Assessing Rat Diaphragm Motor Unit Connectivity Outcome Measures as Quantitative Biomarkers of Phrenic Motor Neuron Degeneration and Compensation

We're interested in understanding normal and maladaptive responses of the neuromuscular system in the context of health, aging, and disease. Specifically, we are aiming to investigate how the respiratory motor system responds to motor neuron losses through compensatory mechanisms such as collateral sprouting. Currently, various labeling techniques such as retrograde tracers, adenoviruses, and immunohistochemistry have been utilized to quantify the number of innovating motor neurons in rodent degenerative models.

Labeling techniques, though valuable for motor neuron evaluations, have limitations in assessing the functionality of motor units and are not suitable for longitudinal assessments. The MuNI technique is a non-invasive approach in preclinical rodent studies that can be used longitudinally to quantify the phrenic motor units. Implementing the MuNI protocol has the potential to significantly aid and accelerate experimental research discoveries toward clinical testing, particularly in non-invasively and longitudinally assessing neuromuscular impairment of the phrenic nerve and diaphragm muscle.

Our eclectic lab group focuses on investigating neuromuscular function in health and preclinical models of neurodegenerative disease. We are striving to utilize translational techniques to better understand whether underlying mechanisms of plasticity can be harnessed and adapted to preserve respiration to improve the quality of life in patients with neurodegenerative disease. To begin, position the anesthetized and prepared rat for compound muscle action potential recording.

Place the active needle electrode subcutaneously over the midclavicular line inferior to the last rib border. Then position the reference needle electrode subcutaneously at the angle between the xiphoid process and the last sternocostal cartilage. Next, use a pair of 28-gauge monopolar needle electrodes for phrenic nerve stimulation at the carotid sheet.

Place the electrode subcutaneously on the lateral neck between the anterior and middle scaly muscles with a separation of about one centimeter. Confirm that the placement of the stimulating needles is at the level below the fourth cervical vertebrae. Then place a disposable surface electrode on the tail for the ground electrode.

To begin, prepare and position the anesthetized rat for electrophysiology. Apply monophasic cathartic square wave pulses to stimulate the phrenic nerve for recording CMAP responses. Increase the stimulus intensity progressively until the amplitude of the response ceases to increase.

Then raise the stimulus intensity to approximately 120%of the level required for a maximal response to ensure supramaximal stimulation and record an additional response. Measure and document the peak-to-peak amplitudes of the CMAP in millivolts. Administer sub-maximal stimulation with a duration of 0.1 milliseconds at a frequency of one hertz.

Gradually increase the intensity in 0.03 milliampere increments to elicit incremental responses until a minimal all-or-none response is achieved. Then acquire the initial response with a stimulus intensity ranging between 2 and 10 milliamperes. After saving the first incremental response, acquire additional increments with progressively higher stimulus intensities in increments of 0.03 milliamperes.

Ensure that the initial negative peak of the incremental responses aligns temporally with the negative peak of the maximal CMAP response. Given the diaphragm's involvement in respiration, check for stability and absence of fractionation in each incremental response to confirm consistency across three duplicate responses. Observe incremental responses in real time and overlay them on previous recordings for visual distinction.

Then confirm that the increment amplitude is at least 25 microvolts. After recording 10 incremental responses, verify that the amplitude of each increment does not exceed one third of the total amplitude of the final response. To estimate the average amplitude of the SMUP, average the values of 10 increments.

Next, divide the maximum CMAP amplitude by the average SMUP amplitude to determine the MUNE. Seven days after intrapleural CTB-SAP injection, MUNE decreased to 60 functional motor units in adult rats compared to 74 in control rats. CMAP amplitude remained relatively stable, suggesting collateral sprouting as a possible compensatory mechanism.

Diaphragm CMAP, SMUP and MUNE were measured over 28 days post-injection. CMAP showed no notable change. The average increase in SMUP for CTB-SAP rats was approximately 50 to 60%The changes were significant over time and attributed to CTB-SAP.

MUNE was reduced by approximately 40%in CTB-SAP rats, indicating significant effects of time and CTB-SAP treatment.