All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation of the jejunal afferent nerve
- Perform rodent euthanasia of the adolescent or adult rodent that has been approved prior to the experiment by the local Ethical Committee (e.g., terminal sedation followed by cardiac puncture, cervical dislocation, etc.).
NOTE: We used cervical dislocation to sacrifice the animals, thus resulting in experiments without the need for further anesthesia or post-surgical care as tissues are further processed in vitro.
NOTE: Age has been shown to attenuate mesenteric afferent mechanosensory functions, we therefore advise researchers to adhere to a specific age group for the duration of a single experiment.
- Place the sacrificed laboratory animal in the supine position and perform an abdominal midline incision through the skin and abdominal muscle layer using a scalpel, extending from the xiphoid process until the pubic bone.
- Bathe the abdominal cavity with cold Krebs solution in order to prevent the intra-abdominal tissues from drying out (Krebs composition: 120.03 mM NaCl, 6.22 mM KCl, 1.57 mM NaH2PO4, 15.43 mM NaHCO3, 1.21 mM MgSO4, 11.52 mM D-glucose and 1.52 mM CaCl2).
- Rapidly excise the entire jejunum using sharp scissors by excising approximately 20 cm of the small bowel starting immediately distally of the duodenojejunal flexure, taking care not to damage surrounding structures and keeping the bowel's mesentery, which contains jejunal blood vessels and afferent nerves, intact.
NOTE: For the mere jejunal dissection in the abdominal cavity, one does not need to use a stereomicroscope, as this can be easily visualized with the naked eye.
- Place the excised jejunum in an ice-cold Krebs solution and keep it on ice while continuously oxygenating the Krebs solution with carbogen (95% O2, 5% CO2).
- Cut the jejunum with sharp scissors in approximately 3 cm long loops. Observe the mesenteric bundle containing the vessels and splanchnic nerve somewhere near the center of the respective loop.
- Flush each segment with Krebs solution using a blunt catheter to remove luminal contents and chyme, as these contain digestive enzymes that will accelerate the deterioration of the tissue sample in vitro.
NOTE: Take care not to damage the lumen of the loop during flushing, as the destruction of the villi will result in the release of mediators that can alter the afferent nerve activity.
- Identify the segment to measure the afferent nerve activity (e.g., the first jejunal segment distally of the ligament of Treitz or the duodenojejunal flexure), and place this in a purpose-built recording chamber coated with a silicone elastomer layer.
NOTE: The beginning of the jejunum is anatomically defined as the part of the small bowel where the ligament of Treitz crosses the small bowel, also called the duodenojejunal flexure.
NOTE: Cover the bottom of the recording chamber with a thin silicone elastomer layer well in advance of the experiment's start. Prepare this elastomer layer according to the manufacturer's instructions.
- Perfuse the chamber constantly with warm, carbogenated Krebs solution at a rate of 10 ml/min and keep the Krebs' temperature in the recording chamber constant at 34 °C.
- 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 4/0 silk ligatures to the in- and outflow ports.
- Attach the syringe driver to the oral end, and perfuse the jejunal segment intraluminally with Krebs solution (non-oxygenated, ambient temperature) at a rate of 10 ml/hr.
- Use insect pins to pin the mesentery of the mounted intestinal segment flat against the silicone elastomer bottom layer. Stretch the mesentery out to optimize visualization of the mesenteric bundle; do not exert strain on the bundle or the jejunum.
- Perform a test ramp distension (vide infra) by closing the output port until the intraluminal pressure of the intestinal segment reaches 60 mmHg to verify that no intraluminal Krebs solution is leaking from the mounted segment. Observe a smooth rise in intraluminal pressure without interruptions.
- Observe small contractions of the segment (peristaltic waves) during the initial distension phase. If required, block peristaltic activity by adding 1 µM of the L-type calcium channel blocker nifedipine to the Krebs solution.
NOTE: Adding 1 µM of atropine to the Krebs solution in addition to nifedipine will completely paralyze the intestinal segment.
- Under a stereomicroscope, 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 afferent nerve that lie buried in the fat tissue.
- Start at a remote distance from the jejunum, and expose both blood vessels in the mesenteric bundle.
- Observe the afferent jejunal nerve in between both vessels as a thin, white thread encapsulated in adipose tissue. Only dissect more proximally towards the jejunum by gently peeling the fat tissue away using tweezers when the initial identification of both mesenteric vessels and/or the afferent nerve is difficult.
- Dissect the jejunal mesenteric nerve of the segment free over a distance of several millimeters, 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 epineuronal sheath, by gently tugging it with the small tweezers.
- Using a micromanipulator, lower the tip of the suction electrode, connected to a syringe with a plunger, into the organ bath; then, by manipulating the plunger, gently aspirate some Krebs solution from the organ bath so that the tip of the electrode is submerged in the Krebs solution (Figure 1). Ensure that the Krebs solution covers the wire electrode inside the suction electrode.
NOTE: Use a pipette puller to prepare the borosilicate glass suction capillary containing the wire electrode before the experiment starts.
- 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 some adipose tissue and aspirate this into the glass capillary while firmly aspirating with the plunger, thereby mechanically 'sealing' the nerve in the capillary from the contents of the organ bath.
- Verify the recording of afferent nerve activity using the data acquisition system, e.g., by performing a ramp distension-induced increase in afferent firing (vide infra). Following the isolation of the nerve into the suction electrode, stabilize the preparation for 15 min in order to obtain a steady state of spontaneous afferent nerve activity before performing the actual experiments.
- To perform ramp distension, distend the intestinal segment by closing the output port, leading to a gradual rise in pressure (up to 60 mmHg). Only perform the desired experimental protocol when three consecutive ramp distensions (with a 15-minute interval) yield a reproducible multi-unit discharge (Figure 2).
