Name: intravascular delivery of biologics to the rat kidney
Uploaded: 2016-09-01T15:00:00.000+00:00
Duration: 7 min 29 s
Description: Watch this Scientific Journal Video about Intravascular Delivery of Biologics to the Rat Kidney at JoVE.com

Gli esperimenti sono stati condotti su ratti femmina Sprague-Dawley, del peso di 250-300 g. Tutte le procedure sugli animali rispettate le norme indicate nella Guida per la cura e l&#39;uso di animali da laboratorio (Istituto di laboratorio risorse animali, National Academy of Sciences, Bethesda, MD, USA) e sono stati approvati dal Mayo Clinic College of Medicine Istituzionale Animal Care e Comitato Usa (IACUC). 1. Preparazione <ol><li> Autoclavare tutti gli strumenti chirurgici prima dell&#39;intervento chirurgico. Se interventi chirurgici multipli su diversi ratti sono in programma nella stessa giornata, lavare gli strumenti dopo ogni procedura di animale e poi sterilizzare utilizzando uno sterilizzatore a caldo tallone. </li><li> Anestetizzare il topo con il 4% isoflurano in 1 L / min O 2. </li><li> Trasferire il ratto ad una piastra elettrica controllata per mantenere la temperatura corporea a 37 ° C. Mantenere l&#39;anestesia con il 1-2% isoflurano in 1 L / min O 2. </li><li> Somministrare il farmaco analgesico (buprenorfina sostenuta di uscita 0,6 mg / kg) subcutaneously. </li><li> Applicare una pomata per gli occhi per evitare l&#39;essiccazione durante la procedura. </li><li> Al fine di compensare la perdita di fluidi corporei causa laparotomia, è importante somministrare 10 ml / kg di 0,9% sottocutanea soluzione fisiologica prima dell&#39;intervento. </li><li> Radere la zona addominale e pulire la pelle con i rilievi etanolo povidone-iodio e il 70%. </li></ol> 2. Procedura chirurgica <ol><li> Assicurarsi che la profondità di sedazione è adeguato per il monitoraggio riflessi fisici, quali il recesso dal pizzico punta, riflesso palpebrale, il tono della mascella, e la respirazione tasso / modello. </li><li> Eseguire una laparotomia attraverso una piccola incisione mediana (2-2,5 cm di lunghezza) con un bisturi chirurgico lama No. 10. </li><li> Estrarre l&#39;intestino e colon al lato destro dell&#39;addome utilizzando tamponi di cotone e coprire con garza sterile imbevuta di 0,9% soluzione salina per mantenere gli organi umido. </li><li> Delicatamente rientrare verso l&#39;alto la milza, fegato, stomaco e del pancreas per esporre la aorta e l&#39;arteria renale sinistra. </li><li> Con l&#39;aiuto di un microscopio operatorio, separare con attenzione l&#39;aorta addominale sopra e sotto il rene sinistro e l&#39;arteria renale sinistra dalle vene, il grasso e il tessuto connettivo circostante con dissezione curvo forcipe smussato e tamponi di cotone sterili. <ol><li> Utilizzare le pinze con un moto (scollamento) open-close ripetuto lungo la lunghezza delle navi per rimuovere il tessuto connettivo e le tamponi di cotone con un moto di rollio laterale per rimuovere il grasso. NOTA: La dissezione della regione peri-aortica è un passo molto delicato come nervi e vasi linfatici potrebbe essere danneggiata. Assicurarsi di mantenere le arterie umido con soluzione salina durante la procedura di dissezione. </li></ol></li><li> Collocare una sutura 4-0 seta sotto l&#39;aorta. </li><li> Utilizzando clip microvascolari, bloccare l&#39;aorta sopra (appena sotto l&#39;arteria mesenterica superiore) e al di sotto della biforcazione dell&#39;arteria renale. </li><li> Puntura dell&#39;aorta a livello della kidn sinistrabiforcazione dell&#39;arteria ey con un catetere endovenoso G 24 e far avanzare il catetere nell&#39;arteria renale. NOTA: questo è un passo fondamentale, come la puntura attraverso l&#39;arteria renale può verificarsi. </li><li> Collegare una siringa riempita con la soluzione farmaco o salina (fino a 500 microlitri) al catetere e profumato rene. </li><li> Subito dopo perfusione, bloccare la vena renale sinistra e dell&#39;uretere sinistro con una clip microvascolare e rimuovere il catetere. Poi, posizionare un pezzo di spugna assorbibile emostatico gelatina, con una piccola goccia di tessuto adesivo, sopra la zona forata dell&#39;aorta e applicare delicatamente pressione con un tampone di cotone. </li><li> Allo stesso tempo, rilasciare il morsetto dall&#39;aorta addominale, sotto fianco biforcazione dell&#39;arteria renale. Dopo 5 minuti, rilasciare il morsetto dalla vena renale e dell&#39;uretere. </li><li> rilasciare con cautela il morsetto dall&#39;aorta, sopra a sinistra biforcazione dell&#39;arteria renale, e consentire la riperfusione renale. L&#39;ischemia renale totale dovrebbe durare non più di 7 minuti. </li><li&gt; garantire che non si verifichino sanguinamento attivo e osservare da vicino l&#39;area per altri 10 min. </li><li> Chiudere l&#39;incisione addominale in due strati (muscoli e la pelle), con 4-0 punti di sutura assorbibili e un modello continuo per prevenire l&#39;infezione. Oltre alla tecnica modello sutura continua, un&#39;altra opzione potrebbe essere quella di utilizzare una tecnica semplice ed interrotta, particolarmente per la chiusura parete del corpo per evitare deiscenza. </li><li> Applicare topico pomata antibiotica sopra l&#39;area di incisione per prevenire le infezioni. </li><li> Trasferire il ratto in una gabbia di osservazione libera assestamento su un rilievo caldo fino al recupero completo con una gamma di temperatura impostata a 35-37 ° C. Assestamento allentato dovrebbe essere coperto (ad esempio con un asciugamano drappo o carta) o rimosso dalla gabbia fino a quando gli animali sono completamente recuperati per impedire il soffocamento o aspirazione di biancheria da letto. </li><li> Dopo l&#39;intervento chirurgico, osservare gli animali continuamente fino a quando la respirazione spontanea, poi ogni ora per un paio d&#39;ore. Re-dosare l&#39;analgesico buprenorfina SR 72 orein seguito, se si osservano segni di disagio, come la letargia, curvo e trasandato, smorfia, non riprendere le normali attività. </li><li> Dopo il completamento di tutti gli studi, eutanasia degli animali con l&#39;inalazione di una dose eccessiva di CO 2 e di raccogliere i tessuti renali di analisi ex vivo come istologia e Western Blotting 5. </li></ol>

<ol>
	<li>Dejani, H., Eisen, T. D., Finkelstein, F. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revascularization+of+renal+artery+stenosis+in+patients+with+renal+insufficiency.">Revascularization of renal artery stenosis in patients with renal insufficiency.</a> Am. J. Kidney Dis. 36 (4), 752-758 (2000).</li><li>Kang, D. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+angiogenesis+in+the+remnant+kidney+model:+I.+Potential+role+of+vascular+endothelial+growth+factor+and+thrombospondin-1.">Impaired angiogenesis in the remnant kidney model: I. Potential role of vascular endothelial growth factor and thrombospondin-1.</a> J. Am. Soc. Nephrol. 12 (7), 1434-1447 (2001).</li><li>Kang, D. H., Hughes, J., Mazzali, M., Schreiner, G. F., Johnson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+angiogenesis+in+the+remnant+kidney+model:+II.+Vascular+endothelial+growth+factor+administration+reduces+renal+fibrosis+and+stabilizes+renal+function.">Impaired angiogenesis in the remnant kidney model: II. Vascular endothelial growth factor administration reduces renal fibrosis and stabilizes renal function.</a> J. Am. Soc. Nephrol. 12 (7), 1448-1457 (2001).</li><li>Kang, D. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+microvascular+endothelium+in+progressive+renal+disease.">Role of the microvascular endothelium in progressive renal disease.</a> J. Am. Soc. Nephrol. 13 (3), 806-816 (2002).</li><li>Chade, A. R., Kelsen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversal+of+renal+dysfunction+by+targeted+administration+of+VEGF+into+the+stenotic+kidney:+a+novel+potential+therapeutic+approach.">Reversal of renal dysfunction by targeted administration of VEGF into the stenotic kidney: a novel potential therapeutic approach.</a> Am. J. Physiol.- Renal Physiol. 302 (10), F1342-F1350 (2012).</li><li>Eirin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+Glomerular+Filtration+Rate+After+Renal+Revascularization+Correlate+With+Microvascular+Hemodynamics+and+Inflammation+in+Swine+Renal+Artery+Stenosis.">Changes in Glomerular Filtration Rate After Renal Revascularization Correlate With Microvascular Hemodynamics and Inflammation in Swine Renal Artery Stenosis.</a> Circ.-Cardiovasc. Interv. 5 (5), 720-728 (2012).</li><li>Chade, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+Renal+Injury+in+Early+Atherosclerosis+and+Renovascular+Disease.">Distinct Renal Injury in Early Atherosclerosis and Renovascular Disease.</a> Circulation. 106 (9), 1165-1171 (2002).</li><li>Seddon, M., Saw, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atherosclerotic+renal+artery+stenosis:+review+of+pathophysiology,+clinical+trial+evidence,+and+management+strategies.">Atherosclerotic renal artery stenosis: review of pathophysiology, clinical trial evidence, and management strategies.</a> Can. J. Cardiol. 27 (4), 468-480 (2011).</li><li>Lao, D., Parasher, P. S., Cho, K. C., Yeghiazarians, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atherosclerotic+renal+artery+stenosis--diagnosis+and+treatment.">Atherosclerotic renal artery stenosis--diagnosis and treatment.</a> Mayo Clin Proc. 86 (7), 649-657 (2011).</li><li>Sharfuddin, A. A., Molitoris, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+ischemic+acute+kidney+injury.">Pathophysiology of ischemic acute kidney injury.</a> Nat. Rev. Nephrol. 7, 189-200 (2011).</li><li>Noiri, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+and+nitrosative+stress+in+acute+renal+ischemia.">Oxidative and nitrosative stress in acute renal ischemia.</a> Am. J. Physiol.- Renal Physiol. 281 (5), F948-F957 (2001).</li><li>Koesters, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tubular+Overexpression+of+Transforming+Growth+Factor-&#206;1+Induces+Autophagy+and+Fibrosis+but+Not+Mesenchymal+Transition+of+Renal+Epithelial+Cells.">Tubular Overexpression of Transforming Growth Factor-&#206;1 Induces Autophagy and Fibrosis but Not Mesenchymal Transition of Renal Epithelial Cells.</a> Am. J. Pathol. 177 (2), 632-643 (2010).</li><li>Shanley, P. F., Rosen, M. D., Brezis, M., Silva, P., Epstein, F. H., Rosen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topography+of+focal+proximal+tubular+necrosis+after+ischemia+with+reflow+in+the+rat+kidney.">Topography of focal proximal tubular necrosis after ischemia with reflow in the rat kidney.</a> Am. J. Pathol. 122 (3), 462-468 (1986).</li></ol>

Questo lavoro è in parte sostenuto da un assegno di ricerca da Astra Zeneca.

The increasing incidence of chronic kidney disease raises the need for novel therapeutic approaches that can promote functional kidney recovery7,8. Traditional therapies include the systemic administration of anti-inflammatory, anti-fibrotic drugs9. However, these strategies are frequently characterized by unwanted side effects due to off-target distribution of the injected drug. Therefore, in this manuscript, we describe a simple procedure for delivering drugs into the renal artery of rats. This protocol will be useful for testing drugs in rodent models of hypertension, renal artery stenosis or any disease that simulate clinical renovascular diseases. However, in preclinical studies, the use of small animal models, though convenient and sometimes beneficial, may bear several disadvantages that need to be taken into consideration. Importantly, the small size of the organs in rodents requires a higher degree of surgical skills, which may be achieved with some practice. Thus, following previously tested critical steps of a surgical technique, may facilitate and aid the learning process. 
A pivotal aspect of the procedure described is the dissection of the renal artery from the renal vein and the surrounding adipose/connective tissue. The use of cotton swabs helps avoid damage to nerves and lymphatic and/or blood vessels, in particular to the thin-walled veins. Another important and delicate step is the cannulation of the renal artery: because of the very small diameter of the vessel, the catheter might puncture through the artery. For this reason, working under a surgical microscope and finding a good angle for the insertion of the catheter is fundamental: the needle should be as much as possible parallel to the renal artery. At the same time, the observation of a distinct, slow color change in the kidney, caused by the infusion of the drug suspension, is essential to be certain that the catheter has been positioned correctly. 
One of the most important issues that might affect the outcome of this surgical procedure is the duration of the ischemia. A prolonged and complete restriction of blood supply to the kidney may cause serious damage to the renal tissue, characterized by increase of reactive oxygen species (ROS) levels and activation of fibrogenic mechanisms and inflammatory mediators within the renal parenchyma10-12. Thus, it is crucial to minimize the time of ischemia in an effort to prevent irreversible interstitial fibrosis as well as cell death. Selective necrosis of the immediate postglomerular portion of a tubular segment sensitive to inhibition of mitochondrial respiration has been previously described after only 15 min of ischemia in rats13. Therefore, we recommend that the total renal ischemia should not exceed 7 min in duration.
Finally, for an optimal recovery, it is very important to keep the animal very well hydrated and warm for the entire duration of the surgery and immediately post op, as laparotomy procedures may result in body fluids loss as well as hypothermia, both of which may increase post op mortality.
In this manuscript, we are showing results obtained from healthy rats to demonstrate the feasibility and the safety of the surgical procedure. We believe that a greater up-regulation of the VEGF pathway would be observed in a disease model. Furthermore, it is important to highlight that, although with the described method biologics can be delivered directly into the renal artery, other organs might be affected via venous outflow and subsequent systemic circulation. However, clamping the vein soon after the infusion for about 5 min, should minimize any potential systemic side effect. In conclusion, the protocol described is safe, and useful for large scale drug testing in rat preclinical studies where high throughput is required before translation to more clinically relevant animal models.

The renal microvasculature is involved in a wide spectrum of kidney diseases. Depending on the pathophysiology of disease, the endothelial cells may present structural or functional impairment, which may play a pivotal role in propagating kidney damage by creating an ischemic microenvironment. This renal microvascular dysfunction may catalyze the onset of a progressive deterioration of renal function over time, leading to chronic kidney disease (CKD), end-stage renal disease, hypertension and cardiorenal syndrome. In fact, untreated hypertension may have implications in renal arterioles, causing nephrosclerosis or glomerulosclerosis with significant reduction in vascular volume fraction, increase in vascular resistance and development of tubulointerstitial fibrosis1.
Loss of renal microvasculature may be due to altered vascular homeostasis induced by local angiogenic/anti-angiogenic factors imbalance. This correlates with attenuated Vascular Endothelial Growth Factor (VEGF) signaling as well as elevated thrombospondin-12-4. Thus, using different animal models (mice, rats and pigs), the therapeutic effect of exogenous administration of VEGF has been recently investigated in some forms of renal disease, showing reduced interstitial fibrosis and stabilized renal and cardiac function3-5. This effect is likely due to actions of VEGF on endothelial cells of the microvascular bed and inflammatory monocyte phenotype switching6.
For some preclinical studies, the use of rodents, the most commonly used laboratory animals, provides a good animal model for high throughput studies due to relatively low costs and ease of handling. Moreover, the use of genetically-altered rats as models of human diseases, such as hypertension, has become more and more frequent in the scientific community. Therefore, the aim of this protocol is to describe a useful intrarenal VEGF delivery technique in rats that is easy to perform and highly reproducible. Moreover, the same method can be used to selectively deliver other drugs.

<table><tbody><tr><td>Surgical Microscope</td><td>Leica</td><td>M125</td><td></td></tr><tr><td>Isoflurane 100 ml</td><td>Cardinal Healthcare</td><td>PI23238</td><td>Anesthetic</td></tr><tr><td>Buprenorphine HCL SR LAB 1 mg/ml, 5 ml</td><td>ZooPharm Pharmacy</td><td></td><td>Buprenorphine narcotic analgesic formulated in a polymer that slows absorption extending duration of action (72 hr duration of activity).&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Liquid is viscous, warming to RT aids in drawing into syringe.&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Recommended dosage: 1 - 1.2 mg/kg SC. DO NOT DILUTE.</td></tr><tr><td>Puralube Vet Ophthalmic Ointment</td><td>Dechra</td><td>NDC17033-211-38</td><td>Sterile ocular lubricant</td></tr><tr><td>Lactated Ringer&#039;s Injection, USP, 250 ml VIAFLEX Plastic Container</td><td>Baxter Healthcare Corp.</td><td>NDC0338-0117-02</td><td>For body fluids replacement</td></tr><tr><td>Sol Povidone-Iodine&amp;nbsp; Swabstick, 3&#039;&amp;nbsp;</td><td>Cardinal Heatlhcare</td><td>23405-010B</td><td></td></tr><tr><td>Sterile cotton tipped applicators</td><td>Kendall</td><td>8884541300</td><td></td></tr><tr><td>4-0 silk suture (without needle)&amp;nbsp;</td><td>Cardinal Heatlhcare</td><td>A183H</td><td></td></tr><tr><td>Vessel Clip, Straight, 0.75 mm x 4 mm Jaw</td><td>World Precision Instruments&amp;nbsp;</td><td>501779-G</td><td></td></tr><tr><td>I.V. Catheter, Straight Hub, Radiopaque, 24 g x 3/4&quot;, FEP Polymer</td><td>Jelco</td><td>4053</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Life Technologies</td><td>10010023</td><td></td></tr><tr><td>SURGIFOAM Absorbable Gelatin Sponge</td><td>Cardinal Healthcare</td><td>179082</td><td></td></tr><tr><td>4-0 VICRYL PLUS (ANTIBACTERIAL) VIOLET 27&quot; RB-1 TAPER</td><td>Ethicon</td><td>VCP304H</td><td>For muscle layer suturing</td></tr><tr><td>4-0 VICRYL PLUS (ANTIBACTERIAL) UNDYED 18&quot; PC-3 CUTTING</td><td>Ethicon</td><td>VCP845G</td><td>For skin layer suturing</td></tr><tr><td>Triple antibiotic ointment</td><td>Actavis</td><td>NDC0472-0179-56</td><td>For topical use on the site of the incision</td></tr><tr><td>Recombinant Rat VEGF 164 Protein</td><td>R&amp;amp;D Sytems</td><td>564-RV</td><td></td></tr><tr><td>Rabbit monoclonal VEGFA</td><td>Abcam</td><td>ab46154</td><td></td></tr><tr><td>Rabbit monoclonal FLK1</td><td>Cell Signaling</td><td>9698</td><td></td></tr><tr><td>Rabbit monoclonal AKT</td><td>Cell Signaling</td><td>4691</td><td></td></tr><tr><td>Rabbit monoclonal phosphoAKT (Ser 473)</td><td>Cell Signaling</td><td>4060</td><td></td></tr><tr><td>Rabbit monoclonal p44/42 MAPK (ERK1/2)</td><td>Cell Signaling</td><td>4695</td><td></td></tr><tr><td>Rabbit monoclonal phospho p44/42 MAPK (Thr202 and Tyr 204)</td><td>Cell Signaling</td><td>4370</td><td></td></tr></tbody></table>

intravascular delivery of biologics to the rat kidney

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage Renal Disease with high risk of death for cardiovascular events. Moreover, recent clinical studies have shown that renovascular structure and function may have a great impact on functional renal recovery after surgery. Here, we describe a protocol for the delivery of drugs into the renal artery of rats. This procedure offers significant advantages over the frequently used systemic administration as it may allow a more localized therapeutic effect. In addition, the use of rodents in pharmacodynamic analysis of preclinical studies may be cost effective, paving the way for the design of translational experiments in larger animal models. Using this technique, infusion of rat recombinant Vascular Endothelial Growth Factor (VEGF) protein in rats has induced activation of VEGF signaling as shown by increased expression of FLK1, pAKT/AKT, pERK/ERK. In summary, we established a protocol for the intrarenal delivery of drugs in rats, which is simple and highly reproducible.

<html> Abbiamo iniettato due diverse dosi di VEGF ricombinante di ratto (rrVEGF, 0,17 mg / kg e 5 mg / kg) o PBS. Gli animali sono stati sacrificati post-chirurgico 8 ore per esaminare l&#39;attivazione della via VEGF. La procedura chirurgica non ha influenzato la morfologia del rene perfuso (Figura 1A) rispetto al controllo (Figura 1B), come mostrato da H &amp; E colorazione. Mentre Sirius colorazione rossa non ha mostrato alcun aumento nella deposizione matrice extracellulare in risposta al tempo ischemico e l&#39;infusione della rrVEGF (Figura 1C) rispetto al controllo (Figura 1D). Con analisi Western Blot, abbiamo osservato un piccolo ma significativo aumento dell&#39;espressione delle proteine ​​coinvolte nel pathway VEGF, come VEGF, Flk1, pAKT / AKT, pERK / ERK (Figura 2). d / 54418 / 54418fig1.jpg &quot;/&gt; Figura 1:. Rappresentativi Immagini di istologica colorazione di renale Tissue ematossilina e eosina (H &amp; E) La colorazione di sezioni di rene non mostra cambiamenti anatomici in ratti che hanno ricevuto VEGF (5 mg / kg, B) rispetto agli animali sham (A) tramite iniezione intrarenale. Considerando che, sirius colorazione rossa mostra differenze significative nella deposizione di collagene in ratti che hanno ricevuto VEGF (5 mg / kg, D) rispetto agli animali sham (C). Barra di scala rappresenta 1 mm e immagini ingrandite rappresentano 400 micron. <a href="http://ecsource.jove.com/files/ftp_upload/54418/54418fig1large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="figura 2" src="/files/ftp_upload/54418/54418fig2.jpg" /> Figura 2:. L&#39;attivazione di VEGF Pathway analisi Western Blot di VEGF, Flk1, phosphoAKT / AKT e phosphoERK / ERK mostra la sovraregolazione di VEGF percorso dopo l&#39;infusione rrVEGF nel rene (n = 4). I dati sono presentati come media ± SEM. * P &lt;0.05 vs PBS. <a href="http://ecsource.jove.com/files/ftp_upload/54418/54418fig2large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a>

Watch this Scientific Journal Video about Intravascular Delivery of Biologics to the Rat Kidney at JoVE.com

Intravascular Delivery of Biologics to the Rat Kidney

La somministrazione di farmaci per il recupero della funzione renale richiede il controllo della localizzazione e distribuzione del composto terapeutico. Qui, descriviamo in dettaglio una semplice tecnica per la consegna intrarenale di farmaci nei ratti. Questa procedura può essere eseguita facilmente senza mortalità ed elevata riproducibilità.

Consegna intravascolare di Biologics al rene di ratto

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage ...

intravascular-delivery-of-biologics-to-the-rat-kidney

Nanyang Technological University

Research

JoVE Journal

Medicine

7.5K Views.  Mayo Clinic.  La somministrazione di farmaci per il recupero della funzione renale richiede il controllo della localizzazione e distribuzione del composto terapeutico. Qui, descriviamo in dettaglio una semplice tecnica per la consegna intrarenale di farmaci nei ratti. Questa procedura può essere eseguita facilmente senza mortalità ed elevata riproducibilità. 

Video: Intravascular Delivery of Biologics to the Rat Kidney

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

Istituzione di un<em&gt; In Vitro</em&gt; Modello di Rat barriera emato-encefalica (BBB): A Focus on BBB impermeabilità e Trasporti recettore-mediata

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

Ricerca

Medicina

Transarterial Administration of Oncolytic Viruses for Locoregional Therapy of Orthotopic HCC in Rats

Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis. In recent years, oncolytic virotherapies have proven themselves to be potentially powerful tools to fight malignancy. Due to the unique dual blood supply in the liver, it is possible to apply therapies locally to orthotopic liver tumors, which are predominantly fed by arterial blood flow. We have previously demonstrated that hepatic arterial delivery of oncolytic viruses results in safe and efficient transduction efficiency of multifocal HCC lesions, resulting in significant prolongation of survival in immune competent rats. This procedure closely mimics the application of transarterial embolization in patients, which is the standard palliative care provided to many HCC patients. The ability to administer tumor therapies through the hepatic artery in rats allows for a highly sophisticated preclinical model for evaluating novel viral vectors under development. Here we describe the detailed protocol for microdissection of the hepatic artery for infusion of oncolytic virus vectors to treat orthotopic HCC.

Oncolytic virotherapies are under development as novel therapeutics for the treatment of hepatocellular carcinoma (HCC). Here we describe a method for locoregional therapy of HCC via hepatic arterial administration of oncolytic virus.

Transarteriosa somministrazione di oncolitici virus per locoregionale Terapia delle ortotopico HCC in Rats

Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis.  In recent years, oncolytic virotherapies have proven ...

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The simple murine AVF model was combined with intraluminal administration of drug solution to the venous endothelium at the same time as fistula creation. Technical aspects of this model are discussed. Under general anesthesia, an abdominal incision is made and the aorta and inferior vena cava (IVC) are exposed. The infra-renal aorta and IVC are dissected for clamping. After proximal and distal clamping, the puncture site is exposed and a 25 G needle is used to puncture both walls of the aorta and into the IVC. Immediately after the puncture, a reporter gene-expressing viral vector was infused in the IVC via the same needle, followed by 15 min of incubation. The intraluminal administration method enabled more robust viral gene delivery to the venous endothelium compared to administration by the external route. This novel method of delivery will facilitate studies that explore the role of the endothelium in AVF maturation and enable intraluminal drug delivery at the time of surgical operation.

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

Intraluminale Drug Delivery per il mouse fistola artero-venosa endotelio

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The ...

Orthotopic Rat Kidney Transplantation: A Novel and Simplified Surgical Approach

Kidney transplantation offers increased survival rates and improved quality of life for patients with end-stage renal disease, as compared to any type of renal replacement therapy. Over the past few decades, the rat kidney transplantation model has been used to study the immunological phenomena of rejection and tolerance. This model has become an indispensable tool to test new immunomodulatory pharmaceuticals and regimens prior to proceeding with expensive preclinical large animal studies.
This protocol provides a detailed overview of how to reliably perform orthotopic kidney transplantation in rats. This protocol includes three distinctive steps that increase the probability of success: perfusion of the donor kidney by flushing through the portal vein and the use of a cuff system to anastomose the renal veins and ureters, thereby decreasing cold and warm ischemia times. Using this technique, we have achieved survival rates beyond 6 months with normal serum creatinine in animals with syngeneic or tolerant kidney transplants. Depending on the aim of the study, this model can be modified by pre- or posttransplant treatments to study the acute, chronic, cellular, or antibody-mediated rejection. It is a reproducible, reliable, and cost-effective animal model to study different aspects of kidney transplantation.

The purpose of this manuscript and protocol is to explain and demonstrate in detail the surgical procedure of orthotopic kidney transplantation in rats. This method is simplified to achieve the correct perfusion of the donor kidney and shorten the reperfusion time by using the venous and ureteral cuff anastomosis technique.

Trapianto di rene ortotopico ratto: un nuovo approccio chirurgico semplificato

Kidney transplantation offers increased survival rates and improved quality of life for patients with end-stage renal disease, as compared to any type of ...

Intraventricular Drug Delivery and Sampling for Pharmacokinetics and Pharmacodynamics Study

Although the blood-brain barrier (BBB) protects the brain from foreign entities, it also prevents some therapeutics from crossing into the central nervous system (CNS) to ameliorate diseases or infections. Drugs are administered directly into the CNS in animals and humans to circumvent the BBB. The present protocol describes a unique way of treating brain infections through intraventricular delivery of antibiotics, i.e., polymyxins, the last-line antibiotics to treat multi-drug resistant Gram-negative bacteria. A straightforward stereotaxic surgery protocol was developed to implant a guide cannula reaching into the lateral ventricle in rats. After a recovery period of 24 h, rats can be injected consciously and repeatedly through a cannula that is fitted to the guide. Injections can be delivered manually as a bolus or infusion using a microinjection pump to obtain a slow and controlled flow rate. The intraventricular injection was successfully confirmed with Evans Blue dye. Cerebrospinal fluid (CSF) can be drained, and the brain and other organs can be collected. This approach is highly amenable for studies involving drug delivery to the CNS and subsequent assessment of pharmacokinetic and pharmacodynamic activity.

Delivery of therapeutics directly into the central nervous system is one way of circumventing the blood-brain barrier. The present protocol demonstrates intracerebroventricular injection for subsequent collection of cerebrospinal fluid and bodily organs. This facilitates the investigation of drug pharmacokinetics and pharmacodynamics in animal models for developing new treatments.

Although the blood-brain barrier (BBB) protects the brain from foreign entities, it also prevents some therapeutics from crossing into the central nervous ...

Neuroscienze

Direct Drug Delivery to Kidney via the Renal Artery

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research field. Accumulation of drugs in certain organs may give rise to adverse and unwanted effects, depending on the nature of injectate. To minimize spillover and/or accumulation in other tissues, the herein described method directs the formulation into the renal artery bloodstream by inserting a catheter in the infra renal aorta, just below where it branches into the renal artery, resulting in the kidney as first reached organ and distributing of formulation throughout the kidney.
This manuscript provides a detailed description of the method, as well as its challenges and difficulties. It guides the experimenter to become skillful with this type of microsurgery that requires accuracy under sterile conditions. Speed is crucial for minimizing the ischemia and practicing the procedure will increase the chance of successful injections without adverse effects. By modulating the time between injection and reperfusion as well as the injected volume, the risk of spillover to other organs is mitigated. 
Note that this technique is suitable for single dosing strategies.

This manuscript describes a method for targeted delivery to a single kidney via a catheter placed in the infrarenal abdominal aorta in the mouse.

Consegna diretta di farmaci ai reni attraverso l'arteria renale

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research ...

A Rat Orthotopic Renal Transplantation Model for Renal Allograft Rejection

Renal allograft rejection limits the long-term survival of patients after renal transplantation. Rat orthotopic renal transplantation is an essential model to investigate the mechanism of renal allograft rejection in pre-clinical studies and could aid in the development of novel approaches to improve the long-term survival of renal allografts. Donor kidney implantation in rat orthotopic renal transplantation is commonly performed by end-to-side anastomosis to recipients' aorta and inferior vena cava. In this model, the donor's kidney was implanted using end-to-end anastomosis to the recipients' renal artery and renal vein. The donor's ureter was anastomosed to the recipient's bladder in an end-to-side 'tunnel' method. This model contributes to better healing of ureter-bladder anastomosis and increases the recipients' survival by avoiding interference with blood supply and venous reflux of the lower body. This model can be used to investigate the mechanisms of acute and chronic immune and pathologic rejection of renal allografts. Here, the study describes the detailed protocols of this orthotopic renal transplantation between rats.

The rat orthotopic renal transplantation model contributes to investigating the mechanism of renal allograft rejection. The current model increases the recipients' survival without interference with blood supply and venous reflux of the lower body using an end-to-end anastomosis of kidney implantation and an end-to-side "tunnel" method of ureter-bladder anastomosis.

Un modello di trapianto renale ortotopico di ratto per il rigetto dell'allotrapianto renale

Renal allograft rejection limits the long-term survival of patients after renal transplantation. Rat orthotopic renal transplantation is an essential ...

Hydrodynamic Renal Pelvis Injection for Non-viral Expression of Proteins in the Kidney

Hydrodynamic injection creates a local, high-pressure environment to transfect various tissues with plasmid DNA and other substances. Hydrodynamic tail vein injection, for example, is a well-established method by which the liver can be transfected. This manuscript describes an application of hydrodynamic principles by injection of the mouse kidney directly with plasmid DNA for kidney-specific gene expression. Mice are anesthetized and the kidney is exposed by a flank incision followed by a fast injection of a plasmid DNA-containing solution directly into the renal pelvis. The needle is kept in place for ten seconds and the incision site is sutured. The following day, live animal imaging, Western blot, or immunohistochemistry may be used to assay gene expression, or other assays suited to the transgene of choice are used for detection of the protein of interest. Published methods to prolong gene expression include transposon-mediated transgene integration and cyclophosphamide treatment to inhibit the immune response to the transgene.

This protocol describes a method to inject plasmid DNA into the mouse kidney via the renal pelvis to produce transgene expression specifically in the kidney.

Iniezione idrodinamica pelvi renale per Non virali espressione delle proteine nel rene

Hydrodynamic injection creates a local, high-pressure environment to transfect various tissues with plasmid DNA and other substances. Hydrodynamic tail ...

Biologia

Hydrodynamic Renal Pelvis Injection: A Technique to Directly Deliver DNA into Kidney Cells of Murine Model

Source: Woodard, L. E. et al. <a href="https://www.jove.com/video/56324" target="_blank">Hydrodynamic Renal Pelvis Injection for Non-viral Expression of Proteins in the Kidney.</a> J. Vis. Exp. (2018)
In this video, we present the protocol for hydrodynamic injection of plasmids into the renal pelvis of the mouse kidney for nucleic acid transfection. This technique allows organ-specific delivery of plasmids by utilizing the principle of high hydrostatic pressure, which forcefully drives the plasmids into the specific site without causing tissue injury.

Iniezione idrodinamica della pelvi renale: una tecnica per fornire direttamente il DNA nelle cellule renali del modello murino

Source: Woodard, L. E. et al. Hydrodynamic Renal Pelvis Injection for Non-viral Expression of Proteins in the Kidney. J. Vis. Exp. (2018)
In this video, ...

JoVE Encyclopedia of Experiments

Ricerca sul cancro

Urinary Tract Cancer

In vivo studies

Intrarenal Drug Delivery in Rats: A Method to Deliver Therapeutic Drugs in Rat Kidney via Intra-arterial Injection

Source: Franchi, et al. <a href="https://www.jove.com/video/54418" target="_blank">Intravascular Delivery of Biologics to the Rat Kidney.</a> J. Vis. Exp.&#160; (2016)
In this video, we demonstrate intra-renal drug administration in rats via the renal artery. This method is beneficial in improving drug targeting, drug potency, and minimizing systemic toxicity.

Somministrazione intrarenale di farmaci nei ratti: un metodo per somministrare farmaci terapeutici nel rene di ratto tramite iniezione intra-arteriosa

Source: Franchi, et al. Intravascular Delivery of Biologics to the Rat Kidney. J. Vis. Exp.&#160; (2016)
In this video, we demonstrate intra-renal drug ...