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

Los experimentos se realizaron en ratas hembra Sprague-Dawley, con un peso de 250-300 g. Todos los procedimientos con animales cumplían con las normas establecidas en la Guía para el Cuidado y Uso de Animales de Laboratorio (Instituto de Recursos Animales de Laboratorio, Academia Nacional de Ciencias, Bethesda, MD, EE.UU.) y fueron aprobados por la Mayo Clinic College de Medicina Institucional Cuidado de Animales y el empleo Comisión (IACUC). 1. Preparación <ol><li> Autoclave todos los instrumentos quirúrgicos antes de la cirugía. Si se han previsto múltiples cirugías en diferentes ratas en el mismo día, enjuague los instrumentos después de cada procedimiento animal y después esterilizar utilizando un esterilizador de cuentas en caliente. </li><li> Anestesiar la rata con 4% de isoflurano en 1 L / min O 2. </li><li> La transferencia de la rata para una resistencia de calentamiento controlado para conservar la temperatura corporal a 37 ° C. Mantener la anestesia con isoflurano 1-2% en 1 L / min O 2. </li><li> Administrar el fármaco analgésico (buprenorfina liberación sostenida de 0,6 mg / kg) Dobcutaneously. </li><li> Aplique un ungüento para los ojos para evitar que se sequen durante el procedimiento. </li><li> Con el fin de compensar la pérdida de fluidos corporales debido a la laparotomía, es importante para administrar 10 ml / kg de 0,9% por vía subcutánea solución salina normal antes de la operación. </li><li> Afeitar el área abdominal y limpiar la piel con almohadillas de etanol povidona yodada y el 70%. </li></ol> 2. Procedimiento Quirúrgico <ol><li> Asegúrese de que la profundidad de la sedación es adecuada mediante el control de los reflejos físicos, como la retirada de la pizca dedo del pie, reflejo palpebral, el tono de la mandíbula, y la respiración tasa / patrón. </li><li> Realizar una laparotomía a través de una pequeña incisión en la línea media (2-2,5 cm de longitud), utilizando una hoja de bisturí quirúrgico Nº 10. </li><li> Tire del delgado y el colon en el lado derecho del abdomen mediante el uso de bastoncillos de algodón y cubrir con una gasa estéril empapada en solución salina normal al 0,9% para mantener los órganos húmedo. </li><li> retraer con suavidad hacia arriba el bazo, el hígado, el estómago y el páncreas para exponer el AORta y la arteria renal izquierda. </li><li> Con la ayuda de un microscopio quirúrgico, separe con cuidado la aorta abdominal por encima y por debajo del riñón izquierdo y la arteria renal izquierda de las venas, la grasa y el tejido conectivo que rodea con unas pinzas de disección romos curvada e hisopos de algodón estériles. <ol><li> Use las pinzas con un movimiento (disección roma) de apertura y cierre repetidos a lo largo de la longitud de los vasos para extraer el tejido conectivo y los bastoncillos de algodón con un movimiento de balanceo lateral para eliminar la grasa. NOTA: La disección de la región peri-aórtica es una etapa muy delicada ya que los nervios y los vasos linfáticos podrían ser dañados. Asegúrese de mantener las arterias húmedo con solución salina durante el procedimiento de disección. </li></ol></li><li> Colocar una sutura de seda 4-0 por debajo de la aorta. </li><li> El uso de clips microvasculares, sujetar la aorta por encima (justo debajo de la arteria mesentérica superior) y por debajo de la bifurcación de la arteria renal. </li><li> La punción de la aorta a nivel del kidn izquierdabifurcación de la arteria ey con un catéter intravenoso 24 G y avanzar el catéter en la arteria renal. NOTA: Este es un paso crítico como punción a través de la arteria renal puede ocurrir. </li><li> Conectar una jeringa llena con la solución de fármaco o solución salina (hasta 500 l) para el catéter y perfundir el riñón. </li><li> Inmediatamente después de la perfusión, la abrazadera de la vena renal izquierda y el uréter izquierdo con un clip microvascular y retirar el catéter. A continuación, coloque un trozo de esponja de gelatina absorbible pinza hemostática, con una pequeña gota de adhesivo tisular, sobre el área de punción de la aorta y aplicar una suave presión con un hisopo de algodón. </li><li> Al mismo tiempo, liberar la abrazadera de la aorta abdominal, por debajo de la bifurcación de la arteria renal izquierda. Después de 5 min, liberar la pinza de la vena renal y el uréter. </li><li> liberar con cuidado la abrazadera de la aorta, por encima de la bifurcación de la arteria renal izquierda, y permitir la reperfusión del riñón. La isquemia renal total debe durar no más de 7 min. </li><li&gt; que no se produzca una hemorragia activa y observar de cerca a la zona durante 10 minutos más. </li><li> Cerrar la incisión abdominal en dos capas (músculo y piel), utilizando 4-0 suturas absorbibles y un patrón continuo para prevenir la infección. Además de la técnica de patrón de sutura continuo, otra opción sería utilizar una técnica simple, interrumpido, especialmente para el cierre de la pared del cuerpo para evitar la dehiscencia. </li><li> Aplique un ungüento antibiótico tópico sobre el área de la incisión para prevenir las infecciones. </li><li> La transferencia de la rata en una jaula de observación libre de la ropa de cama sobre una almohadilla caliente hasta la recuperación completa con un rango de temperatura entre 35-37 ° C. Ropa de cama suelta debe ser cubierto (por ejemplo con un paño o toalla de papel) o retirado de la jaula hasta los animales que se recuperan para evitar la asfixia o aspiración de ropa de cama. </li><li> Después de la cirugía, observar a los animales de forma continua hasta que respira espontáneamente, después cada hora durante unas horas. Vuelva a la dosis del analgésico Buprenorfina SR 72 hrmás adelante, si se observan signos de malestar, como letargo, encorvados y desaliñado, mueca, no reanudar las actividades normales. </li><li> Después de la terminación de todos los estudios, la eutanasia a los animales con la inhalación de una sobredosis de CO2 y obtención de los tejidos renales para ex vivo tales como los análisis de histología y Western Blot 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>

Este trabajo es apoyado en parte por una beca de investigación de 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 pr...

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> Hemos inyectado dos dosis diferentes de VEGF recombinante de rata (rrVEGF, 0,17 mg / kg y 5 mg / kg) o PBS. Los animales se sacrificaron 8 horas después de la cirugía para examinar la activación de la vía VEGF. El procedimiento quirúrgico no afectó la morfología del riñón perfundido (Figura 1A) en comparación con el control (Figura 1B), como se muestra por tinción con H &amp; E. Mientras Sirius tinción con rojo no mostró ningún aument...

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 administración de fármacos para la recuperación de la función renal requiere el control de la localización y distribución del compuesto terapéutico. A continuación, describimos en detalle una técnica sencilla para la entrega intrarrenal de drogas en ratas. Este procedimiento se puede realizar fácilmente sin mortalidad y alta reproducibilidad.

Intravascular de entrega de los productos biológicos para el riñón de rata

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

Research

JoVE Journal

Medicine

7.5K vistas.  Mayo Clinic.  La administración de fármacos para la recuperación de la función renal requiere el control de la localización y distribución del compuesto terapéutico. A continuación, describimos en detalle una técnica sencilla para la entrega intrarrenal de drogas en ratas. Este procedimiento se puede realizar fácilmente sin mortalidad y alta reproducibilidad. 

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.

Establecimiento de un<em&gt; In Vitro</em&gt; Modelo de Rata barrera hematoencefálica (BBB): Un Enfoque en la acreditación Impermeabilidad y transporte mediado por los receptores

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

Investigación

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.

Administración transarterial de virus oncolíticos para locorregional Terapia de HCC ortotópico en ratas

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.

Intraluminal entrega de la droga a la fístula arteriovenosa ratón 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.

Trasplante de riñón de rata ortotópica: un enfoque quirúrgico novedoso y simplificado

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 ...

Neurociencias

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.

Entrega directa de medicamentos al riñón a través de la arteria renal

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 modelo de trasplante renal ortotópico de rata para el rechazo del aloinjerto renal

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.

Inyección hidrodinámica la Pelvis Renal para la no expresión de proteínas en el riñón

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

Biología

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.

Inyección hidrodinámica de pelvis renal: una técnica para administrar directamente el ADN a las células renales de un modelo 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

Investigación sobre el cáncer

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.

Administración intrarrenal de fármacos en ratas: un método para administrar fármacos terapéuticos en riñón de rata mediante inyección intraarterial

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 ...