A simple and reproducible method for quantification of human tear lipids with ultrahigh-performance liquid chromatography-mass spectrometry.

Purpose: The purpose was to select a simple and reproducible method for lipid measurements of human tears with ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS). Two sample preparation procedures were evaluated and compared: the Bligh and Dyer (BD) liquid-liquid extraction method with chloroform and methanol and protein precipitation with isopropanol (IPA). Methods: Reproducibility and recovery efficiencies of 20 non-endogenous internal lipid standards were tested in 10-µl tear samples from healthy subjects. The lipid coverage and the simplicity of execution were also assessed. Lipid profiles of the tear extracts were acquired with UHPLC-MS, uhpland the lipids were identified using SimLipid software. Results: Both methods were robust producing good lipid coverage and reproducibility and high recovery efficiencies. The two protocols identified a 69-feature tear lipidome that covered 11 lipid classes from six different lipid categories. The main differences in recovery were due to the intrinsic lipid selectivity of each solvent. Although both methods were similarly efficient in recovering O-acyl-ω-hydroxy fatty acid (OAHFAs) and non-polar lipids, polar lipids were more efficiently recovered with IPA precipitation, which, in turn, exhibited higher reproducibility. In addition, IPA precipitation is automatable and simpler than the BD approach. Conclusions: IPA precipitation is an excellent procedure for extracting lipids from small tear volumes for quantitative large-scale, untargeted lipid profiling, which may be useful for identifying lipid biomarkers in tears from patients with different ocular surface pathologies, allowing personalized therapies to be designed.

The analysis of human conjunctival epithelium proteome in ocular surface diseases using impression cytology and 2D-DIGE.

Conjunctival impression cytology samples from patients with meibomian gland dysfunction (MGD), dry eye (DE), and healthy subjects (CT) were collected for determination of the degree of squamous metaplasia (SM) by PAS-hematoxylin staining and for comparative proteomic analyses by 2D-DIGE. The protein spots with discriminant expression were identified by MALDI-TOF/TOF mass spectrometry. Three independent statistical studies were conducted: i). Analysis of differential protein expression between study groups: We observed increased expression of proteins S100A4, S100A8, retinal dehydrogenase-1, peroxiredoxin-1, annexin-A1, annexin-A2, α-enolase, and glutathione S-transferase-P in DE, whereas the highest expression of peroxiredoxin-6, actin cytoplasmic-1, peroxiredoxin-2, and heat shock protein HSP-90-α was observed in MGD; ii). Correlation between changes in the proteome profile and the grade of SM: The expression of 5 different cytokeratins (KRT1, KRT4, KRT8, KRT10, and KRT13) correlated with the degree of SM; iii). Proteome profile differences between pathological and CT groups: An overall proteome analysis revealed upregulation of 9 proteins in the pathological groups (Annexin-A1, α-enolase, Annexin-A2, S100A8, cytokeratin-1, Peroxiredoxin-2 and Leukocyte elastase inhibitor) and downregulation of 2 proteins (Galectin-3 and Lipocalin-1). In conclusion, a sensitive proteomic approach to study conjunctival tissue collected from minimally invasive impression cytology was implemented. Differential proteomics analyses showed that in comparison with the MGD, the DE patients presented higher overexpression of proteins related to antimicrobial defense, tissue-damage response, and regulation of body fluid secretions. Changes in MGD proteome were associated with oxidative stress and anti-apoptotic processes. We found a correlation between the grade of SM and expression of proteins associated with cytoskeleton and keratinization. The studied pathological groups shared elements related to the defense and inflammatory responses. Dot blot assays of proteins ANXA1, S100A8, and S100A4 validated the proteomic results obtained from 2D-DIGE experiments and confirmed the correlation between the expression of these proteins and the clinical parameters.

Manual Superficial Keratectomy Is the First Choice Treatment for Salzmann Nodular Degeneration.

PURPOSE: This study aimed to describe the optical and topographic changes after manual superficial keratectomy (MSK) for Salzmann nodular degeneration. METHODS: This was a descriptive, retrospective study. All patients with a clinical diagnosis of Salzmann nodular degeneration were examined at the Cornea Service of the Instituto Clínico Quirúrgico de Oftalmología (ICQO), Bilbao, Spain, and treated with MSK after presenting ocular discomfort and/or decreased visual acuity. The clinical characteristics (including anterior segment optical coherence tomography and Pentacam topography), treatment regimens, surgical procedures, and outcomes were recorded. Descriptive statistics were constructed using mean ± SD, minimum, maximum, and median. The Shapiro-Wilk normality test was used. The Student t test was used to determine significance. RESULTS: Ten eyes of 9 patients were included in this study: 6 patients (66.6%) were female and 3 were male (33.3%). The mean age was 62.2 years, and the follow-up time was between 5 and 21 months. The best-corrected visual acuity before MSK was 0.20 LogMAR (median) and improved to 0.10 after the surgical procedure. The mean spherical equivalent was reduced from -0.23 ± 3.39 D preoperatively to -1.3 ± 3.0 D postoperatively. Astigmatism decreased between 0.5 and 3.75 D. Topographic irregularity normalized total root mean square from 11,596.4 ± 6854.01 to 4817.2 ± 2725.68 µm. CONCLUSIONS: MSK is an effective and safe technique for the treatment of Salzmann nodular degeneration when the Bowman layer is preserved. Anterior segment optical coherence tomography and corneal topography are essential tools for the surgical plan and for the detection of corneal aberrations.

Response and Safety Outcomes in Treatment-Resistant Depression After Subcallosal Cingulate Gyrus Deep Brain Stimulation: Long-term Follow-up Study.

Objective: To replicate previous findings and to investigate related clinical factors of long-term benefits and safety of subcallosal cingulate gyrus deep brain stimulation (SCG-DBS) for treatment-resistant depression (TRD).Methods: Sixteen patients with TRD (with either major depressive disorder or bipolar disorder, DSM-IV and DSM-5 criteria) receiving chronic SCG-DBS were followed for up to 11 years (January 2008 to June 2019). Demographic, clinical, and functioning data were collected pre-surgery and during the follow-up. Response was defined as a ≥ 50% decrease from baseline in the 17-item Hamilton Depression Rating Scale (HAM-D17) score, and remission was defined as ≤ 7 in the HAM-D17 score. The Illness Density Index (IDI) was used as a longitudinal measure of treatment effects. Survival analyses were performed for response outcomes and relapses.Results: Depressive symptoms were significantly decreased over time (F = 2.37; P = .04). Response and remission rates were 75% and 62.5% at individual endpoint. Based on Kaplan-Meier curve analysis, 55% of patients reached remission in 139 days. IDI curves showed sustained clinical improvements as measured with HAM-D17 and Clinical Global Impression and sustained functioning improvement as measured with Global Assessment of Functioning scores. The procedure was generally safe and well tolerated (122 adverse events across 81 patient-years, of which 25 were related to SCG-DBS). Two patients committed suicide long after surgery.Conclusions: SCG-DBS produced a robust and protracted improvement in most patients, which reinforces the possibility that SCG-DBS could be an alternative for patients with treatment-resistant unipolar or bipolar depression. Identification of clinical and neurobiological response predictors should guide the continuation of DBS for TRD, to obtain its indication soon.

Evaluation of inter-day and inter-individual variability of tear peptide/protein profiles by MALDI-TOF MS analyses.

PURPOSE: To characterize the tear film peptidome and low molecular weight protein profiles of healthy control individuals, and to evaluate changes due to day-to-day and individual variation and tear collection methods, by using solid phase extraction coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling. METHODS: The tear protein profiles of six healthy volunteers were analyzed over seven days and inter-day and inter-individual variability was evaluated. The bilaterality of tear film and the effect of tear collection methods on protein profiles were also analyzed in some of these patients. MALDI-TOF MS analyses were performed on tear samples purified by using a solid phase extraction (SPE) method based on C18 functionalized magnetic beads for peptide and low molecular weight protein enrichment, focusing spectra acquisition on the 1 to 20 kDa range. Spectra were analyzed using principal component analysis (PCA) with MultiExperiment Viewer (TMeV) software. Volunteers were examined in terms of tear production status (Schirmer I test), clinical assessment of palpebral lids and meibomian glands, and a subjective OSD questionnaire before tear collection by a glass micro-capillary. RESULTS: Analysis of peptides and proteins in the 1-20 kDa range showed no significant inter-day differences in tear samples collected from six healthy individuals during seven days of monitoring, but revealed subtle intrinsic inter-individual differences. Profile analyses of tears collected from the right and left eyes confirmed tear bilaterality in four healthy patients. The addition of physiologic serum for tear sample collection did not affect the peptide and small protein profiles with respect to the number of resolved peaks, but it did reduce the signal intensity of the peaks, and increased variability. Magnetic beads were found to be a suitable method for tear film purification for the profiling study. CONCLUSIONS: No significant variability in tear peptide and protein profiles below 20 kDa was found in healthy controls over a seven day period, nor in right versus left eye profiles from the same individual. Subtle inter-individual differences can be observed upon tear profiling analysis and confirm intrinsic variability between control subjects. Addition of physiologic serum for tear collection affects the proteome and peptidome in terms of peak intensities, but not in the composition of the profiles themselves. This work shows that MALDI-TOF MS coupled with C18 magnetic beads is an effective and reproducible methodology for tear profiling studies in the clinical monitoring of patients.

Complication Rates after Endoscopic Transsphenoidal Surgery for ACTH-Secreting Pituitary Adenomas: A Comparative Analysis with GH and Nonfunctioning Adenomas.

Objectives Some complications have been more frequently reported after surgery for adrenocorticotropin hormone (ACTH)-secreting pituitary adenomas. We compared complication rates in patients with ACTH-secreting pituitary adenomas with those in patients with growth hormone secreting pituitary adenomas (growth hormone [GH] group) and nonfunctioning adenomas (nonfunctioning group). Design A comparative three-group analysis was performed for all patients who had endoscopic transsphenoidal surgery for pituitary adenomas at our center between January 2011 and May 2019. Variables included demographics, preoperative clinical and radiological characteristics, and postoperative radiological and endocrinological outcomes. Complications were divided into four categories: endocrinological, neurosurgical, medical, and ENT (ear-nose-throat)-related complications. Univariate and multivariate statistical analysis were performed. Results A total of 111 patients with pituitary adenomas and a mean age of 53.7 years were included (25 ACTH, 35 GH, and 51 nonfunctioning adenomas). Overall, 28 patients had microadenomas (25.2%) and 83 had macroadenomas (74.8%). Univariate statistical analysis for complications between groups showed no differences in neurosurgical and medical complications. Transient diabetes insipidus and postsurgical bacterial sinusitis were the only variables more frequently seen in the ACTH group ( p = 0.01 and 0.04, respectively). Multivariate analysis for transient diabetes insipidus showed no differences between groups ( p = 0.58). Conclusion Complication rates were similar in all three adenoma groups, particularly concerning major infections, thrombotic events, postoperative cerebrospinal fluid (CSF) leak, and transient diabetes insipidus. Transient diabetes insipidus was related with adenoma size and intraoperative CSF leak. Despite postoperative bacterial sinusitis was statistically higher in the ACTH group, this data should be interpreted with caution given the low number of patients with this complication.

Inflammatory markers in the tears of patients with ocular surface disease.

PURPOSE: To determine the concentration of interleukins (IL-1beta and -6) and matrix metalloproteinase 9 (pro-MMP-9) in the tears of patients with different ocular surface diseases and to examine the possible relationship between the disorders and molecular inflammation. METHODS: 77 patients diagnosed as having different ocular surface disorders and 18 normal control subjects were studied. Patients were routinely examined and separated into 5 groups: (1) control, (2) blepharitis, (3) ocular allergic disease, (4) dry eye and (5) conjunctivochalasis. Ten microliters of tears were collected by a Weck cell sponge. The concentrations of IL-1beta, IL-6 and pro-MMP-9 were measured by enzyme-linked immunosorbent assay, and the MMP-9 activity was evaluated with gelatin zymography. RESULTS: Levels of IL-1beta and IL-6 in tear fluid were significantly higher in conjunctivochalasis (p = 0.0062 and p = 0.0134) than in the control group. Pro-MMP-9 levels were significantly elevated in blepharitis (p = 0.013), in allergic eye disease, in dry eye and in conjunctivochalasis (all p < 0.001), in comparison to controls. CONCLUSIONS: Pro-MMP-9 levels in tears are elevated in all of the studied pathologies especially in ocular allergy and conjunctivochalasis. However, IL-1beta and IL-6 were only found to be overexpressed in conjunctivochalasis. These findings illustrate the selective implication of different molecules in each disorder.

Corneal lamellar dissection by using suture threads in porcine eyes.

PURPOSE: Lamellar dissection is one of the most delicate maneuvers in corneal surgery. We describe a new method that uses Ferrara ring instrumentation. METHODS: A 360 degrees tunnel is created at half of the stromal depth in porcine corneas. A suture (nylon 6-0 or Vicryl 6-0) was passed through the tunnel by using the Ferrara spatula. By means of alternate traction on the end of the suture thread, complete lamellar dissection was achieved. RESULTS: Light microscopy examination of the cornea confirmed that the dissection had a constant thickness and dissection depth. Scanning electron microscopy showed a homogeneous stromal surface without irregularities. CONCLUSIONS: This technique permits a homogeneous and safe lamellar dissection, which may prove useful in specific surgical procedures of the cornea.

Vector analysis of astigmatic changes and optical quality outcomes after wavefront-guided laser in situ keratomileusis using a high-resolution aberrometer.

PURPOSE: To assess the ocular optical quality results as well as the astigmatic changes after wavefront-guided laser in situ keratomileusis (LASIK) in eyes with myopic astigmatism. SETTING: Instituto Clínico-Quirúrgico Oftalmológico, Bilbao, Vizcaya, Spain. DESIGN: Prospective case series. METHODS: Thirty-three eyes having wavefront-guided LASIK with the Star S4IR excimer laser combined with the iDesign system were enrolled in the study. Changes in visual acuity, refraction, higher-order aberrations, and ocular scattering index (OSI) were evaluated during a 3-month follow-up. The Alpins method was used to analyze the astigmatic changes. RESULTS: A significant reduction was observed in refraction (P < .001), with significant improvement in uncorrected (UDVA) and corrected (CDVA) distance visual acuities (P < .001). All eyes had a spherical equivalent within ±0.50 diopter (D) and UDVA of 20/25 or better at 90 days after surgery. A total of 27.3% of eyes gained lines of CDVA. No significant changes were observed in primary coma and spherical aberration (P ≥ .551). Likewise, no significant changes were observed in OSI (P = .361), with a mean 90-day postoperative value of 0.66 ± 0.58 (SD). A significant change to negative values of the magnitude of error (P = .007) and a significant decrease in the correction index (P = .004) were observed during the follow-up, with mean 90-day postoperative values of -0.24 ± 0.28 D and 0.86 ± 0.17 D, respectively. CONCLUSION: Wavefront-guided LASIK using a high-resolution aberrometer provided safe and efficacious correction of myopic astigmatism, with preservation of the ocular optical quality.

Serum from plasma rich in growth factors regenerates rabbit corneas by promoting cell proliferation, migration, differentiation, adhesion and limbal stemness.

PURPOSE: To evaluate the regenerating potential and the mechanisms through which the autologous serum derived from plasma rich in growth factors (s-PRGF) favours corneal wound healing in vitro and in vivo. METHODS: We compared the effect of various concentrations of s-PRGF versus fetal bovine serum (FBS) and control treatment in rabbit primary corneal epithelial and stromal cells and wounded rabbit corneas. Cell proliferation was measured using an enzymatic colorimetric assay. In vitro and in vivo wound-healing progression was assessed by image-analysis software. Migration and invasion were evaluated using transfilter assays. Histological structure was analysed in stained sections. Protein expression was evaluated by immunohistochemistry. RESULTS: s-PRGF promoted the robust proliferation of epithelial cultures at any concentration, similar to FBS. Likewise, s-PRGF and FBS produced similar re-epithelialization rates in in vitro wound-healing assays. In vivo, s-PRGF treatment accelerated corneal wound healing in comparison with control treatment. This difference was significant only for 100% s-PRGF treatment in our healthy rabbit model. Histological analysis confirmed normal epithelialization in all cases. Immunohistochemistry showed a higher expression of cytokeratins 3/76 and 15, zonula occludens-1 and alpha-smooth muscle actin proteins as a function of s-PRGF concentration. Notably, keratocyte density in the anterior third of the stroma increased with increase in s-PRGF concentration, suggesting an in vivo chemotactic effect of s-PRGF on keratocytes that was further confirmed in vitro. CONCLUSION: s-PRGF promotes proliferation and migration and influences limbal stemness, adhesion and fibrosis during corneal healing.

Ocular Surface Disease Parameters After Collagen Cross-Linking for Keratoconus.

PURPOSE: To analyze the variations in tear quantity and quality after corneal collagen cross-linking (CXL) in patients with keratoconus. METHODS: This is a prospective observational study of 24 eyes with keratoconus that underwent epithelium-off CXL. The variables studied were tear film osmolarity, subjective symptoms (Ocular Surface Disease Index), tear breakup time, Schirmer test (SCH) score, tear clearance, fluorescein staining, and lissamine green staining. Variables were determined preoperatively and 3, 6, and 12 months after CXL. RESULTS: All the studied variables remained stable after 1-year follow-up (no significant difference in comparison with baseline values; Mann-Whitney U test). There was a positive paired correlation between the SCH score and tear clearance at 3; 0.532 (P < 0.01), 6; 0.434 (P = 0.04), and 12 months; 0.675 (P < 0.01). There was no correlation between the SCH score and tear breakup time, apart from a positive correlation at 12 months; 0.601 (P = 0.05). A negative correlation was found between osmolarity and the SCH score at 3; -0.589 (P < 0.01) and 12 months; -0.049 (P = 0.04). The Ocular Surface Disease Index did not correlate with any of the studied variables (Spearman test). CONCLUSIONS: CXL, at least during the first postoperative year, does not modify the parameters currently used to evaluate tear film function. There is no correlation between objective and subjective parameters in the analysis of the tear film function.

Tear proteome analysis in ocular surface diseases using label-free LC-MS/MS and multiplexed-microarray biomarker validation.

We analyzed the tear film proteome of patients with dry eye (DE), meibomian gland dysfunction (MGD), and normal volunteers (CT). Tear samples were collected from 70 individuals. Of these, 37 samples were analyzed using spectral-counting-based LC-MS/MS label-free quantitation, and 33 samples were evaluated in the validation of candidate biomarkers employing customized antibody microarray assays. Comparative analysis of tear protein profiles revealed differences in the expression levels of 26 proteins, including protein S100A6, annexin A1, cystatin-S, thioredoxin, phospholipase A2, antileukoproteinase, and lactoperoxidase. Antibody microarray validation of CST4, S100A6, and MMP9 confirmed the accuracy of previously reported ELISA assays, with an area under ROC curve (AUC) of 87.5%. Clinical endpoint analysis showed a good correlation between biomarker concentrations and clinical parameters. In conclusion, different sets of proteins differentiate between the groups. Apolipoprotein D, S100A6, S100A8, and ceruloplasmin discriminate best between the DE and CT groups. The differences between antileukoproteinase, phospholipase A2, and lactoperoxidase levels allow the distinction between MGD and DE, and the changes in the levels of annexin A1, clusterin, and alpha-1-acid glycoprotein 1, between MGD and CT groups. The functional network analysis revealed the main biological processes that should be examined to identify new candidate biomarkers and therapeutic targets.

Inflammatory molecules in the tears of patients with keratoconus.

PURPOSE: To determine levels of a panel of inflammatory molecules and matrix metalloproteinases in the tears of patients with keratoconus. DESIGN: A prospective, case-control study. PARTICIPANTS: Twenty-eight patients (1 eye from each) diagnosed with keratoconus at the Instituto Galego de Oftalmoloxia, Santiago de Compostela, Spain, during the period from September 2001 to June 2002, and 20 normal control subjects (1 eye each) were studied. METHODS: Patients with keratoconus were examined in a routine fashion, and keratometric readings were taken to monitor the degree of ectasia. Fifteen microliters of tears was collected by capillary flow from each eye. MAIN OUTCOME MEASURES: The concentrations of cytokines (interleukin-4 [IL-4], IL-6, IL-10, and tumor necrosis factor alpha [TNF-alpha]), cell adhesion molecules (intercellular adhesion molecule 1 and vascular cell adhesion molecule 1), and matrix metalloproteinase 9 (MMP-9) were measured by enzyme-linked immunoadsorbent assay. RESULTS: Patients with keratoconus initially had significantly higher levels of IL-6 (6.7 [4.8-10.8] pg/ml vs. 2.2 [1.0-4.1] pg/ml in control subjects [P<0.0001]), TNF-alpha (3.8 [2.9-14.4] pg/ml vs. 1.8 [1.5-2.3] pg/ml in control subjects [P<0.0001]), and MMP-9 (66.5 [49.2-139.3]ng/ml vs. 6.1 [3.9-8.3] ng/ml in control subjects. The extent of the increase was found to be associated with the severity of keratoconus. CONCLUSIONS: Interleukin-6, TNF-alpha, and MMP-9 are overexpressed in the tears of patients with keratoconus, indicating that the pathogenesis of keratoconus may involve chronic inflammatory events.

Optical coherence tomography findings in recurrent corneal erosion syndrome.

PURPOSE: To report the findings of optical coherence tomography (OCT) in the corneas of patients with recurrent corneal erosion syndrome (RCES). METHODS: Ten normal subjects and 25 patients with RCES were recruited for the study. Ten patients with RCES suffered from an acute episode of pain and 15 patients complained of typical chronic symptoms of RCES. All eyes were scanned with the anterior segment 5 line raster acquisition protocol of the Cirrus HD-OCT platform (Zeiss). The etiology of RCES was investigated and treatment was provided following a standard clinical protocol. Scans were obtained at different stages of the pathology and each patient was followed-up after treatment. RESULTS: Tear film, epithelium, basement membrane, Bowman layer, stroma, and Descemet-endothelium complex were identified by OCT in all normal corneas. The same structures were identified in nonaffected areas of RCES corneas. The findings on OCT from patients suffering from acute RCES were as follows: anterior stromal hyper-reflectivity (100%), epithelial edema (100%), and irregular breaks in the epithelium (90%). Undetected epithelial basement membrane (100%), intraepithelial basement membrane (60%), intraepithelial inclusions (60%), and anterior stromal hyper-reflectivity (40%) were seen in patients with chronic RCES. These findings correlated well with the clinical symptoms and with previously described histological reports of RCES. CONCLUSIONS: Corneal OCT can be useful to study and manage RCES. Specific OCT findings may clarify etiology, confirm a suspected diagnosis, and give advice on the decision to treat patients.

Corneal wound healing promoted by 3 blood derivatives: an in vitro and in vivo comparative study.

PURPOSE: The aim of this study was to compare the effect on corneal wound healing of 3 differently manufactured blood derivatives [autologous serum (AS), platelet-rich plasma, and serum derived from plasma rich in growth factors (s-PRGF)]. METHODS: Scratch wound-healing assays were performed on rabbit primary corneal epithelial cultures and human corneal epithelial cells. Additionally, mechanical debridement of rabbit corneal epithelium was performed. Wound-healing progression was assessed by measuring the denuded areas remaining over time after treatment with each of the 3 blood derivatives or a control treatment. RESULTS: In vitro data show statistically significant differences in the healing process with all the derivatives compared with the control, but 2 of them (AS and s-PRGF) induced markedly faster wound healing. In contrast, although the mean time required to complete in vivo reepithelization was similar to that of AS and s-PRGF treatment, only wounds treated with s-PRGF were significantly smaller in size from 2.5 days onward with respect to the control treatment. CONCLUSIONS: All 3 blood derivatives studied are promoters of corneal reepithelization. However, the corneal wound-healing progresses differently with each derivative, being faster in vitro under AS and s-PRGF treatment and producing in vivo the greatest decrease in wound size under s-PRGF treatment. These findings highlight that the manufacturing process of the blood derivatives may modulate the efficacy of the final product.

Clinical presentation and causes of recurrent corneal erosion syndrome: review of 100 patients.

PURPOSE: To study the clinical features and etiology of recurrent corneal erosion syndrome (RCES). METHODS: We examined a total of 100 patients (117 eyes) with the diagnosis of RCES who presented at our institution (Instituto Clinico Quirúrgico de Oftalmología, Bilbao, Spain). Studied data included demography, etiology, corneal location, and association with meibomian gland dysfunction (MGD). RESULTS: The mean age of patients was 44.5 (range, 14-80) years. Attributed causes of RCES were previous minor trauma (46 eyes, 39.3%), epithelial basement membrane corneal dystrophy (20 eyes, 17.1%), photorefractive keratectomy (20 eyes, 17.1%), laser-assisted in situ keratomileusis (9 eyes, 7.7%), and of unknown origin (22 eyes, 18.8%). The most frequent site of RCES was the inferior paracentral cornea (68.4%), followed by the upper cornea (21.3%) and widespread location (21.3%). An association with MGD was found in 59% of patients. CONCLUSIONS: RCES has various etiologies, which explains the variety in the clinical presentation of the disorder. Interestingly, a significant number of RCES patients begin to manifest the syndrome after keratorefractive surgery, and a high percentage of patients also present with MGD.

Tear MMP-9 levels as a marker of ocular surface inflammation in conjunctivochalasis.

PURPOSE: To evaluate the efficacy of surgical treatment for conjunctivochalasis by monitoring matrix metalloproteinase (MMP)-9 levels in the tears of patients with conjunctivochalasis before and after surgery and their correlation with clinical signs and symptoms. METHODS: Twelve eyes of patients with symptomatic conjunctivochalasis were included in this study as well as five eyes of healthy volunteers. Ocular surface inflammation was measured in terms of the concentration of pro-MMP-9 in tears, by enzyme-linked immunosorbent assay and zymography. Tear analysis was performed before and 1 month after surgery. The surgical technique consisted of the excision of redundant tissue and the use of organic glue for wound closure. RESULTS: The concentration of pro-MMP-9 was significantly higher in the conjunctivochalasis eyes than in the healthy controls (223.4 ± 74.53 ng/mL vs. 20.32 ± 5.21 ng/mL; P < 0.001). Tear pro-MMP-9 levels decreased significantly after conjunctival resection in patients with conjunctivochalasis without dry eye compared with patients with conjunctivochalasis and dry eye associated. Zymographic analysis indicated that MMP-9 is present in its active form only in conjunctivochalasis tears. After a follow-up of 4.9 ± 1.3 weeks, all operated eyes were found to have recovered a smooth and stable conjunctival surface, epithelial defects had improved, and epiphora had been resolved in 89% of cases. CONCLUSIONS: Our results indicate that inflammation is likely to play a relevant role in the pathogenesis of conjunctivochalasis. Appropriate surgery decreases inflammatory activity, leading to symptom improvement, and tear analysis may facilitate the treatment of the ocular surface.

Treatment of a large corneal perforation with a multilayer of amniotic membrane and TachoSil.

OBJECTIVE: To describe a novel surgical technique for the sealing of large corneal perforations. METHOD: Case report of a perforated, 3-mm, postherpetic, corneal ulcer. A single layer of amniotic membrane was sewn to the internal surface of the cornea. An absorbable fibrin sealant patch (TachoSil) was placed in the area of the perforation, and a folded double layer of amniotic membrane was finally sewn to the external face of the cornea. RESULT: Surgery without complications, leading to restoration of the anterior chamber and good subjective tolerance. CONCLUSION: The reported technique seems to represent a good alternative emergency procedure for the management of corneal perforations larger than 2 mm.

In vitro effects of three blood derivatives on human corneal epithelial cells.

PURPOSE: We compared the effects of three blood derivatives, autologous serum (AS), platelet-rich plasma (PRP), and serum derived from plasma rich in growth factors (PRGF), on a human corneal epithelial (HCE) cell line to evaluate their potential as an effective treatment for corneal epithelial disorders. METHODS: The concentrations of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), and fibronectin were quantified by ELISA. The proliferation and viability of HCE cells were measured by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay. Cell morphology was assessed by phase-contrast microscopy. The patterns of expression of several connexin, involucrin, and integrin α6 genes were analyzed by real-time RT-PCR. RESULTS: We found significantly higher levels of EGF in PRGF compared to AS and PRP. However, AS and PRGF induced robust proliferation of HCE cells. In addition, PRGF cultured cells grew as heterogeneous colonies, exhibiting differentiated and non-differentiated cell phenotypes, whereas AS- and PRP-treated cultures exhibited quite homogeneous colonies. Finally, PRGF upregulated the expression of several genes associated with communication and cell differentiation, in comparison to AS or PRP. CONCLUSIONS: PRGF promotes biological processes required for corneal epithelialization, such as proliferation and differentiation. Since PRGF effects are similar to those associated with routinely used blood derivatives, the present findings warrant further research on PRGF as a novel alternative treatment for ocular surface diseases.