Centella asiatica (L.)-Neurodifferentiated Mesenchymal Stem Cells Promote the Regeneration of Peripheral Nerve

Abstract

Background: Centella asiatica (L.) is a plant with neuroprotective and neuroregenerative properties; however, its effects on the neurodifferentiation of mesenchymal stem cells (MSCs) and on peripheral nerve injury are poorly explored. This study aimed to investigate the effects of C. asiatica (L.)-neurodifferentiated MSCs on the regeneration of peripheral nerve in a critical-size defect animal model.

Methods: Nerve conduit was developed using decellularised artery seeded with C. asiatica-neurodifferentiated MSCs (ndMSCs). A 1.5 cm sciatic nerve injury in Sprague-Dawley rat was bridged with reversed autograft (RA) (n = 3, the gold standard treatment), MSC-seeded conduit (MC) (n = 4) or ndMSC-seeded conduit (NC) (n = 4). Pinch test and nerve conduction study were performed every 2 weeks for a total of 12 weeks. At the 12th week, the conduits were examined by histology and transmission electron microscopy.

Results: NC implantation improved the rats' sensory sensitivity in a similar manner to RA. At the 12th week, nerve conduction velocity was the highest in NC compared with that of RA and MC. Axonal regeneration was enhanced in NC and RA as shown by the expression of myelin basic protein (MBP). The average number of myelinated axons was significantly higher in NC than in MC but significantly lower than in RA. The myelin sheath thickness was higher in NC than in MC but lower than in RA.

Conclusion: NC showed promising effects on nerve regeneration and functional restoration similar to those of RA. These findings revealed the neuroregenerative properties of C. asiatica and its potential as an alternative strategy for the treatment of critical size nerve defect.

Keywords: Centella; Differentiation; Mesenchymal stem cells; Nerve regeneration.

Fig. 1

NCS. A Mean values of the peak amplitude of CMAP from 2 weeks post-implantation. P value < 0.05. n=3. B. Mean values of latency of CMAP from 2 weeks post-implantation. p value < 0.05. n=3. C. Mean values of NCV from 2 weeks post-implantation. P value < 0.05. n=3

Fig. 2

Sciatic nerve 12 weeks post-implantation. A, B Sciatic nerve in RA treatment (n = 3). C, D Sciatic nerve in MC implantation (n = 4). E, F Sciatic nerve in NC implantation (n = 4)

Fig. 3

H & E staining of the harvested sciatic nerve for RA (n = 3), MC (n = 4) and NC (n = 4) groups (proximal, middle and distal). Arrows show red blood cells on the proximal part of RA and NC similar to those of the native sciatic nerve. Magnification, 20×. Scale bar, 100 μm. T = transverse section, L = longitudinal section

Fig. 4

IHC analysis (ICC) of the sciatic nerve 12 weeks post-implantation for RA, MC and NC groups (proximal and distal). Nuclei were counter-stained with DAPI (blue). Analysis of the longitudinal section. Magnification, 20×. Scale bar, 100 μm. n = 3. A ICC of the sciatic nerve for protein S100β (red), p75NGFR (green), GFAP (red) and MBP (red). B ICC of the sciatic nerve for protein NF (green)

Fig. 5

Identification of engrafted human cells in SD rats by the expression of protein STEM 121 (red). Nuclei were counter-stained with DAPI (blue). Analysis of the longitudinal section. Magnification, 20×. Scale bar, 100 μm. n = 3

Fig. 6

Transverse section of toluidine blue-stained sciatic nerve under a light microscope. Red arrows show myelinated axons, whereas yellow arrows show non-myelinated axons. Magnification, 40×. Scale bar, 200 μm. n = 3

Fig. 7

TEM analysis of the sciatic nerve. The dashed circles in A, D and G show axon in a nerve fascicle. Red arrows show an enlarged myelinated axon. The image in B shows a representative of axonal and fibre diameter. The images in C, F and I show layers of myelin that form myelin sheath. Analysis of the transverse section. A, D, G Magnification, 2550×. Scale bar, 2 μm. B, E, H Magnification, 20,500×. Scale bar, 500 nm. (C, F, I Magnification, 87,000×. Scale bar, 100 nm. n = 3

Fig. 8

Morphometric analysis of sciatic nerve. A Number of myelinated axons. p value: **p = 0.0068, ***p = 0.0006, ****p = < 0.0001. B Axon diameter. C Fiber diameter. D Myelin sheath thickness. p value: *p = 0.0115. E G-ratio. p value: *p = 0.0149, **p = 0.0018. n = 3