Marine Hovakimyan

Imaging and Quantification of Subbasal Nerve Plexus in Healthy Volunteers and Diabetic Patients with or without Retinopathy

Background The alterations of subbasal nerve plexus (SBP) innervation and corneal sensation were estimated non-invasively and compared with the values in healthy volunteers. Additionally, this study addressed the relation of SBP changes to the retinal status, glycemic control and diabetes duration. Methodology/Principal Findings Eighteen eyes of diabetic patients with peripheral diabetic neuropathy aged 68.8±8.8 years and twenty eyes of healthy volunteers aged 66.3±13.3 yrs. were investigated with in vivo confocal laser-scanning microscopy (CLSM). An adapted algorithm for image analysis was used to quantify the morphological and topological properties of SBP. These properties were correlated to incidence of diabetic retinopathy (DR) and corneal sensation (Cochet-Bonnet esthesiometer). The developed algorithm allows a fully automated analysis of pre-segmented SBP structures. Altogether, 10 parameters were analysed, and all of them revealed significant differences between diabetic patients and healthy volunteers. The nerve fibre density, total fibre length and nerve branches were found to be significantly lower in patients with diabetes than those of control subjects (nerve fibre density 0.006±0.002 vs. 0.020±0.007 mm/mm2; total fibre length 6223±2419 vs. 19961±6553 µm; nerve branches 25.3±28.6 vs. 141.9±85.7 in healthy volunteers). Also the corneal sensation was significantly lower in diabetic group when compared to controls (43±11 vs. 59±18 mm). There was found no difference in SBP morphology or corneal sensation in the subgroups with (DR) or without (NDR) diabetic retinopathy. Conclusions/Significance SBP parameters were significantly reduced in diabetic patients, compared to control group. Interestingly, the SBP impairment could be shown even in the diabetic patients without DR. Although automatic adapted image analysis simplifies the evaluation of in vivo CLSM data, image acquisition and quantitative analysis should be optimised for the everyday clinical practice.

Structural-functional correlations of corneal innervation after LASIK and penetrating keratoplasty.

PURPOSE To report a case in which a tissue saving program in an aberrated eye was used. METHODS A new algorithm for the selection of an optimized set of Zernike terms in customized treatments for laser corneal refractive surgery was developed and clinically tested. Ablation was performed using the SCHWIND ESIRIS excimer laser. Pre- and postoperative corneal wave aberrations were analyzed using the Keratron Scout videokeratoscope (Optikon 2000). RESULTS Required ablation was reduced by approximately 15% compared to full customized correction. Refraction was corrected to subclinical levels, uncorrected distance visual acuity improved to 20/20, corrected distance visual acuity gained 2 lines, aberrations were reduced by approximately 40% compared to preoperative baseline levels, and the functional optical zone of the cornea was enlarged by approximately 40% compared to preoperative baseline levels. Trefoil, coma, spherical aberration, and the root-mean-square value of the higher order aberrations were reduced. CONCLUSIONS Eliminating all higher order aberrations may not optimize visual function in highly aberrated eyes. The new algorithm minimized tissue removal in refractive surgery but further clinical evaluations are required to confi rm preliminary results.PURPOSE To compare corneal subbasal nerve fiber distribution and corneal sensation in healthy humans with findings obtained in regenerated subbasal nerves after LASIK and penetrating keratoplasty (PK). METHODS In a comparative case series study, in vivo confocal laser-scanning microscopy was used to investigate subbasal nerve fiber bundles in healthy individuals and at various time points after surgery in patients who had undergone LASIK and corneal grafting. Corneal sensation was measured (Cochet-Bonnet esthesiometer). RESULTS In normal corneas investigated, image superimposition revealed the consistent appearance of curved nerve fibers showing a whorl-like pattern with clockwise orientation. Nerve fibers parallel to Bowmans layer originating peripherally traveled radially inwards to a point located at the lower nasal quadrant. This pattern was not seen in any of the patients after LASIK or PK. Regenerated nerve fibers were thinner, more curved, and showed abnormal branching in nearly all patients. Normal corneal neuro-anatomical architecture remained absent even months after total restoration of corneal sensation. After LASIK, normal sensation was regained independently of normal subbasal nerve anatomy. Corneal grafts have shown some recovery of subbasal nerve morphology, at least in the graft periphery, but not complete recovery of function. CONCLUSIONS It would appear that normal corneal sensation after LASIK or PK does not always depend on normal subbasal nerve anatomy but on the collateral organization of subbasal nerve fibers.PURPOSE To report a case in which a tissue saving program in an aberrated eye was used. METHODS A new algorithm for the selection of an optimized set of Zernike terms in customized treatments for laser corneal refractive surgery was developed and clinically tested. Ablation was performed using the SCHWIND ESIRIS excimer laser. Pre- and postoperative corneal wave aberrations were analyzed using the Keratron Scout videokeratoscope (Optikon 2000). RESULTS Required ablation was reduced by approximately 15% compared to full customized correction. Refraction was corrected to subclinical levels, uncorrected distance visual acuity improved to 20/20, corrected distance visual acuity gained 2 lines, aberrations were reduced by approximately 40% compared to preoperative baseline levels, and the functional optical zone of the cornea was enlarged by approximately 40% compared to preoperative baseline levels. Trefoil, coma, spherical aberration, and the root-mean-square value of the higher order aberrations were reduced. CONCLUSIONS Eliminating all higher order aberrations may not optimize visual function in highly aberrated eyes. The new algorithm minimized tissue removal in refractive surgery but further clinical evaluations are required to confi rm preliminary results.PURPOSE To report a case in which a tissue saving program in an aberrated eye was used. METHODS A new algorithm for the selection of an optimized set of Zernike terms in customized treatments for laser corneal refractive surgery was developed and clinically tested. Ablation was performed using the SCHWIND ESIRIS excimer laser. Pre- and postoperative corneal wave aberrations were analyzed using the Keratron Scout videokeratoscope (Optikon 2000). RESULTS Required ablation was reduced by approximately 15% compared to full customized correction. Refraction was corrected to subclinical levels, uncorrected distance visual acuity improved to 20/20, corrected distance visual acuity gained 2 lines, aberrations were reduced by approximately 40% compared to preoperative baseline levels, and the functional optical zone of the cornea was enlarged by approximately 40% compared to preoperative baseline levels. Trefoil, coma, spherical aberration, and the root-mean-square value of the higher order aberrations were reduced. CONCLUSIONS Eliminating all higher order aberrations may not optimize visual function in highly aberrated eyes. The new algorithm minimized tissue removal in refractive surgery but further clinical evaluations are required to confi rm preliminary results.

Comparative in Vivo Confocal Microscopical Study of the Cornea Anatomy of Different Laboratory Animals

Purpose: The aim of the present study was to analyze and compare in vivo morphology of healthy cornea of six different laboratory animals. Matherials and Methods: One Pomeranian Coarsewool sheep, 5 Beagle dogs, 1 Norwegian and 2 Domestic Short-haired cats, 20 New Zealand White rabbits, 6 Wistar rats, and 10 Balb/c mice were included. The examination was performed bilaterally, using Heidelberg Retina Tomograph equipped with Rostock Cornea Module. The morphology of living corneal layers was visualized and compared between species. The central corneal thickness, density of keratocytes, and endothelial cells were quantified. Results: The epithelial multilayer showed a similarity in morphology between animal types, displaying three clearly distinguishable layers: superficial, intermediate, and basal. Subbasal nerve fibers were displayed as hyperreflective structures underneath basal cells. The subbasal fibers were confirmed in all species, however, the density varied between species. A pronounced Bowman’s membrane was visualized in sheep. In all other species, however, a thin acellular layer with overlying nerve fibers could be seen between basal epithelial cells and anterior stroma. The keratocytes nuclei could be demonstrated in all species except for mice, where no nuclei but only reflective structures resembling keratocytes cell bodies were detected. Overall, the density of keratocytes nuclei was significantly higher in the anterior than in the posterior stroma. Besides endothelial cells density, the endothelial cells morphology was very similar among all species, except for sheep. The endothelial cells were displayed as polygonal structures with bright cytoplasm and dark borders. In sheep, the appearance of the endothelium was very poor because of a thick hyperreflective Descemet’s membrane. Conclusions: The present study will help researchers consider appropriate models for animal experiments, depending on focus of investigation. In vivo CLSM can be used for the characterization of the living cornea over time, thus, reducing the number of animal experiments.

Collagen Cross-Linking: Current Status and Future Directions

Collagen cross-linking (CXL) using UVA light and riboflavin (vitamin B2) was introduced as a clinical application to stabilize the cornea by inducing cross-links within and between collagen fibers. CXL has been investigated extensively and has been shown clinically to arrest the progression of keratoconic or post-LASIK ectasia. With its minimal cost, simplicity, and proven positive clinical outcome, CXL can be regarded as a useful approach to reduce the number of penetrating keratoplasties performed. Small case series have also indicated that CXL is beneficial in corneal edema by reducing stromal swelling behavior and in keratitis by inhibiting pathogen growth. Despite these encouraging results, CXL remains a relatively new method that is potentially associated with complications. Aspects such as side effects and recurrence rates have still to be elucidated. In light of the growing interest in CXL, our paper summarizes present knowledge about this promising approach. We have intentionally endeavored to include the more relevant studies from the recent literature to provide an overview of the current status of CXL.

Biomechanical profile of the cornea in primary congenital glaucoma

Purpose:  The aim of our study was to investigate the biomechanical properties of the cornea in primary congenital glaucoma (PCG) and to identify the potential ocular determinants, which affect the corneal biomechanical metrics.

Short-term corneal response to cross-linking in rabbit eyes assessed by in vivo confocal laser scanning microscopy and histology.

Purpose: Corneal cross-linking for the treatment of keratoconus has been tested in animal trials and proven clinically. A combination of in vivo confocal laser scanning microscopy (CLSM) and histology was used in rabbit corneas to assess early modifications at the cellular level after corneal cross-linking. Methods: Twelve New Zealand male rabbits were tested; in each case, the right eye was the study eye and left eye was the control eye. In vivo CLSM was performed on both eyes before and at 3 days and 1 week after cross-linking. Keratocyte and endothelial cell densities were determined by CLSM before and after cross-linking. After CLSM, the corneas were excised and processed for histology and immunohistochemistry. Results: Massive edema was observed 3 days after cross-linking. The corneal epithelium had already closed again by day 3. No cellular structures were detected in the stroma and endothelium. One week after cross-linking, normal corneal transparency and thickness were restored. The anterior stroma still lacked nuclei. The number of nuclei in the posterior stroma was significantly lower than that in the intact corneas. Highly reflective spindle-shaped structures were detected in the posterior stroma. The endothelial monolayer had closed again but still showed significantly decreased cell density. At 1 week after cross-linking, immunohistochemical staining revealed the presence of proliferating cells in the corneal epithelium, posterior stroma, and endothelium. Conclusions: The early response of the rabbit cornea to cross-linking was successfully characterized at the cellular level by in vivo CLSM and histology, and the results obtained with both techniques correlated positively.

Survival of transplanted human neural stem cell line (ReNcell VM) into the rat brain with and without immunosuppression

Functional replacement of specific neuronal populations through transplantation of neural tissue represents an attractive therapeutic strategy for treating neurodegenerative disorders like Parkinsons disease (PD). Even though the brain is a partially immune privileged site, immunosuppression is still needed for the prevention of host immune response, and thus, xenograft rejection. Here, we investigated the fate of human ventral mesencephalon derived immortalized cell line ReNcell VM upon unilateral transplantation into the intact rat striatum with or without immunosuppression with cyclosporine A (CsA). The status of xenografted human ReNcell VM cells was analysed by immunohistochemistry/immunofluorescence 4 and 6weeks after transplantation. Four weeks after transplantation, ReNcell VM cells could be detected in both groups, although the number of survived cells was significantly higher in brains of immunosuppressed rats. In contrast, only 2 out of 6 brains grafted without immunosuppression revealed human ReNcell VM cells 6weeks post grafting, whereas a considerable number of human cells could still be found in all the brains of immunosuppressed rats. Immunohistochemical analysis of grafted cells showed almost no evidence of neuronal differentiation, but rather astroglial development. In summary, we have shown that the immunosuppression is needed for the survival of human VM derived progenitor cells in the rat striatum. CsA affected cell survival, but not differentiation capacity: in both groups, grafted either with or without immunosuppression, the ReNcell VM cells lacked neuronal phenotype and developed preferentially into astroglia.

Mesencephalic human neural progenitor cells transplanted into the neonatal hemiparkinsonian rat striatum differentiate into neurons and improve motor behaviour

Neural stem cell transplantation is a promising strategy for the treatment of neurodegenerative diseases. To evaluate the differentiation potential of human neural progenitor cells (hNPCs) as a prerequisite for clinical trials, we intracerebrally transplanted in vitro expanded fetal mesencephalic hNPCs into hemiparkinsonian rats. On postnatal day one (P1), 17 animals underwent a unilateral intraventricular 6‐hydroxydopamine injection into the right lateral ventricle. At P3, animals (n = 10) received about 100 000 hNPCs (1 µL) in the right striatum. Five weeks after birth, animals underwent behaviour tests prior to fixation, followed by immunohistochemistry on brain slices for human nuclei, glial fibrillary acidic protein, S100β, neuronal nuclei antigen, neuron‐specific enolase and tyrosine hydroxylase. Compared with the apomorphine‐induced rotations in the lesioned‐only group (7.4 ± 0.5 min−1), lesioned and successfully transplanted animals (0.3 ± 0.1 min−1) showed a significant therapeutic improvement. Additionally, in the cylinder test, the lesioned‐only animals preferred to use the ipsilateral forepaw. Conversely, the lesioned and transplanted animals showed no significant side bias similar to untreated control animals. Transplanted human nuclei‐immunoreactive cells were found to survive and migrate up to 2000 µm into the host parenchyma, many containing the pan‐neuronal markers neuronal nuclei antigen and neuron‐specific enolase. In the striatum, tyrosine hydroxylase‐immunoreactive somata were also found, indicating a dopaminergic differentiation capacity of transplanted hNPCs in vivo. However, the relative number of tyrosine hydroxylase‐immunoreactive neurons in vivo seemed to be lower than in corresponding in vitro differentiation. To minimize donor tissue necessary for transplantation, further investigations will aim to enhance dopaminergic differentiation of transplanted cells in vivo.

Combined nonlinear and femtosecond confocal laser-scanning microscopy of rabbit corneas after photochemical cross-linking.

PURPOSE Photochemical cross-linking of corneal stromal collagen using riboflavin and ultraviolet irradiation is an evolving treatment for keratoconus. The purpose of the present study was to investigate the wound-healing process in rabbit corneas after cross-linking. METHODS Photochemical cross-linking was performed according to a standard protocol on the right eyes of eight male New Zealand White rabbits; the left eyes served as controls. Untreated controls and cross-linked rabbit corneas were imaged 3 days, 6 days, and 6 weeks after treatment using a customized setup for three-dimensional nonlinear microscopy and confocal laser-scanning microscopy of reflected femtosecond light (fs-CLSM). RESULTS The combination of fs-CLSM in reflective mode and two-photon-excited fluorescence permitted differentiation of the following zones in the lamina propria of treated corneas 3 and 6 days after cross-linking: (1) an anterior zone with postapoptotic keratocyte debris, visible only on fs-CLSM in reflective mode; (2) a posterior zone with activated keratocytes with strong autofluorescence; and (3) surviving or restored keratocytes with moderate autofluorescence beyond the intermediate zone. Repopulation with normal keratocytes was achieved by 6 weeks. Bi-directional, second-harmonic generation (SHG) imaging showed no global differences in the fiber orientation and lamellar structure of stromal collagen at any time point. A relatively strong additional two-photon excited fluorescence occurred in the treated corneas with a diffuse three-dimensional spatial distribution. CONCLUSIONS This combination of imaging modalities has the potential to become a new clinical instrument capable of visualizing corneal changes at the cellular and extracellular level.

In vivo confocal laser-scanning microscopy to characterize wound repair in rabbit corneas after collagen cross-linking.

Background:  Collagen cross‐linking using the photosensitizer riboflavin combined with ultraviolet A light was developed to stiffen the cornea by increasing its mechanical and biochemical stability. Investigation of post‐treatment events, such as wound healing, is important to evaluate possible risks and to optimize treatment protocols. This in vivo confocal laser‐scanning microscopy study in rabbits was conducted to provide a quantitative and qualitative analysis of corneal wound repair over 16 weeks following collagen cross‐linking.