Isabel Lema

SUBCLINICAL KERATOCONUS AND INFLAMMATORY MOLECULES FROM TEARS.

Background/aims: Tissue degradation in corneal thinning disorders, such as keratoconus (KC), involves the expression of inflammatory mediators. The purpose of this study was to determine the levels of proinflammatory cytokines and matrix metalloproteinase 9 (MMP-9) in tears from both eyes of unilateral keratoconus (KC) patients. Methods: Thirty patients diagnosed as having asymmetrical KC (30 KC eyes, and 30 subclinical KC eyes) and 20 normal control subjects (one eye) were studied in a prospective, cross-sectional study. Keratoconus screening programmes were performed on these participants. Ten microlitres of tears was collected from each eye. The concentrations of cytokines (interleukin-6 (IL-6) and tumour necrosis factor α (TNF-α)) and MMP-9 were measured by ELISA. Results: Mean values for IL-6 levels were similar in KC and subclinical KC samples (5.5 (4.9 to 6.9) vs 5.7 (4.5 to 6.2) pg/ml, p = 0.131), but significantly higher in relation to the control group (2.2 (1.0 to 4.1) pg/ml, p<0.0001). Significant differences were found in TNF-α levels between KC and subclinical KC eyes (5.4 (4.1 to 6.8) vs 4.8 (4.2 to 6.0) pg/ml, p = 0.032) and control group (1.8 (1.5 to 2.3) pg/ml, p<0.0001). Increased values of MMP-9 were found in KC (59.4 (50.6 to 66.1) ng/ml) vs subclinical KC eye (7.0 (4.8 to 8.6) ng/ml) (p<0.0001). MMP-9 levels in the control group (6.1 (3.9 to 8.3) ng/ml) and subclinical KC were similar (p = 0.203). Conclusions: IL-6 and TNF-α are overexpressed in the tears of subclinical and KC eyes. Increased MMP-9 levels were found only in the KC eye. These results indicate that the pathogenesis of KC may involve chronic inflammatory events.

Inflammatory Markers in the Tears of Patients with Ocular Surface Disease

Purpose: To determine the concentration of interleukins (IL-1β 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-1β, 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-1β 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-1β and IL-6 were only found to be overexpressed in conjunctivochalasis. These findings illustrate the selective implication of different molecules in each disorder.

Inflammatory response to contact lenses in patients with keratoconus compared with myopic subjects.

Purpose: To determine the levels of inflammatory molecules in the tears of patients who wore rigid gas permeable (RGP) contact lenses (CLs) and who had either keratoconus or myopia. Methods: A prospective, case-control study with 4 groups enrolled 20 RGP CL keratoconus wearers and 28 keratoconus non-lens wearers, 20 myopic CL wearers, and 20 subjects with myopia that were non-lens wearers (1 eye per patient). Fifteen microliters of tears were collected by capillary flow. The concentration of cytokines (interleukin-6 [IL-6], IL-10, and tumor necrosis factor [TNF]-α), cell adhesion molecules (intercellular adhesion molecule 1 [ICAM-1] and vascular cell adhesion molecule 1 [VCAM-1]), and matrix metalloproteinase (MMP-9) was measured by enzyme-linked immunosorbent assay. Results: The most significant differences associated with the wearing of RGP CLs in patients with keratoconus were seen in increased levels of proinflammatory cytokines (IL-6, 23.7 vs. 6.4 pg/mL, P = 0.001; TNF-α, 21.3 vs. 3.8 pg/mL, P = 0.028) and cell adhesion molecules (ICAM-1, 32.8 vs. 7.7 ng/mL, P < 0.0001; VCAM-1, 57.4 vs. 29.3 ng/mL, P < 0.0001). In patients with myopia, increased levels of TNF-α (4.2 vs. 1.8 pg/mL, P < 0.0001) and MMP-9 (12.9 vs. 6.1 ng/mL, P < 0.0001) were associated with the wearing of RGP CLs. Conclusions: Wearing RGP CLs induces overexpression of IL-6, TNF-α, ICAM-1, and VCAM-1 in the tears of patients with keratoconus. These increased levels are higher in cases with severe keratoconus.

In vitro and in vivo ocular safety and eye surface permanence determination by direct and Magnetic Resonance Imaging of ion-sensitive hydrogels based on gellan gum and kappa-carrageenan

Gellan gum, kappa-carrageenan and alginates are natural polysaccharides able to interact with different cations that can be used to elaborate ion-activated in situ gelling systems for different uses. The interaction between fluid solutions of these polysaccharides and cations presents into the tear made these biopolymers very interesting to elaborate ophthalmic drug delivery systems. The main purpose of this study is to evaluate the ability of mixtures of these polymers to obtain ion-activated ophthalmic in situ gelling systems with optimal properties for ocular use. To achieve this purpose different proportion of the biopolymers were analyzed using a mixture experimental design evaluating their transparency, mechanical properties and bioadhesion in the absence and presence of simulated tear fluid. Tear induces a rapid sol-to-gel phase transition in the mixtures forming a consistent hydrogel. The solution composed by 80% of gellan gum and 20% kappa-carrageenan showed the best mechanical and mucoadhesive properties. This mixture was evaluated for rheological behavior, microstructure, cytotoxicity, acute corneal irritancy, ex-vivo and in vivo ocular toxicity and in vivo corneal contact time using Magnetic Resonance Images (MRI) techniques. Result indicates that the system is safe at ophthalmic level and produces an extensive ocular permanence higher than 6h.

Corneal descriptive indices in the fellow eye of unilateral keratoconus.

Objectives: To determine whether indices derived from corneal topography could be used to differentiate between three populations at varying degrees of risk for presenting keratoconus, but otherwise unremarkable clinical appearance. Methods: Eighty subjects lacking clinical signs of keratoconus were divided into three groups according to their background. Fifty refractive surgery candidates were used as controls, to be compared with 15 clinically normal fellow eyes of subjects with unilateral keratoconus, and 15 eyes from first and second degree relatives of the former keratoconic patients. Corneal topography indices (curvature, wavefront, and thickness) were obtained from Placido disc (Keratron) and scanning-slit (Orbscan) devices. Receiver operator characteristic curve analysis (sensitivity, specificity, and area under the curve) was performed to highlight the ability of any single index to discriminate between groups, and cutoff values for a fixed 0.80 specificity were determined. Results: Corneal wavefront indices (root mean square and vertical coma) exhibited the best performance for discriminating between controls and the fellow eye of unilateral keratoconus patients. Corneal curvature and thickness indices showed poor capability to differentiate between the groups. Relatives and controls were distinguishable by a single thickness parameter. Conclusions: The proposed cutoff values between controls and patients at risk for keratoconus may prove useful for screening purposes.

Vascular retinal, neuroimaging and ultrasonographic markers of lacunar infarcts.

Background Lacunar infarcts may be caused by macro-or microvascular disease due to several mechanisms. Aim This study aims to demonstrate that retinal vascular disturbances in patients with lacunar infarcts associated with neuroimaging and ultrasound markers can help to identify small-vessel disease. Methods Prospective ambulatory study of patients with ischaemic stroke and a control group. A retinographic study was performed by 20° bilateral optic disc stereophotography and 50° bilateral optic fundus retinography. Microangiopathy was evaluated as the presence of nonparenchymal vascular affectation and retinopathy as at least one retinal disturbance. Ultrasonographic study evaluated carotid disorder parameters and the mean pulsatility index. The MRI protocol included T1-weighted, T2-weighted, DP-weighted and FLAIR. Results We included 156 nonlacunar infarcts, 39 lacunar infarcts and 50 controls. Microangiopathy was more frequent in hypertensive (62.6% vs. 35.7%, P<0.0001) and vascular retinopathy in diabetic patients (11.7% vs. 3.8%, P=0.039). Microangiopathy (97.4% vs. 41.1%, P<0.0001) and leukoaraiosis (94.4% vs. 50.3%, P<0.0001) were more frequent and the mean pulsatility index was higher (1.9±0.2 vs. 1.4±0.5, P<0.0001) in patients with lacunar infarcts. Lacunar infarcts were independently associated with microangiopathy (odds ratio 12.81, 95% CI 1.52–107.86), the mean pulsatility index (odds ratio 8.13, 95% CI 1.17–56.20) and leukoaraiosis (odds ratio 3.45, 95% CI 1.09–10.93). The presence of leukoaraiosis plus microangiopathy was associated with lacunar infarcts with odds ratio 21.31 (95% CI 8.74–51.93). Conclusions The association of retinal microangiopathy (but not vascular retinopathy) and leukoaraiosis is linked to small-vessel disease and may be a useful marker of lacunar infarcts not secondary to a macrovascular lesion.

Videokeratoscopic indices in relation to epidemiological exposure to keratoconus

BackgroundTo establish the relationship between videokeratoscopic indices and the degree of epidemiological exposure to keratoconus in three groups of clinically normal subjects.MethodsCross-sectional study in which 75 subjects lacking clinical signs of keratoconus were divided into three groups according to epidemiological exposure to the condition: 25 fellow eyes of subjects with clinical signs on the contralateral eye only (the “fellow eye” group), to be compared to 25 first-degree relatives of patients with keratoconus (the “relatives” group) and 25 controls without a family history of the disease (the “control” group). Qualitative patterns and quantitative parameters describing curvature (central curvature), irregularity (root mean square of the higher-order corneal wavefront aberration), and asymmetry (inferior-superior dioptric asymmetry, Zernike vertical coma) obtained from videokeratoscopy were used for comparison between groups.ResultsMembers of the fellow eye group featured a greater number of asymmetric curvature patterns and increased values in indices describing asymmetry and irregularity than subjects included in both control and relatives groups. Control and relatives groups were not significantly different. Despite significant differences in the distribution of values between the groups, no single index was able to effectively discriminate between groups using ROC curve analysis.ConclusionsA prior threefold classification of clinically normal subjects according to epidemiological exposure to keratoconus was not sustained by significant differences in videokeratoscopic indices when comparing between groups.

In vivo eye surface residence determination by high-resolution scintigraphy of a novel ion-sensitive hydrogel based on gellan gum and kappa-carrageenan

&NA; In last years, sensitive hydrogels have become a breakthrough in ophthalmic pharmaceutical technology aimed at developing new strategies to increase the residence time of active substances. In a previous paper, we qualitatively demonstrated the capacity of a new ion sensitive hydrogel to increase the residence time. Nevertheless, the clearance of the gel from the ocular surface was not quantifiable with the used methodology. The aim of the present work was to use a well‐established approach based on scintigraphy to quantitatively estimate the residence time of the previously proposed hydrogel. The rat corneal residence time of a topic ophthalmic formulation containing gellan gum and kappa carragenan (0.82% w/v) labeled with 99mTc‐DTPA radiotracer was evaluated and compared with the residence of an aqueous solution. Ophthalmic safety studies such as eye irritation or passage through the cornea were also carried out. After 1.5 h of contact, 77% of the hydrogel remained in the ocular surface, presenting kinetics of disappearance one‐phase decay and a half time of 262 min. We conclude that the novel ophthalmic hydrogel developed with kappa carrageenan and gellan gum remains for long periods of time on the corneal surface, presenting a drop that fits an exponential decay. Graphical abstract Figure. No caption available.

Higher Expression of Toll-Like Receptors 2 and 4 in Blood Cells of Keratoconus Patiens

Inflammation may play a significant role in Keratoconus (KC), but the implication of immunity on this inflammatory response is unknown. Therefore, our aim was to determine the expression levels of Toll-like receptors 2 (TLR2) and 4 (TLR4) in monocytes and neutrophils from patients with KC and control subjects for demonstrating the role of innate immunity in KC. We also study the correlation between TLR2/TLR4 expression and serum levels of proinflammatory markers (IL-1β, IL-6, TNF-α, MMP-9 and NF-κB). Forty patients with bilateral KC (55% males; mean age; 33.1 ± 10.9 years) and 20 control subjects (55% males; mean age; 30.4 ± 7.6 years) were included in the study. Our results showed that mean expression of TLR2 and TLR4 in both neutrophils and monocytes was significantly higher in patients with KC compared to control subjects (all p < 0.0001). Furthermore, KC patients also showed higher serum levels of IL-1B, IL-6, TNF-α, MMP-9 (all p < 0.0001) and NF-κB (p = 0.036). In addition, we found a strong correlation between TLR2 expression in both monocytes and neutrophils (all p < 0.0001), and TLR4 in monocytes (all p < 0.05) with serum levels of IL-1B, IL-6, TNF-α and MMP-9. In conclusion, these findings suggest that TLRs may play an important role in the pathophysiology of KC.

Unilateral keratoconus: videokeratography and Orbscan study--optical correction.

Objectives: To assess the frequency of unilateral keratoconus (KC) and vision correction with a contact lens (CL) in the KC eye. Methods: Transverse study in which we revised the clinical records of 385 KC patients. Of these, 35 (9%) showed clinical unilateral KC. Corneal topography was evaluated by a Placido disk and an elevation topographer. We also determined distance and near visual acuity before and after correction with spectacles or CL in KC eye. Results: Mean central corneal curvature (K) of the KC eyes was 51.45 ± 6.65 D, versus 43.58 ± 1.71 D for the fellow eye (P<0.001). Mean maximum curvature of the inferior cornea was 54.62 ± 5.91 D in the KC eye and 44.66 ± 2.38 in the fellow eye (P<0.001). Mean of the thinnest point pachymetry was 411 ± 55 &mgr;m in the KC eye and 477 ± 42 &mgr;m in the contralateral eye (P<0.0001). In most of the 29 patients in whom CLs were fitted, aspheric rigid gas-permeable CLs were used, corrected visual acuities were significantly better (P<0.0001) than without correction. Conclusions: All unilateral KC patients showed higher corneal curvature in the affected eye. Corneal thickness was likewise clearly reduced in the KC eye, and reduced with respect to normal values in the contralateral eye. Aspheric rigid gas-permeable CL were the most effective for improving distance and near visual acuity in patients with adequate tolerance.