We thank the imaging platform of the Institut de la Vision. We acknowledge the French Muscular Dystrophy Association (AFM) for a PhD fellowship to O.V. and Allergan INC. This work performed in the frame of the LABEX LIFESENSES [reference ANR-10-LABX-65] was supported by French state funds managed by the ANR. We thank Peggy Barbe, and M&#233;lissa Desrosiers for technical assistance with AAV preparations. We are grateful to St&#233;phane Fouquet for excellent technical assistance in confocal microscopy and his expert input with the interpretation of the results.

Tutti gli animali utilizzati in questo studio sono stati curati e trattati secondo la dichiarazione ARVO per l&#39;uso di animali in Oftalmica e Vision Research. 1. Produzione di ricombinante AAV (rAAV) per Transient trasfezione di cellule HEK-293 17,18 NOTA: Vedere McClure C, JoVE (2011) 19. <ol><li> Purificare larga scala Miniprep (almeno 1 mg / ml) dei plasmidi vettore AAV. Utilizzare 3 plasmidi. Il plasmide AAV helper trasportano i geni del capside replica e senza terminale invertita AAV ripete ITR, la cassetta di espressione del transgene trasporta la AAV ITR denominato plasmide di trasferimento, e il plasmide fornendo i geni adenovirus helper E2, E4, e VA geni RNA contemplato come pHELPER. </li><li> Trasfezione 293 cellule con questi 3 plasmidi usano polietilenimina (o altro reagente trasfezione). Sopra 80% di efficienza di trasfezione è necessario per una buona vii rendimenti RAL. </li><li> Purificare AAV ricombinante da 293 lisati cellulari su una pendenza iodixanolo. 15%, 25%, 40% e 60% iodixanolo viene utilizzato per ottenere il gradiente. Colleziona 40% frazione iodixanolo contenente particelle AAV, concentrare la frazione dopo ultracentrifugazione a 500.000 xg per 1 ora e buffer di scambio contro PBS (tampone fosfato salino) con il 0,001% Pluronic. </li><li> Per determinare virale titolo genomico 20, eseguire un DNAse ProteinaseK digest seguito da QPCR. I primer avanti e invertire amplificano la regione 62 bp situata nel AAV2 ITR. Per lo standard plasmide, la sonda è marcato con FAM e ha un buco nero quencher (BHQ). </li><li> Eseguire qPCR (reazione a catena della polimerasi quantitativa) in un volume finale di 25 ml con 340 Nm invertire ITR fondo, 100 nM ITR avanti fondo, 100 nM sonda AAV2 ITR, 0.4 dNTPs mm, 2 mM MgCl2, 1x Platinum Taq Buffer, 1U Platinum Taq e mascherina 5 microlitri (standard, campione e nessun controllo modello). </li><li> Perlo standard, utilizzare il plasmide per la trasfezione in 7 x 10 diluizioni seriali (5 ml ciascuno di 2 x 10 9 2 x 10 3 genomi / ml risultanti in una concentrazione finale 10 ottobre-4 ottobre genomi / ml). </li><li> Iniziare il programma da una fase di denaturazione iniziale a 95 ° C per 15 min seguita da 40 cicli di denaturazione a a 95 ° C per 1 min e ricottura / estensione a 60 ° C per 1 min. Conservare a 4 ° C per una conservazione a breve termine oa -20 ° C per lunghi periodi di stoccaggio. </li></ol> 2. iniezione intravitreale di AAV <ol><li> Anestetizzare topi C57BL6J con ketamina (50 mg / kg) e xilazina (10 mg / kg) e verifica la perdita del riflesso di raddrizzamento, il ritiro e la coda reflex pinch risposta che indica il corretto anestesia. Dilatare gli alunni per l&#39;applicazione della cornea del neosynephrine 5% e il 0,5% mydriaticum collirio. Utilizzare veterinario pomata sugli occhi per prevenire la secchezza mentre sotto anestesia. </li><li> Passare un ultrafine 30 G disposable ago attraverso l&#39;equatore e vicino al limbus, in camera vitreale (vedi Chiu K, JoVE (2007) 21). Iniettare 1 ml magazzino contenente da 1 a 4 x 10 11 vp di ShH10-GFP o AAV5-GFP con l&#39;osservazione diretta dell&#39;ago al centro della cavità vitreale (Figura 1). Utilizzare un occhio come controllo per esperimenti ILM. Iniettare entrambi gli occhi per esperimenti BRB. Applicare un trattamento topico antinfiammatoria e antibatterica sugli occhi iniettati. </li><li> Dilatare alunni neosynephrine e mydriaticum collirio per l&#39;imaging. Eseguire l&#39;esame del fondo con una fotocamera fundus dell&#39;occhio di seguire GFP (Green Fluorescent Protein) espressione 1 settimana, 2 settimane, 1 mese e 2 mesi dopo l&#39;iniezione intravitreale (Figura 1). Sacrificio topi da CO 2 inalazione 1 a 2 mesi dopo l&#39;iniezione. </li></ol> 3. Immunoistochimica <ol><li> Per criosezioni retina (Figura 1), sezionare occhi enucleati per rimuovere lens e della cornea, e l&#39;immersione correzione in 4% paraformaldeide per 1 ora. <ol><li> Cryoprotect gli occhi fissi precedentemente a 10% di saccarosio per 1 ora a temperatura ambiente, 20% di saccarosio per un&#39;altra ora a temperatura ambiente. Cryoprotect in soluzione di saccarosio al 30%, overnight a 4 ° C. Congelare e paraocchi incorporare in embedding resina, come Cryo-matrix. Fare 10 micron criosezioni e montare su vetrini appositamente trattati per sezioni di tessuto congelato e che migliorano l&#39;aderenza del tessuto durante la colorazione. </li><li> Permeabilize sezioni con 0,1% Triton X100 in PBS pH 7,4 per 5 min. Lavare 2x in PBS e blocco per 1 ora a temperatura ambiente in PBS con 1% BSA, 0,1% Tween 20. </li></ol></li><li> Per flatmounts retina (Figura 1), fissare gli occhi enucleati in 4% paraformaldeide per 15 min per facilitare la dissezione. <ol><li> In 15-30 min, sezionare gli occhi per rimuovere la cornea e la lente. Separare la retina da dell&#39;epitelio pigmentato retinico (RPE) e sclera tagliando intorno alla serrata eil nervo ottico. Immergere in 4% paraformaldeide per altri 30 minuti per fissare la retina e la proteina fluorescente in cellule retiniche trasdotte (fissaggio non deve superare 24 ore). Lavare 5 min in PBS sterile, pH 7.4. </li><li> Cambiare il PBS al tampone di bloccaggio (PBS con 1% BSA, 2% di capra normale o siero asino, 0,5% Triton X-100), e incubare in tampone di bloccaggio sia per 4 ore a temperatura ambiente oa 4 ° C durante la notte. </li></ol></li><li> Incubare tessuti con anticorpi primari in tampone di bloccaggio sia per 4 ore a temperatura ambiente o in una notte a 4 ° C. Lavare 3x con PBS. NOTA: Gli anticorpi utilizzati sono i seguenti: anti-laminina (1 / 1.000) etichettatura della ILM, clone anti-rodopsina 4D2 (1/500) aste di etichettatura, anti-glutammina sintetasi clone GS-6 (1 / 1.500) di etichettatura cellule Müller , PNA Lectin (1/40) coni di etichettatura. </li><li> Incubare tessuti con diluizione 1: 500 di anticorpi secondari coniugati fluoro Alexa in tampone di bloccaggio per 1 ora (criosezioni) o 2 ore (flatmounts) presso la sala tempetura e al riparo dalla luce. Lavare 3x con PBS. </li><li> Effettuare tagli alleviare alla retina e flatmount è su un vetrino o con il fotoricettore o cellule gangliari della retina (RGC) lato rivolto verso l&#39;alto. Aggiungi mountingmedium acquosa, applicare il coprioggetto e conservare a 4 ° C. </li><li> Eseguire microscopia confocale a scansione laser confocale. Acquisire le immagini in sequenza, riga per riga, per ridurre l&#39;eccitazione e crosstalk emissione. Definire passo secondo il teorema del campionamento di Nyquist-Shannon. Utilizzare le impostazioni di esposizione che riducono al minimo i pixel troppo saturi nelle immagini finali. Elaborare immagini a 12 bit con le Figi, progetto Z-sezioni su un unico piano con la massima intensità in funzione Z-progetto ed infine convertire in modalità colore RGB a 8 bit. </li></ol> 4. PCR analisi di campioni di sangue del mouse <ol><li> Iniettare 100 microlitri magazzino contenente da 1 a 4 x 10 11 particelle di un AAV-GFP nella vena del pene di topi anestetizzati C57BL6J (5% di isofluranoper l&#39;induzione in una camera stretta e 2,5% di isoflurano attraverso un cono naso durante l&#39;intervento chirurgico) utilizzando una siringa da insulina con un ultra-fine ago 6 millimetri. Questo animale servirà come controllo positivo di far circolare le particelle AAV. </li><li> Campione di sangue dalla coda di topo prima dell&#39;iniezione e 3 ore, 24 ore, 2 giorni e 3 giorni dopo l&#39;iniezione intravitreale ShH10 o dopo l&#39;iniezione intrapenile (Figura 1). Circa il 10-20 microlitri è raccolto in tubi eparina. Sacrificio topi da CO 2 inalazione dopo il completamento della procedura. </li><li> Estratto DNA genomico da campioni di sangue con il kit di estrazione di vostra scelta. </li><li> Eseguire PCR amplificazione del DNA genomico. Per questo protocollo utilizzare la seguente coppia di primer: GFP, senso 5&#39;-CGACACAATCTGCCCTTTCG-3 &#39;, antisenso 5&#39;-CATGGACGAGCTGTACAAGGGA-3&#39;. Il seguente programma PCR è stata effettuata: 95 ° C 2 min (1 ciclo); 95 ° C 45 sec, 55 ° C 1 min, 72 ° C 45 sec (30 cicli); 72 ° C 5 min (1 ciclo);4 ° C in attesa. </li></ol> 5. Opzionale Evans Metodo Blu NOTA: quantificare permeabilità vascolare misurando fuoriuscita di albumina dai vasi sanguigni nella retina con il metodo blu Evans 10. <ol><li> Preparare Evans blu colorante per dissoluzione in una soluzione salina normale (6 mg / ml), sonicazione per 5 minuti in un bagno ad ultrasuoni, e filtrazione attraverso un filtro 5 micron. </li><li> Anestetizzare topi C57BL6J (isoflurano inalazione, vedere il punto 4.1), controllare la perdita del riflesso di raddrizzamento, il riflesso di ritiro e la risposta coda pizzico indica una corretta anestesia, e iniettare Evans blu (45 mg / kg), attraverso la vena del pene (Figura 1). Verificare che le zampe, il muso e le orecchie di topi diventano chiaramente blu dopo l&#39;iniezione blu Evans, confermando l&#39;assorbimento e la distribuzione del colorante. </li><li> Anestetizzare topi C57BL6J 3 ore dopo l&#39;iniezione del colorante con ketamina (50 mg / kg) e xilazina (10 mg / kg) e verifica la perdita di righting reflex indicando una corretta anestesia. Esempio intracardiaca circa 500 ml di sangue in tubi eparina e nei profumi topi per 2 min attraverso il ventricolo sinistro con un tampone citrato (0,05 M, pH 3,5 e sciogliere 7,8 g di acido citrico e 3,8 g di citrato di sodio in 1 L di acqua distillata) preriscaldata a 37 ° C per evitare vasocostrizione. I topi sono sacrificati tramite perfusione. </li><li> Dopo la perfusione, enucleate e con attenzione sezionare entrambi gli occhi sotto un microscopio operatorio per raccogliere le retine (vedi Sezione 3.2). Retine secco in un evaporatore centrifuga durante la notte, pesano, ed estrarre il colorante blu Evans incubando retine con 100 ml di formammide per 18 ore a 65 ° C con agitazione (100 xg). Campioni Centrifuga retina a 30K tubi filtranti omega a 20,798.5 xg per 2 ore a 4 ° C e campioni di sangue centrifugare a 20,798.5 xg per 15 min a 4 ° C. </li><li> Usare entrambe surnatanti per misurare l&#39;assorbanza. Determinare l&#39;assorbanza di ciascun campione sottraendo il massimo assorbanza for Evans blu a 620 nm al minimo assorbanza a 740 nm. Calcola Evans concentrazione blu nel plasma e della retina di una curva standard di Evans blu in formammide. Esprimere BRB permeabilità in microlitro di Evans blu per grammo di retina secca per ora (ml di plasma / g retinica peso secco / hr). </li></ol> 6. Facoltativo Fluoresceina angiografia NOTA: Perdita dai vasi sanguigni della retina nei topi può essere visualizzato mediante iniezione intraperitoneale di fluoresceina sodica. <ol><li> Anestetizzare topi C57BL6J (isoflurano inalazione, vedere il punto 4.1) e dilatare gli alunni con collirio (neosynephrine e mydriaticum). Utilizzare veterinario pomata sugli occhi per prevenire la secchezza mentre sotto anestesia. </li><li> Iniettare intraperitoneale 0,01-0,02 ml 10% fluoresceina sodica in 0,1 ml di soluzione fisiologica sterile per fluorangiografia. </li><li> Raccogliere le immagini di entrambi gli occhi dopo l&#39;iniezione utilizzando una telecamera fundus dell&#39;occhio. Sacrificio topi da CO 2 inalazione dopo la proprocedura. NOTA: 20 sec sono necessari per i vasi retinici a diventare visibile su angiografia. Le immagini devono essere catturato tra 3-10 min massimo dopo l&#39;iniezione di fluorescina. I punti di fuga (se presenti) sono osservabili dopo 2.5 min. Al più tardi momenti la qualità dell&#39;immagine è perso a causa di fluoresceina sodica diffusione nel vitreo, quindi solo immagini poco ottenuti dopo sono utilizzati per l&#39;analisi. </li></ol>

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The authors have nothing to disclose.

La BRB regola lo scambio di molecole tra sangue e retina. La sua composizione è associata a diverse malattie come la retinopatia diabetica o la degenerazione maculare legata all&#39;età (AMD). Abbiamo recentemente dimostrato che in un topo distrofina knock-out, che visualizza permeabile BRB, la retina diventa più permissiva di consegna del gene mediato da vettori virali adeno-associati (AAV). Tuttavia, nonostante BRB particelle permeabilità AAV iniettato intraoculare rimanere confinata al compartimento oculare in qu...

Müller cellule sono il principale componente gliale della retina. Morfologicamente, che abbracciano la retina radiale e la loro endfeet, in contatto con il vitreo, affrontano la ILM e componenti segreti di quest&#39;ultima. La ILM è una membrana basale composta da una decina di diverse proteine ​​della matrice extracellulare (laminina, agrin, perlecan, nidogen, collagene e proteoglicani diversi solfato eparina). Durante lo sviluppo, la sua presenza è indispensabile per istogenesi retinica, la navigazione di assoni ottica, e la sopravvivenza delle cellule gangliari 1-3. Tuttavia, ILM è inessenziale in retina adulta e può essere rimosso chirurgicamente in alcune patologie, senza causare danni alla retina 4. Nella terapia genica, questa membrana diventa una barriera fisica per la trasduzione efficiente della retina mediante AAV per iniezione intravitreale 5. Attraverso l&#39;ampia arborizzazione dei loro processi, le cellule Müller forniscono Suppo nutrizionale e regolamentarert per entrambi i neuroni della retina e cellule vascolari. Cellule Müller sono anche coinvolti nella regolazione dell&#39;omeostasi retinica, nella formazione e mantenimento del BRB 6. Giunzioni strette tra le cellule endoteliali dei capillari della retina, Müller cellule, astrociti e periciti formano la BRB. BRB previene alcuni sostanze di entrare i retina.In molte malattie come la retinopatia diabetica, occlusione venosa retinica e malattie respiratorie, ipossia della retina provoca perdita attraverso la BRB 7-9. Questa rottura è associato ad un aumento della permeabilità vascolare che porta a edema vasogenico, distacco della retina e danni alla retina. Cellule Müller sono strettamente associati con i vasi sanguigni e la membrana basale, svolgendo un ruolo importante sia l&#39;integrità BRB e ILM. Di conseguenza, l&#39;etichettatura cellule gliali Müller è particolarmente rilevante per lo studio dello stato fisico di queste barriere retiniche. Classicoalleato, BRB permeabilità viene misurata usando il saggio blu Evans costituito da iniezione sistemica di Evans colorante blu, che lega non covalente all&#39;albumina plasmatica. Questo test misura la perdita di albumina (proteine ​​di dimensioni intermedie, ~ 66 kDa) dai vasi sanguigni nella retina (vedi Protocolli sezione 5) 10. In alternativa, la perdita vascolare può essere visualizzato con la fluorescenza angiografia retinica attestante l&#39;assenza di perdite di fluoresceina (piccola molecola, ~ 359 Da, vedere Protocolli sezione 6) 11. Tuttavia, entrambi i metodi consentono la valutazione della permeabilità BRB di piccole molecole e proteine, ma non forniscono informazioni circa l&#39;integrità ILM. Quindi, a studiare BRB permeabilità, abbiamo usato un metodo basato AAV che fornisce informazioni sulla permeabilità BRB di molecole più grandi (ad esempio, le particelle di AAV, 25 di diametro nm). In effetti, il nostro metodo in grado di rilevare la presenza di AAV transgene nel sangue, il che suggerisce che le particelle ~ 25 nm di diametro farebberoessere in grado di infiltrarsi nel flusso sanguigno. Questo metodo fornisce anche informazioni sulla struttura del ILM e proteine ​​della matrice extracellulare in condizioni patologiche. Due varianti di AAV sono utili per tale studio: AAV5 e ShH10. Subretinally iniettato, AAV5 ha un tropismo naturale per fotorecettori della retina e dell&#39;epitelio pigmentato 12, ma non si può ottenere attraverso la retina esterna quando somministrato nel vitreo in wild-type retine con ILM intatto 5,13. ShH10 è una variante AAV che è stato progettato per indirizzare specificamente cellule gliali oltre neuroni 14,15. ShH10 etichette selettivamente le cellule Müller sia retine sani e malati con maggiore efficienza in retine con barriere compromesse 16. Questi strumenti virali accoppiato con immuhistochemistry e analisi del sangue-DNA forniscono informazioni sullo stato degli ostacoli retiniche e il loro coinvolgimento nella malattia (Figura 1).

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using adeno-associated virus as a tool to study retinal barriers in disease

Müller cellule sono le principali cellule gliali della retina. I loro finali piedi formano i limiti della retina alle membrane limitanti esterna ed interna (ILM), e in collegamento con astrociti, periciti e cellule endoteliali stabiliscono la barriera emato-retinica (BRB). BRB limita trasporto di materiale tra il flusso sanguigno e la retina mentre la ILM agisce come una membrana basale che definisce istologicamente il confine tra la retina e la cavità vitreo. Etichettatura cellule Müller è particolarmente rilevante per studiare lo stato fisico delle barriere della retina, in quanto queste cellule sono parte integrante della BRB e ILM. Sia BRB e ILM sono spesso alterati nelle malattie della retina e sono responsabili per i sintomi della malattia. Esistono diversi metodi ben stabiliti per studiare l&#39;integrità del BRB, quali il test blu Evans o fluorangiografia. Tuttavia questi metodi non forniscono informazioni sul grado di permeabilità BRB to molecole più grandi, in nanometri. Inoltre, essi non forniscono informazioni sullo stato di altri ostacoli retina, come la ILM. Per studiare BRB permeabilità al fianco della retina ILM, abbiamo usato un metodo basato AAV che fornisce informazioni sulla permeabilità della BRB di molecole più grandi, pur indicando lo stato dei ILM e della matrice extracellulare proteine ​​in stati di malattia. Due varianti di AAV sono utili per tale studio: AAV5 e ShH10. AAV5 ha un tropismo naturale fotorecettori, ma non si può ottenere attraverso la retina esterna quando somministrato nel vitreo quando l&#39;ILM è intatto (cioè, in wild-type retine). ShH10 ha un forte tropismo verso le cellule gliali e selettivamente etichettare glia Müller sia retine sani e malati. ShH10 fornisce consegna del gene più efficiente in retine dove ILM è compromessa. Questi strumenti virali accoppiato con immunoistochimica e sangue-DNA analisi hanno fatto luce sulla condizione delle barriere retina nella malattia.

Probabilmente incrementata trasduzione retinica Müller cellule gliali utilizzando ShH10 se il modello animale mostra perturbazioni nella struttura del ILM (Figura 2A - B). Ad esempio, abbiamo dimostrato che, in assenza di DP71, obiettivi ShH10 particolare, ma in modo più efficiente Müller cellule gliali di iniezione intravitreale, indicando un aumento della permeabilità della ILM in questa linea di topi rispetto ai topi wild-type 16 (Figura 2C - F...

Watch this Scientific Journal Video about Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease at JoVE.com

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

Utilizzando virus adeno-associati come strumento per studiare le barriere della retina nella malattia

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

using-adeno-associated-virus-as-tool-to-study-retinal-barriers

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14.9K Views.  Sorbonne Universtés, UPMC Univ Paris 06, UMR_S 968. To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

Video: Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a number of gene therapy clinical trials. More recently, a number of additional AAV serotypes have also been isolated, which have been shown to exhibit selective tissue-tropism in various small and large animal models1. Of the 10 most commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells and tissues in vitro as well as in vivo.
However, in our recently published studies, we have documented that AAV3 vectors transduce human liver cancer - hepatoblastoma (HB) and hepatocellular carcinoma (HCC) - cell lines extremely efficiently because AAV3 utilizes human hepatocyte growth factor receptor as a cellular co-receptor for binding and entry in these cells2,3.
In this article, we describe the steps required to achieve high-efficiency transduction of human liver cancer cells by recombinant AAV3 vectors carrying a reporter gene. The use of recombinant AAV3 vectors carrying a therapeutic gene may eventually lead to the potential gene therapy of liver cancers in humans.

In this article, we describe the identification of the adeno-associated virus serotype 3 (AAV3) as the most efficient vector for targeting human liver cancer cells.

Ad alta efficienza di trasduzione delle cellule tumorali del fegato da adeno-associato ricombinante sierotipo Virus 3 Vettori

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a ...

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Medicina

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

Cariotipo spettrale allo studio anomalie cromosomiche negli esseri umani e nei topi con malattia del rene policistico

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

Osteopathic Manipulative Treatment as a Useful Adjunctive Tool for Pneumonia

Pneumonia, the inflammatory state of lung tissue primarily due to microbial infection, claimed 52,306 lives in the United States in 20071 and resulted in the hospitalization of 1.1 million patients2. With an average length of in-patient hospital stay of five days2, pneumonia and influenza comprise significant financial burden costing the United States $40.2 billion in 20053. Under the current Infectious Disease Society of America/American Thoracic Society guidelines, standard-of-care recommendations include the rapid administration of an appropriate antibiotic regiment, fluid replacement, and ventilation (if necessary). Non-standard therapies include the use of corticosteroids and statins; however, these therapies lack conclusive supporting evidence4. (Figure 1)

Osteopathic Manipulative Treatment (OMT) is a cost-effective adjunctive treatment of pneumonia that has been shown to reduce patients&#8217; length of hospital stay, duration of intravenous antibiotics, and incidence of respiratory failure or death when compared to subjects who received conventional care alone5. The use of manual manipulation techniques for pneumonia was first recorded as early as the Spanish influenza pandemic of 1918, when patients treated with standard medical care had an estimated mortality rate of 33%, compared to a 10% mortality rate in patients treated by osteopathic physicians6. When applied to the management of pneumonia, manual manipulation techniques bolster lymphatic flow, respiratory function, and immunological defense by targeting anatomical structures involved in the these systems7,8, 9, 10.

The objective of this review video-article is three-fold: a) summarize the findings of randomized controlled studies on the efficacy of OMT in adult patients with diagnosed pneumonia, b) demonstrate established protocols utilized by osteopathic physicians treating pneumonia, c) elucidate the physiological mechanisms behind manual manipulation of the respiratory and lymphatic systems. Specifically, we will discuss and demonstrate four routine techniques that address autonomics, lymph drainage, and rib cage mobility: 1) Rib Raising, 2) Thoracic Pump, 3) Doming of the Thoracic Diaphragm, and 4) Muscle Energy for Rib 1.5,11

Pneumonia is one of the top ten leading causes of death in the U.S. Osteopathic manipulative techniques (OMT) may be utilized as an adjunctive therapy in the treatment of pneumonia to enhance biomechanical and immune function; and ultimately, to reduce hospital stay, duration of antibiotics, incidence of respiratory failure, and mortality. In this video-article, we will review randomized controlled studies on the use of OMT in pneumonia patients, as well as demonstrate specific hands-on techniques that are routinely practiced by osteopathic physicians when treating pulmonary infections.

Trattamento manipolativo osteopatico come strumento aggiuntiva Utile per polmonite

Pneumonia, the inflammatory state of lung tissue primarily due to microbial infection, claimed 52,306 lives in the United States in 20071 and resulted in ...

Fundus Photography as a Convenient Tool to Study Microvascular Responses to Cardiovascular Disease Risk Factors in Epidemiological Studies

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an important role in maintaining cardiovascular health. The retina is a tissue that lines the interior of the eye and it is the only tissue that allows for a non-invasive analysis of the microvasculature. Nowadays, high-quality fundus images can be acquired using digital cameras. Retinal images can be collected in 5 min or less, even without dilatation of the pupils. This unobtrusive and fast procedure for visualizing the microcirculation is attractive to apply in epidemiological studies and to monitor cardiovascular health from early age up to old age.
Systemic diseases that affect the circulation can result in progressive morphological changes in the retinal vasculature. For example, changes in the vessel calibers of retinal arteries and veins have been associated with hypertension, atherosclerosis, and increased risk of stroke and myocardial infarction. The vessel widths are derived using image analysis software and the width of the six largest arteries and veins are summarized in the Central Retinal Arteriolar Equivalent (CRAE) and the Central Retinal Venular Equivalent (CRVE). The latter features have been shown useful to study the impact of modifiable lifestyle and environmental cardiovascular disease risk factors.
The procedures to acquire fundus images and the analysis steps to obtain CRAE and CRVE are described. Coefficients of variation of repeated measures of CRAE and CRVE are less than 2% and within-rater reliability is very high. Using a panel study, the rapid response of the retinal vessel calibers to short-term changes in particulate air pollution, a known risk factor for cardiovascular mortality and morbidity, is reported. In conclusion, retinal imaging is proposed as a convenient and instrumental tool for epidemiological studies to study microvascular responses to cardiovascular disease risk factors.

Retinal image analysis is an unobtrusive procedure for visualizing the microcirculation. The impact of cardiovascular disease risk factors can result in changes of retinal vessel calibers. The procedures to acquire fundus images and the steps for calculating the vessel calibers are described.

Fundus Fotografia come un comodo strumento per studiare le risposte microvascolari a malattie cardiovascolari Fattori di rischio in studi epidemiologici

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an ...

Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research. Successful outcomes are dependent upon three critical components: test chambers that permit self-feeding while standing unrestrained in a confined space, recipes that mask the aversive taste/odor of commercially-available oral contrast agents, and a step-by-step test protocol that permits quantification of swallow physiology. Elimination of one or more of these components will have a detrimental impact on the study results. Moreover, the energy level capability of the fluoroscopy system will determine which swallow parameters can be investigated. Most research centers have high energy fluoroscopes designed for use with people and larger animals, which results in exceptionally poor image quality when testing mice and other small rodents. Despite this limitation, we have identified seven VFSS parameters that are consistently quantifiable in mice when using a high energy fluoroscope in combination with the new murine VFSS protocol. We recently obtained a low energy fluoroscopy system with exceptionally high imaging resolution and magnification capabilities that was designed for use with mice and other small rodents. Preliminary work using this new system, in combination with the new murine VFSS protocol, has identified 13 swallow parameters that are consistently quantifiable in mice, which is nearly double the number obtained using conventional (i.e., high energy) fluoroscopes. Identification of additional swallow parameters is expected as we optimize the capabilities of this new system. Results thus far demonstrate the utility of using a low energy fluoroscopy system to detect and quantify subtle changes in swallow physiology that may otherwise be overlooked when using high energy fluoroscopes to investigate murine disease models.

This study successfully adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research.

Adattamento umani videofluoroscopia Rondine metodi di studio per individuare e caratterizzare disfagia in modelli di malattia Murine

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

Development of an Alpha-synuclein Based Rat Model for Parkinson&#039;s Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

Sviluppo di un Rat modello Alfa-sinucleina per la malattia di Parkinson mediante iniezione stereotassica di un ricombinante vettore virale adeno-associato

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

Using Retinal Imaging to Study Dementia

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central nervous system (CNS) and shares prominent similarities with the brain in terms of embryological origin, anatomical features and physiological properties.&#160; The vascular and neuronal structure in the retina can now be visualized easily and non-invasively using retinal imaging techniques, including fundus photography and optical coherence tomography (OCT), and quantified semi-automatically using computer-assisted analysis programs. Studying the associations between vascular and neuronal changes in the retina and dementia could improve our understanding of dementia and, potentially, aid in diagnosis and risk assessment.&#160; This protocol aims to describe a method of quantifying and analyzing retinal vasculature and neuronal structure, which are potentially associated with dementia. This protocol also provides examples of retinal changes in subjects with dementia, and discusses technical issues and current limitations of retinal imaging.

The retina shares prominent similarities with the brain and thus represents a unique window to study vasculature and neuronal structure in the brain non-invasively. This protocol describes a method to study dementia using retinal imaging techniques. This method can potentially aid in diagnosis and risk assessment of dementia.

Usando la formazione immagine retinica per studiare la demenza

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central ...

Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and living organisms from a wide variety of species. The CRISPR technology has also been explored as novel therapeutics for a number of human diseases. Proof-of-concept data are highly encouraging as exemplified by recent studies that demonstrate the feasibility and efficacy of gene editing-based therapeutic approach for Duchenne muscular dystrophy (DMD) using a murine model. In particular, intravenous and intraperitoneal injection of the recombinant adeno-associated virus (rAAV) serotype rh.74 (rAAVrh.74) has enabled efficient cardiac delivery of the Staphylococcus aureus CRISPR-associated protein 9 (SaCas9) and two guide RNAs (gRNA) to delete a genomic region with a mutant codon in exon 23 of mouse Dmd gene. This same approach can also be used to knock out the gene-of-interest and study their cardiac function in postnatal mice when the gRNA is designed to target the coding region of the gene. In this protocol, we show in detail how to engineer rAAVrh.74-CRISPR vector and how to achieve highly efficient cardiac delivery in neonatal mice.

Here we provide a detailed protocol to carry out in vivo cardiac gene editing in mice using recombinant Adeno-Associated Virus(rAAV)-mediated delivery of CRISPR. This protocol offers a promising therapeutic strategy to treat dystrophic cardiomyopathy in Duchenne muscular dystrophy and can be used to generate cardiac-specific knockout in postnatal mice.

Adeno-associato Virus-mediate consegna di CRISPR per Gene cardiaco nei topi di Editing

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and ...

The Dyspepsia Educational Tool As a Novel Aid in Dyspepsia Management

Digital educational tools have a well-established role in current healthcare. In particular, disorders that are managed non-pharmacologically benefit from this development, as it enables patient engagement in self-management. Dyspepsia is a condition thought to arise from gastric and duodenal perturbations, brain-gut axis disturbances, and dietary factors. Behavioral interventions are a major part of dyspepsia treatment, hence patient engagement and motivation through education is essential. Protocols that describe the development process of such educational tools are scarce. We provide a methodology describing development of a dyspepsia educational tool. Assessment of users' needs is the first step, followed by a literature search. The content is developed based on the main themes and entered into a content management system, to build the program. Final adjustments are made after a pilot test of the tool. The presented protocol can be used as a guide for development of a digital dyspepsia educational tool or as a tool for similar situations.

This protocol describes the development process of a digital dyspepsia educational tool. Assessment of unmet needs and literature, content development, and building of the tool are presented. The methodology can be used as a guide for future development of digital educational tools.

Lo strumento educativo Dyspepsia come un nuovo aiuto nella gestione della dispepsia

Digital educational tools have a well-established role in current healthcare. In particular, disorders that are managed non-pharmacologically benefit from ...

Subconjunctival Administration of Adeno-associated Virus Vectors in Small Animal Models

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ease of accessibility via multiple administration routes. Subconjunctival (SCJ) injections offer advantages over other intraocular administration routes as they are simple, safe, and usually performed in an outpatient setting. SCJ injections in small animals usually require the assistance of an operating microscope due to the size of the eye. Previous work has demonstrated that SCJ injection of specific adeno-associated virus (AAV) serotypes is a valid gene delivery strategy for targeted transduction of the ocular surface, eye muscle, cornea, and optic nerve, providing a potential approach for the treatment of many ocular diseases.
Herein, a detailed protocol is presented for SCJ injections in a mouse model using an injection system consisting of a programmable infusion/withdrawal syringe pump (which allows for consistent and precise injection speed and pressure) and a gastight removable syringe coupled with microinjection needles. The injection system is also adaptable for other intraocular administration routes such as intrastromal, intracameral, intravitreal, and subretinal injections in small animals. Although the delivery of adeno-associated viral vectors for ocular gene therapy studies is described, the protocol herein can also be adapted for a variety of ophthalmic solutions in small animal models. The key practical steps in the administration route, setup for the injection platform, preparation of the injection, and tips from direct experience will be discussed in detail. In addition, common validation techniques for AAV delivery confirmation to the desired tissues will also be briefly discussed.

In this manuscript, subconjunctival injection is demonstrated as a valid vector delivery method for ocular tissues in mice using an injection system consisting of an infusion/withdrawal syringe pump and a gastight removable syringe coupled with microinjection needles. This injection system is also adaptable for other intraocular administration routes.

Somministrazione subcongiuntivale di vettori virali adeno-associati in modelli di piccoli animali

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ...