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 super maximal stimulation and record an additional response. Measure and document the peak-to-peak amplitudes of the CMAP in millivolts. Administer submaximal stimulation with a duration of 0.1 milliseconds at a frequency of 1 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 1/3rd 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 in CTB-SAP treatment.
