Article Figures & Data
Figures
- Figure 1.
Spatiotemporal change for rat sciatic nerve whole ATP levels with fluorescence microscope after axotomy. a, Representative whole nerve intracellular ATP images before (pre) and 5, 30, 60, 120, 180, 240, 300, and 360 min after axotomy in the same individuals. Scale bar, * 1 mm. P, proximal. D, distal. b, The change of ATP levels with six ROIs (from ROI #1 to #6; n = 4 rat per group). Distal nerve stump ATP levels (ROI #5 and #6) were maintained over 360 min. c, Representative whole nerve intracellular ATP images before (pre), and 1 d (24 h), 2 d (48 h), and 14 d after axotomy in the separate individuals. Scale bar, * 1 mm. P, proximal. D, distal. d, The change of ATP levels with six ROIs (from ROI #1 to #6; n = 6 rat per group). Distal nerve stump ATP levels (ROI #5 and #6) were significantly decreased from 2 d after axotomy. The ROI for measuring ATP levels was set on six regions: three proximal (from ROI #1 to #3) and three distal (from ROI #4 to #6) sites for axotomy, and a 1-mm square with 500-μm spacing (Extended Data Fig. 1-1). The OFP/GFP ratios ranged from 1.0 to 2.5. Error bars indicate SD; *p < 0.05, **p < 0.01, ***p < 0.001, two-way ANOVA followed by the Tukey’s post hoc test. ROI, region of interest.
- Figure 2.
Wallerian degeneration and mitochondrial degeneration in axon for rat’s distal nerve stump. a, Representative images of toluidine blue staining, immunohistochemistry (NF-H), electron microscopy, before (pre) and 1, 2, and 14 d after axotomy. Immunohistochemistry was obtained the same results for three individuals. Scale bar, * 10 μm, ** 2 μm. Arrows, myelinated axon; arrowheads, demyelinated Schwann cell. b, Quantification of the myelinated axon. c, d, Quantification of the G-ratio [c: cumulative G-ratio per individual, d: scatter plot, Spearman’s rank correlation coefficient and p values showing G-ratio of individual myelinated axons against axon diameter (n = 400)]. The histologic WD was observed significantly from 2 d after axotomy. In axons, before (pre) and 1 and 2 d after axotomy were evaluated, except for after 14 d with completely collapsed axon morphology (n = 4 rat per group). e, Representative images of mitochondrial findings, before (pre) and 1, 2, and 14 d after axotomy. Evaluation was performed in axons and Schwann cells separately. Scale bar, * 200 nm. f, Violin plot of mitochondrial diameter (nm; n = 200). Cumulative mitochondria diameter per individual are also shown (n = 4 rat per group). g, Quantitative analysis of atypical mitochondria (%; n = 4 rat per group). In axons, mitochondria were significantly degenerated from 1 d after axotomy, whereas in Schwann cells, mitochondria were not significantly degenerated until 14 d after axotomy. All histologic evaluations were performed 3 mm distal to the sectional end and corresponding uninjured nerve. Error bars indicate SD; *p < 0.05, ***p < 0.001, one-way ANOVA followed by the Tukey’s post hoc test (for comparison of axons and Schwann cells, respectively).
- Figure 3.
The gradual decrease of mice longitudinal nerve cross section ATP levels in the distal nerve stump, corresponds to histology. a, Representative ATP images in the longitudinal section before (pre) and 1, 2, and 7 d after axotomy. Scale bar, * 1 mm. P, proximal. D, distal. b, The change of ATP levels with measured ROIs (n = 6 mice per group). Distal nerve stump ATP levels were significantly decreased from 1 d after axotomy. Error bars indicate SD; **p < 0.01, ***p < 0.001, one-way ANOVA followed by the Tukey’s post hoc test. c, Representative enlarged images of two-photon microscopy, immunohistochemistry (left, NF-H/S-100B/DAPI; right, CD68/S-100B/DAPI), and toluidine blue staining, before (pre) and 1, 2, and 7 d after axotomy. Scale bar, * 10 μm. Arrows, macrophage; arrowheads, Schwann cell; double arrows, axonal soma. The ROI for measuring ATP levels was set at 3 mm distal from the sectional end, the center of the longitudinal cross-section, with a 300 × 300-μm square (Extended Data Fig. 3-1). The OFP/GFP ratios ranged from 1.5 to 3.0. All histologic evaluations were performed with the same results for three individuals, 3 mm distal from the sectional end, and the corresponding uninjured nerve. ROI, region of interest.
- Figure 4.
Activation of the glycolytic system. Diagram of energy metabolism pathways and mass spectrometry before and 2 d (48 h) after axotomy. MPC transports pyruvate, the end-product of the glycolytic system, for metabolism in the TCA cycle. Mass spectrometry was performed for the metabolites of glycolysis and the TCA cycle, the end-product ATP, and the coenzyme NAD+. Glycolytic intermediates, NAD+ and ATP were significantly decreased after axotomy. These results are shown in a boxplot containing the interquartile range and centered bars as the median (n = 5 mice per group). All examinations were performed continuously 7 mm from the sectional end of the right sciatic nerve with transection and the corresponding lesion on the left side. *p < 0.05, **p < 0.01, Mann–Whitney U test. CS, citrate synthase; F6P, fructose 6-phosphate; G6P, glucose-6-phosphate; GA3P, glyceraldehyde-3-phosphate; HK, hexokinase; IDH, isocitrate dehydrogenase; LDH, lactate dehydrogenase; NAD+, nicotinamide adenine dinucleotide; PFK, phosphofructokinase; PDH, pyruvate dehydrogenase; TCA cycle, tricarboxylic acid cycle.
- Figure 5.
Activation of the glycolytic system and MCTs in Schwann cell after axotomy. a, c, e, g, Representative cross-section images of immunohistochemistry for glycolytic enzyme (a: HK-1, c: PFK-1, e: LDHA, g: LDHB) before and 2 d after axotomy. b, d, f, h, The number of HK-1 (b), PFK-1 (d), LDHA (f), and LDHB (h) positive Schwann cells (n = 3 mice per group). HK-1, PFK1, and LDHA positive Schwann cells were significantly increased after axotomy, whereas LDHB positive Schwann cells were significantly decreased. i, k, Representative images of immunohistochemistry for MCT (e: MCT-1, g: MCT-4) before and after axotomy. j, l, The number of MCT-1 (j) or MCT-4 (l) positive Schwann cells (n = 3 mice per group). MCT-1 and MCT-4 positive Schwann cells were significantly increased after axotomy. All histologic evaluations were performed 3 mm distal to the sectional end and corresponding uninjured nerve. Error bars indicate SD. Arrows, HK-1/PFK-1/LDHA/LDHB/MCT-1/MCT-4 positive Schwann cells; scale bar, 10 μm. *p < 0.05, **p < 0.01, two-tailed t test. HK, hexokinase; LDHA, lactate dehydrogenase A subunit lactate; LDHB, lactate dehydrogenase B subunit lactate; dehydrogenase; MCT-1, monocarboxylate transporters 1; MCT-4, monocarboxylate transporters 4; PFK, phosphofructokinase.
- Figure 6.
Activation of the glycolytic system and inactivation of the TCA cycle in axons after axotomy. a, c, e, g, Representative cross-section images of immunohistochemistry for glycolytic enzyme (a: HK-1, c: PFK-1, e: LDHA, g: LDHB) before and 2 d after axotomy. The axonal fluorescence intensity in the dashed line was measured. b, d, f, h, The axonal fluorescence intensity of HK-1 (b), PFK-1 (d), LDHA (f), and LDHB (h; n = 3 mice per group). HK-1, PFK1, and LDHA fluorescence intensity was significantly increased after axotomy, whereas LDHB was significantly decreased. i, k, m, Representative images of immunohistochemistry for TCA cycle enzyme (i: PDH-E1α, k: CS, m: IDH3A) before and after axotomy. The axonal fluorescence intensity in the dashed line was measured. j, l, n, The axonal fluorescence intensity of PDH-E1α (j), CS (k), and IDH3A (n; n = 3 mice per group). PDH-E1α, CS, and IDH3A fluorescence intensity was significantly decreased after axotomy. All histologic evaluations were performed 3 mm distal to the sectional end and corresponding uninjured nerve. Error bars indicate SD scale bar, 10 μm. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed t test. CS, citrate synthase; IDH, isocitrate dehydrogenase; HK, hexokinase; LDHA, lactate dehydrogenase A subunit lactate; LDHB, lactate dehydrogenase B subunit lactate; dehydrogenase; PFK, phosphofructokinase; PDH, pyruvate dehydrogenase; TCA cycle, tricarboxylic acid cycle.
- Figure 7.
Inhibition of the glycolytic system or MCTs progress Wallerian degeneration with decreased ATP levels, but MPC does not. a, Representative ATP images for each inhibitor in both sham and transection side. For each experiment, lower figures are enlarged image with measured ROI. Upper figures present overall distal nerve stump ATP image, from the sectional end. Scale bar, * 1 mm, ** 10 μm. P, proximal. D, distal. b, The distal nerve stump ATP levels with measured ROI (n = 6 mice per group). c, Representative images of toluidine blue staining, immunohistochemistry (NF-H/S-100B/DAPI), electron microscopy for each inhibitor in both sham and transection side. Scale bar, * 10 μm, ** 2 μm. d, Quantification of the myelinated axon. e–g, Quantification of the G-ratio [e: cumulative G-ratio per individual; f, g: scatter plot, Spearman’s rank correlation coefficient and p values showing G-ratio of individual myelinated axons against axon diameter (n = 400) in both (f) sham and (g) transection side] (n = 4 mice per group). h, Representative images of immunohistochemistry (MCT-1/S-100B/DAPI) for each inhibitor in both sham and transection side. Scale bar, * 10 μm. i, The number of MCT-1 positive Schwann cells (n = 4 mice per group). j, Representative images of mitochondrial findings for each inhibitor in both sham and transection side. Evaluation was performed in axons and Schwann cells separately. Scale bar, * 200 nm. k, Violin plot of mitochondrial diameter (nm; n = 200). Cumulative mitochondria diameter per individual are also shown (n = 4 rat per group). l, Quantitative analysis of atypical mitochondria (%; n = 4 mice per group). All examinations were performed 1 d (24 h) after inhibitor administration and evaluated 3 mm distal from the sectional end and the corresponding uninjured nerve (Extended Data Fig. 7-1a). The concentrations of all inhibitors were determined from preliminarily experiments (Extended Data Fig. 7-1b–d), by measuring ATP levels 3 mm distal to the sectional end, with a 500 × 500-μm square (Extended Data Fig. 7-2). The ROI for measuring ATP levels was set at 3 mm distal from the sectional end, the center of the longitudinal nerve cross-section, with a 300 × 300-μm square (Extended Data Fig. 3-1). The OFP/GFP ratios ranged from 1.5 to 3.0. Immunohistochemistry showed the same results for the four individuals. A summary of the inhibition experiments is provided in Extended Data Table 7-1. Error bars indicate SD; *p < 0.05, **p < 0.01, ***p < 0.001, two-way ANOVA followed by the Tukey’s post hoc test (for comparison of sham group and transection group, respectively). No statistical data were available for mitochondrial diameter because there was no equal distribution. 2-DG, 2-deoxyglucose; 4-CIN, a-cyano-4-hydroxycinnamic acid; MCT-1, monocarboxylate transporters 1; MSDC-0160, mitochondrial pyruvate carrier (MPC) inhibitors; ROI, region of interest.
- Figure 8.
Ethyl pyruvate (EP) delays Wallerian degeneration with increased ATP levels. a, Representative ATP images for each treatment. Lower figures are enlarged image in the measured ROI. Upper figures present overall distal nerve stump image, from the sectional end. Scale bar, * 1 mm, ** 10 μm. P, proximal. D, distal. b, The distal nerve stump ATP levels with measured ROIs (n = 6 mice per group). c, Representative images of toluidine blue staining, immunohistochemistry (NF-H/S-100B/DAPI), electron microscopy, for each treatment. Scale bar, * 10 μm, ** 2 μm. d, Quantification of the myelinated axon. e, f, Quantification of the G-ratio [e: cumulative G-ratio per individual, f: scatter plot, Spearman’s rank correlation coefficient and p values showing G-ratio of individual myelinated axons against axon diameter (n = 400)] (n = 4 mice per group). g, Representative images of immunohistochemistry (MCT-1/S-100B/DAPI) for each treatment. Scale bar, * 10 μm. h, The number of MCT-1 positive Schwann cells (n = 4 mice per group). i, Representative images of mitochondrial findings for each treatment. Evaluation was performed in axons and Schwann cells separately. Scale bar, * 200 nm. j, Violin plot of mitochondrial diameter (nm; n = 200). Cumulative mitochondria diameter per individual are also shown (n = 4 rat per group). k, Quantitative analysis of atypical mitochondria (%; n = 4 mice per group). All examinations were performed 2 d (48 h) after administration and evaluated 3 mm distal from the sectional end and the corresponding uninjured nerve. The ROI for measuring ATP levels was set at 3 mm distal from the sectional end, the center of the longitudinal nerve cross-section, with a 300 × 300-μm square (Extended Data Fig. 3-1). The OFP/GFP ratios ranged from 1.5 to 3.0. Immunohistochemistry showed the same results for the four individuals. Error bars indicate SD; *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA followed by the Tukey’s post hoc test (for comparison of sham group and transection group, respectively). EP, ethyl pyruvate; MCT-1, monocarboxylate transporters 1; ROI, region of interest.
- Figure 9.
Model depicting changes in the energy metabolism of Schwann cells and axons after axotomy. The proposed metabolic changes in the distal nerve stump. In Schwann cells, the glycolytic system and MCT-induced monocarboxylate transport are activated. In axons, the glycolytic system is activated, whereas TCA cycle is inactivated because of mitochondrial degeneration. The glycolytic intermediate, pyruvate is transported from Schwann cells to axons through MCTs, and both in Schwann cells and axons, pyruvate is metabolized by LDHA to generate NAD+ and promote metabolic shifts toward the glycolytic system to produce ATP. The main metabolic source of ATP is mainly on glycolytic system both in Schwann cells and axons. The excess produced lactate in axons is transported to Schwann cells, subsequently released to extracellular space via MCTs.
Extended Data
Extended Data Figure 1-1
Experimental system for ATP levels with fluorescence microscopy and histological analysis in rats. A schematic illustration of whole nerve ATP levels measurement for GO-ATeam2 rat. All of fluorescence emission in the GO-ATeam2 probe was captured with fluorescence microscopy. ATP levels were intermittently measured in six ROIs (from ROI #1 to #6), three proximal and three distal sites for axotomy, 1-mm square with 500-μm spacing in the same individual. OFP/GFP ratio ranges were from 1.0 to 2.5. The histological analysis was performed 3 mm distal from the sectional end, correspond to ROI #6. Scale bar, * 1 mm,** 1-mm square ROI. P, proximal. D, distal. Download Figure 1-1, TIF file.
Extended Data Figure 3-1
Experimental system for ATP levels with two-photon microscopy and histological analysis in mice. A schematic illustration of center of the longitudinal nerve cross-section ATP levels measurement for GO-ATeam2 mouse. All of fluorescence emission in the GO-ATeam2 probe was captured with two-photon microscopy. ATP levels were measured in each ROIs (*** 300-μm square), 3 mm distal from the sectional end (broken line). OFP/GFP ratio ranges were from 1.5 to 3.0. The histological analysis was performed 3 mm distal from the sectional end. Scale bar, * 1 mm;*** 500-μm square ROI. P, proximal. D, distal. Download Figure 3-1, TIF file.
Extended Data Figure 7-1
Inhibition experiments following the test of two concentrations and measuring ATP levels until 120 min after axotomy. a, A schematic illustration of inhibition experiments. The following inhibitors was used; 2-DG, MSDC-1060, and 4-CIN. The control was set as 0.1% DMSO dissolved in saline. Bilateral sciatic nerves were exposed, and transection side as right, sham side as left. Immediately after the transection of right sciatic nerve, bilateral sciatic nerves were covered with a gelatin sponge soaked with inhibitors. One day after the surgical procedure, ATP levels and histology were evaluated in 3 mm distal from the sectional end. b, Energy metabolic interactions between Schwann cells and axon, and the working point of the inhibitors. Monocarboxylate produced by the Schwann cell glycolytic system is transported to axons through MCTs. Pyruvate, the end product of the glycolytic system, is transported to the mitochondrial matrix through MPC and metabolized in the TCA cycle and electron transport chain. 2-DG is a glycolysis inhibitor, MSDC-0160 is the MPC inhibitor, and 4-CIN is the comprehensive MCTs inhibitor. c, Representative intracellular distal nerve stump ATP levels for each inhibitor compared to the DMSO group, before (pre) and 15, 30, 45, 60, 75, 90, 105, and 120 min after axotomy. Scale bar, * 1mm. P, proximal. D, distal. d, ATP reduction response for two different concentrations of each inhibitor, compared to the DMSO group. Two concentrations of several inhibitors were preliminary tested. *p < 0.05, two-way ANOVA followed by the Tukey’s post hoc test. The ROI for measuring ATP levels was set at 3 mm distal to the sectional end, with a 500 × 500-μm square (Extended Data Fig. 7-2). A higher concentration (150 mm 2DG, 100 μm MSDC-0160, and 10 mm 4-CIN) was applied for experiment in all inhibitors. The OFP/GFP ratios ranged from 1.5 to 3.0. Error bars indicate SD. *p < 0.05, one-way ANOVA followed by Tukey’s post hoc test. Download Figure 7-1, TIF file.
Extended Data Figure 7-2
Experimental system for ATP levels with fluorescence microscopy in mice. A schematic illustration of whole nerve ATP levels measurement for GO-ATeam2 mouse. All of fluorescence emission in the GO-ATeam2 probe was captured with fluorescence microscopy. ATP levels were measured in each ROIs (** 500-μm square), 3 mm distal from the sectional end (broken line). OFP/GFP ratio ranges were from 1.5 to 3.0. The histological analysis was performed 3 mm distal from the sectional end. Scale bar, * 1 mm;** 500-μm square ROI. *** 500-μm square ROI. P, proximal. D, distal. Download Figure 7-2, TIF file.
Extended Data Table 7-1
The summary of the inhibition experiments. The downward arrow means a decrease in the value of each variable relative to the horizontal arrow, whereas upward arrow means an increase. The more numbers of arrows, the greater the change in value. 2-DG, 2-deoxyglucose; 4-CIN, a-cyano-4-hydroxycinnamic acid; MCT-1, monocarboxylate transporters 1; MSDC-0160, mitochondrial pyruvate carrier (MPC) inhibitors. Download Table 7-1, DOCX file.
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