Effect of exogenous spastin combined with polyethylene glycol on sciatic nerve injury

Abstract

Polyethylene glycol can connect the distal and proximal ends of an injured nerve at the cellular level through axonal fusion to avoid Wallerian degeneration of the injured distal nerve and promote peripheral nerve regeneration. However, this method can only prevent Wallerian degeneration in 10% of axons because the cytoskeleton is not repaired in a timely fashion. Reconstruction of the cytoskeletal trunk and microtubule network has been suggested to be the key for improving the efficiency of axonal fusion. As a microtubule-severing protein, spastin has been used to enhance cytoskeletal reconstruction. Therefore, we hypothesized that spastin combined with polyethylene glycol can more effectively promote peripheral nerve regeneration. A total of 120 male Sprague-Dawley rats were randomly divided into sham, suture, polyethylene glycol, and polyethylene glycol + spastin groups. In suture group rats, only traditional nerve anastomosis of the end-to-end suture was performed after transection of the sciatic nerve. In polyethylene glycol and polyethylene glycol + spastin groups, 50 μL of polyethylene glycol or 25 μL of polyethylene glycol + 25 μL of spastin, respectively, were injected immediately under the epineurium of the distal suture. Sensory fiber regeneration distance, which was used to assess early nerve regeneration at 1 week after surgery, was shortest in the suture group, followed by polyethylene glycol group and greatest in the polyethylene glycol + spastin group. Behavioral assessment of motor function recovery in rats showed that limb function was restored in polyethylene glycol and polyethylene glycol + spastin groups at 8 weeks after surgery. At 1, 2, 4 and 8 weeks after surgery, sciatic functional index values and percentages of gastrocnemius muscle wet weight were highest in the sham group, followed by polyethylene glycol + spastin and polyethylene glycol groups, and lowest in the suture group. Masson staining was utilized to assess the morphology of muscle tissue. Morphological changes in skeletal muscle were detectable in suture, polyethylene glycol, and polyethylene glycol + spastin groups at 1, 2, 4, and 8 weeks after surgery. Among them, muscular atrophy of the suture group was most serious, followed by polyethylene glycol and polyethylene glycol + spastin groups. Ultrastructure of distal sciatic nerve tissue, as detected by transmission electron microscopy, showed a pattern of initial destruction, subsequent disintegration, and gradual repair in suture, polyethylene glycol, and polyethylene glycol + spastin groups at 1, 2, 4, and 8 weeks after surgery. As time proceeded, axonal ultrastructure gradually recovered. Indeed, the polyethylene glycol + spastin group was similar to the sham group at 8 weeks after surgery. Our findings indicate that the combination of polyethylene glycol and spastin can promote peripheral nerve regeneration. Moreover, the effect of this combination was better than that of polyethylene glycol alone, and both were superior to the traditional neurorrhaphy. This study was approved by the Animal Ethics Committee of the Second Military Medical University, China (approval No. CZ20170216) on March 16, 2017.

Keywords: Masson staining; Wallerian degeneration; axonal fusion; microtubule; nerve regeneration; neural regeneration; peripheral nerve injuries; peripheral nerves; polyethylene glycol; spastin.

Figure 1

Rat footprint. IT: Inter-toe distance; TW: width between the first and fifth toes; PL: podogram length.

Figure 2

Conditions of rat hindlimbs 1 week after surgery. Red arrows indicate the hindlimb on the surgical side. Hindlimb function in the sham group was normal, with no paralysis or malformation. In suture, PEG, and PEG + spastin groups, varying degrees of paralysis were observed. Especially in the suture group, typical hindlimb paralysis was found after sciatic nerve injury, with the toes curled up and closed. In PEG and PEG + spastin groups, rats also showed paralysis, but the severity of paralysis in these groups was reduced compared with the suture group. The PEG + spastin group presented the lowest degree of paralysis among suture, PEG, and PEG + spastin groups. PEG: Polyethylene glycol.

Figure 3

Masson staining of gastrocnemius muscle of rats in each group at different time points. Images were captured with a DFC 300FX color digital microscope. Cellular structures were normal in the sham group after surgery. In suture, PEG, and PEG + spastin groups, skeletal muscle morphology changed, muscle fiber gaps widened, muscle cells decreased, and extracellular collagen gradually increased with time. Muscular atrophy was most serious in the suture group, followed by PEG and PEG + spastin groups. Scale bar: 50 μm. PEG: Polyethylene glycol.

Figure 4

Ultrastructural changes in axons after sciatic nerve injury in each group. Axon ultrastructure was normal in the sham group after surgery, whereas suture, PEG, and PEG + spastin groups showed a pattern of initial destruction, subsequent disintegration, and gradual repair over 1, 2, 4, and 8 weeks after surgery. One week after surgery in suture, PEG, and PEG + spastin groups, there was obvious destruction of myelin sheaths and disintegration of microtubule and microfilament structures. Two weeks after surgery, regenerated nerve fibers, microtubules and microfilaments gradually became clear, and myelin structures began to appear. As time proceeded, the ultrastructure of axons gradually recovered. The PEG + spastin group was similar to the sham group at 8 weeks after surgery. Scale bars: 20 μm. PEG: Polyethylene glycol.