マウス空腸および結腸セグメントにおける腸間膜求心性神経活動のin vitro記録で

The overall goal of this technique is to record in vitro splanchnic afferent nerve activity from a single jejunal or colonic nerve bundle in response to ramp distensions and the application of compounds. This method can help answer key questions in the field of neuroscience, such as whether afferent nerves supplying the gastrointestinal tract are hypersensitized or desensitized during various disease states. The main advantages of this technique are that it allows nerve discharge recording in vitro and that it allows to quantify the nerve activity without modulation or inputs of the central nervous system.

The implication of this technique extends toward the therapy or diagnosis of several pain syndromes, such as the irritable bowel syndrome, since nerve sensitization plays a major role in the pathogenesis. Generally, individuals new to this method will struggle because the dissection of nerve bundles and the single unit analysis of the recorded signal will take a substantial amount of training. To begin this procedure, place the sacrificed mouse in the supine position.

Perform an abdominal midline incision through the skin and abdominal muscle layer using a scalpel extending from the xiphoid process to the pubic bone. Afterward, rinse the abdominal cavity with cold Krebs solution in order to prevent the intraabdominal tissues from drying out. Then rapidly excise the entire jejunum using sharp scissors by removing approximately 20 centimeters of the small bowel, starting immediately from the duodenojejunal flexure.

Be careful not to damage the surrounding structures, and keep the bowel's mesentery, which contains jejunal blood vessels and afferent nerves, intact. Next, place the excised jejunum in cold Krebs solution on ice while oxygenating it continuously with Carbigen. Then, cut the jejunum in approximately three centimeter long loops.

Locate the mesenteric bundle containing the vessels and splanchnic nerve near the center of the respective loop in order to facilitate the mounting of the loop in the recording chamber. Subsequently flush each segment with Krebs solution using a blunt catheter to remove the luminal contents and chyme, as they contain digestive enzymes that will accelerate the deterioration of the tissue sample in vitro. After that, select a segment for measuring the afferent nerve activity and place it in the recording chamber coded with a silicone elastomer layer.

Keep the Krebs temperature in the recording chamber at 34 degrees Celsius. Then mount the jejunal segment in the organ bath, so that the oral end is connected to the syringe driver providing luminal flow and the aboral end connects to the outflow. Slightly stretch the segment, but take care not to exert excessive tension.

Attach both ends firmly using silk ligatures to the in and outflow ports. Afterward, attach the syringe driver to the oral end and perfuse the jejunal segment intraluminally with Krebs solution at 10 milliliters per hour. Pin the mesentery of the mounted intestinal segment flat against the silicone elastomer bottom layer.

Then, stretch the mesentery out in order to optimize the visualization of the mesenteric bundle. Next, perform a test on ramp distension by closing the output port until the intraluminal pressure of the intestinal segment reaches 60 millimeters of mercury in order to verify that no intraluminal Krebs solution is leaking from the mounted segment. Observe a smooth rise in the intraluminal pressure without interruptions.

Expect small contractions of the segment during the initial distension phase. If required, block the peristaltic activity by adding one micromole of the L-type calcium channel blocker nifedipine to the Krebs solution. Under a stereo microscope, gently start to peel off the fat tissue from the mesentery by gently tugging it with two small tweezers, taking care not to damage the vessels and the afferent nerve that are buried in the fat tissue.

Start peeling off the fat tissue at a distance from the jejunum to expose both blood vessels in the mesenteric bundle. Identify the afferent jejunal nerve in between both vessels as a thin white thread encapsulated in the adipose tissue. Then dissect it more proximally toward the jejunum by gently peeling the fat tissue away using tweezers.

Next, dissect the jejunal mesenteric nerve of the segment by removing the adipose tissue adherent to the nerve. Transect the nerve using sharp tissue scissors. If required, peel off the remaining fat and connective tissue as well as the epineural sheath by gently tugging it.

In this procedure, using a micromanipulator lower the tip of the suction electrode into the organ bath. Then, gently aspirate some Krebs solution from the organ bath, so that the tip of the electrode is submerged in the Krebs solution. Ensure that the Krebs solution covers the wire electrode inside the suction electrode.

Next, position the tip of the suction electrode immediately next to the transected afferent nerve strand and draw the transected nerve strand into the capillary over its entire length. Maneuver the tip of the electrode towards the adipose tissue, and aspirate it into the glass capillary with the plunger, thereby mechanically sealing the nerve in the capillary from the contents of the organ bath. The adequate sealing of the glass capillary from the recording chamber by aspirating some surrounding fat tissue into the capillary is pivotal in order to minimize the redundant background noise that will ultimately hamper the final analysis.

To verify the recording of afferent nerve activity, perform a ramp distension induced increase in afferent firing by closing the output port to lead to a gradual rise in pressure in the intestinal segment. Only perform the desired experimental protocol when three consecutive ramp distensions yield a reproducible multi-unit discharge. Following nerve isolation, stabilize the preparation for 15 minutes in order to obtain steady spontaneous afferent nerve activity before performing the actual experiments.

Shown here is a schematic representation of different afferent fiber units based on their mechanosensitive profile. Low threshold fibers predominantly display an increased nerve activity at low distension pressures, resulting in an LT percentage of over 55%High threshold units on the contrary only display an increase in firing rate at noxious pressures. Wide dynamic range fibers display a gradual increase in nerve activity during the entire distension, whereas mechanically insensitive fibers do not respond to increasing distension pressures.

This figure shows the mesenteric afferent nerve discharge in the wild type mice during ramp distension. Note that in the biphasic distension profile the initial rise in nerve discharge is due to low threshold and wide dynamic range fibers, whereas high threshold and wide dynamic range fibers contribute to the second increase in discharge. Once mastered, the isolation of the jejunal nerve and initial baseline recording can be done in less than one hour if performed properly.

More time is necessary when studying the effect of compounds administered into the organ bath. While attempting this procedure, it's important to remember to maintain a well standardized approach, such as consistently identifying the same segment for all experiments. Following this procedure, other methods like the isolation of dorsal root ganglia with subsequent calcium imaging can be performed in order to assess which relay stations in the gastrointestinal nervous system are involved in the pathogenesis of several diseases.

After its developments, this technique paved the way for researchers in the field of neuroscience to explore the pathogenesis of several pain syndromes in gastrointestinal research. After watching this video, you should have a good understanding of how to isolate jejunal afferent nerves from the mouse gastrointestinal tract, and record activity from it during ramp distensions.