The aim of the following experiment is to analyze schwan cell development along growing axons, their natural habitat. This is achieved by first ex explan superior cervical ganglia, or S cgs of mouse embryos onto collagen matrices as a second step. The S cgs are treated with nerve growth factor or NGF, which facilitates axonal growth from the SCG neurons in a coronal fashion.
In addition to axonal growth from the X explan, endogenous schwan cells can be observed migrating along the extended axons towards the periphery. Schwan cell development can be analyzed by time-lapse imaging and also by endpoint analysis of fixed tissue, which enables distance measurements. The main advantage of this technique over existing methods, like the scratch migration assay, is that the natural habitat of the swan cells is included in this assay.
Visual demonstration of this method is critical. As the correct assembly of the collagen metrics and the correct dissection of the sgs are key to success. These steps are not especially difficult to learn.
They just need practice First. While working in a tissue culture hood understood our conditions with all solutions on ice. Prepare stock culture medium, and rat tail collagen according to the instructions in the written protocol.
Mix, 800 microliters of rat tail collagen with 210 microliters of the stock medium. Without introducing bubbles, the solution should remain a red color. If the solution turns yellow, then the Ratal collagen solution is too acidic and needs to be remade with a larger concentration of sodium hydroxide pipette 100 micro release of the solution into each well of a multi-well slide and make sure that the bottom of each well is completely covered.
Place the dishes into a humidified tissue culture incubator set at 37 degrees Celsius and 5%carbon dioxide for two hours. Our after harvest, E 16 to E 18 embryos are placed in a Petri dish containing PBS then dissected from the amnion and decapitated at a level cordal of the C clavicular. The head and neck regions are then transferred to a silicon bottom dish, and while looking through a dissecting microscope, insect needles are used to pin the tissue in the submandibular and clavicular regions.
Now use forceps to remove the skin and subcutaneous tissue of the submandibular region. Then remove the submandibular salivary glands to reveal the supra hyoid, infra hial, and sternocleidomastoid muscles. Next, use forceps to carefully remove the infra hial and sternocleidomastoid muscles to reveal the larynx and trachea.
Grasp the trachea with the forceps and lift out the trachea and the larynx. Once the larynx and trachea have been removed, the carotid arteries are visible, are both sides of the prevertebral muscles. The superior cervical ganglia are located in the bifurcation of the carotid arteries and can be identified as an oval shape.
Use forceps and a needle holder mounted with an insect needle to remove the superior cervical ganglia and place into a fresh dish containing PBS then dissect any blood vessels or connected tissue from the ganglia. With the help of syringe needles, remove the gelatinized collagen gels from the incubator and use syringe needles mounted onto syringes to transfer the ganglia onto the gels. Place the explants into the tissue culture incubator set at 37 degrees Celsius with 5%carbon dioxide and humid conditions for one to two hours.
After one to two hours of incubation, the superior cervical ganglion explants are retrieved from the tissue culture incubator and 100 microliters of neuro basal medium containing B 27 supplement glutamine and antibiotics is added to each x explan. Subsequently, 200 microliters of neuro basal medium containing 60 nanograms per milliliter. NGF is also added to the wells to facilitate axonal growth.
Test substances may also be added at this time, which is defined as day in vitro zero to study the effect on swan cells that have already begun migrating along. Axons substances can be administered at three days in vitro by removing 180 microliters of the existing media and replacing with 200 microliters of media containing the substance of interest and NGF at double the final concentration, time-lapse imaging is performed using an inverted microscope setup that will enable parallel cell culture incubation and time-lapse imaging to compare different test substances. In one experiment, a multi-position setting is recommended at the end point of the experiment.
After fixing the explants within the culture slides with 4%PFA for three to four hours and washing with PBS perform th immunohistochemistry to validate the X explant as the SCG block the tissue with PBS containing 10%normal donkey serum and 2%tritton X 100 for two hours. Then follow with primary antibody incubation in blocking solution overnight. The next day after primary antibody incubation transfer the X explan containing gels to a 24 well plate after washing with PBS incubate with fluorescent labeled secondary antibodies for two hours while protected from light, then wash with PBS and incubate with dappy solution for 10 minutes.
After washing several times, place the collagen gels containing the X explan onto a microscope, slide, dry the slides and subsequently mount with veal. Use a standard fluorescent microscope to obtain images for later quantitative analysis. It is helpful if the microscope software is capable of recording metadata.
Quantitative analysis of obtained images can be performed with Fiji or ImageJ software, which can be used free of charge for scientific applications. Axonal growth from a schematic SCGX EXPLAN over four days in vitro is demonstrated in this movie. Th immunohistochemistry can be performed on the fourth day in vitro to clearly identify the explanted ganglion.
As a sympathetic ganglion, schwan cells can be observed migrating along the axon, moving from the superior cervical ganglion to the periphery. This video shows the same migration pattern as just seen at a higher magnification. Migration distances can be easily measured on samples counterstain for nuclei with DPI by measuring the distance from the nuclei of the leading schwan cells to the border of the explanted SCG at multiple locations as depicted in the schematic and the image of a DPI labeled NGF treated sample at four days in vitro, this movie shows schwan cell migration at the border of an SCG explan.
The combination of brightfield and fluorescence enables visualization of S 100 GFP positive swan cells and the axons. Finally, this movie shows schwan cell migration at the border of a superior cervical ganglion ex explan. Here only the fluorescence channel is shown enabling easier interpretation of S 100 GFP positive schwan cells.
Following this procedure, additional quantitative analysis can be performed like the quantification of one cell proliferation or the quantification of one cell apoptosis. Of course, the adequate immunohistochemistry has to be performed for endpoint analysis or the specific transgenic animals have to be used for showing the effects in the living context.
