeaux Cedex, France. E-mail: [email protected].
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DakoCytomation automated immunostainer). The reference, and dilution factor of primary antibodies were as follows: AE1/ AE3 cytokeratin (Dako M3515, 1/100), vimentin (Dako M0725, 1/800), and CD34 to detect keratocytes (Dako M7165, QBEnd10, 1/200); collagen IV (Immunotech 1115, 1/100); and a-smooth muscle actin to detect myofibroblasts (Dako M851, 1/100). The CD34 antigen is a 105-120 kDa transmembrane glycoprotein that is expressed on the surface of lymphohematopoietic stem and progenitor cells, small-vessel endothelial cells, embryonic fibroblasts, and some cells in fetal and adult nervous tissues. Its expression is highest in the most primitive stem cells and decreases gradually as lineage-committed progenitors differentiate. The CD34 antigen is also present on capillary endothelial cells and on bone marrow stromal cells.
Conventional Histology
Table 2 shows the results of the conventional histological evaluation. Focal epithelial hypoplasia immediately above the Intacs tunnel was present in 5 eyes. However, epithelial thickness appeared to be normal toward the center of the cornea (Figures 1 and 2). Six eyes had a break in Bowman’s layer, characteristic of advanced keratoconus. Channel haze was observed in the stroma in all eyes except case 4; the haze was at a depth ranging between 50% and 75% (Figure 3). There was no evidence of inflammatory cells, foreign-body granulomas, or neovascularization even though the Intacs inserts had been inserted next to the limbus. All corneas appeared to have a structurally sound stroma with parallel collagen lamellar fibers, except at the site next to insertion of the Intacs segments, where the fibers deviated to give way to the tunnel. As expected, no lipid droplets were observed in the sections because lipids are dissolved during preparation of the tissue for light microscopy. However, acidophilic densification suggestive of collagen synthesis was seen in
J CATARACT REFRACT SURG - VOL 33, FEBRUARY 2007
INTACS IN KERATOCONUS: HISTOPATHOLOGICAL FINDINGS
Table 2. Conventional histology findings.
Case
Epithelial Hypoplasia
Bowman’s Disruption
Channel Localization (%)
Granuloma
1 2 3 4 5 6 7 8
Yes Yes Yes No No Yes Yes Yes
Yes Yes Yes Yes No Yes No Yes
50 75 66 None observed 50–66 66 75 75
No No No No No No No No
2 eyes (cases 3 and 8) adjacent to the tunnel segments (Figure 4). A heterogeneous cellular group was found in 3 eyes (cases 6, 7, and 8) around the channel section (Figure 5). Immunohistochemistry
Immunochemistry of cells inside the tunnel and around the inner edge of the tunnel stained positively for collagen IV in 4 eyes (cases 1, 3, 6, and 8), suggesting that new collagen IV synthesis had occurred (Figure 6). Positive AE1/AE3 cytokeratin staining was limited to the corneal epithelium in all cases. Of particular note, the cellular formations next to the tunnel in samples from cases 6, 7, and 8 were cytokeratin AE1/AE3 negative. All samples stained negatively with a-smooth muscle actin, indicating myofibroblasts were not present. Results for the CD34 stain showed that fewer keratocytes were present above and below the Intacs insertion site than the lateral sides of the insertion site or the center of the cornea (Figures 7 and 8), except in case 4 in which the corneal scar from the insert was no longer visible. All keratocytes and endothelial cells stained positively with vimentin. However, in the deposit areas, a few cells that stained positive with vimentin stained negative with CD34.
Figure 1. Focal hypoplasia of the epithelium overlying the Intacs channel. Note that the epithelial thickness toward the center of the cornea is normal (case 6).
DISCUSSION
Most publications focus on clinical and refractive outcomes after intracorneal ring segment placement without considering the pathology of the corneal wound-healing response. When this issue has been addressed, the histopathological changes have usually been investigated using confocal microscopy,12 optic coherence tomography,13 ultrasound,14 or specular microscopy.15 Moreover, the largest study of the histological effects of intracorneal ring segment implantation conducted to date was performed in 5 New Zealand rabbits.7 Further histological studies in humans are needed because the healing response after Intacs implantation could have profound consequences on both keratoconus progression and the prognosis of future PKP. This study evaluated the histopathological effects of Intacs implantation in 8 keratoconic human corneas. Our findings of epithelial hypoplasia overlying the intracorneal ring segments and maintained stromal structure are in agreement with those in previous studies.7,8 However, normal epithelial thickness above the channel scar was found in cases in which PKP was performed several months
Figure 2. Focal hypoplasia of the epithelium (case 6).
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Figure 3. Channel haze in 50% depth of the stroma (case 1).
(5 to 27) after Intacs explantation. This suggests that hypoplasia of the epithelium over the segments initially regulates the corneal surface but is reversible after insert removal. In addition, the channel fibrous scar eventually heals after insert explantation; evidence in cases 4 and 5 suggest that the healing process requires between 5 months and 15 months. Studies have established that corneal implants evoke the intracellular and extracellular production of lipids, and this is thought to be a nonspecific response to the stress of degenerating cells.7,8 We were unable to identify lipid deposits in our study as we did not perform cryopreservation. However, we identified weak extracellular spaces adjacent to the scar tissue, suggesting an artifact of lipid removal. Our keratocyte density findings are particularly noteworthy. Results of CD34 staining showed that keratocyte density was lower in areas above and below the Intacs insertion site than in the center of the cornea and comparatively lower in the corneal region immediately next to the channel, albeit to a lesser degree. The CD34 antigen is a transmembrane glycophosphoprotein expressed on fibroblast-like cells in connective
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Figure 4. Acidophilic densification adjacent to the segments tunnel suggestive of collagen synthesis (case 3).
tissue.16 It has been known since 2002 that CD34 is expressed on human keratocytes but not by the epithelium or endothelium.17 In 2003, Joseph et al.16 suggested CD34 plays a role in cytoadhesion and signaling related to differentiation and proliferation. To our knowledge, our study is the first to report a decrease in keratocyte density. Twa et al.,7 who evaluated the histology of the corneal stroma after intrastromal corneal ring implantation in 5 New Zealand rabbits, found increased cellular bodies along the inner radius of the ring segments that were shown on conventional histological examination to be keratocytes. As this finding was associated with an elevated calcium level in the 6-month period after surgery, the authors considered it to be an initial response to surgical trauma. However, the study did not report whether keratocytes were present in the stromal tissue above and below the rings. Increased keratocyte density has also been reported in humans. Dawson et al.,11 who used electron microscopy to evaluate a unique corneal button from a patient with a history of previous corneal ring segments, found that activated keratocytes seemed to persist indefinitely.
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Figure 5. Nonhomogeneous cellular formation around the channel section (case 8).
However, neither the duration of corneal ring implantation nor the interval before PKP was reported. In a case report of a patient who had photorefractive keratectomy, Ferrara corneal rings implantation, and PKP with trephination of the corneal tissue containing the implant 2.5 years later, Twa et al.8 identified an increased density of vimentin-positive cells in the deposit region. They considered that the cellular response associated with the deposit was caused by keratocytes, most likely with a myofibroblastic phenotype. Moreover, they found a greater density of cells in the area central to the corneal implant. Spirn et al.9 report similar findings in a patient with progressive corneal keratectasia under the intracorneal ring segment. Ruckhofer,12 who performed confocal microscopy in
Figure 6. Collagen IV synthesis in the channel scar (case 1).
Figure 7. Decrease of CD34 positive cells superiorly and inferiorly to the rings (patient 1).
18 eyes of 12 patients, found that the stroma adjacent to the intracorneal segments appeared to have seemingly compressed layers with many keratocytes, especially underneath the segments.
Figure 8. Decrease of CD34 positive cells superiorly and inferiorly to the rings (patient 6).
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Although these studies show increased keratocyte density in the deposit region, little is known about keratocyte activity in other regions of the cornea. Moreover, there are no objective data comparing cell density in the area next to the rings with that in more distal sites. A decrease in CD34-positive cells could be related to a reduction in keratocytes or to a change in their phenotype via the cell life cycle and apoptosis.18 In primitive fibroblasts, such as dendritic cells in tumors and healing wounds, the CD34 disappears as the cells attain a more differentiated a-smooth muscle actin–positive myofibroblastic or collagenous synthetic phenotype.19 It was based on this understanding of keratocyte phenotypology that we decided to include vimentin staining in our study because the technique enables identification of nonspecific mesenchymatous cells. Although the results for vimentin and CD34 stains were reasonably comparable, some cells, especially in the deposit region, were CD34 negative and vimentin positive. This finding is probably a result of a reduction in keratocytes and a change in keratocyte phenotype. Because all samples stained negatively for a-smooth muscle actin, keratocyte differentiation does not appear to be of a myofibroblastic phenotype and is probably of a collagenous synthetic form. Indeed, our study identified collagen synthesis in the deposit areas. This has been reported,7 but to our knowledge we are the first to characterize the collagen as type IV. Kim et al.20 detected apoptotic keratocytes in the anterior stroma, particularly in the areas with Bowman’s disruption, in 60% of keratoconic corneas and 35% of stromal dystrophy corneas. Fabre et al.21 showed that chronic epithelial injury induces apoptosis via interleukin 1 and that keratoconic eyes may be more susceptible to apoptosis. The authors hypothesize that apoptosis, with normal release of degradative enzymes and other components, would damage surrounding cells. Thus, if Intacs inserts cause keratocyte apoptosis in the area next to the tunnel, it is possible the rings accelerate the progression of corneal ectasia through metalloproteinase release. This hypothesis could explain the cases of progressive keratolysis after Intacs implantation reported by Bourges et al.22 In cases in our study in which PKP was performed several months after Intacs explantation (case 4, 5, and 7), the cornea had become repopulated with keratocytes, probably from neighboring cells. In case 4, the scar was no longer visible. Several reports show that the refractive and topographic changes induced by Intacs inserts are reversible after explantation of the segments. Our study demonstrates that the histological effects of Intacs implantation are also reversible after insert removal. Nevertheless, further studies are needed to establish whether there is an association between Intacs placement and progression of keratoconus
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or graft rejection. Further study is also required evaluating the long-term consequences of reduced peripheral keratocytes associated with Intacs implantation, particularly with regard to axial stromal transparency.
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