The overall goal of the following experiment is to image fluorescently labeled transplanted, neural precursor cell or NPC interactions with fluorescent axons in the mouse spinal cord in real time. This is achieved by carefully removing and embedding the spinal cord from mice transplanted with NPCs into 5%aros and attaching it to a cover slip. The orientation of the spinal cord can be altered to view fluorescence in any region of the spinal cord.
In the second step, the spinal cord preparation is placed on the two photon imaging stage for imaging of viable NPCs while being super fused with warm oxygenated RPMI media for up to 10 hours. An appropriate single edge diic beam splitter is used to separate the YFP and GFP emissions. Ultimately, two photon imaging allows visualization of the interactions of the GFP expressing NPCs with the YFP expressing axons in the damaged spinal cord in real time.
This method can help answer key questions in the field of neurogeneration, such as what are the molecular and cellular signaling cues necessary for transplanted NPC remyelination of damaged axons, and what are the kinetics of these remyelination events? The main advantage of this technique over existing methods such as intertidal two photon imaging, which can be only used to image the dorsal spinal cord, is that this method allows imaging of transplanted NPCs that have migrated deep within the ventral region of the spinal cord. After confirming euthanasia by spinal transection, wet the mouse with 70%ethanol, then use sharp fine scissors to remove the hair and skin from the back of the animal, exposing the spinal column from approximately the cervical lamina one or C one to the sacral lamina four or S four.
Next, using a scalpel with a number 10 blade, make incisions to the left and right of the spinal column to separate it from the surrounding muscle and fat tissue also cut away excess tissue around and on top of the spinal column. This will allow for easier removal of the vertebrae. Then while holding the top of the spinal vertebrae with serrated gray forceps, insert titanium curved van scissors with the curved side up into the exposed spinal column at C one, taking care not to touch the spinal cord.
Slide the scissors all the way to the right and make one small cut. A small crunching sound should be heard and felt as the scissors cut the vertebrae. Repeat this cut on the far left side of the cord, taking care not to work too quickly or to make too large of a cut, as this will risk damaging the cord.
Once the sides of the vertebrae are cut, the single lamini should be able to be lifted with the forceps. Repeat the left and right cuts for each lamina until S four is reached. Continuing to hold and pull back the spinal cord vertebrae.
The vertebrae above the transplant might come off once his spine is cut down to the transplant site. Once the vertebrae are cut down to S four, lay the vertebrae down to help visualize where the transplant site is and cut approximately 20 millimeters rostral and coddle to the transplant site. Now use a scalpel with a number 11 blade to carefully cut the ganglia on the right and left of the ventral side of the spinal cord, starting at the rostral end.
Then invert and hold the animal up so that the spinal cord is facing down and use closed serrated grave forceps to carefully peel out the spinal cord from the coddle end. Then without nicking the spinal cord, carefully cut any of the remaining ganglia to allow the spinal cord to be lifted out in a single intact piece, and place the cord in medium on ice to prepare the spinal cord for imaging. Transfer the nervous tissue to a sheet of param with the ventral side facing up.
Then pipette approximately five milliliters of freshly prepared 37 degrees Celsius, 5%agro solution over the cord, and let it solidify at room temperature. After about five minutes, apply a light coat of tissue adhesive to a 22 millimeter square cover slip and invert the embedded spinal cord ventral side down onto a new piece of perfil. Adhere the cover slip to the exposed dorsal side and submerge the entire preparation in fresh medium to solidify the adhesive.
Then use a razor blade to remove the excess solidified aga rose. Next, insert the tubing connected to the tubing pump into a media bottle, kept in a water bath to super fuse 37 degrees Celsius. Pretty warmed media through a custom built imaging well on the microscope stage at three milliliters per minute.
Then place the spinal cord preparation into the well with the ventral side facing up toward the 25 x dipping objective. To image the ventral spinal cord by two photon microscopy, use an ultra titanium sapphire laser tuned to 900 nanometers with the laser power attenuated at the specimen to less than 5%to ensure minimal phototoxicity. Then using the eyepiece and a bright field light source focus the dipping objective at the ventral edge of the spinal cord.
To set a reference point, make sure the ambient light source is off. Then open the laser shutter to switch the two photonic citation if available. When searching the tissue for areas of interest, use a lower resolution setting a higher volume without digital zoom and a higher scan rate than when acquiring the final images.
To find the cells, it is best to focus on the sample under bright field illumination at the tissue edge, at the transplant site first, and then to switch to fluorescence. A large X and Y field with a low resolution can then be used to enable the quick viewing of a large area as the focus is move deeper into the tissue. Observe the EYFP axons near the ventral edge of the spinal cord.
The second harmonic signal from the collagen observed here in blue will be the brightest at the spinal cord tissue edge. Now, use the appropriate image capture software to acquire the final higher resolution images in sequential focal planes in 2.5 micron increments to compile Z stacks. In this representation, GFP NPCs are observed as blue and damaged YFP axons are observed as yellow pseudo coloring can be performed later using editing software then perform a bi-directional scan with the proper interlay offset to ensure the rapid quantification of any migrating objects in the spinal cord, ensuring that the ideal acquisition frame rate to determine the cellular velocity within the spinal cord is set to approximately 1.7 frames per second.
Finally, analyze crop smooth and pseudo color the image files with the appropriate software, and use the automated processes to comb the consecutive imaging volumes for producing the final time lapse videos. While this explanted spinal cord imaging protocol can be used to visualize any fluorescence within the spinal cord, these images demonstrate representative EGFP neural precursor cell interactions with EYFP axons in a single Z stack within the ventral spinal cord. The acquisition of consecutive Zacks can then be compiled to produce time lapse videos for analyzing the real-time cellular dynamics within the intact tissue.
Indeed use of a 520 nanometer single edge dichroic and a 560 nanometer single edge dichroic beam splitter can separate the EGFP and EYFP signals, allowing pseudo coloring of the individual channels in green and yellow with the imaging software as demonstrated here, While attempting this procedure, it's important to remember to ensure the proper removal of the spinal cord from the mouse without excessive stretching, compressing, or accidental cutting of the nervous tissue. With this Procedure, any combination of fluorescently labeled cells or axons can be imaged in real time to answer additional questions, including what are the kinetics of migration or remyelination under various drug treatments or conditions.
