Regenerative approaches for the cornea.

The cornea is the transparent front part of the eye that transmits light to the back of the eye to generate vision. Loss of corneal transparency, if irreversible, leads to severe vision loss or blindness. For decades, corneal transplantation using human donor corneas has been the only option for treating corneal blindness. Despite recent improvement in surgical techniques, donor cornea transplantation remains plagued by risks of suboptimal optical results and visual acuity, immune rejection and eventually graft failure. Furthermore, the demand for suitable donor corneas is increasing faster than the number of donors, leaving thousands of curable patients untreated worldwide. Here, we critically review the state of the art of biomaterials for corneal regeneration. However, the lessons learned from the use of the cornea as a disease model will allow for extension of the biomaterials and techniques for regeneration of more complex organs such as the heart.

Surface ablation of corneal stroma with few-cycle laser pulses at 800 nm.

We report measurements of crater diameter and surface ablation threshold as a function of laser fluence in porcine corneal stroma and fused silica with pulse durations of 7 fs (2.7 optical cycles), 30 fs and 100 fs at 800 nm. For laser pulses with Gaussian radial intensity profile, we show experimentally that the square of the crater diameter is a linear function of the logarithm of the fluence in fused silica, while it is closer to a linear function of the fluence in corneal stroma. Extrapolating these relations to zero diameter indicates that for both media the minimum fluence required for surface ablation is reduced with shorter pulse duration. A simple theoretical model suggests that this effect is due to a more significant contribution of photoionization as the laser pulse duration shortens.

Determination of porcine corneal layers with high spatial resolution by simultaneous second and third harmonic generation microscopy.

We describe a dual, second harmonic generation (SHG) and third harmonic generation (THG) microscope, with the aim to obtain large-scale images of the cornea that can simultaneously resolve the micron-thick thin layers. We use an Ytterbium femtosecond laser as the laser source, the longer wavelength of which reduces scattering and allows simultaneous SHG and THG imaging. We measure one-dimensional SHG and THG profiles across the entire thickness of pig cornea, detected in both the forward and backward directions. These profiles allow us to clearly distinguish all the porcine corneal layers (epithelium, stroma, Descemet's membrane and endothelium). From these profiles, longitudinal cross sectional images of the corneal layers are generated, providing large scale topographic information with high-spatial resolution. The ability to obtain both SHG and THG signals in epi-detection on fresh eyes gives promising hopes for in vivo applications.

Wavelength dependence of femtosecond laser ablation threshold of corneal stroma.

We measure the surface ablation threshold fluence of porcine corneal stroma for 100 fs laser pulses, with wavelengths between 800 nm and 1450 nm, generated by a Ti:sapphire-pumped optical parametric amplifier. The ablation threshold was found to vary only slightly within this wavelength range, between 1.5 and 2.2 J/cm(2). The data suggest a rapid increase of the ablation threshold for wavelengths up to about 1000 nm, followed by a plateau for longer wavelengths. This behavior is partly confirmed by a simple theoretical model of the ablation process. The influence of the wavelength on the physics of surface ablation is discussed on the basis of the model.

Are There Categories of Corneal Shapes?

This study investigates the possible existence of different natural corneal shape categories. This is important to better describe cornea for both diagnostic and therapeutic assessments. We started by describing corneal shape of different populations as a function of influencing clinical data i.e. age, ametropia and gender. This was done by averaging Zernike polynomial (ZP) decomposition of the anterior surfaces in each subgroup. The results showed small but significant differences of shape that are supported by the literature. This motivated us to examine the feasibility of characterizing the normal corneal shape with an automatic method of clustering independent of any clinical a priori knowledge. Since we did not know beforehand the number of corneal categories, agglomerative hierarchical clustering was applied on ZP coefficients for a large database. The dendrogram based on the Ward's distance was evaluated with two different clustering validity indexes (coefficient of determination R2 and semi partial R2 (SPR2). The optimal number of categories was around four showing corneal shapes ranging from flatter to steeper.

A system for long-term corneal perfusion.

Seventy-two human corneas were maintained in a perfusion system at 37 degrees C and 18 mm Hg intracameral pressure for 1 to 3 weeks. Corneal thickness, which was initially greater than normal because the enucleated eyes were kept at 4 degrees C before excision of the corneas, decreased slowly during the period of incubation. Endothelial removal or perfusion with ouabain (10(-4) M) induced irreversible stromal swelling. Cooling to 4 degrees C for 8 hr during perfusion caused stromal swelling that disappeared after rewarming to 37 degrees C; less stromal swelling occurred with cooling after 3 weeks of perfusion than after 3 days. No enlargement of central endothelial cells was noted in most corneas by serial specular microscopy. Electron microscopy demonstrated reversal of postmortem changes and maintenance of normal intracellular ultrastructure for 3 weeks. This system for long-term corneal perfusion will allow controlled studies of the effects of new methods of corneal preservation and other perturbations upon the corneal endothelium in situ.

Angiogenic factor: a possible mechanism for neovascularization produced by omental pedicles.

To determine possible mechanisms by which omental pedicles protect bronchial anastomoses from ischemia, we studied the angiogenic potential of a lipid extract of omentum. A rabbit cornea model was used to quantify neovascularization produced by methanol-chloroform extract of homogenized autologous omentum or perirenal fat. In 22 anesthetized rabbits, 10 microliters of omental lipid extract was injected into the cornea. In each animal the opposite eye was used as a control and was injected with a similar volume of extract prepared from perirenal fat. The side of injection of autologous omental fat was randomized and was not known to the investigator who assessed neovascularization on days 4, 7, 14, and 21 after injection. Neovascularization was recorded on microphotography and quantified by a point-counting method. Four days after injection, neovascularization in corneas injected with autologous omental fat was significantly (p less than 0.05) greater than in control corneas for all indices of neovascularization including total point count, which was three times greater than control. The angiogenic effect diminished with time, and by 21 days after injection corneal neovascularization was comparable for the two groups. Our results suggest that the lipid fraction of omentum has angiogenic activity that may stimulate neovascularization in ischemic tissues. Lack of sustained activity may be due to washout by neovessels or local tissue metabolism.

Penetrating keratoplasty with anterior or posterior chamber intraocular lens implantation.

OBJECTIVE: To compare the outcome of anterior chamber (AC) intraocular lens (IOL) implantation with the outcome of posterior chamber (PC) IOL implantation at the time of penetrating keratoplasty in the absence of capsular support. DESIGN: Retrospective study of 122 eyes that underwent secondary IOL implantation without capsular support at the time of penetrating keratoplasty. Ninety eyes received an AC IOL and 32 received a PC IOL sutured to the iris or the ciliary sulcus. RESULTS: Survival analysis showed the probability of a clear graft to be 86% for eyes with an AC IOL and 90% for eyes with a PC IOL at 1 year. Intraocular pressure rose by 5 mm Hg or more in 30% of eyes with AC IOLs and 5% of eyes with PC IOLs. Visual acuity was better than 20/200 in 48% of eyes with AC IOLs and 68% of eyes with PC IOLs. CONCLUSION: This study demonstrated a trend for a better outcome with PC IOLs than AC IOLs for secondary implantation at time of penetrating keratoplasty in the absence of capsular support.

Should we patch corneal erosions?

OBJECTIVE: To study the effect of patching on the speed of reepithelialization, slit-lamp signs of epithelial wound healing, and patient discomfort following a corneal abrasion. METHODS: Forty-eight eyes of 46 patients with corneal erosion sparing Bowman membrane were randomized into 2 groups: with or without patching. Slit-lamp examination and photographs of the fluorescein-stained cornea were performed on a daily basis until reepithelialization was complete. Photographs were analyzed using computer-assisted planimetry. RESULTS: No statistically significant difference was found between patched (n = 25) and nonpatched (n = 22) eyes for the mean size of the initial erosion (patched eyes, 23.7 mm2; nonpatched eyes, 18.9 mm2; P = .42), linear speed of reepithelialization (reduction over time of the radius of the largest circle included in the erosion: patched eyes, 0.0375 mm/h; nonpatched eyes, 0.0353 mm/h; P = .78), and surface speed of reepithelialization (reduction over time of the erosion area: patched eyes, 0.6510 mm2/h; nonpatched eyes, 0.5657 mm2/h; P = .60). The power to detect a 12-hour delay of epithelial closure was 95%. There were no significant differences between the 2 groups for pain, analgesia, insomnia, aspect of the epithelial border, intensity and duration of stromal edema, Descemet folds, anterior uveitis, and filaments. CONCLUSIONS: Patching a corneal erosion does not significantly accelerate reepithelialization and does not alter the epithelial wound healing pattern. It does not reduce the incidence and severity of inflammation nor relieve pain when compared with treatment without patching.

Anterior stromal punctures for bullous keratopathy.

OBJECTIVE: To evaluate the therapeutic effects of anterior stromal punctures (ASP) in patients with bullous keratopathy (BK). PATIENTS AND METHODS: Twenty-seven patients awaiting penetrating keratoplasty with a diagnosis of BK were examined. They were seen before treatment with ASP and 1, 4, and 12 weeks after treatment. The examination included slit-lamp examination, photography of the cornea, ultrasonic pachymetry, central esthesiometry, and pneumotonometry. Subjective evaluations of pain, discomfort, and photophobia were also done using a visual scale model. Photographs were analyzed by computer-assisted planimetry and used to measure the corneal surface covered by bullae and microcysts. Pretreatment and posttreatment values (mean +/- SEM) were compared using the Student paired t test. RESULTS: At 3 months, a significant reduction in pain was noted. A decrease in the mean corneal surface covered by bullae (BKPreASP = 2733 +/- 553 microns2; BK3mo = 1006 +/- 356 microns2, P = .004) was observed. A decrease in the esthesiometry (E) measurement (EPreASP = 3.5 +/- 0.4 cm; E3mo = 1.3 +/- 0.3 cm, P < .001), an increase in corneal thickness ([CT] CTPreASP = 869 +/- 24 microns; CT3mo = 902 +/- 21 microns, P < .001), and a decrease in the number of quadrants through which iris (I) details could be seen (IPreASP = 1.7 +/- 0.3; I3mo = 1.2 +/- 0.3, P = .015) were also noted. These findings corroborate the clinical observation of increased subepithelial fibrosis following ASP. CONCLUSIONS: Anterior stromal punctures reduce bullae formation and alleviate pain in patients with BK, and they constitute a valuable alternative to penetrating keratoplasty should surgery be delayed or contraindicated.

The VF-14 index of functional visual impairment in candidates for a corneal graft.

PURPOSE: To validate the Visual Function-14 (VF-14) index of functional visual impairment in candidates for a corneal graft. METHODS: One hundred thirty-four patients who were candidates for a corneal graft participated in this study between August 1996 and February 1997. Demographic, ocular history, best-corrected visual acuity, and detailed ocular examination data were collected. Functional visual impairment information was obtained by telephone interviews using the following: VF-14, SF-36 (Short Form-36, a more generic measure of general health function), and Visual Symptom Score, and four questions measuring the overall amount of trouble with vision, dissatisfaction with vision, ocular pain, and discomfort. RESULTS: The average age of corneal graft candidates was 64 +/- 18 years (range, 18 to 90 years) and 60% were women. The most frequent corneal disease was pseudophakic bullous keratopathy (41%). Ocular comorbidities included glaucoma or ocular hypertension (30%) and cataract (19%). The mean best-corrected visual acuity of the eye scheduled for surgery was 1.33 +/- 0.56 logMAR whereas the best eye best-corrected visual acuity was 0.36 +/- 0.44 logMAR. The mean VF-14 score was 73% +/- 26%, and the internal consistency was high, with a Cronbach alpha value of 0.94. The VF-14 correlated strongly with the best eye best-corrected visual acuity. It also correlated strongly with the Visual Symptom Score, the global measures of trouble and dissatisfaction with vision. Candidates for a corneal graft had low scores for all eight general health concepts evaluated with the SF-36, and the VF-14 correlated with seven of the eight SF-36 subscales. CONCLUSION: The VF-14 is a valid measure of functional visual impairment in candidates for a corneal graft. The Visual Symptom Score and the SF-36 are also useful indices in such patients.

Short-term oxidative status of lens and aqueous humor after excimer laser photorefractive keratectomy.

PURPOSE: Oxidation in the anterior ocular segment is associated with cataractogenesis and possible complications to corneal endothelium. We investigated whether oxidation occurred in the rabbit anterior ocular segment shortly after excimer laser photorefractive keratectomy (PRK). METHODS: Rabbits were treated unilaterally with PRK, the other eye serving as a control. Aqueous humor sampled shortly after treatment was assayed spectrophotometrically for hydrogen peroxide using ferrous oxidation in xylenol in the presence (group 1; n=10), or absence of oxygen (group 2; n=8). Oxidized glutathione and malondialdehyde levels were measured in lenses by spectrophotometry and HPLC. RESULTS: Hydrogen peroxide concentration of aqueous humor was not different between treated (77 +/- 36 microM) and control eyes (88 +/- 34 microM) in the oxygen group or the nonoxygenated group (treated eyes: 6.7 +/- 5.4 microM and control eyes: 5.5 +/- 4.7 microM). Peroxide levels did not correspond to endogenous H2O2 but presumably reflected action on ascorbic acid. There was no difference in the percent of oxidized glutathione between experimental and control eyes. Malondialdehyde could not be detected in the lens of treated or control eyes, despite good sensitivity in recovery assays. CONCLUSION: Based on these assays, there is no evidence that PRK oxidizes the aqueous humor or the lens of treated rabbits within 10 minutes of treatment.

Corneal transplant tolerance of cryopreservation.

PURPOSE: To study the outcome of corneal transplants performed with cryopreserved tissue. METHODS: Maisonneuve-Rosemont Hospital medical records of all corneal transplantations performed with cryopreserved tissue by one surgeon (M.L.F.) between March 1978 and April 1991 were reviewed. The Kaufman--Capella cryopreservation technique was used. Corneas were cryopreserved for periods of 3 days to 16.8 years (mean, 4.6 years) before transplantation. RESULTS: We report a mean follow-up of 54 months (range, 2.8--151.3 months). Survival analysis showed the probability of a clear graft to be 76% at 1 year and 73.2% at 2 years. At the time of the last visit, visual acuity was 20/40 or better in 61 eyes (49.2%). The mean postoperative pachometry was 0.58 mm (range, 0.50--0.75 mm). Specular microscopy performed in 57 eyes showed a mean endothelial cell count of 938 cells/mm(2) 55.1 months (range, 2.9--151.3 months) after surgery. For comparison purposes, the outcome of a subgroup of cryopreserved (n = 33) and noncryopreserved (n = 26) corneas transplanted by the same surgeon between April 1986 and April 1990 was studied. CONCLUSION: Despite an increase in the primary failure rate and higher initial endothelial cell loss, cryopreserved transplants are viable. Although we do not recommend cryopreservation of corneas for elective surgery, we consider that cryopreserved corneas can be very useful in emergency situations when tissue availability is a problem.

Corneal endothelial cell density in glaucoma.

PURPOSE: We studied corneal endothelial cell density in patients with glaucoma. METHODS: One hundred two patients with glaucoma were compared with 52 patients without glaucoma of the same age group. Exclusion criteria included history of either corneal disease, ocular inflammation, trauma, or surgery other than peripheral iridectomy. The following data were extracted from the patient files: glaucoma type and duration, laser treatments, glaucoma medications, and documented intraocular pressure (IOP) measurements. Specular microscopies were performed on central corneas, endothelial images were analyzed by computerized planimetry, and cell counts were calculated. RESULTS: Corneal endothelial cell counts were significantly lower in patients with glaucoma (2,154 +/- 419 cells/mm2) than in controls (2,560 +/- 360 cells/mm2; t test, p < 0.0001). In the glaucoma group, cell counts were inversely proportional to the means of IOPs. Patients receiving three or four glaucoma medications had lower cell counts than those receiving one or two medications. Cell counts were significantly lower both in primary angle-closure glaucoma and in primary open-angle glaucoma. CONCLUSION: This study suggests that patients with glaucoma may have lower corneal endothelial cell density than those without glaucoma of the same age group. The proposed mechanisms are direct damage from IOP, congenital alteration of the corneal endothelium in patients with glaucoma, glaucoma medication toxicity, or a combination of these.

Contrast and glare testing in the assessment of visual performance of candidate eyes for penetrating keratoplasty.

PURPOSE: To determine whether visual acuity (VA) measurements performed at low levels of contrast and glare are a better diagnostic tool for determining whether corneal clouding warrants surgery. METHODS: Fifty-nine subjects were recruited from among the candidates for corneal graft. Monocular VA was measured with three Regan contrast VA charts: 96, 25, and 11%, with and without glare provided by the Brightness Acuity Tester (BAT). The discriminative ability of the tests was estimated using the area (AR) under receiver operating characteristic (ROC) curves. Associations between the different VA tests and the Visual Function Index (VF-14) score were studied, using Spearman coefficients. RESULTS: When comparing candidate eyes with contralateral eyes with corneal disease, lower contrasts VA tests provided greater discriminative power. VA measurements made with glare also tended to provide greater discrimination. In fact, discrimination was best with 11% contrast VA with glare, but "testability" was poor. The most practical test in a clinical setting, which retained high discriminative ability (0.798), was the 25% contrast VA with glare. The eye with the best VA correlated strongly with the VF-14, especially at 25% contrast without glare, resulting in an Rs of -0.729. CONCLUSION: Twenty-five percent contrast VA with BAT could help the practitioner to decide whether a corneal transplant is warranted when symptoms of reduced vision are more important than what high-contrast VA might indicate.

Severe toxic keratopathy secondary to topical anesthetic abuse.

OBJECTIVE: To describe the clinical findings in three cases of abuse of topically administered ophthalmic anesthetics and to review the clinical signs, diagnosis and treatment. DESIGN: Case series. SETTING: Two university-affiliated hospitals in Montreal. PATIENTS: Three patients with toxic keratopathy due to abuse of topically applied anesthetics. RESULTS: The three patients presented with a nonhealing epithelial defect, marked stromal edema, folds in Descemet's membrane and a typical stromal ring infiltrate. All three required a conjunctival flap, and two underwent penetrating keratoplasty. The drugs (0.5% tetracaine and 0.5% proparacaine) were easily obtained, at the workplace in two cases and by stealing from the ophthalmologist's examining room in the third case. CONCLUSIONS: The unrestricted availability of topically applied ophthalmic anesthetics as over-the-counter medications in Canada must be reevaluated.

Automatic 3D reconstruction of quasi-planar stereo Scanning Electron Microscopy (SEM) images.

Scanning Electron Microscopy (SEM) is widely used in science to characterize the surface roughness of materials. Three-dimensional information can be obtained with SEM based on stereovision techniques. A stereo pair is typically obtained by tilting the sample by a few degrees. In this paper we present a fully automated method for 3D reconstruction from a SEM stereo pair without any particular constraint. Results are presented for corneal stromal surfaces.