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    Effects of delayed repair of peripheral nerve injury on the spatial distribution of motor endplates in target muscle
  • Figure 1|Schematic diagram of the measurement of the width of MEP lamellar clusters on cross-sections of the gastrocnemius.

    To observe the spatial distribution of MEPs in gastrocnemius muscle, the intact muscles were imaged under a light-sheeting ultramicroscope (LaVisionBioTec, Bielefeld, Germany) equipped with MV PLAPO 2X/0.5 dry objective (W.D. 20 mm). The fluorescence image of MEPs (633 nm) in control (n = 6), immediate repair (n = 6), 1-month delay (n = 6), and 3-month delay (n = 7) groups was captured by light-sheet microscopy. The Z-axis step size was 5 μm. The acquired images were analyzed using Imaris software (Bitplane, Zurich, Switzerland) to reconstruct the spatial distribution of MEPs and count their number. To obtain the cross-sectional images of the gastrocnemius and measure the width of the lamellar clusters, we resampled the image stacks, and the MIP of Z-stacks (thickness = 250 μm) were made with ImageJ. There are four lamellar clusters of MEPs (medial MEP lamella, and lateral MEP lamellae (LML) 1–3) on cross-sections of the gastrocnemius (Yin et al., 2019) (Figure 1). For each MEP lamella, to calculate the width, we selected one cross-sectional MIP image of the proximal end, middle portion and distal end of the gastrocnemius. We took the average width of these three images as the MEP lamellar width.


    Figure 2|Electrophysiological examination of gastrocnemius muscles in mice with delayed repair after peripheral nerve injury.

    Three months after nerve repair, gastrocnemius CMAPs were recorded in each group. The waveforms in the repair groups (immediate repair, 1-month delay, and 3-month delay) were the same as that in the control group (Figure 2). The amplitudes of the gastrocnemius CMAPs were 16.0 ± 1.6, 10.4 ± 1.1, 1.2 ± 0.5 and 28.5 ± 3.1 mV in the immediate repair, 1-month delay, 3-month delay and control groups, respectively. CMAP amplitudes in the four groups were significantly different (P < 0.05).


    Figure 3|Osmium tetroxide staining of the regenerated tibial nerve in the gastrocnemius of mice with delayed repair after peripheral nerve injury.

    Osmium tetroxide staining revealed regenerated myelinated axons in the distal tibial nerve (Figure 3). The number of regenerated axons decreased with increasing denervation time. Except for the immediate repair and 1-month delay groups, there were significant differences (P < 0.05) in the number of regenerated axons among the groups.


    Figure 4|Confocal microscopy of the MEPs in the gastrocnemius of mice with delayed repair after peripheral nerve injury.


    Confocal microscopic images revealed the structure of regenerating MEPs. The shape of MEPs in the gastrocnemius appeared uniformly pretzel-shaped in the immediate repair and control groups, whereas the shapes of the MEPs in the 1-month delay and 3-month delay groups were irregular and the fluorescent signal was heterogeneous (Figure 4A–D). The mean area of MEPs in the 3-month delay group was significantly smaller than those in the immediate repair and control groups (P < 0.05; Figure 4E). There were no differences between the immediate repair and control groups (P > 0.05). We also assessed maturation of MEPs by counting perforations in single MEPs, and found significant difference among the four groups (P < 0.05; Figure 4F). The maturity of MEPs gradually decreased with increasing denervation time.


    Figure 5|Three-dimensional reconstruction of MEPs in the gastrocnemius in the different groups of mice with delayed repair after peripheral nerve injury.

    Figure 6|Cross-sectional images of MEPs in the gastrocnemius in the different groups of mice with delayed repair after peripheral nerve injury.

    To assess whether delayed repair influences the spatial redistribution of MEPs in gastrocnemius muscle, light-sheet microscopy was used to observe structural changes. Three-dimensional reconstruction (Additional Video 1) showed that the regenerated MEPs were also distributed within lamellar clusters after delayed repair (Figures 5 and 6A–D). However, the homogeneity of MEPs and lamellar cluster dimensions were changed in the 3-month delay group. The width of the lamellar clusters on cross-sections of the gastrocnemius in the 3-month delay group was larger than in the other three groups (P < 0.05). However, there was no difference in width of the lamellar clusters in cross-sections of the gastrocnemius among the immediate repair, 1-month delay and control groups (P > 0.05; Figure 6E). Moreover, we counted the MEPs in each group. This revealed differences among the four groups (P < 0.05; Figure 6F). To examine MEP regeneration in the lamellar clusters, we further counted the MEPs per lamellar cluster. As with the number of total MEPs, the number of MEPs per lamellar cluster also gradually declined with increasing denervation time (P < 0.05; Figure 6G), and the rate of decline was different among the lamellar clusters in the 3-month delay group (Table 1).


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  • 发布日期: 2021-10-11  浏览: 597
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