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

In MN degenerative diseases, such as ALS, it is crucial to assess the MUs involved in ventilation²⁸. Despite the occurrence of respiratory MN degeneration in ALS patients, the specific onset and progression of MN death remain incompletely understood^29,30,31. Recognizing the significance of this aspect, various models, both genetic-based (e.g., SOD1^2,32) and non-genetic-based (e.g., CTB-SAP intrapleural injection^3,27,33), have been employed to emulate respiratory impairment in animal models of neurodegenerative diseases. Accordingly, identifying a biomarker for diagnosing, monitoring, and assessing potential treatment effects is advantageous for these models. Additionally, biomarkers can facilitate the clinical translation of preclinical research findings.

To quantify the number of innervating MNs in rodent degenerative models, different labeling techniques, including retrograde tracers and adenoviruses, have been employed^25,34,35,36. Previously, immunohistochemistry has been utilized to label phrenic motor neurons in the anterior horn of the cervical spinal cord^2,3,33. Labeling techniques, though valuable for MN evaluations, have limitations in assessing the functionality of MUs and are not suitable for longitudinal assessments³⁷. Objective electrophysiological assessments are crucial for successful cross-species translation, especially considering pronounced structural differences in the functional and anatomical features of the motor systems between humans and rodents^38,39. Incorporating neurobiological differences presents a challenge in translating findings from rodent models to patients. MUNE overcomes these obstacles by serving as an objective electrophysiological measure of MN function, allowing the evaluation of functional MU connectivity as a biomarker in MN degenerative model experiments^3,40.

CMAP, SMUP, and MUNE are commonly employed in research studies and for evaluating patients with neuromuscular disorders^9,10,11. These potential biomarkers are minimally invasive, enabling longitudinal assessment of function within the same individual over time. Importantly, while they do not directly gauge the activation or recruitment of the MU by cortical MNs, they do provide a clinically relevant estimation of the MN integrity and its functional counterpart, the MU. Rodent models of MN degenerative diseases are crucial for comprehending the physiopathologic mechanisms underlying human diseases and for the preclinical advance of effective treatments. Developing outcome measures and biomarkers that are translatable across species can streamline and expedite the translation of promising preclinical findings to clinical trials¹¹. Given the relative complexity of the measures, we have adapted and refined these techniques from rat limbs to the diaphragm muscle. This translation has been presented in a visual format to facilitate broader utilization and implementation in rat studies.

From our experience, the clinical electrodiagnostic systems are well-suited for the studies outlined in this context. This suitability stems from enhanced ergonomics in the interface between the examiner and the electrodiagnostic system, facilitating convenient control. In our laboratory group, we utilize a two-channel system featuring two non-switched amplifier channels. These channels are equipped with a 24-bit Analog to Digital Converter and operate at a sampling rate of 48 kHz per channel. The hardware gain is adjustable within a range from 10nV to 100 mV per division. We employed a low-frequency filter with a range spanning from 0.2 Hz to 5 kHz, and the high-frequency filter settings covered a range from 30 Hz to 10 kHz. Additionally, we utilized a constant-current stimulator with adjustable intensity ranging from 0 to 100 mA and duration from 0.02 to 1 ms. The majority of clinical systems offer comparable features that are suitable for recording CMAP, SMUP, and MUNE responses. These systems can be adjusted accordingly to ensure accurate recording of the desired data. For instance, CMAP amplitude can be assessed base-to-peak and peak-to-peak. Clinical electrodiagnostic systems are often defaulted to assess base-to-peak which is calculated from the isoelectric baseline to the initial negative peak.

The process of obtaining CMAP, SMUP, and MUNE responses from the diaphragm involves several crucial steps. Consistent and accurate electrode placement, along with adequate electrode depth, is essential for reliably measuring amplitudes and minimizing background noises during recording. Incorrect placement of stimulating electrodes can lead to unintended consequences. Placing stimulating electrodes too posteriorly can result in exaggerated forelimb muscle contractions to brachial plexus excitation, while higher than C4 level placement can make stimulation of the phrenic nerve more difficult. Deeper placement carries the risk of injuring the carotid artery or evoking the vagus nerve, potentially causing cardiac conductivity issues. Placing recording electrodes higher or lower than the last rib, measurements from intercostal or rectus abdominis stimulation may occur, respectively. To avoid recording from other muscles, notice that the diaphragm negative peak of CMAP latency is typically 1.5-3.5 ms, and its characteristic shape is shown in Figure 2. Our observations indicated that needle electrodes yield more consistent CMAP, SMUP, and MUNE recordings compared to disc-shaped electrodes for diaphragm muscle. This enhanced consistency is attributed to the dome shape of the diaphragm muscle and the presence of other muscles in proximity, such as the intercostal and rectus abdominis muscles. Additionally, flexible placement of the needle electrodes is another advantageous factor. Regarding electrical stimulation intensity, we utilized a range of 60 to 100 mA to stimulate the phrenic nerve, which exceeds the reported ranges for other motor systems. This difference arises from the deeper location of the phrenic nerve in the neck compared to the more superficial locations of the sciatic nerve and brachial plexus in the hindlimb and forelimb, respectively.

Obtaining the average SMUP poses greater technical challenges compared to CMAP. The smaller response size, in the microvolt range rather than millivolts, makes the impact of background noise more pronounced. To mitigate background noise, consider optimizing electrode placement by adjusting the ground electrode, cathode, and anode. Additionally, ensure there is minimal interference from nearby electrical devices in the experimental setup. A Faraday cage, commonly utilized in intracellular electrophysiology applications, is not necessary for this setup. The visual identification of individual SMUP responses is a challenging skill that requires practice for consistent and repeatable results. As the diaphragm is a dynamic muscle, the baseline can shift with each ventilation cycle. Being attentive to the SMUP increments ensuring alignment with the previous SMUP is a critical factor in overcoming this challenge. Additionally, ensuring that the SMUPs initiate within the latency timeframe of the maximal CMAP is important for accurate recordings.

Another challenge of diaphragm recording is the asymmetric nature of this muscle, characterized by varying thicknesses from left to right and influenced by the pressure exerted by the liver on the right side⁴¹. To address this, we conducted electrophysiological measurements separately on the left and right hemidiaphragms. Following that, we calculated the averages for CMAP, SMUP, and MUNE responses separately.

Since the CMAP response reflects the collective depolarization of muscle fibers in a specific muscle, any pathology affecting the phrenic MN to the diaphragm muscle fiber can result in a reduction in CMAP size. Hence, calculating the average CMAP from bilateral hemidiaphragms offers an excellent assessment of the overall functional status of the respiratory neuromuscular system. Compensatory changes, such as collateral sprouting, can lead to the preservation of CMAP size even in the presence of MN or motor axonal loss¹¹. Recording individual increments allows for the estimation of the average output of single MUs (SMUP size), presenting more detailed insights into the functional status of MUs of the respiratory system. Hence, the MUNE technique becomes essential for evaluating the phrenic MNs or axons' input to the diaphragm. Applying these biomarkers in preclinical experiments of therapies for studying MN diseases holds the potential to improve the translation to clinical trials.