Neuroscience
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Quantifying Acute Changes in Renal Sympathetic Nerve Activity in Response to Central Nervous System Manipulations in Anesthetized Rats
Chapters
Summary September 11th, 2018
Methods for measuring sympathetic and cardiovascular responses to central nervous system (CNS) manipulations are important for advancing neuroscience. This protocol was developed to assist scientists with measuring and quantifying acute changes in renal sympathetic nerve activity (RSNA) in anesthetized rats (non-survival).
Transcript
This method can help answer key questions about the autonomic nervous system particularly about the central nervous system mechanisms that regulate sympathetic outflow to the kidney. The main advantage of this technique is that it allows measurement of the impact that central nervous system manipulations have on renal sympathetic outflow to the kidney. Before arterial cannulation, inspect the pressure sensing catheter under high magnification to confirm that the catheter is free of bubbles and debris and has an intact meniscus between the fluid and gel filled components.
Prior to each implantation, refill the gel at the distal tip of the catheter and use a magnet to turn on the transmitter. Place a loose overhand half knot in the proximal suture to briefly occlude the femoral artery. And holding the catheter introducer in the non dominant hand, use vessel cannulation forceps to grasp the tip of the cannula of the telemetry unit to avoid displacing the gel from the tip.
Use a bent 22 gauge needle tip to puncture a hole in the artery and insert the cannula into the artery as far as possible. Then secure the pressure catheter with the proximal and distal suture ties. And tuck the body of the telemetry implant inside the flank adjacent to the incision.
For brain access, gently move the rat into the prone position in a stereotaxic surgery frame with the head between the ear bars and adjust the incisor bar to equalize the height of lambda and bregma. Next make a two centimeter rostral caudal incision through the midline of the scalp and use cotton tipped applicators to firmly remove any connective tissue from the skull surface. Apply hydrogen peroxide to the exposed skull to assist in visualizing the bregma, lambda and midline sutures.
Using an atlas of the rat brain to guide targeting, drill a burr hole osteotomy sized for electrode access through the skull. To isolate the renal sympathetic nerves, connect a wire renal sympathetic nerve activity or RSNA electrode to a 10X pre amplifier and a microelectrode amplifier and make a scalpel incision extending from four to five centimeters below the ribs in the caudal direction slightly lateral to the spine. Blunt dissect to visualize the paraspinal muscles and make a very superficial one to two centimeter rostral caudal incision where the fat meets the muscle.
Using cotton tipped applicators, spread the fat away from the muscle to visualize the kidney without entering the peritoneal space. And use retractors to gently separate the kidney from the paraspinal muscles to visualize the renal artery and abdominal aorta. And use high magnification to identify the renal nerves in the incision pocket.
The nerve bundles are most easily visible in the right angle formed by the aorta and the renal artery closely following the renal artery from the aorta to the kidneys. Then use microdissecting tweezers to gently dissect a segment of the nerve bundle to be placed on the recording electrode from the surrounding tissue. Secure the wire RSNA electrode in a holder and lower the electrode to the level of the nerve segment.
Use a nerve hook to gently lift a segment of the renal nerve onto the electrode without stretching the nerve. And fill the incision with mineral oil to keep the exposed renal sympathetic nerve hydrated. Attach a grounding clip to the animal and the other end to the Faraday cage and use high and low pass filtering to direct the signal to the amplifiers.
Then adjust the gain up to 10 kilohertz including an audio monitor to assess the bursting pattern of the RSNA. To assess the quality of the RSNA recording, evoke the baroreflex with a 100 microliter bolus intravenous injection of 10 micrograms per milliliter phenylephrine to increase the blood pressure and to inhibit the renal sympathetic nerve activity adjusting the position of the electrodes to improve the signal as necessary. Once a clear signal has been obtained, aspirate the mineral oil and secure the electrode in place with silica gel.
In this representative experiment, intravenous injection of phenylephrine was used to induce an increase in the mean arterial pressure and to evoke the baroreflex and transient sympathoinhibition. To quantify the RSNA the raw RSNA was rectified and averaged for non overlapping 10 second segments and the noise estimate was subtracted from each segment. When performing this procedure, it's important to remember to ensure that the rat is adequately anesthetized, that it is properly positioned in the stereotaxic frame and that the system is adequately grounded to reduce electrical noise.
Following this procedure and collection of brain tissue, histology methods can be applied to characterize the types of central system nuclei that control a sympathoexcitatory response. This technique allow neuroscientists to explore autonomic regulation mechanisms in anesthetized rats which informs future studies in animals to understand the control of blood pressure and renal function. Don't forget that it is critical to assess the plane of anesthesia at least every 15 minutes.
And to provide supplemental anesthesia as needed.
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