RNS has been widely applied in the evaluation of NMJ functions. A number of studies have demonstrated that decremental responses are frequently observed in ALS, beginning with the first description from Mulder et al.(1959). However, the results of RNS test and its implications in clinical applications and pathogenic mechanism have not yet been fully elucidated (Alanazy et al., 2017; Fischer et al., 2004; Hegedus et al., 2007; Miyaji et al., 2018; Clark et al., 2016; Iwai et al., 2016; Wainger et al., 2014; Mukhutdinova en al., 2018). So we have conducted a retrospective analysis based on the largest sample size ever to our knowledge, in order to quantify the results of RNS test and find out the respective relationship of CMAP decremental response either to clinical manifestations or to EMG results, so as to substantially advance the understanding of RNS results in ALS patients, effectively instruct clinical application, supply a clue to the establishment of a more reasonable exclusion criterion and enlighten the exploration of pathogenic mechanisms from the perspective of clinical neuroelectrophysiology.
In previous studies, due to the differences in the races, the inconsistency of the instruments and cut-off values in different laboratories as well as the limited number of the samples involved in the test, a large discrepancy exists about the positivity rates in RNS tests, ranging from 29–83% (Sun et al., 2018; Tomoko et al., 2011; Zheng et al., 2017a; Fu et al., 2019; Wang et al., 2017; Alanazy et al., 2017; Zheng et al., 2017b; Hatanaka et al., 2017; Hu et al., 2018). To minimize the effect of potential systemic and random errors, we collected and analyzed an adequately large sample size of case data to improve the generalizability of the results. The present study shows that 3Hz RNS delivered to the accessory nerve has the highest diagnostic sensitivity. The overall positivity rate of 3Hz RNS (decremental response exceeding 10%) for the 449 patients is 49.9%, while that for the 364 clinical definite ALS patients reaches 52.5%. As for the range of decremental CMAP amplitude at RNS test, very few studies have reported the exact values of the range in addition to mean and standard deviation (Sun et al., 2018; Tomoko et al., 2011; Zheng et al., 2017a; Hu et al., 2018). The largest decrement of CMAP amplitude that has ever been reported was 50% in our previous research (Sun et al., 2018) while this study shows that it can reach 55% (Table 1).
According to the Electrophysiological Diagnostic Criteria for ALS revised in 2000 by the World Federation of Neurology Research Group on Motor Neuron Diseases, a significant decrement in CMAP, i.e. when it’s exceeding > 20% in RNS, could be a criterion to exclude ALS (Brook et al., 2000). In Li et al (2020) studies, a significant decrement (≥ 20%) in at least one muscle was observed in 11.3% of the ALS patients, while decrements (≥ 10%) in at least one muscle was observed in 41.3%. However, our study of the 449 cases shows that in 15.5%, 7.6% and 4.0% of the cases RNS decremental responses exceed 20%, 25% and 30% respectively. As far as we can see, the implementation of the current Eelectrophysiological Diagnostic Criteria will result in the misdiagnosis of more than 15.5% of ALS patients. Then, what percentage of RNS decremental response can serve as a criterion to exclude ALS still warrants further investigations.
In addition to RNS test results in ALS patients mentioned above, the results of our statistical analysis also provide robust evidence for the correlations between RNS decremental responses and ALS clinical manifestations. For the first time, a perfect negative linear correlation between disease duratrion and 3Hz RNS decremental responses is established by regression analysis and a significantly higher positivity rate in patients with ΔFS < 0.5 score/month(n = 88) is shown by Chi square. As can be drawn from the ΔFS formula, the longer the disease duration is, the lower the ΔFS will be. Taking the ΔFS formula and the above two results in account, our study indicates that the NMJ dysfunction is deteriorating as the disease progresses, especially after 6 months.
As for the relationship between RNS decremental responses and EMG results, we are the first to establish a regression model (Fig. 2) and compare the sensitivities of two indicators (i.e. MUP duration and duration increment percentage) to RNS test. Our study shows that MUP duration rather than the range of duration increment is more suitable to be used in future studies. The regression model in Fig. 2 confirms the correlation between the impairment of motor neuron and NMJ dysfunction from the perspective of clinical neuroelectrophysiology for the first time. By two intersecting lines, namely x = 20 and y=-10, the coordinate plane is divided into four quadrant (Fig. 2b). It’s worth noting that scatters in the third quadrant satisfying the requirement of RNS(+) and EMG(-) seems to indicates that in some cases, RNS test is more sensitive than EMG and NMJ dysfunction is prior to the loss of motor neuron. Whether this electrophysiological phenomenon can serve as electrophysiological evidence for “dying-back” hypothesis (Michal et al., 2011) still needs further investigations.
This study is not free of limitations. Firstly, the positivity rates of clinically probable cases (n = 46), clinically probable laboratory-supported cases (n = 20) and clinically possible cases (n = 18) are 30.4%, 35.0% and 61.1%, respectively. However, the sample size of the cases at the three levels is relatively small in comparison with that of definite ALS. The difference in positivity rate among these three diagnostic levels has very little meaning. On the one hand, our neurology department is one of the best national medical research institution which mostly receives patients suffering rare and serious diseases and most patients at the early stage of ALS just see a doctor near their hometown as their muscle weakness is not obvious. On the other hand, people are not well informed of ALS knowledge, which makes patients at early stage likely to be missed or even misdiagnosed. That’s why we think multicenter retrospective studies are needed in the future. Secondly, present statistic analysis shows the positivity rate of 3Hz RNS test delivered to the accessory nerve in upper-limb-onset group is 60.6%, leading all the others and followed by bulbar group (45.0%) and lower limb group (32.9%), which is in line with some previous study (Sun et al., 2018; Hatanaka et al., 2017; Hu et al., 2018; Miyaji et al., 2018; Clark et al., 2016). The probable reason for this obviously high positivity rate in the upper-limb-onset group may be ALS’s preference for spreading around its onset site. However, as to whether the positivity rate on the common peroneal nerve at 3Hz RNS might be higher in patients with lower-limb-onset than that in patients with other onset sites, our statistic analysis can hardly lead to a conclusion because of the low positivity rate (3.8%) of RNS test applied to the common peroneal nerve and the lack of positive cases (n = 3). Thirdly, as can be seen from the Fig. 2b, some cases are found with RNS (+) and EMG (-). So some questions remain unanswered. What’s the proportion of these cases in ALS population? Is disease duration of these cases shorter than others? How is their diagnostic levels? To answer them, we’ll keep on collecting data and trying to illuminate these questions in future study.
In conclusion, this is a retrospective study based on 449 cases, with the largest sample size ever reported. It discovers that the overall positivity rate of RNS test is 49.9%. CMAP decremental response at 3Hz RNS applied to the accessory nerve has the best sensitivity and highest positivity rate (52.5% in definite ALS). It obviously increases in response to upper limb onset, ΔFS<0.5 score/month, definite ALS and EMG(+) in sternocleidomastoid muscle. Perfect negative linear correlations exist between either disease duration or MUP duration and decremental percentage of CMAP amplitude. The regression models indicate that the neuromuscular junction (NMJ) dysfunction is deteriorating as the disease progresses; the more serious neurogenic damage is detected by EMG, the more obvious will be the decremental response. Cases which are found with RNS (+) and EMG (-) may enlighten the pathogenic mechanisms from the perspective of clinical neuroelectrophysiology.