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The evoked compound nerve action potential is shaped by the electrical pulse-width

The evoked compound nerve action potential is shaped by the electrical pulse-width DE GRUYTER Current Directions in Biomedical Engineering 2022;8(3): 37-40 Johannes Proksch, Jose Luis Vargas Luna*, Steffen Eickhoff, Winfried Mayr and Jonathan C. Jarvis The evoked compound nerve action potential is shaped by the electrical pulse-width https://doi.org/10.1515/cdbme-2022-2010 cance of the PW in practical applications is often underesti- mated. Grill and Mortimer [2] tested rectangular PW of 500, Abstract: Introduction: Despite its central role in medicine 100, 50, and 10 µs in computer simulations and validated the electrical stimulation (ES) is still limited by its selectivity. Dif- results with in-vitro and in-vivo measurements. They con- ferent reports did assess effects of different waveforms, inten- cluded that a PW below 100μs allows for more spatially selec- sities, and frequency on the activation threshold of nerve fibres tive activation of neurons and increases the threshold differ- with different diameters. We aimed to extend this knowledge ences between fibre types. Gorman and Mortimer [3] con- by investigating the effect of short monophasic rectangular cluded that PW below 10µs would increase the threshold dif- pulses (1, 2, 5, 10, 50, 100 and 200 μs) on the recruitment or- ferences even more, but suggested 10μs as a reasonable limit der. Methods: The sciatic nerve of rats was stimulated, and for neural stimulation [3]. On the other hand, longer PWs pro- the evoked compound nerve action potential (CNAP) meas- duce stronger muscle contractions and deeper penetration be- ured at two sites on the tibialis nerve, using epineural elec- low the skin [4]. trodes. Changes in delay, amplitude, and the shape of the This study aims to provide more in-depth knowledge of the CNAP were analyzed. Results: The amplitude and delay of effect of PW under 10µs and to compare them with more typ- the CNAP were significantly affected by the pulse-width ical durations in terms of their effects on the recruitment of (PW). The delay and duration of the compound nerve action nerve fibres. With this purpose, an exploratory proof of prin- potential increased with longer PW, while the amplitude de- ciple study in an in-vivo model was performed at the Depart- creased. Discussion: Found changes are likely caused by ment for Sport and Exercise Sciences of the John Moores Uni- changes in the time point of excitation of individual neuron versity in Liverpool. fibres, depending on electrical field strength and exposure time. This might be of particular interest when selecting PWs Methods for design and validation of stimulation patterns and analysis All experiments were carried out under strict adherence to the of experimental and clinical observations. Animals (Scientific Procedures) Act of 1986. The procedures were approved by the Home Office (PPL 40/3743) and were Keywords: electric stimulation (ES), compound nerve action conducted in four non-recovery experiments in adult Wistar potential (CNAP), pulse-width (PW), epineural electrodes rats. Anaesthesia was induced using 3% isoflurane in oxygen. To Introduction maintain stable, deep anaesthesia, the respiration rate was One of the fundamental principles of ES is the observation that monitored, and the isoflurane concentration was adjusted be- the excitation threshold of a nerve fibre is inversely propor- tween 1% and 2%. The body temperature was kept between tional to the PW. This was already described by Weiss and 37-38°C with an adjustable heat pad (E-Z Systems Corpora- Lapicque more than 100 years ago [1], and yet, the signifi- tion, Pennsylvania, USA), and the core temperature was mon- itored using a rectal temperature probe. 0.05 mg kg−1 of Bu- prenorphine (Temgesic, Indivior, Slough, UK) was adminis- tered intramuscularly in the contralateral leg for analgesia. For ______ stimulation, a 1.2mm diameter tripolar cuff electrode (Micro *Corresponding author: Jose Luis Vargas Luna: University Cuffe Tunnel, Cortec, Germany) was placed proximal to the Clinic for Physical Medicine, Rehabilitation and Occupational tibialis branch on the sciatic nerve. For monitoring, two Medicine, Medical University of Vienna, Austria, joseluis.vargasluna@gmail.com Johannes Proksch, Winfried 0.6mm diameter bipolar cuff electrodes (Micro Cuffe Tunnel, Mayr: University Clinic for Physical Medicine, Rehabilitation and Cortec, Germany) were placed on the tibialis nerve with a Occupational Medicine, Medical University of Vienna, Austria 1mm distance between them (see Figure 1). Steffen Eickhoff, Jonathan C. Jarvis: School of Sport and Exercise Science, Liverpool John Moores University, Liverpool, UK Open Access. © 2022 The Author(s), published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International Li- cense. 37 Stimulation pulses were generated at a resolution of 1MS/s us- normalised relative to the nearest control response. The nor- ing LabVIEW 2016 (National Instruments Corporation, Aus- malisation responses were checked over time to see if there tin, USA). Voltage-controlled stimulation was applied using was a time-dependent effect of the stimulation or the pro- the analogue output of a NI PCIe 6351 Data Acquisition Card longed anaesthesia. (National Instruments Corporation, Austin, Texas, USA) with The delay between the stimulation onset and P1 was calcu- 10V output range, 5mA output current drive, 20V/μs slew lated. For each subject and PW the difference between the ap- rate and 16bit DAC resolution. The stimulation artefact was pearance of P1 in the proximal and distal channel was calcu- minimised by applying the stimulation via tripolar cuff elec- lated. Three-way ANOVAs were conducted to evaluate the ef- trodes using the middle electrode as cathode and intercon- fect of stimulation amplitude, PW, and subject on the delay nected proximal and distal electrode surfaces as anodes. between stimulation and P1, the time difference between the Stimulation sets with PWs of 100, 50, 20, 10, 5, 2, and 1µs appearance of P1 in the proximal and distal channels (propor- were tested. All pulses were monophasic. After an initial trial tional to the conduction velocity), as well as on the duration with 50 repetitions per duration, the subsequent assessments and amplitude of the CNAP. The duration of the CNAP was were done with 100 repetitions to increase the resolution. Am- defined as the time difference between P1 and P2. (a) (b) plitudes ranged from sub-threshold to full nerve activation. The threshold and supramaximal intensity for each set and subject were determined by single test stimulations. At least 40 or 85 different intensities were then tested within the se- lected range. The different stimulation PW and amplitudes were applied in a randomised order. The data were normalised by applying a supramaximal pulse – monophasic with 100μs th PW – every 20 stimulation. The electroneurogram (ENG) Figure 2: (a) Example of a measured response with the marked signals were recorded using a PowerLab 16/35 (ADINSTRU- points of interest (P1, N1, and P2) in the proximal and distal cuff MENTS Ltd, New Zealand) acquisition unit controlled with electrode (stimulation parameters: PW=100μs; Ampli- tude=1.289mV) (b) Normalized recruitment curve of the proximal LabChart (ADINSTRUMENTS Ltd, New Zealand) at a sam- cuff of the CNAP responses of a single subject for different PW. pling frequency of 200kHz. The black dotted line visualizes that for this subject PW 20μs and 5μs saturate at nearly at the same level Results The delay of the neural responses was between 280µs and 535µs (𝑥 ̅=41438µs n=2114), after the stimulation and lasted between 140µs and 570µs (𝑥 ̅=36974µs n=2114) depending on the subject, PW, and stimulation amplitude. Both the mean Figure 1: Measurement setup with the tripolar stimulation cuff at the sciatic nerve and the two bipolar measurement cuffs at the tib- duration of the CNAP and the mean delay of the response de- ial nerve. creased with decreasing PW. The latencies of P1, N1 and P2 were always longer in the distal channel (see example in Fig- The ENG was differentially amplified, using the proximal ure 2a). electrode as anode and the distal electrode of the same cuff as Delays between stimulation onset and P1 got shorter with de- cathode for both recording cuffs. Shielded copper wire cables creasing PW. However, this effect becomes undetectable for with two conductors were used to connect the cuff electrodes PW smaller or equal to 10µs. Furthermore, this effect de- to the before mentioned amplifier for the ENG recording. All creases with higher recruitment (supra-threshold intensity). shields were connected to a metallic rectal probe which served The calculated conduction speed varied between ~35 and as reference ground for the differential amplifier. ~87m/s. The intersubject variability accounted for most of the Data processing, analyses, and visualisation were done using difference in the speed. This might be due to physiological dif- MATLAB R2019b (The Mathworks, Inc., US). ferences and differences in the distance between the elec- Three points of interest (POI), P1, N1, and P2, according to trodes. Due to poor detection of P1 in the distal cuff within one the expected waveform of an evoked ENG response described subject, the data of this subject was not used to calculated con- by Parker [5], were determined in the recorded traces (see Fig- duction speed. The influence of the PW and of the stimulation ure 2a). amplitde were not significant. On the other hand, the delay of The response amplitude was defined as the difference between P1 was significantly influenced by the PW and the subject, but P1 and N1, similar definitions can be found in [5], and it was 38 not by the stimulation amplitude (see Table 1). The normalised All recorded ENGs in the 4 subjects in the proximal as well as CNAP amplitude and its duration was significantly affected by the distal channel had a similar shape, consistent with pub- all three factors (see Table 1). lished results on CNAP shape and the descriptive parameters amplitude, latency, and duration [5-7]. Assessed conduction velocities were within the expected range for sciatic nerves of Table 1: Three-way ANOVAs conducted on the effect of PW, am- rats [8]. Differences in delay of evoked CNAPs, attributed to plitude and subject on P1 conduction velocity and latency, and the variations in anatomical distances were verified and consid- CNAP amplitude. DF=degrees of freedom. * DF=2 ered in calculations and interpretations. PW stim. amplitude Subject DF=3 Since neither the PW nor the stimulus amplitude affected the DF= 6 DF=1 conduction velocity, we assume that differences in the delay of P1 are mainly due to changes in the time point of excitation Conduction P1 F=0.23 F=1.48 F=6839.39 Error=1378 p=0.97 p=0.22 p<<0.01* between different PW. Hence, the duration of the CNAP, cal- culated via the time difference of P1 and P2 in the proximal delay P1 F=379.97 F=0.15 F=4614.11 Error= 2096 p<<0.01 p=0.70 p<<0.01 channel, and the time difference between P1 and N1, are com- CNAP amplitude F=276.81 F=1188.52 F=25.83 parable across subjects. Error= 2103 p<<0.01 p<<0.01 p<<0.01 The shape of a CNAP is influenced by several factors, as it is a projection of multiple single fibre action potentials (APs) Longer PWs tend to cause a longer delay of P1. With higher with different conduction speed and sagittal distance to the re- recruitment, the duration of the CNAP response increased for cording electrode. The longer the distance from starting point all subjects. to recording site the broader the variance of arrival time of Figure 2b shows the normalised recruitment curve (RC) for all contributing APs, with immediate consequences for P1-la- different stimulation PW in one subject. As results were simi- tency, -amplitude, and the CNAP duration. Another influence lar in both distal and proximal positions, only the normalised on CNAP shape is contributed by the range of distances from CNAP amplitudes from the proximal cuff are reported. In both individual fibres to the recording electrode. An additional fac- cases, the response amplitude of the control pulses did not tor is variation in the time from the stimulus leading edge to change significantly with time, showing that there was no the start of a propagating AP, which depends on fibre size and time-dependent bias of the results. local field strength (see Figure 3b). Stimulation amplitude ranges for each PW to reach certain re- The saturation level in Figure 2b shows a gradual increase of sponse levels are remarkably similar in all subjects. The satu- maximum CNAP amplitude with increasing PWs. This might ration of longer PW is steeper compared with shorter PW. This originate from exciting less small unmyelinated and distant fi- can be seen in a high asymmetry of the recruitment curve, es- bres with lower PW due to extremely increased amplitude pecially for longer PW, caused by a more linear rise, after the threshold. recruitment starts to saturate. Hence, PW do seem to finally Figure 2b also illustrates a significant effect of PW on fibre saturate at nearly the same level (see black dotted line in Fig- threshold and gradient of the RC slope; shortening of PW ure 2b). However, none of the responses reaches 100%. This strongly increases threshold amplitude. Although a similar re- is due to supramaximal stimulation used as normalisation lation can be found in classical strength duration curves, ob- pulses. The stimulation step size increases with shorter PW, as tained via sensory perception feedback or neuromuscular re- the difference between below threshold to "full activation" in- actions [9], the results from direct neural response recording creases with decreasing PW. suggest that more pronounced changes in selectivity neuron types occurs with lower PWs. Discussion For a certain pulse amplitude, an “excitation window” EW can Estimation of recruitment of neurons of different type, size and be defined, showing the range of pulse durations for which, conduction velocity in a mixed nerve by applying specific this amplitude will excite different quality fibres. It starts at electrical stimuli is a complex task and, despite the long his- the threshold of the most sensitive, nearest-to-electrode, large tory of electrical stimulation, not resolved to a sufficient ex- axons and extends to the threshold of the most distant, small tent. Here we present an in-vivo model with cuff electrodes size and/or non-myelinated axons. Two examples are labeled placed along a rat’s sciatic nerve for stimulation and ENG-re- in red and blue (red with 2 different amplitude levels) in the cording, to explore the influence of unusually short PW (1 to threshold curves in Figure 3b. 100µs) on neuron recruitment characteristic by amplitude var- Within an EW a stimulus of a certain length and amplitude iation, in 4 animal subjects. activates action potentials in each reachable neuron. Taking 39 this into account, the observed differences in recruitment and in the CNAP shape can provide additional meaningful infor- CNAP shape, elicited by variation of PW, are most likely not mation for better estimation of recruited neurons in a specific only due to size-dependent fibre involvement, but also by a setup and parameter set. Here we demonstrated that latency distance-to-electrode component. This is probably a specific and duration of the response complement information based feature of epineural electrode placements. Although not eval- on changes in amplitude. The findings are most relevant for uated here, we expect that the distance-to-electrode influence electrodes directly attached to the epineurium. Other more dis- gradually disappears with greater distance. tant electrode configurations require specific studies for better A further influence on CNAP is associated with asynchrony of understanding of anatomical and physiological interaction arrival of contributing single fibre APs, elicited in synchrony with artificially induced ES fields. but traveling with different velocities. This effect is also spe- As suitable methods for selective in-vivo activation of single cific for epineural electrodes and gets more diffused with neurons and recording from single nerve fibres are not in sight larger neuron-to electrode-distance and higher desynchroniza- for the foreseeable future, critical analysis of bio signal record- tion of fibre APs. ings in meticulously target-oriented experimental setups seem currently most promising approaches for gaining more de- tailed insight in mechanisms of ES of neural structures. Author Statement Research funding: The author state that this work was funded by MED-EL Elektro-medizinische Geräte GmbH, Innsbruck, Austria. Authors state no conflict of interest. References [1] Grill, W.M., Nerve Stimulation, in Wiley Encyclopedia of Bio- medical Engineering, M. Akay, Editor. 2006, John Wiley & Sons, Inc: New Jersey. Figure 3: (a) 5: Illustration of strength-duration curves with [2] Grill, W.M. and J.T. Mortimer, The effect of stimulus pulse thresholds for different types of nerve fibres (Ia, Ib, II & III). Two duration on selectivity of neural stimulation. IEEE Trans Bio- different stimulation PW (blue 10μs and orange 50μs) have differ- med Eng, 1996. 43(2): p. 161-6. ent windows of excitation (EW). (b) Exemplary recording of re- [3] Gorman, P.H. and J.T. Mortimer, The Effect of Stimulus Pa- sponses for two different PW in the proximal cuff electrode. rameters on the Recruitment Characteristics of Direct Nerve Stimulation. IEEE Transactions on Biomedical Engineering, Figure 3a represents strength-duration curves for different fi- 1983. BME-30(7): p. 407-414. bre types and helps to identify the excitation windows of PW [4] Milosevic, M., et al., Why brain-controlled neuroprosthetics different intensities (EW20, EW100). The activation window matter: mechanisms underlying electrical stimulation of mus- is defined as the time where the first fibre is triggered (e.g. cles and nerves in rehabilitation. Biomedical engineering online, 2020. 19(1): p. 81-81. fibre Ia) until the last fibre is activated (Fibre III), and it is im- [5] Parker, J.L., et al., Evoked Compound Action Potentials Re- plied in the strength-duration curve described by Lapique veal Spinal Cord Dorsal Column Neuroanatomy. Neuromod- more than a century ago. ulation, 2020. 23(1): p. 82-95. The presence of the excitation window can already be seen in [6] Chapin, J. and K. Moxon, How to Use Nerve Cuffs to Stimu- late, Record or Modulate Neural Activity, in Neural Prosthe- strength-duration behaviour for different sized fibres (see Fig- ses for Restoration of Sensory and Motor Function. 2000, ure 3a). Higher amplitudes (blue rectangle) decrease the delay CRC Press: Boca Raton. between stimulation stimulus onset and threshold (in this ex- [7] Sabetian, P., M.R. Popovic, and P.B. Yoo, Optimising the de- ample Ia fibres) and decrease the length of the excitation win- sign of bipolar nerve cuff electrodes for improved recording dow (e.g. time between reaching the threshold for fibres type of peripheral nerve activity. J Neural Eng, 2017. 14(3): p. Ia and III). Higher synchronisation leads to an additional in- [8] Birren, J.E. and P.D. Wall, Age changes in conduction veloc- crease of CNAP amplitude. ity, refractory period, number of fibers, connective tissue In conclusion, CNAP amplitude alone is not specific enough space and blood vessels in sciatic nerve of rats. J Comp to reliably infer the recruited fibre pool when responses to dif- Neurol, 1956. 104(1): p. 1-16. [9] Tyler, D.J., Peripheral Nerve Stimulation, in Neuroprosthet- ferent stimulus parameters are investigated. Other parameters ics, K. Horch and D. Kipke, Editors. 2017. p. 300-347. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Current Directions in Biomedical Engineering de Gruyter

The evoked compound nerve action potential is shaped by the electrical pulse-width

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DE GRUYTER Current Directions in Biomedical Engineering 2022;8(3): 37-40 Johannes Proksch, Jose Luis Vargas Luna*, Steffen Eickhoff, Winfried Mayr and Jonathan C. Jarvis The evoked compound nerve action potential is shaped by the electrical pulse-width https://doi.org/10.1515/cdbme-2022-2010 cance of the PW in practical applications is often underesti- mated. Grill and Mortimer [2] tested rectangular PW of 500, Abstract: Introduction: Despite its central role in medicine 100, 50, and 10 µs in computer simulations and validated the electrical stimulation (ES) is still limited by its selectivity. Dif- results with in-vitro and in-vivo measurements. They con- ferent reports did assess effects of different waveforms, inten- cluded that a PW below 100μs allows for more spatially selec- sities, and frequency on the activation threshold of nerve fibres tive activation of neurons and increases the threshold differ- with different diameters. We aimed to extend this knowledge ences between fibre types. Gorman and Mortimer [3] con- by investigating the effect of short monophasic rectangular cluded that PW below 10µs would increase the threshold dif- pulses (1, 2, 5, 10, 50, 100 and 200 μs) on the recruitment or- ferences even more, but suggested 10μs as a reasonable limit der. Methods: The sciatic nerve of rats was stimulated, and for neural stimulation [3]. On the other hand, longer PWs pro- the evoked compound nerve action potential (CNAP) meas- duce stronger muscle contractions and deeper penetration be- ured at two sites on the tibialis nerve, using epineural elec- low the skin [4]. trodes. Changes in delay, amplitude, and the shape of the This study aims to provide more in-depth knowledge of the CNAP were analyzed. Results: The amplitude and delay of effect of PW under 10µs and to compare them with more typ- the CNAP were significantly affected by the pulse-width ical durations in terms of their effects on the recruitment of (PW). The delay and duration of the compound nerve action nerve fibres. With this purpose, an exploratory proof of prin- potential increased with longer PW, while the amplitude de- ciple study in an in-vivo model was performed at the Depart- creased. Discussion: Found changes are likely caused by ment for Sport and Exercise Sciences of the John Moores Uni- changes in the time point of excitation of individual neuron versity in Liverpool. fibres, depending on electrical field strength and exposure time. This might be of particular interest when selecting PWs Methods for design and validation of stimulation patterns and analysis All experiments were carried out under strict adherence to the of experimental and clinical observations. Animals (Scientific Procedures) Act of 1986. The procedures were approved by the Home Office (PPL 40/3743) and were Keywords: electric stimulation (ES), compound nerve action conducted in four non-recovery experiments in adult Wistar potential (CNAP), pulse-width (PW), epineural electrodes rats. Anaesthesia was induced using 3% isoflurane in oxygen. To Introduction maintain stable, deep anaesthesia, the respiration rate was One of the fundamental principles of ES is the observation that monitored, and the isoflurane concentration was adjusted be- the excitation threshold of a nerve fibre is inversely propor- tween 1% and 2%. The body temperature was kept between tional to the PW. This was already described by Weiss and 37-38°C with an adjustable heat pad (E-Z Systems Corpora- Lapicque more than 100 years ago [1], and yet, the signifi- tion, Pennsylvania, USA), and the core temperature was mon- itored using a rectal temperature probe. 0.05 mg kg−1 of Bu- prenorphine (Temgesic, Indivior, Slough, UK) was adminis- tered intramuscularly in the contralateral leg for analgesia. For ______ stimulation, a 1.2mm diameter tripolar cuff electrode (Micro *Corresponding author: Jose Luis Vargas Luna: University Cuffe Tunnel, Cortec, Germany) was placed proximal to the Clinic for Physical Medicine, Rehabilitation and Occupational tibialis branch on the sciatic nerve. For monitoring, two Medicine, Medical University of Vienna, Austria, joseluis.vargasluna@gmail.com Johannes Proksch, Winfried 0.6mm diameter bipolar cuff electrodes (Micro Cuffe Tunnel, Mayr: University Clinic for Physical Medicine, Rehabilitation and Cortec, Germany) were placed on the tibialis nerve with a Occupational Medicine, Medical University of Vienna, Austria 1mm distance between them (see Figure 1). Steffen Eickhoff, Jonathan C. Jarvis: School of Sport and Exercise Science, Liverpool John Moores University, Liverpool, UK Open Access. © 2022 The Author(s), published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International Li- cense. 37 Stimulation pulses were generated at a resolution of 1MS/s us- normalised relative to the nearest control response. The nor- ing LabVIEW 2016 (National Instruments Corporation, Aus- malisation responses were checked over time to see if there tin, USA). Voltage-controlled stimulation was applied using was a time-dependent effect of the stimulation or the pro- the analogue output of a NI PCIe 6351 Data Acquisition Card longed anaesthesia. (National Instruments Corporation, Austin, Texas, USA) with The delay between the stimulation onset and P1 was calcu- 10V output range, 5mA output current drive, 20V/μs slew lated. For each subject and PW the difference between the ap- rate and 16bit DAC resolution. The stimulation artefact was pearance of P1 in the proximal and distal channel was calcu- minimised by applying the stimulation via tripolar cuff elec- lated. Three-way ANOVAs were conducted to evaluate the ef- trodes using the middle electrode as cathode and intercon- fect of stimulation amplitude, PW, and subject on the delay nected proximal and distal electrode surfaces as anodes. between stimulation and P1, the time difference between the Stimulation sets with PWs of 100, 50, 20, 10, 5, 2, and 1µs appearance of P1 in the proximal and distal channels (propor- were tested. All pulses were monophasic. After an initial trial tional to the conduction velocity), as well as on the duration with 50 repetitions per duration, the subsequent assessments and amplitude of the CNAP. The duration of the CNAP was were done with 100 repetitions to increase the resolution. Am- defined as the time difference between P1 and P2. (a) (b) plitudes ranged from sub-threshold to full nerve activation. The threshold and supramaximal intensity for each set and subject were determined by single test stimulations. At least 40 or 85 different intensities were then tested within the se- lected range. The different stimulation PW and amplitudes were applied in a randomised order. The data were normalised by applying a supramaximal pulse – monophasic with 100μs th PW – every 20 stimulation. The electroneurogram (ENG) Figure 2: (a) Example of a measured response with the marked signals were recorded using a PowerLab 16/35 (ADINSTRU- points of interest (P1, N1, and P2) in the proximal and distal cuff MENTS Ltd, New Zealand) acquisition unit controlled with electrode (stimulation parameters: PW=100μs; Ampli- tude=1.289mV) (b) Normalized recruitment curve of the proximal LabChart (ADINSTRUMENTS Ltd, New Zealand) at a sam- cuff of the CNAP responses of a single subject for different PW. pling frequency of 200kHz. The black dotted line visualizes that for this subject PW 20μs and 5μs saturate at nearly at the same level Results The delay of the neural responses was between 280µs and 535µs (𝑥 ̅=41438µs n=2114), after the stimulation and lasted between 140µs and 570µs (𝑥 ̅=36974µs n=2114) depending on the subject, PW, and stimulation amplitude. Both the mean Figure 1: Measurement setup with the tripolar stimulation cuff at the sciatic nerve and the two bipolar measurement cuffs at the tib- duration of the CNAP and the mean delay of the response de- ial nerve. creased with decreasing PW. The latencies of P1, N1 and P2 were always longer in the distal channel (see example in Fig- The ENG was differentially amplified, using the proximal ure 2a). electrode as anode and the distal electrode of the same cuff as Delays between stimulation onset and P1 got shorter with de- cathode for both recording cuffs. Shielded copper wire cables creasing PW. However, this effect becomes undetectable for with two conductors were used to connect the cuff electrodes PW smaller or equal to 10µs. Furthermore, this effect de- to the before mentioned amplifier for the ENG recording. All creases with higher recruitment (supra-threshold intensity). shields were connected to a metallic rectal probe which served The calculated conduction speed varied between ~35 and as reference ground for the differential amplifier. ~87m/s. The intersubject variability accounted for most of the Data processing, analyses, and visualisation were done using difference in the speed. This might be due to physiological dif- MATLAB R2019b (The Mathworks, Inc., US). ferences and differences in the distance between the elec- Three points of interest (POI), P1, N1, and P2, according to trodes. Due to poor detection of P1 in the distal cuff within one the expected waveform of an evoked ENG response described subject, the data of this subject was not used to calculated con- by Parker [5], were determined in the recorded traces (see Fig- duction speed. The influence of the PW and of the stimulation ure 2a). amplitde were not significant. On the other hand, the delay of The response amplitude was defined as the difference between P1 was significantly influenced by the PW and the subject, but P1 and N1, similar definitions can be found in [5], and it was 38 not by the stimulation amplitude (see Table 1). The normalised All recorded ENGs in the 4 subjects in the proximal as well as CNAP amplitude and its duration was significantly affected by the distal channel had a similar shape, consistent with pub- all three factors (see Table 1). lished results on CNAP shape and the descriptive parameters amplitude, latency, and duration [5-7]. Assessed conduction velocities were within the expected range for sciatic nerves of Table 1: Three-way ANOVAs conducted on the effect of PW, am- rats [8]. Differences in delay of evoked CNAPs, attributed to plitude and subject on P1 conduction velocity and latency, and the variations in anatomical distances were verified and consid- CNAP amplitude. DF=degrees of freedom. * DF=2 ered in calculations and interpretations. PW stim. amplitude Subject DF=3 Since neither the PW nor the stimulus amplitude affected the DF= 6 DF=1 conduction velocity, we assume that differences in the delay of P1 are mainly due to changes in the time point of excitation Conduction P1 F=0.23 F=1.48 F=6839.39 Error=1378 p=0.97 p=0.22 p<<0.01* between different PW. Hence, the duration of the CNAP, cal- culated via the time difference of P1 and P2 in the proximal delay P1 F=379.97 F=0.15 F=4614.11 Error= 2096 p<<0.01 p=0.70 p<<0.01 channel, and the time difference between P1 and N1, are com- CNAP amplitude F=276.81 F=1188.52 F=25.83 parable across subjects. Error= 2103 p<<0.01 p<<0.01 p<<0.01 The shape of a CNAP is influenced by several factors, as it is a projection of multiple single fibre action potentials (APs) Longer PWs tend to cause a longer delay of P1. With higher with different conduction speed and sagittal distance to the re- recruitment, the duration of the CNAP response increased for cording electrode. The longer the distance from starting point all subjects. to recording site the broader the variance of arrival time of Figure 2b shows the normalised recruitment curve (RC) for all contributing APs, with immediate consequences for P1-la- different stimulation PW in one subject. As results were simi- tency, -amplitude, and the CNAP duration. Another influence lar in both distal and proximal positions, only the normalised on CNAP shape is contributed by the range of distances from CNAP amplitudes from the proximal cuff are reported. In both individual fibres to the recording electrode. An additional fac- cases, the response amplitude of the control pulses did not tor is variation in the time from the stimulus leading edge to change significantly with time, showing that there was no the start of a propagating AP, which depends on fibre size and time-dependent bias of the results. local field strength (see Figure 3b). Stimulation amplitude ranges for each PW to reach certain re- The saturation level in Figure 2b shows a gradual increase of sponse levels are remarkably similar in all subjects. The satu- maximum CNAP amplitude with increasing PWs. This might ration of longer PW is steeper compared with shorter PW. This originate from exciting less small unmyelinated and distant fi- can be seen in a high asymmetry of the recruitment curve, es- bres with lower PW due to extremely increased amplitude pecially for longer PW, caused by a more linear rise, after the threshold. recruitment starts to saturate. Hence, PW do seem to finally Figure 2b also illustrates a significant effect of PW on fibre saturate at nearly the same level (see black dotted line in Fig- threshold and gradient of the RC slope; shortening of PW ure 2b). However, none of the responses reaches 100%. This strongly increases threshold amplitude. Although a similar re- is due to supramaximal stimulation used as normalisation lation can be found in classical strength duration curves, ob- pulses. The stimulation step size increases with shorter PW, as tained via sensory perception feedback or neuromuscular re- the difference between below threshold to "full activation" in- actions [9], the results from direct neural response recording creases with decreasing PW. suggest that more pronounced changes in selectivity neuron types occurs with lower PWs. Discussion For a certain pulse amplitude, an “excitation window” EW can Estimation of recruitment of neurons of different type, size and be defined, showing the range of pulse durations for which, conduction velocity in a mixed nerve by applying specific this amplitude will excite different quality fibres. It starts at electrical stimuli is a complex task and, despite the long his- the threshold of the most sensitive, nearest-to-electrode, large tory of electrical stimulation, not resolved to a sufficient ex- axons and extends to the threshold of the most distant, small tent. Here we present an in-vivo model with cuff electrodes size and/or non-myelinated axons. Two examples are labeled placed along a rat’s sciatic nerve for stimulation and ENG-re- in red and blue (red with 2 different amplitude levels) in the cording, to explore the influence of unusually short PW (1 to threshold curves in Figure 3b. 100µs) on neuron recruitment characteristic by amplitude var- Within an EW a stimulus of a certain length and amplitude iation, in 4 animal subjects. activates action potentials in each reachable neuron. Taking 39 this into account, the observed differences in recruitment and in the CNAP shape can provide additional meaningful infor- CNAP shape, elicited by variation of PW, are most likely not mation for better estimation of recruited neurons in a specific only due to size-dependent fibre involvement, but also by a setup and parameter set. Here we demonstrated that latency distance-to-electrode component. This is probably a specific and duration of the response complement information based feature of epineural electrode placements. Although not eval- on changes in amplitude. The findings are most relevant for uated here, we expect that the distance-to-electrode influence electrodes directly attached to the epineurium. Other more dis- gradually disappears with greater distance. tant electrode configurations require specific studies for better A further influence on CNAP is associated with asynchrony of understanding of anatomical and physiological interaction arrival of contributing single fibre APs, elicited in synchrony with artificially induced ES fields. but traveling with different velocities. This effect is also spe- As suitable methods for selective in-vivo activation of single cific for epineural electrodes and gets more diffused with neurons and recording from single nerve fibres are not in sight larger neuron-to electrode-distance and higher desynchroniza- for the foreseeable future, critical analysis of bio signal record- tion of fibre APs. ings in meticulously target-oriented experimental setups seem currently most promising approaches for gaining more de- tailed insight in mechanisms of ES of neural structures. Author Statement Research funding: The author state that this work was funded by MED-EL Elektro-medizinische Geräte GmbH, Innsbruck, Austria. Authors state no conflict of interest. References [1] Grill, W.M., Nerve Stimulation, in Wiley Encyclopedia of Bio- medical Engineering, M. Akay, Editor. 2006, John Wiley & Sons, Inc: New Jersey. Figure 3: (a) 5: Illustration of strength-duration curves with [2] Grill, W.M. and J.T. Mortimer, The effect of stimulus pulse thresholds for different types of nerve fibres (Ia, Ib, II & III). Two duration on selectivity of neural stimulation. IEEE Trans Bio- different stimulation PW (blue 10μs and orange 50μs) have differ- med Eng, 1996. 43(2): p. 161-6. ent windows of excitation (EW). (b) Exemplary recording of re- [3] Gorman, P.H. and J.T. Mortimer, The Effect of Stimulus Pa- sponses for two different PW in the proximal cuff electrode. rameters on the Recruitment Characteristics of Direct Nerve Stimulation. IEEE Transactions on Biomedical Engineering, Figure 3a represents strength-duration curves for different fi- 1983. BME-30(7): p. 407-414. bre types and helps to identify the excitation windows of PW [4] Milosevic, M., et al., Why brain-controlled neuroprosthetics different intensities (EW20, EW100). The activation window matter: mechanisms underlying electrical stimulation of mus- is defined as the time where the first fibre is triggered (e.g. cles and nerves in rehabilitation. Biomedical engineering online, 2020. 19(1): p. 81-81. fibre Ia) until the last fibre is activated (Fibre III), and it is im- [5] Parker, J.L., et al., Evoked Compound Action Potentials Re- plied in the strength-duration curve described by Lapique veal Spinal Cord Dorsal Column Neuroanatomy. Neuromod- more than a century ago. ulation, 2020. 23(1): p. 82-95. The presence of the excitation window can already be seen in [6] Chapin, J. and K. Moxon, How to Use Nerve Cuffs to Stimu- late, Record or Modulate Neural Activity, in Neural Prosthe- strength-duration behaviour for different sized fibres (see Fig- ses for Restoration of Sensory and Motor Function. 2000, ure 3a). Higher amplitudes (blue rectangle) decrease the delay CRC Press: Boca Raton. between stimulation stimulus onset and threshold (in this ex- [7] Sabetian, P., M.R. Popovic, and P.B. Yoo, Optimising the de- ample Ia fibres) and decrease the length of the excitation win- sign of bipolar nerve cuff electrodes for improved recording dow (e.g. time between reaching the threshold for fibres type of peripheral nerve activity. J Neural Eng, 2017. 14(3): p. Ia and III). Higher synchronisation leads to an additional in- [8] Birren, J.E. and P.D. Wall, Age changes in conduction veloc- crease of CNAP amplitude. ity, refractory period, number of fibers, connective tissue In conclusion, CNAP amplitude alone is not specific enough space and blood vessels in sciatic nerve of rats. J Comp to reliably infer the recruited fibre pool when responses to dif- Neurol, 1956. 104(1): p. 1-16. [9] Tyler, D.J., Peripheral Nerve Stimulation, in Neuroprosthet- ferent stimulus parameters are investigated. Other parameters ics, K. Horch and D. Kipke, Editors. 2017. p. 300-347.

Journal

Current Directions in Biomedical Engineering – de Gruyter

Published: Sep 1, 2022

Keywords: electric stimulation (ES); compound nerve action potential (CNAP); pulse-width (PW); epineural electrodes

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