In vitro Functional Characterization of Mouse Colorectal Afferent Endings

The overall goal of the following experiment is to functionally characterize extrinsic afferent nerve endings in the mouse co rectum in vitro. This is achieved by harvesting the colorectal and the attached pelvic or lumbar Splunk neck nerve in continuum and placing the tissue in an in vitro recording chamber As a second step, the pelvic or lumbar Splunk neck nerve trunk is split into fine filaments so that single fiber electrophysiological recordings can be made of individual nerve axons. Next individual colorectal nerve endings are identified by unbiased electrical stimulation of the receptive fields and functionally classified based on the response profiles to three distinct mechanical stimuli.

In addition, responses to chemicals apply to the nerve endings can be tested by isolating the receptive field with a piece of brass tubing and replacing the solution with the solution containing the chemical or mixture of chemicals. Results show that mouse colorectal is innervated by five different classes of mechano sensitive ens. In addition, about 25%of colorectal ens are mechanically insensitive or silent nociceptors.

This method is used by us and others to study the sensory innervation of the colorectal. We're interested in using this research approach because many gastrointestinal disorders are associated with sensitization or a change in the excitability of the sensory innervation. Our interest in understanding the mechanisms of sensitization relates to our desire to help develop therapeutic strategies that then might be useful to modulate the pain and the discomfort that's typically associated with these gastrointestinal disorders, such as irritable bowel syndrome.

Visual demonstration of this method is critical as the tissue dissection and the nerve splitting steps are very difficult to master. The nerves innovating. The distal colorectal can be easily damaged during dissection.

Thus, a careful dissection and there's splitting steps are really critical for those who want to replicate this experiment. Immediately after euthanizing a mouse exsanguinate it, cut open the thoracic chamber, perforate the right atrium, and immerse the mouse carcass, and about half a liter of ice cold Creb solution. Next, carefully remove all the viscera except the colon and the pelvic organs.

Then transect the body of the mouse at about T 12 slightly above the thoracic diaphragm and transfer the coddle half the body to a dissection chamber containing ice cold bubble crebs solution. Under a stereoscope, remove the bladder and the reproductive organs by transecting at their junctions to the urethra. Also, remove the descending aorta until it bifurcates into the common iliac arteries.

Now, free the pelvic or the lumbar splank neck nerves from the surrounding tissues. Follow the nerves from outside the iliac crest to their entry point into the vertebral column. The pelvic nerves enter into the L six and S one vertebral column and the lumbar splank nerves enter into T 13 and L one demonstrated.

Here is the dissection of the left and right pelvic nerves. Next, cut the pubic synthesis and cut right and left acetabular joints. Then remove the iliac bone After removing the iliac, dissect out the distal colorectal with the attached nerve Free.

The attached nerve from the surrounding muscle and connective tissue. The nerve is susceptible to mechanic damage. We are freed from its surrounding tissue, so special care has to be given to avoid pulling or pinching the nerve.

Then transfer the colorectal with the attached nerve to the tissue chamber. Open the colorectal longitudinally along the anti mesenteric border and position the colorectal mucosal side up. Then extend the nerve into the recording compartment, which is connected to the bath compartment by a mouse hole and gate.

Position the nerve trunk onto a small glass mirror. The mirror is hydrophilic and the nerve will adhere to it. Now next to the recording compartment, pin down the mesenteric edge of the colorectal.

Now super fuse a bath compartment with warm oxygenated Creb solution and fill the recording compartment with mineral oil to complete the setup. Attach the anti mesenteric length of the colorectal to a rake of hooks connected to a force actuator under the stereo microscope at high magnification. Carefully peel back the nerve sheath from the isolated pelvic or lumbar splank neck nerve using fine forceps.

Tease the nerve trunk into five to eight nerve bundles that are each about 100 microns thick. Now put the reference electrode in contact with the Krebs solution in the tissue chamber. Then sequentially position the individual nerve bundles onto the recording electrode to evoke action potentials from the colorectal ens.

Gently stroke the colorectal surface using a soft paintbrush. Thus identify the nerve bundle or bundles that innervate the colon. Now using a pair of 30 gauge needle tips, further split the nerve bundle into 10 micron thick filaments and put one of the filaments onto the recording electrode.

This step demands a high level of eye hand coordination and dexterity. Then to electrically excite the afferent nerve endings position the round tipped concentric electrode perpendicular to the mucosal surface and stimulate at a supra threshold intensity. This produces a current spread over two millimeter radius.

To find receptive endings, move the electrode systematically in 1.5 millimeter steps along the length and width of the flattened co rectum. When an afferent ending is excited, adjust the electrode position and stimulus intensity to pinpoint the receptive field that requires the least stimulus intensity to respond. If the stimulus threshold is greater than three milliamps, discard that nerve ending and move on to study another nerve filament.

Now calculate the conduction velocity from the distance between the stimulating electrode at the receptive field and the recording site, and from the conduction delay between the stimulus artifact and the onsets of the action potential. After locating a receptive field by electrical stimulus, apply three mechanical stimuli. First, apply a probing stimulus by pressing the tip of a calibrated von fray, like nylon monofilament perpendicularly towards the receptive field on the flattened colorectal.

Use monofilaments that produce a 0.4 gram and a one gram force. Second, produce a stroking stimulus by gently stroking the colorectal mucosa with a fine nylon filament strand producing a 10 milligram force to generate a small surface shear stress at the receptive field. Third, conduct the circumferential stretch using a computer controlled force actuator.

Have it deliver a ramp stretched force in a circumferential direction along the anti mesenteric edge of the colorectal. Now classify the ENS as serosal mucosal muscular muscular mucosal, or MIAA mechanically insensitive afferent. Based on the results of the three tests.

Begin by recording a baseline response to a mechanical stimulus as in the previous section by a ramped circumferential, stretching or perpendicular mechanical probing to test for chemical sensitivity. First coat the bottom edge of a piece of brass or stainless steel tubing with petroleum and place it over the receptive field on the colorectal. Now aspirate the Krebs solution from inside the tubing and replace the solution with 170 microliters of test solution.

Leave the tubing in position for three to five minutes while monitoring for responses in the afferent. Immediately after removing the tubing within four to six minutes, test the afferent response to the same mechanical stimulus as in the baseline response. After 15 or more minutes, the reagents can be considered washed out.

Then the mechanical stimulus is applied again. To compare with the baseline ants, a dissected nerve was stimulated to find a receptive field. The receptive field was identified by an action potential response using conduction delay of the action potential.

The calculated conduction velocity was point 43 meters per second, which is well in the range of an unmyelinated C fiber. After identifying the receptive field, it was probed perpendicularly by handheld von fray like monofilaments, and also gently stroked by a handheld fine nylon filament. AFR responses to probing were also assessed by a computer controlled force actuator that delivers to the same site on the colorectal, a series of precisely timed and reproducible mechanical forces.

Similarly, circumferential stretch of the colorectal was delivered by the same actuator effort. Responses can be distinguished based on the responses to probing, stroking, and stretch responses from a stretch sensitive afferent were evoked by three consecutive circumferential stretches separated by five minutes. The spike numbers were evenly pinned and displayed as stimulus response functions revealing high reproducibility and both response magnitude and response threshold.

Next chemicals were applied. Direct activation of ens occurred after an application of an acid hypertonic solution after application of an inflammatory soup to a mechanically insensitive afferent. No activation of the ending occurred, but acquisition of meno sensitivity was apparent.

A stretch sensitive, meno sensitive afferent was also sensitized to mechanical stretch of the receptive field after application of an inflammatory soup Once mastered, it usually takes three to four hours to complete the tissue dissection and achieve single fiber recording. A typical experiment takes a whole day from start to finish. After having seen this video, you should understand how to surgically remove and harvest the colorectal with nerves attached.

Then be able to record from single Ahern fibers in either the pelvic nerve or the lumbar splank nerve, innervating the colon using an unbiased electrical search strategy. You should then be able to identify receptive endings in the co rectum and test them for mechano sensitivity as well as chemo sensitivity by application of chemicals directly to the receptive endings. This preparation is a powerful preparation that allows you to study pathophysiological mechanisms as well as basic physiological questions in the colorectal.