脊髓损伤

    Chronic spinal cord compression associated with intervertebral disc degeneration in SPARC-null mice
  • Figure 4|Distribution of spinal cord cells in the L2/3 level in SPARC-null mice. 

    Compression of the spinal cord was confirmed by hematoxylin and eosin staining (Figure 4A). Western blot showed that the expressions of NeuN and Olig2 in SPARC-null mice were significantly decreased, while Iba1 and GFAP expressions were significantly increased compared with levels in WT mice (Figure 4B). In addition, triple immunofluorescence staining and quantitative analysis demonstrated that neurons in the compression center were significantly reduced in SPARC-null mice. In addition, microglia/macrophages in WT mice were individually and sporadically scattered in the spinal cord, while those in SPARC-null mice were mostly limited in the injury center. Unlike microglia/macrophages, astrocytes were mainly expressed in the white matter of WT mice, while in SPARC-null mice, astrocytes were significantly increased in both gray and white matter and were not restricted to the injury center, above and below the compression center, respectively (Figure 4C and D). SPARC-null mice showed neuronal depletion, inflammation, and astrocyte hyperplasia.


    Figure 5|Apoptosis and demyelination in the spinal cord at L2/3 level caused by the absence of SPARC.  

    TUNEL-positive cells were observed in neurons and oligodendrocytes of SPARC-null mice, while few TUNEL-positive cells were observed in WT mice (Figure 5A and B). Immunofluorescence staining indicated that the expressions of apoptosis-related proteins Bax and cleaved caspase-3 were increased in SPARC-null mouse neurons and increased cleaved caspase-3 was observed in oligodendrocytes compared with WT mice (Figure 5A and B). In addition, MBP is a protein widely distributed in oligodendrocytes and contains a variety of basic amino acids to maintain the stability of the structure and function of myelin sheath in the CNS (Müller et al., 2013), was less integrated in SPARC-null mice compared with WT mice (Figure 5C). The western blot results were consistent with the above results (Figure 5D). Therefore, our results suggested that the absence of SPARC in mice leads to apoptosis and demyelination in the spinal cord, which is one of the characteristics of CSCC.


    Figure 6|Neuroinflammation at the L2/3 level caused by the absence of SPARC.   

    After spinal cord compression, microglia/macrophages are activated and recruited to produce iNOS, IL-6, IL-1β, and other cytokines, which leads to neuroinflammation (Pei et al., 2017). As shown by immunofluorescence staining, few inactive microglia were distributed in the spinal cord of WT mice, while the levels of Iba1, IL-1β, and IL-6 (markers of M1 phenotype microglia/macrophages) were significantly increased in SPARC-null mice. Western blot results were consistent with the immunofluorescence results (Figure 6A and B). CD206 expression was also increased in SPARC-null mice compared with expression in WT mice, suggesting an increase in M2 phenotype microglia/macrophages (Ip et al., 2008; Bhargava et al., 2021) (Figure 6C and D). CD3+ T lymphocytes and CD11b+ macrophage-like positive cells also increased in SPARC-null mice (Figure 6E). These results indicate that the SPARC-null mice exhibited neuroinflammation; however, both M1 and M2 microglia/macrophages were polarized.


    Figure 7|Astrocyte activation in L2/3 level in SPARC-null mice. 

    Astrocytes are the most widely distributed group of cells in the mammalian CNS and are involved in the formation of the blood-brain barrier. They also have an important role in neuroinflammation (Giovannoni and Quintana, 2020). Immunofluorescence staining showed that the expression of astrocyte marker GFAP was increased both in white and gray matter in SPARC-null mice compared with WT mice. PCNA (a hyperplasia marker (González-Maga?a and Blanco, 2020)) and cytokines such as C3, IL-1β, and tumor necrosis factor-α (which represent A1 phenotype (Escartin et al., 2021)), S100A10, IL-10, and TGF-β (which represent the A2 type (Eroglu, 2009)) were significantly increased in SPARC-null mice (Figure 7). 


    Figure 8|The absence of SPARC activates NLRP3 inflammasomes at the L2/3 level in spinal cord.  

    The inflammasome is a cellular signal receptor that senses neuropathological conditions. NLRP3 is an inflammasome that contains apoptosis-associated microprotein ASC and whose activation can lead to proteolysis of caspase-1. This, in turn, activates pro-inflammatory cytokines, such as IL-1β and IL-18, and causes neuroinflammation (Riaz et al., 2021). Immunofluorescence staining showed that NLRP3 expression was low in WT mice, while it was increased in SPARC-null mice, especially in the compression part (in both gray matter and white matter) (Figure 8A and B). These data were further confirmed by western blot (Figure 8C). Therefore, we concluded that the absence of SPARC in mice activates NLRP3 inflammasomes, which is one of the characteristics of CSCC. 


    Figure 9|The absence of SPARC inhibits the BDNF/TrkB signaling pathway at the L2/3 level in spinal cord.  

    BDNF is the most abundant neurotrophic factor in the body and promotes the growth of neurons and the formation and stability of synapses in nerve cells in the brain. It also participates in cell differentiation, cell viability, and synaptic plasticity by binding to tyrosine kinase B (TrkB) (Numakawa and Odaka, 2021). Next, we examined whether spinal cord compression caused by the absence of SPARC inhibits the BDNF/TrkB pathway. Immunofluorescence staining results showed lower BDNF expression in the whole spinal cord of SPARC-null mice compared with WT mice. In SPARC-null mice, BDNF was decreased at the non-compression part, while the expression of BDNF was observed at the compression site; however, overall, it was declined in SPARC-null mice (Figure 9A and B). Western blot results also indicated a down-regulation of TrkB and BDNF expression in SPARC-null mice vs. WT mice (Figure 9C). In summary, our results suggested that the absence of SPARC inhibits the BDNF/TrkB signaling pathway.


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  • 发布日期: 2022-08-29  浏览: 167
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