Figure 1｜Diagrams and maps of the stimulating electrode.
Figure 2｜Scanning electron microscopy analysis of the electrode.
The size of the spinal cord of the rats was measured for electrode design after the operation. The designed length and width of two high-density flexible electrode arrays (Figures 1 and 2) were 5.8 × 2.2 mm2. The electrode arrays shown in Figure 1A and 1B were 9 × 3 and 9 × 2, respectively. Because of the limitation of the electrode technology, we could not realize 9 × 5 array in this area. The 9 × 2 array can fill the unused space in the 9 × 3 array.
Figure 3｜Schematic diagram of the experimental process.
Charge-balanced biphasic current stimulation signals were generated by a Master 9 multi-channel programmable stimulator (AMPI, Jerusalem, Israel) and two ISO-Flex stimulus isolation units (AMPI). The current amplitude of the negative pulse was five times greater than the amplitude of the positive pulse. Because the threshold of SCS at the cathode was lower than that at the anode (Holsheimer et al., 2002; Lavrov et al., 2008), the stimulation effect was mainly the effect of the negative pulse within the first phase. The smaller current of the positive pulse was unable to activate the nerves, and did not interfere with the experimental results. To keep the charge balance and protect the nerves, the width of the negative pulse (200 μs) was one fifth of that of the positive pulse. As shown in Figure 3, the cathode was moved sequentially to all electrode sites in the two arrays, and the anode was placed subcutaneously on the side of the rats. Because the threshold of the response to single-synaptic stimulation during repeated stimulation, the interval between stimulating pulses was at least 2 seconds. The stimulation signal was recorded using a 16-channel data acquisition system (AD Instruments, Bellavista, New South Wales, Australia).
Figure 4｜Regions of the muscles activated upon epidural stimulation of the spinal cord.
Figure 4 is the ESCSMFR map drawn from the experimental results of eight SCI rats. The lengths and widths of the vertebral and spinal-cord segments were averaged for the eight rats. In the map, the electrode is round for the sake of clarity. As shown in Figure 4, the ESCSMFRs of the VL were in the L1 vertebral level, whereas the ESCSMFRs of the MG were in the L2 vertebral level. This means that the L1 and L2 vertebral segments were the optimal stimulation regions for VL and MG, respectively.
Figure 5｜Stimulation-response heat maps in the regions activated by spinal-cord stimulation.
As illustrated in Figure 5, even in one ESCSMFR, the stimulation responses differed among different stimulation sites for a given current. The RMS of the yellow areas was higher (meaning the response to stimulation was stronger) than that of the blue areas. In the ESCSMFRs of the VL, the stimulation responses at the different sites were significantly different (P < 0.001). The stimulation responses in the lateral sites of the spinal cord were much stronger than those in the medial sites. By contrast, there was little difference among sites in the MG, with the stimulation responses in the medial sites of the spinal cord being slightly stronger than those in the lateral sites. In Figure 5, the highlighted sites in the heat map are those with the strongest stimulation response in the ESCSMFRs. These sites required the smallest stimulation current for a given response and the least unnecessary activation of spinal neuronal circuits. Therefore, stimulation in the highlighted areas was able to accurately control lower-limb movement.
Figure 7｜Stimulation responses as a function of stimulating current.
We presented the relationship between the stimulation current and the muscle responses as the RMS of the stimulation response, as described by Equation (1). The average and variance of the 10 stimulation responses at the selected sites in the eight SCI rats are shown in Figure 7. The responses of the muscles to stimulation increased with increasing current. When the current exceeded the maximum threshold, the rate of increase of the response with current substantially decreased, and other muscles (in particular, antagonist muscles) began to respond, which is an undesirable side effect. Thus, there are maximum and minimum current thresholds for effective stimulation.
Figure 8｜Stimulation response as a function of frequency.
As shown in Figure 8, the peak-to-peak intensity of the response increased with increasing stimulation frequency. Hence, the intensity of the response was enhanced by ESCS. The rate of change in the intensity decreased with increasing frequency. When the frequency was lower than 25 Hz, the lower-limb muscles of the rats experienced unfused tetanus; at frequencies above 25 Hz, tonic contraction occurred.
Figure 9｜Stimulation response of the left vastus lateralis of rats with spinal cord injury for different frequencies at a current of 380 μA.
The response of the left VL to stimulation in the rat model is shown in Figure 9. These results correspond with those of Figure 8A. Specifically, a higher stimulation frequency resulted in a shorter response interval. Moreover, the peak-to-peak intensity of the stimulation response for a constant current increased from 4 to 7 Hz as the frequency was increased from 20 to 80 Hz.