The experiments were performed on female Sprague-Dawley rats, weighing 250-300 g. All animal procedures complied with the standards stated in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Academy of Sciences, Bethesda, MD, USA) and were approved by the Mayo Clinic College of Medicine Institutional Animal Care and Use Committee (IACUC).
1. Preparation
<ol>
	<li>Autoclave all surgical instruments before surgery. If multiple surgeries on different rats are planned in the same day, rinse instruments after each animal procedure and then sterilize using a hot bead sterilizer.</li>
	<li>Anesthetize the rat with 4% isoflurane in 1 L/min O2.</li>
	<li>Transfer the rat to a controlled heating pad to preserve the body temperature at 37 &#176;C. Maintain anesthesia with 1-2% isoflurane in 1 L/min O2.</li>
	<li>Administer the analgesic drug (Buprenorphine Sustained Release 0.6 mg/kg) subcutaneously.</li>
	<li>Apply ointment to the eyes to prevent drying during the procedure.</li>
	<li>In order to compensate for loss of body fluids due to laparotomy, it is important to administer 10 ml/kg of 0.9% normal saline subcutaneously preoperatively.</li>
	<li>Shave the abdominal area and clean the skin with povidone-iodine and 70% ethanol pads.</li>
</ol>
2. Surgical Procedure
<ol>
	<li>Ensure that the depth of sedation is adequate by monitoring physical reflexes, such as withdrawal from toe pinch, palpebral reflex, jaw tone, and respiration rate/pattern.</li>
	<li>Perform a laparotomy through a small midline incision (2-2.5 cm in length) using a surgical scalpel blade No. 10.</li>
	<li>Pull the intestine and colon to the right side of the abdomen by using cotton swabs and cover them with sterile gauze soaked in to 0.9% normal saline to maintain the organs moist.</li>
	<li>Gently retract upward the spleen, liver, stomach and pancreas to expose the aorta and the left kidney artery.</li>
	<li>With the help of a surgical microscope, carefully separate the abdominal aorta above and below the left kidney and the left renal artery from the veins, the fat and the surrounding connective tissue with blunt dissecting curved forceps and sterile cotton swabs.
	<ol>
		<li>Use the forceps with a repeated open-close motion (blunt dissection) along the length of the vessels to remove the connective tissue and the cotton swabs with a lateral rolling motion to remove the fat. 
		NOTE: The dissection of the peri-aortic region is a very delicate step as nerves and lymphatic vessels might be damaged. Make sure to keep the arteries moist with saline during the dissection procedure.</li>
	</ol>
	</li>
	<li>Place a 4-0 silk suture underneath the aorta.</li>
	<li>Using microvascular clips, clamp the aorta above (just below the superior mesenteric artery) and below the renal artery bifurcation.</li>
	<li>Puncture the aorta at the level of the left kidney artery bifurcation with a 24 G intravenous catheter and advance the catheter into the renal artery. 
	NOTE: this is a critical step as puncture through the renal artery may occur.</li>
	<li>Connect a syringe filled with the drug solution or saline (up to 500 &#956;l) to the catheter and perfuse the kidney.</li>
	<li>Immediately after perfusion, clamp the left renal vein and the left ureter with a microvascular clip and remove the catheter. Then, place a piece of absorbable hemostat gelatin sponge, with a small drop of tissue adhesive, over the punctured area of the aorta and gently apply pressure with a cotton swab.</li>
	<li>At the same time, release the clamp from the abdominal aorta, below the left renal artery bifurcation. After 5 min, release the clamp from renal vein and ureter.</li>
	<li>Carefully release the clamp from the aorta, above the left renal artery bifurcation, and allow for kidney reperfusion. The total renal ischemia should last no longer than 7 min.</li>
	<li>Ensure that no active bleeding occurs and closely observe the area for 10 more min.</li>
	<li>Close the abdominal incision in two layers (muscle and skin), using 4-0 absorbable sutures and a continuous pattern to prevent infection.&#160;In addition to the continuous pattern suture technique, another option would be to use a simple, interrupted technique, especially for body wall closure to prevent dehiscence.</li>
	<li>Apply topical antibiotic ointment over the incision area to prevent infections.</li>
	<li>Transfer the rat into a bedding-free observation cage on a warm pad until complete recovery with a temperature range set at 35-37 &#176;C. Loose bedding should be covered (e.g. with a drape or paper towel) or removed from the cage until animals are fully recovered to prevent suffocation or aspiration of bedding.</li>
	<li>After surgery, observe the animals continuously until breathing spontaneously, then hourly for a few hours. Re-dose the analgesic Buprenorphine SR 72 hr later if signs of discomfort are observed, such as lethargy, hunched and scruffy, grimace, not resuming normal activities.</li>
	<li>After completion of all studies, euthanize animals with the inhalation of an overdose of CO2 and harvest the renal tissues for ex vivo analyses such as histology and Western Blotting5.</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>

This work is partly supported by a research grant from 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 border="0" cellpadding="0" cellspacing="0" width="928">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Surgical Microscope</td>
			<td style="width:176px;">Leica</td>
			<td style="width:119px;">M125</td>
			<td style="width:376px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Isoflurane 100 ml</td>
			<td style="width:176px;">Cardinal Healthcare</td>
			<td style="width:119px;">PI23238</td>
			<td>Anesthetic</td>
		</tr>
		<tr height="133">
			<td height="133" style="height:133px;width:257px;">Buprenorphine HCL SR LAB 1mg/ml, 5 ml</td>
			<td style="width:176px;">ZooPharm Pharmacy</td>
			<td style="width:119px;"></td>
			<td style="width:376px;">Buprenorphine narcotic analgesic formulated in a polymer that slows absorption extending duration of action (72 hours duration of activity).&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Liquid is viscous, warming to room temperature aids in drawing into syringe.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Recommended dosage: 1-1.2 mg/kg SC. DO NOT DILUTE.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Puralube Vet Ophthalmic Ointment</td>
			<td style="width:176px;">Dechra</td>
			<td style="width:119px;">NDC17033-211-38</td>
			<td>Sterile ocular lubricant</td>
		</tr>
		<tr height="56">
			<td height="56" style="height:56px;width:257px;">Lactated Ringer&#39;s Injection, USP, 250 mL VIAFLEX Plastic Container</td>
			<td style="width:176px;">Baxter Healthcare Corp.</td>
			<td style="width:119px;">NDC0338-0117-02</td>
			<td>For body fluids replacement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Sol Povidone-Iodine&#160; Swabstick, 3&#39;&#160;</td>
			<td style="width:176px;">Cardinal Heatlhcare</td>
			<td style="width:119px;">23405-010B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Sterile cotton tipped applicators</td>
			<td style="width:176px;">Kendall</td>
			<td style="width:119px;">8884541300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">4-0 silk suture (without needle)&#160;</td>
			<td style="width:176px;">Cardinal Heatlhcare</td>
			<td style="width:119px;">A183H</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Vessel Clip, Straight, 0.75 x 4mm Jaw</td>
			<td style="width:176px;">World Precision Instruments&#160;</td>
			<td style="width:119px;">501779-G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">I.V. Catheter, Straight Hub, Radiopaque, 24g x 3/4&#34;, FEP Polymer</td>
			<td style="width:176px;">Jelco</td>
			<td align="right" style="width:119px;">4053</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Phosphate Buffered Saline</td>
			<td style="width:176px;">Life Technologies</td>
			<td align="right" style="width:119px;">10010023</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">SURGIFOAM Absorbable Gelatin Sponge</td>
			<td style="width:176px;">Cardinal Healthcare</td>
			<td align="right" style="width:119px;">179082</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">4-0 VICRYL PLUS (ANTIBACTERIAL) VIOLET 27&#34; RB-1 TAPER</td>
			<td style="width:176px;">Ethicon</td>
			<td style="width:119px;">VCP304H</td>
			<td>For muscle layer suturing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">4-0 VICRYL PLUS (ANTIBACTERIAL) UNDYED 18&#34; PC-3 CUTTING</td>
			<td style="width:176px;">Ethicon</td>
			<td style="width:119px;">VCP845G</td>
			<td>For skin layer suturing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Triple antibiotic ointment</td>
			<td style="width:176px;">Actavis</td>
			<td style="width:119px;">NDC0472-0179-56</td>
			<td>For topical use on the site of the incision</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Recombinant Rat VEGF 164 Protein</td>
			<td style="width:176px;">R&#38;D Sytems</td>
			<td style="width:119px;">564-RV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Rabbit monoclonal VEGFA</td>
			<td style="width:176px;">Abcam</td>
			<td style="width:119px;">ab46154</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Rabbit monoclonal FLK1</td>
			<td style="width:176px;">Cell Signaling</td>
			<td align="right" style="width:119px;">9698</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Rabbit monoclonal AKT</td>
			<td style="width:176px;">Cell Signaling</td>
			<td align="right" style="width:119px;">4691</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Rabbit monoclonal phosphoAKT (Ser 473)</td>
			<td style="width:176px;">Cell Signaling</td>
			<td align="right" style="width:119px;">4060</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Rabbit monoclonal p44/42 MAPK (ERK1/2)</td>
			<td style="width:176px;">Cell Signaling</td>
			<td align="right" style="width:119px;">4695</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Rabbit monoclonal phospho p44/42 MAPK (Thr202 and Tyr 204)</td>
			<td style="width:176px;">Cell Signaling</td>
			<td align="right" style="width:119px;">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.

We injected two different doses of recombinant rat VEGF (rrVEGF, 0.17 &#956;g/kg and 5 &#956;g/kg) or PBS. The animals were euthanized 8 hr post-surgery to examine the activation of the VEGF pathway. The surgical procedure did not affect the morphology of the perfused kidney (Figure 1A) when compared to control (Figure 1B), as shown by H&#38;E staining. While Sirius red staining did not show any increase in extracellular matrix deposition in response to the ischemic time and the infusion of the rrVEGF (Figure 1C) when compared to control (Figure 1D). By Western Blot analysis, we observed a small but significant increase in the expression of proteins involved in the VEGF pathway, such as VEGF, FLK1, pAKT/AKT, pERK/ERK (Figure 2).
<img alt="Figure 1" src="/files/ftp_upload/54418/54418fig1.jpg" /> 
Figure 1: Representative Images of Histological Staining of Renal Tissue. Hematoxilin and Eosin (H&#38;E) staining of kidney sections shows no anatomical changes in rats that received VEGF (5 &#956;g/kg, B) compared to sham animals (A) via intrarenal injection. Whereas, sirius red staining shows no significant differences in collagen deposition in rats that received VEGF (5 &#956;g/kg, D) compared to sham animals (C). Scale bar represents 1 mm and magnified images represent 400 &#956;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54418/54418fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54418/54418fig2.jpg" /> 
Figure 2: Activation of VEGF Pathway. Western Blot analysis of VEGF, FLK1, phosphoAKT/AKT and phosphoERK/ERK showing the upregulation of VEGF pathway after rrVEGF infusion in the kidney (n = 4). Data are presented as mean &#177; SEM. *p &#60;0.05 vs. PBS. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54418/54418fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</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

The administration of drugs for recovery of kidney function requires control of the localization and distribution of the therapeutic compound. Here, we describe in detail a simple technique for intrarenal delivery of drugs in rats. This procedure may be easily performed with no mortality and high reproducibility.

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

7.4K Views.  Mayo Clinic. The administration of drugs for recovery of kidney function requires control of the localization and distribution of the therapeutic compound. Here, we describe in detail a simple technique for intrarenal delivery of drugs in rats. This procedure may be easily performed with no mortality and high reproducibility.

Video: Intravascular Delivery of Biologics to the Rat Kidney

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these diseases. Chronic retinal diseases such as retinal oedema or neovascularization usually require multiple intraocular injections to effectively treat the condition. However, the risks associated with these injections increase with repeated delivery of the drug. Therefore, alternative delivery methods need to be established in order to minimize the risks of reinjection. Several other investigations have developed methods to deliver drugs over extended time, through materials capable of releasing chemicals slowly into the eye. In this investigation, we outline the use of Elvax 40W, a copolymer resin, to act as a vehicle for drug delivery to the adult rat retina. The resin is made and loaded with the drug. The drug-resin complex is then implanted into the vitreous cavity, where it will slowly release the drug over time. This method was tested using 2-amino-4-phosphonobutyrate (APB), a glutamate analogue that blocks the light response of the retina. It was demonstrated that the APB was slowly released from the resin, and was able to block the retinal response by 7 days after implantation. This indicates that slow-release drug delivery using this copolymer resin is effective for treating the retina, and could be used therapeutically with further testing.

This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is described.

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a serious problem. Therefore, the acceptance criteria for organ donors have been extended leading to the usage of marginal kidney grafts. These marginal organs tolerate cold storage poorly resulting in increased preservation injury and higher rates of delayed graft function. To overcome the limitations of cold storage, extensive research is focused on alternative normothermic preservation methods.
Ex vivo normothermic organ perfusion is an innovative preservation technique. The first experimental and clinical trials for ex vivo lung, liver, and kidney perfusions demonstrated favorable outcomes.
In addition to the reduction of cold ischemic injury, the method of normothermic kidney storage offers the opportunity for organ assessment and repair. This manuscript provides information about kidney retrieval, organ preservation techniques, and isolated ex vivo normothermic kidney perfusion (NEVKP) in a porcine model. Surgical techniques, set up for the perfusion solution and the circuit, potential assessment options, and representative results are demonstrated.

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

The severe organ shortage has resulted in increased use of marginal kidney grafts for transplantation. This has triggered interest in alternative storage methods, since marginal grafts especially tolerate cold storage poorly. The technique of normothermic ex vivo kidney perfusion (NEVKP) represents a novel preservation method for kidney grafts prior to &#160;transplantation.

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a ...

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.

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

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

Surgical Labyrinthectomy of the Rat to Study the Vestibular System

To study the vestibular system or the vestibular compensation process, a number of methods have been developed to cause vestibular damage, including surgical or chemical labyrinthectomy and vestibular neurectomy. Surgical labyrinthectomy is a relatively simple, reliable, and rapid method. Here, we describe the surgical technique for rat labyrinthectomy. A postauricular incision is made under general anesthesia to expose the external auditory canal and the tympanic membrane, after which the tympanic membrane and the ossicles are removed without the stapes. The stapes artery, which is located between the stapes and the oval window, is a vulnerable structure and must be preserved to obtain a clear surgical field. A hole to fenestrate the vestibule is made with a 2.1-mm drill bur superior to the stapes. Then, 100% ethanol is injected through this hole and aspirated several times. Meticulous dissection under a microscope and careful bleeding control are essential to obtain reliable results. Symptoms of vestibular loss, such as nystagmus, head tilting, and a rolling motion, are seen immediately after surgery. The rotarod or rotation chair test can be used to objectively and quantitatively evaluate the vestibular function.

This protocol describes the surgical labyrinthectomy of a rat, which is a useful method for studying the vestibular system.

To study the vestibular system or the vestibular compensation process, a number of methods have been developed to cause vestibular damage, including ...

A Clinical Trial Assessing the Safety, Efficacy, and Delivery of Olive-Oil-Based Three-Chamber Bags for Parenteral Nutrition

Limited evidence exists to precisely estimate efficacy and safety differences between parenteral nutrition (PN) prepared using olive-oil-based three-chamber bags (3CBs) and soybean-oil-based compounded bags (CoBs) in hospitalized adult patients. We designed a multicenter, randomized, prospective, open-label, noninferiority protocol to compare the efficacy, safety, and distribution of olive-oil-based 3CBs and soybean-oil-based CoB formulations in adult Chinese patients requiring PN during surgical intervention. Subjects were randomized to receive either one of the study treatments using an interactive voice or web-based recognition system in accordance with the randomization code. Randomization was further stratified based on the study site and surgical category. Both treatment groups received similar amounts of calories and protein. In addition, the two study treatments contained a similar composition of the amino-acid component. The only difference between the two PN formulations was the lipid constitution. The duration of administration of study treatments was a minimum of 5 days up to a maximum of 14 days after the surgical procedure. The primary efficacy endpoint was serum prealbumin levels on day 5 of the study. Noninferiority was proved if the anti-log of the lower bound of the 95% confidence interval (CI) of the treatment difference was at least 0.80. Other efficacy measures included treatment preparation time; duration to achieve tolerability of oral nutrition; associated infectious complications; length of hospitalization; and laboratory assessment of markers of nutrition, inflammation, metabolism, and oxidative stress. A total of 458 patients were enrolled in the study. The results showed that olive-oil-based 3CBs were non-inferior to soybean-based CoBs, besides being well tolerated. The infection rate was found to be significantly lower in the olive-oil-based 3CB group. Thus, this study may be used as a reference for future research on lipid emulsion and 3CBs.

Here, we present a protocol for a study comparing the efficacy, safety, and delivery of olive-oil-based 3CB and soybean-oil-based CoB formulations in adults requiring parenteral nutrition. The results revealed that olive-oil-based 3CBs is non-inferior and well tolerated compared to soybean formulations.

Limited evidence exists to precisely estimate efficacy and safety differences between parenteral nutrition (PN) prepared using olive-oil-based ...

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

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.

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

Intratracheal Administration of Dry Powder Formulation in Mice

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper introduces a noninvasive method of intratracheal delivery of dry powder formulation in mice. A dry powder loading device that consists of a 200 &#181;L gel loading pipette tip connected to an 1 mL syringe via a three-way stopcock is presented. A small amount of dry powder (1-2 mg) is loaded into the pipette tip and dispersed by 0.6 mL of air in the syringe. Because pipette tips are disposable and inexpensive, different dry powder formulations can be loaded into different tips in advance. Various formulations can be evaluated in the same animal experiment without device cleaning and dose refilling, thereby saving time and eliminating the risk of cross-contamination from residual powder. The extent of powder dispersion can be inspected by the amount of powder remaining in the pipette tip. A protocol of intubation in mouse with a custom-made light source and a guiding cannula is included. Proper intubation is one of the key factors that influences the intratracheal delivery of dry powder formulation to the deep lung region of the mouse.

Dry powder formulations for inhalation have great potential in treating respiratory diseases. Before entering human studies, it is necessary to evaluate the efficacy of the dry powder formulation in preclinical studies. A simple and noninvasive method of the administration of dry powder in mice through the intratracheal route is presented.

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper ...