周围神经损伤

    Role of microtubule dynamics in Wallerian degeneration and nerve regeneration after peripheral nerve injury
  • Figure 1|The effects of paclitaxel and nocodazole on microtubule dynamics in nerve explant Schwann cells. 

    First we tested the effect of microtubule-targeting agents on microtubule stabilization and destabilization. Based on concentrations used in a previous report (Imai et al., 2017), we treated nerve explants with a series of concentrations of paclitaxel (0, 1, 10, 50, 100, or 200 nM) or nocodazole (0, 1, 10, 100, 500, or 1000 nM). As expected, expression of acetylated tubulin (ace-tub), a marker of stable microtubules (Xiong et al., 2019), was up-regulated with increasing paclitaxel concentrations, while expression of tyrosinated tubulin (tyr-tub), a marker of unstable microtubules (Hu et al., 2017), was down-regulated with increasing paclitaxel concentrations. In contrast, treatment with nocodazole led to a decrease in ace-tub levels and an increase in tyr-tub levels (P < 0.05; Figure 1A–F). 
    Next, primary Schwann cells were isolated from neonatal rats, and passage 3 cells were treated with paclitaxel (1,10, 50, 100, or 200 nM) or nocodazole (1, 10, 100, 500, or 1000 nM) for 24 hours. As the drug concentration increased, the changes in cell morphology became more and more pronounced, and cell growth was increasingly hampered (Figure 1G & H). However, when the drugs were washed out, even the cells that had been treated with the highest concentrations recovered their normal morphology and regrew quickly (Figure 1I & J). These results indicate that the highest drug concentrations tested, 200 nM paclitaxel and 1000 nM nocodazole, safely regulate the microtubule dynamics of Schwann cells without inducing cell death; therefore, we chose these concentrations for use in subsequent experiments. 

    Figure 2|Paclitaxel and nocodazole affect axon and myelin degeneration in nerve explants. 

    Nerve explant cultures have been demonstrated to be a reliable in vitro model for studying WD (Shin et al., 2014; Park et al., 2015; Li et al., 2021). This simplified model eliminates unpredictable factors that can interfere with in vivo experiments. The nerve explants were cultured for 5 or 8 days, after which longitudinal sections were stained with NF200 (an axonal marker (Hu et al., 2019)) and MBP (a myelin marker (Li et al., 2021)). The immunostaining results showed that both the axons and myelin were undergoing degeneration, which is characteristic of WD. We found that treatment with paclitaxel significantly accelerated axon and myelin degeneration, while treatment with nocodazole significantly inhibited this process (both P < 0.05; Figure 2A–J). Similar trends were observed when NF200 and MBP levels were assessed by western blot (P < 0.05; Figure 2K–N).

    Figure 3|The effects of paclitaxel and nocodazole on myelin degeneration in nerve explants. 

    Figure 4|Teased nerve explant fibers exhibit myelin ovoid formation.

    ORO staining is used to label lipid droplets in adipocytes (Schachner-Nedherer et al., 2019; Velickovic et al., 2020). Recent studies have indicated that degenerated myelin fibers, which have a shrunken shape, also stain heavily with ORO, while normal myelin fibers, which have a circular shape, only stain faintly (Wang et al., 2020; Zou et al., 2020). Therefore, we stained cross sections of nerve explants cultured for 5 or 8 days with ORO and calculated the proportion of degenerated nerve fibers (shrunken myelin fibers with high fluorescence intensity) in each sample, as well as the mean intensity of ORO fluorescence. The proportion of degenerated myelin in the paclitaxel group was significantly higher than that in the vehicle group, and was the lowest in the nocodazole group (P < 0.05; Figure 3). Next, the nerves were teased into single nerve fibers and immunostained for α-tubulin to facilitate visualization of the myelin sheath by phase contrast microscopy. Myelin ovoids, which are the typical structure of a fragmented myelin sheath, was easily identified and quantified using this method (Jung et al., 2011) (Figure 4A and B). Consistent with the immunohistochemistry results, the number of myelin ovoids present in every 200-μm length of teased fiber (the ovoid index) (Jung et al., 2011) was highest in the paclitaxel group, followed by the vehicle group and then the nocodazole group (P < 0.05 among the three groups; Figure 4C).

    Figure 5|Effects of paclitaxel and nocodazole on Schwann cell dedifferentiation in sciatic nerve explants.  

    A WD progresses after PNI, mature Schwann cells undergo dedifferentiation. The dedifferentiated Schwann cells play a crucial role in WD; for example, they release chemoattractants to recruit macrophages that clear the debris of degenerated axons and myelin, and can phagocytose and degrade this debris themselves (Jang et al., 2017). Thus, we next performed immunostaining and western blotting with c-Jun (a marker of immature Schwann cells (Scapin et al., 2020)) and MAG (a marker of mature Schwann cells (Bolívar et al., 2020)) antibodies to evaluate Schwann cell dedifferentiation in nerve explants cultured for 5 or 8 days. The ratio of c-Jun/DAPI double-positive cells to all DAPI-positive cells and the level of c-Jun expression were dramatically higher in the cells induced with paclitaxel than in the vehicle and nocodazole groups, while both values were lowest in the nocodazole group. In contrast, the ratio of MAG/DAPI double-positive Schwann cells to all dapi-positive Schwann cells and the level of MAG expression were significantly higher in the paclitaxel group than in the vehicle group, and both values were lowest in the nocodazole group (P < 0.05; Figure 5). 

    Figure 6|Paclitaxel and nocodazole affect axonal regeneration in the distal trunk of the injured sciatic nerve at 3 dpi.

    To assess the role of microtubule dynamics on nerve regeneration, rats were subjected to sciatic nerve crush injury and immediately treated with paclitaxel, nocodazole, or saline (vehicle). Three days later, immunohistochemistry for GAP43 (a marker of axonal regeneration (Romeo-Guitart et al., 2020)) or SCG10 (a marker for regenerating sensory axons (Lai et al., 2020)) was performed on the injured nerves to detect regenerating axons. On cross sections taken 5 mm distal to the injured site, we found the highest density of both GAP43- and SCG10-positive axons in the paclitaxel group and the lowest density in the nocodazole group (P < 0.05; Figure 6A–D). To verify these results, total protein isolated from a 1-cm segment of the nerve spanning the injured site and the distal trunk was subjected to western blot. GAP43 and SCG10 expression in the injured nerve were significantly higher in the paclitaxel group compared with the vehicle group, and significantly lower in the nocodazole group (both P < 0.05; Figure 6E–G). 

    Figure 7|Effects of paclitaxel and nocodazole on nerve regeneration of the injured sciatic nerve at 28 dpi.

    Figure 8|Effects of paclitaxel and nocodazole on gastrocnemius muscle myoatrophy in rats with sciatic nerve injury. 

    Based on our and others’ previous studies, we selected 28 dpi as the time point for evaluating nerve recovery outcomes. At this time point, the numbers of NF200-positive axons and MBP/NF200 double positive myelinated axons in a cross section taken 5 mm distal to the injured site were highest in the paclitaxel group and lowest in the nocodazole group (P < 0.05; Figure 7). Next, we assessed the wet weight and total myofiber area of the gastrocnemius muscle, a key target muscle of the sciatic nerve, as these measures are widely used to assess nerve regeneration (Hu et al., 2020). Gross observation of the gastrocnemius muscles and closet observation of the hematoxylin-eosin-stained cross sections showed that myoatrophy was attenuated in the paclitaxel group compared with the vehicle group; however, myoatrophy was increased in the nocodazole group compared with the vehicle group (P < 0.05; Figure 8).

    Figure 9|Effects of paclitaxel and nocodazole on functional recovery after sciatic nerve injury. 

    The ultimate aim of nerve repair is to restore nerve conduction and reverse functional losses (Wang et al., 2014; Li et al., 2019). SFI assessment is widely used to test the functional recovery of the injured hindlimb after sciatic nerve injury in animal models (Hu et al., 2020; de Oliveira Marques et al., 2021). Therefore, we performed this assessment using a footprint test system, and found that there was significantly greater motor function recovery in the paclitaxel group compared with the vehicle group, and significantly lower recovery in the nocodazole group (P < 0.05; Figure 9A–C). CMAP amplitude and latency can be evaluated to assess nerve conduction (Pan et al., 2017; Park et al., 2018) (Figure 9D). Statistical analysis of the CMAP images generated in our study showed that, compared with the vehicle group, treatment with paclitaxel resulted in a higher amplitude (which indicates that more axons regenerated and arrived at the muscles measured in the paws (Zhan et al., 2013)), as well as a shorter latency (which corresponds to quicker nerve conduction (Pan et al., 2017)). The nocodazole group exhibited the lowest amplitude and longest latency among all three groups (P < 0.05; Figure 9E & F). 


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  • 发布日期: 2021-10-16  浏览: 535
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