Article Figures & Data
Figures
- Figure 1.
PEGPH20 decreases voluntary wheel running but does not cause acute mechanical hypersensitivity. A, Voluntary wheel running is significantly decreased up to 3 d after the administration of PEGPH20 (n = 12), compared with vehicle controls (n = 12). B, Withdrawal thresholds to muscle squeezing are significantly lower 5 d after the administration of PEGPH20 (n = 8) compared with vehicle-treated mice (n = 8). C, There are no significant differences in grip strength between mice treated with PEGPH20 (n = 8) versus vehicle-treated mice (n = 8). Two-way RM ANOVA (Bonferroni post hoc); A, F(1,14) = 6.654, p = 0.218; B, F(4,56) = 5.515, p = 0.0008; C, F(4,56) = 0.68. A–C, *p < 0.05. **p <0.01 versus vehicle.
- Figure 2.
PEGPH20 induces increased response to noxious metabolites in metabo-nociceptive primary muscle afferents. A–D, We did not observe changes in the mechanical thresholds or response patterns of mechanically sensitive neurons after PEGPH20 (n = 18) administration, compared with vehicle controls (n = 18). The same can be said of the responses to heat (n = 3 per group) or cold (n = 4 per group) stimulation. E–F, While there were no changes in the response to chemical stimulation in neurons sensitive to a low concentration of metabolites (vehicle, n = 5; PEGPH20 n = 12), injection of PEGPH20 caused a significant increase in the instantaneous frequency of firing in high metabolite responsive neurons (metabo-nociceptors; vehicle n = 8, PEGPH20 n = 9). G, Representative traces of mechanical, thermal, and chemical responses from neurons recorded from either vehicle or PEGPH20-treated mice. Arrows represent application of stimulus. A–F, Mann–Whitney U test, *p < 0.02 versus vehicle.
- Figure 3.
PEGPH20 disrupts of skeletal muscle architecture that is accompanied by macrophage infiltration. A, Muscle extracellular membrane (green, WGA) is disrupted 3 d after the administration of PEGPH20. EBD (red) usually remains in intravascular spaces in intact tissue but after hyaluronidase administration it leaks into the affected skeletal muscle. B, Percentage of myofibers positive for EBD 3 d after PEGPH20 administration. C, At the same time point macrophages (red, LysM/tdTomato) infiltrate the connective tissue surrounding the skeletal muscle in contrast to the almost nonpresent macrophages in the vehicle-treated animals. White scale bar: 50 μm. ***p < 0.001 χ2 test.
- Figure 4.
Ablation of macrophages does not prevent decreased voluntary wheel running after administration of PEGPH20. A, Administration of AP for 7 d starting 3 d before PEGPH20 injection removes macrophages from all hindpaw tissues as revealed by the lack of GFP signal (arrows) in the treated animals compared with vehicle-treated controls. B, Quantification of GFP signal reveals significantly less signal in AP-treated mice (n = 3) versus vehicle-treated animals (n = 3). C, Ablation of macrophages in MaFIA mice does not prevent the development of decreased voluntary wheel running distance after PEGPH20 (n = 12) administration compared with vehicle-treated mice (n = 12). In fact, the combination of PEGPH20+AP (n = 8) used to ablate macrophages prevents the slow recuperation that initiates around 4 d after PEGPH20 administration. D, Total wheel running distance is significantly lower in PEPH20, AP alone (n = 8), or PEGPH20+AP-treated mice compared with just vehicle-treated mice. The total running distance of PEGPH20+AP-treated mice was also significantly lower than in animals treated with just PEGPH20 or AP alone. White scale bar: 50 μm. B, Unpaired t test; C, Mixed-effects analysis (F(2,29) = 19.16, p < 0.0001) with Bonferroni post hoc; D, One-way ANOVA (F(3,24) = 38.87, p < 0.001) with Bonferroni post hoc. #p < 0.05, ^p < 0.01 versus PEGPH20+AP; *p < 0.05, **p < 0.01 versus vehicle.
Tables
Data structure Type of test Comparison 95% confidence interval a Normally distributed Two-way RM ANOVA Bonferroni post hoc test PEGPH20 vs API buffer day 1 368.4–7286 Bonferroni post hoc test PEGPH20 vs API buffer day 2 67.36–6985 Bonferroni post hoc test PEGPH20 vs API buffer day 3 695.6–7613 b Normally distributed Two-way RM ANOVA Bonferroni post hoc test PEGPH20 vs vehicle day 5 11.85–146.1 c Normally distributed Two-way RM ANOVA Bonferroni post hoc test −8.005–12.52 d Non-normally distributed Kruskal–Wallis test Test does not generate confidence interval e Non-normally distributed Mann–Whitney U test −1.000–3.000 f Non-normally distributed Mann–Whitney U test −1.000–2.000 g Non-normally distributed Mann–Whitney U test −80.66–89.10 h Non-normally distributed Mann–Whitney U test 1.700–24.62 i Non-normally distributed Mann–Whitney U test −12.70–24.30 j Non-normally distributed Mann–Whitney U test 1.700–24.62 k Normally distributed χ2 test Test does not generate confidence interval l Normally distributed Unpaired t test 22,155–108,900 m Normally distributed Mixed-effects analysis PEGPH20 vs vehicle day 1 −7169 to −963.0 Bonferroni post hoc test PEGPH20 vs vehicle day 2 −6877 to −456.6 Bonferroni post hoc test PEGPH20 vs vehicle day 3 −8772 to −279.7 Bonferroni post hoc test PEGPH20 vs PEGPH20+AP day 4 110.9–2674 Bonferroni post hoc test PEGPH20 vs PEGPH20+AP day 5 309.0–2507 Bonferroni post hoc test PEGPH20 vs PEGPH20+AP day 6 389.1–3866 Bonferroni post hoc test PEGPH20 vs PEGPH20+AP day 7 580.0–3549 n Normally distributed One-way RM ANOVA Bonferroni post hoc test PEGPH20 vs PEGPH20+AP 395.7–2221 Bonferroni post hoc test PEGPH20 vs vehicle −4459 to −2177 Bonferroni post hoc test PEGPH20+AP vs vehicle −5256 to −3997 Bonferroni post hoc test AP vs vehicle 1464–4666 - Table 2
General electrophysiological parameters from recordings 1 and 3 d after administration of PEGPH20 or vehicle
Condition Vehicle 1 d Vehicle 3 d Conduction 10.56 ± 1.99 6.71 ± 1.37 velocity (n = 27) (n = 21) Mechanical threshold 2.77 ± 1.08 4.29 ± 1.52 (n = 9) (n = 7) Mechanical response 44.02 ± 18.57 108.80 ± 50.19 (peak IF, Hz) (n = 9) (n = 7) Heat response 3.03 ± 0.95 4.7 ± 0.0 (peak IF, Hz) (n = 2) (n = 1) Cold response 63.53 ± 18.53 34.90 ± 33.20 (peak IF, Hz) (n = 2) (n = 2) Low metabolite response 13.4 ± 0.0 26.58 ± 23.24 (peak IF, Hz) (n = 1) (n = 4) Hight metabolite response 1.39 ± 0.64 8.37 ± 6.19 (peak IF, Hz) (n = 3) (n = 4) Condition PEGPH20 1d PEGPH20 3d Conduction 6.487 ± 1.18 10.33 ± 1.92 velocity (n = 24) (n = 24) Mechanical threshold 5.2 ± 1.11 1.92 ± 1.17 (n = 9) (n = 7) Mechanical response 77.41 ± 9.95 88.07 ± 31.53 (peak IF, Hz) (n = 9) (n = 7) Heat response 26.7 ± 0.0 7.9 ± 1.5 (peak IF, Hz) (n = 1) (n = 2) Cold response No cold responses
recorded39.48 ± 18.73 (peak IF, Hz) (n = 4) Low metabolite response 3.9 ± 1.45 30.66 ± 12.09 (peak IF, Hz) (n = 5) (n = 7) Hight metabolite response 13.16 ± 4.62 37.82 ± 14.05 (peak IF, Hz) (n = 4) (n = 5) We do not observe any significant differences between electrophysiological recordings performed either 1 or 3 d after treatment in each condition.
Gene D1 D3 D5 D7 TrkA 21.9 ± 13.5 70.2 ± 8.0 −60.7 ± 13.4* −52.2 ± 30.3 P2X3 49.8 ± 13.1 458.3 ± 13.8*** 114.8 ± 29.2 459.6 ± 25.9** P2X5 −4.4 ± 10.2 −63.9 ± 39.1 −37.0 ± 38.9 −62.8 ± 15.9 TRPV1 9.3 ± 15.2 58.6 ± 9.0 −66.3 ± 20.1 −63.4 ± 49.4 TRPA1 −23.8 ± 12.6 −13.66 ± 113.03 58.3 ± 28.6 −32.99 ± 37.53 ASIC3 0.8 ± 7.2 7.5 ± 5.0 −82.77 ± 110.1 −76.33 ± 61.62 GFRα1 −2.8 ± 7.3 −81.8 ± 12.1** −93.1 ± 26.8*** −97.6 ± 56.1*** GFRα2 30.3 ± 18.8 −44.8 ± 95.9 −55.9 ± 41.4 −69.2 ± 29.4 GFRα3 −7.6 ± 5.5 −60.5 ± 120.6 −18.5 ± 37.31 −37.8 ± 14.72 IL1r1 −17.9 ± 11.9 −90.1 ± 85.3* −83.3 ± 28.5 −89.1 ± 7.9* Values indicate % change versus vehicle-treated controls. Mean ± SEM n = 4 per group per time point. One-way ANOVA (TrkA F(4,14) = 10.57, p = 0.0004; P2X3 F(4,14) = 17.27, p < 0.0001; GFRα1 F(4,13) = 29.57, p < 0.0001; LI1r1 F(4,12) = 8.272, p = 0.0019) with Bonferroni post hoc. *p < 0.05, **p < 0.01, ***p < 0.001 versus vehicle-treated controls. GAPDH CT (mean ± SEM) CTRL: 20.97 ± 0.26; D1 19.651 ± 0.07; D3 20.295 ± 0.45 D5 21.2 ± 0.39 D7 22.32 ± 0.82. No significant differences were detected between the CTRL or treatment groups (one-way ANOVA F(4,14) = 3.8, p = 0.026).
Vehicle PEGPH20 PEGPH20+AP AP 1d 5100.08 ± 972.65 m 1034.07 ± 507.76 m* 297.27 ± 63.98 m** 2118.52 ± 622.81 m 2d 4314.84 ± 1028.88 m 647.95 ± 237.69 m* 97.19 ± 92.70 m* 529.68 ± 225.32 m* 3d 5617.62 ± 1360.53 m 1091.83 ± 372.54 m 319.24 ± 113.41 m* 4541.73 ± 2236.27 m 4d 4566.33 ± 987.13 m 1457.09 ± 454.28 m* 64.62 ± 22.11 m* 419.32 ± 340.40 m* 5d 4308.31 ± 793.60 m 1624.41 ± 386.50 m* 216.20 ± 85.72 m** # 1819.74 ± 1368.44 m* 6d 4795.18 ± 1037.20 m 2278.82 ± 616.13 m 151.05 ± 40.59 m* # 646.48 ± 496.81 m* 7d 5052.14 ± 1275.32 m 2393.98 ± 522.54 m 329.55 ± 108.71 m* # 2223.02 ± 1497.26 m Administration of AP alone (n = 6) induced changes similar to vehicle (n = 8), PEGPH20 (n = 12), and PEGPH20+AP (n = 12). the combination of PEGPH20+AP produced significantly lower levels of activity during the first 4 d after administration but did not show the recuperation observed by 7 d in both the PEGPH20 or AP alone treated groups. Data from AP alone for day 3 only includes n = 2 because of equipment failure that did not allow capture of data for the single time point. Two-way ANOVA, F(3,34) = 12.54, p < 0.0001 with Bonferroni post hoc test. *p < 0.05, **p < 0.01 versus vehicle, #p < 0.05 versus PEGPH20.
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