Corneal Epithelium In Patients Research Articles

Association between Moraxella keratitis and advanced glycation end products

Diabetes mellitus is recognized as a major predisposing factor for Moraxella keratitis. However, how diabetes mellitus contributes to Moraxella keratitis remains unclear. In this study, we examined Moraxella keratitis; based on the findings, we investigated the impact of advanced glycation end products (AGEs) deposition in the cornea of individuals with diabetic mellitus on the adhesion of Moraxella isolates to the cornea. A retrospective analysis of 27 culture-proven cases of Moraxella keratitis at Ehime University Hospital (March 2006 to February 2022) was performed. Moraxella isolates were identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Among the patients, 30.4% had diabetes mellitus and 22.2% had the predominant ocular condition of using steroid eye drops. The species identified were Moraxella nonliquefaciens in 59.3% and Moraxella lacunata in 40.7% of patients. To investigate the underlying mechanisms, we assessed the effects of M. nonliquefaciens adherence to simian virus 40-immortalized human corneal epithelial cells (HCECs) with or without AGEs. The results demonstrated the number of M. nonliquefaciens adhering to HCECs was significantly increased by adding AGEs compared with that in controls (p < 0.01). Furthermore, in the corneas of streptozotocin-induced diabetic C57BL/6 mice treated with or without pyridoxamine, an AGE inhibitor, the number of M. nonliquefaciens adhering to the corneas of diabetic mice was significantly reduced by pyridoxamine treatment (p < 0.05). In conclusion, the development of Moraxella keratitis may be significantly influenced by the deposition of AGEs on the corneal epithelium of patients with diabetes mellitus.

New characterization and safety evaluation of human limbal stem cells used in clinical application: fidelity of mitotic process and mitotic spindle morphologies

BackgroundLimbal stem cells (LSCs) are crucial for the regeneration of the corneal epithelium in patients with limbal stem cell deficiency (LSCD). Thus, LSCs during cultivation in vitro should be in highly homogeneous amounts, while potency and expression of stemness without tumorigenesis would be desirable. Therefore, further characterization and safety evaluation of engineered limbal grafts is required to provide safe and high-quality therapeutic applications.MethodsAfter in vitro expansion, LSCs undergo laboratory characterization in a single-cell suspension, cell culture, and in limbal grafts before transplantation. Using a clinically applicable protocol, the data collected on LSCs at passage 1 were summarized, including: identity (cell size, morphology); potency (yield, viability, population doubling time, colony-forming efficiency); expression of putative stem cell markers through flow cytometry, immunofluorescence, and immunohistochemistry. Then, mitotic chromosome stability and normal mitotic outcomes were explored by using live-cell imaging. Finally, impurities, bacterial endotoxins and sterility were determined.ResultsExpression of the stemness marker p63 in single-cell suspension and in cell culture showed high values by different methods. Limbal grafts showed p63-positive cells (78.7 ± 9.4%), Ki67 proliferation (41.7 ± 15.9%), while CK3 was negative. Impurity with 3T3 feeder cells and endotoxins was minimized. We presented mitotic spindles with a length of 11.40 ± 0.54 m and a spindle width of 8.05 ± 0.55 m as new characterization in LSC culture. Additionally, live-cell imaging of LSCs (n = 873) was performed, and only a small fraction < 2.5% of aberrant interphase cells was observed; 2.12 ± 2.10% of mitotic spindles exhibited a multipolar phenotype during metaphase, and 3.84 ± 3.77% of anaphase cells had a DNA signal present within the spindle midzone, indicating a chromosome bridge or lagging chromosome phenotype.ConclusionThis manuscript provides, for the first time, detailed characterization of the parameters of fidelity of the mitotic process and mitotic spindle morphologies of LSCs used in a direct clinical application. Our data show that p63-positive CK3-negative LSCs grown in vitro for clinical purposes undergo mitotic processes with extremely high fidelity, suggesting high karyotype stability. This finding confirms LSCs as a high-quality and safe therapy for eye regeneration in humans.

Targeting Ca2+-dependent pathways to promote corneal epithelial wound healing induced by CISD2 deficiency

Chronic epithelial defects of the cornea, which are usually associated with severe dry eye disease, diabetes mellitus, chemical injuries or neurotrophic keratitis, as well as aging, are an unmet clinical need. CDGSH Iron Sulfur Domain 2 (CISD2) is the causative gene for Wolfram syndrome 2 (WFS2; MIM 604928). CISD2 protein is significantly decreased in the corneal epithelium of patients with various corneal epithelial diseases. Here we summarize the most updated publications and discuss the central role of CISD2 in corneal repair, as well as providing new results describing how targeting Ca2+-dependent pathways can improve corneal epithelial regeneration. This review mainly focuses on the following topics. Firstly, an overview of the cornea and of corneal epithelial wound healing. The key players involved in this process, such as Ca2+, various growth factors/cytokines, extracellular matrix remodeling, focal adhesions and proteinases, are briefly discussed. Secondly, it is well known that CISD2 plays an essential role in corneal epithelial regeneration via the maintenance of intracellular Ca2+ homeostasis. CISD2 deficiency dysregulates cytosolic Ca2+, impairs cell proliferation and migration, decreases mitochondrial function and increases oxidative stress. As a consequence, these abnormalities bring about poor epithelial wound healing and this, in turn, will lead to persistent corneal regeneration and limbal progenitor cell exhaustion. Thirdly, CISD2 deficiency induces three distinct Ca2+-dependent pathways, namely the calcineurin, CaMKII and PKCα signaling pathways. Intriguingly, inhibition of each of the Ca2+-dependent pathways seems to reverse cytosolic Ca2+ dysregulation and restore cell migration during corneal wound healing. Notably, cyclosporin, an inhibitor of calcineurin, appears to have a dual effect on both inflammatory and corneal epithelial cells. Finally, corneal transcriptomic analyses have revealed that there are six major functional groupings of differential expression genes when CISD2 deficiency is present: (1) inflammation and cell death; (2) cell proliferation, migration and differentiation; (3) cell adhesion, junction and interaction; (4) Ca2+ homeostasis; (5) wound healing and extracellular matrix; and (6) oxidative stress and aging. This review highlights the importance of CISD2 in corneal epithelial regeneration and identifies the potential of repurposing venerable FDA-approved drugs that target Ca2+-dependent pathways for new uses, namely treating chronic epithelial defects of the cornea.

Increased Matrix Metalloproteinase-2 and Matrix Metalloproteinase-3 Concentrations in Corneal Epithelium of Patients with Recurrent Corneal Erosions.

Purpose To assess the role of selected matrix metalloproteinases in defective corneal re-epithelization in patients with recurrent corneal erosions. Subjects The study group (group 1) included patients with recurrent corneal erosions qualified for phototherapeutic keratectomy. The group 1 was divided into two subgroups regarding the etiology of recurrent corneal erosions: group 1A, Cogan's basement membrane dystrophy, and group 1B, trauma. The control group (group 2) included patients with healthy eyes qualified for Epi-Bowman Keratectomy. Methods The analyzed material was the corneal epithelium collected during phototherapeutic keratectomy or Epi-Bowman Keratectomy in the study or control group, respectively. Matrix metalloproteinases concentration was determined by an immunohistochemical method using Human Magnetic Luminex® Assay. Results The study revealed a statistically significantly higher concentration of matrix metalloproteinase-2 in group 1 compared to the control and a statistically significantly higher concentration of matrix metalloproteinase-3 in group 1 compared to the control. Conclusions The results obtained in the study can prove that matrix metalloproteinase-2 and matrix metalloproteinase-3 having the ability to dissolve anchoring fibers and the corneal epithelial basement membrane could be responsible for epithelial instability and their accumulation in the corneal epithelium may induce recurrence of erosion.

Deciphering the mechanoresponsive role of \u03b2-catenin in keratoconus epithelium

Keratoconus (KC) is a corneal dystrophy characterized by progressive ectasia that leads to severe visual impairment and remains one of the leading indications for corneal transplantation. The etiology is believed to be multifactorial and alterations have been documented in the biomechanical, biochemical and ultrastructural characteristics of the cornea. While the exact site of disease origin is still debated, changes in the corneal epithelium are believed to occur even before the disease is clinically manifested. In this study we investigate the possible role of β-catenin as mechanotransducer in KC corneal epithelium. The sheets of corneal epithelium removed from keratoconic eyes when they underwent collagen crosslinking as a therapeutic procedure were used for this study. The healthy corneal epithelium of patients undergoing Laser Refractive Surgery for the correction of their refractive error, served as controls. Immunoblotting and tissue immunofluorescence studies were performed on KC epithelium to analyse the expression and localization of β-catenin, E-cadherin, ZO1, α-catenin, Cyclin D1, α-actinin, RhoA, and Rac123. Co-immunoprecipitation of β-catenin followed by mass spectrometry of KC epithelium was performed to identify its interacting partners. This was further validated by using epithelial tissues grown on scaffolds of different stiffness. Histology data reported breaks in the Bowman’s layer in KC patients. We hypothesize that these breaks expose the epithelium to the keratoconic corneal stroma, which, is known to have a decreased elastic modulus and that β-catenin acts as a mechanotransducer that induces structural changes such as loss of polarity (Syntaxin3) and barrier function (ZO1) through membrane delocalization. The results of our study strongly suggest that β-catenin could be a putative mechanotransducer in KC epithelium, thus supporting our hypothesis.

Phenotypic Characterization of Corneal Epithelium in Long-Term Follow-Up of Patients Post-Autologous Cultivated Oral Mucosal Epithelial Transplantation.

To analyze the phenotype of the corneal epithelium in patients with long-term follow-up who underwent autologous cultivated oral mucosal epithelial transplantation (COMET) using in vivo confocal microscopy (IVCM) and impression cytology with immunofluorescence staining (ICIF). Thirteen eyes from patients with severe limbal stem cell deficiency, who underwent COMET at least 48 months before, were recruited in this noncomparative cohort study. After eye examination, IVCM and ICIF were performed. Clinical manifestations of the cornea were evaluated and compared with epithelial findings detected by IVCM and ICIF [cytokeratin (CK) 3, CK7, and CK12]. Two corneal buttons derived from patients receiving the corneal transplantation post-COMET were sent for immunohistochemistry (CK3, CK6, CK7, CK12, paired box gene 6, p63, zonula occludens-1, and integrin β -1). The mean age of patients was 51.2 ± 20.6 years, and the mean follow-up time since COMET was 78.7 ± 16.3 months. Six of 13 eyes showed clinically successful COMET. In these eyes, IVCM demonstrated predominant cornea-like epithelium and ICIF reported positivity for CK3 and CK12, confirming the presence of oral mucosal and corneal epithelium. Meanwhile, 7 eyes showed total conjunctivalization, corresponding with substantial conjunctival epithelium detected by IVCM and positivity for conjunctival (CK7) and oral mucosal epithelial (CK3) markers detected by ICIF. The immunohistochemistry of corneal buttons stained positive for oral mucosal, corneal epithelial, and stem cell markers (CK3, CK12, and p63). In long-term follow-up of COMET, epithelium of successful patients demonstrated cornea-like phenotype, whereas failed cases revealed mainly conjunctival phenotype. However, there were evidences that oral mucosal epithelial cells remained across the cornea in both successful and failed COMET as detected by IVCM and ICIF.

Optimization of Human Limbal Stem Cell Culture by Replating a Single Limbal Explant.

Cultured limbal epithelial stem cell transplantation is a clinical procedure used to regenerate the corneal epithelium in patients with limbal stem cell deficiency. The protocols used to expand limbal epithelial cells in vitro need to be optimized, since the scarcity of human ocular tissue donors is limiting the potential use of this procedure. Here, we describe a method to consecutively expand a single human limbal explant. With this method it is possible to obtain up to three limbal epithelial primary cultures from the same explant, thus increasing the efficiency of the in vitro cell culture.

Исследование состояния слезопродукции и эпителия роговицы у пациентов с возрастной катарактой

Исследование состояния слезопродукции и эпителия роговицы у пациентов с возрастной катарактой

Limbal Stem Cell Transplantation: Clinical Results, Limits, and Perspectives.

Limbal stem cell deficiency (LSCD) is a clinical condition characterized by damage of cornea limbal stem cells, which results in an impairment of corneal epithelium turnover and in an invasion of the cornea by the conjunctival epithelium. In these patients, the conjunctivalization of the cornea is associated with visual impairment and cornea transplantation has poor prognosis for recurrence of the conjunctivalization. Current treatments of LSCD are aimed at replacing the damaged corneal stem cells in order to restore a healthy corneal epithelium. The autotransplantation of limbal tissue from the healthy, fellow eye is effective in unilateral LSCD but leads to depauperation of the stem cell reservoir. In the last decades, novel techniques such as cultivated limbal epithelial transplantation (CLET) have been proposed in order to reduce the damage of the healthy fellow eye. Clinical and experimental evidence showed that CLET is effective in inducing long-term regeneration of a healthy corneal epithelium in patients with LSCD with a success rate of 70%–80%. Current limitations for the treatment of LSCD are represented by the lack of a marker able to unequivocally identify limbal stem cells and the treatment of total, bilateral LSCD which requires other sources of stem cells for ocular surface reconstruction.

Effects of optimal nursing in improving postoperative complications of Epi-LASIK

Objective To investigate effects of optimal nursing in improving postoperative complications of epipolis laser in situ ketatomileusis (Epi-LASIK) . Methods A total of 80 patients (160 eyes) with myopia undergoing Epi-LASIK operation in our hospital from June 2010 to May 2014 were included in the study. Control group contained 40 patients from June 2010 to August 2012 and received routine nursing, while observation group contained 40 patients from September 2012 to May 2014 and were given additional optimal nursing. Postoperative pain symptoms, healing time of corneal epithelium and corneal haze (Haze) were compared between two groups. Results After 1 day and 3 days post operation, the scores of pain of patients in observation group were significant lower than those of control group (P<0.05) . Average healing time of corneal epithelium of patients in observation group (5.05±0.58) days was shorter than control group (5.59±0.98) days. The difference was statistically significant (P<0.05) . After 1, 3 and 6 months, incidence rate of Haze in observation group was significantly lower than control group (P<0.05) . Conclusions Optimal nursing effectively prevents and reduces postoperative complications of Epi-LASIK. Key words: Nursing care; Pain; Epithelium, corneal; Epi-LASIK

Preservation of Ocular Epithelial Limbal Stem Cells: The New Frontier in Regenerative Medicine.

Significant advances have been made in the field of ocular regenerative medicine. Promising stem cell-based therapeutic strategies have been translated into the clinical practice over the last few decades. These new stem cell-based therapies offer the possibility of permanently restoring corneal epithelium in patients with severe disabling and blinding ocular surface disease. The European Union has already classified stem cell-based therapies as "medicinal products". Therefore, manipulation is strictly regulated according to the defined conditions of good manufacturing practice, with the production of stem cell therapeutics at only accredited production sites authorized by the national regulatory agencies. In this regard, as first medical products are licensed for commercial use in Europe enabling a more widespread access to a stem cell-based therapy, the need for safe, validated and reproducible techniques for ex vivo cultured tissue preservation and distribution are coming to the forefront of research. However, these provide various new challenges for biobanking industry such as the retention of viability, good functionality of stem cells and sterility issues. This chapter provides an overview of the current advances in the field of corneal/limbal epithelial stem cell culture preservation techniques using either hypothermic storage or cryopreservation methods, that were used in different culturing steps (from stem cell isolation to the ex vivo epithelial graft preparation), with the reported impact on the post-thawing product recovery.

In vivo confocal microscopy of early corneal epithelial recovery in patients with chemical injury.

To investigate the early recovery of corneal epithelium in patients with chemical injuries by the application of in vivo laser scanning confocal microscopy (LSCM). LSCM was performed on 26 eyes of 18 consecutive patients at 1, 2, and 3 months post injury. The morphology of central corneal epithelium and limbus was evaluated. Analysis was performed to compare the densities of corneal apical surface cells (ASCs) and basal epithelial cells (BECs) among different injury severity and time points after injury. The comparisons were also made on inflammatory cells (ICs) and dendritic cells (DCs) infiltrating at the limbus. Moreover, the presence rate of palisades of Vogt (POV) was analyzed at 3 months post injury. Corneal re-epithelialization was found within 3 months post injury in all eyes, except that persistent epithelium defect was identified in grade IV injured eyes even at 3 months after injury. The injury severity had a significantly negative correlation with the densities of ASCs. However, it had no relationship with the densities of BECs. The density of ICs at the limbus decreased significantly with the prolongation of follow-up, except in the grade I injured eyes. The presence rate of POV also had a significantly negative correlation with injury severity. Epithelial recovery after chemical injury varied in cellular morphology and the densities of ASCs among eyes with different injury severity. The presence rates of POV decreased with the aggravation of injury. LSCM is a feasible method for observing the early recovery of corneal epithelium in patients with chemical injuries.

Biological Parameters Determining the Clinical Outcome of Autologous Cultures of Limbal Stem Cells

Limbal cultures restore the corneal epithelium in patients with ocular burns. We investigated the biological parameters instrumental for their clinical success. We report a long-term multicenter prospective study on 152 patients carrying corneal destruction due to severe ocular burns, treated with autologous limbal cells cultured on fibrin and clinical-grade 3T3-J2 feeder cells. Clinical results were statistically evaluated both by parametric and nonparametric methods. Clinical outcomes were scored as full success, partial success and failure in 66.05, 19.14 and 14.81% of eyes, respectively. The total number of clonogenic cells, colony size, growth rate and presence of conjunctival cells could not predict clinical results. Instead, the clinical data provided conclusive evidence that graft quality and likelihood of a successful outcome rely on an accurate evaluation of the number of stem cells detected before transplantation as holoclones expressing high levels of the p63 transcription factor. No adverse effects related to the feeder layer have been observed and the regenerated epithelium was completely devoid of any 3T3-J2 contamination. Cultures of limbal stem cells can be safely used to successfully treat massive destruction of the human cornea. We emphasize the importance of a discipline for defining the suitability and the quality of cultured epithelial grafts, which are relevant to the future clinical use of any cultured cell type.

Differences in corneal epithelium in patients with diagnosed macular and granular corneal dystrophy

AbstractPurpose To describe severity and forms of epithelial changes in patients suffering from macular and granular corneal dystrophy.Methods In examined group 9 patients were included, 4 with diagnosis of macular and 5 of granular corneal dystrophy. Patients underwent basic slit lamp examination and in next order in vivo corneal confocal microscopy. Full thickness corneal scans were made and compared with corresponding images from healthy objects. Additional imaging of epithelial layer with higher magnification was made to compare detailed changes within single cells.Results On a slit lamp examination we described: in case of granular dystrophy macroscopic, polymorphic deposits of crystal structure, located in full thickness of corneal stroma and in epithelium. In macular dystrophy cornea stromal and epithelial changes took more diffused form. The corneal confocal microscopy images showed different intensity of typical stromal changes (crystalic or diffused depends on dystrophy type). In epithelium, in cases of macular dystrophy, we found different stages of intracellular changes, which secondary involved intercellular space. In granular dystrophy changes localized mostly in intercellular space with preserved cellular architectonic.Conclusion However both described dystrophies are classified as stromal, in different way involve cells in epithelial layer. Corneal confocal microscopy can be useful for detailed examination of corneal dystrophic changes.

Clinical transplantation ofex vivoexpanded autologous limbal epithelial cells using a culture medium with human serum as single supplement: a retrospective case series

Presently, our clinic is the only centre in Scandinavia that offers patients with corneal surface pathology including limbal stem cell deficiency (LSCD) transplantation of ex vivo expanded limbal epithelial cells (LECs). We here present clinical data of the first nine patients with LSCD who were transplanted with autologous LECs expanded in medium completely free of any animal-derived products and non-human/recombinant growth factors (including Cholera Toxin), and with autologous human serum as the only growth supplement. We conducted a noncomparative retrospective study of patients with LSCD at our centre between 2009 and 2011. The diagnosis was based on history and clinical signs. A biopsy was taken from healthy limbus, and the epithelium was expanded on amniotic membrane (AM) in medium containing autologous serum and subsequently transplanted to the affected eye. Successful outcome was defined as relief of pain and photophobia and/or improved best corrected visual acuity (BCVA) and/or reestablishment of a stable corneal epithelium and regression of corneal vascularization. Five of the nine transplanted patients (55.6%) had an improvement in either subjective symptoms or objective findings (11- to 28-month follow-up). Our clinical study shows that patients with LSCD can be treated successfully with transplantation of LECs expanded ex vivo in a medium with autologous serum as the only growth supplement. The use of this novel culture system, which is devoid of animal-derived products and non-human/recombinant growth factors (including Cholera Toxin), reduces the risks of inter-species disease transmission and host immune responses to xenogenic proteins, both obvious advantages for the patient.

Long‐term effectiveness of autologous cultured limbal stem cell grafts in patients with limbal stem cell deficiency due to chemical burns

Chemical burns cause depletion of limbal stem cells and eventually lead to corneal opacity and visual loss. We investigated the long-term effectiveness of autologous cultured limbal stem cell grafts in patients with limbal stem cell deficiency. Prospective, non-comparative interventional case series. Sixteen eyes from 16 patients with severe, unilateral limbal stem cell deficiency caused by chemical burns. Autologous ex vivo cultured limbal stem cells were grafted onto the recipient eye after superficial keratectomy. Clinical parameters of limbal stem cell deficiency (stability/transparency of the corneal epithelium, superficial corneal vascularization and pain/photophobia), visual acuity, cytokeratin expression on impression cytology specimens and histology on excised corneal buttons. At 12 months post-surgery, evaluation of the 16 patients showed that 10 (62.6%) experienced complete restoration of a stable and clear epithelium and 3 (18.7%) had partially successful outcomes (re-appearance of conjunctiva in some sectors of the cornea and instable corneal surface). Graft failure (no change in corneal surface conditions) was seen in three (18.7%) patients. Penetrating keratoplasty was performed in seven patients, with visual acuity improving up to 0.8 (best result). For two patients, regeneration of the corneal epithelium was confirmed by molecular marker (p63, cytokeratin 3, 12 and 19, mucin 1) analysis. Follow-up times ranged from 12 to 50 months. Grafts of autologous limbal stem cells cultured onto fibrin glue discs can successfully regenerate the corneal epithelium in patients with limbal stem cell deficiency, allowing to perform successful cornea transplantation and restore vision.

Evaluation of Molecular Markers in Corneal Regeneration by Means of Autologous Cultures of Limbal Cells and Keratoplasty

To determine the epithelial phenotype in patients with a limbal stem cell deficiency (LSCD) after ocular surface reconstruction with autologous cultured stem cells. To correlate the epithelial phenotype with the clinical outcome. Six eyes affected by LSCD, verified and graded by impression cytology, were treated with an autologous fibrin-cultured limbal stem cell graft. The clinical outcome was defined as a "success" or a "failure," depending on ocular surface stability. To improve their visual function, 4 patients underwent lamellar or penetrating keratoplasty after the stem cell graft. The phenotype of the regenerated corneal epithelium was determined by immunofluorescence of the corneal button to detect CK12, CK3, CK19, and Muc1 as corneal and conjunctival markers. After a mean follow-up of 24 months, 5 cases were defined as successes; 1 case presented an epithelial defect 4 months after grafting and was defined as a failure. Immunofluorescence performed on 4 patients after lamellar and penetrating keratoplasty confirmed the presence of epithelial corneal markers (CK12 and CK3) in 2 of the success cases and the presence of conjunctival markers (CK19 and Muc1) in the 1 failure case. In one of the success cases, both corneal and conjunctival markers were detected on the corneal button. All success cases showed maintenance of marker accounting for high proliferative potential (DeltaNp63alpha) after transplantation. Autologous cultures of limbal stem cells can regenerate a functional corneal epithelium in patients affected by unilateral LSCD. We showed a correlation between the clinical outcome and the molecular marker expression.

Techniques for Culture and Assessment of Limbal stem Cell Grafts

The therapeutic use of limbal cultures for the permanent regeneration of corneal epithelium in patients with limbal stem cell deficiency (LSC D) has been reported in many studies. A ccording to the guidelines for good manufacturing practice (GMP), strictly regulated procedures and stringent quality control tests are now required to manipulate stem cells as “medicinal products” and make engraftment safer and eventually more successful. This paper describes techniques for optimal preparation of limbal stem cell grafts, including 1) a reliable impression cytology assay for the grading of LSCD, 2) culture methods that maintain high percentages of limbal stem cells, 3) the use of specific markers for the detection of corneal, conjunctival, and limbal stem cells, namely keratin 12, mucin 1, and ΔNp63α, and 4) assays to assess the presence of contaminants, such as murine fibroblasts, endotoxins, mycoplasmae, and viral particles, in the cultured graft. The use of some of these assays allowed us to obtain a regenerated normal corneal epithelium in approximately 80% of 166 LSCD patients who received transplants from 2004 to 2008.

Changes in Corneal Epithelial Layer Inflammatory Cells in Aqueous Tear–Deficient Dry Eye

To investigate the morphology, distribution, and density of inflammatory cells in the corneal epithelium of aqueous tear-deficient dry eye. Thirty-two patients with non-Sjögren's syndrome (NSS) dry eye, 14 patients with Sjögren's syndrome (SS) dry eye, and 33 healthy volunteers were studied. In vivo laser scanning confocal microscopy was used to investigate both Langerhans cell (LCs) and leukocyte distribution and density in the peripheral and central corneal epithelium. LC morphology was also evaluated. Multifactor regression analysis assessed whether there is a correlation between clinical manifestations and inflammatory cell densities. LCs were present in both central (34.9 +/- 5.7 cells/mm(2)) and peripheral (90.7 +/- 8.2 cells/mm(2)) parts of the normal corneal epithelium. Moreover, LC density increased dramatically in the central corneal epithelium in patients with NSS (89.8 +/- 10.8 cells/mm(2)) and SS (127.9 +/- 23.7 cells/mm(2)). The ratio of LCs with obvious processes was much higher in patients with dry eye than in healthy volunteers. LC density also increased in peripheral corneal epithelium in patients with SS, but not in those with NSS. Leukocyte density in normal corneal epithelium was very low, whereas it increased in the central corneal epithelium (4.6 +/- 1.0 cells/mm(2)) in NSS and in both central (49.0 +/- 12.9 cells/mm(2)) and peripheral (84.2 +/- 36.8 cells/mm(2)) corneal epithelium in SS. Densities of LCs and leukocytes showed significant correlation with the severity found in clinical evaluation. The LC and leukocyte changes in the corneal epithelium suggest their involvement in aqueous tear-deficient dry eye pathophysiology. In vivo dynamic assessment of central corneal inflammatory cell density may serve as an indicator of dry eye severity and provide new insight for dry eye treatment.

Corneal Epithelial Opacity in Dysfunctional Tear Syndrome

To compare the appearance of the superficial corneal epithelium in patients with dysfunctional tear syndrome (DTS) and that of an asymptomatic control group using laser scanning confocal microscopy and to determine the correlations between confocal microscopic findings and clinical severity parameters. Prospective case-control study. Thirty-one patients with newly diagnosed DTS and 21 asymptomatic control subjects were evaluated for this study. Subjects with DTS were classified into 4 levels of clinical severity (DTS 1 through 4) based on the Delphi dry eye panel report criteria. The Heidelberg Retina Tomograph 2 Rostock Cornea Module (Heidelberg Engineering GmbH, Heidelberg, Germany) laser scanning confocal microscope was used to image the superficial corneal epithelium. Areas of single or multiple opaque superficial epithelial cells were measured as a percentage of the 400 x 400-microm(2) field area in 4 randomly selected confocal images from each eye. Spearman correlations between the confocal findings and severity of symptoms, visual acuity, and ocular surface signs were calculated. The mean area of opaque superficial corneal epithelial cells was significantly greater in DTS patients than in normal subjects (P < .0001). Significant differences were observed between the DTS severity groups and the control group (P < .001), except for the DTS 1 group. The area of opaque cells significantly increased with level of clinical severity. The confocal findings showed significant correlation with clinical severity parameters, including blurred vision symptoms (r = 0.86; P = .0001), best-corrected visual acuity (Spearman r = 0.4; P = .03), conjunctival lissamine green staining scores (Spearman r = 0.4; P = .026), corneal fluorescein staining scores (Spearman r = 0.5; P = .002), and videokeratoscopic surface regularity index (Spearman r = 0.5; P = .02). Morphologic changes in the superficial corneal epithelium of DTS patients detected by laser scanning confocal microscopy correlates with blurred vision symptoms and objective severity parameters. Objective confocal image analysis of the superficial corneal epithelium may prove useful for classifying DTS severity and for monitoring the efficacy of therapies.