Richard Funk

Progression of Parkinson's Disease Pathology Is Reproduced by Intragastric Administration of Rotenone in Mice

In patients with Parkinsons disease (PD), the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS), the dorsal motor nucleus of the vagus (DMV), the intermediolateral nucleus of the spinal cord and the substantia nigra, providing the basis for the neuropathological staging of the disease. Here we report that intragastrically administered rotenone, a commonly used pesticide that inhibits Complex I of the mitochondrial respiratory chain, is able to reproduce PD pathological staging as found in patients. Our results show that low doses of chronically and intragastrically administered rotenone induce alpha-synuclein accumulation in all the above-mentioned nervous system structures of wild-type mice. Moreover, we also observed inflammation and alpha-synuclein phosphorylation in the ENS and DMV. HPLC analysis showed no rotenone levels in the systemic blood or the central nervous system (detection limit [rotenone]<20 nM) and mitochondrial Complex I measurements showed no systemic Complex I inhibition after 1.5 months of treatment. These alterations are sequential, appearing only in synaptically connected nervous structures, treatment time-dependent and accompanied by inflammatory signs and motor dysfunctions. These results strongly suggest that the local effect of pesticides on the ENS might be sufficient to induce PD-like progression and to reproduce the neuroanatomical and neurochemical features of PD staging. It provides new insight into how environmental factors could trigger PD and suggests a transsynaptic mechanism by which PD might spread throughout the central nervous system.

Environmental toxins trigger PD-like progression via increased alpha-synuclein release from enteric neurons in mice

Pathological studies on Parkinsons disease (PD) patients suggest that PD pathology progresses from the enteric nervous system (ENS) and the olfactory bulb into the central nervous system. We have previously shown that environmental toxins acting locally on the ENS mimic this PD-like pathology progression pattern in mice. Here, we show for the first time that the resection of the autonomic nerves stops this progression. Moreover, our results show that an environmental toxin (i.e. rotenone) promotes the release of alpha-synuclein by enteric neurons and that released enteric alpha-synuclein is up-taken by presynaptic sympathetic neurites and retrogradely transported to the soma, where it accumulates. These results strongly suggest that pesticides can initiate the progression of PD pathology and that this progression is based on the transneuronal and retrograde axonal transport of alpha-synuclein. If confirmed in patients, this study would have crucial implications in the strategies used to prevent and treat PD.

Corneal Endothelial Cytotoxicity of Riboflavin/UVA Treatment in vitro

Recently, we have developed collagen crosslinking induced by combined riboflavin/UVA treatment, thus increasing the biomechanical rigidity of the cornea to treat progressive keratoconus. The present safety study was performed to evaluate possible cytotoxic effects of combined riboflavin/UVA treatment on the corneal endothelium in vitro. Endothelial cell cultures from porcine corneas were treated with 500 µM riboflavin solution, exposed to various endothelial UVA irradiances (370 nm) ranging from 0.1 to 1.6 mW/cm2 for 30 min and evaluated 24 h later using trypan blue staining and Yopro fluorescence staining. The effect of either treatment alone (UVA irradiation ranging from 0.2 to 6 mW/cm2) was also tested. An abrupt cytotoxic threshold irradiance level was found at 0.35 mW/cm2 after combined treatment with riboflavin plus UVA irradiation and at 4 mW/cm2 with UVA irradiation alone. Riboflavin alone was not toxic. A cytotoxic effect of the combined riboflavin/UVA treatment on corneal endothelial cells is to be expected with a corneal thickness of less than 400 µm. Therefore, pachymetry should be routinely performed before riboflavin/UVA treatment to exclude patients at risk.

Mapping of single cells by near infrared Raman microspectroscopy

Objective of our work is the development of new diagnostic methods for detection of tissue states (e.g. tumors, necrosis) in vivo. Therefore, it is important to understand the spectral features of pure individual components of tissues, that means cells and subcellular components. Raman spectroscopy has promising potential as an analytical tool for clinical applications because it can probe the chemical composition and molecular structure of such complex systems. Furthermore, the spatial resolution of Raman microspectroscopy in the low micrometer scale and its ability to probe samples under in vivo conditions allow new insights into living single cells without the need for fixatives, markers or stains. In a first set of experiments we prepared human embryonic lung epithelial fibroblasts, human osteogenic sarcoma cells and human astrocytoma cells. Raman mapping data sets were acquired with 1 μm step size and 1 min exposure time per spectrum using 785 nm excitation wavelength. Principal component analysis (PCA) was used for evaluation of the spectral maps. Bands in individual spectra were assigned to proteins, lipids, cholesterol and nucleic acids. Based on this information, the main cellular constituents of freeze dried cells and living cells in media were identified in score plots of principal components.

Effects of Electromagnetic Fields on Cells: Physiological and Therapeutical Approaches and Molecular Mechanisms of Interaction

This review concentrates on findings described in the recent literature on the response of cells and tissues to electromagnetic fields (EMF). Models of the causal interaction between different forms of EMF and ions or biomolecules of the cell will be presented together with our own results in cell surface recognition. Naturally occurring electric fields are not only important for cell-surface interactions but are also pivotal for the normal development of the organism and its physiological functions. A further goal of this review is to bridge the gap between recent cell biological studies (which, indeed, show new data of EMF actions) and aspects of EMF-based therapy, e.g., in wounds and bone fractures.

Synergistic Effect of Titanium Alloy and Collagen Type I on Cell Adhesion, Proliferation and Differentiation of Osteoblast-Like Cells

A number of studies have demonstrated the pivotal role of collagen in modulating cell growth and differentiation. In bone, where the extracellular matrix is composed of approximately 85% type I collagen, cellular interaction with matrix components has been shown to be important in the regulation of the osteoblast phenotype. Preservation or enhancement of normal osteoblast function and appositional bone formation after implant placement represents a strategy that can be useful for the purpose of improving osseointegration. In order to further improve biocompatibility, we combined two known favorable compounds, namely the titanium alloy, Ti6A14V, with type I collagen. We assessed the in vitro behavior of primary osteoblasts grown on both fibrillar collagen-coated and tropocollagen-coated Ti6A14V in comparison with uncoated titanium alloy, using an improved adsorption procedure. As parameters of biocompatibility, a variety of processes, including cell attachment, spreading, cytoskeletal organization, focal contact formation, proliferation and expression of a differentiated phenotype, were investigated. Our results demonstrated for the first time that in comparison to uncoated titanium alloy, collagen-coated alloy enhanced spreading and resulted in a more rapid formation of focal adhesions and their associated stress fibers. Growing on collagen-coated Ti6A14V, osteoblasts had a higher proliferative capacity and the intracellular expression of osteopontin was upregulated compared to uncoated titanium alloy. Type I collagen-coated titanium alloy exhibits favorable effects on the initial adhesion and growth activities of osteoblasts, which is encouraging for its potential use as bone graft material. Moreover, collagen type I may serve as an excellent biocompatible carrier for osteotropic factors such as cell adhesion molecules (e.g. fibronectin) or bone-specific growth factors.

The rapid release of corticosterone from the adrenal induced by ACTH is mediated by nitric oxide acting by prostaglandin E2

The adrenal cortex is a major stress organ in mammals that reacts rapidly to a multitude of external and internal stressors. Adrenocorticotropin (ACTH) is the main stimulator of the adrenal cortex, activating corticosteroid synthesis and secretion. We evaluated the mechanism of action of ACTH on adrenals of male rats, preserving the architecture of the gland in vitro. We demonstrated that both sodium nitroprusside (NP), a nitric oxide (NO) donor, and ACTH stimulate corticosterone release. NO mediated the acute response to ACTH because Nω-nitro-l-arginine methyl ester, a NO synthase inhibitor, and hemoglobin, a NO scavenger, blocked the stimulation of corticosterone release induced by ACTH. NP stimulated prostaglandin E release, which in turn stimulated corticosterone release from the adrenal. Additionally, indomethacin, which inhibits cyclooxygenase, and thereby, prostaglandin release, prevented corticosterone release from the adrenal induced by both NP and ACTH, demonstrating that prostaglandins mediate acute corticosterone release. Corticosterone content in adrenals after incubation with ACTH or NP was lower than in control glands, indicating that any de novo synthesis of corticosterone during this period was not sufficient to keep up with the release of the stored hormone. The release induced by ACTH or NP depleted the corticosterone content in the adrenal by ≈40% compared with the content of glands incubated in buffer. The mechanism of rapid release is as follows: NO produced by NO synthase activation by ACTH activates cyclooxygenase, which generates PGE2, which in turn releases corticosterone stored in microvesicles and other organelles.

Immunohistochemical detection of human skin nerve fibers

The autonomic nervous system is involved in different functions such as transduction of afferent sensory inputs, trophic actions, modulation of immunologic events and thermoregulation. In the present investigation, we studied the pattern of human autonomic skin innervation with special reference to its relation to blood vessels, hair follicles, sweat glands and sensory receptors. For the first time, two clinically important areas have been compared: the skin of the forearm and of the face. Using indirect immunohistochemistry, we analyzed the distribution of calretinin (CR), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), substance P (SP), neurokinin A (NKA), vasoactive intestinal peptide (VIP), nitric oxide synthase (NOS), tyrosine hydroxylase (TH), histamine, serotonin, enkephalin, and, enzyme histochemically, NADPH-diaphorase (NADPH-d). In the epidermis, we found nerve fibers containing SP, NKA and CGRP. In the dermis, SP-, CR-, VIP-, CGRP- and NKA-positive nerve fibers were detected. Particularly the large nerve fibers contained CR. VIP-positive fibers occurred especially around hair follicles and sweat glands. CGRP-positive nerve fibers were located close to the epidermal basal membrane, in the wall of blood vessels, and to a lesser extent around hair follicles. Immunoreactivity for SP and NKA in the dermis was observed predominantly in the papillary layer near the epidermal basal membrane. All neuropeptides tested in this study were also detected in the nerve fibers of the subcutis. Most of them were CGRP- and VIP-positive. They occurred in association with sweat glands and large arteries. NPY-positive nerve fibers are predominant in the wall of arteries, arterioles and veins. Nerve fibers containing NKA and SP were less common and identified only in the walls of large arteries in deeper dermal layers. In double-staining experiments, the NADPH-d reaction and reactivity to tubulin revealed a partial co-localization in nerve fibers, blood vessel walls, around glands and ganglionic cells. VIP-positive fibers were more common in the face skin than in the forearm. However, in forearm we detected more NPY-, CGRP-, NKA- and SP-positive nerve fibers than in face skin. These findings are important for future studies on skin disorders, such as sensory neuropathies, inflammatory reactions or allergic responses of human skin.

Blood Supply of the Retina

An overview of the functional anatomy of the retinal microcirculatory system and its regulation mechanisms is presented. The retinal microvasculature is characterized by thin capillaries which leave large vessel-free spaces compared to other microvascular beds. Despite high blood flow velocities, the blood flow volume within the capillaries is relatively low. This results in a high arteriovenous pO2 difference and a small capacity to tolerate periods of low perfusion. Furthermore, from the optic nerve head on there is no autonomic perivascular innervation to control the microvascular tone. A control via the bloodstream (mediators, e.g. O2, CO2, hormones), astrocytes, neurites and Müller cells (mediators, e.g. NO, prostaglandins, neuropeptides) takes over. Finally, the role of pericytes in the control of retinal hemodynamics is discussed.

Age-related changes in cells and tissues due to advanced glycation end products (AGEs)

Advanced glycation end products (AGEs) formed by nonenzymatic glycation and oxidation of proteins accumulate during normal aging and at accelerated rate during the course of diabetes. They play a role in the pathogenesis of several other chronic diseases such as Alzheimers disease, arthritis, atherosclerosis, pulmonary fibrosis and renal failure. AGE-formation changes the chemical and biological properties of proteins inside and outside of the cell. Binding to specific cell surface receptors induces activation of cellular signaling pathways leading to cellular dysfunction and cell death. AGEs are inducible by oxidative stress and induce oxidative stress. Subject of current studies of cell biologists is the intracellular processing of AGEs, which is accompanied by changes of the endolysosomal compartment.