The overall goal of the following experiment is to evaluate neurite outgrowth in stato acoustic ganglion neurons grown in culture when various growth factors are added to the media. This is achieved by first dissecting the stato acoustic ganglion and control spinal cords from the embryo to create X explants for culture. As a second step X explants are placed in collagen, either with soluble proteins to test for overall outgrowth or with protein coated beads to test for directional outgrowth.
Next X explants are labeled with anti beta tubulin and imaged with a confocal microscope. In order to visualize the neurites emerging from the ganglion results are obtained that show the effects that secreted ligands can exert on neuron survival and neurite outgrowth. This method can help to answer key questions in the field of auditory research, such as what molecules are important for otic neuron survival and axon guidance as sensory organs become innervated.
To begin this procedure, place the beads in a micro tube with one milliliter of sterile PBS and mix them well. Then wait for the beads to settle to the bottom of the tube. Next, wash the beads by removing the supernatant after the beads have settled and resus suspend in PBS.
After that, incubate the beads in purified protein or PBS alone as the control at room temperature for one hour after that, rinse the beads in PBS. Finally store them in a 24 well plate with one milliliter of PBS until they are placed in collagen. In this step, remove the embryo from the egg.
At E four, place it in a dish with chick ringer solution to remove embryonic membranes. Next, place the embryo in a cigar lined Petri dish. With cold HBSS position the embryo on its side with the otic vesicle facing up.
Then pin the embryo to the dish using the dissecting pins. After that, using two dissecting pins, make a horizontal cut through the skin along the ventral edge of the odo cyst. Next, make a vertical cut anterior and posterior to the SAG.
Pull the odo cyst in a posterior direction to separate it from the SAG, displace the tissue surrounding the SAG with dissection pins and carefully remove the SAG from the embryo using number 55 forceps. Subsequently, remove large chunks of mesenchymal tissue and protruding nerve bundles from the SAG. Then use a wide mouth pipette tip to transfer the X explan to a 24 well plate with 0.5 milliliters of SAGX explan holding medium.
Keep the x explan on ice or at room temperature for up to four hours. Begin this procedure by removing the E six embryo from the egg and place it in a dish containing chick ringer solution. Then remove the head and embryonic membranes.
Place the body in a cigar dissecting dish containing cold spinal cord dissection. Medium position, and pin the embryo ventral side down. Next, place the dissecting pins through each limb and the anterior end.
Using a pair of forceps or the dissecting pins, carefully remove the skin and tissue by moving in a ventral direction until the dorsal surface of the spinal cord is visible. Then cut the dorsal midline of the spinal cord with a dissecting pin from anterior to posterior along the entire length of the spinal cord. This creates an open book as the left and right sides of the spinal cord separate.
This is usually done in three parts. Remove the surrounding tissue laterally to isolate the spinal cord and expose the dorsal root ganglia. Next, remove the DRG and meninges by rubbing a dissecting pin between the spinal cord and the DRG.
After that, remove the spinal cord from the body by holding the comos end of the spinal cord with the forceps and lifting it up and away from the experimenter. Subsequently, remove the remaining DRG and meninges. Hold one end of the spinal cord with the forceps.
Use a pair of Venus scissors to cut along the ventral midline and to bisect the spinal cord. Then hold one end of the explan with the forceps to immobilize the tissue and use the Venus scissors to cut small implants to about 100 to 500 microns long along the length of the tissue. Finally, use a wide mouth pipette tip to transfer the implants to a 24 well plate with 0.5 milliliters of cold dissection medium.
Keep the implants on ice for up to four hours to maintain tissue viability to culture X explan with purified proteins. First, prepare a 1.5 milligrams per milliliter collagen solution in a 15 milliliter conical tube according to the accompanied manuscript and place it on ice. Then verify the pH of seven to 7.4 of the collagen solution.
Using pH indicator paper. Adjust the pH by adding sodium hydroxide in one microliter volumes. Next, transfer the explan to a 24 well plate using a wide mouth pipette tip and aspirate any excess liquid.
Multiple x explan can be cultured in one well, but should be placed at least 500 microns apart from each other. Add 0.5 milliliters of collagen to the well containing the explan. Position the explan on the bottom surface of the well.
Be sure to set each culture completely before proceeding with the next well because the collagen will begin to polymerize at room temperature. The next step is to place the culture plate on a 37 degrees Celsius Slide warmer for 30 to 45 minutes to polymerize the collagen. When the collagen has polymerized, it will appear as a gel add 0.5 milliliters of warm explan culture medium supplemented with either purified proteins or PBS and incubate for 24 hours effects on neurite outgrowth should be visible by 24 hours to co-culture explants with protein coated beads.
First, repeat the procedures for the explan culture preparation. Then transfer one to five beads from PBS to the well containing the collagen solution and X explan using a pipette. Next, use a pair of forceps to position the beads 50 to 500 microns from the edge of the x explan.
After that, place the plate on a 37 degrees Celsius. Slide warmer for 30 to 45 minutes to polymerize the collagen. Check the cultures and reposition the tissue and beads during the first five minutes as the collagen polymerizes.
Then add 0.5 milliliters of SAG media to each well and incubate for 24 hours. In this procedure, rinse the cultures in PBS and fix them in 4%para formaldehyde for one hour at room temperature. Then release the gels from the walls of the wells by tracing around the edge of the gels with the rounded end of a Teflon micros spatula To perform immuno staining.
Rinse the gels several times in PBS afterward, incubate them in 0.5 milliliters of blocking solution overnight at four degrees Celsius the next day. Incubate the gels in 0.5 milliliters of primary antibody overnight at four degrees Celsius. After that, rinse them several times in PBS, then incubate them in 0.5 milliliters of secondary antibody overnight at four degrees Celsius due to light sensitivity of secondary antibodies.
Keep the plates in dark or rapid foil. Next, rinse the gels several times in PBS. Then store them in PBS at four degrees Celsius for up to one week here.
SAG neuron cell bodies and neurites were immuno stained with beta tubulin and imaged with a 10 x objective on a confocal microscope after 24 hours. In vitro, SAG explants displayed greater neurite outgrowth in the presence of 100 nanograms per milliliter of purified human NT three in the cell culture medium shown in B when compared to the control in a, in another experiment, SAG neurons were co cultured with the beads soaked in 100 nanograms per milliliter of NT three. To demonstrate that a point source of NT three can locally promote nerite outgrowth.
The explants displayed longer and denser neurite outgrowth on the side facing the bead when compared to the opposite side as shown in D and to the control cultures with beads soaked in PBS indicated in C.Here the spinal cord explan were immuno stain with beta tubulin and imaged with a confocal microscope after 24 hours of growth in the presence of 400 nanograms per milliliter of purified mouse wint five. A neurite outgrowth from chick spinal cord implants was increased as shown in B when compared to the controls cultured without wint five A in a. In another experiment, the beads which had been soaked in 500 nanograms per milliliter of wind five A and had been placed 300 to 500 microns from the edge of the spinal cord.
Explan also promoted neurite outgrowth as shown in D when compared to the control cultures in c Following this procedure. Other methods like tunnel assays on cryo sections of collagen gel cultures can be performed in order to answer additional questions such as how the level of cell survival correlates with the amount of neuro outgrowth from the same sample.
