Low-energy extracorporeal shock wave therapy promotes vascular endothelial growth factor expression and improves locomotor recovery after spinal cord injury
Authors: Yamaya Seiji , Ozawa Hiroshi , Kanno Haruo , Kishimoto Koshi N. , Sekiguchi Akira , Tateda Satoshi , Yahata Kenichiro , Ito Kenta , Shimokawa Hiroaki , Itoi Eiji 
Published: October 2014

Object
Extracorporeal shock wave therapy (ESWT) is widely used for the clinical treatment of various human diseases. Recent studies have demonstrated that low-energy ESWT upregulates the expression of vascular endothelial growth factor (VEGF) and promotes angiogenesis and functional recovery in myocardial infarction and peripheral artery disease. Many previous reports suggested that VEGF produces a neuroprotective effect to reduce secondary neural tissue damage after spinal cord injury (SCI). The purpose of the present study was to investigate whether lowenergy ESWT promotes VEGF expression and neuroprotection and improves locomotor recovery after SCI.

Methods
Sixty adult female Sprague-Dawley rats were randomly divided into 4 groups: sham group (laminectomy only), sham-SW group (low-energy ESWT applied after laminectomy), SCI group (SCI only), and SCI-SW group (low-energy ESWT applied after SCI). Thoracic spinal cord contusion injury was inflicted using an impactor. Low-energy ESWT was applied to the injured spinal cord 3 times a week for 3 weeks. Locomotor function was evaluated using the Basso, Beattie, and Bresnahan (BBB) Scale (open field locomotor score) at different time points over 42 days after SCI. Hematoxylin and eosin staining was performed to assess neural tissue damage in the spinal cord. Neuronal loss was investigated by immunostaining for NeuN. The mRNA expressions of VEGF and its receptor, Flt-1, in the spinal cord were assessed using real-time polymerase chain reaction. Immunostaining for VEGF was performed to evaluate VEGF protein expression in the spinal cord.

Results
In both the sham and sham-SW groups, no animals showed locomotor impairment on BBB scoring. Histological analysis of H & E and NeuN stainings in the sham-SW group confirmed that no neural tissue damage was induced by the low-energy ESWT. Importantly, animals in the SCI-SW group demonstrated significantly better locomotor improvement than those in the SCI group at 7, 35, and 42 days after injury (p < 0.05). The number of NeuN-positive cells in the SCI-SW group was significantly higher than that in the SCI group at 42 days after injury (p < 0.05). In addition, mRNA expressions of VEGF and Flt-1 were significantly increased in the SCI-SW group compared with the SCI group at 7 days after injury (p < 0.05). The expression of VEGF protein in the SCI-SW group was significantly higher than that in the SCI group at 7 days (p < 0.01).

Conclusions
The present study showed that low-energy ESWT significantly increased expressions of VEGF and Flt-1 in the spinal cord without any detrimental effect. Furthermore, it significantly reduced neuronal loss in damaged neural tissue and improved locomotor function after SCI. These results suggested that low-energy ESWT enhances the neuroprotective effect of VEGF in reducing secondary injury and leads to better locomotor recovery following SCI. This study provides the first evidence that low-energy ESWT can be a safe and promising therapeutic strategy for SCI.