Promotive effect of skin precursor-derived Schwann cells on brachial plexus neurotomy and motor neuron damage repair through milieu-regulating secretome

Abstract

Brachial plexus injury (BPI) with motor neurons (MNs) damage still remain poor recovery in preclinical research and clinical therapy, while cell-based therapy approaches emerged as novel strategies. Previous work of rat skin precursor-derived Schwann cells (SKP-SCs) provided substantial foundation for repairing peripheral nerve injury (PNI). Given that, our present work focused on exploring the repair efficacy and possible mechanisms of SKP-SCs implantation on rat BPI combined with neurorrhaphy post-neurotomy. Results indicated the significant locomotive and sensory function recovery, with improved morphological remodeling of regenerated nerves and angiogenesis, as well as amelioration of target muscles atrophy and motor endplate degeneration. Besides, MNs could restore from oxygen-glucose-deprivation (OGD) injury upon SKP-SCs-sourced secretome treatment, implying the underlying paracrine mechanisms. Moreover, rat cytokine array assay detected 67 cytokines from SKP-SC-secretome, and bioinformatic analyses of screened 32 cytokines presented multiple functional clusters covering diverse cell types, including inflammatory cells, Schwann cells, vascular endothelial cells (VECs), neurons, and SKP-SCs themselves, relating distinct biological processes to nerve regeneration. Especially, a panel of hypoxia-responsive cytokines (HRCK), can participate into multicellular biological process regulation for permissive regeneration milieu, which underscored the benefits of SKP-SCs and sourced secretome, facilitating the chorus of nerve regenerative microenvironment. Furthermore, platelet-derived growth factor-AA (PDGF-AA) and vascular endothelial growth factor-A (VEGF-A) were outstanding cytokines involved with nerve regenerative microenvironment regulating, with significantly elevated mRNA expression level in hypoxia-responsive SKP-SCs. Altogether, through recapitulating the implanted SKP-SCs and derived secretome as niche sensor and paracrine transmitters respectively, HRCK would be further excavated as molecular underpinning of the neural recuperative mechanizations for efficient cell therapy; meanwhile, the analysis paradigm in this study validated and anticipated the actions and mechanisms of SKP-SCs on traumatic BPI repair, and was beneficial to identify promising bioactive molecule cocktail and signaling targets for cell-free therapy strategy on neural repair and regeneration.

Keywords: Branchial plexus injury; Hypoxia-responsive cytokines; Motor neurons; Regenerative microenvironment; Secretome; Skin precursor-derived Schwann cells.

Graphical abstract

Fig. 1

Identification of rat SKP-SCs and cell implantation treatment to lower brachial plexus injury (LBPI). (A, B) SKP-SCs showed a typical bipolar spindle-shape with side by side alignment. Immuno-stained SKP-SCs positively expressed Schwann cell markers S100β (red) and GFAP (green), with DAPI stained cell nuclei (blue). Scale bar, 100 μm. (C) Anatomic morphology of branchial plexus in Sprague–Dawley rats. (D) Photograph showing the operative region at inferior trunk of branchial plexus, the green and blue circle represented the exposed surgical field and the damaging position respectively, the blue and green arrow represented the transection injury location and cell injection point respectively.

Fig. 2

Functional recovery evaluation of regenerated nerves. (A, B) Evaluation of Terzis grooming test score and cold sensitivity score increased with time by week, showing significant recovery in SKP-SC group than in PBS group in six weeks. (C) Representative electrophysiological CMAP curves of the injured side at six weeks after surgery, the significance difference of CMAP amplitudes was statistically analyzed. Data are presented as Mean ± SEM, n = 6; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Fig. 3

The pro-regenerative effect of SKP-SCs on injured branchial plexus. (A) Representative image of nerve regeneration in three groups in vivo. NF200 (green) stained regenerating neurofilaments and S100β (purple) stained Schwann cells showed the higher fluorescence intensity expressed in SKP-SC group than that in PBS group. Scale bar, 250 μm. (B, C) Statistical histograms showed the average fluorescence intensity of NF200 and S100β of regenerated nerves among three groups. Data are presented as Mean ± SEM, n = 3; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. (D) The regeneration of neurovascular bundles in three groups in vivo showed the expression CD31 (red) in three groups, with NF200 (green) labeled neurofilaments and Hoechst (blue) labeled nucleus. Scale bar, 75 μm.

Fig. 4

The evaluation of the ultrastructure of the regenerated nerve fibers under transmission electron microscope. (A) Representative images of the regenerated nerve fibers (Scale bar, 5 μm) and (B) the myelin lamellar in three groups (Scale bar, 500 nm). Statistical analysis of (C) the diameter of myelinated nerve fibers (n > 40), (D) the thickness of myelin sheath (n > 40), and (E) the number of myelin lamellar (n = 14) of the regenerated nerve fibers in three groups. Data are presented as Mean ± SEM. ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Fig. 5

Analysis of the gross morphology and Masson's trichrome staining of target muscles paraffin section. (A) Representative gross photograph of bilateral biceps muscles of three groups at six weeks postoperatively. (B) Histograms showed the wet weight ratios of targeted muscles (right injured side/left contralateral uninjured side) in three groups (n = 6). (C) Representative overall images of Masson's trichrome staining biceps muscles paraffin sections in three groups (Scale bar, 500 μm). Statistical histograms showed (D) the percentage of muscle fiber area and (E) the collagen fiber area in the whole muscle (n = 3; 3 fields per rat). (F) Representative images of regional biceps muscles paraffin sections in three groups (Scale bar, 100 μm). (G) Statistical histograms showed the mean cross-sectional area of muscle fibers and (H) the average percentage of collagen fiber area in the regional muscle (n = 3; not less than 4 random fields per rat). Data are presented as Mean ± SEM. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Fig. 6

Cross-sectional area of muscle fibers and neuro-muscular junction analysis of target muscles freezing section. (A) Statistical histograms showed the average cross-sectional area of targeted muscle fibers (n = 3; not less than 9 random fields per rat). (B) Representative images of laminin immunostaining of cross-section of targeted muscles in different groups. (C) Representative images of α-bungarotoxin staining of motor endplates in three groups, showing mature pretzel, intermediate morphology, or immature plaque. Data are presented as Mean ± SEM. ∗, p < 0.05; ∗∗∗, p < 0.001. Scale bar, 50 μm.

Fig. 7

Identification of motor neurons and treatment of OGD-injured neurons with SKP-SC sourced conditioned medium (SKP-SC-CM). (A) Morphology of primarily cultured motor neurons with extended neurites forming a network at day 4. Scale bar, 100 μm. (B) Immunofluorescence stained motor neurons positively expressed ChAT (green) and TUJ1 (red) with Hoechst labeled nuclei (blue). Scale bar, 75 μm. (C) Statistical histograms showed the increasing neuronal cell viability in a dose-dependent manner. (D) Immunofluorescence stained motor neurons positively expressed TUJ1 (green) in each group (control, OGD, OGD + SKP-SC-CM). Scale bar, 75 μm. (E, F) Statistical histograms of the average longest neurite length and the number of primary neurites per cell was better in OGD + SKP-SC-CM group than that in OGD group. For each group and experiment, about 100 neurons per condition were assessed. Data are presented as Mean ± SEM, n = 3; ∗∗∗, p < 0.001.

Fig. 8

Bioinformatic cluster analysis and classification of screened cytokines contained in SKP-SC-CM. (Upper panel) The histograms showed the involved cell type number of (Bottom panel) 32 informative cytokines respectively. (Middle panel) The different candidate cytokines involved in 5 cell types, associated cellular biological processes were presented in the scatter plots. (Right panel) the implicated cytokine number of each cellular biological process, involving with neurons, Schwann cells, inflammatory cells, VECs, and implanted SKP-SCs, were displayed in histograms, corresponding to biological process terms (Left panel) respectively.

Fig. 9

Expression of the hypoxia responsive cytokines at mRNA level in SKP-SCs. The histograms showed that at 24 h after hypoxia, the mRNA expression of PDGF-AA, VEGF-A and IL-1β in SKP-SCs were significantly elevated, other cytokines did not show marked increase in hypoxia SKP-SCs than that in normoxia SKP-SCs.