Transplantation of Schwann cells co-cultured with brain-derived neurotrophic factor for the treatment of experimental autoimmune neuritis

https://doi.org/10.1016/j.jneuroim.2013.08.004Get rights and content

Highlights

  • We study the effects of Schwann cells (SCs) with BDNF for the treatment of EAN.

  • BDNF promotes the proliferation and secretory function of SCs in vitro.

  • SCs injected through cisterna magna can migrate to the injured peripheral nerves.

  • BDNF-treated SCs can reduce paralysis, inflammation, and demyelination in EAN.

  • BDNF-treated SCs can improve the self-repair capability of body in EAN.

Abstract

The aim of this study was to investigate the effects of transplantation of Schwann cells (SCs) co-cultured with brain-derived neurotrophic factor (BDNF) for the treatment of experimental autoimmune neuritis (EAN). Primary SCs were co-cultured with various BDNF concentrations, and the optimum concentration was determined by cell proliferation, and NGF and FGF levels. A rat model of EAN was established by immunization with 400 μg of P2 peptide dissolved in Freund's complete adjuvant. SCs were labeled with CFSE and injected into the cisterna magna 14 days after immunization. We found proliferation of SCs, and NGF and FGF levels were highest at a BDNF concentration of 50 ng/mL. Compared with EAN group, SCs + BDNF group showed the lower paralysis scores from day 34 to day 45, and in sciatic nerves showed a significant decrease in inflammatory cell infiltration (involved CD4-, CD8- and CD68-positive cells) at days 25 and 35, an alleviated demyelination at days 35 and 45, and an increase in S-100-positive cells and a decrease in NGF-positive cells at each time point (P < 0.05). Compared with the EAN group, the SCs + BDNF group showed, in sciatic nerves, the mRNA level of NGF was significantly decreased but that of S-100 was increased at day 25, the mRNA level of CCL3 was also remarkably reduced at day 35, and the mRNA level of CD11a, CCL3 and NGF was reduced but that of S-100 was elevated at day 45 (P < 0.05). There were no differences in results between the SCs group and EAN group. In the end, we draw the conclusions that the exogenous SCs injected through cisterna magna can migrate to the injured peripheral nerves, BDNF promotes the proliferation and secretory function of SCs in vitro, and BDNF-treated SCs in vivo can reduce paralysis, inflammation, and demyelination and improve the self-repair capability of body in EAN.

Introduction

Guillain–Barré syndrome (GBS) is a common paralytic disease in healthy adults with a prevalence of 1–4/100,000 (Vucic et al., 2009). Plasma exchange (PE) and intravenous immunoglobulin (IVIg) are the most effective treatments for GBS at present. Although PE and IVIg shorten the duration of GBS, 4–15% of patients die, and up to 20% of patients are left disabled despite modern treatment (Rees et al., 1998, Visser et al., 1999). Therefore, further research is needed to find ways to prevent the 20% of patients from being left with continuous disability and a better or cocktail therapy may be explored.

Experimental autoimmune neuritis (EAN) is a widely used animal model of GBS (Maurer and Gold, 2002). In 2007, the first therapy for EAN with cell transplantation was performed by Xu et al. (Xu et al., 2007). This study initiated new ideas for the treatment of EAN with cell transplantation.

Many neurons in the peripheral nervous system (PNS) are insulated with a myelin sheath formed by Schwann cells surrounding the axons. During nerve regeneration, SCs regulate demyelination through their connection with axons (Jessen and Mirsky, 1999). After peripheral nerve injury, SCs proliferate to scavenge the dead cells together with macrophages. During these processes, several neurotrophic factors are produced to facilitate nerve regeneration (Syroid et al., 1996, Murwani and Armati, 1998). Currently, SCs are widely used as seed cells in tissue engineering. Brain derived neurotrophic factor (BDNF) plays an important role in neuron degeneration and regeneration. Some studies have shown that BDNF can promote the survival of SCs in vitro through inhibition of apoptosis. Therefore, the treatment of SCs in vitro with suitable neurotrophic factors may improve the survival of transplanted SCs. In this study, we exposed cultured SCs to BDNF and then transplanted the SCs into rats to investigate their effects on EAN.

Section snippets

Isolation and characterization of SCs

Under aseptic conditions, the epineurium was dissected bilaterally from the sciatic nerves in neonatal SD rats (4–6 days after birth). SCs were cultured and passaged based on the method used by Qin et al. (Qin et al., 2012). Cells of passage 3 were plated onto glass coverslips in 24-well plates and incubated in a 5% CO2 incubator at 37 °C for 24 h. Then, the cultures were fixed (30 min, 4% paraformaldehyde in PBS) and stoved at 37 °C. The cells were washed in PBS for 3 times and then treated with 1 

Characterization of SCs

SCs could be visualized by immunohistochemistry using antibodies against S-100. They grew in a shoulder-to-shoulder and head-to-head manner. After DAB staining, SCs stained brown when observed under a phase contrast microscope (Fig. 1. A).

Determination of the optimal concentration of BDNF

After stimulation with different concentrations of BDNF, the proliferation of SCs was detected with a MTT assay. A concentration of 50 ng/mL induced the highest proliferation activity of SCs (Fig. 1. B). Furthermore, after a 24 h-stimulation, the peak NGF and

Discussion

Recently, a report from Zujovic V (Zujovic et al., 2012) has demonstrated a striking therapeutic effect of SCs in DA rats with MOG-induced EAE (a model of MS). By contrast, we have shown that allogenic SCs, transplanted into Lewis rats with P2 57–81 peptide-induced EAN at the acute phase of disease, do not lead to any distinguished neurological, histopathological improvement or expression change of CD11a, CCL3, NGF and S-100 mRNA in sciatic nerves, despite exogenous SCs injected through

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgments

This study was supported by the Natural Science Foundation of China (project number: 30901331) and Heilongjiang youth science fund (project number: QC2009C21).

References (20)

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  • A comparative morphological, electrophysiological and functional analysis of axon regeneration through peripheral nerve autografts genetically modified to overexpress BDNF, CNTF, GDNF, NGF, NT3 or VEGF

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    Schwann cells express p75, a low-affinity receptor for all neurotrophins, trkB (Frisén et al., 1993), ret1/GDNFRalpha1 (Hase et al., 2005) and trkC, the receptor for NT3 (Cosgaya et al., 2002). BDNF and GDNF promote the proliferation of cultured Schwann cells in vitro (Hou et al., 2013) and GDNF induced Schwann cell proliferation in intact sciatic nerves (Höke et al., 2003). Interestingly, Schwann cell proliferation was not observed in chronically denervated autografts transduced with LV-GDNF.

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