脊髓损伤

    Microglial voltage-gated proton channel Hv1 in spinal cord injury
  • Figure 1|Hv1 and Nox2 are upregulated in two independent models of spinal cord injury (SCI). 

    Increased expression of Hv1 is noted in several animal models of SCI. In a mouse contusion model, Hvcn1 mRNA from injured spinal cord tissue was elevated at 3 days after injury and persisted up to 28 days in both sexes; consistently, western blot analysis showed a nearly four-fold increase in Hv1 protein at 3 days post SCI compared with the sham control. To confirm whether the increase of tissue Hv1 is contributed by myeloid cells, CD11b+ cells were sorted from injured spinal cord tissue for qRT-PCR. The results show a significant increase in Hvcn1 mRNA in isolated CD11b+ cells at 3 days post injury compared to cells from the sham group (Li et al., 2021). In line with this, we found a significant increase in the expression of Hv1 in sorted microglia/macrophages after SCI lasting up to 2 weeks by performing bioinformatics analysis of a recently published RNA sequencing dataset (Noristani et al., 2017). More specifically, the published study used Cx3cr1gfp/+ mice to perform laminectomy at thoracic 9 (T9) level to obtain either complete paraplegia or hemi paraplegia (Figure 1A). Subsequently, 1 cm length of tissue containing the epicenter of injury was collected at 72 hours, 1 and 2 weeks and Cx3cr1+ cells were isolated for RNA sequencing (Noristani et al., 2017). Analysis of the Noristani et al. (2017) dataset revealed a significant increase in Hcvn1 mRNA in Cx3cr1+ microglia/macrophages at 1 week and 2 weeks after both hemisection and full transection of the spinal cord (Figure 1B). This confirms that local and peripheral immune response in the epicenter of injury has remarkably high expression of Hv1 channels presumably by a combination of resident activated microglia and infiltrated macrophages that contribute to the secondary damage after SCI. 
    To address the function of Hv1 in SCI, we performed a laminectomy over the dorsal portion of T9 in both WT and Hv1–/– mice. Using a modified NYU impactor, mice were subjected to a 3-g drop with a tip diameter of 0.5-mm flat surface from a height of 6.25 mm, resulting in a moderate contusion injury. Basso mouse scale (BMS) scoring (Basso et al., 2006) for locomotion was performed after the injury and weekly thereafter for up to 8 weeks (Figure 1D). We observed significant improvement of motor recovery in the Hv1–/– mice with SCI compared to the WT/SCI mice (Figure 1E; Murugan et al., 2020). This robust rescue of behavioral outcomes in Hv1–/– mice compared to WT mice after SCI was also observed independently by two other research groups in similar SCI models (Li et al., 2020b, 2021). Additional readouts of attenuated neuronal damages in the Hv1–/–/SCI from these three groups include attenuated neuronal loss, increased white matter sparing, and reduced demyelination. The following paragraph will discuss the current understanding of Hv1-mediated mechanism following SCI. All studies consistently showed the Hv1–/–/SCI developed alleviated secondary damages (Li et al., 2020b, c, 2021; Murugan et al., 2020).
    Several studies have demonstrated that Hv1 channel activity is coupled to NOX-dependent pH regulation, membrane depolarization, and ROS production. This Hv1-NOX coupling has been shown in a number of immune cells including microglia (Wu, 2014b), neutrophils (El Chemaly et al., 2010), B cells (Capasso et al., 2010), and eosinophils (Zhu et al., 2013). Since there is a potent immune response following SCI, it is no surprise that Hv1-NOX mediated ROS is elevated in injured tissue. Indeed, our analysis of RNA sequencing dataset from Noristani et al. (2017) confirmed a significant increase in Cybb gene that encodes the NOX2 protein lasting at 1 week and 2 weeks after SCI (Figure 1C). 


    Figure 2|Microglial Hv1 mechanisms in SCI. 

    Depletion of Hv1 reduces NOX-dependent ROS production. ROS concentration in Hv1–/–/SCI spinal tissue homogenates is lower than the WT/SCI at early time points. Flow cytometry revealed that both microglia and infiltrating myeloid cells have attenuated ROS production in the Hv1–/–/SCI compared to the WT/SCI. In line with the other studies, immunofluorescent staining of 8-hydroxyguanosine, a marker of oxidative damage in cellular DNA, showed less co-localization with ionized calcium binding adaptor molecule 1 positive (Iba1+ - a microglia/macrophage marker) cells in the Hv1–/– in the early time points. In agreement with this, ROS production is attenuated in the Hv1–/–/SCI compared to the sham (Li et al., 2020b, 2021; Murugan et al., 2020; Figure 2). 


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  • 发布日期: 2021-12-17  浏览: 405
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