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. 2019 Dec 6:13:548.
doi: 10.3389/fncel.2019.00548. eCollection 2019.

Extracellular Vesicles Derived From Olfactory Ensheathing Cells Promote Peripheral Nerve Regeneration in Rats

Bing Xia  1 , Jianbo Gao  1 , Shengyou Li  1 , Liangliang Huang  2 , Teng Ma  1 , Laihe Zhao  1 , Yujie Yang  1 , Jinghui Huang  1 , Zhuojing Luo  1
Affiliations

Extracellular Vesicles Derived From Olfactory Ensheathing Cells Promote Peripheral Nerve Regeneration in Rats

Bing Xia et al. Front Cell Neurosci. .

Abstract

Accumulating evidence showed that extracellular vesicles (EVs) and their cargoes are important information mediators in the nervous system and have been proposed to play an important role in regulating regeneration. Moreover, many studies reported that olfactory ensheathing cells (OECs) conditioned medium is capable of promoting nerve regeneration and functional recovery. However, the role of EVs derived from OECs in axonal regeneration has not been clear. Thereby, the present study was designed to firstly isolate EVs from OECs culture supernatants, and then investigated their role in enhancing axonal regeneration after sciatic nerve injury. In vitro studies showed that OECs-EVs promoted axonal growth of dorsal root ganglion (DRG), which is dose-dependent and relies on their integrity. In vivo studies further demonstrated that nerve conduit containing OECs-EVs significantly enhanced axonal regeneration, myelination of regenerated axons and neurologically functional recovery in rats with sciatic nerve injury. In conclusion, our results, for the first time, demonstrated that OECs-EVs are capable of promoting nerve regeneration and functional recovery after peripheral nerve injuries in rats.

Keywords: extracellular vesicles; functional recovery; nerve regeneration; olfactory ensheathing cells; peripheral nerve injury.

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Figures

Figure 1
Figure 1
Characterization of olfactory ensheathing cells and isolated EVs. Notes: double immunofluorescent staining of cultured OECs showed the expression of SMA (green; A) and p75NTR (red; B) with DAPI nuclear counterstaining (blue; C). Merge images revealed an OECs purity of more than 96% (D). Western blot analysis of EVs isolated from the supernatant of cultured OECs confirmed the presence of EVs marker proteins of Alix, TSG101, CD63, and the absence of non-exosomal protein (calnexin; E). The TEM analysis of isolated EVs (F). Representative traces from nanoparticle tracking analysis (NTA; G). The upper right corner of the images (A–D,F) is a higher magnification of the boxed area in (A–D,F). Scale bars: (A–D) 100μm; (F) 200 nm. Abbreviations: OECs, olfactory ensheathing cells; SMA, smooth muscle α-actin; DAPI, 4′,6-diamidino-2-phenylindole; EVs, extracellular vesicles; TEM, transmission electron microscopy; NTA, nanoparticle tracking analysis.
Figure 2
Figure 2
Internalization of OECs-derived EVs by DRG neurons and axons. Notes: the obtained OECs-EVs were labeled by PKH26 (red) and DRG neurons were immunofluorescent stained for β-tubulin III (green) with DAPI nuclear counterstaining (blue). Representative images showed no EVs in the cytoplasm and axons of DRG neurons (A–C,G–I) in the control groups, while labeled EVs (red) can be found within the cytoplasm and axons of DRG neurons in the OECs-EVs treated groups (B–F,J–L). Scale bars: (A–F) 10 μm; (G,H) 50 μm; (J,K) 25 μm. Abbreviations: OECs, olfactory ensheathing cells; EVs, extracellular vesicles; DRG, dorsal root ganglion; GFAP, glial fibrillary acidic protein; DAPI, 4′,6-diamidino-2-phenylindole.
Figure 3
Figure 3
EVs derived from OECs can enhance axonal elongation of DRG neurons and explants. Notes: Representative images of DRG neurons stained for β-tubulin III (red) and NeuN (green), the DRG explants were stained for β-tubulin III (green) with nuclear DAPI counterstaining. Axonal elongation of DRGs after 3 days PBS (A,E), OECs-EVs (107 particles/ml; B,F), OECs-EVs (108 particles/ml; C,G) and OECs-EVs (109 particles/ml; D,H) daily treatment. Treatment of OECs-EVs with different concentrations increased the average length of the longest neurite and the ratio of total area of neurites/total area of explant body (I,J). Effect of pre-treated OECs-EVs on axonal elongation with 3 days daily treatment (K,L). OECs-EVs were incubated with trypsin, lysed by water or treated with trypsin followed by water lysis, control represents PBS treatment; n = 4 per group; scale bars: (A–D) 100 μm; (E,F) 300 μm; (G,H) 1,000 μm. The results are expressed as the mean ± SEM. One-way analysis of variance (ANOVA) test with Tukey’s post hoc test was used to examine the significance of results. **p < 0.01 for comparison with control group, $p < 0.05 and $$p < 0.01 for comparison with OECs-EVs (107 particles/ml) group, ##p < 0.01 for comparison with intact EVs group. Abbreviations: EVs, extracellular vesicles; OECs, olfactory ensheathing cells; DRG, dorsal root ganglion; DAPI, 4′,6-diamidino-2-phenylindole.
Figure 4
Figure 4
Nerve regeneration in the grafts at 4 weeks. Notes: (A) gross appearance of the graft conduit. (B) Sciatic nerve graft conduit after implantation. Regenerated nerves were stained for NF160 in the autograft group (C), EVs+conduit group (D) and PBS+conduit group (E). The representative images of proximal, middle and distal section of the graft in the autograft (C1–C3), EVs+conduit group (D1–D3) and PBS+conduit group (E1–E3); in the EVs+conduit group and PBS+conduit group, in addition to the same volume of EVs and PBS, the same volume of Matrigel® was added to provide a supportive environment; n = 4 per group, scale bars: (C–E) 500 μm. Abbreviations: EVs, extracellular vesicles.
Figure 5
Figure 5
Cross-sections of the distal regenerated nerve segments at 4 and 8 weeks. Notes: representative images of regenerated axons at the distal nerve segment stained for NF-160 (green) in the groups of autograft (A), EVs+conduit (B) and PBS+conduit (C) at 4 weeks and autograft (D), EVs+conduit (E) and PBS+conduit (F) at 8 weeks. The upper right corner of the images (A–F) is higher magnifications of the boxed area in (A–F). Quantifications of the total number of the regenerated axons in the cross-sections at 4 weeks (G) and 8 weeks (H); n = 4 per group, scale bars: (A–F) 100 μm. The results are expressed as the mean ± SEM. One-way analysis of variance (ANOVA) test with Tukey’s post hoc test was used to examine the significance of results. ***p < 0.005 for comparison with the PBS+conduit, $p < 0.05 and $$p < 0.01 for comparison with the autograft group. Abbreviations: EVs, extracellular vesicles.
Figure 6
Figure 6
Morphological appearance and morphometric assessments of regenerated nerves in each group. Notes: representative images of toluidine blue staining of regenerated axons in the autograft (A), EVs+conduit (B) and PBS+conduit (C) in the distal part of the graft of each group at 8 weeks after surgery, respectively. Representative TEM micrographs of regenerated axons (D–F) and myelin sheath (G–I) in the distal part of the graft in the autograft group (D,G), EVs+conduit group (E,H) and PBS+conduit group (F,H) at 8 weeks after surgery, respectively. Quantification of the average number of myelinated axons (J), average diameter of myelinated axons (K), myelin sheath thickness (L) and G-ratio (M); n = 6 per group, scale bars: (A–C) 50 μm; (D–F) 5 μm; (G–I) 5 nm. The results are expressed as the mean ± SEM. One-way analysis of variance (ANOVA) test with Tukey’s post hoc test was used to examine the significance of results. *p < 0.05, **p < 0.01 and ***p < 0.005 for comparison with Conduit group, $p < 0.05 and $$p < 0.01 for comparison with autograft group. Abbreviations: EVs, extracellular vesicles; TEM, transmission electron microscopy.
Figure 7
Figure 7
Functional assessment for regenerated nerves. Notes: representative images of operative left footprints (A) in the autograft group (a), EVs+conduit group (b) and PBS+conduit group (c) at 8 weeks postoperatively. (B) The sciatic function index (SFI) values of each group at 2, 4, and 8 weeks after surgery. Representative light micrographs of gastrocnemius muscles after Masson trichrome staining of the control group (normal muscle; C), the autograft group (D), EVs+conduit group (E) and PBS+conduit group (F). (G) Quantification of average percentage of muscle fiber area in each group. (H) Ratio of injured muscle (ipsilateral) weight to normal muscle (contralateral) weight. The electrophysiological assessment of each group (I–K); the peak amplitude of CMAP (I), the nerve conduction velocity (J) and latency of CMAP onset (K); n = 6 per group, scale bars: (C–F) 100 μm. All data are expressed as the mean ± SEM. One-way analysis of variance (ANOVA) test with Tukey’s post hoc test was used to examine the significance of results. *p < 0.05, **p < 0.01 and ***p < 0.005 for comparison with the PBS+conduit group, $p < 0.05 for comparison with the autograft group and ##p < 0.01 for comparison with the control (normal muscle) group. Abbreviations: EVs, extracellular vesicles; CMAP, compound muscle action potential.
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