culturing-and-electrophysiology-of-cells-on-nrcc-patch-clamp-chips

Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard ...

The Cl(-) channel blockers, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) or 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) dose-dependently protected against oxygen-glucose deprivation in cultured rat cortical neurons. DIDS or SITS attenuated oxygen-glucose deprivation-induced increases in extracellular glutamate concentrations and intracellular Ca(2+). DIDS or SITS provided moderate protection against N-methyl-D-aspartate (NMDA) toxicity and decreased NMDA receptor-mediated increases in intracellular Ca(2+). Whole-cell NMDA receptor currents were attenuated 39+/-2% and 21+/-3% by 1 mM DIDS and SITS, respectively. Other Cl(-) channel blockers as equipotent as DIDS and SITS did not decrease oxygen-glucose deprivation- or NMDA-mediated neuronal Ca(2+) influx or toxicity. Neurotoxicity by exogenous glutamate was not prevented by SITS and was exacerbated by DIDS. Reductions in oxygen-glucose deprivation-induced increases in intracellular Ca(2+) levels underlie neuroprotection by DIDS and SITS. This was a reflection of lower extracellular [glutamate], direct inhibition of Ca(2+) influx through postsynaptic NMDA receptors, and possibly through other protective properties associated with DIDS and SITS.

Protection of cortical neurons against oxygen-glucose deprivation and N-methyl-D-aspartate by DIDS and SITS.

Schutz der kortikalen Neuronen gegen Sauerstoff-Glukose-Entzug und N-Methyl-D-Aspartat durch DIDS und sitzt.

Schutz durch Cholesterin-extrahierende Cyclodextrine: eine Rolle f&#xFC;r N-Methyl-D-aspartat-Rezeptor Umverteilung.

Cyclodextrins (CDs) are cyclic oligosaccharides composed of a lipophilic central cavity and a hydrophilic outer surface. Some CDs are capable of extracting cholesterol from cell membranes and can affect function of receptors and proteins localized in cholesterol-rich membrane domains. In this report, we demonstrate the neuroprotective activity of some CD derivatives against oxygen-glucose deprivation (OGD), N-methyl-D-aspartic acid (NMDA) and glutamate in cortical neuronal cultures. Although all CDs complexed with NMDA or glutamate, only beta-, methylated beta- and sulfated beta-CDs displayed neuroprotective activity and lowered cellular cholesterol. Only CDs that lowered cholesterol levels redistributed the NMDA receptor NR2B subunit, PSD-95 (postsynaptic density protein 95 kDa) and neuronal nitric oxide synthase (nNOS) from Triton X-100 insoluble membrane domains to soluble fractions. Cholesterol repletion counteracted the ability of methylated beta-CD to protect against NMDA toxicity, and reversed NR2B, PSD-95 and nNOS localization to Triton X-100 insoluble membrane fraction. Surprisingly, neuroprotective CDs had minimal effect on NMDA receptor-mediated increases in intracellular Ca(2+) concentration ([Ca(2+)](i)), but did suppress OGD-induced increases in [Ca(2+)](i). beta-CD, but not Mbeta-CD, also caused a slight block of NMDA-induced currents, suggesting a minor contribution to neuroprotection by direct action on NMDA receptors. Taken together, data suggest that cholesterol extraction from detergent-resistant microdomains affects NMDA receptor subunit distribution and signal propagation, resulting in neuroprotection of cortical neuronal cultures against ischemic and excitotoxic insults. Since cholesterol-rich membrane domains exist in neuronal postsynaptic densities, these results imply that synaptic NMDA receptor subpopulations underlie excitotoxicity, which can be targeted by CDs without affecting overall neuronal Ca(2+) levels.

Protection by cholesterol-extracting cyclodextrins: a role for N-methyl-D-aspartate receptor redistribution.

This study challenges the conventional view that metalloporphyrins protect cultured cortical neurons in models of cerebral ischemia by acting as intracellular catalytic antioxidants [superoxide dismutase (SOD) mimetics]. High SOD-active Mn(III)porphyrins meso-substituted with N,N'-dimethylimidazolium or N-alkylpyridinium groups did not protect neurons against oxygen-glucose deprivation (OGD), although lower SOD-active and -inactive para isomers protected against N-methyl-D-aspartate (NMDA) exposure. Mn(III)meso-tetrakis(4-benzoic acid)porphyrin (Mn(III)TBAP), as well as SOD-inactive metalloTBAPs and other phenyl ring- or beta-substituted metalloporphyrins that contained redox-insensitive metals, protected cultures against OGD and NMDA neurotoxicity. Crucially, neuroprotective metalloporphyrins suppressed OGD- or NMDA-induced rises in intracellular Ca2+ concentration in the same general rank order as observed for neuroprotection. Results from paraquat toxicity, intracellular fluorescence quenching, electrophysiology, mitochondrial Ca2+, and spontaneous synaptic activity experiments suggest a model in which metalloporphyrins, acting at the plasma membrane, protect neurons against OGD by suppressing postsynaptic NMDA receptor-mediated Ca2+ rises, thereby indirectly preventing accumulation of neurotoxic mitochondrial Ca2+ levels. Though neuroprotective in a manner not originally intended, SOD-inactive metalloporphyrins may represent promising therapeutic agents in diseases such as cerebral ischemia, in which Ca2+ toxicity is implicated. Conventional syntheses aimed at improving the catalytic antioxidant capability and/or intracellular access of metalloporphyrins may not yield improved efficacy in some disease models.

An alternative Ca2+-dependent mechanism of neuroprotection by the metalloporphyrin class of superoxide dismutase mimetics.

Eine alternative Ca2 +-abh&#xE4;ngige Mechanismen der Neuroprotektion durch die Metalloporphyrin Klasse von Superoxiddismutase Mimetika.

Minocycline is a potent neuroprotective tetracycline in animal models of cerebral ischemia. We examined the protective properties of chlortetracycline (CTC) and demeclocycline (DMC) and showed that these two tetracyclines were also potent neuroprotective against glutamate-induced neuronal death in vitro and cerebral ischemia in vivo. However, CTC and DMC appeared to confer neuroprotection through a unique mechanism compared with minocycline. Rather than inhibiting microglial activation and caspase, CTC and DMC suppressed calpain activities. In addition, CTC and DMC only weakly antagonized N-methyl-D-aspartate (NMDA) receptor activities causing 16 and 14%, respectively, inhibition of NMDA-induced whole cell currents and partially blocked NMDA-induced Ca2+ influx, commonly regarded as the major trigger of neuronal death. In vitro and in vivo experiments demonstrated that the two compounds selectively inhibited the activities of calpain I and II activated following glutamate treatment and cerebral ischemia. In contrast, minocycline did not significantly inhibit calpain activity. Taken together, these results suggested that CTC and DMC provide neuroprotection through suppression of a rise in intracellular Ca2+ and inhibition of calpains.

Chlortetracycline and demeclocycline inhibit calpains and protect mouse neurons against glutamate toxicity and cerebral ischemia.

Chlortetracyclin und Demeclocyclin Calpainen hemmen und zu sch&#xFC;tzen Maus Neuronen gegen Glutamat-Toxizit&#xE4;t und zerebraler Isch&#xE4;mie.

Neural networks are formed by accurate connectivity of neurons and glial cells in the brain. These networks employ a three-dimensional bio-surface that both assigns precise coordinates to cells during development and facilitates their connectivity and functionality throughout life. Using specific topographic and chemical features, we have taken steps towards the development of poly(dimethylsiloxane; PDMS) neurochips that can be used to generate and study synthetic neural networks. These neurochips have micropatterned structures that permit adequate cell positioning and support cell survival. Within days of plating, cells differentiate into neurons displaying excitability and communication, as evidenced by intracellular calcium oscillations and action potentials. The structural and functional capacities of such simple neural networks open up new opportunities to study synaptic communication and plasticity.

Neurogenesis and neuronal communication on micropatterned neurochips.

Neurogenese und neuronale Kommunikation auf mikrostrukturierten Neurochips.

The goal of this study was to determine if novel porphyrins protect cultured cortical neurons from excitotoxic NMDA exposure or oxygen-glucose deprivation (OGD), which model key aspects of cerebral ischemia. Porphyrins were chosen based on conventional and unconventional criteria. Metalloporphyrin catalytic antioxidants possessing a redox-sensitive metal core can exhibit potent and wide-ranging catalytic antioxidant abilities, which are conventionally believed to underlie neuroprotection. We report here that a recent-generation potent peroxynitrite decomposition catalyst, FP-15, protected a majority of neurons against OGD and NMDA toxicity, without suppressing NMDA-mediated intracellular Ca2+ (Cai2+) elevations or whole-cell currents. We have previously shown that neuroprotection against OGD and NMDA toxicity correlated with an ability to suppress neurotoxic Cai2+ elevations and not antioxidant ability. We now evaluate if this unconventional mechanism extends to inert metal-free porphyrins. Neuron cultures were completely protected against OGD and NMDA toxicity by H2-meso-tetrakis(3-benzoic acid)porphyrin (H2-TBAP(3)) or H2-meso-tetrakis(4-sulfonatophenyl)porphyrin (H2-TPPS(4)), although only H2-TPPS(4) suppressed (completely) NMDA-induced Cai2+ rises. H2-meso-tetrakis(3,3'-benzoic acid)porphyrin (H2-TBAP(3,3')) or H2-meso-tetrakis(N-methylpyridynium-4-yl)porphyrin (H2-TM-PyP(4)) provided at least partial protection against OGD and NMDA toxicity and partially suppressed NMDA-induced Cai2+ elevations. Despite the complexity of Ca2+-independent and -dependent based mechanisms, the inventory of porphyrins demonstrating neuroprotection in ischemia-relevant insults is now expanded to include FP-15 and inert metal-free compounds, although with no apparent advantage gained by using FP-15.

Competing approaches to excitotoxic neuroprotection by inert and catalytic antioxidant porphyrins.

Das Ziel dieser Studie war es, festzustellen, ob neuartige Porphyrins kultivierte kortikale Neuronen vor excitotoxischem NMDA Exposition oder Sauerstoff-Glucose Entbehrung (OGD), die wesentliche Aspekte der zerebralen Isch&auml;mie model sch&uuml;tzen. Porphyrins wurden basierend auf konventionellen und unkonventionellen Kriterien ausgew&auml;hlt. Metalloporphyrin katalytische Antioxidantien besitzen einen Metallkern Redox-Sensitive k&ouml;nnen potent und weitreichenden katalytische antioxidative F&auml;higkeiten aufweisen, die konventionell geglaubt werden, um die Neuroprotektion zugrunde liegen. Wir berichten hier, die j&uuml;ngste Generation potenter Peroxynitrite Zersetzung Katalysator, FP-15, gesch&uuml;tzt, eine Mehrheit von Neuronen gegen OGD und NMDA-Toxizit&auml;t, ohne Unterdr&uuml;ckung NMDA-vermittelte intrazellul&auml;ren Ca2 + (Cai2 +) Hochheben oder ganze Zellen str&ouml;men. Wir haben zuvor gezeigt, dass Neuroprotection gegen OGD und NMDA Toxizit&auml;t korreliert mit einer F&auml;higkeit, neurotoxische Cai2 + Erhebungen zu unterdr&uuml;cken und nicht Antioxidans. Wir bewerten jetzt, falls dieser unkonventionelle Mechanismus zum inerten metallfreie Porphyrins erstreckt. Neuron-Kulturen waren v&ouml;llig gesch&uuml;tzt gegen OGD und NMDA-Toxizit&auml;t von H2-Meso-tetrakis(3-benzoic acid) Porphyrin (H2-TBAP(3)) oder H2-Meso-tetrakis(4-sulfonatophenyl) Porphyrin (H2-TPPS(4)), obwohl nur H2-TPPS(4) (vollst&auml;ndig) NMDA-induzierte Cai2 + steigt unterdr&uuml;ckt. H2-Meso-tetrakis(3,3&#39;-benzoic acid) Porphyrin (H2-TBAP(3,3&#39;)) oder H2-Meso-tetrakis(N-methylpyridynium-4-yl) Porphyrin (H2-TM-PyP(4)) bei einer zumindest teilweisen Schutz gegen OGD und NMDA Toxizit&auml;t zur Verf&uuml;gung gestellt und teilweise unterdr&uuml;ckt NMDA-induzierte Cai2 + Erhebungen. Trotz der Komplexit&auml;t der Ca2 +-unabh&auml;ngige und -abh&auml;ngige basierte Mechanismen, das Inventar des Porphyrins demonstrierende Neuroprotection in Isch&auml;mie-relevante Beleidigungen jetzt erweitert ist, um FP-15 und inerte metallfreie Verbindungen, obwohl mit keinen offensichtlichen Vorteil gewonnen mit FP-15.

Konkurrierende Ans&auml;tze zur excitotoxischem Nervenschutz durch inerte und katalytische Antioxidans Porphyrins.

Antifreeze glycoproteins (AFGPs) are a subclass of biological antifreezes found in deep sea Teleost fish. These compounds have the ability to depress the freezing point of the organism such that it can survive the subzero temperatures encountered in its environment. This physical property is very attractive for the cryopreservation of cells, tissues, and organs. Recently, our laboratory has designed and synthesized a functional carbon-linked (C-linked) AFGP analogue (1) that demonstrates tremendous promise as a novel cryoprotectant. Herein we describe the in vitro effects and interactions of C-linked AFGP analogue 1 and native AFGP 8. Our studies reveal that AFGP 8 is cytotoxic to human embryonic liver and human embryonic kidney cells at concentrations higher than 2 and 0.63 mg/mL, respectively, whereas lower concentrations are not toxic. The mechanism of this cytotoxicity is consistent with apoptosis because caspase-3/7 levels are significantly elevated in cell cultures treated with AFGP 8. In contrast, C-linked AFGP analogue 1 displayed no in vitro cytotoxicity even at high concentrations, and notably, caspase-3/7 activities were suppressed well below background levels in cell lines treated with 1. Although the results from these studies limit the human applications of native AFGP, they illustrate the benefits of developing functional C-linked AFGP analogues for various medical, commercial and industrial applications.

In vitro studies of antifreeze glycoprotein (AFGP) and a C-linked AFGP analogue.

Frostschutzmittel Glykoproteine (AFGPs) sind eine Unterklasse der biologischen Frostschutzmittel in Tiefsee Teleost Fische gefunden. Diese Verbindungen haben die M&ouml;glichkeit, den Gefrierpunkt des Organismus zu dr&uuml;cken, so dass es die Subzero &uuml;berleben kann Temperaturen in seiner Umgebung. Diese physikalische Eigenschaft ist sehr attraktiv f&uuml;r die Kryokonservierung von Zellen, Geweben und Organen. Vor kurzem hat unser Labor entwickelt und synthetisiert ein funktionale gebundene Kohlenstoff (C-verlinkt) AFGP Analog (1), das gro&szlig;e Versprechen als eine neuartige Cryoprotectant veranschaulicht. Hier beschreiben wir die in-vitro-Effekte und Wechselwirkungen zwischen C-chromosomale AFGP analog 1 und native AFGP-8. Unsere Studien zeigen, dass AFGP 8 bzw. zytotoxischen menschlichen embryonalen Leber und menschlichen embryonalen Nierenzellen bei Konzentrationen h&ouml;her als 2 und 0,63 mg/mL, ist, w&auml;hrend niedrigere Konzentrationen nicht giftig sind. Der Mechanismus der diese Zytotoxizit&auml;t entspricht Apoptosis da Caspase-3/7 Ebenen in Zellkulturen behandelt mit AFGP 8 deutlich erh&ouml;ht sind. Im Gegensatz dazu C-chromosomale AFGP analog 1 keine in-vitro-Zytotoxizit&auml;t angezeigt, selbst bei hohen Konzentrationen und insbesondere Caspase-3/7-Aktivit&auml;ten deutlich unter Hintergrundwerte in Zelllinien mit 1 behandelt unterdr&uuml;ckt wurden. Obwohl die Ergebnisse aus diesen Studien die menschlichen Anwendungen von native AFGP begrenzen, erl&auml;utern sie die Vorteile der Entwicklung von funktionaler C-chromosomale AFGP Analogien f&uuml;r verschiedene medizinische, kommerzielle und industrielle Anwendungen.

In vitro Studien von Frostschutzmittel Glykoprotein (AFGP) und ein C-chromosomale AFGP analog.

Interface-Ger&auml;te wie z. B. integrierte planare Patch-Clamp-Chips werden entwickelt, um die elektrophysiologische Aktivit&auml;t neuronaler Netzwerke gewachsen in-vitro-Studie. Das Dienstprogramm solcher Ger&auml;te werden abh&auml;ngig von der F&auml;higkeit, Neuronen mit Features f&uuml;r die Benutzeroberfl&auml;che auf dem Chip durch die Kontrolle der neuronalen Platzierung und durch leitende Verbindungen der Zelle ausrichten. In diesem Beitrag pr&auml;sentieren wir eine Strategie, um dieses Ziel zu erreichen. Gemusterte chemische Modifikation von S&uuml;nde Oberfl&auml;chen mit Poly-d-Lysin von PDMS Briefmarken &uuml;bertragen wurde verwendet, um die Adh&auml;sion und Leitung von Cryo erhaltenen prim&auml;ren Ratte kortikale Neuronen zu f&ouml;rdern. Wir zeigen, dass diese Neuronen positioniert und in Microhole-Features, die letztlich als Patch-Clamp-Schnittstellen auf dem Chip dient gewachsen werden k&ouml;nnen.

Zelle Platzierung und Leitlinien f&uuml;r die Substrate f&uuml;r Neurochip-Schnittstellen.

Interface devices such as integrated planar patch-clamp chips are being developed to study the electrophysiological activity of neuronal networks grown in vitro. The utility of such devices will be dependent upon the ability to align neurons with interface features on the chip by controlling neuronal placement and by guiding cell connectivity. In this paper, we present a strategy to accomplish this goal. Patterned chemical modification of SiN surfaces with poly-d-lysine transferred from PDMS stamps was used to promote adhesion and guidance of cryo-preserved primary rat cortical neurons. We demonstrate that these neurons can be positioned and grown over microhole features which will ultimately serve as patch-clamp interfaces on the chip.

Cell placement and guidance on substrates for neurochip interfaces.

We report on a simple and high-yield manufacturing process for silicon planar patch-clamp chips, which allow low capacitance and series resistance from individually identified cultured neurons. Apertures are etched in a high-quality silicon nitride film on a silicon wafer; wells are opened on the backside of the wafer by wet etching and passivated by a thick deposited silicon dioxide film to reduce the capacitance of the chip and to facilitate the formation of a high-impedance cell to aperture seal. The chip surface is suitable for culture of neurons over a small orifice in the substrate with minimal leak current. Collectively, these features enable high-fidelity electrophysiological recording of transmembrane currents resulting from ion channel activity in cultured neurons. Using cultured Lymnaea neurons we demonstrate whole-cell current recordings obtained from a voltage-clamp stimulation protocol, and in current-clamp mode we report action potentials stimulated by membrane depolarization steps. Despite the relatively large size of these neurons, good temporal and spatial control of cell membrane voltage was evident. To our knowledge this is the first report of recording of ion channel activity and action potentials from neurons cultured directly on a planar patch-clamp chip. This interrogation platform has enormous potential as a novel tool to readily provide high-information content during pharmaceutical assays to investigate in vitro models of disease, as well as neuronal physiology and synaptic plasticity.

A novel silicon patch-clamp chip permits high-fidelity recording of ion channel activity from functionally defined neurons.

Wir berichten &uuml;ber eine einfache und hochverzinsliche Herstellungsprozess f&uuml;r Silizium planar Patch-Clamp-Chips, die niederohmigen Kapazit&auml;ts- und Serie von individuell bestimmte kultivierten Neuronen erm&ouml;glichen. &Ouml;ffnungen sind in einem qualitativ hochwertige Silizium-Nitrid-Film auf einem Silizium-Wafer ge&auml;tzt; Brunnen sind auf der R&uuml;ckseite des Wafers durch Nass&auml;tzen ge&ouml;ffnet und passiviert ein dickes hinterlegten Siliciumdioxid-Film, die Kapazit&auml;t des Chips zu reduzieren und die Bildung einer Zelle hochohmige auf Blende Siegel zu erleichtern. Die Chip-Oberfl&auml;che eignet sich f&uuml;r die Kultur der Neuronen &uuml;ber eine kleine &Ouml;ffnung in das Substrat mit aktuellen minimal Leck. Diese Features werden kollektiv, High-Fidelity-elektrophysiologische Aufnahme Transmembran Str&ouml;mungen aus Ion Channel-Aktivit&auml;ten in kultivierten Neuronen aktivieren. Mit kultivierte Lymnaea Neuronen zeigen wir ganze Zellen aktuelle Aufnahmen ein Klemm-Spannung Anregung Protokoll entnommen und im Klemm-Strom-Modus berichten wir Aktionspotentiale durch Membrane Depolarisierung Schritte angeregt. Trotz der relativ gro&szlig;en Gr&ouml;&szlig;e dieser Neuronen zeigte gute zeitliche und r&auml;umliche Kontrolle der Zellmembran Spannung. Unseres Wissens ist dies der erste Bericht der Aufnahme von Ion Channel-Aktivit&auml;ten und Aktionspotentiale von Neuronen, die direkt auf eine planare Patch-Clamp-Chip kultiviert. Dieses Verh&ouml;r-Plattform hat enormes Potential als neuartige Werkzeug leicht hoher Informationsgehalt w&auml;hrend pharmazeutische Proben zur Untersuchung von in-vitro-Modelle von Krankheit, sowie neuronale Physiologie und synaptische Plastizit&auml;t bereitstellen.

Eine neuartige Patch-Clamp-Siliziumchip erlaubt originalgetreue Aufnahme von Ion Channel-Aktivit&auml;ten von funktional definierte Neuronen.

We present a polymer microchip capable of monitoring neuronal activity with a fidelity never before obtained on a planar patch-clamp device. Cardio-respiratory neurons Left Pedal Dorsal 1 (LPeD1) from mollusc Lymnaea were cultured on the microchip's polyimide surface for 2 to 4 hours. Cultured neurons formed high resistance seals (gigaseals) between the cell membrane and the surface surrounding apertures etched in the polyimide. Gigaseal formation was observed without applying external force, such as suction, on neurons. The formation of gigaseals, as well as the low access resistance and shunt capacitance values of the polymer microchip resulted in high-fidelity recordings. On-chip culture of neurons permitted, for the first time on a polymeric patch-clamp device, the recording of high fidelity physiological action potentials. Microfabrication of the hybrid poly(dimethylsiloxane)-polyimide (PDMS-PI) microchip is discussed, including a two-layer PDMS processing technique resulting in minimized shrinking variations.

High-fidelity patch-clamp recordings from neurons cultured on a polymer microchip.

Wir pr&auml;sentieren einen Polymer-Mikrochip &Uuml;berwachung neuronaler Aktivit&auml;t mit einer Treue f&auml;hig, noch nie auf eine planare Patch-Clamp-Ger&auml;t erh&auml;ltlich. Kardio-respiratorische Neuronen linke Pedal Dorsal 1 (LPeD1) von Weichtieren Lymnaea wurden auf dem Mikrochip-Polyimid-Oberfl&auml;che f&uuml;r 2 bis 4 Stunden kultiviert. Kultivierte Neuronen gebildet Hochwiderstandsf&auml;hige Dichtungen (Gigaseals) zwischen der Zellmembran und die Oberfl&auml;che rund um &Ouml;ffnungen in die Polyimid ge&auml;tzt. Gigaseal Bildung wurde beobachtet, ohne &auml;u&szlig;ere Kraft, wie z. B. Absaugung, auf Neuronen zu &uuml;bernehmen. Die Bildung von Gigaseals, sowie die niedrigen Zugang Widerstand und Shunt Kapazit&auml;tswerte von Polymer-Mikrochip f&uuml;hrten zu High-Fidelity-Aufnahmen. Auf dem Chip Kultur von Neuronen erlaubt, zum ersten Mal auf einem Polymeren Patch-Clamp-Ger&auml;t, die Aufzeichnung von High Fidelity physiologische Aktionspotentiale. Microfabrication des Hybrid Poly (Dimethylsiloxane)-Polyimid (PDMS-PI) Mikrochip wird diskutiert, einschlie&szlig;lich eine zweischichtige PDMS Verarbeitung-Technik, was zu schrumpfen Variationen minimiert.

High-Fidelity-Patch-Clamp-Aufnahmen von Neuronen, die auf einem Mikrochip Polymer kultiviert.

Understanding mechanotransduction pathways leading to thrombosis will require progressive steps, including determination of the mechanical behavior of the platelet membrane in response to applied loads. The platelet membrane deformation capacity, as quantified by membrane progression into a borosilicate glass micropipette of defined internal diameter, was probed in murine platelets using a controlled range of negative pressure (0-7Â cmÂ H(2)O). Based on our observations that the platelet portion outside the micropipette was mostly spherical and that the platelet volume did not change upon aspiration, a novel continuum mechanics-based model of the platelet micropipette aspiration experiment was created, and a new hyperelastic isotropic material model including membrane residual tension was proposed for the platelet membrane. Murine platelet membranes maintained an average linear deformation behavior: L (p)/R (p) =Â 146,100p (i)Â Ã— R (p) + 19.923, where L (p) is the platelet length aspirated in the micropipette (m), R (p) is micropipette radius (m) and p (i) is the aspiration pressure (Pa). The theoretical model was used to generate material constants for the murine platelet membrane that allowed for an accurate simulation of the micropipette aspiration experiments. From published results, another set of material constants was established for the human platelet membrane. Limited cases of platelet lysis upon aspiration were analyzed using the theoretical model to determine preliminary membrane tension strength values.

A mechanobiological investigation of platelets.

Verst&auml;ndnis Mechanotransduction &Uuml;bertragungswege bei der Thrombose wird progressive Ma&szlig;nahmen, einschlie&szlig;lich der Bestimmung des mechanischen Verhaltens der Thrombozyten-Membran als Reaktion auf die aufgebrachten Lasten erfordern. Die Thrombozyten Membran Verformung Kapazit&auml;t, wie mit Membran Fortschreiten in einer Mikropipette Borosilikatglas von definierten Innendurchmesser, quantifiziert wurde sondiert in murinen Thrombozyten mit einem kontrollierten Bereich von Unterdruck (0-7 cm H(2)O). Basierend auf unsere Beobachtungen, dass der Thrombozyten-Teil au&szlig;erhalb der Mikropipette meist sph&auml;risch war und die Thrombozyten-Lautst&auml;rke nicht auf Wunsch ge&auml;ndert haben, ein neuartiges Kontinuum Mechanik basierendes Modell des Experiments Thrombozyten Mikropipette Bestreben entstand, und ein neues hyperelastische isotrope Materialmodell einschlie&szlig;lich Membrane verbleibende Spannung wurde vorgeschlagen, f&uuml;r die Thrombozyten-Membran. Murine Thrombozyten Membranen verwaltet eine durchschnittliche lineare Verformungsverhaltens: L (p) / r (p) = 146.100 p (i) &times; R (p) + 19.923, wo L (p) ist die L&auml;nge des Pl&auml;ttchens aspiriert in die Mikropipette (m), R (p) Mikropipette Radius (m) und p (i) ist der Anspruch-Druck (Pa). Das theoretische Modell wurde zur Materialkonstanten f&uuml;r die murinen Thrombozyten-Membran zu generieren, die f&uuml;r eine genaue Simulation der Mikropipette streben Experimente erlaubt. Aus ver&ouml;ffentlichten Ergebnisse entstand eine weitere Reihe von Materialkonstanten f&uuml;r die menschlichen Thrombozyten-Membran. Begrenzte F&auml;llen der Thrombozyten-Zerst&ouml;rung auf Bestreben wurden analysiert mit das theoretische Modell vorl&auml;ufige Membrane St&auml;rke Spannungswerten bestimmen.

Article	Year
Protection of cortical neurons against oxygen-glucose deprivation and N-methyl-D-aspartate by DIDS and SITS. European journal of pharmacology\| PubMed ID: 12600690	2003
Protection by cholesterol-extracting cyclodextrins: a role for N-methyl-D-aspartate receptor redistribution. Journal of neurochemistry\| PubMed ID: 15748165	2005
An alternative Ca2+-dependent mechanism of neuroprotection by the metalloporphyrin class of superoxide dismutase mimetics. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 16081500	2005
Chlortetracycline and demeclocycline inhibit calpains and protect mouse neurons against glutamate toxicity and cerebral ischemia. The Journal of biological chemistry\| PubMed ID: 16091365	2005
Neurogenesis and neuronal communication on micropatterned neurochips. Biotechnology and bioengineering\| PubMed ID: 16094670	2005
Competing approaches to excitotoxic neuroprotection by inert and catalytic antioxidant porphyrins. Neuroscience letters\| PubMed ID: 16631306	2006
In vitro studies of antifreeze glycoprotein (AFGP) and a C-linked AFGP analogue. Biomacromolecules\| PubMed ID: 17411090	2007
Cell placement and guidance on substrates for neurochip interfaces. Biotechnology and bioengineering\| PubMed ID: 19753615	2010
A novel silicon patch-clamp chip permits high-fidelity recording of ion channel activity from functionally defined neurons. Biotechnology and bioengineering\| PubMed ID: 20648547	2010
High-fidelity patch-clamp recordings from neurons cultured on a polymer microchip. Biomedical microdevices\| PubMed ID: 20694518	2010
A mechanobiological investigation of platelets. Biomechanics and modeling in mechanobiology\| PubMed ID: 20706764	2011

Geoff Mealing

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