Intrathecal administration of antisense oligonucleotide against p38α but not p38β MAP kinase isoform reduces neuropathic and postoperative pain and TLR4-induced pain in male mice

Abstract

p38 mitogen-activated protein kinase (MAPK) consists of two major isoforms: p38α and p38β; however, it remains unclear which isoform is more important for chronic pain development. Recently, we developed potent, long-lasting, and p38 MAPK subtype-specific antisense oligonucleotides (ASOs). We examined the therapeutic effects of isoform-specific ASOs in several chronic pain models following single intrathecal injection (300 μg/10 μl) in CD1 mice. In the chronic constriction injury (CCI) model, p38α MAPK ASO, given on post-operative day 5, reduced CCI-induced mechanical allodynia in male but not female mice. In contrast, mechanical allodynia after CCI in both sexes was not affected by p38β MAPK ASO. Intrathecal injection of p38α or p38β ASO resulted in a partial reduction (≈ 50%) of spinal p38α or p38β mRNA level, respectively, in both sexes at two weeks. In contrast, intrathecal injection of the ASOs did not affect p38α and p38β MAPK mRNA levels in dorsal root ganglia. Intrathecal p38α ASO also reduced postoperative pain (mechanical and cold allodynia) in male mice after tibia fracture. However, intrathecal p38α ASO had no effect on mechanical allodynia in male mice after paclitaxel treatment. Intrathecal p38α MAPK ASO pre-treatment also prevented TLR4-mediated mechanical allodynia and downregulated levels of p38α MAPK and phosphorylated p38 MAPK following intrathecal treatment of lipopolysaccharide. In summary, our findings suggest that p38α MAPK is the major p38 MAPK isoform in the spinal cord and regulates chronic pain in a sex and model-dependent manner. Intrathecal p38α MAPK ASO may offer a new treatment for some chronic pain conditions.

Keywords: Antisense oligonucleotides (ASOs); Microglia; Neuropathic pain; P38 mitogen-activated protein kinase (MAPK); Sex; Spinal cord.

Figure 1. Intrathecal injection of p38α but not p38β MAPK ASO alleviates mechanical allodynia in male mice with CCI injury

(A, B) Intrathecal injection of 300 µg p38α but not p38β MAPK ASO 5 days after CCI increased paw withdrawal threshold (A) and decreased paw withdrawal frequency (B) in male mice. **p<0.01 and *p<0.05 in comparison to vehicle (PBS). Two-Way ANOVA with Bonferroni’s post-hoc test, n=6–7 mice/per group. Paw withdrawal frequency was induced by a 0.4 g von Frey hair.

Figure 2. Intrathecal injection of p38α or p38β MAPK ASO fails to affect mechanical allodynia in CCI-injured female mice

(A, B) Intrathecal injection of 300 µg p38α or p38β MAPK ASO 5 days after CCI did not alter paw withdrawal threshold (A) or paw withdrawal frequency (B) in CCI-injured female mice. Two-Way ANOVA with Bonferroni’s post-hoc test, n=6–7 mice per group. Paw withdrawal frequency was induced by a 0.4 g von Frey hair.

Figure 3. Intrathecal injection of p38α or p38β MAPK ASO reduces the respective expression of p38α or p38β mRNA in spinal cord of mice with CCI injury

(A–D) Intrathecal injection of p38α MAPK ASO only reduced spinal mRNA levels of p38α MAPK, but not p38β MAPK, on both sides and in both males (A,B) and females (C,D). ****p<0.0001, ***p<0.001, **p<0.01. C, contralateral; I, ipsilateral to CCI. One-way ANOVA with Bonferroni’s post-hoc test, n=6–7 mice/group. Spinal cord tissues were collected two weeks after the ASO treatment.

Figure 4. Intrathecal injection of p38α or p38β MAPK ASO does not alter p38α or p38β MAPK mRNA expression in DRG tissues after CCI

(A–D) Intrathecal injection of p38α or p38β MAPK ASO did not affect p38α or p38β MAPK mRNA levels in lumbar (L3–L5) DRG tissues on both sides and in male mice (A, B) and female mice (C, D). ****p<0.0001 and **p<0.01. C, contralateral; I, ipsilateral to CCI. One-way ANOVA with Bonferroni’s post-hoc test, n=6–7 mice/group. Note that nerve injury increased p38β mRNA in the ipsilateral DRGs of vehicle- and p38α ASO-treated females, compared to contralateral DRGs (D). However, p38β MAPK ASO-treated females, p38β MAPK mRNA levels did not change, compared to vehicle-treated females (D). DRG tissues were collected two weeks after the ASO treatment.

Figure 5. Intrathecal injection of p38α but not p38β MAPK ASO reduces postoperative pain in male mice with tibia fracture

(A, B) Tibia fracture induced mechanical allodynia in male mice. Intrathecal injection of 300 µg p38α but not p38β MAPK ASO, given 15 days after surgery, increased paw withdrawal threshold (A) and reduced cold allodynia score (B) in male mice with bone fracture. *p<0.05 in comparison to respective vehicle (PBS). Two-Way ANOVA with Bonferroni’s post-hoc test, n = 4–5 mice per group.

Figure 6. Intrathecal injection of p38α or p38β MAPK ASO fails to affect neuropathic pain in male mice after chemotherapy-induced neuropathy

Paclitaxel (2 mg/kg, i.p., 4 injections on day 0, 2, 4, and 6) induced robust mechanical allodynia in male mice. Intrathecal injection of 300 µg p38α or p38β MAPK ASO 3 days after the first paclitaxel treatment had no effects on paw withdrawal threshold in these animals. Two-Way ANOVA with Bonferroni’s post-hoc comparison, n = 6–7 mice per group.

Figure 7. Intrathecal p38α MAPK ASO prevents the TLR4-induced mechanical allodynia and decreases protein levels of p38α MAPK and phosphorylated p38 MAPK in spinal cord following intrathecal LPS treatment

(A–C) Mice received intrathecal 300 µg p38α or p38β MAPK ASO 5 days before intrathecal injection of LPS (10 µg). (A) LPS-induced mechanical allodynia was blocked by p38α but not p38β MAPK ASO. Two-Way ANOVA with Bonferroni’s pro-test, n=5 per group. ***p<0.001 in comparison to vehicle (PBS). (B) p38α MAPK ASO downregulated the levels p38α MAPK and phosphorylated p38 MAPK. Student’s t- test, n=3 per group, *p<0.05.