Femtosecond laser cutting of human corneas for the subbasal nerve plexus evaluation.

Assessment of various morphological parameters of the corneal subbasal nerve plexus is a valuable method of documenting the structural and presumably functional integrity of the corneal innervation in health and disease. The aim of this work is to establish a rapid, reliable and reproducible method for visualization of the human corneal SBP using femtosecond laser cut corneal tissue sections. Trephined healthy corneal buttons were fixed and processed using TissueSurgeon-a femtosecond laser based microtome, to obtain thick tissue sections of the corneal epithelium and anterior stroma cut parallel to the ocular surface within approximately 15 min. A near infrared femtosecond laser was focused on to the cornea approximately 70-90 µm from the anterior surface to induce material separation using TissueSurgeon. The obtained corneal sections were stained following standard immunohistochemical procedures with anti-neuronal ß-III tubulin antibody for visualization of the corneal nerves. Sections that contained the epithelium and approximately 20-30 µm of anterior stroma yielded excellent visualisation of the SBP with minimal optical interference from underlying stromal nerves. In conclusion, the results of this study have demonstrated that femtosecond laser cutting of the human cornea offers greater speed, ease and reliability than standard tissue preparation methods for obtaining high quality thick sections of the anterior cornea cut parallel to the ocular surface.

Comparative quantitative assessment of the human corneal sub-basal nerve plexus by in vivo confocal microscopy and histological staining.

PurposeThis study was designed to compare and contrast quantitative data of the human corneal sub-basal nerve plexus (SBP) evaluated by two different methods: in vivo confocal microscopy (IVCM), and immunohistochemical staining of ex vivo donor corneas.MethodsSeven parameters of the SBP in large-scale IVCM mosaicking images from healthy subjects were compared with the identical parameters in ex vivo donor corneas stained by ß-III-tubulin immunohistochemistry. Corneal nerve fiber length (CNFL), corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD), average weighted corneal nerve fiber tortuosity (CNFTo), corneal nerve connection points (CNCP), average corneal nerve single-fiber length (CNSFL), and average weighted corneal nerve fiber thickness (CNFTh) were calculated using a dedicated, published algorithm and compared.ResultsOur experiments showed significantly higher values for CNFL (50.2 vs 21.4 mm/mm2), CNFD (1358.8 vs 277.3 nerve fibers/mm2), CNBD (847.6 vs 163.5 branches/mm2), CNFTo (0.095 vs 0.081 µm-1), and CNCP (49.4 vs 21.6 connections/mm2) in histologically staining specimens compared with IVCM images. In contrast, CNSFL values were higher in IVCM images than in histological specimens (32.1 vs 74.1 µm). No significant difference was observed in CNFTh (2.22 vs 2.20 µm) between the two groups.ConclusionsThe results of this study have shown that IVCM has an inherently lower resolution compared with ex vivo immunohistochemical staining of the corneal SBP and that this limitation leads to a systematic underestimation of several SBP parameters. Despite this shortcoming, IVCM is a vital clinical tool for in vivo characterization, quantitative clinical imaging, and evaluation of the human corneal SBP.

[Characterisation of tear film dynamics after application of trehalose for treatment of dry eye]. / Charakterisierung der Tränenfilmdynamik am Beispiel einer Trehaloseapplikation zur Behandlung des Trockenen Auges.

The implementation of additional modalities for tear film break-up time characterisation expands the application range of the Oculus® Keratograph 5M. The aim of the present study was to evaluate the possibilities for non-invasive break-up time analysis using this device. Furthermore we applied the Oculus® Keratograph 5M to characterise possible modifications of tear break-up time after application of Thealoz® eye drops (Théa Pharma). The device allowed for a precise and solid topographical analysis of tear film dynamics. We could show that at four weeks after treatment, trehalose solution was a better treatment for dry eye compared with saline. These results are in agreement with our previous in vitro findings concerning the protective role of trehalose on desiccation-caused cell death in a corneal epithelial model.

Combined therapy with cyclodextrin/allopregnanolone and miglustat improves motor but not cognitive functions in Niemann-Pick Type C1 mice.

Niemann-Pick Type C1 (NPC1) is an autosomal recessive disorder characterized by the accumulation of cholesterol and glycosphingolipids. Combination-treatment utilizing cyclodextrin, allopregnanolone and miglustat (CYCLO/ALLO/miglustat) can ameliorate NPC1 disease in a mutant mouse model. The present study was designed to add behavioral analysis in NPC1 mutant mice upon CYCLO/ALLO/miglustat therapy. NPC1 mutant (BALB/cJ NPC1NIH) and control mice were used. For the combination treatment mice were injected with CYCLO/ALLO weekly, starting at P7. The miglustat injection was performed daily from P10 till P23. Starting at P23, miglustat was added to the powdered chow. For the sham treatment of control and mutant mice the same schedule was used with 0.9% NaCl injection. Locomotor activity was assessed in open field, elevated plus maze and accelerod tests. For assessment of spatial learning and memory the Morris water maze test was conducted. Electron microscopy has been performed to support the behavioral data. The sham-treated mutant mice exhibited motor impairments in all performed tests. In the water maze the sham-treated mutants exhibited impairment in remembering the location of the hidden platform. CYCLO/ALLO/miglustat treatment positively influenced motor dysfunction: total distance and number of visits significantly increased, and accelerod performance improved. The spatial learning, however, did not benefit from therapy. At the morphological level, an excessive accumulation of electron-dense material was seen in the cerebellar Purkinje cells of mutant mice. A regression of these autophagosomal inclusions was seen upon therapy. CYCLO/ALLO/miglustat therapy ameliorates motor but not cognitive deficits in NPC1 mutant mice, suggesting unequal vulnerability of different brain areas to the treatment.

[Pathological conditions of the ocular surface -- a clinical and confocal laser-scanning microscopy study]. / Oberflächenerkrankungen des Auges unterschiedlicher Genese - klinische und konfokalmikroskopische Untersuchungen.

Confocal in vivo laser scanning microscopy is an established technique to visualise morphology of the cornea and conjunctiva, whereby the image interpretation needs experience. We report about changes of the ocular surface in the pathological conditions of infectious, metabolic and traumatic genesis and discuss their relevance. The micromorphology of the corneal epithelium and stroma in respect to pathogens (bacterial, fungal) is discussed. Metabolic disease induces multifaceted corneal alterations which can be visualised and used for assessment of the disease progression. Follow-up microscopic investigations allow for an assessment of the wound healing dynamics and enable a prognosis to be made for corneal recurrence. Taken together, confocal in vivo microscopy allows a non-invasive microscopy on the cellular level and thus complements clinical diagnostics.

Ex vivo measurement of postmortem tissue changes in the crystalline lens by Brillouin spectroscopy and confocal reflectance microscopy.

Use of Brillouin spectroscopy in ophthalmology enables noninvasive, spatially resolved determination of the rheological properties of crystalline lens tissue. Furthermore, the Brillouin shift correlates with the protein concentration inside the lens. In vitro measurements on extracted porcine lenses demonstrate that results obtained with Brillouin spectroscopy depend strongly on time after death. The intensity of the Brillouin signal decreases significantly as early as 5 h postmortem. Moreover, the fluctuation of the Brillouin frequency shift inside the lens increases with postmortem time. Images of lens tissue taken with a confocal reflectance microscope between measurements reveal a degenerative aging process. These tissue changes correlate with our results from Brillouin spectroscopy. It is concluded that only in vivo measurements appropriately reflect the rheological properties of the eye lens and its protein concentration.

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 Parkinson's 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.

In vitro differentiation of the immortalized mesencephalic progenitor cell line CSM14.1 occurs independently from haematopoietic cytokines.

In vitro expanded neural precursor cells could provide a renewable source of dopaminergic (DAergic) neurons for cell replacement therapy. In the present study immortalized cell line CSM14.1 was investigated in vitro. Cells were derived from the ventral mesencephalic area of a 14-day-old rat embryo and immortalized retrovirally with the temperature-sensitive mutant of the SV40 Large T-antigen. We investigate the proliferation and differentiation of these cells under various culture conditions, at different temperatures and serum conditions. For differentiation were propagated cells at 39 degrees C in medium supplemented with 1% FCS with or without cytokines. At chosen time points cells were investigated for the expression of different markers by western blot and immunocytochemistry. As controls cells cultured at 33 degrees C with 10% FCS for 3 days were used. We have shown that serum reduction alone is not sufficient for CSM14.1-cells to stop proliferating and begin differentiation. Following serum reduction and elevation of the temperature cells changed their morphology began to express specific band of the neuronal marker NeuN. Following cytokines treatment the mean length of cellular processes increased from 319 to 385 microm per cell, whereas the expression of neuronal markers such as NeuN and TH was not markedly changed. In conclusion, the differentiation cocktail consisting of interleukin 1(Il-1), Il-11, leukaemia inhibitory factor (LIF) and GDNF, does influence the outgrowth of neuritis but does not change the expression of mature neuronal markers at the protein level in CSM14.1 cells.

[Two-photon microscopy of the cornea using intrinsic contrast]. / Farbstofffreie Zweiphotonenmikroskopie der Augenhornhaut.

BACKGROUND: Three-dimensional imaging of the cornea under physiological conditions is best performed with intrinsic contrast mechanisms for the visualisation of cells and extracellular matrix. However, the unique transparency of the cornea goes along with a lack of contrast for the extracellular matrix (ECM) in reflective mode microscopy and optical coherence tomography. METHODS: Femtosecond laser-based non-linear microscopy provides novel contrast mechanisms for the visualisation of ECM. The confinement of the non-linear contrast to the focus volume provides an intrinsic sectioning property for 3D imaging. Further advantages of the infrared light are lower phototoxicity and higher penetration depth into the tissue. For the visualisation of the cornea and its layered substructures two non-linear contrast mechanisms are of main interest: Two-photon excited autofluorescence of NAD(P)H in the cytoplasma and second harmonic generation (SHG) in the collagen-I fibres of the stroma. Ex-vivo corneas of the rabbit were imaged to demonstrate the abilities of non-linear microscopy. RESULTS: Using the autofluorescence of NAD(P)H the corneal epithelium with squamous cells, wing cells and basal cells is visualised in three dimensions without additional exogenoeus staining. Stromal keratocytes are also imaged using the NAD(P)H autofluoresecence. The layered structure of lamella in the stroma is visible after virtual resclicing of the 3D volume data. The en-face SHG images detected through the transparent cornea in forward direction show areas of parallel streaks, which increase in size and periodically alter in orientation (90 degrees , 45 degrees) with increasing depth from anterior to posterior. These streaks are not visible in the backward SHG signal. First results on rabbit corneas, which were cross-linked with Rivoflavin and UV application showed a signature of treatment five weeks post treatment. There were zones in the stroma totally lacking NAD(P)H autofluorescence and the abundance of keratocytes was less homogeneous than in control corneas. CONCLUSION: These results and current reports on applications in the literature show that femtosecond laser-based non-linear microscopy is an emerging imaging modality which provides dye-free imaging of the corneal ECM and therefore complements scattering imaging modalities such as optical coherence tomography and confocal laser scanning microscopy in the reflective mode.