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. Consegna idrodinamica di sRANKL MC DNA <ol><li> Consegna idrodinamica via Tail Vena del mouse <ol><li> Pesare il mouse prima dell&#39;iniezione coda vena. Diluire sRANKL o proteina fluorescente verde (GFP) MC in soluzione di Ringer (pre-riscaldare a 37 ° C) in un volume totale di circa 10% del peso corporeo del topo. </li><li> Riscaldare il topo in una gabbia per 10 min prima dell&#39;iniezione per dilatare i vasi sanguigni e rendere vene laterali (LV) visibili. Monitorare con attenzione il mouse per evitare la disidratazione e ipertermia. </li><li> Non appena i VL sono dilatati e visibili, trasferire il mouse per il limitatore e inserire la spina abbastanza lontano nel barile per frenare il movimento. Monitorare il mouse per il normale attività come la respirazione. Regolare la spina del dispositivo di immobilizzazione, se necessario. </li><li> Disinfettare la zona di iniezione 3/4 dalla punta della coda con un batuffolo imbevuto di alcool. Utilizzare un ago G 27-30 per l&#39;iniezione coda vena mouse. Tenere la coda saldamente con una mano e inserire l&#39;ago inla vena della coda. Applicare una pressione sulla siringa e completare l&#39;iniezione entro 5-7 sec. </li><li> Rimuovere l&#39;ago dalla vena della coda e applicare una pressione con un batuffolo di cotone sul sito di iniezione per fermare l&#39;emorragia. Monitorare il mouse per 15-30 minuti, quindi trasferire il mouse fino al vivaio una volta che il tasso di respirazione è ridotta al suo tasso normale. </li></ol></li><li> Conferma di espressione sistemica mouse sRANKL e quantificazione dei mouse Serum TRACP5b post Gene Transfer <ol><li> Riscaldare il topo in una gabbia per 5-10 minuti, al fine di dilatare i vasi sanguigni e rendere visibile LV. Monitorare con attenzione il mouse per evitare la disidratazione e ipertermia. </li><li> Fare una piccola incisione con una lama di coda del topo in un sito un quarto dalla punta della coda. </li><li> Raccogliere circa 100 ml di sangue in provette di siero di separazione. </li><li> Incubare le provette di siero di separazione a temperatura ambiente per 30 minuti. </li><li> Centrifugare i campioni a 10.000 xg per 5 minuti e raccogliere il siero. Store siero in -80 ° C per ulteriori analisi. </li><li> Eseguire sRANKL ELISA e mouse dosaggio TRACP5b siero su campioni di siero utilizzando un kit commerciale disponibile. </li></ol></li></ol> 2. In vitro Osteoclasta generazione di mouse BMMs <ol><li> Isolamento e Coltura di BMMs <ol><li> Isolamento della tibia e femore ossa: Euthanize il mouse donatore con CO 2, e disinfettare le gambe con il 70% di etanolo. Sezionare dal pube all&#39;osso calcagno, per rimuovere femore e tibia ossa intatte. </li><li> Rimuovere la pelle ei muscoli attenzione senza danneggiare l&#39;osso. Posizionare femore e tibia trasformati in una capsula di Petri contenente tampone fosfato salino (PBS). </li><li> Rimozione di midollo osseo: Tagliare la punta su un lato del femore e della tibia ossa e lavare il midollo osseo in una provetta da 50 ml con una siringa da 1 ml con un ago G 25, caricato con α-MEM contenente 1% di penicillina / streptomicina (osteoclasti cultura medio / OCM). </li><li&gt; Processo midollo osseo: Mescolare e trasferire sospensione midollo osseo in un tubo da 50 ml facendola passare attraverso un filtro 70 micron cella nylon. Centrifugare le cellule a 300 xg e risospendere in α-MEM contenente 1% di penicillina / streptomicina e 10% FBS (Osteoclasta colturale / OCM (+)). </li></ol></li><li> Cultura dei macrofagi <ol><li> Contare le cellule utilizzando un emocitometro o contatore di cellule automatico. </li><li> Piastra 1 x 10 6 cellule per bene per un 6-pozzetti o 3 x 10 5 cellule per vetrino (5 mm) o fette di dentina collocati in piastra a 96 pozzetti ed incubare a 37 ° C. </li><li> Aspirare tutti i supporti contenenti cellule non aderenti e Incubare le cellule a 37 ° C con OCM fresco (+) contenente topo 25 ng / ml M-CSF per 48 ore. </li><li> Aspirare tutti i media e incubare le cellule a 37 ° C con nuova OCM (+) supporti contenenti il ​​mouse 25 ng / ml M-CSF e 30 ng / ml il mouse sRANKL per avviare osteoclastogenesis. Rifornire i media ogni 3 giorni come richiesto. </li><li> Crescere celluleper circa 6-8 giorni per formare celle multi-nucleari giganti in grado di riassorbimento osseo. Non appena compaiono le cellule multinucleate, eseguire TRAP (fosfatasi acida tartrato resistente) colorazione utilizzando un kit commerciale disponibile, e quando è completamente maturato, eseguire test funzionali. Visualizza anelli di F-actina su vetrini con macchia phalloidin e immagine le cellule attaccate alla dentina fette da microscopia elettronica a scansione (SEM), come descritto in precedenza 19. </li></ol></li></ol> 3. In vitro Osteoclasta generazione da PBMC umano <ol><li> Isolamento di PBMC umano <ol><li> Aggiungere 10 ml di pre-riscaldato Histopaque-1077 in una provetta da 50 ml. </li><li> Filtro leucociti vuoto ottenuto dalla banca del sangue con PBS sterile in una provetta da 50 ml (rapporto 1:1 di sangue con PBS). Strato il sangue diluito su tutto la soluzione HISTOPAQUE molto lentamente, facendo in modo che il sangue non si mescola con il HISTOPAQUE. </li><li> Centrifugare i campioni a 1000 xg per 20 min a 18 ° C. Seguireing centrifugazione, isolare attentamente il buffy bianco strato di rivestimento contenente i globuli bianchi e le trasferisce in un nuovo tubo da 50 ml. </li><li> Diluire le cellule con PBS e centrifugare di nuovo a 650 xg per 5 minuti per raccogliere le cellule. </li><li> Rimuovere il surnatante e risospendere le cellule in OCM (-) i media e contare le cellule utilizzando contatore di cellule. </li></ol></li><li> Placcatura e Coltura PBMC <ol><li> Risospendere le cellule in OCM (+) Media e piastra 8 x 10 6 cellule / ml per pozzetto in una piastra da 6 pozzetti o 1 x 10 6 cellule / ml per pozzetto in piastre da 96 pozzetti. Cellule piastra su vetrini (5 mm) come richiesto per l&#39;imaging immunofluorescenza nonché sulle fette di osso adatti per saggi di riassorbimento osseo come precedentemente descritto 20. </li><li> Cambia terreni di coltura, rifornire le cellule umane con M-CSF ogni 2-3 giorni o secondo necessità. Quindi procedere alla differenziazione passo il giorno 5 aggiungendo 30 ng / ml di sRANKL umana con 25 ng / ml M-CSF umano nella OCM (+) mezzi di avviare osteoclastogenesè. </li><li> Crescere cellule per circa 14 a 21 giorni per formare cellule multi-nucleari giganti. Non appena compaiono le cellule multinucleate, queste cellule possono quindi essere etichettati per TRAP, e quando è completamente maturato, test funzionali possono essere eseguiti. Anelli di F-actina possono essere visualizzati su vetrini con macchia falloidina. La morfologia cellulare può essere esaminato in cellule attaccate alla dentina fette mediante SEM. </li></ol></li></ol>

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The authors have nothing to disclose.

Condizioni muscolo-scheletriche sono le cause principali di morbilità e di disabilità e sono composti da oltre 150 malattie e sindromi; che colpisce circa 90 milioni di americani oggi. L&#39;infiammazione articolare e la distruzione ossea sono caratteristiche predominanti di disturbi muscolo-scheletrici, tra cui l&#39;artrite e l&#39;osteoporosi. L&#39;osteoporosi è una condizione che indebolisce l&#39;integrità ossea, causando spesso fratture delle ossa. L&#39;artrite è una malattia cronica, debilitante malattia c...

Malattie muscolo-scheletriche colpiscono milioni di persone negli Stati Uniti e presentare gravi conseguenze per i sistemi sanitari nazionali e locali 1. Questi disturbi sono caratterizzati da perdita di osso e funzione articolare che richiedono un trattamento estensivo e lunghi periodi di recupero. Comunemente, un relativo aumento del numero e / o attività di osteoclasti, cellule specializzate a riassorbire osso, osteoporosi ed artrite si osserva 2. In condizioni fisiologiche il numero e l&#39;attività degli osteoclasti è regolata da attivatore del recettore del fattore nucleare κ-B ligando (RANKL), che è prodotto dagli osteoblasti. Osteoprotegerina (OPG), un recettore decoy per RANKL è anche prodotta dagli osteoblasti 3 In modelli animali in vivo che coinvolgono la sovraespressione sistemica di sRANKL, o la cancellazione di OPG sono molto prezioso nella ricerca sull&#39;osteoporosi.; Tuttavia, questi metodi richiedono la generazione di topi transgenici 4,5. Qui, una nuova alternativametodo di iperespressione sRANKL per lo studio dei disturbi muscoloscheletrici legati è descritto. In particolare, minicircle (MC) tecnologia del DNA e metodi di consegna idrodinamiche sono stati usati per conseguire trasferimento genico di sRANKL in vivo e iperespressione del mouse sRANKL sistemicamente 6. Questo metodo è anche complementare ad altri modelli in vivo di osteoporosi, come la modulazione ormonale degli osteoclasti seguenti ovariectomia 7 e intervento dietetico da basso contenuto di calcio dieta 8. Questi modelli sono molto utili per studiare diversi aspetti dei disturbi muscoloscheletrici legati tuttavia essi richiedono procedure chirurgiche e può richiedere fino a diversi mesi, ad un costo significativo 9. Ovariectomizzate (OVX) modello di roditore è un modello animale sperimentale in cui la rimozione delle ovaie porta alla mancanza di estrogeni mimando così postmenopausale umano 10. Umano post-menopausa osteoporosi, una condizione in cui gli estrogeni deficirenza porta ad un aumento del rischio di fratture ossee e l&#39;osteoporosi colpisce circa otto milioni di donne negli Stati Uniti da soli. Anche se il modello OVX è utile per l&#39;osteoporosi post-menopausale che offre vantaggi limitati a studiare l&#39;osteoporosi in generale. Estrogeni sopprime perdita ossea, inducendo osteoclasti e osteoblasti inibizione dell&#39;apoptosi, quindi in sua assenza un aumento dell&#39;attività degli osteoclasti si osserva 10-12. Un rapporto di squilibrio RANKL-OPG che favorisce riassorbimento osseo si osserva anche 13. Tuttavia, la carenza di estrogeni in vivo è accompagnato anche da una diminuzione dei livelli di fattore di crescita trasformante β (TGF β), aumento della interleuchina-7 (IL-7) e TNF, IL-1 e IL-6 14,15. Poiché queste citochine hanno conosciuto funzioni modulatori rimodellamento osseo indipendente dal percorso che RANKL, è impossibile attribuire qualsiasi osteoclasti esclusivamente all&#39;asse RANKL-RANK. Il modello descritto in questo documento consente ai ricercatori di studiare in vivo l&#39;asse RANKL-RANK in osteoclastogenesi e la perdita di osso senza citochine pro-infiammatorie rispetto ai modelli di roditori OVX. Inoltre, le tecniche osteoclastogenesi in vitro sono strumenti essenziali per studiare l&#39;attivazione degli osteoclasti per i potenziali trattamenti terapeutici delle malattie muscolo-scheletriche. Precedenti studi hanno inoltre dimostrato che la coltura di midollo osseo di topo macrofagi derivati ​​(BMMs) con il mouse macrofagi fattore stimolante le colonie (M-CSF) e il mouse sRANKL può portare a osteoclasti differenziazione 3,16,17. Qui sono descritti i protocolli per generare cellule osteoclasti come multinucleate da midollo osseo di topo e di cellule mononucleari del sangue periferico (PBMC) in vitro 18. I saggi cellulari necessari per definire un maturo osteoclasti terminalmente differenziate e pienamente funzionale sono anche descritte brevemente. Queste tecniche in vitro completano il romanzo di approccio vivo e insieme fungono da pstrumenti investigativi owerful per studiare la differenziazione degli osteoclasti e l&#39;attivazione. L&#39;utilizzo di questi sistemi, gli scienziati sono in grado di generare osteoclasti in vivo e in vitro e definire gli stimoli e segnali necessari per la proliferazione e l&#39;attivazione e testare l&#39;efficacia di inibitori farmacologici e biologici.

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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.

Qui, una tecnica di trasferimento nuovo gene per la differenziazione degli osteoclasti in vivo e protocolli di coltura cellulare per differenziare le cellule precursori in osteoclasti in vitro come metodo per studiare gli effetti di citochine sulla osteoclastogenesis sono descritti. In Figura 1, sono mostrati i risultati rappresentativi di successo trasferimento genico di GFP e mouse sRANKL MC nei topi. Nella figura 2, vengono mostrate le immagini rappresentative del 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; Tecnica Gene Transfer e<em&gt; In vitro</em&gt; test cellulari per lo studio della perdita ossea nei disturbi muscoloscheletrici

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 Views.  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. 

Video: 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.

Una strategia per identificare le mutazioni de novo nei disturbi comuni come autismo e schizofrenia

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, ...

Ricerca

Medicina

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.

Istituzione e Propagazione dei Tumori retinoblastoma umani in topi privi di sistema immunitario

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.

La valutazione longitudinale di perdita dell&#39;arto posteriore ossea del mouse dopo la lesione del midollo spinale tramite Novel, In vivo, Metodologia

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.

Produzione e di rilevamento di specie reattive dell&#39;ossigeno (ROS) in Cancro

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.

Biomarcatori in un modello animale per Rivelare correlati neurali, ematologiche, e comportamentali di 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.

Un nuovo approccio chirurgico per somministrazione endotracheale di agenti bioattivi in ​​un modello murino fetale

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.

Sviluppo di un audio-based ambiente di gioco virtuale per assistere con doti di navigazione 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 ...

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.

Un romanzo ad alta risoluzione<em&gt; In vivo</em&gt; Tecnica di imaging per studiare la risposta dinamica di strutture intracraniche alla crescita del tumore e Terapeutica

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.

Una applicazione Novel di muscoloscheletrico Ultrasound Imaging

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.

Basata-Cell Assay Protocollo per la previsione prognostica della scoliosi idiopatica Uso Cellular dielettrica Spettroscopia

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