周围神经损伤

    Characteristics of neural growth and cryopreservation of the dorsal root ganglion using three-dimensional collagen hydrogel culture versus conventional culture
  • Figure 1|Experimental procedures of the study and images showing the morphology of T-DRG and C-DRG. 

    We harvested DRG from E7–9 chicken embryos and seeded them in TCP or collagen hydrogel, respectively, for T-DRG and C-DRG. The experimental protocol is shown in Figure 1. After 7 days of culture, we observed the extension of nerve bundles in both T-DRG and C-DRG. The nerve bundles in C-DRG were denser than those in T-DRG.

    Figure 2|RNA sequencing analysis of T-DRG and C-DRG.  

    We then extracted RNA from T-DRG and C-DRG for transcriptomic analysis. The original read count data are shown in Additional Table 2. A Pearson correlation heatmap and principal component analysis plot revealed that both T-DRG and C-DRG samples had good biological reproducibility (Additional Figure 1). T-DRG was considered the control group. In the C-DRG group, 1866 genes were upregulated and 1154 genes were downregulated. We labeled the 30 DEGs that had the highest counts per million (Figure 2A). Among these DEGs, downregulated genes were related to extracellular matrix (ECM) organization and actin cytoskeleton organization, while upregulated genes were related to processes such as axonogenesis and synaptic plasticity (Figure 2B). We performed GO biological process enrichment on all DEGs, and selected the top 15 GO terms with the largest significances. Compared with the T-DRG group, the upregulated terms in C-DRG included synapse organization, axonogenesis, axon development, cell morphogenesis involved in neuron differentiation, and synaptic signaling. The downregulated terms included mitotic nuclear division, ECM organization, nuclear division, extracellular structure organization, and cell cycle G2/M phase transition (Figure 2C). In the KEGG analysis, the 15 signaling pathways that were most significantly different between the C-DRG and T-DRG groups are shown in Figure 2D. Cell cycle-related genes were the most downregulated, while genes related to the synaptic vesicle cycle, retrograde endocannabinoid signaling, MAPK signaling pathway, neuroactive ligand–receptor interaction, cAMP signaling pathway, and calcium signaling pathway were upregulated. In particular, ECM–receptor interaction had the highest-fold enrichment. All results of the DEGs and the GO and KEGG enrichment analyses are listed in Additional Tables 3–5.

    Figure 3|Nerve bundle extension in T-DRG and C-DRG. 

    To compare the morphological characteristics of DRG in TCPs and collagen gel, we performed immunofluorescence staining of T-DRG and C-DRG after 7 days of culture. Neurons were labeled with βIII-tubulin antibodies (Varderidou-Minasian et al., 2020). The nerve bundles of T-DRG were thicker and sparser, while those of C-DRG were thinner and denser (Figures 1B and 3A). The images were skeletonized and quantitatively analyzed (n = 15). The number of branches per C-DRG was significantly larger than that per T-DRG (38 230 ± 20 987 vs. 6384 ± 2615, P < 0.001) (Figure 3B). Similarly, the number of junctions per C-DRG was markedly larger than that per T-DRG (17 843 ± 11 239 vs. 3418 ± 1311, P < 0.001) (Figure 3C). There were also more end-point voxels per C-DRG than T-DRG (8674 ± 4254 vs. 1939 ± 678, P < 0.001) (Figure 3D). Moreover, the average branch length per C-DRG was much shorter than that per T-DRG (16.2 ± 2.0 μm vs. 32.4 ± 4.7 μm, P < 0.001) (Figure 3E). We selected axon guidance-related DEGs for heatmap plotting (Figure 3F). The resulting plot revealed that, compared with the T-DRG group, most DEGs in axon guidance were upregulated in the C-DRG group (especially GAP43 and TUBB3). The qPCR results verified that the axon guidance- and axonogenesis-related genes TUBB3, GAP43, NEFM, and NEFL were significantly upregulated (Figure 3G). These results suggest that C-DRG has a denser nerve bundle than T-DRG.

    Figure 4|Cell proliferation, fibrosis, and Schwann cells in T-DRG and C-DRG. 

    To evaluate the proliferation and expression of Schwann cell biomarkers in T-DRG and C-DRG, we performed immunofluorescence staining. Our results revealed that the migration ability of Schwann cells in C-DRG was lower than that in T-DRG. Cells were therefore more scattered in T-DRG and more concentrated in C-DRG (Figure 4A and B). Additionally, the fluorescent density of the Schwann cell marker S100B (Tucker et al., 2011) in T-DRG was significantly lower than in C-DRG (P < 0.01) (Figure 4A and C). Furthermore, T-DRG expressed a large amount of the fibrotic marker α-SMA (Lan et al., 2018), and α-SMA expression was significantly lower in C-DRG (P < 0.05) (Figure 4B and C). The ratio of EdU-positive cells in C-DRG was markedly lower than that in T-DRG (0.094 ± 0.014 vs. 0.298 ± 0.051, P < 0.001) (Figure 4D). Moreover, RNA-seq heatmaps showed that, compared with the T-DRG group, DEGs involved in the positive regulation of epithelial-mesenchymal transition (EMT) and the regulation of mitotic nuclear division were downregulated in the C-DRG group. In contrast, myelination-related DEGs were upregulated in the C-DRG group (especially S100B and NRG1) (Figure 4E). The qPCR results also showed that the Schwann cell-related genes S100B and NRG1 were upregulated in C-DRG compared with T-DRG, while the proliferation-related genes MKI67 and PCNA were downregulated. The EMT transcription factor ZEB1 was significantly downregulated (Figure 4F).


    Figure 5| Apoptosis in T-DRG and C-DRG. 

    To evaluate the anti-apoptotic abilities of T-DRG and C-DRG Schwann cells, we performed serum starvation. After 48 hours of serum withdrawal, apoptotic cells were labeled using TUNEL. Most TUNEL-positive cells were Schwann cells (Figure 5A). The ratio of TUNEL-positive cells in C-DRG was significantly lower than that in T-DRG (0.073 ± 0.019 vs. 0.422 ± 0.201, P < 0.01) (Figure 5B). DEGs related to the apoptotic process were also downregulated (Figure 5C). Moreover, qPCR revealed that the apoptosis-related genes FAS, CASP2, and FADD were significantly downregulated in C-DRG compared with T-DRG (Figure 5D).


    Figure 6|Morphological characteristics and viability of cryo-T-DRG and cryo-C-DRG. 

    After cryopreservation and thawing, T-DRG and C-DRG were cultured for 7 days (cryo-T-DRG and cryo-C-DRG, respectively). We performed a morphological analysis of cryo-T-DRG and cryo-C-DRG. Neurons were labeled with βIII-tubulin antibody. Similar to pre-cryopreservation, the nerve bundles of cryo-T-DRG were thicker and sparser, whereas the nerve bundles of cryo-C-DRG were thinner and denser (Figure 6A). The images were skeletonized and quantitatively analyzed [n(cryo-T-DRG) = 16, n(cryo-T-DRG) = 12]. The number of branches per cryo-C-DRG was significantly greater than that per cryo-T-DRG (3422 ± 1265 vs. 1300 ± 1326, P < 0.001) (Figure 6B). Similarly, the number of junctions per cryo-C-DRG was markedly higher than that per cryo-T-DRG (1505 ± 659 vs. 683 ± 727, P < 0.01) (Figure 6C). There were also more end-point voxels per cryo-C-DRG than per cryo-T-DRG (896 ± 318 vs. 331 ± 299, P < 0.001) (Figure 6D). Finally, the average branch length per cryo-C-DRG was much shorter than that per cryo-T-DRG (18.6 ± 4.2 μm vs. 36.5 ± 3.1 μm, P < 0.001) (Figure 6E). Overall, compared with pre-cryopreservation, the values of these indicators were lower after thawing. The viability of cryo-C-DRG was also higher than that of cryo-T-DRG (84.2 ± 4.0% vs. 46.3 ± 1.6%, P < 0.001) (Figure 6F). Our results therefore revealed that cryo-C-DRG have a denser nerve bundle and better viability compared with cryo-T-DRG.


    Figure 7| Changes in cryo-T-DRG and cryo-C-DRG after 7 days of culture. 

    To evaluate the expression of Schwann cell biomarkers in cryo-T-DRG and cryo-C-DRG, we performed immunofluorescence staining. Apoptotic cells were labeled with TUNEL. The ratio of TUNEL-positive cells in cryo-T-DRG was significantly higher than in cryo-C-DRG (P < 0.05) (Figure 7A and B). In addition, compared with cryo-T-DRG, cryo-C-DRG had significantly higher S100B fluorescent density (P < 0.001) (Figure 7A and C). We also observed that the nerve bundles of cryo-C-DRG disintegrated after being damaged by low temperatures, but Schwann cells remained present, and the newly formed nerve bundles were able to grow along the existing Schwann cells, which functioned as axon guidance. On day 2 after thawing, newly formed nerve bundles appeared and began to slowly extend in the direction of existing Schwann cells. On day 7 after thawing, newly formed nerve bundles mostly overlapped with the existing Schwann cells (Figure 7D). The qPCR results revealed that, compared with cryo-T-DRG, the Schwann cell-related genes S100B and NRG1 were upregulated in cryo-C-DRG. The axon guidance- and axonogenesis-related genes TUBB3, GAP43, NEFM, and NEFL, as well as the proliferation-related gene PCNA, were also significantly upregulated in cryo-C-DRG. In contrast, the fibrosis-related genes ACTA2 and FN1 and the apoptosis-related gene FAS were downregulated compared with cryo-T-DRG. Finally, the neurodevelopmental transcription factor gene TCF3 was upregulated in cryo-C-DRG (Figure 7E). Compared with pre-cryopreservation, both cryo-T-DRG and cryo-C-DRG had downregulation of proliferation-related genes and of biomarker genes of Schwann cells and neurons; in contrast, genes for transcription factors involved in fibrosis, apoptosis, and neurodevelopment were upregulated (Figure 7F). The detailed qPCR statistics are displayed in Additional Figure 2.


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  • 发布日期: 2021-02-06  浏览: 574
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