A preclinical model of non‐invasive stimulation reduces spinal cord injury initiated spasticity

Spasticity is a velocity-dependent increase in muscle tone caused by a hyperexcitable stretch reflex. The stretch reflex is monosynaptic and consists of two parts: (i) Ia afferents that wrap around intrafusal muscle fibres, depolarize when deformed (i.e. when the muscle is stretched) and synapse directly in the ventral horn with (ii) α-motoneurons that innervate the muscle. The Ia afferents will have either a direct excitatory effect or indirect inhibitory effect (through the activation of inhibitory interneurons that synapse with the α-motoneurons) depending on whether the α-motoneurons innervate either the same or the antagonizing muscle that is being extended. The net effect being that as a muscle is stretched a constant length is maintained. Spasticity is a prevalent secondary outcome following spinal cord injury (SCI), although the exact mechanism underpinning how a spinal lesion leads to an overexcitable stretch reflex has yet to be determined conclusively. Currently, the most common therapeutic used to treat SCI-induced spasticity is an orally administered GABAB agonist (baclofen), despite the prevalence of adverse side effects. The upshot is that an alternative therapy for treating SCI-induced spasticity without significant drawbacks is needed. Recently, Mekhael et al. (2019) published a study in the Journal of Physiology demonstrating the functional benefits of direct current spinal stimulation on SCI-induced spasticity in a preclinical animal model. Mice underwent a moderately severe (60 kilodyne) low-thoracic (T13) spinal cord contusion, which reliably produces a murine analogue of human spasticity. Mekhael et al. (2019) designed a unique apparatus that provides direct current stimulation from the spinal cord to the sciatic nerve (i.e. anodal trans spinal subthreshold stimulation) or vice versa (i.e. cathodal trans spinal subthreshold stimulation). Importantly, this apparatus allows for stimulation in awake animals and for the non-invasive measurement of muscle tone and electromyogram (EMG) data, thus making a repeated course of stimulation possible. Mice received either spinal-to-sciatic or sciatic-to-spinal stimulation for 20 min each day for 7 days and were compared with non-injured or sham-treated groups. In both muscle tone and EMG readouts, Mekhael et al. (2019) show that spinal-to-sciatic stimulation significantly reduces spasticity to near-uninjured levels for up to 4 weeks following injury. Alterations to locomotor patterns were observed in both modes of stimulation, although these results need to be interpreted carefully because it is not clear whether these changes in locomotor pattern necessarily correspond to a functional improvement. It is also interesting that the pretesting baseline appears to vary between the injured controls and the treatment groups. Using a motorized ladder wheel, it is shown that, at the 4 week timepoint, the left hindlimb had significantly improved fine motor skills in the spinal-to-sciatic stimulation group, although it is not obvious which leg was stimulated as part of the intervention. Taken together, these functional results suggest that a non-invasive spinal-to-sciatic stimulation significantly reduces spasticity after SCI, showing substantial translational promise. Several interesting and clinically relevant follow-up questions can be proposed, including would the same spasticity attenuation be observed with the stimulation delivered with a delay between the injury and therapy and would the reduction in spasticity be maintained at timepoints greater than 4 weeks? After muscle stimulation, two distinct electromyographic waveforms are observed: (i) the M-wave that is produced from the direct electrical excitation of the motoneuron and (ii) the H-wave that is produced by depolarizing afferents synapsing with the motoneurons in the ventral horn. In healthy conditions, as the frequency of stimulation is increased, the magnitude of the H-wave diminishes; this phenomenon is known as rate-dependent depression (RDD). A characteristic trait of spasticity is a reduced RDD (i.e. an increase of stimulation frequency does not diminish the H-wave). The spinal-to-sciatic stimulation used in the study remarkably restores the RDD of the H-reflex to mimic uninjured levels. This further bolsters the evidence provided that stimulation therapy attenuates SCI-induced spasticity. Baclofen has also been shown to restore RDD of the H-reflex in spastic mice (Lee et al. 2014). It would be of interest to compare the magnitude of effects with respect to spinal-to-sciatic stimulation to this common pharmacological intervention. Additionally, administering both pharmacological and stimulatory interventions to a single group could identify whether both anti-spastic treatments converge on the same mechanism or whether they have synergistic benefits. To determine the molecular mechanisms responsible for the reduction in spasticity, Mekhael et al. (2019) investigated the expression levels of two ion channels involved in maintaining intracellular Cl− concentration. The K+-Cl− (KCC2) and Na+-K+-Cl− (NKCC1) cotransporters have been implicated in producing motoneuronal hyperexcitability involved in the overexcitable stretch reflex. By taking whole sublesion spinal lysates, Mekhael et al. (2019) measured the gross transcript and protein levels of NKCC1 and KCC2. There were no significant differences in the mRNA levels of either transporters or of KCC2 protein levels following SCI or stimulation. Using a different experimental model but still assessing whole spinal lysates, Boulenguez et al. (2010) reported decreased KCC2 expression following SCI. To better understand how Cl− cotransporter expression contributes to SCI-induced spasticity, future experiments should aim to avoid the use of whole spinal lysates. Whole spinal lysates assess mRNA or protein expression in a large span of sublesion spinal cord tissue that may not directly contribute to the spinal nerve stretch reflex (which would be limited to the ventral motor pools in segments L4–L6). Additionally, a large number of cells, both neuronal and glial, that are not directly involved in the stretch reflex could differentially express the measured Cl− cotransporters following SCI, which would obfuscate the readout of total protein expression. A preferential technique for avoiding these issues would be to assess the expression of KCC2 and NKCC1 in the motoneurons that directly contribute to the sciatic stretch reflex by immunolabelling the cotransporters and quantifying fluorescence intensities between groups. Alternatively, laser-assisted microdissection could be employed to isolate the L4–L6 ventral motor pools followed by a western blot on the proteins of interest to more specifically investigate the relevant tissue area. Mekhael et al. (2019) go on to show that the SCI-induced upregulation and subsequent sciatic-to-spinal stimulation mediated downregulation of NKCC1 was the result of protein degradation probably dependent on HSP70. It would be of interest to mechanistically connect the effect of stimulation to the expression of the Cl− cotransporters. An interesting hypothesis involving neurotrophic factors potentially emerges to complete the story: contraction of the hindlimb, through exercise, elevates the transcription of the neurotrophin brain-derived neurotrophic factor (BDNF) in hindlimb muscles and protein levels in the ventral motor pools (Gomez-Pinilla et al. 2001) and exogenous BDNF delivery increases KCC2 expression and decreases NKCC1 expression (Eftekhari et al. 2014). Therefore, the spinal-to-sciatic stimulation used by Mekhael et al. (2019) could be inducing BDNF production, which then alters the expression of Cl− cotransporters, thus restoring normal excitability of the motoneurons and diminishing spasticity. Testing this hypothesis would involve spinal-to-sciatic stimulation following SCI in a BDNF conditional knockout line (Jax 004339) crossed with inducible Cre mice specific to muscles (Jax 025667) or motoneurons (Jax 008364). Should BDNF production and downstream signalling be necessary for the observed altered Cl− cotransporter expression levels, then the effect of the intervention would be abolished in the BDNF deficient mice. Mekhael et al. (2019) provide exciting and novel preclinical results on a readily translatable therapeutic intervention: spinal-to-sciatic trans spinal subthreshold direct current stimulation. The stimulation produces a long-term reduction in spasticity and improvement of locomotor function with a non-invasive and feasible intervention comprising a 20 min bout of trans spinal stimulation for 7 days immediately following SCI. An indication of the complete mechanism is provided by demonstrating a reduction in NKCC1 expression levels in stimulated whole spinal lysates mediated by HSP70 degradation. From this work, several interesting future research directions emerge with respect to the contribution of neurotrophic factors, in addition to the possibility that direct current stimulation could be beneficial in other hyperexcitable conditions, including spastic cerebral palsy, post-stroke spasticity and even neuropathic pain. No competing interests declared. Sole author. No funding.

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