Questo lavoro è stato sostenuto dal Karolinska Institutet KID-finanziamento, Autorità svedese per la protezione dalle radiazioni, Karolinska Institutet Eye Research Foundation, Gun och Bertil Stohnes Stiftelse, Occhio di St. Erik Hospital Research Foundation, Ögonfonden, Konung Gustav V: s och Drottning Viktorias Frimurarstiftelse.

1. L&#39;esposizione alle radiazioni ultraviolette <ol><li> 15 minuti prima dell&#39;esposizione, anestetizzare una femmina Sprague-Dawley di ratto con una miscela di 90 mg / kg Ketalar (ketamina) e 10 mg / kg Rompun (xilazina) mediante iniezione intraperitoneale. </li><li> Posizionare l&#39;animale in un dispositivo di immobilizzazione ratto e tendere le cinghie fino immobilizzazione del ratto senza provocare una compressione del tronco 13. </li><li> Instillare Mydriacyl (tropicamide), 10 mg / ml, in entrambi gli occhi di ratto per indurre midriasi. </li><li> Posizionare l&#39;animale in modo che un occhio è posizionato contro un fascio di raggi UV a 300 nm 14 con larghezza di 10 nm a metà del massimo e schermare l&#39;occhio controlaterale con nastro nero. </li><li> Esporre il ratto unilateralmente sub-dose soglia 1 kJ / m 2 raggi UV-B a 300 nm per 15 min 15. </li></ol> 2. Dissezione <ol><li> Dopo intervallo predeterminato post esposizione (1, 5, 24 e 120 ore), sacrificare il topo sei settimane di età (peso &lt;200 g) da carbon biossido di asfissia e dislocazione delle vertebre cervicali. </li><li> Enucleare gli occhi. Successivamente, inserire la cornea degli occhi verso il basso, lato posteriore in alto. Poi, un buco minima applicando una cannula 27 gauge e spingere tangenzialmente alla sclera per evitare di danneggiare la lente che è molto vicino alla sclera. Quindi utilizzare un paio di forbici per tagliare chirurgia oftalmica circonferenzialmente appena dietro il limbus finché la parte posteriore della sclera può essere sollevato. Quindi, sollevare la lente con una pinza contundenti curve. </li><li> Rimuovere residui del corpo ciliare dall&#39;equatore lente al microscopio, mantenendo la lente in soluzione salina bilanciata di non più di 5-10 minuti. </li></ol> 3. RT-PCR quantitativa <ol><li> Inserire un obiettivo in una miscela di 350 microlitri RA1 tampone di lisi (II NucleoSpin RNA totale isolamento dell&#39;RNA kit) e 3,5 microlitri β-mercaptoetanolo in una provetta Eppendorf da 2 ml. </li><li> Mantenere l&#39;obiettivo in questa miscela per 30 min a temperatura ambiente. </li><li> Omogeneizzare la lente conun ago fino solo nucleo duro della lente viene lasciata dalla lente (corteccia e della capsula della lente vengono lisate in tampone di lisi). Rimuovere il nucleo dal mix. </li><li> Conservare i campioni immediatamente a -70 ° C. </li><li> Scongelare i campioni e seguire la &quot;purificazione RNA totale da cellule in coltura e tessuti&quot; protocollo (NucleoSpin RNA II RNA totale Kit di isolamento). </li><li> Conservare i campioni di RNA a -70 ° C. </li><li> Per verificare sufficiente asportazione di DNA da ogni campione, eseguire una PCR nelle seguenti condizioni (passo 1: 95 ° C per 2 min; step 2 per 40 cicli: 95 ° C per 20 sec, 55 ° C per 20 sec, 72 ° C per 20 sec; passaggio 3: 72 ° C per 7 min) con primers specifici DNA di p53, forward 5&#39;-ACCCTCTGACCTTTTTCCCA-3 &#39;e 5&#39;-inversa TGCTGGGATCTTAGGCACTC-3&#39; e la DNA polimerasi Taq (dNTPack), secondo la fabbricazione protocollo. Il prodotto di PCR previsto è lungo 243 coppie di basi. </li><li> Eseguire un 1,5% elettroforesi su gel di agarosio per rilevare il DNA specifico prodotto di PCR di 243 paia di basi. Per preparare1,5% gel di agarosio in tampone TBE tener 3,75 g di agarosio e risolvere in 250 ml di tampone TBE. Aggiungi etidio bromuro (0,5 mg / ml) 500 microlitri in 500 ml di soluzione 1,5% gel di agarosio. Riscaldare il miscuglio in un forno a microonde e mescolare per risolvere l&#39;agarosio. </li></ol> Nessuno dei campioni deve rivelare eventuali specifiche di DNA prodotti PCR su 1,5% gel di agarosio. <ol start="9"><li> Misurare la concentrazione di campioni di RNA (1 microlitri) e il rapporto di assorbanza del campione a 260 nm (RNA) e 280 nm (proteina) in una NanoDrop ND-1000 spettrofotometro. Se l&#39;RNA rapporto alle proteine ​​di densità ottica dei campioni di RNA è di 2,0 o più, quindi il campione di RNA è puro. Se il rapporto di assorbanza del campione RNA è inferiore a 2,0, ripetere RNA purificazione 3,5. </li><li> Prendere un volume di campione di RNA corrispondenti a 1 ug e sintetizzare cDNA seguito Kit 1 Strand protocollo di sintesi di cDNA st (1 st Strand cDNA Kit sintesi per RT-PCR). </li><li> Conservare campioni di cDNA a -20 ° C(Per un periodo di 1 o più anni, conservare a -70 ° C). </li><li> Eseguire quantitativa real-time PCR su iCycler Colore unico MyiQ sistema in tempo reale di rilevamento PCR. Triplica carico su piastra a 96 pozzetti con 1 campione microlitri di cDNA, TaqMan Gene Expression Master Mix, TaqMan Gene Expression Assay per caspasi 3, secondo le istruzioni fabbricazione. Carico triplica su un&#39;altra piastra a 96 pozzetti con 1 campione microlitri di cDNA, TaqMan Gene Expression Master Mix, saggio TaqMan Gene Expression per minori di 18 anni, secondo le istruzioni per la produzione (Protocollo TaqMan Gene Expression Assay). Diluire in serie una frazione di cDNA da tre scelte a caso non esposti lenti. Eseguire diluizioni seriali insieme con il cDNA da campioni in una piastra a 96 pozzetti. </li><li> Utilizzare il metodo standard della curva per la quantificazione dei risultati. Il numero di cicli a fluorescenza soglia viene utilizzata come misura nel software MyiQ. Una curva standard che esprime il numero di cicli al livello di soglia in funzione della concentrazione relativa di calibratore è esnoltre stabilito per le diluizioni seriali in ogni piatto. cDNA da tre campioni non trattati lente è stato utilizzato per la calibrazione. Riguardano il numero di cicli di soglia per ciascun campione per la curva standard per ottenere la concentrazione relativa del campione di cDNA misurata. Infine, ottenere il livello di espressione dei geni bersaglio mediante la relativa concentrazione relativa del bersaglio cDNA ai 18s controllo interno cDNA. </li></ol> 4. Colorazione immunoistochimica <ol><li> Fissazione <ol><li> Staccare l&#39;occhio in PBS (tampone fosfato salino, pH 7,4). </li><li> Messo l&#39;occhio in un tubo riempito con 4% paraformaldeide (PFA) in freddo ghiaccio per 20 min. Preparare PFA 4% il giorno prima. Conservare a +4 ° C fino all&#39;uso. PFA è fresco per circa una settimana. </li><li> Rimuovere PFA con una micropipetta. Poi, lavare con PBS freddo ghiaccio per 20 min. </li><li> Mettere l&#39;occhio in saccarosio 30% a +4 ° C durante la notte. </li><li> Mettere l&#39;occhio in una tazza piena di temperatura di taglio ottimale (OCT)-medio (Tissue-Tek). Messo l&#39;occhio in una posizione adatta per il sezionamento. </li><li> Congelare in OCT-media a ghiaccio secco. </li><li> Conservare a -70 ° C fino all&#39;uso. </li></ol></li><li> Sezionamento <ol><li> Posizionare l&#39;occhio per la crio-sezionamento in un piano adeguato. </li><li> Tagliare tre 5 micron di spessore sagittale sezioni da una porzione centrale di ciascuna lente. Scartare almeno 6 sezioni tra sezioni sequenziali per evitare di macchiare i nuclei stessa in diverse sezioni. </li><li> Una volta che una sezione è stata tagliata, delicatamente posizionare un vetrino su di esso. La sezione deve quindi attenersi alla diapositiva. </li><li> Lasciare i vetrini asciugare all&#39;aria prima dell&#39;uso. Vetrini possono essere conservati a -20 ° C fino all&#39;utilizzo. </li></ol></li><li> Fluorescenza immunoistochimica <ol><li> Lasciate che i campioni raggiungere temperatura ambiente (5-10 minuti). </li><li> Disegnare bordi attorno ai campioni con un PAP-pen (Invitrogen). </li><li> Lasciate che il confine a secco per un po &#39;(10 min). </li><li> Etichettare i vetrini da microscopio. </li><li> Reidratare in 1 × PBS per 15 min. </li><li> Permeabilize in soluzione standard di blocco per 30 min. </li><li> Aggiungere l&#39;anticorpo primario (policlonale di coniglio, spaccati caspasi-3 anticorpo Asp175 9661; segnalazione Cell Technology, Inc) diluito 1:3000 in soluzione standard di blocco. </li><li> Conservare in una camera umidificata a 4 ° C durante la notte. </li><li> Lavare in PBS pipettando (3 × 5 min) o immergendolo in un bagno di grandi dimensioni (&gt; 15 min, 1-2 modifiche). </li><li> Aggiungi anticorpo secondario (anticorpo di coniglio anti secondaria con un assorbimento specifico / spettro di emissione) diluito 1:300 nella soluzione standard di blocco. </li><li> Lavare in PBS pipettando (3 × 5 min) o immergendolo in un bagno di grandi dimensioni (&gt; 15 min, 1-2 modifiche). </li><li> Pulire PAP-penna strisci. </li><li> Memorizzare in una camera umidificata a temperatura ambiente per 3 ore. Evitare l&#39;esposizione alla luce. </li><li> Aggiungere una goccia di Vectashield e mettere un coprioggetto sul vetrino. Evita di fare le bolle. </li><li> Lasciare indurire Vectashield (~ 20 min). </li><li> Tenere sezioni in al buio fino al momento dell&#39;analisi. </li><li> Tutte le sezioni di controllo vengono elaborati in assenza di anticorpo primario. </li><li> Cercare i risultati in poche ore sotto un microscopio a fluorescenza. </li><li> Contare nuclei delle cellule epiteliali &#39;da un fiocco laterale nucleare a prua dall&#39;altra parte nucleare di ogni lente. Applicare il filtro blu standard per vedere tutte nuclei epiteliale lente in blu e un filtro standard che si inserisce l&#39;emissione dell&#39;anticorpo secondario per vedere la caspasi-3 nuclei positivi in ​​verde. </li><li> Registrare il numero di tutti i nuclei lente epiteliale e il numero di nuclei positivi. Contare le cellule tre volte per ogni sezione. </li></ol></li></ol>

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<html> Questo articolo descrive i metodi che consentono di studiare gli eventi molecolari che si verificano durante raggi UV-B indotta da cataratta. Considerando, che più le informazioni disponibili in vivo UVR cataratta indotta è stato derivato da esperimenti su albino Sprague-Dawley 7, 16, 17, 18, ​​19, abbiamo deciso di utilizzare l&#39;albino ratto Sprague-Dawley nel presente studio. Età dei ratti aveva sei settimana fa. Il genere è stato scelto per essere femm...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> Ketalar </td><td> Pfizer </td><td> 150086 </td><td> 50 mg / ml </td></tr><tr><td> Rompun </td><td> Bayer </td><td> 022545 </td><td> 20 mg / ml </td></tr><tr><td> Oculentum Simplex </td><td> Apoteket, Svezia </td><td> 336164 </td><td> 5 g </td></tr><tr><td> Mydriacyl </td><td> Alcon </td><td> 00352 </td><td> 10 mg / ml </td></tr><tr><td> Soluzione salina bilanciata </td><td> Alcon </td><td> 0007950055 </td><td></td></tr><tr><td> 3,5 ul β-mercaptoetanolo </td><td></td><td></td><td></td></tr><tr><td> NucleoSpin RNA II RNA totale Kit di isolamento </td><td> Macherey-Nagel GmbH &amp; Co, Düren, Germania </td><td> 740.955,50 </td><td></td></tr><tr><td> p53 primer specifici del DNA </td><td> biomers.net GmbH </td><td> Una soluzione su misura </td><td></td></tr><tr><td> Taq DNA polimerasi, dNTPack </td><td> Roche </td><td> 04 728 866 001 </td><td></td></tr><tr><td> Agarosio </td><td> Sigma </td><td> A5093 </td><td></td></tr><tr><td> Bromuro di etidio soluzione 0,5 mg / ml </td><td> Sigma </td><td> E1385 </td><td></td></tr><tr><td> Nano-Drop ND-1000 spettrofotometro </td><td> Prodotti NanoDrop </td><td></td><td></td></tr><tr><td> 1 ° Strand cDNA kit di sintesi per RT-PCR (AMV) </td><td> Roche Diagnostics GmbH </td><td> 11 483 188 001 </td><td></td></tr><tr><td> iCycler MyiQ Colore unico reale sistema di rilevamento Time PCR </td><td> Bio-Rad Laboratories </td><td></td><td></td></tr> &lt;tr&gt; <td> Piastra a 96 pozzetti </td><td> Sarstedt </td><td> 72.1979.202 </td><td></td></tr><tr><td> TaqMan Gene Expression Master Mix </td><td> Applied Biosystems </td><td> 4369016 </td><td></td></tr><tr><td> TaqMan Gene Expression Assay per caspasi 3 </td><td> Applied Biosystems </td><td> Rn00563902_m1 </td><td></td></tr><tr><td> TaqMan Gene Expression Assay per minori di 18 anni </td><td> Applied Biosystems </td><td> Hs99999901_s1 </td><td></td></tr><tr><td> MyiQ software </td><td> Bio-Rad Laboratories </td><td></td><td></td></tr><tr><td> Cleaved caspasi-3 (Asp175) </td><td> Tecnologia delle celle di segnalazione </td><td> 9661 </td><td></td></tr><tr><td> Anti IgG di coniglio </td><td> Abcam </td><td> Ab6798 </td><td></td></tr><tr><td> Saccarosio </td><td> Sigma Aldrich </td><td> 84097 </td><td></td></tr&gt; <tr><td> Vectashield </td><td> Vector Laboratories </td><td></td><td></td></tr><tr><td> Microscopio Universale Axioplan 2 Imaging </td><td> Carl Zeiss </td><td></td><td></td></tr><tr><td> Paraformaldeide </td><td> Sigma Aldrich </td><td> 441244 </td><td></td></tr><tr><td> Buffer TBE 10x </td><td> Promega </td><td> V4251 </td><td></td></tr></tbody></table>

characterization of molecular mechanisms of in vivo uvr induced cataract

La cataratta è la principale causa di cecità nel mondo 1. L&#39;Organizzazione Mondiale della Sanità definisce cataratta come una opacità del cristallino dell&#39;occhio che impedisce il trasferimento della luce. La cataratta è una patologia multifattoriale associata al diabete, il fumo, le radiazioni ultraviolette (UVR), alcool, radiazioni ionizzanti, gli steroidi e l&#39;ipertensione. Vi è una forte evidenza sperimentale 2-4 e 5,6 epidemiologica che UVR causa della cataratta. Abbiamo sviluppato un modello animale per UVR B cataratta indotta sia anestetizzati 7 e non animali anestetizzati 8. L&#39;unica cura per la cataratta è l&#39;intervento chirurgico, ma questo trattamento non è accessibile a tutti. È stato stimato che un ritardo di insorgenza della cataratta per 10 anni potrebbe ridurre la necessità di chirurgia della cataratta del 50% 9. Per ritardare l&#39;incidenza di cataratta, è necessario per comprendere i meccanismi di formazione della cataratta e trovare strategia efficace di prevenzionegie. Tra i meccanismi di sviluppo della cataratta, apoptosi svolge un ruolo cruciale nella apertura di cataratta nell&#39;uomo e negli animali 10. La nostra attenzione è stata recentemente apoptosi nella lente come meccanismo per la cataratta sviluppo 8,11,12. Si prevede che una migliore comprensione dell&#39;effetto della UVR sul percorso apoptosi fornirà possibilità per la scoperta di nuovi farmaci per prevenire la cataratta. In questo articolo, si descrive come la cataratta può essere indotta sperimentalmente dalla esposizione in vivo a raggi UV-B. Inoltre RT-PCR e immunoistochimica sono presentati come strumenti per lo studio dei meccanismi molecolari di raggi UV-B indotta da cataratta.

Le varie fonti di variazione delle misurazioni sono stati stimati con l&#39;analisi della varianza e si è constatato che, considerando tre misurazioni per animale, la varianza per misure era dell&#39;ordine del 15% di quella per gli animali. Pertanto, considerando intera analisi lente, non è possibile aumentare la precisione. Test ortogonale chiarito un contrasto statisticamente significativo per la caspasi-3 messaggio tra 120 intervallo di latenza ore rispetto intervalli di latenza inferiori. <p class="jove_conte...

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Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

La cataratta è la principale causa di cecità nel mondo. Radiazione ultravioletta solare (UVR) è il principale fattore di rischio per lo sviluppo della cataratta. Un modello animale di gran lunga raggi UV-B indotta da cataratta è stato sviluppato. In questo articolo si descrivono i metodi di indagine della cataratta: l&#39;esposizione a raggi UV, RT-PCR e immunoistochimica.

Caratterizzazione dei meccanismi molecolari di<em&gt; In vivo</em&gt; UVR indotta cataratta

Cataract is the leading cause of blindness in the world 1. The World Health Organization defines cataract as a clouding of the lens of the eye which ...

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JoVE Journal

Medicine

Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

9.8K Views.  Karolinska Institutet. La cataratta è la principale causa di cecità nel mondo. Radiazione ultravioletta solare (UVR) è il principale fattore di rischio per lo sviluppo della cataratta. Un modello animale di gran lunga raggi UV-B indotta da cataratta è stato sviluppato. In questo articolo si descrivono i metodi di indagine della cataratta: l'esposizione a raggi UV, RT-PCR e immunoistochimica.

Video: Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

In vivo Near Infrared fluorescenza (NIRF) intravascolare Imaging Molecolare della placca infiammatoria, un approccio multimodale per Imaging di aterosclerosi

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

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Medicina

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

Un In vivo Modello di roditore della contrazione indotta Lesioni e monitoraggio non invasivo di recupero

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19 F MRI per il tracciamento cellulare

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

Molecular Profiling del microambiente tumorale invasiva in un modello 3-Dimensional delle cellule del cancro colorettale e<em&gt; Ex vivo</em&gt; fibroblasti

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemioterapia-indotta vascolare Tossicità - in tempo reale<em&gt; In vivo</em&gt; Imaging di Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

L&#39;uso di ultra-alto campo MRI in piccolo roditore modelli di malattia del rene policistico per<em&gt; In Vivo</em&gt; Fenotipizzazione and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

Mouse Model of Alloimmune-induced Vascular Rejection and Transplant Arteriosclerosis

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune system of the recipient causes damage of the arterial wall and dysfunction of endothelial cells and smooth muscle cells. This triggers a pathological repair response that is characterized by intimal thickening and luminal occlusion. Understanding the mechanisms by which the immune system causes vasculature rejection and TA may inform the development of novel ways to manage graft failure. Here, we describe a mouse aortic interposition model that can be used to study the pathogenic mechanisms of vascular rejection and TA. The model involves grafting of an aortic segment from a donor animal into an allogeneic recipient. Rejection of the artery segment involves alloimmune reactions and results in arterial changes that resemble vascular rejection. The basic technical approach we describe can be used with different mouse strains and targeted interventions to answer specific questions related to vascular rejection and TA.

We describe a protocol for aortic interposition grafting in mice. The goal of the protocol is to provide a model with which to study pathological processes and therapeutic strategies relevant to alloimmune reactions in arteries and the resultant arterial changes that contribute to organ transplant failure.

Modello murino della alloimmune indotta Rifiuto vascolare e trapianto Arteriosclerosi

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

Istituzione e caratterizzazione di UTI e CAUTI in un modello murino

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

L&#39;esaurimento delle cellule di topo da xenotrapianti tumorali umani migliora in modo significativo Analisi A valle delle cellule bersaglio

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Ileectomy-induced Bile Overaccumulation in Mouse Intestine

Intestinal resection is a common therapeutic approach for human diseases such as obesity, inflammatory bowel disease, Crohn's disease, and colon cancer that often results in severe short bowel syndrome-like adverse effects including bile acid diarrhea, dehydration, electrolyte disturbances, and nutrient malabsorption. Here we introduce a murine ileal resection model, termed ileectomy, to evaluate tissue communication and the maintenance of systemic homeostasis. After ileal resection, circulating blood is permanently devoid of the ileum-specific endocrine hormone fibroblast growth factor 15 (FGF15), which releases its endocrinal inhibition of bile acid synthesis in the liver. In combination with the increased production and abolished reabsorption of bile acids after removing the ileum, mice that underwent surgery suffer from bile salt overaccumulation in the intestine and associated diarrhea, morbidity, and mortality. Novel usage of the surgery model introduced in this study may provide mechanistic and functional insights into ileal control of systemic metabolic regulation in physiology and disease.

Small intestine-dependent bile acid reabsorption and feedback inhibition of hepatic bile acid synthesis is important for systemic homeostasis and health. In this study, we describe a mouse model for ileal resection to evaluate ileectomy-induced bile malabsorption, overaccumulation, and toxicity in mouse intestine.

Overaccumulation ileectomy-indotta della Bile nell'intestino del Mouse

Intestinal resection is a common therapeutic approach for human diseases such as obesity, inflammatory bowel disease, Crohn's disease, and colon cancer ...