Intraspinal microstimulation preferentially recruits fatigue-resistant muscle fibres and generates gradual force in rat

Abstract

Intraspinal microstimulation (ISMS), a novel rehabilitative therapy consisting of stimulation through fine, hair-like microwires targeted at the ventral spinal cord, has been proposed for restoring standing and walking following spinal cord injury. This study compared muscle recruitment characteristics of ISMS with those produced by peripheral nerve cuff stimulation (NCS). Thirty-three minutes of either ISMS or NCS at 1, 20 or 50 s(-1) and 1.2 x threshold (T) amplitude depleted glycogen from muscle fibres of vastus lateralis and rectus femoris. ISMS and NCS were also carried out at 20 s(-1) and 3.0T. Muscle serial sections were stained for glycogen and for myosin heavy chain (MHC)-based fibre types using a panel of monoclonal antibodies. The results of this study show that ISMS recruited fatigue-resistant (FR) fibres at 2.9, 1.9, 1.7 and 2.5 times their relative MHC content at 1, 20 and 50 s(-1) 1.2T and 20 s(-1) 3.0T, respectively. In contrast, NCS recruited FR fibres at 1.2, 1.0, 2.1 and 0.0 times their MHC content at 1, 20 and 50 s(-1) 1.2T and 20 s(-1) 3.0T, respectively. The proportion of FR fibres recruited by ISMS and NCS was significantly different in the 20 s(-1) 3.0T condition (P < 0.0001). We also report that force recruitment curves were 4.9-fold less steep (P < 0.019) for ISMS than NCS. The findings of this study provide evidence for the efficacy of ISMS and further our understanding of muscle recruitment properties of this novel rehabilitative therapy.

Figure 1. ISMS electrode locations

Frozen spinal cords were sectioned on a cryostat and representative photomicrographs (A) were taken in order to verify electrode placement within the ventral grey matter. A composite schematic for electrode tip locations (B) was generated by plotting 17 ISMS electrodes used in this study.

Figure 2. Glycogen depletion protocol

Representative photomicrographs of histological and immunohistochemical stains were used to identify glycogen content (periodic acid Schiff's) (A), type-I fibres (anti-MHCI) (B), type-IIA fibres (anti-MHCIIa) (C) and type-IIB fibres (anti-MHCIIb) (D). The scale bar in D applies to panels A–D. Control stains for glycogen (E) were performed on sham control hindlimb muscle in order to verify that neither the ischaemia nor the extraction procedure produced glycogen depletion. Fibres were identified by their reaction with the corresponding MHC antibody, while type-IID/X fibres were identified by the absence of immunohistochemical staining. Although the IIA fibre marked with an asterisk in A appears depleted to the naked eye, it fell just below the threshold for detection according to computer analysis.

Figure 3. Glycogen depletion results

Graphs show MHC type-I, -I/IIA, -IIA, -IID/X and -IIB fibres depleted by either NCS or ISMS as a proportion of the total fibres depleted in 1 s⁻¹ 1.2T (A), 20 s⁻¹ 1.2T (B), 50 s⁻¹ 1.2T (C) and 20 s⁻¹ 3.0T (D) conditions. Six animals were used for each stimulation condition.

Figure 4. Fatigue-resistant fibres depleted in each condition

Proportion of glycogen-depleted fibres that were FR type (MHC type-I, -I/IIA and -IIA) in each experimental group (n = 6) by either NCS or ISMS. Results are shown as means ± s.e.m. for both 1.2T and 3.0T. Statistical significance was set at P < 0.05.

Figure 5. Depletion of fatigue-resistant fibres as a function of myosin heavy chain content

Relative MHC isoform content in 8 rectus femoris and 8 vastus lateralis muscles from 8 different animals as determined by one-dimensional electrophoresis. A, representative gel blot of rectus femoris and vastus lateralis muscles with type-IIa, -IId/x, -IIb and -I MHC bands delineated. B, bar-plot showing results of SDS-PAGE for MHC isoform content in rectus femoris (RF) and vastus lateralis (VL) muscles. C, after grouping MHC isoform content results into FR and FF categories the total number of FR fibres depleted by each condition was divided by the FR MHC content yielding fold differences in the recruitment of FR fibre types. Note that statistical significance in B was set at P < 0.05.

Figure 6. Peak twitch force results

Force recruitment curves produced by either NCS of the femoral nerve (A) or ISMS in the quadriceps motoneurone pool in the ventral grey matter (B). Force is normalized to the peak twitch force produced by the nerve cuff in each hindlimb and is expressed as a function of pulse amplitude in microamps. The same symbols in A and B indicate that the NCS and ISMS curves were obtained from the same animals.