Herbert A. Reitsamer

Advances in broad bandwidth light sources for ultrahigh resolution optical coherence tomography

Novel ultra-broad bandwidth light sources enabling unprecedented sub-2 microm axial resolution over the 400 nm-1700 nm wavelength range have been developed and evaluated with respect to their feasibility for clinical ultrahigh resolution optical coherence tomography (UHR OCT) applications. The state-of-the-art light sources described here include a compact Kerr lens mode locked Ti:sapphire laser (lambdaC = 785 nm, delta lambda = 260 nm, P(out) = 50 mW) and different nonlinear fibre-based light sources with spectral bandwidths (at full width at half maximum) up to 350 nm at lambdaC = 1130 nm and 470 nm at lambdaC = 1375 nm. In vitro UHR OCT imaging is demonstrated at multiple wavelengths in human cancer cells, animal ganglion cells as well as in neuropathologic and ophthalmic biopsies in order to compare and optimize UHR OCT image contrast, resolution and penetration depth.

Brain and Retinal Pericytes: Origin, Function and Role

Pericytes are specialized mural cells located at the abluminal surface of capillary blood vessels, embedded within the basement membrane. In the vascular network these multifunctional cells fulfil diverse functions, which are indispensable for proper homoeostasis. They serve as microvascular stabilizers, are potential regulators of microvascular blood flow and have a central role in angiogenesis, as they for example regulate endothelial cell proliferation. Furthermore, pericytes, as part of the neurovascular unit, are a major component of the blood-retina/brain barrier. CNS pericytes are a heterogenic cell population derived from mesodermal and neuro-ectodermal germ layers acting as modulators of stromal and niche environmental properties. In addition, they display multipotent differentiation potential making them an intriguing target for regenerative therapies. Pericyte-deficiencies can be cause or consequence of many kinds of diseases. In diabetes, for instance, pericyte-loss is a severe pathological process in diabetic retinopathy (DR) with detrimental consequences for eye sight in millions of patients. In this review, we provide an overview of our current understanding of CNS pericyte origin and function, with a special focus on the retina in the healthy and diseased. Finally, we highlight the role of pericytes in de- and regenerative processes.

The differential gene expression profiles of proximal and distal muscle groups are altered in pre-pathological dysferlin-deficient mice

The selective pattern of muscle involvement is a key feature of muscular dystrophies. Dysferlinopathy is a good model for studying this process since it shows variable muscle involvement that can be highly selective even in individual patients. The transcriptomes of proximal and distal muscles from wildtype C57BL/10 and dysferlin deficient C57BL/10.SJL-Dysf mice at a prepathological stage were assessed using the Affymetrix oligonucleotide-microarray system. We detected significant variation in gene expression between proximal and distal muscle in wildtype mice. Dysferlin defiency, even in the absence of pathological changes, altered this proximal distal difference but with little specific overlap with previous microarray analyses of dysferlinopathy. In conclusion, proximal and distal muscle groups show distinct patterns of gene expression and respond differently to dysferlin deficiency. This has implications for the selection of muscles for future microarray analyses, and also offers new routes for investigating the selectivity of muscle involvement in muscular dystrophies.

Imaging ex vivo and in vitro brain morphology in animal models with ultrahigh resolution optical coherence tomography

The feasibility of ultrahigh resolution optical coherence tomography (UHR OCT) to image ex vivo and in vitro brain tissue morphology on a scale from single neuron cells to a whole animal brain was investigated using a number of animal models. Sub-2-microm axial resolution OCT in biological tissue was achieved at different central wavelengths by separately interfacing two state-of-the-art broad bandwidth light sources (titanium:sapphire, Ti:Al2O3 laser, lambdac=800 nm, Deltalambda=260 nm, Pout=50 mW and a fiber laser light source, lambdac=1350 nm, Deltalambda=470 nm, Pout=4 mW) to free-space or fiber-based OCT systems, designed for optimal performance in the appropriate wavelength regions. The ability of sub-2-microm axial resolution OCT to visualize intracellular morphology was demonstrated by imaging living ganglion cells in cultures. The feasibility of UHR OCT to image the globular structure of an entire animal brain as well as to resolve fine morphological features at various depths in it was tested by imaging a fixed honeybee brain. Possible degradation of OCT axial resolution with depth in optically dense brain tissue was examined by depositing microspheres through the blood stream to various depths in the brain of a living rabbit. It was determined that in the 1100 to 1600-nm wavelength range, OCT axial resolution was well preserved, even at depths greater than 500 microm, and permitted distinct visualization of microspheres 15 microm in diameter. In addition, the OCT image penetration depth and the scattering properties of gray and white brain matter were evaluated in tissue samples from the visual cortex of a fixed monkey brain.

TGF-beta signalling in the adult neurogenic niche promotes stem cell quiescence as well as generation of new neurons.

Members of the transforming growth factor (TGF)‐β family govern a wide range of mechanisms in brain development and in the adult, in particular neuronal/glial differentiation and survival, but also cell cycle regulation and neural stem cell maintenance. This clearly created some discrepancies in the field with some studies favouring neuronal differentiation/survival of progenitors and others favouring cell cycle exit and neural stem cell quiescence/maintenance. Here, we provide a unifying hypothesis claiming that through its regulation of neural progenitor cell (NPC) proliferation, TGF‐β signalling might be responsible for (i) maintaining stem cells in a quiescent stage, and (ii) promoting survival of newly generated neurons and their functional differentiation. Therefore, we performed a detailed histological analysis of TGF‐β1 signalling in the hippocampal neural stem cell niche of a transgenic mouse that was previously generated to express TGF‐β1 under a tetracycline regulatable Ca‐Calmodulin kinase promoter. We also analysed NPC proliferation, quiescence, neuronal survival and differentiation in relation to elevated levels of TGF‐β1 in vitro and in vivo conditions. Finally, we performed a gene expression profiling to identify the targets of TGF‐β1 signalling in adult NPCs. The results demonstrate that TGF‐β1 promotes stem cell quiescence on one side, but also neuronal survival on the other side. Thus, considering the elevated levels of TGF‐β1 in ageing and neurodegenerative diseases, TGF‐β1 signalling presents a molecular target for future interventions in such conditions.

Femtosecond laser–assisted small-aperture corneal inlay implantation for corneal compensation of presbyopia: Two-year follow-up

Purpose To report the 2‐year postoperative safety and efficacy outcomes after monocular Kamra corneal inlay (ACI7000PDT) implantation in femtosecond laser–created corneal pockets of emmetropic presbyopic patients to improve near and intermediate vision. Setting University Eye Clinic, Paracelsus Medical University, Salzburg, Austria. Design Prospective interventional case series. Methods Patients had corneal inlay implantation in the nondominant eye. Emmetropic presbyopic patients between 45 and 60 years old with an uncorrected distance visual acuity (UDVA) of 20/20 or better in both eyes and without additional ocular pathology were eligible. Contrast sensitivity, visual field examinations, endothelial cell count (ECC), and central corneal thickness (CCT) measurements were assessed preoperatively and 12 and 24 months postoperatively. The UDVA, uncorrected intermediate visual acuity (UIVA), and near visual acuity (UNVA) were assessed preoperatively and 1 day, 1 week, and 1, 3, 6, 9, 12, 18, and 24 months postoperatively. The minimum postoperative follow‐up was 24 months. Results After 24 months, the mean binocular UNVA improved from 20/50 to 20/25; 20 patients (83%) had a UNVA of 20/25 or better. The mean binocular UIVA was 20/20. The mean UDVA was 20/20 in the surgical eye and 20/16 binocularly after 24 months. Contrast sensitivity under photopic and mesopic conditions remained in the range of the normal population. No patient had detectable central visual field defect. No inlay was explanted. No inflammatory reactions were observed. The ECC and CCT remained stable. Conclusion The corneal inlay implanted in femtosecond laser–created pockets was effective and safe for the corneal compensation of presbyopia in emmetropic patients after 24 months. Financial Disclosure Acufocus Inc., Irvine, California, USA, financially supports the Research Foundation for Promoting Ophthalmology, Salzburg, Austria, as the clinical research center of the University Eye Clinic (Paracelsus Medical University). Dr. Grabner received travel expenses from Acufocus, Inc. Dr. Riha currently also works as a clinical application specialist for Acufocus, Inc. No other author has a financial or proprietary interest in any material or method mentioned.

Mitochondrial haplogroups and control region polymorphisms in age-related macular degeneration: a case-control study.

Background Onset and development of the multifactorial disease age-related macular degeneration (AMD) are highly interrelated with mitochondrial functions such as energy production and free radical turnover. Mitochondrial dysfunction and overproduction of reactive oxygen species may contribute to destruction of the retinal pigment epithelium, retinal atrophy and choroidal neovascularization, leading to AMD. Consequently, polymorphisms of the mitochondrial genome (mtDNA) are postulated to be susceptibility factors for this disease. Previous studies from Australia and the United States detected associations of mitochondrial haplogroups with AMD. The aim of the present study was to test these associations in Middle European Caucasians. Methodology/Principal Findings Mitochondrial haplogroups (combinations of mtDNA polymorphisms) and mitochondrial CR polymorphisms were analyzed in 200 patients with wet AMD (choroidal neovascularization, CNV), in 66 patients with dry AMD, and in 385 controls from Austria by means of multiplex primer extension analysis and sequencing, respectively. In patients with CNV, haplogroup H was found to be significantly less frequent compared to controls, and haplogroup J showed a trend toward a higher frequency compared to controls. Five CR polymorphisms were found to differ significantly in the two study populations compared to controls, and all, except one (T152C), are linked to those haplogroups. Conclusions/Significance It can be concluded that haplogroup J is a risk factor for AMD, whereas haplogroup H seems to be protective for AMD.

Ciliary Blood Flow and Aqueous Humor Production

Aqueous humor production is a metabolically active process sustained by the delivery of oxygen and nutrients and removal of metabolic waste by the ciliary circulation. This article describes our investigations into the relationship between ciliary blood flow and aqueous humor production. The results presented indicate that there is a dynamic relationship between ciliary blood flow and aqueous humor production, with production being blood flow independent above a critical level of perfusion, and blood flow dependent below it. The results also show that the plateau portion of the relationship shifts up or down depending on the level of secretory stimulation or inhibition, and that oxygen is one critical factor provided by ciliary blood flow. Also presented is a theoretical model of ocular hydrodynamics incorporating these new findings.

Brain pericyte plasticity as a potential drug target in CNS repair

Brain pericytes (BrPCs) are essential cellular components of the central nervous system neurovascular unit involved in the regulation of blood flow, blood-brain barrier function, as well as in the stabilization of the vessel architecture. More recently, it became evident that BrPCs, besides their regulatory activities in brain vessel function and homeostasis, have pleiotropic functions in the adult CNS ranging from stromal and regeneration promoting activities to stem cell properties. This special characteristic confers BrPC cell plasticity, being able to display features of other cells within the organism. BrPCs might also be causally involved in certain brain diseases. Due to these properties BrPCs might be potential drug targets for future therapies of neurological disorders. This review summarizes BrPC properties, disorders in which this cell type might be involved, and provides suggestions for future therapeutic developments targeting BrPCs.

Neural Crest Origin of Retinal and Choroidal Pericytes

PURPOSE The origin of pericytes (PCs) has been controversially discussed and at least three different sources of PCs are proposed: a neural crest, mesodermal, or bone marrow origin. In the present study we investigated a potential neural crest origin of ocular PCs in a transgenic Rosa26-YFP-Sox10-Cre neural crest-specific reporter mouse model at different developmental stages. METHODS The Rosa26-YFP-Sox10-Cre mouse model expresses the yellow fluorescent protein (YFP) reporter in cells with an active Sox10 promoter and was here used for cell fate studies of Sox10-positive neural crest derived progeny cells. Detection of the YFP signal in combination with double and triple immunohistochemistry of chondroitin sulfate proteoglycan (NG2), platelet derived growth factor receptor β (PDGFRβ), α smooth muscle actin (αSMA), oligodendrocyte transcription factor 2 (Olig2), and lectin was performed and analyzed by confocal microscopy. RESULTS Sox10-YFP-positive cells and profiles were detected in the inner nuclear layer, the ganglionic cell layer, and the axons of the nerve fiber layer in postnatal retinas. An additional population has been identified in the retina, optic nerve, and choroid that displays strong perivascular localization. These cells were colocalized with the PC-specific markers NG2 and PDGFRβ in embryonic (E14.5) as well as postnatal (P4, P12, 6-week-old) vasculature. Beside PCs, vascular smooth muscle cells (vSMCs) were also labeled by the Sox10-YFP reporter protein in all ocular tissues investigated. CONCLUSIONS Since YFP-positive PCs and vSMCs are colocalized with NG2 and PDGFRβ, we propose that capillary PCs and vSMCs in the retina and the optic nerve, both parts of the central nervous system, as well as in the choroid, a tissue of mesodermal origin, derive from the neural crest.