Novel Approach for Simultaneous Recording of Renal Sympathetic Nerve Activity and Blood Pressure with Intravenous Infusion in Conscious, Unrestrained Mice.

The overall goal of this novel surgical and experimental method is to evaluate renal sympathetic activity in conscious, unrestrained mice without confounding effects of anesthesia. Given the recent interest in mouse models, this approach provides an accessible technique by which autonomic tone can be reliably assessed in conscious mice. The main advantage of this technique is that nerve activity can be assessed in the conscious, comfortably-resting mouse without confounding factors such as anesthesia or restraint.

After preparing the mouse for surgery, position it on its right side with the rostral end pointing to the surgeon's left. First, make a five millimeter incision in the mid-dorsal region with the scalpel. Secondly, make a larger incision in the skin overlying the left flank perpendicular to the spine in two millimeters caudal to the rib cage.

From there, tunnel a 13 gauge stainless steel needle subcutaneously to the dorsal exit site. Next, use a prepared RSNA electrode. Pass it through the needle and pull the needle back so the electrode tip lies on the abdominal muscle of the left flank.

Part of the electrode lead should extend under the skin. Now, expose the left kidney, move aside the electrode tip, and make an incision in the abdominal muscle, directly underlying the skin incision. Next, open the surgical field with micro retractors and retract the kidney.

Using applicators, separate the fat and connective tissue along the back muscle to view the kidney. Do not stretch the renal neurovascular bundle, as this will cause irreversible damage to the nerve and compromise the experiment. To visualize the renal neurovascular bundle, use a high power objective.

It usually resides alongside the renal vessels. Now very carefully dissect the nerve bundle from the surrounding tissues with fine, straight forceps. Do not touch the nerve bundle or disrupt the vessels that supply the nerve.

It is vital to keep the nerve bundle in tact through this process. Next, introduce the RSNA electrode tip into the abdomen. Position the tip and ground wire perpendicularly to the renal neurovascular bundle.

The ground wire must make good contact with the underlying tissues. Then carefully lift the renal nerve bundle with an angled forceps, and slide the electrode tip beneath the nerve, and put the nerve so that it is in direct contact with both wires, but do not allow the electrode to compress the renal vessels. Next, slip a small piece of sterilized paraffin film between the nerve wires and the ground wire.

Then soak up the blood and fluids using small absorbent spears. To electrically isolate the electrode tip, carefully apply just enough two component silicon elastomer under and around the nerve. After a few minutes of curing, carefully lift the silicon with forceps and secure it with a small amount of liquid surgical adhesive.

Then close the abdominal incision with discontinuous absorbable 50 sutures and do the same with the overlying skin. The next section describes implanting the blood pressure telemeter. Begin with repositioning the mouse on its back with the rostral end pointing toward the surgeon.

Adjust the anesthesia as needed, and administer a second dose of glycopyrrolate. Then make a midline incision in the neck region with the scalpel, beginning from just below the lower jaw and extending to just above the ribcage. Now separate the glandular tissue to expose the underlying neck muscles.

Being very careful to avoid damaging the vagus nerve, proceed by exposing the left common carotid artery and separating it from the surrounding tissues. Now pass three pieces of 60 silk suture under the artery, spanning the full length of its exposure. Position one suture rostrally and tie it to occlude the vessel.

Position a second suture at the middle and tie loosely and position the third suture caudally and tie it loosely. Next, retract the rostral-most suture and secure it to the nose cone using a small piece of umbilical tape. Then with micro mosquito forceps, retract the caudal-most suture to restrict the blood flow.

Now with fine spring scissors, make a small incision into the vessel wall as rostrally as possible. Through the incision, introduce the radiotelemeter catheter and advance it to the caudal suture. Tighten the middle suture to stabilize it.

Next, release the caudal retraction and advance the catheter 10 millimeters. Then secure the catheter with the suture. Finally tunnel the telemeter body to a subcutaneous pocket along the right flank and proceed with implanting in the jugular venous catheter.

Begin with using cotton-tipped applicators to expose the right jugular vein. Then pass two pieces of 60 silk suture around the vein. Position one far rostrally and tie it to occlude the vein.

Position the other far caudally and retract it gently to stop the blood flow in the vessel. Next use fine spring scissors to make a small incision in the vessel wall as close to the rostral suture as possible. Then catheterize the vein with heat stretched tubing filled with saline.

Now advance the catheter eight millimeters into the vein and secure it with the silk sutures and a small drop of gel formula cyanoacrylate. Now rotate the mouse to its left side and use a 13 gauge needle to tunnel the catheter from the neck to the exit prepared at the dorsal mid-scapular region. Next reposition the mouse on its back and close the neck incision with discontinuous 50 sutures.

Now put the animal in the prone position and slip a small subcutaneous button in stainless steel spring onto the venous catheter. Secure the button under the skin with sutures. Then secure the polyethylene pedestal protecting the electrode leads to the underlying muscle using tissue adhesive.

Lastly, suture the overlying skin over the flange for additional support. House the mice at the same temperature and humidity as they will experience during the RSNA recordings. Allow the mice at least 30 minutes of stabilization in the recording set up before recording one hour of baseline blood pressure and RSNA data.

Make sure that the mouse is resting quietly during the recordings, since natural movements are associated with increases in sympathetic tone. During the recording, it is important to take note of the animal's movements. Such notes can be made directly onto the digital trace.

Now proceed with measuring the response to intravenously administered substrates. Later, after euthanasia, continue taking measurements for 30 minutes. Following the described protocol, 12 of 12 mice survived and recovered well.

Within 24 hours, the mice behaved normally and showed no signs of distress. 48 hours after the surgery, a verifiable and clear RSNA signal was recorded in 10 of the 12 mice. The typical increases of RSNA expected with normal activity such as eating and grooming were observable.

By 120 hours, five mice still showed a high quality RSNA signal. Blood pressure and heart rate remained stable for the five day investigation period and values were not different from those recorded over 10 days of post-surgical recovery. To verify that RSNA was entrained with the arterial baro reflex, the mice were treated with sodium nitroprusside and phenylephrine.

RSNA characteristically increased in response to the induced reduction of arterial pressure and conversely was virtually silenced with increased arterial pressure. Furthermore, RSNA was completely eliminated following a ganglionic blockade with hexamethonium. This observation supports the post-ganglionic nature of the RSNA signal.

Once mastered, this surgical technique can be completed in under three hours when performed properly. When attempting this procedure, it's important to always remember to take great care when handling the renal nerves in order to preserve their viability for later recording. Following this procedure, other methods such as air jet stress can be used to further assess autonomic tone in conscious mice.

With its development, this technique opens new possibilities for researchers to understand the important contributions of the autonomic nervous system to key areas of cardiovascular physiology and pathophysiology such as long-term blood pressure control, hypertension, and obesity.