Andreas Wree

Epithelial innervation of human cornea: a three-dimensional study using confocal laser scanning fluorescence microscopy.

Purpose: Evaluation of a new method to visualize distribution and morphology of human corneal nerves (Aδ- and C-fibers) by means of fluorescence staining, confocal laser scanning microscopy, and 3-dimensional (3D) reconstruction. Methods: Trephinates of corneas with a diagnosis of Fuchs corneal dystrophy were sliced into layers of 200 μm thickness using a Draeger microkeratome (Storz, Germany). The anterior lamella was stained with the Life/Dead-Kit (Molecular Probes Inc.), examined by the confocal laser scanning microscope “Odyssey XL,” step size between 0.5 and 1 μm, and optical sections were digitally 3D-reconstructed. Results: Immediate staining of explanted corneas by the Life/Dead-Kit gave a complete picture of the nerves in the central human cornea. Thin nerves running parallel to the Bowman layer in the subepithelial plexus perforate the Bowman layer orthogonally through tube-like structures. Passing the Bowman layer, Aδ- and C-fibers can be clearly distinguished by fiber diameter, and, while running in the basal epithelial plexus, by their spatial arrangement. Aδ-fibers run straight and parallel to the Bowman layer underneath the basal cell layer. C-fibers, after a short run parallel to the Bowman layer, send off multiple branches penetrating epithelial cell layers orthogonally, ending blindly in invaginations of the superficial cells. In contrast to C-fibers, Aδ-fibers show characteristic bulbous formations when kinking into the basal epithelial plexus. Conclusions: Ex-vivo fluorescence staining of the cornea and 3D reconstructions of confocal scans provide a fast and easily reproducible tool to visualize nerves of the anterior living cornea at high resolution. This may help to clarify gross variations of nerve fiber patterns under various clinical and experimental conditions.

Estimation of volume fractions in nervous tissue with an image analyzer

The calculation of volume fractions in nervous tissue is a method often used in neuroanatomy. In ontogenetic studies and problems concerning aging, as well as after experimental procedures, volume fractions are calculated in order to quantify the results. Grey level index (GLI) and grey cell coefficient (GCC) are different parameters correlated to the volume density of cellular elements in nervous tissue. The grey cell coefficient (GCC) is defined as the volume fraction of cellular elements in a griseum (grey matter area) and is estimated by a basic stereological method (point counting) from observations in two dimensions. The grey level index (GLI), measured with an automatic image analyzer, is the areal proportion of projected profiles of all stained elements within a volume given by the area of the measuring field and the thickness of section. GLI and GCC are compared in specimens obtained by a special histological procedure, and a method to estimate volume fractions by means of GLI-measurement is described.

Small molecule GSK-3 inhibitors increase neurogenesis of human neural progenitor cells

Human neural progenitor cells provide a source for cell replacement therapy to treat neurodegenerative diseases. Therefore, there is great interest in mechanisms and tools to direct the fate of multipotent progenitor cells during their differentiation to increase the yield of a desired cell type. We tested small molecule inhibitors of glycogen synthase kinase-3 (GSK-3) for their functionality and their influence on neurogenesis using the human neural progenitor cell line ReNcell VM. Here we report the enhancement of neurogenesis of human neural progenitor cells by treatment with GSK-3 inhibitors. We tested different small molecule inhibitors of GSK-3 i.e. LiCl, sodium-valproate, kenpaullone, indirubin-3-monoxime and SB-216763 for their ability to inhibit GSK-3 in human neural progenitor cells. The highest in situ GSK-3 inhibitory effect of the drugs was found for kenpaullone and SB-216763. Accordingly, kenpaullone and SB-216763 were the only drugs tested in this study to stimulate the Wnt/β-catenin pathway that is antagonized by GSK-3. Analysis of human neural progenitor differentiation revealed an augmentation of neurogenesis by SB-216763 and kenpaullone, without changing cell cycle exit or cell survival. Small molecule inhibitors of GSK-3 enhance neurogenesis of human neural progenitor cells and may be used to direct the differentiation of neural stem and progenitor cells in therapeutic applications.

Cerebral amyloid-β proteostasis is regulated by the membrane transport protein ABCC1 in mice

In Alzheimer disease (AD), the intracerebral accumulation of amyloid-β (Aβ) peptides is a critical yet poorly understood process. Aβ clearance via the blood-brain barrier is reduced by approximately 30% in AD patients, but the underlying mechanisms remain elusive. ABC transporters have been implicated in the regulation of Aβ levels in the brain. Using a mouse model of AD in which the animals were further genetically modified to lack specific ABC transporters, here we have shown that the transporter ABCC1 has an important role in cerebral Aβ clearance and accumulation. Deficiency of ABCC1 substantially increased cerebral Aβ levels without altering the expression of most enzymes that would favor the production of Aβ from the Aβ precursor protein. In contrast, activation of ABCC1 using thiethylperazine (a drug approved by the FDA to relieve nausea and vomiting) markedly reduced Aβ load in a mouse model of AD expressing ABCC1 but not in such mice lacking ABCC1. Thus, by altering the temporal aggregation profile of Aβ, pharmacological activation of ABC transporters could impede the neurodegenerative cascade that culminates in the dementia of AD.

A convenient approach for finite-element-analyses of orthopaedic implants in bone contact: Modeling and experimental validation

With regard to the growing potential of finite-element-analysis (FEA) in the field of orthopaedic biomechanics, we present an approach helping in the development of appropriate models of the implant-bone compound. The algorithm is based on computed-tomography data of the bone and accordant computer-aided-design (CAD) data of the implant and aims at predicting the bone strains and interface mechanics of the included parts. The developed algorithm was validated exemplary using an acetabular cup in combination with a left and a right fresh-frozen human hemipelvis. The strains under maximum loads during the gait cycle as well as the micromotion in the bone-implant interface were measured and compared to results from equivalent finite-element-analyses. Thereby, we found strong correlation between the calculated and measured principal strains with correlation coefficients of r(2)=0.94 (left side) and r(2)=0.86 (right side). A validation of micromotion was not possible due to limited accuracy of the motion tracking system.

Immunocytochemical characterization of in vitro PKH26-labelled and intracerebrally transplanted neonatal cells

The lipophilic dye PKH26 that binds irreversibly to cell membranes has been used to label various cell types in vitro prior to transplantation in order to recognize grafted cells posttransplantationally in the host tissue by fluorescence microscopy. The purpose of the present study was to optimize immunocytochemical staining procedures for PKH26-containing specimens in cell culture and after transplantation into rat brain. We demonstrated that freeze-thawing allowed for proper immunostaining of intracellular epitopes whereas PKH26-labelling was preserved. PKH26-labelled donor cells were detectable at least up to 4 months after transplantation in the host brain.

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.

Orexinergic innervation of the extended amygdala and basal ganglia in the rat

The orexinergic system interacts with several functional states of emotions, stress, hunger, wakefulness and behavioral arousal through four pathways originating in the lateral hypothalamus (LH). Hundreds of orexinergic efferents have been described by tracing studies and direct immunohistochemistry of orexin in the forebrain, olfactory regions, hippocampus, amygdala, septum, basal ganglia, thalamus, hypothalamus, brain stem and spinal cord. Most of these tracing studies investigated the whole orexinergic projection to all regions of the intracranial part of the CNS. To identify the orexinergic efferents at the subnuclear level of resolution, we focussed on the orexinergic target in the amygdala, which is substantially involved in the LH output and contributes mostly to the functional outcome of the orexinergic system and the basal ganglia. Immunohistochemical identification of axonal orexin A and orexin B in male adult rats has been performed on serial sections. In the extended amygdala many new orexinergic targets were found in the anterior amygdaloid area (dense), anterior cortical nucleus (moderate), amygdalostriatal transition region (moderate), basolateral regions (moderate), basomedial nucleus (moderate), several bed nucleus of the stria terminals regions (few to dense), central amygdaloid subdivisions (dense), posteromedial cortical nucleus (moderate) and medial amygdaloid subnuclei (dense). Furthermore, the entopeduncular nucleus has been newly identified as another target for orexinergic fibers with a high density. These results suggest that subdivisions and subnuclei of the extended amygdala are specific targets of the orexinergic system.

Differential astroglial activation in 6-hydroxydopamine models of Parkinson’s disease

In rat models of Parkinsons disease, injections of 6-hydroxydopamine (6-OHDA) into different areas of the basal ganglia result in dopaminergic neurodegeneration in the substantia nigra. The extent and time course of the dopaminergic lesions varies between the models. While the effects on neurons have been extensively studied, little is known about the effects on astrocytes. We compared astrocytic activation (i.e. increase in number and staining intensity of glial fibrillary acidic protein immunoreactive cells) at the injection site and in downstream structures of the motor loop, i.e. the globus pallidus (GP) and the subthalamic nucleus (STN) following 6-OHDA lesion of the medial forebrain bundle (MFB) or the striatum. Lesions in both regions resulted in astrocytic activation at the lesion site, but their remote effects varied. MFB injections caused astrocytic activation in the ipsi- and contralateral striatum, whereas striatal injections resulted in astrocytic activation in the GP and STN. Since 6-OHDA injections into the MFB and the striatum result in complete and partial SNc lesions, respectively, we hypothesize that communication links exist between astrocytes, or between neurons and astrocytes, along neuronal pathways that transmit activating signals in response to neuronal damage-but only if the neuronal pathways are at least partially intact.

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.