Longitudinal assessment of neuropathy in diabetes using novel ophthalmic markers (LANDMark) : baseline findings

Purpose Over the past decade, corneal nerve morphology and corneal sensation threshold have been explored as potential surrogate markers for the evaluation of diabetic neuropathy. We present the baseline findings of a Longitudinal Assessment of Neuropathy in Diabetes using novel ophthalmic Markers (LANDMark). Methods The LANDMark Study is a 5-year, two-site, natural history (observational) study of individuals with Type 1 diabetes stratified into those with (T1W) and without (T1WO) neuropathy according to the Toronto criteria, and control subjects. All study participants undergo detailed annual assessment of neuropathy including corneal nerve parameters measured using corneal confocal microscopy and corneal sensitivity measured using non-contact corneal esthesiometry. Results 396 eligible individuals (208 in Brisbane and 188 in Manchester) were assessed: 76 T1W, 166 T1WO and 154 controls. Corneal sensation threshold (mbars) was significantly higher in T1W (1.0 ± 1.1) than T1WO (0.7 ± 0.7) and controls (0.6 ± 0.4) (P=0.002); post-hoc analysis (PHA) revealed no difference between T1WO and controls (Tukey HSD, P=0.502). Corneal nerve fiber length (mm/mm2) (CNFL) was lower in T1W (13.8 ± 6.4) than T1WO (19.1 ± 5.8) and controls (23.2 ± 6.3) (P<0.001); PHA revealed CNFL to be lower in T1W than T1WO, and lower in both of these groups than controls (P<0.001). Corneal nerve branch density (branches/mm2) (CNBD) was significantly lower in T1W (40 ± 32) than T1WO (62 ± 37) and controls (83 ± 46) (P<0.001); PHA showed CNBD was lower in T1W than T1WO, and lower in both groups than controls (P<0.001). Alcohol and cigarette consumption did not differ between groups, although age, BMI, BP, waist circumference, HbA1c, albumin-creatinine ratio, and cholesterol were slightly greater in T1W than T1WO (p<0.05). Some site differences were observed. Conclusions The LANDMark baseline findings confirm that corneal sensitivity and corneal nerve morphometry can detect differences in neuropathy status in individuals with Type 1 diabetes and healthy controls. Corneal nerve morphology is significantly abnormal even in diabetic patients ‘without neuropathy’ compared to control participants. Results of the longitudinal trial will assess the capability of these tests for monitoring change in these parameters over time as potential surrogate markers for neuropathy.

New technologies for assessing the contact lens wearing eye

New technologies for examination of the anterior eye in contact lens practice don’t appear to have taken a huge leap in the past decade however there a several novel adaptations of existing technology worthy of note. In other areas of health we have self-diagnosis via smartphone or other gadgets adapted as medical devices. In practice and research in vitro and in vivo new adaptive technologies have expanded our capabilities in assessing the anterior eye, in particular corneal and conjunctival confocal microscopy.

Longitudinal Assessment of Neuropathy in Diabetes using novel ophthalmic MARKers (LANDMark): Baseline findings

Purpose:Over the past decade, corneal nerve morphology and corneal sensation threshold have been explored as potential surrogate markers for the evaluation of diabetic neuropathy. We present the baseline findings of a Longitudinal Assessment of Neuropathy in Diabetes using novel ophthalmic Markers (LANDMark). Methods:The LANDMark Study is a 5-year, two-site, natural history (observational) study of individuals with Type 1 diabetes stratified into those with (T1W) and without (T1WO) neuropathy according to the Toronto criteria, and control subjects. All study participants undergo detailed annual assessment of neuropathy including corneal nerve parameters measured using corneal confocal microscopy and corneal sensitivity measured using non-contact corneal esthesiometry. Results:396 eligible individuals (208 in Brisbane and 188 in Manchester) were assessed: 76 T1W, 166 T1WO and 154 controls. Corneal sensation threshold (mbars) was significantly higher in T1W (1.0 ± 1.1) than T1WO (0.7 ± 0.7) and controls (0.6 ± 0.4) (P=0.002); post-hoc analysis (PHA) revealed no difference between T1WO and controls (Tukey HSD, P=0.502). Corneal nerve fiber length (mm/mm2) (CNFL) was lower in T1W (13.8 ± 6.4) than T1WO (19.1 ± 5.8) and controls (23.2 ± 6.3) (P<0.001); PHA revealed CNFL to be lower in T1W than T1WO, and lower in both of these groups than controls (P<0.001). Corneal nerve branch density (branches/mm2) (CNBD) was significantly lower in T1W (40 ± 32) than T1WO (62 ± 37) and controls (83 ± 46) (P<0.001); PHA showed CNBD was lower in T1W than T1WO, and lower in both groups than controls (P<0.001). Alcohol and cigarette consumption did not differ between groups, although age, BMI, BP, waist circumference, HbA1c, albumin-creatinine ratio, and cholesterol were slightly greater in T1W than T1WO (p<0.05). Some site differences were observed. Conclusions:The LANDMark baseline findings confirm that corneal sensitivity and corneal nerve morphometry can detect differences in neuropathy status in individuals with Type 1 diabetes and healthy controls. Corneal nerve morphology is significantly abnormal even in diabetic patients ‘without neuropathy’ compared to control participants. Results of the longitudinal trial will assess the capability of these tests for monitoring change in these parameters over time as potential surrogate markers for neuropathy.

Utility of assessing nerve morphology in central cornea versus whorl area for diagnosing diabetic peripheral neuropathy

Purpose To compare small nerve fiber damage in the central cornea and whorl area in participants with diabetic peripheral neuropathy (DPN) and to examine the accuracy of evaluating these 2 anatomical sites for the diagnosis of DPN. Methods A cohort of 187 participants (107 with type 1 diabetes and 80 controls) was enrolled. The neuropathy disability score (NDS) was used for the identification of DPN. The corneal nerve fiber length at the central cornea (CNFLcenter) and whorl (CNFLwhorl) was quantified using corneal confocal microscopy and a fully automated morphometric technique and compared according to the DPN status. Receiver operating characteristic analyses were used to compare the accuracy of the 2 corneal locations for the diagnosis of DPN. Results CNFLcenter and CNFLwhorl were able to differentiate all 3 groups (diabetic participants with and without DPN and controls) (P < 0.001). There was a weak but significant linear relationship for CNFLcenter and CNFLwhorl versus NDS (P < 0.001); however, the corneal location x NDS interaction was not statistically significant (P = 0.17). The area under the receiver operating characteristic curve was similar for CNFLcenter and CNFLwhorl (0.76 and 0.77, respectively, P = 0.98). The sensitivity and specificity of the cutoff points were 0.9 and 0.5 for CNFLcenter and 0.8 and 0.6 for CNFLwhorl. Conclusions Small nerve fiber pathology is comparable at the central and whorl anatomical sites of the cornea. Quantification of CNFL from the corneal center is as accurate as CNFL quantification of the whorl area for the diagnosis of DPN.

Corneal recovery after uncomplicated and complicated PRK and LASIK

Refractive errors, especially myopia, seem to increase worldwide. Concurrently, the number of surgical refractive corrections has increased rapidly, with several million procedures performed annually. However, excimer laser surgery was introduced after a limited number of studies done with animals and to date there still are only few long-term follow-up studies of the results. The present thesis aims to evaluate the safety and functional outcome of, as well as to quantify the cellular changes and remodelling in the human cornea after, photorefractive keratectomy (PRK) and laser assisted in situ keratomileusis (LASIK). These procedures are the two most common laser surgical refractive methods. In Study I, myopic ophthalmic residents at Helsinki University Eye Hospital underwent a refractive correction by PRK. Five patients were followed up for 6 months to assess their subjective experience in hospital work and their performance in car driving simulator and in other visuomotor functions. Corneal morphological changes were assessed by in vivo confocal microscopy (ivCM). Study II comprised 14 patients who had undergone a PRK operation in 1993-1994. Visual acuity was examined and ivCM examinations performed 5 years postoperatively. In Study III 15 patients received LASIK refractive correction for moderate to high myopia (-6 - -12 D). Their corneal recovery was followed by ivCM for 2 years. Diffuse lamellar keratitis (DLK) is a common but variable complication of LASIK. Yet, its aetiology remains unknown. In Study IV we examined six patients who had developed DLK as a consequence of formation of an intraoperative or post-LASIK epithelial defect, to assess the corneal and conjunctival inflammatory reaction. In the whole series, the mean refractive correction was -6.46 diopters. The best spectacle corrected visual acuity (BSCVA) improved in 30 % of patients, whereas in four patients BSCVA decreased slightly. The mean achieved refraction was 0.35 D undercorrected. After PRK, the stromal scar formation was highest at 2 to 3 months postoperatively and subsequently decreased. At 5 years increased reflectivity in the subepithelial stroma was observed in all patients. Interestingly, no Bowman s layer was detected in any patient. Subbasal nerve fiber bundle(snfb) regeneration could be observed already at 2 months in 2 patients after PRK. After 5 years, all corneas presented with snfb, the density of which, however, was still lower than in control corneas. LASIK induced a hypocellular area on both sides of the flap interface. A decrease of the most anterior keratocyte density was also observed. In the corneas that developed DLK, inflammatory cell-type objects were present in the flap interface in half of the patients. The other patients presented only with keratocyte activation and highly reflective extracellular matrix. These changes resolved completely with medication and time. Snfb regeneration was first detected at one month post-LASIK, but still after two years the density of snfb, however, was only 64 % of the preoperative values. The performance of ophthalmological examinations and microsurgery without spectacles was easier postoperatively, which was appreciated by the residents. Both PRK and LASIK showed moderate to good accuracy and high safety. In terms of visual perception and subjective evaluation, few patients stated any complaints in the whole series of studies. Instead, the majority of patients experienced a marked improvement in everyday life and work performance. PRK and LASIK have shown similar results, with good long term morphological healing. It seems evident that, even without the benefit of over-20-year follow-up results, these procedures are sufficiently safe and accurate for refractive corrections and corneal reshaping.

Corneal nerves in refractive surgery and dry eye

This study aimed to investigate the morphology and function of corneal sensory nerves in 1) patients after corneal refractive surgery and 2) patients with dry eye due to Sjögren's syndrome. A third aim was to explore the possible correlation between cytokines detected in tears and development of post-PRK subepithelial haze. The main methods used were tear fluid ELISA analysis, corneal in vivo confocal microscopy, and noncontact esthesiometry. The results revealed that after PRK a positive correlation exists between the regeneration of subbasal nerves and the thickness of regenerated epithelium. Pre- or postoperative levels of the tear fluid cytokines TGF-β1, TNF-α, or PDGF-BB did not correlate with the development of corneal haze objectively estimated by in vivo confocal microscopy 3 months after PRK. After high myopic LASIK, a discrepancy between subjective dry eye symptoms and objective signs of dry eye was observed. The majority of patients reported ongoing dry eye symptoms even 5 years after LASIK, although no objective clinical signs of dry eye were apparent. In addition, no difference in corneal sensitivity was observed between these patients and controls. Primary Sjögren's syndrome patients presented with corneal hypersensitivity, although their corneal subbasal nerve density was normal. However, alterations in corneal nerve morphology (nerve sprouting and thickened stromal nerves) and an increased number of antigen-presenting cells among subbasal nerves were observed, implicating the presence of an ongoing inflammation. Based on these results, the relationship between nerve regeneration and epithelial thickness 3 months after PRK appears to reflect the trophic effect of corneal nerves on epithelium. In addition, measurement of tear fluid cytokines may not be suitable for screening patients for risk of scar (haze) formation after PRK. Presumably, at least part of the symptoms of "LASIK-associated dry eye" are derived from aberrantly regenerated and abnormally functioning corneal nerves. Thus, they may represent a form of corneal neuropathy or "phantom pain" rather than conventional dry eye. Corneal nerve alterations and inflammatory findings in Sjögren's syndrome offer an explanation for the corneal hypersensitivity or even chronic pain or hyperalgesia often observed in these patients. In severe cases of disabling chronic pain in patients with dry eye or after LASIK, when conventional therapeutic possibilities fail to offer relief, consultation of a physician specialized in pain treatment is recommended.

Confocal and two-photon microscopy : image enhancement /

Confocal and two-photon microcopy have become essential tools in biological research and today many investigations are not possible without their help. The valuable advantage that these two techniques offer is the ability of optical sectioning. Optical sectioning makes it possible to obtain 3D visuahzation of the structiu-es, and hence, valuable information of the structural relationships, the geometrical, and the morphological aspects of the specimen. The achievable lateral and axial resolutions by confocal and two-photon microscopy, similar to other optical imaging systems, are both defined by the diffraction theorem. Any aberration and imperfection present during the imaging results in broadening of the calculated theoretical resolution, blurring, geometrical distortions in the acquired images that interfere with the analysis of the structures, and lower the collected fluorescence from the specimen. The aberrations may have different causes and they can be classified by their sources such as specimen-induced aberrations, optics-induced aberrations, illumination aberrations, and misalignment aberrations. This thesis presents an investigation and study of image enhancement. The goal of this thesis was approached in two different directions. Initially, we investigated the sources of the imperfections. We propose methods to eliminate or minimize aberrations introduced during the image acquisition by optimizing the acquisition conditions. The impact on the resolution as a result of using a coverslip the thickness of which is mismatched with the one that the objective lens is designed for was shown and a novel technique was introduced in order to define the proper value on the correction collar of the lens. The amoimt of spherical aberration with regard to t he numerical aperture of the objective lens was investigated and it was shown that, based on the purpose of our imaging tasks, different numerical apertures must be used. The deformed beam cross section of the single-photon excitation source was corrected and the enhancement of the resolution and image quaUty was shown. Furthermore, the dependency of the scattered light on the excitation wavelength was shown empirically. In the second part, we continued the study of the image enhancement process by deconvolution techniques. Although deconvolution algorithms are used widely to improve the quality of the images, how well a deconvolution algorithm responds highly depends on the point spread function (PSF) of the imaging system applied to the algorithm and the level of its accuracy. We investigated approaches that can be done in order to obtain more precise PSF. Novel methods to improve the pattern of the PSF and reduce the noise are proposed. Furthermore, multiple soiu'ces to extract the PSFs of the imaging system are introduced and the empirical deconvolution results by using each of these PSFs are compared together. The results confirm that a greater improvement attained by applying the in situ PSF during the deconvolution process.

Corneal graft survival after therapeutic keratoplasty for Acanthamoeba keratitis

Purpose: To describe corneal graft survival and visual outcome after therapeutic penetrating keratoplasty in patients with Acanthamoeba keratitis (AK) that is unresponsive to clinical treatment. Methods: Retrospective study. Thirty-two patients with AK who underwent therapeutic penetrating keratoplasty (tPK) from August 1996 to August 2005 were included. Data relating to clinical features, visual acuity, surgical technique, graft survival and complications were collected. Graft survival was evaluated by the Kaplan-Meier method and comparisons were performed using the Log-rank test. Results: Most patients (62.5%) were female. Mean age [+/- standard deviation (SD)] was 35 (+/- 13) years (range 15-68 years). All patients were contact lens wearers. Eighteen patients (56%) presented paralytic mydriasis and glaucoma during the treatment. Thirteen patients (40%) developed glaucoma after surgery; eight of them (61%) required a second PK because of graft failure. Of the 32 keratoplasty eyes, 56.2% presented graft failure at any follow-up point. Forty-five per cent of graft failures occurred before the 12 month follow-up, so 55% remained clear in the first year after surgery. Twelve patients underwent a second PK; seven of them failed and 45% were clear at 1 year. Two patients presented graft recurrence of amoebic infection. There was no significant difference in graft survival when eyes with or without mydriasis were compared (P = 0.40). Eyes with glaucoma presented a significantly shorter graft survival (P = 0.01). Conclusion: Penetrating keratoplasty is a treatment option for eyes that are unresponsive to clinical treatment infections. However, graft survival is poor; postoperative glaucoma is frequent and is associated with shorter graft survival.

The application of Cd Se/ZnS quantum dots and confocal laser scanning microscopy for three-dimensional imaging of nanfibrous structures

This paper reports a fast, accurate, and non-destructive three-dimensional imaging approach based on using quantum dots and confocal laser scanning microscopy to get three-dimensional images of internal pore structure of the nanofibrous materials. A practical method of making the fiber fluorescent using quantum dots was applied before three-dimensional imaging by confocal laser scanning microscopy. Fibrous scaffolds with different porosity parameters produced by electrospinning and their three-dimensional pore structure was evaluated by this approach. Furthermore, the introduced approach can be used to measure the pore interconnectivity of the scaffold

Human corneal epithelial cell shedding and fluorescein staining in response to silicone hydrogel lenses and contact lens disinfecting solutions

A pilot study was conducted to evaluate human corneal epithelial cell shedding in response to wearing a silicone hydrogel contact lens/solution combination inducing corneal staining. The nature of ex vivo collected cells staining with fluorescein was also examined. A contralateral eye study was conducted in which up to eight participants were unilaterally exposed to a multipurpose contact lens solution/silicone hydrogel lens combination previously shown to induce corneal staining (renu® fresh™ and balafilcon A; test eye), with the other eye using a combination of balafilcon A soaked in a hydrogen peroxide care system (Clear Care®; control eye). Lenses were worn for 2, 4 or 6 hours. Corneal staining was graded after lens removal. The Ocular Surface Cell Collection Apparatus was used to collect cells from the cornea and the contact lens. In the test eye, maximum solution-induced corneal staining (SICS) was observed after 2 hours of lens wear (reducing significantly by 4 hours; p < 0.001). There were significantly more cells collected from the test eye after 4 hours of lens wear when compared to the control eye and the collection from the test eye after 2 hours (for both; n = 5; p < 0.001). The total cell yield at 4 hours was 813 ± 333 and 455 ± 218 for the test and control eyes, respectively (N = 5, triplicate, p = 0.003). A number of cells were observed to have taken up the fluorescein dye from the initial fluorescein instillation. Confocal microscopy of fluorescein-stained cells revealed that fluorescein was present throughout the cell cytoplasm and was retained in the cells for many hours after recovery from the corneal surface. This pilot study indicates that increased epithelial cell shedding was associated with a lens-solution combination which induces SICS. Our data provides insight into the transient nature of the SICS reaction and the nature of fluorescein staining observed in SICS.

Proliferation of the vascular endothelium of the iris following total debridement of the corneal epithelium and limbal excision of rabbits

Background Damage to the corneal epithelium causes not only a reaction for its repair but also affects other parts of the cornea as well as different components of the anterior segment of the eye. The purpose of this investigation was to analyze the consequences, following epithelial and limbal damage, to the iris of rabbits (Oryctolagus cuniculus).Methods The corneal epithelium was thoroughly scraped followed by surgical excision of the limbus. Next, (3)H-thymidine ((3)H-TdR) was injected intravitreally both into the right (experimental) and left (control) eyes which had their anterior segments processed for autoradiography at intervals of 2, 7 and 21 days after surgery (three rabbits per interval). The irises were also examined with scanning-electron and confocal microscopy after Evans blue injection.Results There was a high frequency of labeling in the cells of the iris blood vessels in the experimental eye, particularly the endothelial ones. The ratio of labeled cells between experimental and control irises was 40:1, with a population of nuclei increasing by 25% and remaining labeled up to 21 days. There was also an increase in the volume of the iris vasculature as shown by confocal microscopy. The high labeling frequencies of the vascular cells were observed throughout the iris from the ciliary to the pupillary regions.Conclusions The lesions on the corneal epithelium elicit proliferation of the iris vascular cells, mainly its endothelium, as well as an early breakdown of the blood-aqueous barrier. The daughter cells resulting from the damage to the eye surface were detected up to 21 days after a single injection of (3)H-TdR, most likely due to their slow turnover. As a consequence of this proliferation, the vasculature of the iris increased in volume.

Effect of bleaching on sound enamel and with early artificial caries lesions using confocal laser microscopy

The aim of this study was to evaluate effect of bleaching agents on sound enamel (SE) and enamel with early artificial caries lesions (CL) using confocal laser scanning microscopy (CLSM). Eighty blocks (4 × 5 × 5 mm) of bovine enamel were used and half of them were submitted to a pH cycling model to induce CL. Eight experimental groups were obtained from the treatments and mineralization level of the enamel (SE or CL) (n=10). SE groups: G1 - unbleached (control); G2 - 4% hydrogen peroxide (4 HP); G3 - 4 HP containing 0.05% Ca (Ca); G4 - 7.5% hydrogen peroxide (7.5 HP) containing amorphous calcium phosphate (ACP). CL groups: G5 - unbleached; G6 - 4 HP; G7 - 4 HP containing Ca; G8 - 7.5 HP ACP. G2, G3, G6, G7 were treated with the bleaching agents for 8 h/day during 14 days, while G4 and G8 were exposed to the bleaching agents for 30 min twice a day during 14 days. The enamel blocks were stained with 0.1 mM rhodamine B solution and the demineralization was quantified using fluorescence intensity detected by CLSM. Data were analyzed using ANOVA and Fisher's tests (α=0.05). For the SE groups, the bleaching treatments increased significantly the demineralization area when compared with the unbleached group. In the CL groups, no statistically significant difference was observed (p>0.05). The addition of ACP or Ca in the composition of the whitening products did not overcome the effects caused by bleaching treatments on SE and neither was able to promote remineralization of CL.

Effect of Er:YAG laser and diamond drill on hybrid layer morphology obtained with self-etch adhesive: analysis by SEM and confocal laser scanning microscopy (CLSM)

This study aimed to evaluate the effect of Er:YAG (L) and diamond drills (DD) on: 1) the microshear bond strength (MPa); 2) the adhesive interface of two-step (TS) – Adper Scotchbond Multipurpose and one-step (OS) adhesives – Adper EasyOne, both from 3M ESPE. Material and methods: According to the preparation condition and adhesives, the samples were divided into four groups: DD_TS (control); DD_OS; L_TS and L_OS. 60 bovine incisors were randomly divided into experimental and groups: 40 for microshear bond strength (n = 10) and 20 for the adhesive interface morphology [6 to measure the thickness of the hybrid layer (HL) and length of tags (t) by CLSM (n = 3); 12 to the adhesive interface morphology by SEM (n = 3) and 2 to illustrate the effect of the instruments on dentine by SEM (n = 1)]. To conduct the microshear bond strength test, four cylinders (0.7 mm in diameter and 1 mm in height with area of adhesion of 0.38 mm) were constructed with resin composite (Filtek Z350 XT – 3M ESPE) on each dentin surface treated by either L or DD and after adhesives application. Microshear bond strength was performed in universal testing machine (EMIC 2000) with load cell of 500 kgf and a crosshead speed of 0.5 mm / min. Adhesive interface was characterized by thickness of hybrid layer (HL) and length of tags (t) in nm, with the aid of UTHSCSA ImageTool software. Results: Microshear bond strength values were: L_TS 34.10 ± 19.07, DD_TS 24.26 ± 9.35, L_OS 33.18 ± 12.46, DD_OS 21.24 ± 13.96. Two-way ANOVA resulted in statistically significant differences only for instruments (p = 0.047). Mann-Whitney identified the instruments which determined significant differences for HL thickness and tag length (t). Concerning to the adhesive types, these differences were only observed for (t). Conclusion: It can be concluded that 1) laser Er:YAG results in higher microshear bond strength values regardless of the adhesive system (TS and OS); 2) the tags did not significant affect the microshear bond strength; 3) the adhesive interface was affected by both the instruments for cavity preparation and the type of adhesive system used.

Confocal laser scanning microscopy investigation of bond interfaces involved in fiber glass post cementation

Objective: This confocal microscopy study evaluated the cement/dentin and cement/post interfaces along theroot canalwallswhenfiberglasspostswerebonded to dentin using different types of cements. Material & Methods: Thirty endodontically treated premolars were divided into 3 groups according to the adhesive materials used in the bonding procedure: Prime & Bond 2.1/Self Cure + Enforce, RelyX Unicem and RelyX Luting. Rhodamine B dye was incorporated in the luting materials for the cementation of the fiber glass posts (Exacto, Angelus) to dentin. Three transversal slices (apical, middle and coronal) were examined under confocal laser scanning microscopy. Statistical analysis was performed using the Kappa, Kruskal-Wallis and Dunnet tests, in a significance level of 5%. Results: The Prime & Bond 2.1/Self Cure + Enforce presented a uniform formation of tags in the dentin but gaps in the cement/dentin interface. The RelyX Unicem and RelyX Luting presented an adhesive interface with a fewer amount of gaps, but showed shorter tag formation than the Enforce system. All cements presented the same pattern of bubbles inside the cements. The RelyX Luting presented a greater amount of cracks inside the cement in comparison with the other cements in the coronal third, while no difference was observed between RelyX Unicem and Enforce. The RelyX Luting showed the lowest quantity of cement penetration into the post. Conclusion: In general, the quality of bonding interfaces of fiber posts luted to root canals was affected by both location and type of cement.