the compound muscle action potential ( cmap ) scan is a noninvasive diagnosis technique for neurodegenerative pathologies , such as amyotrophic lateral sclerosis ( als ) .
it allows a quick analysis of the muscle action potentials in response to motor nerve stimulation , by electrical stimulation applied on the surface of the motor nerve and response evaluation by surface electromyography ( semg ) at muscle level .
each motor unit ( mu ) of muscles has a different stimulus intensity ( si ) at which it is activated , meaning that mus have different thresholds .
varying the intensity of the stimuli applied , gradually increasing from subthreshold to supramaximal values , will sequentially activate all mus in the muscle .
this way , it is possible to obtain a graphical representation of the evoked action potentials amplitude in the muscle versus the stimulation intensity .
this record will show a sigmoid tendency which is called the cmap scan [ 13 ] .
henderson et al .   examined the variability of the cmap scan between healthy and als subjects .
evidences were found for a significant difference in relation to cmap scan evolution , number of steps ( visible jumps in cmap amplitude within consecutive stimuli ) , and size , since als patients present more and larger steps on the stimulus - response curve than healthy controls .
a healthy cmap scan has traditionally a sigmoid tendency curve for the motor unit action potential amplitude in response to a linear increase of current intensity .
this technique provides clinically relevant information about reinnervation processes , number and size of functional motor units , and neuromuscular activity / excitability .
moreover , cmap amplitude has significant correlation with muscle strength , motor unit number estimation , and functional disability in als . to be used as a clinical tool , stimulation parameters must be standardized and quantified in order to enable uniform collection and comparison of data .
several studies have been made recently to verify the potentiality of this technique , investigating the influence of different parameters in the quality of the cmap scan . a consensual way is based on the analysis of key points like the maximum cmap amplitude , s5 , s50 , and s95 ( the stimulus intensity that elicited 5% , 50% , and 95% of the maximum cmap amplitude , resp . ) , si range ( the difference between s95 and s5 ) , and step percentage , despite the fact that the physiological influence of different stimulation parameters remains unknown .
an untested parameter of the cmap is the waveform of the electrical pulse used in the nerve stimulation .
the waveform is a graphical representation over time of a signal , as it reflects its shape . in electrical stimulation ,
the waveform represents the variation over time of the applied current or voltage on the muscle or nerve
. it can be monophasic or biphasic and also have different shapes like sine and square , among others .
this work aims to study the influence of different pulse modulated waveforms in peripheral nerve excitability , by cmap scan technique , on healthy subjects for future comparison in als patients . to accomplish that , an electrical stimulation protocol was developed and biosignals from healthy subjects were acquired , analyzed , and processed , in order to extract features for the analysis of the different waveforms ' influence in the stimulation of the peripheral nerve .
the waveforms tested in the stimulation protocol were monophasic square , triangular , and quadratic waves , and also a biphasic square wave .
in this study a total of 13 healthy subjects were submitted to the same test .
this group was composed of 7 males and 6 females , mean age of 26.00 ( 3.63 ) years , range from 20 to 36 years , and without clinical history on neurologic disorders .
the stimuli were applied in the median nerve on the right wrist and emg signal was collected on the abductor pollicis brevis muscle surface on the right thumb .
self - adhesive pregelled ag / agcl , 30  24  mm disposable electrodes were attached to the skin for acquisition and stimulation and placed according to seniam   standards .
the ground electrode was placed on the left wrist ulnar styloid process . during the test ,
the subjects were seated , motionless and relaxed , with thumb fixation to minimize movement artifacts .  
different number of stimuli and current increment steps were tested in order to have a stimulation protocol that would allow obtaining a curve with enough resolution but not excessive test duration .
after extensive testing , it was perceived that only 3 stimuli per step were sufficient to have high resolution in the cmap . to optimize the relation between curve definition and test duration , the current increment steps have different values depending on the correspondent curve zone .
this way , a small step increment of current intensity is defined to increase the definition of the sigmoid curve only on the transition zones .
on the initial ( 4 to 6  ma ) and final ( 25 to 30  ma ) stages the current increment would be of 1  ma for each step . from 6 to 25 
ma the increment was 0.5  ma . to properly cancel noise and mechanical artifacts ,
the pulses were randomly distributed in frequencies from 1.8 to 2.2  hz . the emg signal was acquired with a 3000  hz sampling frequency , amplified with a gain of 201 and 12 bits of resolution . a combined wireless ,
miniaturized and synchronized unit was specifically developed for emg acquisition and nerve stimulation [ 7 , 8 ] .
the system was previously validated in comparison with a clinical system installed in hospital santa maria , lisbon .
the procedure was repeated 4 times per subject , each repetition using a different single pulse stimulation waveform .
a standard square wave was applied in test 1 , a triangular wave in test 2 , and a quadratic wave in test 3 . in all these 3 protocols ,
a 4th protocol was tested with a biphasic single pulse square wave , with the same intensities of the other tests . the current charge difference from each waveform
the stimulation charge was computed according to the stimulation intensity and waveform , based on the following formula : ( 
 1 
 ) q=t1t2i  dt , where i represent the current intensity and t1 to t2 is the stimulus time range .
the different single pulses total charge has been equalized for each current intensity , maintaining the amplitude and varying the pulse - width time . from the theory ,
the total charge of the electrical impulse is the most reliable measurement of the biological reaction to the electrical stimulation .
the collected biosignals were processed and features were extracted , according to the following steps : detecting peak - to - peak amplitude of the stimulus - response m-wave.cmap scan composition via interpolation.extracting s5 , s50 , s95 , and si range and stimulus - response amplitude elicited by these parameters.detecting the beginning ( sigmoid trigger intensity ) , final ( sigmoid plateau first intensity ) , and slope of the resulting sigmoid.computing the intersubject mean and standard deviation of the computed parameters.computing the differences in the computed parameters for each waveform , when comparing with the traditional square wave.these steps were repeated for each waveform type and all subjects .
all data had posterior visual validation by two specialists . detecting peak - to - peak amplitude of the stimulus - response m - wave .
extracting s5 , s50 , s95 , and si range and stimulus - response amplitude elicited by these parameters . detecting the beginning ( sigmoid trigger intensity ) ,
computing the differences in the computed parameters for each waveform , when comparing with the traditional square wave .
each subject was analyzed regarding the maximum cmap amplitudes , the excitability parameters ( s5 , s50 , and s95 , stimulus current intensity in ma that elicited correspondent response amplitude in mv ) , the sigmoid slope , and current intensity differences of the cmap scan , between each different waveform . 

figure 3(a ) presents a cmap wave elicited by the four different waveforms with the same pulse amplitude ( 10.5  ma ) .
differences in the responses ' peak - to - peak amplitude can be observed , using the same current intensity and charge .  
figure 3(b ) shows , for one example subject , four cmap scans generated with four waveforms . like in figure 3(a ) , the differences between the waveforms in the stimulation intensities to generate the same response amplitude can be observed . 

table 1 presents the mean cmap scan sigmoid slope differences between each waveform in comparison to the square wave .
the value is given in percentage , assuming that the square wave 's sigmoid slope is 100% . in table 2
the mean intensity differences regarding the stimulation parameters s5 , s50 , and s95 between each waveform and the square wave are presented . as the stimulation parameters increase in amplitude ( from s5 to s50 and s50 to s95 ) , the difference between each wave ( from square to triangular , triangular to quadratic , and quadratic to biphasic square ) also increases .
. however , there are also several constraints which can not be simulated , like the patient comfort to some type of stimulation , the nervous fiber fatigue associated with the habituation to the electrical pulse , or the possible changes of tissue impedance during stimulation ( increase of temperature , increase of skin sudation , microirritations to the tissue after consecutive stimulus application , etc . ) .
some effects are also impossible to contemplate in a theoretical model , such as the study in injured patients or with an early diagnosis for the disease .
peripheral nerve stimulation is influenced by external variables that are hard to control , like the adipose tissue layer that the electrical current has to pass , the distance between the stimulation electrodes and the nerve to be stimulated , among other body inhomogeneities .
taking this into consideration , the chosen analysis parameters were the ones that allowed a more objective assessment of the considered effects among different subjects .
the results show that the square pulse , besides needing less current intensity to generate the same response amplitude as the other waves , is also the one that presents a steeper curve slope .
this means that , for the square wave , the intensities range between the beginning and final of the cmap scan is usually shorter than for the other waves .
the quadratic wave , among the monophasic waves group , represents the stimulation pulse that needs a larger current intensity to elicit the same response amplitude in comparison with the other waves .
this happens possibly due to a nervous fiber sensibility to charge transfer rate , since in the used setup all single pulses have the total charge equalized . concerning the biphasic square pulse , it is possible to verify that it has a distinct behavior from the monophasic pulses , with activation intensities of the response levels s5 , s50 , and s95 quite superior and also a rather inferior sigmoid slope .
this indicates that , possibly , only one of the flanks of the biphasic waveform is activating the nerve fibers .
the monophasic waveforms have a more linear behavior , while the biphasic waveform presents a more unstable behavior with greater variations .
the analysis of the influence of the waveform on the peripheral nerve stimulation reveals new effects in the context of the nerves ' excitability .
electrophysiology studies reveal significant differences between healthy subjects and neurodegenerative patients . upon this , when a variable that is able to manipulate or change the sensibility of the cmap stimulus - response curve is introduced
, a question arises : will this variable have the same behavior when evaluating patients ?
is it possible that subjects with pathology or propensity to develop will show differences in the sensibility to the electrical charge ?
these are the relevant questions that this work starts to address , studying healthy subjects .
the evaluation of new wave parameterizations in healthy subjects , in addition to being a basis for comparison for future patients evaluation , also represents a contribute for research associated with the cmap scan methodology and a step forward on the understanding of the electrical pulse waveform influence on peripheral nerve excitability .
 
 introduction . compound muscle action potential ( cmap ) 
 scan is a noninvasive promissory technique for neurodegenerative pathologies diagnosis . in this work new cmap scan protocols 
 were implemented to study the influence of electrical pulse waveform on peripheral nerve excitability . 
 methods . a total of 13 healthy subjects were tested . 
 stimulation was performed with an increasing intensities range from 4 to 30  ma . 
 the procedure was repeated 4 times per subject , using a different single pulse stimulation waveform : monophasic square and triangular and quadratic and biphasic square . 
 results . 
 different waveforms elicit different intensity - response amplitude curves . 
 the square pulse needs less current to generate the same response amplitude regarding the other waves and this effect is gradually decreasing for the triangular , quadratic , and biphasic pulse , respectively . 
 conclusion . 
 the stimulation waveform has a direct influence on the stimulus - response slope and consequently on the motoneurons excitability . 
 this can be a new prognostic parameter for neurodegenerative disorders . 
1. Introduction
2. Methods
3. Results
4. Discussion