Abstract
Limb spasms are phenomena of hyperreflexia that occur after spinal cord injury. Currently, the clinical treatment is less than ideal. Our goal is to develop a combination therapy based on individualized medicine to reduce spasticity after spinal cord injury. In this study, rats received a severe contusive injury at the T9 segment of the spinal cord, followed by gene therapy with adenoassociated virus encoding human neurotrophic factor 3 (AAV-NT3) and a 2-week exercise program starting at 4 weeks after injury. We quantified the frequency of spasms during a swimming test at 4 and 6 weeks after injury and confirmed the results of the swimming test by measuring the H-reflex of the plantar muscle. We obtained weekly hind limb exercise scores to assess the effect of the interventions in hind limb motor function improvement. Then, we used immunofluorescence to observe the immunoreactivity of spinal motor neurons, synaptophysin, cholinergic interneurons, and GABAergic interneurons. We also measured the expression of KCC2 in the spinal cord by western blot. We found that AAV-NT3 gene therapy, exercise, and combination therapy all attenuated the frequency of spasms in the swimming test conducted at 6 weeks after spinal cord injury and increased rate-dependent depression of H-reflex. Combination therapy was significantly superior to AAV-NT3 alone in protecting motor neurons. Recovery of KCC2 expression was significantly greater in rats treated with combination therapy than in the exercise group. Combination therapy was also significantly superior to individual therapies in remodeling spinal cord neurons. Our study shows that the combination of AAV-NT3 gene therapy and exercise can alleviate muscle spasm after spinal cord injury by altering the excitability of spinal interneurons and motor neurons. However, combination therapy did not show a significant additive effect, which needs to be improved by adjusting the combined strategy.
Highlights
Limb spasticity is one of the most common complications after spinal cord injury
We determined the level of NT-3 in the muscle and the corresponding spinal segment dorsal root ganglia (DRG) by western blot at 4 weeks after spinal cord injury (Figures 2(b) and 2(d))
In the group of rats in which adenoassociated virus encoding human neurotrophic factor 3 (AAV-NT3) encoding human NT-3 was injected into the gastrocnemius muscle, the level of human NT-3 was significantly increased in the ipsilateral muscle or ipsilateral DRG (p < 0 001; one-way analysis of variance (ANOVA) with the post hoc Bonferroni test; Figures 2(c) and 2(e))
Summary
Limb spasticity is one of the most common complications after spinal cord injury. It has been reported that 12%–37% of patients with acute spinal cord injury have spasms, and the incidence of limb spasms in patients with chronic spinal cord injury is 65%–78% [1]. The symptoms of spasticity include muscle hypertonia, hyperstimulation of the body, clonus, and muscle spasms accompanied with severe pain [2]. These symptoms are usually caused by peripheral stimulation, such as muscle stretching or tactile stimulation, resulting in an increased myoelectrical response in the skin. There is still no effective method to treat the cause of spasms, only to reduce spasm symptoms. We chose to focus on treatments that improve motor neuron and interneuron survival and function These include gene therapies that increase the expression of growth factors, as well as exercise, which improves motor and neuronal function. We investigated the effects of a combination of neurotrophin-3 (NT-3) treatment with exercise, to determine if it would be more efficacious than either therapy alone
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