Blood-CNS barriers in neurodegenerative diseases

The blood-brain barrier (BBB) and the blood-spinal cord barrier (BSCB) separate the brain and the spinal cord from the circulating blood and are important for the maintenance of the CNS homeostasis. They build a physical barrier thereby protecting the CNS from pathogens and toxic agents, and their disruption plays a crucial role in the pathogenesis of several CNS disorders. In this thesis, the blood-CNS-barriers were studied via in vitro models in two case studies for neurodegenerative disorders, in particular Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS). The first model evaluates treatment possibilities of AD using nanotechnology-based strategies. Since the toxic amyloid-β42 (Aβ42) peptide plays a crucial role in the pathogenesis of AD, reduced generation or enhanced clearance of Aβ42 peptides are expected to modify the disease course in AD. Therefore, several Aβ42-lowering drugs like flurbiprofen had been tested in clinical trials, but most of them failed due to their low brain penetration. Here, flurbiprofen was embedded in polylactide (PLA) nanoparticles and its transport was examined in an in vitro BBB model. The embedding of flurbiprofen into the nanoparticles disguised its cytotoxic potential and enabled the administration of higher drug concentrations which resulted in a sufficient transport of the drug across an endothelial cell monolayer. These results demonstrate that non-permeable drugs can be transported efficiently via nanoparticles and that these nanotechnology-based strategies are a promising tool to generate novel therapeutic options for AD and other CNS diseases. rnThe focus of the second project was to investigate the impaired integrity of the BSCB in a mouse model for ALS. About 20% of all familial ALS cases are associated with missense mutations or small deletions in the gene that encodes Cu/Zn-superoxide dismutase 1 (SOD1). To date, the molecular mechanisms resulting in ALS are still unknown, but there is evidence that the disruption of the BSCB is one of the primary pathological events. In both familial and sporadic ALS patients, loss of endothelial integrity and endothelial cell damage was observed, and studies with SOD1 transgenic mice demonstrated that the BSCB disruption was found prior to motor neuron degeneration and neurovascular inflammation. Thus, an in vitro model for ALS endothelial cells was generated which exhibited comparable integrity characteristics and tight junction (TJ) protein expression profiles as isolated primary endothelial cells of the BSCB of SOD1 transgenic mice. In this, an alteration of the βcat/AKT/FoxO1 pathway, which regulates the expression of the TJ protein claudin-5, could be observed. These data furthermore indicate that ALS is a neurovascular disease, and understanding of the primary events in ALS pathogenesis will hopefully provide ideas for the development of new therapeutic strategies. rn

Control over microstructure evolution and device performance in solution processable organic field-effect transistors

Die Arbeit beschäftigt sich mit der Kontrolle von Selbstorganisation und Mikrostruktur von organischen Halbleitern und deren Einsatz in OFETs. In Kapiteln 3, 4 und 5 eine neue Lösungsmittel-basierte Verabeitungsmethode, genannt als Lösungsmitteldampfdiffusion, ist konzipiert, um die Selbstorganisation von Halbleitermolekülen auf der Oberfläche zu steuern. Diese Methode als wirkungsvolles Werkzeug erlaubt eine genaue Kontrolle über die Mikrostruktur, wie in Kapitel 3 am Beispiel einer D-A Dyad bestehend aus Hexa-peri-hexabenzocoronene (HBC) als Donor und Perylene Diimide (PDI) als Akzeptor beweisen. Die Kombination aus Oberflächenmodifikation und Lösungsmitteldampf kann die Entnetzungseffekte ausgleichen, so dass die gewüschte Mikrostruktur und molekulare Organisation auf der Oberfläche erreicht werden kann. In Kapiteln 4 und 5 wurde diese Methode eingesetzt, um die Selbstorganisation von Dithieno[2, 3-d;2’, 3’-d’] benzo[1,2-b;4,5-b’]dithiophene (DTBDT) und Cyclopentadithiophene -benzothiadiazole copolymer (CDT-BTZ) Copolymer zu steuern. Die Ergebnisse könnten weitere Studien stimulieren und werfen Licht aus andere leistungsfaähige konjugierte Polymere. rnIn Kapiteln 6 und 7 Monolagen und deren anschlieβende Mikrostruktur von zwei konjugierten Polymeren, Poly (2,5-bis(3-alkylthiophen-2-yl)thieno[3,2-b]thiophene) PBTTT und Poly{[N,N ′-bis(2-octyldodecyl)-naphthalene-1,4,5,8-bis (dicarboximide)-2,6-diyl]-alt-5,5′- (2,2′-bithiophene)}, P(NDI2OD-T2)) wurden auf steife Oberflächen mittels Tauchbeschichtung aufgebracht. Da sist das erste Mal, dass es gelungen ist, Polymer Monolagen aus der Lösung aufzubringen. Dieser Ansatz kann weiter auf eine breite Reihe von anderen konjugierten Polymeren ausgeweitet werden.rnIn Kapitel 8 wurden PDI-CN2 Filme erfolgreich von Monolagen zu Bi- und Tri-Schichten auf Oberflächen aufgebracht, die unterschiedliche Rauigkeiten besitzen. Für das erste Mal, wurde der Einfluss der Rauigkeit auf Lösungsmittel-verarbeitete dünne Schichten klar beschrieben.rn

Modelling, simulation and characterization of epithelial cell culture biochip

A microfluidic Organ-on-Chip has been developed for monitoring the epithelial cells monolayer. Equivalent circuit Model was used to determine the electrical properties from the impedance spectra of the epithelial cells monolayer. Black platinum on platinum electrodes was electrochemically deposited onto the surface of electrodes to reduce the influence of the electrical double layer on the impedance measurements. Measurements of impedance with an Impedance Analyzer were done to validate the equivalent circuit model and the decrease of the double layer effect. A Lock-in Amplifier was designed to measure the impedance.

pH-dependent distribution of chlorin e6 derivatives across phospholipid bilayers probed by NMR spectroscopy

The pH-dependent membrane adsorption and distribution of three chlorin derivatives, chlorin e6 (CE), rhodin G7 (RG), and monoaspartyl-chlorin e6 (MACE), in the physiological pH range (pH 6-8) were probed by NMR spectroscopy. Unilamellar vesicles consisting of dioleoyl-phosphatidyl-choline (DOPC) were used as membrane models. The chlorin derivatives were characterized with respect to their aggregation behavior, the pK(a) values of individual carboxylate groups, the extent of membrane adsorption, and their flip-flop rates across the bilayer membrane for pH 6-8. External membrane adsorption was found to be lower for RG than for CE and MACE. Both electrostatic interactions and the extent of aggregation seemed to be the main determinants of membrane adsorption. Rate constants for chlorin transfer across the membrane were found to correlate strongly with the pH of the surrounding medium, in particular, for CE and RG. In acidic solution, CE and RG transfer across the membrane was strongly accelerated, and in basic solution, all compounds were retained, mostly in the outer monolayer. In contrast, MACE flip-flop across the membrane remained very low even at pH 6. The protonation of ionizable groups is suggested to be a major determinant of chlorin transfer rates across the bilayer. pK(a) values of CE and RG were found to be between 6 and 8, and two of the carboxylate groups in MACE had pK(a) values below 6. For CE and RG, the kinetic profiles at acidic pH indicated that the initial fast membrane distribution was followed by secondary steps that are discussed in this article.

Population doublings and percentage of S100-positive cells as predictors of in vitro chondrogenicity of expanded human articular chondrocytes

The aim of this study was to investigate the interconnection between the processes of proliferation, dedifferentiation, and intrinsic redifferentiation (chondrogenic) capacities of human articular chondrocyte (HAC), and to identify markers linking HAC dedifferentiation status with their chondrogenic potential. Cumulative population doublings (PD) of HAC expanded in monolayer culture were determined, and a threshold range of 3.57-4.19 PD was identified as indicative of HAC loss of intrinsic chondrogenic capacity in pellets incubated without added chondrogenic factors. While several specific gene and surface markers defined early HAC dedifferentiation process, no clear correlation with the loss of intrinsic chondrogenic potential could be established. CD90 expression during HAC monolayer culture revealed two subpopulations, with sorted CD90-negative cells showing lower proliferative capacity and higher chondrogenic potential compared to CD90-positive cells. Although these data further validated PD as critical for in vitro chondrogenesis, due to the early shift in expression, CD90 could not be considered for predicting chondrogenic potential of HAC expanded for several weeks. In contrast, an excellent mathematically modeled correlation was established between PD and the decline of HAC expressing the intracellular marker S100, providing a direct link between the number of cell divisions and dedifferentiation/loss of intrinsic chondrogenic capacity. Based on the dynamics of S100-positive HAC during expansion, we propose asymmetric cell division as a potential mechanism of HAC dedifferentiation, and S100 as a marker to assess chondrogenicity of HAC during expansion, of potential value for cell-based cartilage repair treatments.

Structure-activity relationship and mechanism of action studies of manzamine analogues for the control of neuroinflammation and cerebral infections

Structure-activity relationship studies were carried out by chemical modification of manzamine A (1), 8-hydroxymanzamine A (2), manzamine F (14), and ircinal isolated from the sponge Acanthostrongylophora. The derived analogues were evaluated for antimalarial, antimicrobial, and antineuroinflammatory activities. Several modified products exhibited potent and improved in vitro antineuroinflammatory, antimicrobial, and antimalarial activity. 1 showed improved activity against malaria compared to chloroquine in both multi- and single-dose in vivo experiments. The significant antimalarial potential was revealed by a 100% cure rate of malaria in mice with one administration of 100 mg/kg of 1. The potent antineuroinflammatory activity of the manzamines will provide great benefit for the prevention and treatment of cerebral infections (e.g., Cryptococcus and Plasmodium). In addition, 1 was shown to permeate across the blood-brain barrier (BBB) in an in vitro model using a MDR-MDCK monolayer. Docking studies support that 2 binds to the ATP-noncompetitive pocket of glycogen synthesis kinase-3beta (GSK-3beta), which is a putative target of manzamines. On the basis of the results presented here, it will be possible to initiate rational drug design efforts around this natural product scaffold for the treatment of several different diseases.

Microfluidic wound-healing assay to assess the regenerative effect of HGF on wounded alveolar epithelium

We present a microfluidic epithelial wound-healing assay that allows characterization of the effect of hepatocyte growth factor (HGF) on the regeneration of alveolar epithelium using a flow-focusing technique to create a regular wound in the epithelial monolayer. The phenotype of the epithelial cell was characterized using immunostaining for tight junction (TJ) proteins and transmission electron micrographs (TEMs) of cells cultured in the microfluidic system, a technique that is reported here for the first time. We demonstrate that alveolar epithelial cells cultured in a microfluidic environment preserve their phenotype before and after wounding. In addition, we report a wound-healing benefit induced by addition of HGF to the cell culture medium (19.2 vs. 13.5 μm h(-1) healing rate).

Adsorption behavior of redox-active suppressor additives: Combined electrochemical and STM studies

The redox chemistry and the related surface phase behavior of Safranine (SAF) and Janus Green B (JGB) have been studied by means of cyclic voltammetry in combination with in situ Scanning Tunneling Microscopy using HOPG (Highly Oriented Pyrolytic Graphite) and single crystalline Cu(1 0 0) as model substrates, both revealing different widths of the accessible potential windows. JGB and SAF serve as prototypical heterocyclic suppressor/leveler additives that are used for the metallization of 3D-TSVs (3D Through Silicon Vias) following a classical "leveling" concept. SAF can be considered as the reductive decomposition product of JGB that is formed at the copper/electrolyte interface upon electroplating. Both additives reveal a pronounced pH-dependent redox-chemistry with redox-transitions lying close to or even beyond the anodic limit of the copper potential window. Affected by these redox-processes are in particular the aromatic cores of those heterocycles that can be (quasi)reversibly reduced by a two electron transfer process within the potential window of copper. Therefore we identify the reduced form of those dyes as the active components for the suppressing/leveling effect in copper plating. STM data clearly shows a dye surface phase behavior that is crucially determined by its potential-dependent redox-chemistry. This will be exemplarily discussed for the SAF dye. On chloride-modified Cu(1 0 0) mono-reduced SAF forms a structurally well-defined monolayer of cationic stacking polymers. However, this coupled anion/cation layer reveals only minor suppressing capabilities with respect to the copper dissolution and deposition processes. Complete reduction of the aromatic heterocycle finally leads to the 3D precipitation of hydrophobic reaction products. 3D clusters of this SAF precipitate are discussed as the active structural motif for the suppressing effect of these dyes. (C) 2011 Elsevier Ltd. All rights reserved.

Rapid cytopathic effects of Clostridium perfringens beta-toxin on porcine endothelial cells

Clostridium perfringens type C isolates cause fatal, segmental necro-hemorrhagic enteritis in animals and humans. Typically, acute intestinal lesions result from extensive mucosal necrosis and hemorrhage in the proximal jejunum. These lesions are frequently accompanied by microvascular thrombosis in affected intestinal segments. In previous studies we demonstrated that there is endothelial localization of C. perfringens type C beta-toxin (CPB) in acute lesions of necrotizing enteritis. This led us to hypothesize that CPB contributes to vascular necrosis by directly damaging endothelial cells. By performing additional immunohistochemical studies using spontaneously diseased piglets, we confirmed that CPB binds to the endothelial lining of vessels showing early signs of thrombosis. To investigate whether CPB can disrupt the endothelium, we exposed primary porcine aortic endothelial cells to C. perfringens type C culture supernatants and recombinant CPB. Both treatments rapidly induced disruption of the actin cytoskeleton, cell border retraction, and cell shrinkage, leading to destruction of the endothelial monolayer in vitro. These effects were followed by cell death. Cytopathic and cytotoxic effects were inhibited by neutralization of CPB. Taken together, our results suggest that CPB-induced disruption of endothelial cells may contribute to the pathogenesis of C. perfringens type C enteritis.

Continuous Formation and Separation of Calcium-Alginate Capsules Containing Viable Mammalian Cells in Microfluidic Devices

Microfluidic devices can be used for many applications, including the formation of well-controlled emulsions. In this study, the capability to continuously create monodisperse droplets in a microfluidic device was used to form calcium-alginate capsules.Calcium-alginate capsules have many potential uses, such as immunoisolation of cells and microencapsulation of active drug ingredients or bitter agents in food or beverage products. The gelation of calcium-alginate capsules is achieved by crosslinking sodiumalginate with calcium ions. Calcium ions dissociated from calcium carbonate due to diffusion of acetic acid from a sunflower oil phase into an aqueous droplet containing sodium-alginate and calcium carbonate. After gelation, the capsules were separated from the continuous oil phase into an aqueous solution for use in biological applications. Typically, capsules are separated bycentrifugation, which can damage both the capsules and the encapsulated material. A passive method achieves separation without exposing the encapsulated material or the capsules to large mechanical forces, thereby preventing damage. To achieve passiveseparation, the use of a microfluidic device with opposing channel wa hydrophobicity was used to stabilize co-laminar flow of im of hydrophobicity is accomplished by defining one length of the channel with a hydrogel. The chosen hydrogel was poly (ethylene glycol) diacrylate, which adheres to the glass surface through the use of self-assembled monolayer of 3-(trichlorosilyl)-propyl methacrylate. Due to the difference in surface energy within the channel, the aqueous stream is stabilized near a hydrogel and the oil stream is stabilized near the thiolene based optical adhesive defining the opposing length of the channel. Passive separation with co-laminar flow has shown success in continuously separating calcium-alginatecapsules from an oil phase into an aqueous phase. In addition to successful formation and separation of calcium alginate capsules,encapsulation of Latex micro-beads and viable mammalian cells has been achieved. The viability of encapsulated mammalian cells was determined using a live/dead stain. The co-laminar flow device has also been demonstrated as a means of separating liquid-liquidemulsions.

The neurovascular unit as a selective barrier to polymorphonuclear granulocyte (PMN) infiltration into the brain after ischemic injury

The migration of polymorphonuclear granulocytes (PMN) into the brain parenchyma and release of their abundant proteases are considered the main causes of neuronal cell death and reperfusion injury following ischemia. Yet, therapies targeting PMN egress have been largely ineffective. To address this discrepancy we investigated the temporo-spatial localization of PMNs early after transient ischemia in a murine transient middle cerebral artery occlusion (tMCAO) model and human stroke specimens. Using specific markers that distinguish PMN (Ly6G) from monocytes/macrophages (Ly6C) and that define the cellular and basement membrane boundaries of the neurovascular unit (NVU), histology and confocal microscopy revealed that virtually no PMNs entered the infarcted CNS parenchyma. Regardless of tMCAO duration, PMNs were mainly restricted to luminal surfaces or perivascular spaces of cerebral vessels. Vascular PMN accumulation showed no spatial correlation with increased vessel permeability, enhanced expression of endothelial cell adhesion molecules, platelet aggregation or release of neutrophil extracellular traps. Live cell imaging studies confirmed that oxygen and glucose deprivation followed by reoxygenation fail to induce PMN migration across a brain endothelial monolayer under flow conditions in vitro. The absence of PMN infiltration in infarcted brain tissues was corroborated in 25 human stroke specimens collected at early time points after infarction. Our observations identify the NVU rather than the brain parenchyma as the site of PMN action after CNS ischemia and suggest reappraisal of targets for therapies to reduce reperfusion injury after stroke.

An optimized in vitro model of the respiratory tract wall to study particle cell interactions

As a part of the respiratory tissue barrier, lung epithelial cells play an important role against the penetration of the body by inhaled particulate foreign materials. In most cell culture models, which are designed to study particle-cell interactions, the cells are immersed in medium. This does not reflect the physiological condition of lung epithelial cells which are exposed to air, separated from it only by a very thin liquid lining layer with a surfactant film at the air-liquid interface. In this study, A549 epithelial cells were grown on microporous membranes in a two chamber system. After the formation of a confluent monolayer the cells were exposed to air. The morphology of the cells and the expression of tight junction proteins were studied with confocal laser scanning and transmission electron microscopy. Air-exposed cells maintained monolayer structure for 2 days, expressed tight junctions and developed transepithelial electrical resistance. Surfactant was produced and released at the apical side of the air-exposed epithelial cells. In order to study particle-cell interactions fluorescent 1 microm polystyrene particles were sprayed over the epithelial surface. After 4 h, 8.8% of particles were found inside the epithelium. This fraction increased to 38% after 24 h. During all observations, particles were always found in the cells but never between them. In this study, we present an in vitro model of the respiratory tract wall consisting of air-exposed lung epithelial cells covered by a liquid lining layer with a surfactant film to study particle-cell interactions.

Equine peripheral blood-derived progenitors in comparison to bone marrow-derived mesenchymal stem cells

Fibroblast-like cells isolated from peripheral blood of human, canine, guinea pig, and rat have been demonstrated to possess the capacity to differentiate into several mesenchymal lineages. The aim of this work was to investigate the possibility of isolating pluripotent precursor cells from equine peripheral blood and compare them with equine bone marrow-derived mesenchymal stem cells. Human mesenchymal stem cells (MSCs) were used as a control for cell multipotency assessment. Venous blood (n = 33) and bone marrow (n = 5) were obtained from adult horses. Mononuclear cells were obtained by Ficoll gradient centrifugation and cultured in monolayer, and adherent fibroblast-like cells were tested for their differentiation potential. Chondrogenic differentiation was performed in serum-free medium in pellet cultures as a three-dimensional model, whereas osteogenic and adipogenic differentiation were induced in monolayer culture. Evidence for differentiation was made via biochemical, histological, and reverse transcription-polymerase chain reaction evaluations. Fibroblast-like cells were observed on day 10 in 12 out of 33 samples and were allowed to proliferate until confluence. Equine peripheral blood-derived cells had osteogenic and adipogenic differentiation capacities comparable to cells derived from bone marrow. Both cell types showed a limited capacity to produce lipid droplets compared to human MSCs. This result may be due to the assay conditions, which are established for human MSCs from bone marrow and may not be optimal for equine progenitor cells. Bone marrow-derived equine and human MSCs could be induced to develop cartilage, whereas equine peripheral blood progenitors did not show any capacity to produce cartilage at the histological level. In conclusion, equine peripheral blood-derived fibroblast-like cells can differentiate into distinct mesenchymal lineages but have less multipotency than bone marrow-derived MSCs under the conditions used in this study.

Cartilage tissue engineering by expanded goat articular chondrocytes

In this study we investigated whether expanded goat chondrocytes have the capacity to generate cartilaginous tissues with biochemical and biomechanical properties improving with time in culture. Goat chondrocytes were expanded in monolayer with or without combinations of FGF-2, TGF-beta1, and PDGFbb, and the postexpansion chondrogenic capacity assessed in pellet cultures. Expanded chondrocytes were also cultured for up to 6 weeks in HYAFF-M nonwoven meshes or Polyactive foams, and the resulting cartilaginous tissues were assessed histologically, biochemically, and biomechanically. Supplementation of the expansion medium with FGF-2 increased the proliferation rate of goat chondrocytes and enhanced their postexpansion chondrogenic capacity. FGF-2-expanded chondrocytes seeded in HYAFF-M or Polyactive scaffolds formed cartilaginous tissues with wet weight, glycosaminoglycan, and collagen content, increasing from 2 days to 6 weeks culture (up to respectively 2-, 8-, and 41-fold). Equilibrium and dynamic stiffness measured in HYAFF M-based constructs also increased with time, up to, respectively, 1.3- and 16-fold. This study demonstrates the feasibility to engineer goat cartilaginous tissues at different stages of development by varying culture time, and thus opens the possibility to test the effect of maturation stage of engineered cartilage on the outcome of cartilage repair in orthotopic goat models.

Expansion on specific substrates regulates the phenotype and differentiation capacity of human articular chondrocytes

In this study, we investigated if monolayer expansion of adult human articular chondrocytes (AHAC) on specific substrates regulates cell phenotype and post-expansion multilineage differentiation ability. AHAC isolated from cartilage biopsies of five donors were expanded on plastic dishes (PL), on dishes coated with collagen type II (COL), or on slides coated with a ceramic material (Osteologic, OS). The phenotype of expanded chondrocytes was assessed by flow cytometry and real-time RT-PCR. Cells were then cultured in previously established conditions promoting differentiation toward the chondrogenic or osteogenic lineage. AHAC differentiation was assessed histologically, biochemically, and by real-time RT-PCR. As compared to PL-expanded AHAC, those expanded on COL did not exhibit major phenotypic changes, whereas OS-expanded cells expressed (i) higher bone sialoprotein (BSP) (22.6-fold) and lower collagen type II (9.3-fold) mRNA levels, and (ii) lower CD26, CD90 and CD140 surface protein levels (1.4-11.1-fold). Following chondrogenic differentiation, COL-expanded AHAC expressed higher mRNA levels of collagen type II (2.3-fold) and formed tissues with higher glycosaminoglycan (GAG) contents (1.7-fold), whereas OS-expanded cells expressed 16.5-fold lower collagen type II and generated pellets with 2.0-fold lower GAG contents. Following osteogenic differentiation, OS-expanded cells expressed higher levels of BSP (3.9-fold) and collagen type I (2.8-fold) mRNA. In summary, AHAC expansion on COL or OS modulated the de-differentiated cell phenotype and improved the cell differentiation capacity respectively toward the chondrogenic or osteogenic lineage. Phenotypic changes induced by AHAC expansion on specific substrates may mimic pathophysiological events occurring at different stages of osteoarthritis and may be relevant for the engineering of osteochondral tissues.