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In healthy eyes, the tear film lipid layer (TFLL) is considered to act as an evaporation resistant barrier, which prevents eyes from drying. Seeking to understand the mechanisms behind the evaporation resistance of the TFLL, we studied mixtures of lipid layer wax esters and O-acyl-omega-hydroxy fatty acids. Analyzing their self-assembly and biophysical properties led to new discoveries concerning the structure and function of the TFLL. We discovered how these lipids self-assemble at the air-water interface and form an efficient antievaporative barrier, demonstrating for the first time how the interaction of different tear film lipid species can improve the evaporation resistance compared with individual lipid classes on their own. These results provide a potential mechanism for the evaporation resistance of the lipid layer. In addition, the results serve as a base for the future development of improved dry eye treatments and other applications where the evaporation of water represents a significant challenge.

The marked sexual dimorphism prevalent in inflammatory/autoimmune diseases is mostly due to sex hormone actions. One common eye disease that disproportionately affects women is dry eye. Thus, our aim was to optimise our highly sensitive liquid chromatography–tandem mass spectrometry method for steroid hormone quantification in tear fluid (TF). We used tears and matched serum samples from 10 heathy individuals. Estrone, estradiol testosterone, progesterone, androstenedione, and dehydroepiandrosterone, were quantified with an HPLC coupled with a Triple Quad 5500 MS. Estrone was measured in 80% of female and 20% of male TF samples (mean ± SD, 68.9 ± 62.2 pmol/L), whereas estradiol was undetectable in tears. Progesterone was identified in half of the female tear samples (2.91 ± 3.47 nmol/L) but in none of the male samples, whereas testosterone was quantifiable only in male tears (0.24 ± 0.1 nmol/L). TF hormone levels were, on average, from 1.4% to 55% of systemic values. Estrone, progesterone, and testosterone levels in tears correlated with the matching serum samples (r = 0.82, 0.79, and 0.85, respectively), but androstenedione and dehydroepiandrosterone showed no correlations. Our LC–MS/MS method could detect five out of the six steroid hormones studied in individual human TF samples and could therefore be used to analyse the role of sex steroids in eye diseases.

In the research field of extracellular vesicles (EVs), the use of fetal bovine serum (FBS) depleted of EVs for in vitro studies is advocated to eliminate the confounding effects of media derived EVs. EV-depleted FBS may either be prepared by ultracentrifugation or purchased commercially. Nevertheless, these preparations do not guarantee an RNA-free FBS for in vitro use. In this study we address the RNA contamination issue, of small non-coding (nc)RNA in vesicular or non-vesicular fractions of FBS, ultracentrifugation EV-depleted FBS, commercial EV-depleted FBS, and in our recently developed filtration based EV-depleted FBS. Commercially available serum- and xeno-free defined media were also screened for small ncRNA contamination. Our small ncRNA sequencing data showed that all EV-depleted media and commercially available defined media contained small ncRNA contaminants. Out of the different FBS preparations studied, our ultrafiltration-based method for EV depletion performed the best in depleting miRNAs. Certain miRNAs such miR-122 and miR-203a proved difficult to remove completely and were found in all media. Compared to miRNAs, other small ncRNA (snRNA, Y RNA, snoRNA, and piRNA) were difficult to eliminate from all the studied media. Additionally, our tested defined media contained miRNAs and other small ncRNAs, albeit at a much lower level than in serum preparations. Our study showed that no media is free of small ncRNA contaminants. Therefore, in order to screen for baseline RNA contamination in culturing media, RNA sequencing data should be carefully controlled by adding a media sample as a control. This should be a mandatory step before performing cell culture experiments in order to eliminate the confounding effects of media.

Dry eye syndrome (DES) is a prevalent disease in which the tear film homeostasis is compromised. One of the main causes of DES is thought to be an alteration in the composition of the outermost layer of the tear film, the tear film lipid layer (TFLL), resulting in an increased evaporation of water from the tear film and subsequent drying of the ocular surface. Recent studies have suggested that the specific TFLL lipids, namely, O-aryl-omega-hydroxy fatty acids (OAHFAs) and diesters (DiEs), may play a role in the development of DES. However, their specific connection to DES has remained largely unknown until now because of the lack of information on their biophysical properties and their role in the TFLL. Herein, we have addressed this issue by studying the biophysical properties and evaporation resistance of a library containing 10 synthetic analogues of TFLL OAHFAs and DiEs. Our results show how the variations of chain length and polar groups affect the phase behavior of these lipids at the tear film surface. In addition, the results revealed that the OAHFAs exhibiting a liquid-expanded to solid phase transition formed films with high evaporation resistance, whereas the DiEs were found to have no evaporation resistance. Altogether, our results shed new light on the role of the OAHFAs and DiEs in the TFLL and their connection to DES, suggesting that OAHFAs are likely a key lipid class in maintaining the TFLL evaporation resistance.

Objective To evaluate visual performance after unilateral photorefractive keratectomy (PRK) performed for ≥2.75-diopter (D) myopic anisometropia. Design Prospective observational case series. Participants Eleven patients exhibiting ≥2.75-D myopic anisometropia appropriate for PRK. Methods Photorefractive keratectomy was performed on 11 eyes of 11 patients for myopic anisometropia ranging from –2.75 to –9.00 D. Best spectacle-corrected visual acuity (VA), contrast sensitivity (CS), and pattern visual evoked potentials (pVEPs) were measured preoperatively and at 3 and 6 months. Main outcome measures Best spectacle-corrected VA; pVEPs; and CS, measured as the area under the CS curve. Results Best spectacle-corrected VA increased nonsignificantly 6 months after PRK. A tendency towards decreased latency time of pVEPs for both eyes was observed after PRK. A significant increase in CS occurred after PRK in the operated eye at 6 months, but a tendency toward decreased CS appeared in the unoperated eye at 3 months. Preoperative CS levels, however, were reached at 6 months. Conclusions The adult central visual system is sensitive to visual deprivation caused by anisometropia. Imbalance between eyes may improve visual performance in the more emmetropic eye. Anisometropia appears to reduce visual performance in the more myopic eye, but this can probably be reversed by refractive correction.

PURPOSE: To evaluate the time course of changes in corneal sensitivity to mechanical and chemical stimuli produced by laser in situ keratomileusis (LASIK) in humans. METHODS: We performed a cross-sectional study of 17 LASIK-operated eyes (VisX S2, equipped with version 2.50-3.10 software) and 15 control eyes of 17 individuals to evaluate regeneration of corneal sensitivity after LASIK. Gas pulses of variable flow and compositions were applied to the cornea by a non-contact gas esthesiometer. Mechanical stimuli consisted of air puffs at flows from 0 to 200 ml/min. Chemical stimulation was made with gas pulses containing 0% to 80% CO2 in air at subthreshold flow. Mechanical and chemical thresholds and intensity-response curves for the evoked sensations were determined prior to surgery, and 7 to 9 days, 3 to 5 months, and 1.5 to 2.5 years after surgery. RESULTS: Corneal sensitivity to mechanical stimulation was enhanced 7 to 9 days after surgery but subsequently dropped markedly and remained significantly below control levels 3 to 5 months after LASIK. Sensitivity to both mechanical and chemical types of stimuli was close to normal 2 years postoperatively. CONCLUSIONS: Corneal sensitivity decreased immediately after LASIK but mechanical sensitivity showed a transient hyperesthesia 7 to 9 days afterward. Subsequently, a long-lasting and deep hypoesthesia to mechanical and chemical stimuli was observed. Gas esthesiometry revealed that disturbances of corneal sensation still exist at times when coarse mechanical sensitivity appeared to be normal. [J Refract Surg 2004;20:229-235]

Purpose: To examine the inflammatory reaction in acute or late-onset post-laser in situ keratomileusis (LASIK) diffuse lamellar keratitis (DLK) associated with an epithelial defect. Setting: Department of Ophthalmology, Helsinki University Central Hospital, Helsinki, Finland. Methods: Six consecutive LASIK patients presented with stage 2 to 3 unilateral DLK 1 to 4 days after formation of an epithelial detachment (intraoperatively or up to 19 months postoperatively). Five corneas of 5 DLK patients, 1 eye twice, were examined by corneal in vivo confocal microscopy 1 to 8 days after the appearance of the epithelial defect. Confocal microscopy of conjunctival venules was performed in 2 of 6 DLK patients to quantify leukocyte rolling and extravasation. Corneas of 5 patients and conjunctival venules of 4 patients who had uneventful LASIK served as controls. Results: Two of the 4 patients examined 0 to 1 day after the onset of DLK presented with small objects, presumably inflammatory cells (diameter 6.0 to 10.0 μm), in the LASIK flap interface. A third patient examined 1 day after the onset of DLK had larger objects (approximately 13.0 μm in diameter) in the interface. Three other cases (1 to 7 days after the onset of DLK) showed changes typical of keratocyte activation and altered extracellular matrix. All cases healed completely following treatment with steroids. Control LASIK subjects showed some keratocyte activation on day 5. Conclusions: Neither uneventful LASIK nor DLK induced an inflammatory reaction displaying leukocyte rolling in conjunctival venules or extravasation into the conjunctival stroma. Diffuse lamellar keratitis related to an epithelial defect does not always lead to the appearance of inflammatory cells in the flap interface. The corneal manifestations of epithelial defect-related DLK may originate from sterile epithelial–stromal or inflammatory cell–stromal cell interactions, leading to alteration of the keratocyte phenotype.

Purpose: To study the effect of photorefractive keratectomy (PRK) on the concentration of group IIA phospholipase A 2 (GIIAPLA 2 ) in tears. Setting: Departments of Ophthalmology and Pathology, University of Turku, Turku, and Helsinki University Eye Hospital, Helsinki, Finland. Methods: Tear samples were collected from 25 eyes of 23 patients (mean age 32.3 years ± 8.6 [SD]) preoperatively and 2 and 7 days after PRK. The GIIAPLA 2 concentration in the tears was measured by time-resolved fluoroimmunoassay. Results: The GIIAPLA 2 concentration was significantly lower and the tear fluid flow rate significantly higher 2 days after PRK than preoperatively. At 7 days, the GIIAPLA 2 concentration and the tear fluid flow-corrected excretion of GIIAPLA 2 were significantly higher than preoperatively and at 2 days. The tear flow rate was also significantly higher than preoperatively. Conclusions: The GIIAPLA 2 content in tears decreased 2 days after PRK due to dilution of the GIIAPLA 2 content during hypersecretion of reflex tears. Photorefractive keratectomy caused an increase in the tear flow rate, GIIAPLA 2 concentration, and tear fluid flow-corrected excretion of GIIAPLA 2 in tears 7 days after surgery, enhancing the protection of tears against bacterial infections.

Purpose To assess the value of in vivo confocal microscopy (CM) in the diagnosis of Fleck dystrophy and pre-Descemet's membrane corneal dystrophy. Methods Case report of two patients. Standard slit-lamp and ophthalmic examination and in vivo CM were performed on both patients. The thickness of the cornea and the morphology of the corneal epithelium, stroma, endothelium, and subbasal nerves were evaluated by confocal microscopy. Results Biomicroscopy revealed bilateral, fine, dust-, and flour-like opacities in the corneal stroma for the Fleck dystrophy patient. In the pre-Descemet's membrane corneal dystrophy patient, biomicroscopy showed opacities larger than those in the first patient. Both patients were then examined by in vivo CM. Confocal microscopy of the Fleck dystrophy showed intracellular deposits throughout the stroma. In pre-Descemet's membrane corneal dystrophy, however, these and the extracellular deposits were observed immediately anterior to Descemet's membrane. The thicknesses of the corneas were 560 and 650 microm for Fleck and pre-Descemet's membrane corneal dystrophy, respectively. The surface epithelium, subbasal nerves, and endothelium showed normal morphology in both patients. Conclusion In vivo CM is a valuable tool in diagnosing rare corneal dystrophies when the final diagnosis is difficult to obtain with conventional methods.

We describe four patients who developed corneal erosion after an exposure to a pepper spray containing toxic carriers. Two of these patients were exposed to a pepper gas containing 5% oleoresin capsicum (OC) as an irritant and 92% trichlorethylene or unknown amount of dichloromethane as a carrier. One patient was exposed to a mock (containing 92% trichlorethylene as a carrier) training pepper gas without OC. The fourth patient was exposed to an unidentified Russian pepper gas spray. Two of the patients were examined by in vivo confocal microscopy to demonstrate the depth and quality of the stromal damage. To test the toxicity of the commercial tear spray, it was analyzed and test sprayed on a soft contact lens and into a plastic cup. Visual acuity was measured and the eyes were examined with a slit-lamp up to 5 months. Physical damage to a soft contact lens was visually acquired. All patients showed a long-lasting, deep corneal and conjuctival erosion, which resolved partly with medical therapy during the following weeks/months. Confocal microscopy revealed corneal nerve damage, and keratocyte activation reaching two-thirds of stroma for one patient. The spray caused serious damage to both the soft contact lens and the plastic cup. The safety of the commercially available pepper sprays should be assessed before marketing, and a list of acceptable ingredients created. The sprays should also have instructions on the use of the compound as well as on the first aid measures after the exposure. Solvents known to be toxic should not be used.

Hydrocortisone reduces the number of inflammatory leukocytes within tissues, but thus far the site of action on the multistep adhesion cascade leading to leukocyte extravasation has not been identified. We have recently developed a noninvasive in vivo reflected-light confocal microscopy technique to study this at sites of inflammation in human patients. In the present study, we evaluated the effect of preoperative intravenous hydrocortisone treatment on leukocyte trafficking after conjunctival inflammation induced by cataract surgery in human subjects in vivo. The surgery generated leukocyte rolling along the endothelial lining of conjunctival vessels. While preoperative hydrocortisone did not reduce the number of rolling cells, it significantly raised the velocity of individual rolling leukocytes and concomitantly reduced leukocyte emigration into conjunctival tissue. Immunohistology of conjunctival biopsies excised from the individuals studied provided circumstantial evidence that endothelial P-selectin might play a role in the surgery-induced up-regulation of the leukocyte rolling. Furthermore, hydrocortisone reduced surgery-induced P-selectin induction, suggesting a role for this selectin in the regulation of local leukocyte traffic into sites of inflammation in human conjunctiva. Taken together, these results suggest that control of the rolling velocity might be an effective way to adjust leukocyte traffic in vivo in human subjects.

To study the effect of inlay implantation on corneal shape, assessing the changes using optical coherence tomography (OCT) and a customized image analysis software.Thirteen rabbit eyes were operated on, with positive-powered corneal inlays implanted into 11 eyes and 2 eyes serving as flap-only controls. Cross-sectional OCT images were obtained using Optovue preoperatively, and 1 hour, 1 week, and 1 month postoperatively. Topography maps were obtained preoperatively using Medmont E300. Image analysis software was built to extract corneal thickness and radius of curvature. Anterior corneal radius of curvature values obtained from the OCT images were compared with the corneal topography maps to assess the validity of the method.Corneal thickness increased more than predicted immediately after the implantation. However, by 1 month, it matched closer to the added thickness of the corneal inlays. An overall pattern of epithelial thinning was observed of up to 25% at 1 month. The anterior corneal surface had steepened up to 15% at 1 month, whereas inconsistent changes for the posterior corneal surface were observed.After corneal inlay implantation, an immediate response was observed in the cornea. Corneal swelling because of surgical trauma was the most likely cause for the observed thickening of the cornea up to 1 week. By 1 month, the epithelial layer measured thinner for most eyes. Most of the added thickness was transferred to the central anterior corneal surface, causing steepening at 1 month. The anterior corneal radius of curvature values obtained from OCT images were in broad agreement with Medmont topography.

Purpose: Climatic droplet keratopathy (CDK) is an acquired corneal disease characterized by progressive scarring of the cornea. In several corneal diseases, matrix metalloproteinases (MMPs) are upregulated during the degradation of epithelial and stromal tissues. We investigated the levels, degree of activation and molecular forms of MMP-2, MMP-9, MMP-8 and MMP-13 and their tissue inhibitors TIMP-1 and TIMP-2 in tear fluid of patients with CDK. Methods: Seventeen CDK patients and 10 controls living in Argentine Patagonia received a complete eye examination, and MMPs and TIMP-1 ⁄ 2 were determined by immunofluorometric assay (IFMA), gelatin zymography and quantitative Western immunoblot analysis in tear samples. Results: The MMPs were detected mostly in their latent forms. The levels of MMP-9 and MMP-2 were found to be significantly elevated in CDK patients, whereas latent and active MMP-8 levels were significantly enhanced in controls. There was no significant difference in the level of MMP-13. TIMPs were found as part of complexes, and the TIMP-1 levels were significantly lower in patients than controls. Conclusion: Elevated MMP-2 and MMP-9 levels have been implicated in the failure of corneal re-epithelialization, and enhanced MMP-2 and MMP-9 levels in CDK patients suggest that these MMPs may play a role in corneal scarring in CDK. Elevated levels of MMP-8 suggest a defensive role for this MMP in inflammatory reactions associated with recurring corneal traumas. Decreased expression of TIMP-1 in CDK patients suggest deficient antiproteolytic shield likely to render the corneas of CDK patients vulnerable to enhanced MMPs. Overall, these data suggest a mechanistic link between MMPs and TIMP-1 level in cornea and tears with corneal scarring in CDK.

PURPOSETo study the results of late photorefractive keratectomy (PRK) in corneas originally subjected to LASIK. METHODSSeven eyes of seven patients who had LASIK for myopia were retreated with PRK at least 2 years after LASIK. Uncorrected visual acuity (UCVA), best spectacle-corrected visual acuity (BSCVA), refraction, and haze were evaluated before and after LASIK and after retreatment by PRK. RESULTSPhotorefractive keratectomy retreatment improved BSCVA in six (86%) of seven eyes, and one (14%) eye showed no changes. None of the eyes lost lines of BSCVA. Five of seven eyes developed mild haze, which disappeared before the last postoperative follow-up. CONCLUSIONSPhotorefractive keratectomy retreatment performed at least 2 years after LASIK can improve visual acuity. We hypothesize that LASIK-induced corneal nerve damage disturbs corneal wound healing by increasing the tendency for development of haze. [J Refract Surg. 2008;24:710-712.] ABOUT THE AUTHORS From the Department of Ophthalmology, University of Helsinki (Neira-Zalentein, Moilanen, Tuisku, Holopainen, Tervo); and Centre for Military Medicine, Aero Medical Center (Moilanen), Helsinki, Finland. This study was supported by the Helsinki University Research Fund TYH 3316 and 6243, Evald and Hilda Nissi Foundation, Mary och Georg C. Ehrnrooths Foundation; Research Foundation of Orion Corporation; Friends of the Blind Foundation; Leo, Mary and Mary-Ann Hackman Foundation; and the Finnish Eye Foundation. The authors have no financial interest in the materials presented herein. Correspondence: Timo M.T. Tervo, MD, Dept of Ophthalmology, University of Helsinki, PO Box 220, 00029 HUS, Finland. Tel: 358 9 471 73109; Fax: 358 9 471 73162; E-mail: timo.tervo@hus.fi Received: June 20, 2007 Accepted: April 1, 2008 Posted online: May 15, 2008

Purpose To evaluate the long-term effects of excimer laser treatment for ametropia after surgical treatment of rhegmatogenous retinal detachment (RRD) with scleral buckles. Setting Helsinki University Eye Hospital, Helsinki, Finland. Methods Ten patients (10 eyes) who had 1 or more surgeries for RRD followed by refractive surgery for myopia were retrospectively enrolled in this study and were examined after excimer laser refractive surgery. Photorefractive keratectomy (PRK) or laser in situ keratomileusis (LASIK) was performed using a Star S2 excimer laser system (Visx). The best spectacle-corrected visual acuity (BSCVA), refraction, degree of anisometropia, and topographical changes were evaluated before and after the surgeries. Results All patients were free of asthenopic symptoms after refractive surgery. At the end of the follow-up, a mean of 67 months ± 14 (SD) after refractive surgery, 6 patients were within ±1.0 diopter of the intended correction. Compared with previously reported cohorts of myopic patients, the achieved refraction in patients who previously had a scleral buckling procedure was worse. The postoperative refraction was stable, and corneal topography did not show induced scar formation, keratectasia, or irregular astigmatism. After refractive surgery, the BSCVA improved 1 Snellen line in 3 patients and 2 lines in 1 patient. One patient lost 1 Snellen line and another patient lost 2 lines. Four patients showed no changes. New retinal complications were not observed. Conclusions In the long-term, PRK and LASIK were safe methods for the treatment of myopia in RRD patients after scleral buckling. The predictability of myopic LASIK/PRK may be worse than generally reported in myopic cohorts.

PURPOSE: To report a method of treatment for through-the-flap multibacterial ulcerative keratitis after laser in situ keratomileusis (LASIK). METHODS: Bacterial ulcerative keratitis after LASIK was treated with topical and systemic antibiotics followed by flap lifting, cleaning, and phototherapeutic keratectomy (PTK). Follow-up examinations included in vivo confocal microscopy, corneal topography, and wavefront analysis. RESULTS: Rapid recovery of the ulcerative keratitis was observed after flap lifting and cleaning of the interface and PTK combined with topical and systemic antibiotics. Two years postoperatively, corneal topography showed a slight depression of the ulcer area and decentration of the photoablation. Wavefront analysis revealed an irregular scan with a pronounced coma-like aberration, which with a wavefront-guided custom test lens correction provided 20/16 visual acuity. CONCLUSIONS: Ulcerative bacterial keratitis is a possible sight-threatening complication of LASIK refractive surgery. Lifting and rinsing the flap combined with cleaning of the flap interface with PTK may be helpful in these conditions when regression of the ulcer does not occur with topical and oral antibiotic treatment. [J Refract Surg. 2005;21:404-406.]

To evaluate visual performance after unilateral photorefractive keratectomy (PRK) performed for/=2.75-diopter (D) myopic anisometropia.Prospective observational case series.Eleven patients exhibiting/=2.75-D myopic anisometropia appropriate for PRK.Photorefractive keratectomy was performed on 11 eyes of 11 patients for myopic anisometropia ranging from -2.75 to -9.00 D. Best spectacle-corrected visual acuity (VA), contrast sensitivity (CS), and pattern visual evoked potentials (pVEPs) were measured preoperatively and at 3 and 6 months.Best spectacle-corrected VA; pVEPs; and CS, measured as the area under the CS curve.Best spectacle-corrected VA increased nonsignificantly 6 months after PRK. A tendency towards decreased latency time of pVEPs for both eyes was observed after PRK. A significant increase in CS occurred after PRK in the operated eye at 6 months, but a tendency toward decreased CS appeared in the unoperated eye at 3 months. Preoperative CS levels, however, were reached at 6 months.The adult central visual system is sensitive to visual deprivation caused by anisometropia. Imbalance between eyes may improve visual performance in the more emmetropic eye. Anisometropia appears to reduce visual performance in the more myopic eye, but this can probably be reversed by refractive correction.

Purpose. To describe the confocal microscopic findings, with special reference to corneal subbasal nerves, after herpes simplex virus (HSV) keratitis. Methods In this study, 16 HSV eyes and 14 contralateral eyes of 16 patients, diagnosed with unilateral HSV keratitis 1–12 months earlier by the presence of dendritic corneal ulceration or microbiologic confirmation, were examined by in vivo confocal microscopy for evaluation of corneal morphology. Results. Herpes simplex virus eyes: In 2 eyes the surface epithelial cells appeared large, and no abnormalities were observed in the basal epithelial cells. In 2 eyes subbasal nerve fiber bundles were completely absent, in 3 eyes there was a reduced number of long nerve fiber bundles, and in 11 eyes the subbasal nerve plexus appeared normal. In 10 corneas, highly reflective dendritic structures were found at the level of the basal epithelial cells. Frequently these structures were found in the vicinity of stromal fibrosis. Areas with increased abnormal extracellular matrix were found in 11 eyes. Stromal nerves were not visualized in all corneas, but appeared normal when observed. Results. Contralateral eyes: No abnormalities were observed in the epithelium. All corneas presented with a normal subbasal nerve plexus, but in 2 eyes dendritic particles were observed. Three corneas presented with activated keratocytes and increased amounts of abnormal extracellular matrix. Conclusions. When visualized by confocal microscopy, the subbasal nerve plexus appears relatively unaffected in cases with resolved HSV keratitis. Unidentified dendritic structures, presumably Langerhans cells, are frequently seen at the level of the basal epithelium in corneas with a history of herpetic disease.

Corneal sensory nerves are involved not only in corneal healing1–5 but they also participate in the reflex arc regulating tear fluid flow.6–8 Recent studies have also revealed that both stromal keratocytes and basal epithelial cells are innervated,9 an observation that further underlines the potential importance of neural input in corneal healing. In vivo confocal microscopy (CM) has proven to be a relatively reliable method for studying corneal nerves in clinical patient materials.10–13 The recovery of the normal morphology of the subbasal nerve bundles correlates with the restoration of corneal sensitivity.11 Unfortunately the Cochet-Bonnet esthesiometer seems to be relatively insensitive in detecting sensory alterations when compared with a non-contact gas esthesiometer.12 Nonhealing corneal ulcers are frequently associated with impaired corneal sensitivity and ocular surface pathology.14,15 Consequently, clinical tools that enable the direct evaluation of corneal innervation are needed. The current chapter is a review on our confocal microscopical findings on some post-surgical conditions such as PRK10 and LASIK,11,13,16,17 as well as some corneal diseases: familial amyloidosis type II/Meretoja disease,18 autosomal dominant cornea plana19, basement membrane dystrophy/recurrent erosion syndrome,20,21 diabetic neuropathy,20,22 herpes keratitis20 and Sjogren’s syndrome.

A novel non-invasive, in vivo technique for the quantification of leukocyte rolling and extravasation at sites of inflammation in human patients

Understanding of corneal wound healing plays an important role, not only in management of corneal infections, but especially in refractive surgery. A better control of wound healing mechanisms might improve the results of such resculpturing techniques and help to avoid complications arising from these procedures. While studies have been focused in different aspects of corneal wound healing, our knowledge has increased greatly during the last years. Many problems associated with corneal healing also contribute to clinical pathology following corneal surgery. Understanding of such conditions has been augmented by the continuously developing corneal imaging techniques. We have used in vivo confocal microscopy (IVCM) for assessing corneas subjected to refractive surgery as well as corneas with common complications resulting from such procedures. IVCM has become a powerful tool for examining corneal cells, nerves, inflammations and infections. It allows information to be acquired repeatedly and at subbiomicroscopic levels that earlier had been obtainable only by invasive microscopic methods. Pre-examining corneas preoperatively by IVCM in order to reveal diseases or conditions in which elective refractive surgical procedures should not be undertaken or to select the ideal operation technique may help to avoid complications in the future. Measurement of the thickness of corneal sublayers or estimation of the thickness of a laser in situ keratomileusis flap or wound bed are other applications in which confocal microscopy may be valuable. In this article we attempt to describe the in vivo confocal findings of common refractive procedures and their complications, and discuss their biology in light of the existing knowledge on wound healing phenomena.

Purpose To analyze the in vivo morphology of the different corneal sublayers and corneal nerves in primary Sjogren's syndrome (SS). Methods Ten eyes of 10 patients with primary SS and 10 eyes of 10 sex- and age-matched control subjects were investigated. Diagnosis was based on American-European consensus criteria. In vivo confocal microscopy with through-focusing was used to investigate corneal morphology and to measure corneal sublayer thickness. Results Epithelial punctate staining with fluorescein was observed in 6 of 10 SS and none of 10 control corneas. In addition, Schirmer I test results were significantly lower in SS. Epithelial thickness did not differ between the SS and control groups. Confocal microscopy revealed patchy alterations or irregularities in surface epithelial cells in 6 of 10 SS corneas, whereas the basal epithelium appeared normal in all corneas. Average corneal thickness was lower in the SS group (515.9 +/- 22.0 micro m) than in the control (547.4 +/- 42.0 micro m; P = 0.050, t-test). Accordingly, the mean intraocular pressure was lower in the SS group (13.9 +/- 2.1 mm Hg) than in the control (16.7 +/- 2.9 mm Hg; P = 0.022). The subbasal nerve plexus and stromal nerve fiber bundles were present in all corneas. No difference was noted in nerve density. However, in 4 of 10 SS eyes, the subbasal nerve plexus showed structures resembling nerve sprouting, suggesting ongoing active neural growth. None of the control corneas exhibited such features. Signs of anterior keratocyte activation were observed in 5 of 10 SS corneas. Conclusions In SS, the corneal surface epithelium was irregular and patchy. Anterior keratocytes frequently showed morphologic features of activation. The subbasal nerve fiber bundles revealed abnormal morphology, and the central corneal thickness was reduced by stromal thinning. The findings confirm epithelial, stromal, and neural abnormalities in the corneas of patients with SS.

Matrix metalloproteinases (MMPs) play a central role in the wound-healing process. The objective of this study was to identify and characterize the levels and molecular forms of human tear fluid collagenase-2 (MMP-8) and membrane type 1-MMP (MT1-MMP or MMP-14) in patients who had undergone excimer laser photorefractive keratectomy (PRK) and in healthy subjects. Whether MT1-MMP activates pro-MMP-8 was also determined.Tear fluid samples were collected with scaled and blunted microcapillaries from healthy control subjects and, on the second postoperative day, from patients who had undergone PRK. Time and the volume collected were registered. Molecular forms and levels of pro and active MMP-8 and MT1-MMP in these samples were determined by Western immunoblot analysis, quantitated by computer scanning. The concentration of MMP-8 was also determined by immunofluorescence assay. The conversion of pure human polymorphonuclear neutrophil (PMN) pro-MMP-8 to the active form by the catalytic domain of MT1-MMP was studied by Western immunoblot analysis.The tear fluid flow was increased after PRK. Tear fluid flow-corrected excretion of MMP-8 was significantly higher in PRK-treated patients, as assessed by immunofluorescence assay and quantitative Western immunoblot analysis. The major MMP-8 species detected in tears of both PRK-treated patients and healthy control subjects represented latent and active 75- and 65-kDa highly glycosylated MMP-8 isoforms. The less-glycosylated 45- to 55-kDa MMP-8 isoform was not detectable. Tear fluid flow-corrected secretion of MT1-MMP was significantly higher in PRK-treated patients. Soluble 80-kDa MT1-MMP immunoreactivities were detected in tears of both healthy control subjects and PRK-treated patients, and may represent a complex captured by tissue inhibitor of metalloproteinase (TIMP)-2. Human PMN pro-MMP-8 was converted to the active form by MT1-MMP, and TIMP-2 prevented this activation.Corneal renewal eventually occurs at a high rate and is affected by the rate of corneal collagen and other matrix protein breakdown. Accordingly, tear fluid MMP-8 and MT1-MMP levels were shown to be constantly high in normal subjects. With PRK, a fast wound-healing process was associated with even higher MMP-8 and MT1-MMP levels and their activation. The results suggest a role for a MMP-8 and MT1-MMP network in the corneal wound-healing cascade. Furthermore, MT1-MMP (MMP-14) seems to activate pro-MMP-8.

PURPOSE. To examine human corneal morphology and nerve recovery 5 years after photorefractive keratectomy (PRK). METHODS. Fourteen eyes of 14 patients (ages, 27-53 years) who underwent 6-mm diameter PRK for low to moderate myopia (spherical equivalent [SE] -2.5 to - 8.0 D) were examined once 5 years after surgery. Nine healthy individuals served as control subjects. Standard biomicroscopy, manifest refraction, and visual acuity tests were performed. The morphology of the corneas was examined by in vivo confocal microscope. Thicknesses of the epithelium and stroma, as well as the density of corneal opacity (haze) were obtained from digital image analysis of the confocal microscopy through-focusing (CMTF) scans. RESULTS. Confocal microscopy revealed increased reflectivity in the subepithelial extracellular matrix, keratocyte nuclei and processes in all patients. The mean objective haze estimate was 166.7 U (range, 50-390) in control corneas compared with a mean of 225.9 U (range, 125-430, P = 0.15) in the post-PRK corneas. The density of the subbasal nerve fiber bundles in post-PRK corneas (mean, n = 4.2; range, n = 1-7 per field of view) was not significantly lowered from that in control subjects (mean, n = 4.9; range, n = 3-6; P = 0.56). Bowman's layer was undetectable in all post-PRK corneas. Clinically, slit-lamp- observed trace of haze in four corneas correlated positively with the ablation depth (P = 0.016) and the thickness of the haze area (P = 0.006) in the confocal microscope. CONCLUSIONS. In vivo confocal microscopy demonstrates the presence of morphologic alterations even 5 years after PRK. However, these alterations are overcome by cellular and neural recovery and do not seem to interfere with visual performance.