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

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
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	<li>Autoclave all surgical instruments before surgery. If multiple surgeries on different rats are ...

<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>

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.

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			<td height="21" style="height:21px;width:257px;">Surgical Microscope</td>
			<td style="width:176px;">Leica</td>
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			<td height="21" style="height:21px;width:257px;">Isoflurane 100 ml</td>
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			<td>Anesthetic</td>
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			<td height="133" style="height:133px;width:257px;">Buprenorphine HCL SR LAB 1mg/ml, 5 ml</td>
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			<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>
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			<td height="42" style="height:42px;width:257px;">Puralube Vet Ophthalmic Ointment</td>
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			<td>Sterile ocular lubricant</td>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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		<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>
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		<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>
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			<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>
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		<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>
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		<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>
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			<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>
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			<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>
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			<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>
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			<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>
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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 t...

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 ...

The overall goal of this procedure is to deliver biologics directly into the renal artery in order to induce a localized therapeutic effect in the renal microvascular compartment in rats. The main advantage of this technique over existing methods such as systemic administration of anti-inflammatory or antifibrotic drugs is that the intrarenal delivery technique minimizes any potential systemic side effects. The implications of this technique extend to a therapy of a variety of renal pathologies, because the renal microvascular function is involved in a wide spectrum of kidney diseases.Playing a key role in the progression of kidney damage. To prepare for surgery, begin by autoclaving all surgical instruments. After anesthetizing a rat, according to the text protocol, transfer the animal to a controlled heating pad, to preserve the animal's body temperature at 37 degrees Celsius.Use 1%to 2%isoflourane in one liter per minute of oxygen to maintain anesthesia. Administer an analgesic subcutaneously, and apply eye ointment to prevent dryness. To compensate for loss of body fluids, administer 10 milliliters per kilogram of 0.9%normal saline subcutaneously.Then, shave the abdominal area, and use povidone iodine and 70%ethanol pads to clean the skin. To begin surgery, ensure that the depth of sedation is adequate by monitoring physical reflexes such as a withdrawal from a toe pinch. Use a number 10 surgical scalpel blade to perform a laparotomy through a small midline incision.With cotton swabs, pull the intestine and colon to the right side of the abdomen, and cover them with sterile gauze, soaked in 0.9%normal saline to keep the organs moist. Gently retract upward the spleen, liver, stomach, and pancreas to expose the aorta and the left kidney artery. Next, under a surgical microscope, use blunt dissecting curved forceps with a repeated open close motion along the length of the vessels and sterile cotton swabs, to 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.The use of cotton swabs helps avoid damage to nerves and infected blood vessel, in particular to thin-walled veins. Then, place a 4-0 silk suture underneath the aorta, and using microvascular clips, clamp the aorta above and below the renal artery bifurcation. Now, using a 24 gauge intravenous catheter, puncture the aorta at the level of the left kidney artery bifurcation, and carefully advance the catheter into the renal artery.Because of the small size of the renal artery, the catheter might puncture through the vessel. It is important to work under a surgical microscope and to insert the needle as parallel to the artery as possible. Then, connect a syringe filled with the drug solution or saline to the catheter, and profuse the kidney.Immediately after profusion, use a microvascular clip to clamp the left renal vein and the left ureter before removing 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 with a cotton swap, gently apply pressure. At the same time, release the clamp from the aorta below the left renal artery bifurcation.After five minutes, release the clamp from the renal vein and ureter. Carefully release the clamp from the aorta above the left renal artery bifurcation, and allow the kidney to reperfuse. The total renal ischemia should last no longer than seven minutes.After ensuring that no active bleeding occurs, and closely observing the area for 10 more minutes, use 4-0 absorbable sutures and a continuous pattern to prevent infection, and close the abdominal incision in two layers. Apply topical antibiotic ointment over the incision area to prevent infections. Then, transfer the rat into the observation cage on a warm pad until complete recovery.After completion of all of the studies, euthanize the animals according to the text protocol. Two different doses of recombinant rat VEGF, or PBS as control, were injected into rats, and then animals were euthanized eight hours post surgery to examine the activation of the VEGF pathway. As seen here, by H&E staining, the surgical procedure did not effect the morphology of the perfused kidney, as compared to the control organ.In addition, serious red staining did not show an increase in extracellular matrix deposition in response to the ischemic time and the infusion of rrVEGF, as compared to control tissue. Finally, by Western blot analysis, a small but significant increase in the expression of proteins involved in the VEGF pathways was observed, including VEGF, FLK-1, Phospho-AKT and AKT, and Phospho-ERK and ERK. Once mastered, this technique can be done in about 45 minutes if it is performed properly.While attempting this procedure, it's important to follow institutional and national guidelines for the animal care and use, in particular, to alleviate the pain and discomfort by the administration of analgesics. Following this procedure, other methods, such as Western blotting, or histological staining of the kidney can be performed to determine the efficacy of different therapies as well as activation of specific pathways.

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Medicine

5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements of homeostasis, integrative animal models are necessary to study development and progression of CKD. To study development of CKD and novel therapeutic interventions in CKD, we use the 5/6th nephrectomy ablation model, a well known experimental model of progressive renal disease, resembling several aspects of human CKD. The gross reduction in renal mass causes progressive glomerular and tubulo-interstitial injury, loss of remnant nephrons and development of systemic and glomerular hypertension. It is also associated with progressive intrarenal capillary loss, inflammation and glomerulosclerosis. Risk factors for CKD invariably impact on endothelial function. To mimic this, we combine removal of 5/6th of renal mass with nitric oxide (NO) depletion and a high salt diet. After arrival and acclimatization, animals receive a NO synthase inhibitor (NG-nitro-L-Arginine) (L-NNA) supplemented to drinking water (20 mg/L) for a period of 4 weeks, followed by right sided uninephrectomy. One week later, a subtotal nephrectomy (SNX) is performed on the left side. After SNX, animals are allowed to recover for two days followed by LNNA in drinking water (20 mg/L) for a further period of 4 weeks. A high salt diet (6%), supplemented in ground chow (see time line Figure 1), is continued throughout the experiment. Progression of renal failure is followed over time by measuring plasma urea, systolic blood pressure and proteinuria. By six weeks after SNX, renal failure has developed. Renal function is measured using 'gold standard' inulin and para-amino hippuric acid (PAH) clearance technology. This model of CKD is characterized by a reduction in glomerular filtration rate (GFR) and effective renal plasma flow (ERPF), hypertension (systolic blood pressure&gt;150 mmHg), proteinuria (&gt; 50 mg/24 hr) and mild uremia (&gt;10 mM). Histological features include tubulo-interstitial damage reflected by inflammation, tubular atrophy and fibrosis and focal glomerulosclerosis leading to massive reduction of healthy glomeruli within the remnant population (&lt;10%). Follow-up until 12 weeks after SNX shows further progression of CKD.

A two-stage method to establish chronic kidney disease (CKD) in the Lewis rat by surgically removing 5/6th of renal mass is described. Combination of the surgical procedure, NOS-inhibition and a high-salt diet leads to a model resembling human CKD, allowing study of causal mechanisms and development of novel therapeutic interventions.

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements ...

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 ...

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 ...

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 ...

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.

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

Delivery of Exogenous Artificially Synthesized miRNA Mimic to the Kidney Using Polyethylenimine Nanoparticles in Several Kidney Disease Mouse Models

microRNAs&#160;(miRNAs), small noncoding RNAs (21-25 bases) that are not translated into proteins, inhibit lots of target messenger RNAs (mRNAs) by destabilizing and inhibiting their translation in various kidney diseases. Therefore, alternation of miRNA expression by exogenous artificially synthesized miRNA mimics is a potentially useful treatment option for inhibiting the development of many kidney diseases. However, because serum RNAase immediately degrades systematically administered exogenous miRNA mimics in vivo, delivery of miRNA to the kidney remains a challenge. Therefore, vectors that can protect exogenous miRNA mimics from degradation by RNAase and significantly deliver them to the kidney are necessary. Many studies have used viral vectors to deliver exogenous miRNA mimics or inhibitors to the kidney. However, viral vectors may cause an interferon response and/or genetic instability. Therefore, the development of viral vectors is also a hurdle for the clinical use of exogenous miRNA mimics or inhibitors. To overcome these concerns regarding viral vectors, we developed a nonviral vector method to deliver miRNA mimics to the kidney using tail vein injection of polyethylenimine nanoparticles (PEI-NPs), which led to significant overexpression of target miRNAs in several mouse models of kidney disease.

Here, we deliver exogenous artificially synthesized miRNA mimics to the kidney via tail vein injection of a nonviral vector and polyethylenimine nanoparticles in several kidney disease mouse models. This led to significant overexpression of target miRNA in the kidney, resulting in inhibited progression of kidney disease in several mouse models.

microRNAs&#160;(miRNAs), small noncoding RNAs (21-25 bases) that are not translated into proteins, inhibit lots of target messenger RNAs (mRNAs) by ...