The overall goal of this procedure is to isolate functionally viable neurons and CLIA from the adult mouse myenteric plexus. This is accomplished by first coating the sterile cell culture surface with polylysine and laminin. The second step is to prepare solutions and surgical area for myenteric plexus isolation.
Next, remove the gastrointestinal tract and isolate the desired section to create longitudinal muscle myenteric plexus preparation. The final step is to sequentially digest the LMMP in collagenase and trypsin, followed by plating the cells on the pre-coded cell culture surfaces. Ultimately, electrophysiology, immuno cyto chemistry and single cell PCR can be used to study the effects of enteric neurons, glia, or the interaction between these two cell types.
Demonstrating the procedure will be Dr.Trisha Har Smith, a postdoctoral fellow in all laboratory, and assisting her will be Joy Gombe, a PhD candidate in all laboratory. The main advantage of this technique over existing techniques is that the neurons and glia come from the adult mouse. This allows for fully functional neurons and glia, which can potentially come from genetically Modified animals.
Though this method can provide insight into the electrical properties of neurons. It can also be applied to other methodologies such as immuno cyto chemistry, single cell PCR, and real-time calcium imaging. Begin this procedure by placing a euthanized mouse in dorsal recumbent on the surgical surface.
Next, clean the abdominal skin with 70%ethanol. Then lift it with a pair of forceps and open the abdominal cavity with scissors to reveal the internal digestive organs. After that, lift a section of ileum to reveal the mesentary.
Remove the gastrointestinal tract and snip through the mesentery with scissors. Then gently remove and unravel the ileum and colon. Be careful not to pull the mesentery from the ileum and colon after the full length intestine is unraveled.
Remove the ileum by cutting through the intestine distal to the stomach and proximal to the cecum. This procedure can also be performed with a colonic tissue by removing the colon from the distal to the cecum to the proximal to anus. Next section the ileum into three or more large pieces.
Gently rub kreb solution through the gut section until all fecal matter is removed into a separate waste container. Place the clean section into the container of Krebs labeled clean and repeat the procedures until the entire ileum is cleaned. To remove the LMMP, cut the ileum into two to four centimeter small segments.
Afterward, place a segment of ileum on a plastic or glass rod. The ileum should fit snuggly but not loose or taut. Prevent the GI tract from rotating around the rod by gently pinning the tube to the rod with the thumb.
Then gently remove bits of mesentery that are still attached to the GI tract with forceps. After that, separate the LMMP from the underlying circular muscle. By first gently rubbing the edge of the forceps along the entire line where the mesentery was attached from top to bottom of the segment.
Subsequently, gently create a gap in the longitudinal muscle. Then gently tease away the longitudinal muscle using a cotton swab wedded with crebs beginning at the top of the gap, using the lightest horizontal stroke while applying very light pressure until the longitudinal muscle just begins to separate from the circular muscle. Do this down the entire strip along the mesentary attachment point.
Next, gently work around the GI tube by moving from top to bottom and back. As the longitudinal muscle is slowly separated from the circular muscle all the way around the tube. When complete, the LMMP will naturally come off from the remaining GI tube.
Then place the resulting thin strip of longitudinal muscle in the beaker labeled LMMP and repeat the procedures for all the segments In this procedure. Place the rinse to LMMP segments into the digestion solution. Then snip the LMMP into tiny pieces with scissors.
Next, digest them for 60 minutes. In a 37 degree Celsius water bath with a shaker while being gently bubbled with carbogen. After the digestion is complete, gather the cells by centrifugation for eight minutes at 356 Gs in a centrifuge, cool to four degrees Celsius.
In the meantime, prepare 0.05%trypsin solution in a cell culture hood by adding one milliliter warmed 0.25%trypsin and four milliliters warmed HBSS into a sterile 50 milliliter cell culture tube. After centrifugation, discard the supernatant carefully remove the cell pellet and place it into a clean tube with five milliliters of 0.05%tryin solution. Next, digest the cells in 0.05%tryin solution in a 37 degree Celsius water bath with shaking for seven minutes, neutralize tryin with 10 milliliters of cold rinse media.
After seven minutes of tripsin treatment, then centrifuge the cells for eight minutes at 356 gs. In the meantime, balance section of sterilized TX mesh on top of a sterile 15 milliliter cell culture tube. After centrifugation, discard the supernatant gently resuspend the cell mixture by triggering it in three milliliters.
Complete neuron media. All alterations should be done very gently To avoid generating air bubbles, then filter the cell solution through NYX mesh into a 15 milliliter clean cell culture tube. Gather the cells by centrifugation for eight minutes at 356 gs.
After that, remove and discard the supernatant. Re suspend the cells in 1200 microliters complete neuron media. Next, t the cells gently using a one milliliter plastic pipette tip.
Be careful not to generate air bubbles pipette slowly and gently until most chunks are broken up and the cells are suspended into liquid. Now, add 750 microliters of the complete media to each of the 12 wells containing a pre-coded glass cover slip. Then add 100 microliters of the rated cell solution.
Incubate the neurons in a cell culture incubator at 37 degrees Celsius with 5%carbon dioxide change. Half of the cell media every two days. Neurons are ready for electrophysiological recordings after one to two days in culture.
Shown here is the immunohistochemical characterization of enteric neurons. Lea isolated from the mouse. Longitudinal muscle confocal microscopy revealed the neuronal specific beta three tubulin staining in the whole Mount ileal longitudinal muscle preparation from the mouse.
And these are the cells isolated from the LMMP preparations, which contain the neurons that stained positively for cal binding and Retin glia cells shown here were visualized with the glia specific marker GFAP. However, no staining was seen when primary antibody was omitted. Here are the neurons and glia isolated from the mouse longitudinal muscle growing in close proximity to one another.
The confocal microscopy images indicate the green neurons and red ggl readily growing adjacent to one another and appear to interact in vitro. This figure shows the electrophysiology of the cultured enteric neurons and CLIA in the current clamp mode. All neurons displayed action potentials upon current injection.
CLIA do not have action potentials, but do display large electrotonic potentials in response to current injection. The neurons cultured from the mouse ileum are an electrophysiological heterogeneous population. In the current clamp mode, a current injection of 0.09 nano amps into neurons results in action potentials.
A HP negative neurons immediately return to the resting membrane potential following stimulation. While a HP positive neurons display an A HP following the stimulation in which the resting membrane potential falls below baseline before slowly returning to the initial value. Once mastered, this technique can be done in four hours if performed properly.
When attempting this procedure, it's important to remember to keep glassware and surgical instruments as clean as possible. Also iterate and bubble the cells gently and change half of the cell culture media every Two days following this procedure. Methods like electrophysiology and immuno cyto chemistry can be performed in order to answer additional questions like how ion channels in the gut response to drug treatment, and to learn about the expression of proteins in neurons and lia, and how the interaction of these two cell types are altered by various treatments After its development.
This technique has paved the way for researchers in the field of neurobiology to explore the basic functions of neuropathies and g neuropathies of the enteric nervous system in genetically modified animals. After watching this video, you should have a good understanding of how to isolate neurons and glia from the TER plexus of the adult mouse enteric nervous system.
