Research was partly supported by NIH research grants R01 AR062173 and SHC 250862 to IEA. ES is the recipient of NIH T32 CTSC predoctoral fellowship.

1. Hydrodynamische Lieferung der sRANKL MC DNA <ol><li> Hydrodynamische Lieferung über Mausschwanzvene <ol><li> Wiegen Sie die Maus vor der Schwanzveneninjektion. Verdünnen sRANKL oder grün fluoreszierende Protein (GFP) MC in Ringer-Lösung (pre-warmen bei 37 ° C) in einem Gesamtvolumen von ca. 10% der Mauskörpergewicht. </li><li> Wärmen Sie die Maus in einem Käfig für 10 Minuten vor der Injektion, um die Blutgefäße erweitern und machen seitlichen Venen (LVs) sichtbar. Überwachen Sie die Maus sorgfältig zu Dehydrierung und Hyperthermie zu vermeiden. </li><li> Sobald die LVs sind erweitert und sichtbar, übertragen Sie die Maus auf den Restrainer und den Stecker weit genug in den Lauf, um die Bewegung zurückzuhalten. Überwachen Sie die Maus für die normale Aktivität wie Atmung. Stellen Sie den Stecker des Restrainer, wenn nötig. </li><li> Desinfizieren Sie die Injektionsbereich 3/4 von der Spitze des Schwanzes mit einem Alkoholtupfer. Verwenden Sie eine 27 bis 30 G-Nadel für die Mausschwanzveneninjektion. Halten Sie den Schwanz mit einer Hand fest und legen Sie die Nadel inin die Schwanzvene. Üben Sie Druck auf die Spritze und füllen Sie die Injektion innerhalb von 5-7 Sekunden. </li><li> Entfernen Sie die Nadel aus der Schwanzvene und Druck mit einem Wattebausch auf die Injektionsstelle, um Blutungen zu stoppen. Überwachen Sie die Maus für 15-30 min, übertragen Sie dann die Maus wieder auf, sobald das Vivarium Atemfrequenz wird auf seiner normalen Geschwindigkeit reduziert. </li></ol></li><li> Bestätigung der Maus Systemische sRANKL Expression und Quantifizierung der Maus Serum TRACP5b Beitrag Gentransfer <ol><li> Wärmen Sie die Maus in einem Käfig für 5-10 min, um die Blutgefäße erweitern und machen LVs sichtbar. Überwachen Sie die Maus sorgfältig zu Dehydrierung und Hyperthermie zu vermeiden. </li><li> Machen Sie einen kleinen Schnitt mit einem Messer an der Maus den Schwanz an einem Ort, ein Viertel von der Spitze des Schwanzes. </li><li> Sammeln Sie etwa 100 ul Blut im Serum Trennung Röhren. </li><li> Serumtrennröhrchen bei Raumtemperatur inkubiert für 30 min. </li><li> Zentrifugieren Sie die Proben bei 10.000 xg für 5 min und sammeln Serum. Store Serum -80 ° C zur weiteren Analyse. </li><li> Führen sRANKL ELISA und Mausserum TRACP5b Assay auf Serum-Proben unter Verwendung eines kommerziell erhältlichen Kits. </li></ol></li></ol> 2. Osteoklasten in vitro Generierung von Maus BMM <ol><li> Isolierung und Kultivierung von BMM <ol><li> Die Isolierung des Schien-und Oberschenkelknochen: Euthanize den Spender-Maus mit CO 2, und desinfizieren Sie die Beine mit 70% Ethanol. Präparieren Sie vom Schambein zu dem Fersenbein, um die Oberschenkel und Schienbein Knochen entfernen intakt. </li><li> Entfernen Sie die Haut-und Muskel sorgfältig, ohne die Knochen. Platzieren verarbeitet Femur und Tibia Knochen in eine Petrischale mit phosphatgepufferter Salzlösung (PBS). </li><li> Entfernung von Knochenmark: Schneiden Sie die Spitze auf der einen Seite der Oberschenkel und Schienbein Knochen und spülen, die Knochenmark in einen 50-ml-Tube mit einer 1 ml-Spritze mit einer 25 G-Nadel, mit α-MEM mit 1% Penicillin / Streptomycin belastet (Osteoklasten Kultur Medium / OCM). </li><li&gt; Prozessknochenmark: Mix und übertragen Knochenmark-Suspension auf einen neuen 50-ml-Tube, indem es durch einen 70 um Nylon Zellsieb. Zentrifugation der Zellen bei 300 × g und resuspendieren in α-MEM, das 1% Penicillin / Streptomycin und 10% FBS (Osteoklasten-Kulturmedium / OCM (+)). </li></ol></li><li> Kultur von Makrophagen <ol><li> Zählen von Zellen mit einer Zählkammer oder automatische Zellzähler. </li><li> Platte 1 x 10 6 Zellen pro Well einer 6-Well-Platte oder 3 × 10 5 Zellen auf Glasdeckgläschen (5 mm) oder Dentinscheiben in 96-Well-Platte gelegt und bei 37 ° C </li><li> Saugen Sie alle Medien, die nicht-adhärenten Zellen und Inkubation Zellen bei 37 ° C mit frischem OCM (+) mit 25 ng / ml Maus M-CSF für 48 Stunden. </li><li> Saugen Sie alle Medien Zellen und Inkubation bei 37 ° C mit frischem OCM (+)-Medien mit 25 ng / ml Maus M-CSF und 30 ng / ml Maus sRANKL Osteoklastogenese zu initiieren. Tanken Medien alle 3 Tage nach Bedarf. </li><li> Zellen wachsenca. 6-8 Tage, um das riesige Mehrkernzellen, die Resorption Knochen zu bilden. Sobald kernige Zellen erscheinen, führen TRAP (Tartrat-resistente saure Phosphatase)-Färbung mit einem kommerziell erhältlichen Kit, und als voll ausgereift, führen Funktionstests. Visualisieren F-Aktin-Ringe auf Deckgläsern mit Phalloidin-Färbung und Bild die Zellen angebracht, um Scheiben durch Rasterelektronenmikroskopie (SEM), wie zuvor beschrieben 19 Dentin. </li></ol></li></ol> 3. Osteoklasten in vitro-Erzeugung aus Menschen PBMCs <ol><li> Isolierung von humanen PBMCs <ol><li> 10 ml vorgewärmten Histopaque-1077 in ein 50-ml-Tube. </li><li> Leere Leukozytenfilter aus Blutbank mit sterilem PBS erhalten in einen 50-ml-Tube (1:1-Verhältnis von Blut mit PBS). Schicht die verdünnte Blut über die Histopaque Lösung sehr langsam, dafür sorgen, dass das Blut nicht mit dem Histopaque mischen. </li><li> Zentrifuge Proben bei 1000 g für 20 min bei 18 ° C. Folgening Zentrifugation sorgfältig isolieren weiß Buffy-Coat-Schicht, die die weißen Blutkörperchen und übertragen auf ein neues 50-ml-Tube. </li><li> Zellen mit PBS verdünnen und Zentrifuge wieder bei 650 g für 5 min, um die Zellen zu sammeln. </li><li> Überstand entfernen und resuspendieren Zellen in OCM (-) Medien und zählen Zellen mit Zellzähler. </li></ol></li><li> Plating und Kultivierung PBMCs <ol><li> Resuspendieren der Zellen in OCM (+)-Medium und der Platte 8 x 10 6 Zellen / ml pro Vertiefung einer 6-Well-Platte oder 1 x 10 6 Zellen / ml pro Vertiefung in 96-Well-Platte. Platten Zellen auf Glasdeckgläschen (5 mm), die für Immunfluoreszenz-Bildgebung als auch auf Knochenscheiben für Knochenresorption Assays, wie zuvor beschrieben 20 erforderlich. </li><li> Änderung Kulturmedien, Auffüllen der Zellen mit menschlichen M-CSF alle 2-3 Tage oder nach Bedarf. Dann Differenzierungsschritt am Tag 5 gehen durch Zugabe von 30 ng / ml humanes sRANKL zusammen mit 25 ng / ml menschlichen M-CSF in OCM (+)-Medium zu initiieren osteoclastogenesist. </li><li> Wachsen Zellen für ca. 14 bis 21 Tage, um das riesige Mehrkern Zellen zu bilden. Sobald kernige Zellen angezeigt werden, können diese Zellen dann für TRAP beschriftet werden, und als voll ausgereift, können funktionelle Assays durchgeführt werden. F-Actin-Ringe können auf Deckgläsern mit Phalloidin-Färbung sichtbar gemacht werden. Die Morphologie der Zellen in Zellen angebracht, um Scheiben von SEM Dentin untersucht werden. </li></ol></li></ol>

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The authors have nothing to disclose.

Erkrankungen des Bewegungsapparats sind die führenden Ursachen von Morbidität und Behinderung und werden von über 150 Krankheiten und Syndrome besteht; Auswirkungen auf heute etwa 90 Millionen Amerikaner. Gelenkentzündungen und Knochenzerstörung wiegen Merkmale Erkrankungen des Bewegungsapparats, einschließlich Arthritis und Osteoporose. Osteoporose ist eine Erkrankung, die Knochen schwächt Integrität, die häufig Frakturen der Knochen führt. Arthritis ist eine chronische, schwächende Erkrankung, die durch Ent...

Muskel-Skelett-Erkrankungen betreffen Millionen von Menschen in den Vereinigten Staaten und Gegenwart schwerwiegende Folgen für die nationalen und lokalen Gesundheitssysteme ein. Diese Erkrankungen sind durch den Verlust von Knochen-und Gelenkfunktion, die umfangreiche Behandlung und lange Erholungsphasen erfordern gekennzeichnet. Üblicherweise wird eine relative Erhöhung der Anzahl und / oder Aktivität von Osteoklasten, spezialisierte Zellen zu Knochenresorption, Osteoporose und Arthritis beobachtet 2. Unter physiologischen Bedingungen ist die Anzahl und Aktivität von Osteoklasten durch Rezeptor-Aktivator-von Kernfaktor κ-B-Liganden (RANKL), die von Osteoblasten produziert wird geregelt. Osteoprotegerin (OPG), wird ein Köder Rezeptor für RANKL auch durch Osteoblasten 3 erzeugten In-vivo-Tiermodelle, die systemische Überexpression von sRANKL oder Deletion von OPG einzubeziehen sind sehr wertvoll bei Osteoporose-Forschung. Allerdings erfordern diese Verfahren die Herstellung von transgenen Mäusen 4,5. Hier wird eine neuartige AlternativeVerfahren überexprimieren sRANKL für die Untersuchung von Muskel-Skelett-bedingten Erkrankungen beschrieben. Insbesondere Minicircle (MC) DNA-Technologie und hydrodynamischen Versandmethoden wurden verwendet, um Gen-Transfer von sRANKL in vivo zu erreichen und überexprimieren Maus sRANKL systemisch 6. Diese Methode ist auch eine Ergänzung zu anderen in-vivo-Modelle von Osteoporose, wie hormonelle Modulation der Osteoklasten folgenden ovariectomy 7 und diätetische Intervention durch kalziumarme Ernährung 8. Diese Modelle sind sehr nützlich, um verschiedene Aspekte des Bewegungsbedingten Störungen aber sie erfordern chirurgische Verfahren und kann bis zu mehreren Monaten, was mit erheblichen Kosten 9 studieren. Ovariektomierte (OVX) Nagetier-Modell ist ein Modell, bei dem Versuchstier Entfernung der Eierstöcke führt zu Östrogenmangel, wodurch menschliche postmenopausalen Osteoporose 10 imitiert. Menschen postmenopausalen Osteoporose, eine Erkrankung, bei Östrogen deficirenz führt zu einem erhöhten Risiko von Knochenbrüchen und Osteoporose rund acht Millionen Frauen allein in den Vereinigten Staaten. Obwohl die OVX Modell ist nützlich für postmenopausalen Osteoporose bietet begrenzte Vorteile bei der Untersuchung von Osteoporose im Allgemeinen. Östrogen unterdrückt Knochenverlust, durch Induktion von Osteoklasten und Osteoblasten Hemmung der Apoptose, also in seiner Abwesenheit eine erhöhte Aktivität der Osteoklasten beobachtet 10-12. Ein RANKL-OPG-Verhältnis Ungleichgewicht, das die Knochenresorption begünstigt wird auch beobachtet, 13. Jedoch Östrogenmangel in vivo auch begleitet von verringerten Niveaus des transformierenden Wachstumsfaktor β (TGF β), erhöhte Interleukin-7 (IL-7) und TNF, IL-1 und IL-6 14,15. Da diese Zytokine Knochenumbau modulatorische Funktionen der RANKL Weg unabhängig bekannt ist, ist es unmöglich, eine Osteoklastenaktivierung nur auf die RANKL-RANK-Achse zuzuordnen. Die in diesem Dokument beschriebene Modell ermöglicht es Forschern, in viv studierenRANKL-RANK-o-Achse in Osteoklastogenese und Knochenverlust ohne pro-inflammatorischen Zytokinen im Vergleich zu OVX Nagetiermodellen. Zusätzlich kann in vitro Osteoklastogenese Techniken sind wesentliche Instrumente zur Aktivierung der Osteoklasten für mögliche therapeutische Behandlungen von Muskel-Skelett-Erkrankungen zu studieren. Frühere Studien haben auch gezeigt, dass die Kultivierung Knochenmark der Maus Makrophagen (BMM) mit Maus-Makrophagen-Kolonie-stimulierenden Faktor (M-CSF) und Maus sRANKL können, um die Differenzierung von Osteoklasten 3,16,17 führen. Hier werden die Protokolle kernige Osteoklasten-ähnliche Zellen aus Maus-Knochenmark sowie aus menschlichen peripheren mononukleären Blutzellen (PBMCs) in vitro zu erzeugen 18 beschrieben. Die zellbasierte Assays erforderlich, um eine reife terminal differenzierten und voll funktions Osteoklasten definieren ebenfalls kurz beschrieben. Diese in-vitro-Techniken ergänzen den Roman in vivo Ansatz zusammen und dienen als powerful Untersuchungswerkzeuge, um die Differenzierung von Osteoklasten und Aktivierung zu studieren. Mit Hilfe dieser Systeme sind die Wissenschaftler in der Lage, die Osteoklasten in vivo und in vitro zu erzeugen und definieren Sie die Reize und Signale für ihre Verbreitung und Aktivierung erforderlich sowie testen die Wirksamkeit von pharmakologischen und biologischen Inhibitoren.

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a novel in vivo gene transfer technique and in vitro cell based assays for the study of bone loss in musculoskeletal disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

Hier wird ein neuartiges Gentransfertechnik zur Differenzierung von Osteoklasten in vivo Zellkulturprotokolle zur Differenzierung in Osteoklasten-Vorläuferzellen in vitro als ein Verfahren, um die Wirkungen von Cytokinen auf die Entstehung von Osteoclasten zu studieren beschrieben. In Fig. 1 sind die repräsentativen Ergebnisse erfolgreicher Gentransfer von GFP und Maus sRANKL MC in Mäusen gezeigt. In Abbildung 2 sind die repräsentativen Bilder von Knochenmark der M...

Watch this Scientific Journal Video about A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders at JoVE.com

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

A Novel<em&gt; In vivo</em&gt; Gentransfer-Technik und<em&gt; In-vitro-</em&gt; Zellbasierte Assays für das Studium der Knochenverlust in Skeletterkrankungen

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

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Research

JoVE Journal

Medicine

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

11.6K Aufrufe.  University of California, Davis. Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described. 

Serie: A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

Eine Strategie, um de novo Mutationen in Common Störungen wie Autismus und Schizophrenie Identifizieren

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

Forschung

Medizin

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Aufbau und Verbreitung der Menschenrechte Retinoblastoma Tumore in immundefizienten Mäuse

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

Longitudinal Evaluation of Mouse Hind Limb Knochenverlust nach Rückenmarksverletzungen mit Novel, In vivo Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

Produktion und Nachweis von reaktiven Sauerstoffspezies (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarker in einem Tiermodell für die Aufdeckung Neural, hämatologischen und Behavioral Korrelate der PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

A Novel Surgical Ansatz intratracheale Verabreichung von Wirkstoffen in einem Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Development of an Audio-based Virtual Gaming Environment to Assist with Navigation Skills in the Blind

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio based cues and set within the context of a video game metaphor, users gather relevant spatial information regarding a building's layout. This allows the user to develop an accurate spatial cognitive map of a large-scale three-dimensional space that can be manipulated for the purposes of a real indoor navigation task. After game play, participants are then assessed on their ability to navigate within the target physical building represented in the game. Preliminary results suggest that early blind users were able to acquire relevant information regarding the spatial layout of a previously unfamiliar building as indexed by their performance on a series of navigation tasks. These tasks included path finding through the virtual and physical building, as well as a series of drop off tasks. We find that the immersive and highly interactive nature of the AbES software appears to greatly engage the blind user to actively explore the virtual environment. Applications of this approach may extend to larger populations of visually impaired individuals.

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind.

Entwicklung eines Audio-based Virtual Gaming Umwelt mit Navigation Skills in the Blind Assist

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

A Novel Hochauflösende<em&gt; In vivo</em&gt; Bildtechnik, um die Dynamik der intrakranielle Strukturen zu Tumorwachstum und Therapeutics Studieren

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

A Novel Application of Musculoskeletal Ultrasound Imaging

Ultrasound is an attractive modality for imaging muscle and tendon motion during dynamic tasks and can provide a complementary methodological approach for biomechanical studies in a clinical or laboratory setting. Towards this goal, methods for quantification of muscle kinematics from ultrasound imagery are being developed based on image processing. The temporal resolution of these methods is typically not sufficient for highly dynamic tasks, such as drop-landing. We propose a new approach that utilizes a Doppler method for quantifying muscle kinematics. We have developed a novel vector tissue Doppler imaging (vTDI) technique that can be used to measure musculoskeletal contraction velocity, strain and strain rate with sub-millisecond temporal resolution during dynamic activities using ultrasound. The goal of this preliminary study was to investigate the repeatability and potential applicability of the vTDI technique in measuring musculoskeletal velocities during a drop-landing task, in healthy subjects. The vTDI measurements can be performed concurrently with other biomechanical techniques, such as 3D motion capture for joint kinematics and kinetics, electromyography for timing of muscle activation and force plates for ground reaction force. Integration of these complementary techniques could lead to a better understanding of dynamic muscle function and dysfunction underlying the pathogenesis and pathophysiology of musculoskeletal disorders.

We describe a new ultrasound-based vector tissue Doppler imaging technique to measure muscle contraction velocity, strain and strain rate with sub-millisecond temporal resolution during dynamic activities. This approach provides complementary measurements of dynamic muscle function and could lead to a better understanding of mechanisms underlying musculoskeletal disorders.

A Novel Anwendung von Muskel-Skelett-Ultraschall-Bildgebung

Ultrasound is an attractive modality for imaging muscle and tendon  motion during dynamic tasks and can provide a complementary methodological  approach ...

Cell-based Assay Protocol for the Prognostic Prediction of Idiopathic Scoliosis Using Cellular Dielectric Spectroscopy

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the prognosis of idiopathic scoliosis in asymptomatic and affected children. The assay consists of the evaluation of the functional status of Gi and Gs proteins in peripheral blood mononuclear cells (PBMCs) by cellular dielectric spectroscopy (CDS), using an automated CDS-based instrument, and the classification of children into three functional groups (FG1, FG2, FG3) with respect to the profile of imbalance between the degree of response to Gi and Gs proteins stimulation. The classification is further confirmed by the differential effect of osteopontin (OPN) on response to Gi stimulation among groups and the severe progression of disease is referenced by FG2. Approximately, a volume of 10 ml of blood is required to extract PBMCs by Ficoll-gradient and cells are then stored in liquid nitrogen. The adequate number of PBMCs to perform the assay is obtained after two days of cell culture. Essentially, cells are first incubated with phytohemmaglutinin (PHA). After 24 hr incubation, medium is replaced by a PHA-free culture medium for an additional 24 hr prior to cell seeding and OPN treatment. Cells are then spectroscopically screened for their responses to somatostatin and isoproterenol, which respectively activate Gi and Gs proteins through their cognate receptors. Both somatostatin and isoproterenol are simultaneously injected with an integrated fluidics system and the cells&#39; responses are monitored for 15 min. The assay can be performed with fresh or frozen PBMCs and the procedure is completed within 4 days.

A structured protocol is presented for a cell-based assay as a functional test to predict the prognosis of idiopathic scoliosis using cellular dielectric spectroscopy (CDS). The assay can be performed with fresh or frozen peripheral blood mononuclear cells (PBMCs) and the procedure is completed within 4 days.

Zellbasierte Assay-Protokoll für die prognostische Prognose der idiopathischen Skoliose Mit Cellular dielektrischer Spektroskopie

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the ...