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

Overview

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.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Surgical Procedure

1. Anesthetize the mouse and 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.
2. Perform a laparotomy through a small midline incision (2–2.5 cm in length) using a surgical scalpel blade No. 10.
3. Pull the intestine and colon to the right side of the abdomen by using cotton swabs and cover them with sterile gauze soaked into 0.9% normal saline to maintain the organs moist.
4. Gently retract upward the spleen, liver, stomach, and pancreas to expose the aorta and the left kidney artery.
5. 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.
   1. 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.
6. Place a 4-0 silk suture underneath the aorta.
7. Using microvascular clips, clamp the aorta above (just below the superior mesenteric artery) and below the renal artery bifurcation.
8. Puncture the aorta at the level of the left kidney artery bifurcation with a 24G intravenous catheter and advance the catheter into the renal artery.
   NOTE: This is a critical step as puncture through the renal artery may occur.
9. Connect a syringe filled with the drug solution or saline (up to 500 μL) to the catheter and perfuse the kidney.
10. 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.
11. At the same time, release the clamp from the abdominal aorta, below the left renal artery bifurcation. After 5 min, release the clamp from the renal vein and ureter.
12. 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.
13. Ensure that no active bleeding occurs and closely observe the area for 10 more min.
14. Close the abdominal incision in two layers (muscle and skin), using 4-0 absorbable sutures and a continuous pattern to prevent infection. 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.
15. Apply topical antibiotic ointment over the incision area to prevent infections.
16. Transfer the rat into a bedding-free observation cage on a warm pad until complete recovery with a temperature range set at 35–37 °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.
17. 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.

Materials

Name	Company	Catalog Number	Comments
Surgical Microscope	Leica	M125
Isoflurane 100 ml	Cardinal Healthcare	PI23238	Anesthetic
Buprenorphine HCL SR LAB 1 mg/ml, 5 ml	ZooPharm Pharmacy		Buprenorphine narcotic analgesic formulated in a polymer that slows absorption extending duration of action (72 hr duration of activity). Liquid is viscous, warming to RT aids in drawing into syringe. Recommended dosage: 1-1.2 mg/kg SC. DO NOT DILUTE.
Lactated Ringer's Injection, USP, 250 ml VIAFLEX Plastic Container	Baxter Healthcare Corp.	NDC0338-0117-02	For body fluids replacement
Sol Povidone-Iodine  Swabstick, 3'	Cardinal Heatlhcare	23405-010B
Sterile cotton tipped applicators	Kendall	8884541300
4-0 silk suture (without needle)	Cardinal Heatlhcare	A183H
Vessel Clip, Straight, 0.75 mm x 4 mm Jaw	World Precision Instruments	501779-G
I.V. Catheter, Straight Hub, Radiopaque, 24 g x 3/4", FEP Polymer	Jelco	4053
Phosphate Buffered Saline	Life Technologies	10010023
SURGIFOAM Absorbable Gelatin Sponge	Cardinal Healthcare	179082
4-0 VICRYL PLUS (ANTIBACTERIAL) VIOLET 27" RB-1 TAPER	Ethicon	VCP304H	For muscle layer suturing
4-0 VICRYL PLUS (ANTIBACTERIAL) UNDYED 18" PC-3 CUTTING	Ethicon	VCP845G	For skin layer suturing
Triple antibiotic ointment	Actavis	NDC0472-0179-56	For topical use on the site of the incision