Riconosciamo il lavoro del dottor Adlah Sukkar, MD nell&#39;assistere con fusione del polmone. Questo lavoro è stato finanziato dal NHLBI, HL088005.

Tutti i protocolli eseguiti su ratti sono stati approvati dalla Johns Hopkins Animal Care University e del Comitato uso e in conformità con le linee guida NIH. Quando possibile, l&#39;animale deve essere preparata chirurgicamente in una zona separata dalla zona chirurgica per ridurre al minimo la contaminazione del sito chirurgico. 1. Anestesia / analgesia <ol><li> Posto ratto (ratti Sprague Dawley maschi, 125-150 g; Harlan, Indianapolis, IN) in una camera di induzione infuso con 3% isoflurano. </li><li> Posizionare ratto anestetizzato a bordo chirurgia attaccato al cono e ventilatore con il 3% anestesia isoflurano. Utilizzare la tecnica sterile per tutte le procedure. Drappeggio ratto garantire campo sterile operativo. </li></ol> 2. Intubazione <ol><li> Immobilizzare appendici in posizione supina con nastro adesivo chirurgico. </li><li> Rimuovere ratto dal naso a cono, estendere la lingua con le pinze imbottite. </li><li> Utilizzare 14 intracath calibro con stiletto in metallo smussato come guida. Far scorrere dietro aNgue, nella trachea. </li><li> Rimuovere stiletto in metallo bianco lasciando intracath plastica in trachea. Garantire il ratto respira e l&#39;aria fluisce attraverso il tubo. </li><li> Sostituire il cono con adattatore diretto al catetere, collegare ratto di ventilatore (90 atti / min, 8 ml / kg di volume corrente; Roditore Ventilatore modello 683, Harvard Apparatus, Holliston, Massachusetts). </li><li> Applicare Puralube (Butler Schein, Dublin, OH) unguento veterinaria sugli occhi. </li></ol> 3. Toracotomia <ol><li> Luogo ratto lato destro verso il basso, immobilizzare appendici con nastro adesivo chirurgico. </li><li> Shave sinistra nella cassa toracica laterale. </li><li> Rimuovere pelo in eccesso con il vuoto di piccole dimensioni. </li><li> Fai campo sterile pulendo giù con l&#39;alcol seguita da Povidone-Iodio swabstick (Dynarex Corporation, Orangebur, NY). Ripetere questa operazione altre due volte (per un totale di tre scrub). </li><li> Fai incisione trasversale con le forbici sterili dissezione o bisturi sterile nel centro del campo. </li><li> Blunt sezionare attraverso layers del tessuto e grasso fino a nervature (ultimo strato è sottile membrana che copre le costole). </li><li> Contare costole per determinare 3 ° spazio intercostale. </li><li> Utilizzando sterili 45 ° Graefe pinza, fare un&#39;incisione smussato tra il 3 ° e 4 ° costola. </li><li> Inserire separatori costola, tirare leggermente la creazione di vuoto aperto con visualizzazione completa di polmone, nastro adesivo posto su suture di tenere aperto. </li></ol> 4. Sinistra legatura dell&#39;arteria polmonare <ol><li> Con 90 ° pinza Graefe, spostare polmone sinistro posteriore con la mano destra. </li><li> Utilizzando i modelli Dumont N ° 5 pinze diritte, prendere a sinistra dell&#39;arteria polmonare e delle vie aeree con la mano sinistra. Sinistro dell&#39;arteria polmonare si stese sopra delle vie aeree. Garantire che pinze sono direttamente perpendicolare al tavolo. Inoltre, è più efficace per raccogliere l&#39;arteria polmonare sinistra / bronco principale sinistro nella posizione più distale (più vicino al parenchima). Spingere il polmone sinistro di ventilazione da parte con questa manovra la mano sinistra. </li><li> Utilizzare Dumontpattern # 5 45 ° pinze curve per separare l&#39;arteria polmonare sinistra dal bronco principale di sinistra alle frontiere naturali. Questa linea di separazione appare sottile e bianca tra le due strutture singole. </li><li> Separare direttamente sotto l&#39;arteria, senza passare attraverso la nave, senza problemi far scorrere le punte pinze tenute insieme lungo la separazione. </li><li> Continuare fino a punte della pinza sono visibilmente separati l&#39;arteria polmonare sinistra e il bronco principale di sinistra. Solo un piccolo punto della punta deve essere pienamente attraverso. Se il sangue può essere visualizzata sulla punta della pinza, allora non è completamente tramite legatura e non deve essere tentata. Una volta nello spazio, l&#39;arteria polmonare sinistra porrà sulla curva della pinza. Tenere in questa posizione. </li><li> Delicatamente rilasciare dritto smussato presa pinza (mano sinistra) e prendere parte di un pre-taglio sutura (~ 2-3 cm; sutura in polipropilene formato 6-0, Myco medici, Cary, NC). </li><li> Aprire pinze curve cullano sinistro dell&#39;arteria polmonare e grab sutura. Estrarre delicatamente sutura attraverso lo spazio tra sinistra dell&#39;arteria polmonare e bronco principale di sinistra in un movimento verso l&#39;alto rispetto alla curva della pinza. </li><li> Fissate occludere la polmonare sinistra con un nodo quadrato, resto attentamente snip di sutura. </li><li> Chiudi costole con una pinzetta smussate per tenere costola e due volte con sutura in polipropilene (monofilamento blu) taglia 4-0 collegato a un 19 mm, 3/8 retromarce cerchio di taglio dell&#39;ago (Myco Medical, Cary, NC) in una pinza emostatica, facendo attenzione a non sutura della pelle (solo costole). </li><li> Completare un nodo allentato quadrato, gonfiare il polmone, luogo positiva di fine espirazione (PEEP; 2-5 cm H 2 O), hyperinflate nodo polmonare poi sicuro strettamente e fare un altro nodo pieno prima snipping resto di sutura. Rimuovere dalla PEEP e visualizzare per 30 sec per garantire che il polmone non collassa. Applicare 5 gocce Bupivicaine (APP Pharmaceuticals, Schaumbur, IL). </li><li> Chiudi la pelle mettendo colla tessuto sulla ferita e spingere indietro la pelle insieme usando end di cotone punta dell&#39;applicatore. Dare Bupivicaine (2,0 mg / kg per via sottocutanea) al sito di incisione successivamente ogni 8 ore per 24 ore o fino a quando l&#39;animale riprende la normale attività. Come strato sottocutaneo in questa zona è molto sottile, non è facile da suturare da solo. Quando la colla è applicata tessuto e la pelle viene accostati chiude anche lo strato sottocutaneo. </li><li> Spegnere gas isoflurano, ma continuano a ventilare il ratto per 1-2 minuti in aria ambiente fino al ritorno del movimento volontario. Scollegare il tubo tracheale dal ventilatore e assicurarsi che il ratto è in respiro spontaneo prima di rimuoverlo. </li><li> Pulire Puralube da occhi con un batuffolo di cotone e il movimento del monitor e di recupero. Iniettare buprenorfina cloridrato (0,05 mg / kg intraperitoneale, Butler Schein, Dublino OH). Continua la consegna di ogni analgesico ore 12 per 48 ore dopo l&#39;intervento chirurgico. </li></ol> 5. Sinistra incannulamento arteria carotide Per valutare l&#39;entità del perfus bronchialiione del polmone ischemico sinistra a tempi desiderati sinistra dopo legatura dell&#39;arteria polmonare, iniettare microsfere marcate attraverso l&#39;arteria carotide sinistra nella aortico. Preparare ratti come sopra 1-2. <ol><li> Tagliare linea mediana lungo il collo, smussare sezionare per rivelare trachea e sinistra arteria carotide (al sito di iniezione microsfere). </li><li> Inserire il catetere riempito con soluzione fisiologica eparinizzata, punta smussata di PE20 tubi (Becton Dickinson, Sparks MD) nel serbatoio collegato a 25 g di aghi, 4 vie rubinetto, siringa da 1 ml. </li><li> Bottiglia Luogo di microsfere (15 micron cremisi polistirene microsfere fluorescenti, 1 x 10 6 sfere / ml; Invitrogen, Eugene, OR) in sonicatore acqua per 30 sec. </li><li> Rimuovere bottiglia, vortice e redigere 0,5 ml (500.000 microsfere), in un ml 1 siringa di vetro Hamilton (Hamilton Company, Reno, NV) attraverso un ago 20 g. </li><li> Collegare la siringa Hamilton a 4-way rubinetto e infondere microsfere con pompa a siringa (rate: 500 microlitri / min; Genie Plus, Kent scientifico, Torringtsu, CT). </li><li> Rimuovere la siringa Hamilton e apparecchi filo con 1 ml di soluzione fisiologica heparized a 500 microlitri / min. </li><li> Eseguire toracotomia pieno petto e exsanguinate il ratto dal recidendo la vena cava inferiore. </li><li> Rimuovere il polmone sinistro e altri tessuti di interesse. </li><li> Per estrarre microsfere dal tessuto, prendere l&#39;intero polmone sinistro del ratto dopo dissanguamento e trasferirla in KOH 2M (4-6 ml). Mettere in un bagno d&#39;acqua ° 55 C e lasciare per una notte per la digestione dei tessuti. Aggiungere Tween 80 (0,25%) per lavare le perline, vortice (10 sec) e centrifugare (2000 rpm, 20 ° C per 10 min). Rimuovere il surnatante, aggiungere 2-etossietil acetato (1 ml), vortex e lasciare riposare per 1 ora. Vortex e centrifugare la sospensione (2.000 rpm; (20 ° C per 10 min) Rimuovere il 2-etossietil strato acquoso contenente l&#39;acetato, fluorescenza posto in una cuvetta e misurare usando un Hitachi F-2500 spettrofotometro a fluorescenza (eccitazione 612/emission 618. , Digilab, Holliston, MA). </li></ol>

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Sinistra legatura dell&#39;arteria polmonare in tutte le specie testate porta alla robusta neovascolarizzazione sistemica del polmone ischemica. Abbiamo presentato i dettagli del metodo chirurgico in un modello di ratto. I nostri risultati prodotti da fusione vascolare, istopatologia, e in vivo dimostrano che l&#39;etichettatura arterie bronchiali proliferano e perfusione del parenchima polmonare. Così, i meccanismi di angiogenesi bronchiale può essere studiata in un modello animale che affianca la condizione...

Angiogenesi sistemica nel polmone è ben riconosciuta. In condizioni patologiche quali asma 2, fibrosi polmonare interstiziale 3, cancro 4, tromboembolia polmonare cronica e 5, il sistema vascolare sistemica e circonda i prolifera polmonari e invade il parenchima polmonare. Tuttavia, i modelli animali per studiare questa attivazione differenziale del sistemica piuttosto che la circolazione polmonare sono pochi. Forse il modello più riproducibile di neovascolarizzazione sistemica nel polmone del mammifero adulto è quella che si verifica dopo induzione cronica ischemia arteriosa polmonare. La risposta a sinistra ostruzione dell&#39;arteria polmonare nell&#39;uomo 5-7, 8 cani, maiali, pecore 9 10, cavie, ratti 11 1, 12, 13, 14 e topi è la rapida proliferazione dell&#39;arteria bronchiale e arterie intercostali. I meccanismi responsabili della sistemica neovascularizione del polmone dopo ischemia polmonare non sono noti e non sono state ampiamente studiate. La durata di angiogenesi bronchiale nel ratto dopo sinistra ostruzione dell&#39;arteria polmonare è stata descritta nel lavoro istologica di Weibel 1. Estendere questo lavoro nel ratto, il nostro laboratorio ha focalizzato su entrambi i fattori di crescita importanti in questo processo, nonché l&#39;esito fisiologico di questo neovascolarizzazione nel polmone. Risultati dimostrano la chemochina CXC CINC-3 è elevata precoce dopo ischemia e trattando ratti con un anticorpo neutralizzante per CXCR2, il recettore per CINC-3, attenua angiogenesi 13. Il sistema vascolare di nuova costituzione bronchiale 14 giorni dopo l&#39;insorgenza di ischemia polmonare ha dimostrato di essere anormale, con un significativo aumento della permeabilità delle proteine ​​15. Funzione polmonare sinistra non stava mostrando normale diminuita capacità di diffusione e una diminuzione del volume polmonare 15. Sebbene la neovascolarizzazione può avere contributed alla conservazione del tessuto polmonare durante ischemia cronica polmonare, sembra non essere normale e può contribuire ad un prolungato decremento della funzione polmonare. Forse uno degli aspetti più curiosi di questo modello riguarda la distribuzione spaziale della proliferazione dei vasi sanguigni. Nonostante il rilascio di fattori di crescita all&#39;interno del parenchima polmonare da ischemia, la neovascolarizzazione originario monte relativamente grandi arterie bronchiali. L&#39;arteria bronchiale normale si pone come un piccolo ramo della aorta e invade l&#39;albero delle vie aeree alla carena. Così il meccanismo con cui i fattori di crescita indurre la fase iniziale di arteriogenesi non è chiara. Suggeriamo che il topo, con una anatomia vascolare simili agli esseri umani, fornisce un&#39;opportunità unica di studiare i meccanismi responsabili per l&#39;angiogenesi sistemico polmonare durante l&#39;ischemia. Sebbene completa ostruzione dell&#39;arteria polmonare sinistra è un evento raro in soggetti umani,aumentata vascolarizzazione bronchiale sembra essere similmente indotta in pazienti indipendentemente dalla dimensione del sito e ostruzione dell&#39;arteria polmonare 16. Così, forniamo una descrizione dettagliata del metodo chirurgico per legare l&#39;arteria polmonare sinistra in ratti e un mezzo per quantificare la grandezza di angiogenesi.

<table border="1"><tbody><tr><td> Reagenti: </td><td> Azienda </td><td></td><td></td></tr><tr><td> buprenorfina cloridrato, Puralube </td><td> Butler Schein </td><td></td><td></td></tr><tr><td> bupivicaine </td><td> APP Pharmaceuticals </td><td></td><td></td></tr><tr><td> Povidone-iodio swabstick </td><td> Dynarex Corporation </td><td></td><td></td></tr><tr><td> polipropilene dimensioni sutura 6-0, 3/8 cerchio ago inversione di taglio </td><td> Myco Medical </td><td></td><td></td></tr><tr><td> PE20 tubi </td><td> Becton Dickinson </td><td></td><td></td></tr><tr><td> 15 micron fluorosfere polistirene cremisi </td><td> Invitrogen </td><td></td><td></td></tr><tr><td> 1 ml di vetro siringa Hamilton </td><td> Hamilton Azienda </td><td> &lt;/ Td&gt; <td></td></tr><tr><td> Attrezzatura: </td><td></td><td></td><td></td></tr><tr><td> Genie Inoltre siringa pompa </td><td> Kent scientifico </td><td></td><td></td></tr><tr><td> Spettrofotometro fluorescenza </td><td> Digilab </td><td></td><td></td></tr><tr><td> Roditore Ventilatore Modello 683 </td><td> Harvard Apparatus </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> Tabella 1. Tabella di reagenti e attrezzature specifiche. </td></tr></tbody></table>

angiogenesis in the ischemic rat lung

Il polmone adulto è perfuso sia l&#39;arteria sistemica bronchiale e il ritorno venoso intero che scorre attraverso le arterie polmonari. Nella maggior parte delle patologie polmonari, è il più piccolo vascolare sistemica che risponde ad una esigenza di perfusione polmonare avanzata e mostra neovascolarizzazione robusta. Ischemia vascolare polmonare indotta da ostruzione arteriosa polmonare ha dimostrato di provocare una rapida angiogenesi arteriosa sistemica nell&#39;uomo e in modelli animali. Sebbene la valutazione istologica del corso tempo di proliferazione dell&#39;arteria bronchiale nei ratti è stato accuratamente descritto da Weibel 1, meccanismi responsabili di questa crescita di nuovi vasi organizzata non sono chiare. Noi forniamo i dettagli chirurgici di indurre sinistra ischemia arteriosa polmonare nel ratto che porta alla neovascolarizzazione bronchiale. Quantificazione del grado di angiogenesi presenta una sfida aggiuntiva per la presenza dei due letti vascolari all&#39;interno del polmone. Metodideterminare angiogenesi funzionale sulla base iniezioni microsfere sono etichettati.

Espressi vascolare: Risultati degli effetti di sinistra ischemia dell&#39;arteria polmonare nel ratto sono rappresentati in figura 1. Viene mostrato un cast in metacrilato del circolo bronchiale e la vascolarizzazione estesa della struttura delle vie aeree sinistra 28 giorni dopo la LPAL. Per ottenere questo cast, il sistema vascolare sistemica è stata iniettata una miscela metacrilato (rosso), retrogrado nell&#39;aorta discendente e la trachea è stata incannulata e iniettato con un materiale a base d...

Watch this Scientific Journal Video about Angiogenesis in the Ischemic Rat Lung at JoVE.com

Angiogenesis in the Ischemic Rat Lung

Il polmone è perfuso sia l&#39;arteria sistemica bronchiale e arterie polmonari. Nella maggior parte delle patologie polmonari, è il più piccolo vascolare sistemica che mostra neovascolarizzazione robusta. La cessazione del flusso ematico polmonare promuove l&#39;angiogenesi vivace bronchiale. Noi forniamo i dettagli chirurgici di indurre ischemia arteriosa polmonare sinistra che promuove la neovascolarizzazione bronchiale.

L&#39;angiogenesi nel polmone del ratto ischemica

The  adult lung is perfused by both the systemic bronchial artery and the entire  venous return flowing through the pulmonary arteries. In most lung ...

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15.5K Views.  Johns Hopkins University. Il polmone è perfuso sia l'arteria sistemica bronchiale e arterie polmonari. Nella maggior parte delle patologie polmonari, è il più piccolo vascolare sistemica che mostra neovascolarizzazione robusta. La cessazione del flusso ematico polmonare promuove l'angiogenesi vivace bronchiale. Noi forniamo i dettagli chirurgici di indurre ischemia arteriosa polmonare sinistra che promuove la neovascolarizzazione bronchiale.

Video: Angiogenesis in the Ischemic Rat Lung

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Multi-modal Imaging di angiogenesi in un modello di ratto Nudo di metastasi ossee cancro al seno utilizzando la risonanza magnetica, tomografia computerizzata volumetrica e Ultrasuoni

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Ricerca

Medicina

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

Distacco della retina Modello in Roditori da iniezione sottoretinico di ialuronato di sodio

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and repair damage in simple tissues in the orthopedic and periodontal fields. However, attempts to regenerate the structures and functions of more complex three-dimensional (3D) organs such as lungs have not been very successful because the biological processes of organ regeneration have not been well explored. It is becoming clear that angiogenesis, the formation of new blood vessels, plays key roles in organ regeneration. Newly formed vasculatures not only deliver oxygen, nutrients and various cell components that are required for organ regeneration but also provide instructive signals to the regenerating local tissues. Therefore, to successfully regenerate lungs in an adult, it is necessary to recapitulate the lung-specific microenvironments in which angiogenesis drives regeneration of local lung tissues. Although conventional in vivo angiogenesis assays, such as subcutaneous implantation of extracellular matrix (ECM)-rich hydrogels (e.g., fibrin or collagen gels or Matrigel - ECM protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells), are extensively utilized to explore the general mechanisms of angiogenesis, lung-specific angiogenesis has not been well characterized because methods for orthotopic implantation of biomaterials in the lung have not been well established. The goal of this protocol is to introduce a unique method to implant fibrin gel on the lung surface of living adult mouse, allowing for the successful recapitulation of host lung-derived angiogenesis inside the gel. This approach enables researchers to explore the mechanisms by which the lung-specific microenvironment controls angiogenesis and alveolar regeneration in both normal and pathological conditions. Since implanted biomaterials release and supply physical and chemical signals to adjacent lung tissues, implantation of these biomaterials on diseased lung can potentially normalize the adjacent diseased tissues, enabling researchers to develop new therapeutic approaches for various types of lung diseases.

Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse.

L&#39;impianto di fibrina gel sul mouse polmone per studiare angiogenesi Lung-specifica

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

Metodo di isolato<em&gt; Ex Vivo</em&gt; Lung Perfusion in un modello di ratto: Lezioni apprese da Sviluppo di un programma EVLP Rat

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

Rat Model of Photochemically-Induced Posterior Ischemic Optic Neuropathy

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history have&#160;remained poorly understood. Recently, we developed a reliable, reproducible animal model of PION and tested the treatment effect of some neurotrophic factors in this model1. The purpose of this video is to demonstrate our photochemically induced model of posterior ischemic optic neuropathy, and to evaluate its effects with retrograde labeling of retinal ganglion cells. Following surgical exposure of the posterior optic nerve, a photosensitizing dye, erythrosin B, is intravenously injected and a laser beam is focused onto the optic nerve surface. Photochemical interaction of erythrosin B and the laser during irradiation damages the vascular endothelium, prompting microvascular occlusion mediated by platelet thrombosis and edematous compression. The resulting ischemic injury yields a gradual but pronounced retinal ganglion cell dieback, owing to a loss of axonal input - a remote, injury-induced and clinically relevant outcome. Thus, this model provides a novel platform to study the pathophysiologic course of PION,&#160;and can be further optimized for testing therapeutic approaches for optic neuropathies as well as other CNS ischemic diseases.

The goal of this protocol is to photochemically induce ischemic injury to the posterior optic nerve in rat. This model is critical to studies of the pathophysiology of posterior ischemic optic neuropathy, and therapeutic approaches for this and other optic neuropathies, as well as of other CNS ischemic diseases.

Rat Modello di fotochimica-Induced posteriore Neuropatia ottica ischemica

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history ...

Rat Heterotopic Abdominal Heart/Single-lung Transplantation in a Volume-loaded Configuration

Herein, we describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The configuration of the transplant graft involves anastomosis of donor inferior vena cava (IVC) to recipient IVC, and donor ascending aorta (Ao) to recipient abdominal Ao. The right upper and middle lung lobes are preserved and function as conduits for blood flow from right heart to left heart.
There are several advantages to using this technique, and it lends itself to a broad range of applications. Because the graft is transplanted in a configuration that allows for dyamic volume-loading, cardiac function may be directly assessed in vivo. The use of pressure-volume conductance catheters permits characterization of load-dependent and load-independent hemodynamic parameters. The graft may be converted to a loaded configuration by applying a clamp to the recipient&#8217;s infra-hepatic IVC. We describe modified surgical techniques for both donor and recipient operations, and an ideal myocardial protection strategy. Depending on the experimental aim, this model may be adapted for use in both acute and chronic studies of graft function, immunologic status, and variable ventricular loading conditions. The conducting airways to the transplanted lung are preserved, and allow for acute lung re-ventilation. This facilitates analysis of the effects of the mixed venous and arterial blood providing coronary perfusion to the graft.
A limitation of this model is its technical complexity. There is a significant learning curve for new operators, who should ideally be mentored in the technique. A surgical training background is advantageous for those wishing to apply this model. Despite its complexity, we aim to present the model in a clear and easily applicable format. Because of the physiologic similarity of this model to orthotopic transplantation, and its broad range of study applications, the effort invested in learning the technique is likely to be worthwhile.

We describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The transplant configuration results in a partially loaded graft circulation, allowing direct functional assessment. This model may be employed for acute or chronic studies of function and immunologic status of the transplanted graft.

Rat eterotopico addominale Cuore / Trapianti Single-polmone in una configurazione del volume-caricato

Herein, we describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The configuration of the transplant graft ...

Demonstration of the Rat Ischemic Skin Wound Model

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and unrelieved pressure due to immobility. Animal models have been developed that attempt to mimic these conditions for the purpose of furthering our understanding of the complexity of chronic wounds. The model described herein is a rat ischemic skin flap model that permits a prolonged reduction of blood flow resulting in wounds that become ischemic and resemble a chronic wound phenotype (reduced vascularization, increased inflammation and delayed wound closure). It consists of a bipedicled dorsal flap with 2 ischemic wounds placed centrally and 2 non-ischemic wounds lateral to the flap as controls. A novel addition to this ischemic skin flap model is the placement of a silicone sheet beneath the flap that functions as a barrier and a splint to prevent revascularization and reduce contraction as the wounds heal. Despite the debate of using rats for wound healing studies due to their quite distinct anatomic and physiologic differences compared to humans (i.e., the presence of a panniculus carnosus muscle, short life-span, increased number of hair follicles, and their ability to heal infected wounds) the modifications employed in this model make it a valuable alternative to previously developed ischemic skin flap models.

The rat, due to its size, availability, and rather docile behavior, has been utilized as a research model for many years. The goal of this protocol is to utilize the rat as an ischemic skin wound healing model to provide valuable insight into the pathophysiology of chronic wounds.

Dimostrazione del Topo ischemica pelle ferita Modello

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and ...

In Vitro Model of Coronary Angiogenesis

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects in these vessels represent severe health risks such as in atherosclerosis, which can lead to myocardial infarctions and heart failures in patients. Consequently, coronary artery disease is one of the leading causes of death worldwide. Despite its clinical importance, relatively little progress has been made on how to regenerate damaged coronary arteries. Nevertheless, recent progress has been made in understanding the cellular origin and differentiation pathways of coronary vessel development. The advent of tools and technologies that allow researchers to fluorescently label progenitor cells, follow their fate, and visualize progenies in vivo have been instrumental in understanding coronary vessel development. In vivo studies are valuable, but have limitations in terms of speed, accessibility, and flexibility in experimental design. Alternatively, accurate in vitro models of coronary angiogenesis can circumvent these limitations and allow researchers to interrogate important biological questions with speed and flexibility. The lack of appropriate in vitro model systems may have hindered the progress in understanding the cellular and molecular mechanisms of coronary vessel growth. Here, we describe an in vitro culture system to grow coronary vessels from the sinus venosus (SV) and endocardium (Endo), the two progenitor tissues from which many of the coronary vessels arise. We also confirmed that the cultures accurately recapitulate some of the known in vivo mechanisms. For instance, we show that the angiogenic sprouts in culture from SV downregulate COUP-TFII expression similar to what is observed in vivo. In addition, we show that VEGF-A, a well-known angiogenic factor in vivo, robustly stimulates angiogenesis from both the SV and Endo cultures. Collectively, we have devised an accurate in vitro culture model to study coronary angiogenesis.

In vitro models of coronary angiogenesis can be utilized for the discovery of the cellular and molecular mechanisms of coronary angiogenesis. In vitro explant cultures of sinus venosus and endocardium tissues show robust growth in response to VEGF-A and display a similar pattern of COUP-TFII expression as in vivo.

Modello in vitro dell'angiogenesi coronarica

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects ...

A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods for choosing appropriate cuff sizes for the pulmonary vein (PV), pulmonary artery (PA), or bronchus (Br) are varied, thus making the determination of proper cuff size during rat lung transplantation an exercise of trial and error. By standardizing the cuffing technique to use the smallest effective cuff appropriate for the size of the vessel or bronchus, one can make the transplantation procedure safer, faster, and more successful. Since diameters of the PV, PA, and Br are related to the body weight of the rat, we present a strategy to choosing an appropriate size using a weight-based guide. Since lung volume is also related to body weight, we recommend that this relationship should also be considered when choosing the proper volume of air for donor lung inflation during warm ischemia as well as for the proper volume of PBS to be instilled during bronchoalveolar lavage (BAL) fluid collection. We also describe methods for 4th intercostal space dissection, wound closure, and sample collection from both the native and transplanted lobes.

Here, we present optimizations to a rat lung transplantation model that serve to improve outcomes. We provide a size guide for cuffs based on body weight, a measurement strategy to ascertain the 4th intercostal space, and methods of wound closure and BAL (bronchoalveolar lavage) fluid and tissue collection.

Un modello di trapianto di polmone di ratto di ischemia calda / danno da riperfusione: ottimizzazioni per migliorare i risultati

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods ...

<meta charset="utf8"></head><body>Monitoraggio in tempo reale delle dinamiche cellulari nel trapianto di polmone di topo

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by crosstalk between donor and recipient cells. A better understanding of these processes relies on the use of preclinical animal models and is aided by an ability to study intra-graft immune cell trafficking in real-time. Intravital two-photon microscopy can be used to image tissues and organs for depths up to several hundred microns with minimal photodamage, which affords a great advantage over single-photon confocal microscopy. Selective use of transgenic mice with promoter-specific fluorescent protein expression and/or adoptive transfer of fluorescent dye-labeled cells during intravital two-photon microscopy allows for the dynamic study of single cells within their physiologic environment. Our group has developed a technique to stabilize mouse lungs, which has enabled us to image cellular dynamics in na&#239;ve lungs and orthotopically transplanted pulmonary grafts. This technique allows for detailed assessment of cellular behavior within the vasculature and in the interstitium, as well as for examination of interactions between various cell populations. This procedure can be readily learned and adapted to study immune mechanisms that regulate inflammatory and tolerogenic responses after lung transplantation. It can also be expanded to the study of other pathogenic pulmonary conditions.

<meta charset="utf8"></head><body>Autore in primo piano: Avanzamento dell'immunologia dei trapianti di polmone attraverso l'imaging intravitale

Here, we present a protocol to intravitally image the transplanted mouse left lung using two-photon microscopy. This represents a valuable tool for studying cellular dynamics and interactions in real-time following murine lung transplantation.

Microscopia intravitale a due fotoni del polmone di topo trapiantato

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by ...