The overall goal of this protocol is to demonstrate the methodology for the electrophysiological recordings of sensory nerves of the colorectum in vitro. This method can help answer key questions in vitro pen field such as the contribution of the outer peripheral efferens sensitivity to correct hyperalgesia. The main advantage of this technique is that it allows for the distinction of the colorectum in the similar way as the carotid distinction is performed in vivo, in animals or in patients.
Visual demonstration of this method is critical because locating the ganglia and dissecting the nerves are relatively difficult. To begin this procedure, expose the abdominal cavity of a deeply anesthetized rat by performing a midline incision on the abdominal wall using a scalpel. Afterward, pull the mesentery and other tissues aside to expose the colorectum.
Then, place the animal under the dissecting microscope. Through careful dissection, locate the left pelvic ganglion and identify the pelvic nerves and the lumbar splanchnic nerves joining it. Subsequently, cut these nerves a few millimeters away from the pelvic ganglion.
Locating pelvic ganglion and dissecting nerves is the most critical and difficult step of this procedure. Next, cut the symphysis bone to expose the rectum. Remove the tissues above the colorectum and be careful to leave the pelvic ganglion intact.
After sacrificing the rat by intravenous injection of an overdose of pentobarbital, transect the colon about three centimeters above the pelvic ganglion and remove the colorectum from the animal using forceps. Subsequently, transfer the colorectum to a Petri dish filled with pre-cooled Krebs solution. Remove the feces by gently flushing the colon.
Next, remove the remnant urinary bladder and other tissues carefully, without compromising the pelvic ganglion. In this procedure, place the colon into a recording chamber and profuse the tissue continuously with carbogenated Krebs solution. Next, cannulate the colorectum at both the oral and anal ends, then start the intraluminal infusion of the colon with saline in the oral to anal direction, after that, locate the major pelvic ganglion under the dissecting microscope and use insect pins to expose the ganglion.
With careful dissection, find a fine branch of nerve emanating from the ganglion and running towards the colon, then, cut the nerve close to the ganglion. Before the next step, turn on the heating bath and keep the Krebs solution at 34 degrees celsius. Now, inspect a pre-pulled glass pipette under the dissecting microscope.
Break the tip of the electrode with a pair of forceps so that it is of a size compatible with the diameter of the nerve to be recorded. Following that, bevel the tip by placing it close to a lighter flare. New to this methodology may also find it tricky to bevel the tip of the electrode compatible with the size of the nerve.
Next, place the beveled electrode in the electrode holder, then connect a 10 milliliter syringe to the side port of the holder for the application of negative or positive pressure to the electrode. After that, connect the holder to the head stage of the bio amplifier, and mount the head stage onto a manipulator. Following this, move the electrode to the tissue bath and fill the electrode with the Krebs solution by applying gentle suction until it contacts the silver wire of the holder.
Next, place the electrode tip close to the cut end of the nerve and apply negative pressure to suck the nerve into the electrode. Then, apply more negative pressure, so that about one millimeter of the nerve is pulled into the electrode and forms a tight seal. Now, turn on the bio amplifier and set the filter to 300 to three thousand hertz.
Monitor the signal on the oscilloscope and record the nerve signal and the intraluminal pressure signal in the spike data processing software. Next, apply ramp distention of the colon by closing the three way tap on the outlet cannula while continuously infusing intraluminally. Monitor the intraluminal pressure until it reaches 60 millimeters mercury, then open the three way tap on the draining cannula.
Repeat this procedure at regular intervals of fifteen minutes and apply drugs extra or intraluminally to test the chemical sensitivity of the afferent nerves. Here is the schematic illustration of the experimental setup for the ex vivo tube colorectum preparation with a representative recording from a nerve distal to the pelvic ganglion. In the preparations from normal rats, the colonic afferent nerves typically have a low level of a regular spontaneous activity.
Ramp distention of the colon induces a gradual increase in the firing rate. The mechanosensory property of each nerve is represented by plotting the intraluminal pressure afferent nerve response curve. Alternatively, recording can be made from a branch of the pelvic nerves proximal to the pelvic ganglion.
The profile of the mechanosensory activity of the proximal pelvic nerves is usually similar to that seen in the nerve distal to the pelvic ganglion. Principal component analysis of the waveforms of the action potentials from the multiunit recording can be made to discriminate single unit activity. Here are the superimposed waveforms of two individual pelvic afferens.
Once mastered, this technique can be done in two hours if it's performed properly. After watching this video, you should have a good understanding of the anatomy of the major pelvic ganglia and the other nerves and be able to locate the ganglia to dissect the nerves and record the afferent activity. An advantage of this protocol is that it may allow the comparison of the electrophysiological properties and the pharmacological profiles of the pelvic and the splanchnic afferent nerves.
