Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons

The overall goal of the following experiment is to increase the efficacy of the retrograde transport of neuro anatomical tracers to spinal cord motor neurons following intramuscular injection. This is achieved by initiating an acetylcholinesterase histochemical reaction in a mocked up tissue of interest to identify the location of the motor end plates on the animal musculature. In the second step, intramuscular injections of the retrograde tracer are performed along the entire span of the motor end plate region.

On the muscles of interest, the animals are then kept for 14 days to allow the transport of the tracer into the spinal cord. Motor neurons ultimately targeting the entire length of the motor endplate region yields more labeled motor neurons that extend across multiple segments of the rodent spinal cord when visualized using fluorescence microscopy. The implications of this study extend towards the development of therapies for neuromuscular dysfunction, which includes spinal cord injury and amyotrophic lateral sclerosis.

As this is a minimally invasive way, and therefore a clinically relevant way to deliver therapeutic genes into the spinal cord, motor neurons, Generally individuals new to this method will struggle because the motor endplate regions cannot be visualized in the musculature of living animals. Visual demonstration of this technique is critical as the intramuscular injections require the mental transposition of the motor endplate onto the muscles To create a motor endplate map for the intramuscular injection begin by immersing the skinned body or limb of interest into the reaction mixture for an overnight incubation at four degrees Celsius. The next morning, wash the carcass for two minutes in distilled water.

At this stage, the muscles will be a blue color and the motor end plates will appear as white dots dip the carcass in ammonium sulfide, wash the carcass three times, pet the limb dry and detect the MEP region, and then analyze the contrast to inject the live muscle at the motor end plates. First, use a micropipet puller to pull graded glass micro equipped with plungers. Next, using a dissecting microscope, break the tips of the micro pipettes with a pair of forceps such that the internal diameter of each lumen is approximately 0.5 millimeters.

Then fill the pool pipettes with flora gold. Now apply eye lubricant to an anesthetized animal and shave the limb to be injected. Use gauze to wipe the shaved area with three alternating scrubs of chlorhexidine and 70%alcohol.

Then place the animal on a clean underpad to allow access to the targeted muscle. When the animal's in position, use a pair of forceps with tooth grips to lift the skin over the targeted muscle away from the underlying musculature, and use surgical scissors to make an incision in the skin. Take care that the incision is large enough to completely expose the muscle of interest with a minimal disruption to the fascia.

Then using the motor end plate photographs to mentally transpose the location to and shape of the motor end plate region of interest onto the muscle and administer three to four injections of one to two microliters of floral gold along the full length of the motor end plate region. After the last injection, gently wipe the muscle to remove any seepage. Then use blunt forceps to bring the two ends of the incised skin close together, close the wound with surgical clips and infiltrate the entire span of the wound with the appropriate local anesthetic.

Two weeks after the injections, intracardiac perfuse the animal with fixative. To dissect the cervical spinal cord, lay the animal in the prone position and use a scalpel to cut the skin at the body's midline. Reflect the skin from the base of the skull to the level of the iliac bone.

Then cut through and reflect the paravertebral muscles to expose the dorsal aspect of the vertebral column. After identifying the bony spinous process of axis, remove the segment with the pair of forceps to locate the corresponding underlying dorsal root. Label the right C two root with a permanent felt tip marker.

Then remove the successive vertebrae one by one marking the right dorsal roots in alternating colors. For example, here, C 2 4, 6, and eight have been colored blue while C 3 5, 7 and T one have been labeled black. Next, use a 30 gauge surgical needle to gently pierce through the dura covering the lower end of the spinal cord with the bevel of the needle facing up.

Lift the dura away from the cord while moving the needle roly. To make a longitudinal slit and reflect the Dora, use a new scalpel blade to cut the spinal cord transversely into one to two segment blocks to aid in the orientation of the tissue segment following dissection. Leave the segments in place and for each block, use the scalpel to carefully make a small fiducial mark on the right side of each block, halfway between the two adjacent roots.

Then to prepare the tissues for histological analysis, transfer the individual blocks into small, clearly labeled bottles containing paraldehyde for an overnight incubation at room temperature. The next morning, transfer the blocks into new bottles containing sucrose solution at four degrees Celsius. After at least two days, move the segments into individual cryo molds.

Orient the blocks with the dorsal aspect facing up and cover them with tissue freezing medium. Then freeze the cryo molds at negative 20 degrees Celsius and cut 50 micron sections with a cryostat. To dissect the lumbar spinal cord, place the animal in the supine position and make a midline incision along the abdomen.

Remove the viscera and dissect out the muscles of the posterior abdominal wall. To expose the ventral aspect of the vertebral column, locate the short coddle mosts rib and its adjoining T 13 vertebrae where the T 13 ventral root exits the bone. Then using fine surgical Ron jurors consecutively, remove up to three vertebrae rostral to T 13 to follow the T 13 ventral root until its point of entry and the ventral aspect of the ventral cord.

Mark the ventral root with a permanent felt tip marker. Then remove the vertebrae as just demonstrated marking L one to L six and S one with color to eight in their easy identification. Finally, dissect the blocks and prepare them for histology as just demonstrated in this representative mocked up tissue during acetylcholinesterase histochemical staining the motor end plates for the acromial trapezius, spinal delius and the triceps brachii are apparent as white speckled dots that form a continuous line crossing the entire width of the muscle while the muscle fibers adopt the green blue hue.

After the immersion and ammonium sulfide, the motor end plates turned black over the brown muscle fibers. A retrograde neuronal tracer was then injected along the length of the motor end plate region of a live mouse triceps brachii muscle. As expected, the tracer labeled motor neurons form a longitudinal column that extended across multiple segments of the spinal cord, targeting only one part of the motor end plate region, as shown here by the green, blue, or red areas of the triceps.

Brachii motor endplate region result in reduced motor neuron labeling as shown by the green, blue, or red motor columns. Therefore, for maximal uptake of the tracer into the innervating motor neurons, the entire motor end plate region must be injected Following this procedure. Other methods like immunohistochemistry or stereo logical cell counting can be performed in order to answer additional questions like antigen antibody reactions and the morphology of labeled modern neurons After its development.

This technique has paved the way for researchers in the field of neuroscience to develop therapeutic strategies like gene therapy, ER rodent models of neuromuscular dysfunction.