[The role of the gut microbiome in idiopathic Parkinson's disease]. / Die Rolle des Darmmikrobioms beim idiopathischen Parkinson-Syndrom.

BACKGROUND: In recent years studies have provided increasing evidence suggesting an association between the (gut) microbiome and idiopathic Parkinson's disease (IPD). OBJECTIVE: The aim of this article is to summarize and evaluate existing evidence with respect to the relevance of the (gut) microbiome for IPD. MATERIAL AND METHODS: An analysis and critical review of studies in the field of IPD and (gut) microbiome were carried out. The resulting potential perspectives and therapeutic strategies are discussed. RESULTS: Despite partially divergent results between different studies (potentially due to the applied methods and variance in the composition of the investigated cohorts), there is an overlap between studies indicating an association between IPD, the microbiome and microbial metabolites. Nevertheless, the cause-effect relationship between IPD and the microbiome has still not been clarified. Taken together, existing evidence supports a potentially relevant role for the microbiome with respect to typical disease symptoms and pathogenesis of the disease. CONCLUSION: Over the past 5 years there has been an enormous increase in the evidence with respect to the relevance of the microbiome for IPD. While early work in this field was mainly descriptive, new diagnostic methods provide evidence for the underlying mechanisms and the complex interactions between man as the host, the human immune system, the enteric nervous system, gut microbiota and microbial metabolites. A relatively novel and clinically relevant field of research is how the gut microbiome can influence the success of oral pharmacotherapy and whether substitution of specific microbiome components might be used either for future therapeutic or prophylactic strategies.

Simulation of in-vivo-equivalent epithelial barriers using a micro fluidic device.

In biomedical approaches cell culture models do often not fully represent their biological counterparts. Often the methods used do not completely mimic the in-vivo situation, either by using only single-cell-type culture approaches, or by using inadequate culture conditions. We therefore developed a variable system based on individual modules to simulate in vitro equivalent cell-barriers (e.g. for mucous layers). This system allows the growth of different communicating cell types in micro channels. Hot embossing is used to fabricate the micro structured polymer sheets. The stamp for hot embossing is fabricated by UV-lithography/electroforming or by micro milling. The system consists of a container with micro fluidic modules and a pump-system for a continuous medium-supply. An individual module is made of two micro-structured polycarbonate-sheets separated by a transmissible polycarbonate membrane. The two sheets are arranged orthogonally to induce a cross flow. The system is highly variable by channel-geometry (height and width), capacity (number of micro fluidic modules), and pore sizes of the transmissible membranes. In a first approach we simulated the intestinal mucosa. Epithelial cells and primary neurons of the enteric nervous system were cultured on both sides of the transmissible membrane within the two different compartments. So the cells could be supplied with two different media. We kept a mono-culture of primary neurons or epithelial cells for 5 days and a co-culture between these two cell-types was established for 4 days. The proposed system delivers a sophisticated model for the simulation of various epithelial layers which takes the specific biological properties into account.

Gastrointestinal involvement in Parkinson's disease: pathophysiology, diagnosis, and management.

Growing evidence suggests an increasing significance for the extent of gastrointestinal tract (GIT) dysfunction in Parkinson's disease (PD). Most patients suffer from GIT symptoms, including dysphagia, sialorrhea, bloating, nausea, vomiting, gastroparesis, and constipation during the disease course. The underlying pathomechanisms of this α-synucleinopathy play an important role in disease development and progression, i.e., early accumulation of Lewy pathology in the enteric and central nervous systems is implicated in pharyngeal discoordination, esophageal and gastric motility/peristalsis impairment, chronic pain, altered intestinal permeability and autonomic dysfunction of the colon, with subsequent constipation. Severe complications, including malnutrition, dehydration, insufficient drug effects, aspiration pneumonia, intestinal obstruction, and megacolon, frequently result in hospitalization. Sophisticated diagnostic tools are now available that permit more detailed examination of specific GIT impairment patterns. Furthermore, novel treatment approaches have been evaluated, although high-level evidence trials are often missing. Finally, the burgeoning literature devoted to the GIT microbiome reveals its importance for neurologists. We review current knowledge about GIT pathoanatomy, pathophysiology, diagnosis, and treatment in PD and provide recommendations for management in daily practice.

Synaptophysin is an autoantigen in paraneoplastic neuropathy.

Paraneoplastic neurological syndromes (PNS) are often associated with antineuronal autoantibodies and many of them could be identified in the recent years. However, there are still new antineuronal binding patterns with yet unidentified autoantigens. We here describe a new autoantibody associated with paraneoplastic sensorimotor and autonomic neuropathy in a patient with small cell lung cancer. In indirect immunofluorescence test, the patient's serum colocalised with the synaptic protein synaptophysin in the cerebellum and myenteric plexus of the gut. Immunoblotting showed a 38 kDa reactivity, which is also the molecular weight of synaptophysin. Therefore a Western Blot with recombinant synaptophysin has been used and revealed reactivity of the serum against synaptophysin. In patients with non-paraneoplastic neuropathies or healthy controls, anti-synaptophysin autoantibodies were not detectable. In 20 SCLC patients without neurological syndromes, two patients had low-titer anti-synaptophysin autoantibodies. The patient's serum and IgG fraction showed cytotoxicity to primary cultured myenteric plexus neurons. We conclude that synaptophysin is an autoantigen in paraneoplastic neurological syndromes.

Nerve growth factor secretion in cultured enteric glia cells is modulated by proinflammatory cytokines.

The enteric nervous system is composed of neurones and glial cells. These enteric glia cells (EGC) appear to be essential for the maintenance of gut homeostasis and mucosal integrity. Neurotrophin nerve growth factor (NGF) also plays an important role for the gut integrity by regulating sensory and inflammatory processes in the intestines. Here, we demonstrate EGCs as one source of NGF and show increased levels of NGF mRNA/protein and tropomyosin receptor kinase A (TrkA) mRNA in cultured EGCs upon stimulation with proinflammatory cytokines and lipopolysaccharides. NGF is continuously secreted from cultured EGCs and proinflammatory cytokines and lipopolysaccharides stimulate the secretion of this neurotrophin in a time- and dose- dependent manner, whereas interleukin-4 had no effect on NGF expression. Furthermore, NGF secretion was sustained for more than 12 h after withdrawal of the proinflammatory cytokines, suggesting the involvement of transcriptional and/or translational processes. Thus, the release of proinflammatory cytokines can increase NGF secretion by EGCs and leads to a higher expression of TrkA in EGCs. NGF, in turn, can increase visceral sensitivity and, on the other hand, appears to improve gut inflammation. Therefore, NGF secreting EGCs may play a key role in modulating visceral sensitivity and might be involved in inflammatory processes of the gut.

Neurodegeneration through oxidative stress: monitoring hydrogen peroxide induced apoptosis in primary cells from the subventricular zone of BALB/c mice using field-effect transistors.

Dementia is one of the big medical challenges of our time with Alzheimer's, Huntington's and Parkinson's disease among its most common forms. In year 2000, 4.5 million people were diagnosed with Alzheimer's disease in the United States. In the case of Alzheimer's disease one of many contributing factors is a metabolic imbalance that leads to elevated oxidative stress levels. Consequences of this imbalance can be symptoms like apraxia, agnosia or sundowning. The use of field-effect transistors is a novel approach to study the effects of external stimuli on cells in vitro to provide researchers with a new tool for high resolution and high throughput studies to better understand cellular interaction and the effects of pharmacological compounds. In our study we use ion-sensitive field-effect transistors (FETs) to analyze the apoptosis inducing effects of hydrogen peroxide treatment on primary cells obtained from the subventricular zone of postnatal BALB/c mice. Upon apoptosis, the cell-substrate adhesion of the neurons is gradually weakened until complete detachment. In former studies we used our FET devices to conduct Electrical Cell-substrate Impedance Sensing (ECIS) experiments on the single cell level using morphologically different cell lines. Here we demonstrate that our novel approach of ECIS using FET devices can be expanded to primary neuronal tissue with high prospects for further studies in the field of pharmacological research.

The IL-6/sIL-6R fusion protein hyper-IL-6 promotes neurite outgrowth and neuron survival in cultured enteric neurons.

The undisturbed development of the enteric nervous system depends on the supply of various neurotrophic factors during ontogenesis. Besides glial cell line-derived neurotrophic factor (GDNF), leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) take part in its development. CNTF and LIF belong to the interleukin-6 (IL-6) family of cytokines. The combination of IL-6 and the soluble IL-6 receptor accelerates peripheral nerve regeneration. In this study, we examined the effect of the fusion protein Hyper-IL-6, which consists of IL-6 and the soluble receptor sIL-6R, on neurite outgrowth and neuronal survival in vitro. Myenteric plexus of newborn rats was dissected and dissociated. Cells were grown in either serum-free chemically defined medium alone or medium supplemented with sIL-6R, IL-6, sIL-6+IL-6, Hyper-IL-6, CNTF, LIF, or GDNF. Average neurite outgrowth per neuron was highest in GDNF-treated and Hyper-IL-6-treated cultures. The number of neurite-bearing neurons was reduced in GDNF cultures compared with Hyper-IL-6-treated cells, so that the total neurite outgrowth was maximal after Hyper-IL-6 stimulation. Hyper-IL-6 furthermore stimulated neuronal survival and morphologic differentiation of the enteric glia.

Trophic actions of 2-chloroadenosine and bFGF on cultured myenteric neurones.

The effects of the stable adenosine analogue, 2-chloroadenosine (2-CA) and basic fibroblast growth factor (bFGF) on myenteric neurones in dissociated cell culture were examined. 2-CA had no effect on neuronal numbers, but increased neurite length, in a dose-dependent manner. bFGF increased both the number of myenteric neurones and neurite length. When 2-CA was applied together with bFGF, an enhanced increase in neurite outgrowth, but no additional increase in neuronal numbers was observed. 2-CA-induced effects were blocked by the adenosine antagonist 8-(p-sulphophenyl)theophylline. These results show, for the first time, that both purines and bFGF may have trophic actions on myenteric neurones and also indicate that purines enhance some effects of bFGF, in a synergistic manner.

Chronic electroconvulsive shock treatment elicits up-regulation of CRF and AVP mRNA in select populations of neuroendocrine neurons.

The effects of repeated electroconvulsive seizures (ECS) on expression of mRNAs coding for corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) in neuroendocrine neurons of the hypothalamo-pituitary-adrenocortical (HPA) axis and hypothalamo-neurohypophysial system (HNS) were assessed via semi-quantitative in situ hybridization histochemical analysis. Measures of mRNA content were accompanied by measurement of peptide- and hormone-expression in the relevant neuroendocrine systems. Following 7 daily ECS treatments, CRF mRNA was significantly increased in the medial parvocellular paraventricular nucleus (PVN) of treated rats relative to controls. CRF peptide content of whole PVN homogenates was decreased to 50% of control levels. Changes in CRF message and peptide levels were accompanied by increases in pituitary ACTH content and by elevated plasma corticosterone, suggesting ECS elicits long-term up-regulation of the HPA axis. AVP mRNA in the medial parvocellular PVN, which is known to up-regulate in response to HPA challenge by adrenalectomy, was not increased by ECS. Chronic ECS causes a clear up-regulation of HNS neurons of the supraoptic nucleus, characterized by increased AVP mRNA content, decreased AVP peptide content, and depletion of neurohypophysial AVP. However, no changes were observed in magnocellular vasopressinergic neurons of the PVN, indicating that magnocellular SON and PVN neurons respond differentially to stimulation by ECS. The data indicate that ECS is a potent stimulus for activation of select components of both the HPA axis and the HNS. As such, ECS provides a useful tool for examining mechanisms underlying neuroendocrine processes.

Protein extracts from the gut wall influence the postnatal development of rat myenteric neurons in vitro.

Neurons migrating from the neural crest into the presumptive gut wall need an appropriate microenvironment to survive and differentiate during ontogenesis. The rat enteric nervous system (ENS) keeps developing beyond birth. The aim of this study was to investigate the neurotrophic effects of the postnatal microenvironment. Myenteric plexus was isolated from the smooth muscle layer of newborn rats at different ages (postnatal day (p)1, p7, p14) and grown as dissociated cell cultures. The culture medium (hormone-supplemented, serum-free) was supplemented either with glial-cell-line-derived-neurotrophic factor (GDNF) or with protein extracts from homogenized smooth muscle layer of p7 rats. Cultures kept in defined medium alone were used as controls. After 18 h in vitro both GDNF and protein extract had a neuritogenic effect upon p1 and p7 neurons, while p14 neurons were only stimulated by the protein extracts. The GDNF effect upon these neurons did not differ significantly from the effects in defined medium alone. The average neurite outgrowth in extract-supplemented cultures was always longer than that seen in those treated with GDNF. Although GDNF influences the postnatal development of myentric neurons in vitro it could not be detected immunohistochemically either in Western blots of the protein extracts or in cryostat sections of the gut.

A new method for the isolation of myenteric plexus from the newborn rat gastrointestinal tract.

The myenteric plexus is not only essential for gastrointestinal functions, but it is also a very interesting model for the study of neuronal circuits and neuron-glial interrelationships and may be a valuable source of donor tissue, for grafting into different regions of the central nervous system. For both grafting and culture procedures it is a great advantage to obtain the maximum amount of tissue. To date, most studies have isolated the myenteric plexus by manual microdissection after collagenase digestion. Using this method, it has only been possible to obtain relatively small amounts of the myenteric plexus, mostly from the cecum and proximal colon of the guinea-pig or rat. We present here a new method, which enables much greater quantities of the plexus from the small intestine and colon to be obtained. The myenteric plexus of the entire small intestine can be isolated by a combination of enzymatic digestion and mechanical agitation. The method works from birth up to 3 week old pups, and with some modifications tissue from older or even adult animals can also be processed. Another advantage over the microdissection method is that the myenteric plexuses of the different parts of the intestine can be cultured and studied separately.

Improved osmication, dehydration and flat embedding of vibratome sections and delicate whole mount preparations.

A simple method for flat embedding vibratome sections or delicate whole mount preparations in epoxy resin is described that allows thin layers of interest within the specimen to be cut and studied as a whole in one plane. The fatal curling that occurs during dehydration was avoided or at least minimized by dehydrating the specimens in a layer of filter paper and a plastic meshwork under permanent slight pressure. The flatness of the structures was maintained through a graded series of ethanols followed by acetone. The specimens were transferred to the resin and polymerized between two coated glass slides making it possible to view and select the material without direct handling of the thin resin layer.

The extracellular matrix and its role in cell migration and development of the enteric nervous system.

The extracellular matrix (ECM), a network consisting of many different macromolecules, fulfils many important functions in every multicellular organism, especially during their development. Among other factors, ECM molecules are necessary for cell migration and also regulate cell differentiation, as could be shown in a wide range of animals. The enteric nervous system (ENS) is built up by neural crest cells (NCC) migrating along predetermined pathways into the developing gut. Studies done for example in mice and chickens did not only enable scientists to reconstruct these routes but also to demonstrate their dependence on ECM molecules such as laminin. Currently we are investigating the influence of different ECM constituents, growth factors and noxious factors on NCC migration and differentiation in the developing chicken gut. The easy handling of the chicken embryo and the use of different methods will give us valuable insights for further investigations.

What do knockout models teach us about the enteric nervous system?

Since the first histological studies, enormous strides have been made in understanding the genetics and cell biology of enteric nervous system (ENS) formation. Several mitogenic and trophic factors have been implicated in the process of neural cell proliferation and differentiation. A number of natural (piebald-lethal mice [s l], lethal spotting mice [ls], spotting lethal rats [sl]) or target (Gfralpha1-deficient mice, ret.k - mice, and NT-4 knockout mice) mutations have been reported to produce developmental defects in neural crest cell migration, differentiation or survival. Study of these mutations continues to provide new insights into this complex system. In the present investigation, we showed that a lack of basic fibroblast growth factor (FGF) or growth hormone (GH) leads to morphological abnormalities of the enteric nervous system. Because knockouts, neither of FGF nor of GH, produce enteric nervous system defects substantial enough to compromise the ability of the gut to support life, we postulate that FGF and GH affect only a relatively small subset of neurons and/or that compensatory effects of other growth factors might occur.

Mouse-isolated plexus differentiates neural crest precursors into enteric neuroblasts.

Aim of this study was to investigate, for the first time, whether isolated newborn mouse enteric plexus could induce in vitro differentiation of the vagal neural crest-derived cells into enteric neuroblasts. Fragments of the myenteric plexus were isolated from the small intestine of 6-day-old Swiss mice and were collected and stored in DMEM-F12 medium, then cultured on polymerized human fibronectin layer. The vagal portion of the neural tube, isolated from a 9.5-day-old Swiss mouse embryo, was put in the same chamber slides where the isolated myenteric plexus had been cultured for 3 days. The vagal neural crest-derived cells migrated onto the polymerized human fibronectin layer and formed a crown of cells around the neural tube. After 6 days, the cultures were stopped and studied immunohistochemically for anti-NF160 KD, anti-TH, and RetR5 antibodies to analyse the differentiation stage of the cultured cells. Analysis of results included the comparison of two culture groups: Group 1, used as control, in which vagal neural crest-derived cells were put in DMEM-F12, supplemented only with 10 % of FCS; Group 2, in which vagal neural crest-derived cells were put in the same medium as Group 1, with the addition of myenteric plexus fragments isolated from newborn mice to form the co-culture. The following results were obtained: in Group 1 the neural tubes originated a cell population strongly positive for anti-NF160 and anti-TH Ab, but negative for RetR5 Ab. This positivity was found both in the cells adjacent to the neural tube and in those migrating from it distally. The Group 2 originated cells, which after migration were positive for anti-NF160 and for anti-TH antibodies. In addition, in this culture group, the cells which migrated from the neural tube were positive for anti-RetR5 antibody. The co-culture used in this study induces the differentiation of vagal stem cells into enteric neuroblasts, cells TH+ and RetR5+. These cells, after reaching the embryonic intestine, migrate to colonize the hindgut and form the ENS. Therefore this biotechnology seems a good method to obtain in vitro enteric precursors of ENS.

How to approach the ENS: various ways to analyse motility disorders in situ and in vitro.

Motility disorders of the human intestine are so variable that they cannot be diagnosed by just one technique. Their aetiology is obviously so varied that they have to be approached with a broad range of technical methods. These reach from the simple haematoxylin-stained section to the isolation of stem or precursor cells. In this study, various methods to investigate the enteric nervous system and its surrounding tissue are demonstrated. While sections from paraffin-embedded material or cryostat sections provide only a two-dimensional perspective of the ENS, the whole-mount method yields three-dimensional perspectives of large areas of the gut wall. The three-dimensional impression can even be enhanced by electron microscopy of the isolated ENS. Dynamical aspects of ENS development can be tackled by in vitro studies. The myenteric plexus can be isolated and cultivated under the influence of the microenvironment (protein extracts). Although the postnatal myenteric plexus is not fully developed, the choice of embryological neuronal cells seems to be more effective for certain approaches. They can be isolated from the embryonic mouse gut and cultivated under the influence of various factors. This method seems to us a valuable tool for the investigation of the aetiology of motility disorders, although only a "complete" approach which considers all available methods will yield at the end a clear understanding which might lead to new therapeutical concepts.

Cell specific ultrasound effects are dose and frequency dependent.

Ultrasound is widely used in clinical practice, mostly in diagnostic studies, but increasingly in therapeutic applications as well. This may be the case in acceleration of wound healing or treatment of cancer. Still, little is known about the direct effect of frequency or energy density of the ultrasound upon the cells themselves. We therefore investigated the impact of three different protocols using high, medium and low energy densities at three different frequencies on normal endothelial and epithelial as well as carcinoma cell lines (neuroblastoma and adenocarcinoma cell lines). Proliferation of endothelial and epithelial cell lines was significantly increased depending on the frequency and energy density applied. No influence on actin cytoskeleton formation was seen in these cells after treatment, while a significant decrease in the density of microvilli and the length of filopodia in the epithelial cell line could be noted. The proliferation rate of the carcinoma cell lines was reduced and cells destroyed. Apoptosis was induced in the adenocarcinoma cells after ultrasound exposure. Additionally, the expression of neurofilament was increased in neuroblastoma cells as evidence of beginning differentiation. So, different settings of frequency and energy density in an ultrasonic treatment protocol lead to different impacts on proliferation, morphology and differentiation and might be used to stimulate or inhibit the growth of individual cell types.

Expression and function of the Transforming Growth Factor-b system in the human and rat enteric nervous system.

BACKGROUND: Transforming growth factor-betas (TGF-bs) are pleiotropic growth factors exerting neurotrophic functions upon various neuronal populations of the central nervous system. In contrast, the role of TGF-b isoforms in the enteric nervous system (ENS) is largely unknown. We therefore analyzed the gene expression pattern of the TGF-b system in the human colon and in rat myenteric plexus, and smooth muscle cell cultures and determined the effect of TGF-b isoforms on neuronal differentiation. METHODS: Human colonic samples as well as cultured rat myenteric plexus, and smooth muscle cells were assessed for mRNA expression levels of the TGF-b system (TGF-b1-3, TbR-1-3) by qPCR. The colonic wall was separated into mucosa and tunica muscularis and enteric ganglia were isolated by laser microdissection (LMD) to allow site-specific gene expression analysis. Effects of TGF-b isoforms on neurite outgrowth and branching pattern of cultured myenteric neurons were monitored. KEY RESULTS: mRNA expression of the TGF-b system was detected in all compartments of the human colonic wall as well as in LMD-isolated myenteric ganglia. Cultured myenteric neurons and smooth muscle cells of rat intestine also showed mRNA expression of all ligands and receptors. Transforming growth factor-b2 treatment increased neurite length and branching pattern in cultured myenteric neurons. CONCLUSIONS & INFERENCES: The TGF-b system is abundantly expressed in the human and rat ENS arguing for an auto-/paracrine function of this system on enteric neurons. Transforming growth factor-b2 promotes neuronal differentiation and plasticity characterizing this molecule as a relevant neurotrophic factor for the ENS.

Enteric neurons from postnatal Fgf2 knockout mice differ in neurite outgrowth responses.

The enteric nervous system (ENS) consists of several neuronal subclasses with distinct functional properties. The formation and maintenance of these distinct populations during development and aging is dependent on the support of appropriate neurotrophic factors. During early postnatal development, the ENS has to adept continuously to changing alimentation situations, which might also affect neuronal maturation and differentiation. There is evidence that basic-fibroblast-growth-factor (Fgf2) exerts neurotrophic effects in the ENS. In this study primary myenteric plexus cultures from both wild type and Fgf2-knockout mice were investigated under the influence of Fgf2 and glial-cell-line-derived-factor (GDNF). It could be demonstrated, that the influence of neurotrophic support is decreased in the Fgf2-knockouts, while the neuronal cultures of wild type revealed a more pronounced receptiveness for trophic support. These data show that Fgf2 plays a role in the development of the ENS.

The microenvironment in chronic pancreatitis and pancreatic cancer induces neuronal plasticity.

BACKGROUND: Pancreatic neuropathy in chronic pancreatitis (CP) and pancreatic cancer (PCa) is characterized by pancreatic neuropathy, i.e. increased neural density and hypertrophy, which are associated with neuropathic pain. To better understand the mechanism of these neuropathic alterations, we aimed at achieving an in-vitro simulation of the intrapancreatic neuroplasticity. METHODS: Dissociated myenteric plexus (MP) and dorsal root ganglia (DRG) neurons of newborn rats were treated with normal human pancreas (NP), CP or PCa tissue extracts. Furthermore, MP and DRG neurons were cultured in supernatants from different pancreatic cancer cell lines (PCC) and human pancreatic stellate cells (hPSC) obtained from either CP or PCa tissues. For analysis, the neurite density, outgrowth, neuronal branching capacity and perikaryonal size were quantified. KEY RESULTS: Myenteric plexus and DRG neurons grown in CP and PCa tissue extracts built denser networks than in NP extracts. Both neuronal types showed a strong neurite outgrowth, more complex branching pattern and a somatic hypertrophy in CP and PCa extracts. Pancreatic cancer cell supernatants induced a prominent neurite outgrowth, increased neurite density and perikaryonal hypertrophy in MP and DRG neurons. Supernatants of CP-derived hPSC strongly stimulated neurite outgrowth. Glial density in MP cultures was strikingly increased by PCa tissue extracts. CONCLUSIONS & INFERENCES: Intrapancreatic microenvironment in CP and PCa induces neuroplastic alterations under in-vitro conditions, leading to increased neural density and hypertrophy. Thus, due to its neurotrophic attributes, the intrapancreatic microenviroment in CP and PCa seems to be a key player in the generation of pancreatic neuropathy and neuroplasticity.