Prevention of vincristine-induced peripheral neuropathy by genetic deletion of SARM1 in mice

Abstract

Peripheral polyneuropathy is a common and dose-limiting side effect of many important chemotherapeutic agents. Most such neuropathies are characterized by early axonal degeneration, yet therapies that inhibit this axonal destruction process do not currently exist. Recently, we and others discovered that genetic deletion of SARM1 (sterile alpha and TIR motif containing protein 1) dramatically protects axons from degeneration after axotomy in mice. This finding fuels hope that inhibition of SARM1 or its downstream components can be used therapeutically in patients threatened by axonal loss. However, axon loss in most neuropathies, including chemotherapy-induced peripheral neuropathy, is the result of subacute/chronic processes that may be regulated differently than the acute, one time insult of axotomy. Here we evaluate if genetic deletion of SARM1 decreases axonal degeneration in a mouse model of neuropathy induced by the chemotherapeutic agent vincristine. In wild-type mice, 4 weeks of twice-weekly intraperitoneal injections of 1.5 mg/kg vincristine cause pronounced mechanical and heat hyperalgesia, a significant decrease in tail compound nerve action potential amplitude, loss of intraepidermal nerve fibres and significant degeneration of myelinated axons in both the distal sural nerve and nerves of the toe. Neither the proximal sural nerve nor the motor tibial nerve exhibit axon loss. These findings are consistent with the development of a distal, sensory predominant axonal polyneuropathy that mimics vincristine-induced peripheral polyneuropathy in humans. Using the same regimen of vincristine treatment in SARM1 knockout mice, the development of mechanical and heat hyperalgesia is blocked and the loss in tail compound nerve action potential amplitude is prevented. Moreover, SARM1 knockout mice do not lose unmyelinated fibres in the skin or myelinated axons in the sural nerve and toe after vincristine. Hence, genetic deletion of SARM1 blocks the development of vincristine-induced peripheral polyneuropathy in mice. Our results reveal that subacute/chronic axon loss induced by vincristine occurs via a SARM1 mediated axonal destruction pathway, and that blocking this pathway prevents the development of vincristine-induced peripheral polyneuropathy. These findings, in conjunction with previous studies with axotomy and traumatic brain injury, establish SARM1 as the central determinant of a fundamental axonal degeneration pathway that is activated by diverse insults. We suggest that targeting SARM1 or its downstream effectors may be a viable therapeutic option to prevent vincristine-induced peripheral polyneuropathy and possibly other peripheral polyneuropathies.

Keywords: CIPN; SARM1; axonal degeneration; chemotherapy-induced peripheral neuropathy; vincristine.

Figure 1

SARM1 knockout blocks vincristine-induced degeneration of IENF. (A) IENFs stained with the pan-axonal marker PGP 9.5 (red) extend at a ∼90° angle from the subdermal plexus into the stratum spinosum of the epidermis (nuclei stained with DAPI in blue). (B) Same image as A, without overlay of the epidermis. Note that IENF extend into superficial layers of the stratum spinosum. (C) In vincristine-treated wild-type mice fewer fibres cross into the epidermis and many IENFs appear short. (D) Quantification of IENF density. There are significantly fewer IENFs in vincristine-treated wild-type mice as compared to vehicle-treated wild-type and SARM1 knockout mice as well as SARM1 knockout treated vincristine mice. Data are presented as mean ± SEM [one-way ANOVA, F(3,41) = 6.870; P = 0.0007; post hoc Tukey’s; *P < 0.05; **P < 0.01; n = 8–9 for vehicle-treated wild-type (WT Veh) and SARM1 knockout (SARM1 KO Veh) mice and n = 14 for vincristine-treated wild-type (WT Vinc) and SARM1 knockout mice (SARM1 KO Vinc)]. For clarity in the figure, only significant results are indicated. (E) IENFs in vehicle-treated SARM1 knockout mice look similar to IENFs in vehicle-treated wild-type mice (B). (F) Representative image of IENFs in vincristine-treated SARM1 knockout mice. Note that many fibres cross into the epidermis and extend to the superficial layers of the stratum spinosum. Scale bar = 25 μm.

Figure 2

SARM1 knockout prevents degeneration of myelinated axons in the distal sural nerve in VIPN. (A, B, D and E) Photomicrographs of toluidine blue stained semithin cross sections of the distal sural nerve. There was no apparent difference in gross morphology between vehicle-treated wild-type (A) and SARM1 knockout (D) mice. In vincristine-treated wild-type mice, axons appear less densely packed (B). Some myelinated axons exhibit a homogenous pale or dark cytoplasm and disrupted myelin sheath (B, arrows, insert), likely representing degenerating axons. When axons with this morphology were evaluated at the electron microscopy level, they indeed show morphological signs of axonal degeneration (C, arrow). In contrast, distal sural nerves of vincristine-treated SARM1 knockout mice (E) look very similar to vehicle-treated sural nerves of SARM1 knockout mice. At the electron microscopy level, myelinated and unmyelinated axons of vincristine-treated SARM1 knockout mice appear morphologically intact (F). (G) When the same sural nerve is analysed at proximal and distal levels, there are significantly fewer axons distally in vincristine-treated wild-type mice, but not in vincristine-treated SARM1 knockout and vehicle-treated wild-type and SARM1 knockout mice [two-way repeated measures ANOVA, there was a significant main effect of genotype × proximal/distal localization F(3,32) = 4.287; P = 0.0119; Sidak’s post hoc ***P < 0.001; n = 7–12 per group]. There was no difference in axon number of the proximal sural nerve between vehicle and vincristine-treated wild-type and SARM1 knockout mice [one-way ANOVA, F(3,32) = 2.736, P = 0.0597]. (H) Vincristine treated wild-type mice have significantly decreased axon density in the distal sural nerve compared to vehicle-treated wild-type and SARM1 knockout mice as well as vincristine-treated SARM1 knockout mice [one-way ANOVA; F(3,41) = 4.361; P = 0.0094; Tukey’s post hoc *P < 0.05; n = 9–13 per group]. (I and J) Analysis of axon size of the distal sural nerve reveals a decrease in the proportion of small myelinated axons in vincristine-treated wild-type mice (I, multiple t-tests with Bonferroni correction; ***P < 0.001; n = 9–10), but not in vincristine-treated SARM1 knockout mice (H; multiple t-tests; n = 8–11). In A, the asterisk marks a granular cell; in B and C, arrows point to myelinated axons with signs of degeneration; in C, arrowhead points to a degenerating Remak bundle. I and J analysed to axon size of 38 μm, shown to size of 20 μm for clarity. Scale bar in A, B, D, and E = 10 μm; C and F = 2 μm. KO = knockout.

Figure 3

Genetic deletion of SARM1 blocks vincristine-induced axonal degeneration in nerve fascicles of the toe. (A–D) Photomicrographs of toluidine blue-stained semithin sections of nerve fascicles in the toe demonstrate morphological changes and axonal degeneration in vincristine-treated wild-type mice (B, arrows), whereas nerve fascicles of vincristine-treated SARM1 knockout (D) and vehicle-treated wild-type (A) and SARM1 knockout (C) appear morphologically similar. (E) There are significantly more axons with signs of degeneration in vincristine-treated wild-type mice when compared to vehicle-treated wild-type and SARM1 knockout as well as vincristine-treated SARM1 knockout mice [one-way ANOVA F(3,40) = 13.34; P < 0.0001; Tukey’s multiple comparison test; ****P < 0.0001; ***P < 0.001, n = 7 for vehicle-treated wild-type and SARM1 knockout mice, n = 13 for vincristine-treated wild-type and SARM1 knockout mice]. For clarity in the figure, only significant results are indicated. Scale bar = 10 μm. KO = knockout.

Figure 4

SARM1 knockout preserves the tail action potential amplitude following vincristine treatment. (A) Depicted are representative tracings from one wild-type and one SARM1 knockout mouse before and after vincristine. Note the decreased tail amplitude after vincristine treatment in wild-type, but not SARM1 knockout mice. (B) The tail amplitude is not statistically significantly different between groups at baseline [one-way ANOVA, P = 0.1031, F(3,46) = 2.181; n = 10–15 per group]. (C) After vincristine, wild-type mice show a decreased tail amplitude [one-way ANOVA, P = 0.0091 F(3,46) = 4.325, **P < 0.01, Tukey’s post hoc; n = 10–15 per group], whereas the tail amplitude of vincristine-treated SARM1 knockout mice is not significantly different. (D) There was no change in conduction velocity in any of the groups [two-way ANOVA, there was no significant main effect between groups F(3,46) = 2.787, P = 0.0512 and between pre and post vincristine treatment F(1,46) = 0.9229, P = 0.3417; n = 10–15 per group]. KO = knockout.

Figure 5

SARM1 knockout prevents the development of hyperalgesia in VIPN. (A) The mechanical withdrawal threshold is significantly reduced after vincristine in wild-type mice, but not in SARM1 knockout mice. There was a significant main effect between groups [two-way repeated measures ANOVA F(1,15) = 9.718; P = 0.0071), time F(1,15) = 19.74, P < 0.0005 and time × group interaction F(1,15) = 8.035, P = 0.0126; Sidak’s multiple comparison test ***P < 0.001 n = 7–10 per group]. (B) After vincristine treatment, the heat withdrawal threshold decreases significantly in wild-type mice, but not SARM1 knockout mice [two-way repeated measures ANOVA, significant main effect on time × genotype interaction F(1,15) = 10.69; P = 0.0052; Sidak’s post hoc *P < 0.05; n = 7–10 per group]. (C) There was no difference in rotarod performance in any of the groups (two-way repeated measures ANOVA, there was no main effect between groups, time and group × time interaction). (D) There was no difference in the time mice are able to hang on to an inverted screen between wild-type and SARM1 knockout mice (two-way repeated measures ANOVA, n = 7–10 per group). KO = knockout.