Maria João Quadrado

Diabetes and corneal cell densities in humans by in vivo confocal microscopy.

Purpose: Diabetes is accompanied by an increased autofluorescence of the cornea, probably because of accumulation of advanced glycation end products (AGEs). The pathogenic mechanism is still unknown. This study aimed to quantify differences in corneal cell densities between diabetic patients and healthy controls. Methods: The left cornea of 15 patients with non-insulin-dependent diabetes mellitus (NIDDM) with level of retinopathy 20 according to the Early Treatment of Diabetic Retinopathy Study (ETDRS) and of 15 healthy controls were examined by noninvasive in vivo confocal microscopy in an observational prospective study. The cell densities in 6 corneal layers were determined along the optical axis of the cornea by using stereologic methods. Results: The average cell density per unit area in the superficial and basal epithelium and the endothelial layer was 725 ± 171, 5950 ± 653, and 2690 ± 302 cells/mm2 in controls and 815 ± 260, 5060 ± 301, and 2660 ± 364 cells/mm2 in diabetic patients. The cell density per unit volume in the anterior, mid-, and posterior stroma was 26,300 ± 4090, 19,390 ± 3120, and 25,700 ± 3260 cells/mm3 in controls and 27,560 ± 3880, 21,930 ± 2110, and 25,790 ± 3090 cells/mm3 in patients with diabetes. In both groups, the density in the midstroma was significantly lower than in both the anterior stroma and the posterior stroma (P < 0.02). The cell density in the basal layer of diabetic patients was significantly lower than in healthy controls (−15.0%, P < 0.0004). In the other layers, no significant differences between both groups (P > 0.07) were observed. Conclusions: The lower basal cell density found in patients with diabetes may result from a combination of different mechanisms including decreased innervation at the subbasal nerve plexus, basement membrane alterations, and higher turnover rate in basal epithelial cells. The lower cell density in the midstroma of diabetic patients and healthy controls may be attributed in part to differences in oxygen concentration in the stromal layers (depths). Changes in cellular density did not seem to be responsible for the increased autofluorescence in diabetes.

Femtosecond laser versus mechanical microkeratomes for flap creation in laser in situ keratomileusis and effect of postoperative measurement interval on estimated femtosecond flap thickness

PURPOSE: To prospectively compare laser in situ keratomileusis (LASIK) flaps created with a femtosecond laser or a mechanical microkeratome and analyze the effect of the postoperative measurement interval on estimated femtosecond flap thickness using ultrasound (US) pachymetry. SETTING: Department of Ophthalmology, University Hospital of Coimbra, and Coimbra Surgical Centre, Coimbra, Portugal. METHODS: Flaps were created with a Hansatome Zero Compression microkeratome (Group 1), Zyoptix XP keratome (Group 2), or IntraLase 60 kHz femtosecond laser (Groups 3 and 4). Flap thickness was determined by intraoperative US pachymetry immediately after flap creation in Groups 1, 2, and 3. In Group 4, pachymetry was performed 20 minutes after the laser treatment. The main outcome measures were flap thickness and deviation from the target value. RESULTS: Eighty patients had LASIK for myopia or myopic astigmatism. The mean flap thickness was 149.1 μm ± 24.9 (SD) in Group 1, 124.7 ± 23.8 μm in Group 2, 143.1 ± 18.4 μm in Group 3, and 115.5 ± 12.5 μm in Group 4. The difference in flap thickness between Group 3 and Group 4 was statistically significant (P<.01). The flap thickness deviation from the target value was 22.8 μm in Group 1, 19.0 μm in Group 2, 26.1 μm in Group 3, and 10.4 μm in Group 4. CONCLUSIONS: Results indicate that the time of measurement after femtosecond affects the estimated flap thickness. Waiting 20 minutes after laser treatment permitted easier separation and may eliminate the effect of variable corneal dehydration on flap measurement by subtraction pachymetry.

Corneal Cell Density Measurement in vivo by Scanning Slit Confocal Microscopy: Method and Validation

Purpose: Method and validation of a technique to quantify cell density in vivo in 6 corneal layers with a scanning slit confocal microscope (SSCM). Method: A confocal image of a small volume in a corneal layer is registered on videotape. Cells or nuclei according to a layer classification are counted manually using an unbiased frame. Surface cell density is calculated from an image on the screen, and volumetric density is obtained using stereological methods. Results: Image distortion on the screen is less than 3%. The classification of a cell layer is verified by determining the position of the measurement volume in the cornea. Validation of density measurements is performed by comparing confocal results with those obtained by histology. The difference between the two methods varies from –24.1% (posterior stroma) to +16.4% (basal layer). Intersession and intrasession repeatability are 8.3 and 5.8%, respectively. The cell density (mean ± SD) in 20 healthy controls in the superficial, basal and endothelial layers was 759 ± 162, 5,817 ± 632 and 2,743 ± 285 cells·mm–2 (surface), and in the anterior, mid and posterior stroma 28,616 ± 3,081, 19,578 ± 4,421 and 26,073 ± 5,962 cells·mm–3 (volumetric). These results are in accordance with those of other investigators. Conclusions: The SSCM can produce repeatable quantitative measurements of corneal cell density in conscious humans.

Evaluation of treatment with cysteamine eyedrops for cystinosis with confocal microscopy.

Purpose: The purpose of this study was to evaluate the efficacy of cysteamine eyedrops 0.1136% (new formulation, now stable at room temperature without the need for refrigeration for up to 2 months) for the treatment of cystine crystals in the cornea using slit lamp biomicroscopy and confocal microscopy. Methods: A 20-year-old woman with infantile cystinosis, with a history of kidney transplantation at age 10, was studied. She applied cysteamine eyedrops (0.1136%) 10 times a day (a new formulation, now stable at room temperature without the need for refrigeration for up to 2 months, was prepared for compassionate use). The density of the cystine crystals in the cornea was evaluated using slit lamp biomicroscopy and confocal microscopy (Heidelberg Retina Tomograph 2 equipped with the Rostock Module for the Cornea). Results: The deposits were absent in the surface epithelium and basal cells of the central cornea before and after treatment. We found crystals mainly in the anterior and medium stroma; they had various shapes; were intracellular, rectangular, or fusiform; and showed hyperreflectivity. The posterior stroma showed lower density of the crystals. No deposits in the endothelium were found. Therapy with cysteamine eyedrops reduced the density of the crystals and lessened the photophobia. Conclusion: Confocal microscopy is a valuable technique to study cystine crystals of the cornea in vivo. Cysteamine eyedrops appear to be very useful in the treatment of these crystals, especially for photophobic complaints.

Estudio multicéntrico retrospectivo sobre trasplante de limbo

PURPOSE To report the results of limbal transplantation (LT) in patients with limbal stem cell deficiency (LSCD) in the context of ocular surface diseases. MATERIALS AND METHODS A multicenter (5 centers) retrospective case series analysis of patients who underwent LT between 1996 and 2004 was performed. Data were collected by the same researcher using a customized database. Success was defined by the absence of a persistent corneal epithelial defect, on-going inflammation or recurrence of a pterygium. RESULTS Data from 72 LT performed in 61 patients (65 eyes) with a mean follow-up of 20.8 months (SD 23.5; range, 3-115) were analyzed. There were 33 males and 28 females with a mean age of 55.8 years (SD: 15.6; range, 20-89). Fifty-eight (80.6%) LT were autografts (40 pterygia, 12 alkali burns, 3 iatrogenic cases, 2 viral infections, 1 neoplasia case) and 14 (19.4%) were allografts from cadaveric donors (7 immune-based disorders, 6 alkali burns, 1 iatrogenic case); all patients receiving allografts also received systemic immunosuppression. Of the total number of LT, 48 (66.7%) were successful. This proportion increased to 81.0% (47/58) when autografts were used. However, only 7.1% (1/14) of all allografts were successful. The success rate was higher (80.0%) when performed for a pterygium and lower when done for immune-based inflammation (14.3%). CONCLUSION Autograft tissue for LT is always preferable to allografts to surgically treat LSCD, as clinical success is significantly higher, and systemic immunosuppression is avoided. As expected, immune-based disorders are the most difficult cases to treat. LT has been shown to be an excellent option for recurrent pterygium, although prospective studies need to be performed to further corroborate these results.espanolObjetivo: Revisar el resultado de los trasplantes de limbo (TL) realizado en pacientes con Sindrome de insuficiencia limbica (SIL) en el contexto de varias enfermedades de la superficie ocular. Materiales y metodos: Se realizo un estudio retrospectivo y multicentrico (cinco centros) de los TL realizados entre 1996 y 2004. Los datos fueron recogidos por el mismo investigador, en una base de datos especialmente disenada para el estudio. Se considero como «exito» del TL a la ausencia de: defectos epiteliales, inflamacion y recurrencia del pterigion cuando este fue la causa del TL. Resultados: Se analizaron un total de 72 TL realizados en 61 pacientes (65 ojos) con tiempo de seguimiento de 20,8 meses (DS 23,5; rango, 3-115). Hubo 33 hombres y 28 mujeres, con una media de 55,8 anos (DS: 15,6; rango, 20-89). Se realizaron 58 (80.6%) TL autologos (40 pterigion, 12 causticaciones, tres iatrogenicas, dos infecciones virales, una neoplasia) y 14 (19,4%) TL alogenicos de donante cadaver (siete inflamaciones inmunes, seis causticaciones, uno iatrogenico). Todos los pacientes a los que se les realizo TL alogenicos recibieron inmunosupresion sistemica. Al final del seguimiento, 48 (66,7%) TL se consideraron un exito. Este porcentaje fue del 81,0% (47/58) en los TL autologos y del 7,1% (1/14) en los TL alogenicos. El porcentaje de exito tambien dependio de la etiologia, siendo alto en los casos de pterigion (80,0%) y menor en las patologias inmunologicas (14,3%). Conclusiones: El TL autologo es preferible al alogenico, pues la tasa de fracasos disminuye notablemente y, ademas, se evita el uso de inmunosupresion oral mantenida. Ademas, el pronostico es siempre peor en los casos de patologia inflamatoria inmune. El TL autologo parece ser una buena eleccion para el tratamiento del pterigion recurrente, aunque se necesitan estudios prospectivos que corroboren estos resultados. EnglishPurpose: To report the results of limbal transplantation (LT) in patients with limbal stem cell deficiency (LSCD) in the context of ocular surface diseases. Materials and methods: A multicenter (5 centers) retrospective case series analysis of patients who underwent LT between 1996 and 2004 was performed. Data were collected by the same researcher using a customized database. Success was defined by the absence of a persistent corneal epithelial defect, on-going inflammation or recurrence of a pterygium. Results: Data from 72 LT performed in 61 patients (65 eyes) with a mean follow-up of 20.8 months (SD 23.5; range, 3-115) were analyzed. There were 33 males and 28 females with a mean age of 55.8 years (SD: 15.6; range, 20-89). Fifty-eight (80.6%) LT were autografts (40 pterygia, 12 alkali burns, 3 iatrogenic cases, 2 viral infections, 1 neoplasia case) and 14 (19.4%) were allografts from cadaveric donors (7 immune-based disorders, 6 alkali burns, 1 iatrogenic case); all patients receiving allografts also received systemic immunosuppression. Of the total number of LT, 48 (66.7%) were successful. This proportion increased to 81.0% (47/58) when autografts were used. However, only 7.1% (1/14) of all allografts were successful. The success rate was higher (80.0%) when performed for a pterygium and lower when done for immune-based inflammation (14.3%). Conclusion: Autograft tissue for LT is always preferable to allografts to surgically treat LSCD, as clinical success is significantly higher, and systemic immunosuppression is avoided. As expected, immune-based disorders are the most difficult cases to treat. LT has been shown to be an excellent option for recurrent pterygium, although prospective studies need to be performed to further corroborate these results.

Activation of MAPK Signaling Pathway and NF-κB Activation in Pterygium and Ipsilateral Pterygium-Free Conjunctival Specimens

PURPOSE To evaluate mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) signaling pathways in pterygium and pterygium-free conjunctivas. METHODS Primary pterygia (n = 21), ipsilateral superior-temporal bulbar conjunctivas (n = 8), and healthy conjunctival (n = 5) biopsy specimens were analyzed. Total and phosphorylated (phospho) levels of extracellular-regulated 1/2 (ERK1/2), p38, and c-jun N-terminal (JNK) MAPKs and NF-κB inhibitor-alpha (IκΒ-α) were analyzed by immunobead-based assay. Tissue phospho-, total protein, and activation values determined by phospho/total ratios were compared. Correlation among those values and clinical parameters were determined. Average-linkage hierarchical cluster analysis identified patients with similar protein activation values. The k-nearest neighbor classifier predicted the origin of specimens based on protein levels. RESULTS Pterygium samples had significantly lower total JNK and IκΒ-α levels than did healthy conjunctivas. Decreased total JNK and IκΒ-α and increased phospho-IκΒ-α levels and phospho/total ratio of JNK and IκΒ-α were present in ipsilateral conjunctivas compared with healthy conjunctivas. Protein levels were correlated among them in pterygium, ipsilateral, and healthy conjunctivas and with sun exposure, pterygium grade, and pterygium measurements. Cluster analysis of activation values and ratios in pterygium and ipsilateral-conjunctiva revealed different groups of patients with similar values. Prediction accuracy was 70% to 80% for the classifiers phospho- and total protein levels and phospho/total ratio. CONCLUSIONS Pterygium and pterygium-free ipsilateral conjunctivas had alterations in MAPK and NF-κB pathways not present in healthy conjunctivas. The high prediction accuracy based on phospho- and total protein levels and phospho/total ratio of ERK1/2, p38, JNK, and IκB-α suggests these molecules as potential biomarkers of inflammation in pterygia.

Irregular Astigmatism After Corneal Transplantation--Efficacy and Safety of Topography-Guided Treatment.

Purpose: To analyze the efficacy and safety of topography-guided photorefractive keratectomy (TG-PRK) to treat irregular astigmatism after corneal transplantation. Methods: This was a retrospective observational case series. Eyes with irregular astigmatism after penetrating keratoplasty treated with TG-PRK (Allegretto Wave Eye-Q) with the topography-guided customized ablation treatment protocol were included. All treatments had been planned to correct the topographic irregularities, as well as to reduce the refractive error after neutralizing the induced refractive change. Clinical records, treatment plan, and the examinations performed were reviewed and the following data were collected: corrected and uncorrected distance visual acuities; manifest refraction; topographic parameters, and corneal endothelial cell count. Results: We included 31 eyes [30 patients; mean age 45.0 ± 13.4 (SD) years]. At the last postoperative follow-up (mean 9.2 ± 8.2 months), we observed a significant improvement in corrected (P = 0.001) and uncorrected distance visual acuities (P < 0.001). There was a gain of ≥1 uncorrected distance visual acuity line in 96.8% (n = 30) of the eyes. Similarly, the refractive parameters also improved (cylinder P < 0.001; spherical equivalent P = 0.002). At the last visit, 54.8% (n = 17) of the patients presented a spherical equivalent of ±1 D. The 3-mm topographic irregularity also decreased significantly (P < 0.001). There was no significant variation of the corneal endothelial cell count. Conclusions: This is the largest case series of TG-PRK to treat irregular astigmatism in postcorneal transplantation eyes. Our results confirm that TG-PRK is an efficient treatment, associated with significant improvements of both visual acuity and refractive parameters.

Femtosecond laser and microkeratome-assisted Descemet stripping endothelial keratoplasty: first clinical results

Aim To perform Descemet stripping automated endothelial keratoplasty (DSAEK) using a novel technique to obtain very thin (<100 µm) posterior corneal disks. Methods Twenty five DSAEK grafts were prepared with two sequential cuts: the first cut, of variable thickness, was made with a femtosecond laser and the second with a 300 µm microkeratome head. Spectacle corrected visual acuity, endothelial cell density evaluation with specular microscopy and anterior segment optical coherence tomography to measure central and peripheral graft thickness was performed preoperatively and postoperatively at 1, 3 and 6 months. Results There were no irregular cuts or perforations during tissue preparation. Central graft thickness was 79.6 µm (SD±14.5; range 54–98) and 69.3 µm (SD±14.2; range 49–96) at 3 and 6 months. Corrected distance visual acuity improved from 0.91 logMAR preoperatively to 0.11 logMAR at 6 months. Donor endothelial cells averaged 2675 cells/mm2 preoperatively and 1729 cells/mm2 at 6 months. There were no graft detachments. Conclusions This new technique consistently yielded very thin grafts (<100 µm), excellent visual acuity results and good endothelial cell counts. No donor tissue was wasted.

Anterior Chamber Epithelial Cyst After Uneventful Deep Anterior Lamellar Keratoplasty.

Purpose: To report the case of an anterior chamber epithelial cyst after uncomplicated deep anterior lamellar keratoplasty (DALK) treated with a tissue-sparing surgical technique. Methods: A 35-year-old female patient underwent DALK in her OS because of stage IV keratoconus. The surgery was uneventful, and 3 months postoperatively, best-corrected visual acuity (BCVA) was 20/25. By the sixth postoperative month, a cystic iris lesion was noticed. The lesion grew progressively larger, and BCVA dropped to 20/63 in OS. Cyst excision was performed. Results: No complications possibly related to the surgery were observed. Two years later, BCVA was 20/25 in OS, and there were no signs of cyst recurrence. Histopathological examination of the lesion showed an epithelial implantation cyst. Conclusions: An anterior chamber epithelial cyst is a sight-threatening condition that may occur after DALK. In selected cases, a conservative surgical approach is an effective treatment option, conveying excellent functional and anatomical outcomes.

Contact lenses as drug controlled release systems: a narrative review

Topically applied therapy is the most common way to treat ocular diseases, however given the anatomical and physiological constraints of the eye, frequent dosing is required with possible repercussions in terms of patient compliance. Beyond refractive error correction, contact lenses (CLs) have, in the last few decades emerged as a potential ophthalmic drug controlled release system (DCRS). Extensive research is underway to understand how to best modify CLs to increase residence time and bioavailability of drugs within therapeutic levels on the ocular surface.These devices may simultaneously correct ametropia and have a role in managing ophthalmic disorders that can hinder CL wear such as dry eye, glaucoma, ocular allergy and cornea infection and injury. In this narrative review the authors explain how the ocular surface structures determine drug diffusion in the eye and summarize the strategies to enhance drug residence time and bioavailability. They synthesize findings and clinical applications of drug soaked CLs as DCRS combined with delivery diffusion barriers, incorporation of functional monomers, ion related controlled release, molecular imprinting, nanoparticles and layering. The authors draw conclusions about the impact of these novel ophthalmic agents delivery systems in improving drug transport in the target tissue and patient compliance, in reducing systemic absorption and undesired side effects, and discuss future perspectives.