Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

The overall goal of this procedure is to quantitatively assess both functional and morphological innervation of the diaphragm by phrenic motor neurons of the cervical spinal cord. This is accomplished by conducting compound muscle action potential recording from the hemi diaphragm in anesthetized animals following supra maximal stimulation of the ipsilateral phrenic nerve. The next step is to conduct whole mount diaphragm immunohistochemistry to morphologically assess the innervation of individual neuromuscular junctions.

Ultimately, combining compound muscle action potential and neuromuscular junction analyses provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease. This method can help answer key questions in the spinal cord injury field, such as can therapeutic interventions be used to successfully restore diaphragmatic function? The implications of this technique extend towards therapy for diseases such as traumatic spinal cord injury and ILS because diaphragmatic respiratory function plays a key role in patient outcome in both of these debilitating conditions, Demonstrating the procedure will be Dr.Kali, a postdoctoral fellow in my laboratory, Melanie Martin, an undergraduate student in Dr.Wright's laboratory, and Tamara holla a research technician in my laboratory.

After anesthetizing the animal, place it on the surgical board, dorsal side down and secure the forelimbs to the board. Then shave the ventral surface from the base of the skull towards the abdomen on both sides of the midline. Once the skin has been cleaned, position the electrodes attach the ground to the tail and place the reference electrode into the contralateral lower abdomen subcutaneously.

The recording electrode should be prepared with conductive gel on the self-adhesive portion and then attached along the unilateral costal margin. Now place the stimulating electrodes, transcutaneous half a centimeter apart, lateral to the trachea and superior to the clavicle. They should enter perpendicular to the skin and go about one centimeter deep.

These electrodes must be well secured, best done by hand. Attach all the electrodes to their appropriate stimulators and amplifiers. The collected data must be recorded to a computer.

For analysis, set the stimulus parameters of the single pole stimulation to 0.5 milliseconds, one hertz super maximal pulses. Amplitude can range from six to eight volts. Set the interest stimulation time to 30 seconds, and average the response from 10 consecutive stimulations using the baseline to peak amplitude record from each animal about once per week if desired.

Animal handling is detailed in the text protocol. After euthanizing an animal using an overdose of injectable anesthesia, conduct a laparotomy using scalpel or scissors. Make an incision from the xiphoid process coddly along the midline.

Then using a scalpel and forceps, carefully separate the skin and connective tissue from the musculature. Remove the diaphragm by pulling up the xiphoid process and cutting out the diaphragm from the surrounding bone. Be careful not to damage the diaphragm muscle.

Transfer the diaphragm superior side up to a silicone line dish filled with 4%paraform aldehyde in PBS under a stereoscope. Pin down the tissue surrounding the diaphragm, stretching the diaphragm taut. Then remove the connective tissue from the superior surface using forceps and scissors.

Let the fixative work for 20 minutes and then remove it using three 10 minute washes in PBS. Next, apply 0.1 molar glycine with 2%BSA in PBS for 30 minutes. Then incubate the diaphragm in rod Domine conjugated alpha bungalow toin solution for 15 minutes.

Wash off the bungalow toxin with PBS as done for the fixative. Next, change the bath to cold methanol and chill the tissue for five minutes at negative 20 degrees Celsius. After five minutes promptly, remove the methanol and wash the tissue with PBS as before.

Now, apply a block using 0.2%TBP solution for one hour. Change the solution to the primary antibody in 0.2%TBP and let the tissue incubate overnight at four degrees Celsius with gentle agitation. The next day, use three 10 minute washes in 0.2%TBP to remove the primary antibody, and then incubate the tissue in the secondary antibody solution for an hour with rocking and in the dark later, use three washes in PBS to remove the secondary antibody and keep the tissue in the dark.

Then under a stereoscope, remove more connective tissue on the superior surface of the muscle. Once cleaned off mount and image the tissue under an upright epi fluorescence microscope quantitatively analyze the morphology of the stained hemi diaphragm. Use 20 and 40 x magnification Because the diaphragm is analyzed as a whole mount.

Most of the fluorescent staining is out of focus. Using a standard microscope, it is best to conduct the analysis in real time. In order to properly score the morphology of the individual NM js, Divide the muscle into three sections for analysis based on the topographical innervation patterns of the spinal cord.

Starting in the ventral mosts region, analyze all the visible njs along the muscle fibers do not analyze any deeper than the first three to four fibers from the surface because the antibodies penetration is never complete. An intact NMJ has a pre terminal axon that is thick and all regions of the nerve terminal completely overlap with the underlying muscle acetylcholine receptors. A partially de unnerved NMJ will have some regions of the underlying acetylcholine receptors unoccupied.

A completely denervated NMJ will show no correspondence with the acetylcholine receptors at the nerve terminal. Other njs in the region should show another morphology or the success of the antibodies Penetration would be in question if more than one pre terminal axon innervates an NMJ. The NMJ is multiply.

Innervated denervation may have occurred in this case, followed by reinnervation. If there is overlap at the nerve terminal with the underlying acetylcholine receptors and the pre terminal axon is very thin, then the NMJ is thinly innervated. This is also an indication of reation.

In an experiment involving adult spra, Dolly rats controls received a laminectomy while others received a unilateral hemi contusion SCI. At C four. Five weeks later, the peak cmap amplitude was recorded from the hemi diaphragm ipsilateral to the laminectomy or injury site.

It was significantly reduced in the SCI rats as compared to the laminectomy only control rats in a different experiment involving rats expressing mutated human, so D one protein to serve as a model of a LS.The NMJ morphology in the hemi diaphragm was completely intact in wild type control animals. However, in the transgenic rats, the njs showed significant pathology. Some showed complete denervation marked by the arrowhead.

Other njs were partially denervated as marked by the arrowhead, and as seen in the NMJ on the right, other njs were multiply innervated, and others had thin pre terminal axons here marked with an arrowhead head. After watching this video, you should have a good understanding of how to quantitatively assess both functional and morphological innervation of the diaphragm by phrenic motor neurons. Thanks for watching and good luck with all your experiments.