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    Robust temporal changes of cellular senescence and proliferation after sciatic nerve injury
  • Figure 1|Cellular senescence in sciatic nerves post-injury.

    On investigating the involvement of cellular senescence post-injury, KEGG results showed that stress signals elicit the activation of various intracellular cascades, including mitogen-activated protein kinases and checkpoint kinases, suppress the activation of cyclin-dependent kinases, induce an irreversible cell-cycle arrest, and cause cellular senescence (Figure 1A). The abundances of genes involved in the cellular senescence pathway in intact or injured rat sciatic nerves were screened according to previously obtained sequencing data (0, 1, 4, 7, and 14 days post-injury; Yi et al., 2015) to identify significantly differentially expressed genes. MEK (MAP2K1, mitogen-activated protein kinase kinase 1) and GADD45G (growth arrest and DNA damage inducible gamma) were increased at 1 day post-injury. CDKN1A p21 (cyclin-dependent kinase inhibitor 1A), CDKN2A p16 (cyclin-dependent kinase inhibitor 2A), ATM (ataxia-telangiectasia mutated), RAD1 (cell cycle checkpoint protein RAD1), and CHK1/2 (checkpoint kinase 1/2) were decreased post-injury. Increased amount of these genes might contribute to cellular senescence. On the contrary, FOXO3 (forkhead box O3), a gene that functions as a trigger for cellular apoptosis, was slightly decreased at day 1 post-injury. Down-regulated FOXO3 might exert an inhibition effect on cellular senescence. Moreover, many cell cycle-related genes, including CDC25A (cell division cycle 25A), CDK1/2/4 (cyclin-dependent kinase 1/2/4), CCND1 (cyclin D1), CCNA2 (cyclin A2), and CCNE1 (cyclin E1), showed elevated expressions, which indicates that cell division was robustly activated in the injured nerve stumps (Figure 1A). The dynamic patterns of genes in KEGG cellular senescence showed that genes coding for essential factors of senescence, such as p16 and p21, were up-regulated post-injury. Genes coding for checkpoint kinases, cyclin-dependent kinases, and cyclin family members were also increased (Figure 1B).
    Other than the identification of transcriptome signatures, sciatic nerve sections were subjected to immunostaining to visualize β-galactosidase activity. Some β-galactosidase signals could be observed in the day 0 control group. The signals of β-galactosidase seemed to be attenuated at day 1 post-injury. However, much more intense signals were detected at later time points, especially at day 4 and day 7 post-injury (P < 0.05 at days 4 and 7; Figure 1C and D).


    Figure 2|Validation of the expression patterns of senescence-associated genes after sciatic nerve injury using real-time quantitative polymerase chain reaction.

    Gene expressions were additionally examined using real-time polymerase chain reaction (RT-PCR). RT-PCR results showed elevated mRNA expression levels of Cdkn1a, Cdkn2a, Atm, and Cdk1 in the injured sciatic nerves as compared with the day 0 group (P < 0.05; Figure 2). These observations, together with sequencing outcomes, indicated a significant involvement of senescence-associated genes post-injury.


    Figure 3|Senescence activities of Schwann cells, macrophages, and fibroblasts in sciatic nerves post-injury.

    Immunopositivity of cell senescence marker p16 (Uyar et al., 2020) in rat sciatic nerve stumps were further examined using immunostaining. Consistent with the β-galactosidase signals, the immunopositivity of p16 seemed to be robustly increased at multiple time points post-injury, particularly at days 4 and 7, as compared with day 0 (Figure 3A–C). Sciatic nerve stumps were further co-immunostained with p16 and Schwann cell marker S100β (Zhang et al., 2021a ) (Figure 3A), macrophage marker CD68 (Alves et al., 2018) (Figure 3B), as well as fibroblast marker P4HB (Schmid et al., 2020) (Figure 3C). Co-immunostaining of injured sciatic nerve stumps with p16 and S100β showed that the number of Schwann cells first decreased and then gradually increased post-injury, and the number of p16-positive Schwann cells was increased at days 4 and 7 post-injury as compared with the day 0 control (Figure 3A). Immunostaining with CD68 indicated that the number of macrophages was greatly increased immediately post-injury (day 1 post-injury), while the number of p16-positive macrophages seemed to be elevated at 4 and 7 days (Figure 3B). p16-positive fibroblasts also appeared to be increased post-injury (Figure 3C).


    Figure 4|Cellular senescence signaling in rat dorsal root ganglia post-injury.

    Cellular senescence status of dorsal root ganglia was also examined according to genetic and morphological aspects. Gene expression levels in rat dorsal root ganglia were screened according to previously obtained sequencing data (Gong et al., 2016). Gene changes were less robust in dorsal root ganglia than in injured sciatic nerves. Only GADD45, p21, RAD9 (cell cycle checkpoint protein RAD9), and CycD (cyclin D1) were significantly altered post-injury (Figure 4A). In addition, many genes in dorsal root ganglia showed reduced expressions post-injury (Figure 4B).
    Morphological immunostaining of β-galactosidase revealed the presence of cellular senescence in dorsal root ganglia of both uninjured and injured rats. Consistent with sequencing data that showed that the gene expressions of few cellular senescence-related genes were significantly changed in dorsal root ganglia, signals of β-galactosidase in dorsal root ganglia were not obviously altered post-injury (Figure 4C).


    Figure 5|Cell proliferation in sciatic nerves post-injury.

    Given that senescence is typically associated with the loss of replicative potential (Birch et al., 2018), other than cellular senescence, we also determined changes of cellular proliferation using sequencing data of sciatic nerves after nerve injury. Here, the temporal expression profiles of a series of proliferation marker genes were investigated to determine cellular proliferation status (Whitfield et al., 2006). Many proliferation marker genes, including MKI-67, which is a gene coding for proliferating cell nuclear antigen Ki67, were elevated following peripheral nerve injury, especially at days 1 and 4 (Figure 5A). 
    Immunostaining of sciatic nerve stumps also revealed higher abundances of Ki67 than in uninjured nerves (P < 0.05 at day 7; Figure 5B and C). Sciatic nerves were co-immunostained with S100β, a marker of Schwann cells. Some co-localized S100β and Ki67 signals were detected in Ki67-positive cells at days 0-14 (Figure 5B and C). 
    To further visualize cell proliferation status, rats were injected with EdU. EdU incorporation was observed in day 0 sciatic nerve samples. Stronger EdU signals were detected after nerve injury. Summarized data showed that the relative number of EdU colocalized with DAPI increased at days 1, 4, and 7 post-injury (P < 0.05) and recovered to baseline levels at day 14 (Figure 5C–E). Immunostaining outcomes, consistent with genetic signatures, showed that peripheral nerve injury induced cellular proliferation at injured sites, especially at early time points.


    Figure 6|Validation of the expression patterns of proliferation-associated genes after sciatic nerve injury by real-time quantitative polymerase chain reaction.

    Similar to the senescence-associated genes, the temporal expressions of proliferation-associated genes were also validated using RT-PCR. RT-PCR showed that, consistent with RNA deep sequencing outcomes, mRNA expression levels of Mcm5 (minichromosome maintenance complex component 5), Ccnf (cyclin F), Dhfr (dihydrofolate reductase), and Timp1 (TIMP metallopeptidase inhibitor 1) were augmented in sciatic nerves post-injury (Figure 6). 


    Figure 7|Cell proliferation in rat dorsal root ganglia after sciatic nerve injury.

    Cellular proliferation in dorsal root ganglia was assessed based on sequencing data of dorsal root ganglia at 0, 3, 9 hours, 1, 4, and 7 days after nerve injury (Gong et al., 2016). Unlike sciatic nerve stumps, the majority of proliferation marker genes, including MKI-67, were not significantly changed. Only UNG (uracil-DNA glycosylase) and CCNF were elevated at 1, 4 and 7 days post-injury, MCM5 and DHFR were elevated at 4 and 7 days, and TIMP1 was elevated at 4 days. Some proliferation marker genes even exhibited reduced expressions. For instance, in dorsal root ganglia, PLK1 (polo-like kinase 1) was down-regulated at 9 hours post-injury and MAPK13 (mitogen activated protein kinase 13) was down-regulated at 1 day post-injury (Figure 7A).
    Ki67 and EdU staining showed that although a larger number of proliferating cells appeared to exist, changes of cellular proliferation in dorsal root ganglia were less noticeable than in sciatic nerves (Figure 7B and C).


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  • 发布日期: 2022-01-12  浏览: 426
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