Noninvasive corneal stromal collagen imaging using two-photon-generated second-harmonic signals

PURPOSE: To investigate the feasibility of using femtosecond-pulse lasers to produce second-harmonic generated (SHG) signals to noninvasively assess corneal stromal collagen organization. SETTING: The Eye Institute, University of California, Irvine, California, USA. METHODS: Mouse, rabbit, and human corneas were examined by two-photon confocal microscopy using a variable-wavelength femtosecond lasers to produce SHG signals. Two types were detected: forward scattered and backward scattered. Wavelength dependence of the SHG signal was confirmed by spectral separation using the 510 Meta (Zeiss). To verify the spatial relation between SHG signals and corneal cells, staining of cytoskeletons and nuclei was performed. RESULTS: Second-harmonic-generated signal intensity was strongest with an excitation wavelength of 800 nm for all 3 species. Second-harmonic-generated forward signals showed a distinct fibrillar pattern organized into bands suggesting lamellae, while backscattered SHG signals appeared more diffuse and indistinct. Reconstruction of SHG signals showed two patterns of lamellar organization: highly interwoven in the anterior stroma and orthogonally arranged in the posterior stroma. Unique to the human cornea was the presence of transverse, sutural lamellae that inserted into Bowman's layer, suggesting an anchoring function. CONCLUSIONS: Using two-photon confocal microscopy to generate SHG signals from the corneal collagen provides a powerful new approach to noninvasively study corneal structure. Human corneas had a unique organizational pattern with sutural lamellae to provide important biomechanical support that was not present in mouse or rabbit corneas.     

Structural analysis of normal corneas and diseased corneas by applying second harmonic generation

We have established a second harmonic generation (SHG) microscopy system for imaging of the human cornea with a mode-locked femtosecond laser and a laser confocal microscope. This SHG microscopy system has allowed us to scan corneal tissue noninvasively ex vivo and to obtain three-dimensional images of corneal collagen lamellae. Such three-dimensional imaging of the normal anterior cornea revealed that collagen lamellae at the anterior stroma are inter-woven and adhere to Bowman membrane with these adherent lamellae being designated "sutural lamellae." Sutural lamellae adhere to Bowman membrane at an angle of approximately 19 degrees, whereas the angle of lamellae in the mid-stroma relative to Bowman membrane is smaller. We hypothesize that the structural unit consisting of both Bowman membrane and the sutural lamellae contributes to the rigidity and anterior curvature of the cornea. SHG imaging of keratoconic corneas revealed an either abnormal or a total lack of structure of the sutural lamellae, suggesting that this abnormality might be related to that of the corneal anterior curvature in such corneas. Furthermore, SHG imaging of corneas affected by stromal edema showed that the structure of the sutural lamellae was maintained, although abnormal collagen signals both above and below Bowman membrane were detected in corneas affected by clinical stromal edema for more than 12 months. SHG imaging of the structure of collagen lamellae in normal and diseased corneas thus has the potential to provide insight both into the mechanism for maintenance of corneal curvature as well as into the pathophysiology of corneal diseases.     

Quantitative analysis of collagen fiber in keratoconus

In order to identify the direct pathogenic factors involved in the stromal thinning of keratoconus, quantitative analysis of keratocytes, collagen fibers and collagen lamellae in keratoconus cornea was performed histologically by light and electron microscopy. Both normal and keratoconus corneas showed a similar cell density of keratocytes in the central stroma, therefore the total number of keratocytes in keratoconus cornea might be smaller than that of controls, because of the thinning of stroma in the keratoconus. The collagen lamellae in keratoconus corneas showed a significant decrease in number compared with controls. There was a direct relationship between the stromal thickness and number of collagen lamellae. On the other hand, there was no statistical significance between normal and keratoconus corneas in terms of the thickness of collagen lamellae. These results suggest that the thinning of the cornea in keratoconus might occur as the result of a defect of some collagen lamellae due to a disorganization during the process of collagen lamellae formation.     

Histology after lamellar keratoplasty and corneal excimer laser ablation.

PURPOSE: To correlate clinical and histological findings after lamellar keratoplasty, phototherapeutic keratectomy, and application of a donor lenticule on a human cornea. METHODS: A cornea was obtained during penetrating keratoplasty. The specimen was fixated, dehydrated and embedded in Epon resin. The tissue was cut in 0.5-microm-thick semi-thin sections, stained with toluidine blue, and studied with light microscopy. RESULTS: The central part of the photoablated cornea, which was covered by the donor lenticule, did not differ from a normal cornea. Peripherally, a hazy ring was found clinically. Histology showed an irregular epithelium. Where it was thickened, the epithelium was hyperplastic and showed an increased number of cell layers. In the hazy region, Bowman's layer was absent, indicating that the donor lenticule did not cover this part of the photokeratectomized cornea. The anterior-most part of the corneal stroma was vacuolized and contained amorphous extracellular material; swollen keratocytes were present in this region. Beneath this layer, collagen lamellae were wavy and interwoven and keratocytes were increased in number, appeared swollen, and some had assumed an atypical shape. Peripheral to the haze, the cornea was clear. Histologically, the epithelium was irregular and hyperplastic, Bowman's layer was absent, and stromal collagen lamellae were abnormally organized, but no vacuolization was found. CONCLUSIONS: The formation of haze after excimer laser photokeratectomy can be minimized if the ablated stroma is covered by a corneal lenticule.     

Histopathological variation in keratoconus.

During examination of 131 penetrating keratoplasty specimens from patients with keratoconus obtained in an 11-year period, we observed two histopathologic variants based on the appearance of Bowman's layer and the corneal epithelium. "Typical" keratoconus specimens had multiple breaks in Bowman's layer and central epithelial thinning, whereas "atypical" corneas lacked breaks in Bowman's layer and had less thinning of the central epithelium. Ninety-five corneas were from patients who underwent grafting in only one eye. Seventy-six (80%) of these corneas were "typical" and 19 corneas (20%) were "atypical" in appearance. Both variants had similar degrees of central stromal thinning. Patients with "typical" and "atypical" corneas differed demographically by race only; 49% of "typical" and 95% of "atypical" corneas were from white individuals. Thirty-six corneas were from 18 patients who underwent bilateral penetrating keratoplasty. The histologic appearance of these corneal pairs was concordant in 13 patients and discordant (one "typical" and one "atypical" cornea) in five patients. Statistical analysis indicated that this distribution is not significantly different from that predicted by chance and suggests that "typical" and "atypical" corneas are manifestations of the same disease process.     

圆锥角膜中细胞凋亡及Fas-L蛋白的表达

目的 探讨凋亡与圆锥角膜发病的关系及凋亡相关蛋白Fas-L的表达.方法 对20例圆锥角膜及5例正常角膜用原位末端标记法(TUNEL)检测凋亡,用免疫组织化学SP法检测Fas-L蛋白的表达;透射电镜观察凋亡细胞的形态学变化.结果 TUNEL染色示圆锥角膜组中上皮层、基质层及内皮层中细胞凋亡与正常角膜组比较差异均有统计学意义(P＜0.05);免疫组织化学示圆锥角膜组与正常角膜组基质层间Fas-L表达比较差异有统计学意义(P＜0.05);透射电镜可见圆锥角膜中存在凋亡特征的细胞.结论 圆锥角膜中存在凋亡,Fas-L蛋白的表达存在异常,Fas-FasL系统可能在圆锥角膜细胞凋亡中发挥了重要作用.     

Changes in collagen orientation and distribution in keratoconus corneas

PURPOSE: To map the collagen orientation and relative distribution of collagen fibrillar mass in keratoconus corneal buttons. METHODS: Structural analysis was performed by obtaining synchrotron x-ray scattering patterns across the samples at 0.25-mm intervals. The patterns were analyzed to produce two-dimensional maps of the orientation of the lamellae and of the distribution of total and preferentially aligned lamellae. RESULTS: Compared with normal corneas, in keratoconus the gross organization of the stromal lamellae was dramatically changed, and the collagen fibrillar mass was unevenly distributed, particularly around the presumed apex of the cone. CONCLUSIONS: The development of keratoconus involves a high degree of inter- and probably intralamellar displacement and slippage that leads to thinning of the central cornea and associated changes in corneal curvature. This slippage may be promoted by a loss of cohesive forces and mechanical failure in regions where lamellae bifurcate.     

Biomechanical and wound healing characteristics of corneas after excimer laser keratorefractive surgery: is there a difference between advanced surface ablation and sub-Bowman's keratomileusis?

PURPOSE: To describe the biomechanical and wound healing characteristics of corneas after excimer laser keratorefractive surgery. METHODS: Histologic, ultrastructural, and cohesive tensile strength evaluations were performed on 25 normal human corneal specimens, 206 uncomplicated LASIK specimens, 17 uncomplicated sub-Bowman's keratomileusis (SBK) specimens, 4 uncomplicated photorefractive keratectomy (PRK) specimens, 2 uncomplicated advanced surface ablation (ASA) specimens, 5 keratoconus specimens, 12 postoperative LASIK ectasia specimens, and 1 postoperative PRK ectasia specimen and compared to previously published studies. RESULTS: Histologic and ultrastructural studies of normal corneas showed significant differences in the direction of collagen fibrils and/or the degree of lamellar interweaving in Bowman's layer, the anterior third of the corneal stroma, the posterior two-thirds of the corneal stroma, and Descemet's membrane. Cohesive tensile strength testing directly supported these morphologic findings as the stronger, more rigid regions of the cornea were located anteriorly and peripherally. This suggests that PRK and ASA, and secondarily SBK, should be biomechanically safer than conventional LASIK with regard to risk for causing keratectasia after surgery. Because adult human corneal stromal wounds heal slowly and incompletely, all excimer laser keratorefractive surgical techniques still have some distinct disadvantages due to inadequate reparative wound healing. Despite reducing some of the risk for corneal haze compared to conventional PRK, ASA cases still can develop corneal haze or breakthrough haze from the hypercellular fibrotic stromal scarring. In contrast, similar to conventional LASIK, SBK still has the short- and long-term potential for interface wound complications from the hypocellular primitive stromal scar. CONCLUSIONS: Ophthalmic pathology and basic science research show that SBK and ASA are improvements in excimer laser keratorefractive surgery compared to conventional LASIK or PRK, particularly with regard to maintaining corneal biomechanics and perhaps moderately reducing the risk of corneal haze. However, most of the disadvantages caused by wound healing issues remain.     

Ultrastructural analysis of collagen fibrils and proteoglycans in keratoconus

Purpose: To investigate ultrastructural alterations in the distribution of collagen fibrils (CFs) and proteoglycans (PGs) in the keratoconus cornea. Methods: Four normal corneas (donor age 24–75 years) and four severe and one mild keratoconus corneas (donor age 24–47 years) were fixed in 2.5% glutaraldehyde containing 0.05% cuprolinic blue dye for electron microscopy. Analyses were carried out on approximately 39 000 CF and 66 000 PG filaments in the anterior, middle and posterior stroma, using analySIS^® soft imaging software. Results: In severe keratoconus, stromal lamellae were seen to undulate in most regions, whereas in mild keratoconus only the middle and posterior lamellae were affected. In keratoconus corneas the mean diameter and interfibrillar spacing of CFs was reduced in all zones (p < 0.0001) and the CF and PG number density and area fractions were significantly increased (p < 0.0001) compared with in normal corneas and were higher (p < 0.0001) in the corneas with severe keratoconus than in that with mild keratoconus. The lamellae contained microfibrils (8–9 nm wide) and, in addition, PGs embedded within CFs. Degenerate keratocytes containing PGs were found in all keratoconus corneas. Conclusions: These studies suggest that as keratoconus progresses, the PG content of the stroma increases, whereas fibril diameter is reduced. The altered stromal content of PGs may influence CF diameters and their organization in keratoconus, weakening lateral cohesion and resulting in significant disorder of CF packing.     

Expression of type XII collagen and hemidesmosome-associated proteins in keratoconus corneas

PURPOSE: Keratoconus is a disease characterized by thinning of the central and paracentral cornea and scarring in advanced cases. This study was performed to examine the expression of type XII collagen, proteins associated with hemidesmosomes, and beta1 integrin in keratoconus corneas. METHODS: Corneal buttons were collected from normal subjects and patients with keratoconus and other corneal diseases. Immunofluorescence staining was performed on frozen sections for type XII collagen, bullous pemphigoid antigen (BP180), and integrin subunits alpha6, beta4, and beta1. RESULTS: To varying degrees, all proteins examined were expressed in normal human corneas. The staining intensity of type XII collagen was diminished in keratoconus corneas in the epithelial basement membrane zone and the stromal matrix. No significant variation was found in either the staining patterns or intensities for BP180, or integrins alpha6, beta4, and beta1. CONCLUSIONS: The level of type XII collagen was reduced in the epithelial basement membrane zone and stromal matrices in keratoconus corneas. These alterations may affect critical interactions of the corneal epithelium with the under-lying basement membrane, and cell-matrix interactions and matrix organization in the stroma.     

Keratectasia after laser in situ keratomileusis. Clinicopathological case report

PURPOSE: To describe the morphological features of a prominent ectasia of the cornea after laser in situ keratomileusis (LASIK). METHODS: The morphology of the ectatic corneas was examined using corneal topography, light microscopy and transmission electron microscopy in 2 cases who underwent penetrating keratoplasty due to poor visual acuity induced by progressive corneal ectasia after LASIK. RESULTS: On topographic examination, the apex of the corneal surface was observed within the central 3-mm zone, and the smallest thickness was 0.116 and 0.271 mm in each case. On histological examination, the epithelial layer became thinner and detached easily. Bowman's membrane was broken down and folded. An irregular arrangement of the stromal lamellae with fibroblastic keratocytes was found. The fulled fiber cell, a transformed epithelial cell, was visible in a plane on Bowman's layer in the central region. In contrast, the corneal endothelium was intact, and no abnormality was found in both cases. CONCLUSION: On morphological examination of 2 cases with corneal ectasia, a forward protrusion of both the anterior and posterior corneal surfaces occurred, and epithelial detachment, Bowman's membrane breakage and folding and irregular lamellae were found. The 2 cases had greatly thinned and protruding corneas, yet there was no abnormality in the corneal endothelium.     

Keratocyte apoptosis associated with keratoconus.

Keratoconus is an ectatic corneal dystrophy associated with stromal thinning and disruption of Bowman's layer. The purpose of this study was to explore a possible association between keratocyte apoptosis and keratoconus. Keratocyte apoptosis was evaluated in corneas of patients with keratoconus, corneas of patients with stromal dystrophies, and normal donor corneas using the transferase-mediated dUTP-digoxigenin nick and labeling (TUNEL) assay. Keratocyte apoptosis was also studied in keratoconus and normal corneas using transmission electron microscopy. TUNEL-stained keratocytes were detected in 60% of corneas with keratoconus, but only 35% of corneas with stromal dystrophies (P =0.03). The number of TUNEL-positive keratocytes detected in the keratoconus, stromal dystrophy, and normal corneas was 7+/-1 (mean+/-standard error, range 0-20), 2+/-0. 8 (range 0-9), and 0+/-0 (range 0-0) TUNEL-positive cells per section, respectively. The differences between the keratoconus and the stromal dystrophy (P =0.0097) or the normal cornea (P =0.01) groups were statistically significant. The difference between the stromal dystrophy and normal cornea groups was not statistically significant (P =0.45). The stromal dystrophy group was included to account for surgery-associated keratocyte apoptosis. No TUNEL-stained keratocytes were detected in normal corneas. Cell morphologic changes consistent with apoptosis were detected by transmission electron microscopy (TEM) in keratocytes of keratoconus corneas, but not in keratocytes in normal corneas. Chronic keratocyte apoptosis associated with ongoing epithelial injury may link risk factors associated with keratoconus such as chronic eye rubbing, contact lens wear, or atopic eye disease. Similarly, increases that have been detected in several different degradative enzymes in keratoconus corneas could be associated with chronic keratocyte apoptosis and less than perfect control of release of intracellular contents.     

Conductive keratoplasty: histological study of human corneas

PURPOSE: To determine the morphologic changes in human corneas over time following radiofrequency-based conductive keratoplasty (CK) treatment. DESIGN: Prospective, observational case series. METHODS: In a single-center study six human corneas of six patients with localized peripheral keratoconus underwent CK treatment followed by penetrating keratoplasty. Three spots were applied in the periphery of each cornea (6 mm optical zone). Corneal buttons were examined with light and electron microscopy at different postoperative intervals up to 6 months post-CK. RESULTS: In samples assessed on day one post-CK, small areas of detachment between the basal layer of epithelial cells and Bowman's layer were observed. At 1 week after the CK procedure, the epithelium appeared almost normal. Endothelium and Descemet's membrane had no alterations. In all samples, thermally induced misconfiguration of collagen fibers, described as "crumpled" changes of collagen layers, was observed reaching 75% to 80% of the stromal depth. The area of alterations had a cylindrical shape with a diameter of 120 mum. CONCLUSIONS: The conductive keratoplasty procedure produced collagen "crumpling and splitting" changes in human corneas, which were observed during the follow-up of 6 months. Areas adjacent to treatment site were minimally damaged.     

Report of a new family with dominant congenital heredity stromal dystrophy of the cornea.

PURPOSE: To report a new family with the rare form of congenital and hereditary stromal dystrophy of the cornea. METHODS: A mother and son, showing a bilateral congenital clouding of the cornea, were studied clinically and by biomicroscopy. After corneal transplantation, light microscopy and electron microscopy were performed. RESULTS: The stroma of the cornea was bilaterally and symmetrically thickened with diffuse and homogeneous small opacities. The opacities were present at birth and slowly progressive. Visual acuity was reduced to 2/10. Electron microscopy of the excised corneas showed a thickened stroma owing to cleaving of the lamellae by alternating layers of small-diameter collagen fibrils arranged in a random fashion. The epithelium, Bowman's membrane, the endothelium, and Descemet's membrane were normal. CONCLUSIONS: This family presents with a congenital stromal dystrophy of the cornea not linked to endothelial defects and thus differs from the more common form of congenital hereditary corneal endothelial dystrophy.     

Rabbit cornea microstructure response to changes in intraocular pressure visualized by using nonlinear optical microscopy

PURPOSE: To characterize the microstructural response of the rabbit cornea to changes in intraocular pressure (IOP) by using nonlinear optical microscopy (NLOM). METHODS: Isolated rabbit corneas were mounted on an artificial anterior chamber in series with a manometer and were hydrostatically pressurized by a reservoir. The chamber was mounted on an upright microscope stage of a custom-built NLOM system for corneal imaging without using exogenous stains or dyes. Second harmonic generation in collagen was used to image through the full thickness of the central corneal stroma at IOPs between 5 and 20 mm Hg. Microstructural morphology changes as a function of IOP were used to characterize the depth-dependent response of the central cornea. RESULTS: Regional collagen lamellae architecture through the full thickness of the stroma was specifically imaged as a function of IOP. Hypotensive corneas showed gaps between lamellar structures that decreased in size with increasing IOP. These morphologic features appear to result from interwoven lamellae oriented along the anterior-posterior axis and parallel to the cornea surface. They appear throughout the full thickness and disappear with tension in the anterior but persist in the posterior central cornea, even at hypertensive IOP. CONCLUSIONS: NLOM reveals interwoven collagen lamellae sheets through the full thickness of the rabbit central cornea oriented along the anterior-posterior axis and parallel to the surface. The nondestructive nature of NLOM allows 3-dimensional imaging of stromal architecture as a function of IOP in situ. Collagen morphologic features were used as an indirect measure of depth-dependent mechanical response to changes in IOP.     

Histopathology of epi-LASIK in eyes with virgin corneas and eyes with previously altered corneas

PURPOSE: To perform a histological analysis of free epithelial flaps that were intentionally created with an Epi-K epikeratome (Moria S.A.) during epi-LASIK in eyes with virgin corneas and eyes with previous corneal surgery or keratoconus. SETTING: Vissum-Instituto Oftalmológico de Alicante, Alicante, Spain. METHODS: This prospective and consecutive case series comprised 18 free flaps obtained from 18 patients. Twelve patients had virgin corneas, and 6 had altered corneas from previous surgery, trauma, or keratoconus. The flaps were fixed in 4% buffered formaldehyde (pH 7) for posterior histopathological analysis. Serial cuts of each flap were performed, and the sheets were stained with hematoxylin-eosin and periodic acid-Schiff. The main outcome measure was the histopathology of the corneal flaps. RESULTS: All flaps from virgin corneas consisted entirely of epithelium without residual stromal tissue or Bowman's layer. Histopathological analysis of the flaps after epi-LASIK in patients with previously altered corneas showed varying levels of stroma in all cases. CONCLUSION: Epi-LASIK with the Epi-K epikeratome effectively cleaved the epithelium from Bowman's layer in healthy corneas; however, when the integrity of Bowman's layer is compromised, epi-LASIK should be avoided as stromal invasion will likely occur.     

Excimer laser-assisted anterior lamellar keratoplasty for keratoconus, corneal problems after laser in situ keratomileusis, and corneal stromal opacities

PURPOSE: To evaluate excimer laser-assisted anterior lamellar keratoplasty to augment thin corneas as in keratoconus (<350 microm) and corneal ectasia after laser in situ keratomileusis (LASIK) and to treat anterior stromal opacities. SETTING: Ophthalmology Department, School of Medicine, Gazi University, Ankara, Turkey. METHODS: Thirteen eyes (5 keratoconus, 3 macular dystrophies, 1 post-LASIK ectasia, 1 post-LASIK interstitial keratitis, 3 post-herpetic keratitis sequelae) of 13 patients were included in this prospective study. The treatment group was divided into corneal ectasia and stromal opacity groups. A donor stromal button approximately 350 microm thick received a 100 microm excimer laser ablation on the endothelium. The remaining cornea (epithelium, Bowman's membrane, and stroma) was punched with a 7.5 or 7.7 mm trephine. After transepithelial ablation of the host cornea to 200 mum thickness, the corneal button was sutured with interrupted 10-0 monofilament nylon. Sutures were removed between 3 months and 6 months postoperatively. Preoperative and postoperative simulated keratometric cylinders and corneal thickness values were compared using the Wilcoxon signed rank test. The postoperative spherical equivalent refraction and best spectacle-corrected visual acuity (BSCVA) between the groups were compared using the Mann-Whitney U test. RESULTS: The mean follow-up was 27.6 months +/- 8.3 (SD). All patients gained 2 lines or more of BSCVA, and no patient lost a line. The mean corneal thickness was 381.2 +/- 88.2 microm preoperatively, which significantly increased to 534.9 +/- 96.6 microm postoperatively (P < .05). The mean preoperative simulated keratometric cylinder was 7.44 +/- 7.18 diopters (D); postoperatively, it decreased to 2.61 +/- 1.73 D (P < .05). There was no significant difference in postoperative spherical equivalent refraction or BSCVA between the groups (P > .05). CONCLUSIONS: This technique presents a different modality for the treatment of keratoconus, post-LASIK corneal problems, and other corneal stromal opacities with anterior lamellar keratoplasty. Additional studies with more patients and longer follow-up will help determine the role of this technique as a substitute for penetrating keratoplasty in these patients.     

A study of corneal thickness, shape and collagen organisation in keratoconus using videokeratography and X-ray scattering techniques

In keratoconus, the cornea becomes progressively ectactic resulting in severe visual impairment. Here, we use a combination of videokeratography and synchrotron X-ray diffraction to investigate the relationship between corneal shape and thickness, and the distribution and predominant orientation of stromal fibrillar collagen in five keratoconus corneas. In all but the least advanced case, the thinning and ectasia measured in vivo using corneal videokeratography was accompanied by corresponding changes in the relative distribution and orientation of stromal collagen in the excised corneal buttons. Although the most severe case of keratoconus possessed the most pronounced stromal collagen alterations, and only a minor disruption to stromal collagen arrangement was seen in the least advanced case, a variability in the extent of stromal collagen alteration was seen between these clinical extremes. The observed abnormalities in collagen distribution and orientation are consistent with a mechanism of keratoconus progression that involves inter-fibrillar or inter-lamellar slippage causing a redistribution of tissue within the cornea.     

Clinical and histopathologic changes in the host cornea after epikeratoplasty for keratoconus.

Five consecutive patients underwent epikeratoplasty for keratoconus. Postoperatively, four patients had poor visual acuity (average, 20/200) secondary to folds in Descemet's membrane and interface scarring. Two underwent penetrating keratoplasty eight months later. Histopathologic examination of the host corneas and the overlying lenticules disclosed epithelial irregularity and subepithelial fibrosis. The host corneas showed folds in Descemet's membrane and focal posterior stromal fibrosis. Electron microscopy disclosed breaks in Bowman's membrane with irregular collagen, posterior aggregates of amorphous material, and focal endothelial degeneration. The fifth patient had graft ulceration and vascularization that required removal of the lenticule. She underwent a penetrating keratoplasty five months later and histopathologic examination demonstrated persistent folds in Descemet's membrane. Immunostaining of specimens from three cases disclosed a reduced expression of sulfated epitopes of keratan sulfate and an increase in sulfated dermatan sulfate in the lenticule and host corneal tissues. These alterations in stromal proteoglycans are characteristic of stromal scars and keratoconus and provide evidence of pathologic processes in the graft tissue. Because of potential complications, epikeratoplasty should be considered only for those patients who are unsuitable candidates for contact lenses or penetrating keratoplasty.     

Reduced expression of the gap junction protein Connexin 43 in keratoconus

PURPOSE: The purpose of the present study was to examine whether keratoconus, which is a bilateral noninflammatory corneal ectasia with multifactorial aetiology, shows altered expression of Connexin (Cx43). Cx43 is an important gap junction protein that contributes crucially to epithelial and stromal integrity of cornea. METHODS: Eight keratoconic human corneal buttons were examined with immunohistochemistry and Western blotting and compared with eight normal human corneal buttons, to unravel changes in Cx43 expression. RESULTS: All normal corneas exhibited similar epithelial Cx43 expression patterns, with the protein located in the basal epithelial layer. In contrast, some keratoconic corneas showed an altered pattern of immunostaining and Western blotting confirmed a decreased expression of Cx43 in keratoconic corneas. CONCLUSIONS: Our results indicate that a decrease in Cx43 amount together with functional alteration of the protein is associated with keratoconus pathophysiology However, these changes apply only to some of the corneas examined and may not generally account for the development of keratoconus.