Korean J Pain.  2022 Oct;35(4):391-402. 10.3344/kjp.2022.35.4.391.

The role of botulinum toxin type A related axon transport in neuropathic pain induced by chronic constriction injury

• Bu H
• Jiao P
• Fan X
• Gao Y
• Zhang L
• Guo H

• KMID: 2533979
• DOI: http://doi.org/10.3344/kjp.2022.35.4.391

Abstract

Background
The mechanism of peripheral axon transport in neuropathic pain is still unclear. Chemokine ligand 13 (CXCL13) and its receptor (C-X-C chemokine receptor type 5, CXCR5) as well as GABA transporter 1 (GAT-1) play an important role in the development of pain. The aim of this study was to explore the axonal transport of CXCL13/CXCR5 and GAT-1 with the aid of the analgesic effect of botulinum toxin type A (BTX-A) in rats.
Methods
Chronic constriction injury (CCI) rat models were established. BTX-A was administered to rats through subcutaneous injection in the hind paw. The pain behaviors in CCI rats were measured by paw withdrawal threshold and paw withdrawal latencies. The levels of CXCL13/CXCR5 and GAT-1 were measured by western blots.
Results
The subcutaneous injection of BTX-A relieved the mechanical allodynia and heat hyperalgesia induced by CCI surgery and reversed the overexpression of CXCL13/CXCR5 and GAT-1 in the spinal cord, dorsal root ganglia (DRG), sciatic nerve, and plantar skin in CCI rats. After 10 mmol/L colchicine blocked the axon transport of sciatic nerve, the inhibitory effect of BTX-A disappeared, and the levels of CXCL13/CXCR5 and GAT-1 in the spinal cord and DRG were reduced in CCI rats.
Conclusions
BTX-A regulated the levels of CXCL13/CXCR5 and GAT-1 in the spine and DRG through axonal transport. Chemokines (such as CXCL13) may be transported from the injury site to the spine or DRG through axonal transport. Axon molecular transport may be a target to enhance pain management in neuropathic pain.

Keyword

Axonal Transport; Botulinum Toxins; Type A; Chemokines; Colchicine; GABA Plasma Membrane Transport Proteins; Hyperalgesia; Neuralgia; Receptors; Chemokine; Sciatic Nerve

Figure

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  Fig. 1 In chronic constriction injury (CCI) rats, the paw withdrawal thresholds (PWTs) and paw withdrawal latencies (PWLs) in the ipsilateral were decreased from day 3 after modeling and reached a stable level from day 7 (A, C). The PWTs and PWLs in the contralateral side in each group had no significant changes in time points (B, D). The error bar means mean ± standard deviation. aP < 0.05 vs. sham; Two-way ANOVA; n = 11 rats in each group.

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  Fig. 2 Chronic constriction injury (CCI) of the sciatic nerve upregulated the expression of CXCL13, CXCR5, and GAT-1 from day 3 or 7 in the spinal cord, DRG, sciatic nerve, and plantar skin by western blot. (A, E, I, M) The expression of CXCL13, CXCR5, and GAT-1 in the spinal cord. (B, F, J, N) The expression of these proteins in the DRG. (C, G, K, O) The expression of these proteins in the sciatic nerve. (D, H, L, P) The expression of these proteins in the plantar skin. The error bar means mean ± standard deviation. CXCL13: chemokine ligand 13, CXCR5: C-X-C chemokine receptor type 5, GAT-1: GABA transporter 1, DRG: dorsal root ganglia. aP < 0.05 vs. sham; One-way ANOVA; n = 3 in each group.

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  Fig. 3 In chronic constriction injury (CCI) rats, the paw withdrawal thresholds (PWTs) and paw withdrawal latencies (PWLs) in the ipsilateral side were decreased from day 3 after modeling. Botulinum toxin type A (BTX-A) (4, 7, 10 U/kg) reversed these decreases from day 10 in a dose-dependent manner (A, C). The PWTs and PWLs of the contralateral side in each group exhibited no significant changes in time points (B, D). The error bar means mean ± standard deviation. aP < 0.05 vs. CCI + normal saline (NS) group, Two-way ANOVA, n = 6 in each group; bP < 0.05 vs. CCI + 4 U/kg BTX-A group, Two-way ANOVA, n = 6 in each group; cP < 0.05 vs. CCI + 7 U/kg BTX-A group, Two-way ANOVA, n = 6 in each group.

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  Fig. 4 The botulinum toxin type A (BTX-A) inhibited the overexpression of CXCL13, CXCR5, and GAT-1 in chronic constriction injury (CCI) rats, and the effect of BTX-A is dose-dependent. The BTX-A reversed the increased expression of CXCL13, CXCR5, and GAT-1 induced by CCI in the spinal cord tested by western blot (A, E, I, M), DRG (B, F, J, N), sciatic nerve (C, G, K, O), and plantar skin (D, H, L, P). CXCL13: chemokine ligand 13, CXCR5: C-X-C chemokine receptor type 5, GAT-1: GABA transporter 1, DRG: dorsal root ganglia. The error bar means mean ± standard deviation. aP < 0.05 vs. sham group, bP < 0.05 vs. CCI + normal saline group, cP < 0.05 vs. CCI + 4 U/kg BTX-A group, dP < 0.05 vs. CCI + 10 U/kg BTX-A group; One-way ANOVA; n = 3 in each group.

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  Fig. 5 Botulinum toxin type A (BTX-A) injection reversed the decrease of paw withdrawal thresholds (PWTs) in the ipsilateral hind paw of chronic constriction injury (CCI) rats from day 10 after modeling (A). However, the PWTs in the contralateral side had no significant changes in each group (B). The error bar means mean ± standard deviation. aP < 0.05 vs. CCI + normal saline (NS) group, Two-way ANOVA, n = 6 in each group.

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  Fig. 6 Western blot analysis showing changes in CXCL13, CXCR5, and GAT-1 levels of the spinal cord at 10, 14, and 21 days after surgery. Treatment with botulinum toxin type A (BTX-A) prevented the increase in CXCL13, CXCR5, and GAT-1 proteins as graphed in A, B, C, and D. The error bar means mean ± standard deviation. CCI: chronic constriction injury, CXCL13: chemokine ligand 13, CXCR5: C-X-C chemokine receptor type 5, GAT-1: GABA transporter 1. aP < 0.05 vs. CCI + normal saline (NS) group, One-way ANOVA, n = 3 in each group.

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  Fig. 7 (A–C) Western blot data showing that the increased expression of CXCL13, CXCR5, and GAT-1 proteins after chronic constriction injury (CCI) surgery in the DRG, sciatic nerve, and plantar skin was reversed by botulinum toxin type A (BTX-A). CXCL13: chemokine ligand 13, CXCR5: C-X-C chemokine receptor type 5, GAT-1: GABA transporter 1, DRG: dorsal root ganglia. The error bar means mean ± standard deviation. aP < 0.05 vs. CCI + normal saline (NS) group, One-way ANOVA, n = 3 in each group.

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  Fig. 8 The mechanical hypersensitivity after chronic constriction injury (CCI) surgery was attenuated after the sciatic nerve was treated with 10 mmol/L colchicine (COL) (A). The paw withdrawal thresholds (PWTs) in the contralateral hind paw of each group had no significant change at time points (B). The dose of botulinum toxin type A (BTX-A) which was used was 10 U/kg. The error bar means mean ± standard deviation. aP < 0.05 vs. CCI + normal saline (NS) + NS group, Two-way ANOVA, n = 6 in each group. bP < 0.05 vs. CCI + NS + 10 U/kg BTX-A group, Two-way ANOVA, with n = 6 in each group.

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  Fig. 9 Rats were treated with colchicine (COL, 10 mmol/L) in the sciatic nerve, and (or) botulinum toxin type A (BTX-A, 10 U/kg) injected to the plantar surface of the hind paw. The levels of CXCL13, CXCR5, and GAT-1 were increased in the spinal cord, DRG, sciatic nerve and hind paw of chronic constriction injury (CCI) rats. Both COL and BTX-A attenuated these increases. But in the spinal cord (A, E, I, M) and DRG (B, F, J, N), combination of COL and BTX-A could partly reversed BTX-A-induced CXCL13, CXCR5, and GAT-1 inhibition. The effect of combination of COL and BTX-A on the BTX-A-induced inhibition of GAT-1 in sciatic nerve (O) and CXCR5 in hind paw (L) was not significant. The trends of the expression of CXCL13 in sciatic nerve (C, G) and hind paw (D, H), CXCR5 in sciatic nerve (K), and GAT-1 in hind paw (P) were similar with those in spinal cord and DRG. These suggest that blocking axonal transport by COL can reverse BTX-A-induced inhibition neuro-inflammation. The error bar means mean ± standard deviation. CXCL13: chemokine ligand 13, CXCR5: C-X-C chemokine receptor type 5, GAT-1: GABA transporter 1, DRG: dorsal root ganglia, NS: normal saline. aP < 0.05 vs. the sham group, bP < 0.05 vs. the CCI-NS-NS group, cP < 0.05 vs. CCI-COL-NS group, One-way ANOVA, n = 3 in each group.

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