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. 2023 Oct 3;147(3):1025–1042. doi: 10.1093/brain/awad339

Figure 3.

Figure 3

TLR4 antisense attenuates TLR4 protein expression in L4 and L5 dorsal root ganglia and oxaliplatin-induced CIPN. Western blot analysis of dorsal root ganglia (DRG) extracts from male rats treated intrathecally with antisense-oligodeoxynucleotides (AS-ODN) against TLR4 mRNA, once a day for 4 days (120 µg in 20 µl/day). (A) TLR4 AS-ODN-treatment significantly decreased anti-TLR4 immunoreactivity 24 h after the last treatment (−28.06 ± 1.29%, unpaired Student's t-test, n = 3, *P < 0.05). Of note, the magnitude of the attenuation of TLR4 in DRG neurons is probably an underestimate, as TLR4 levels in other cells in the DRG are not affected by intrathecal antisense but are measured on the western blots. (B) Nine days after the last administration of TLR4 AS-ODN, anti-TLR4 immunoreactivity was not significantly different from the levels in DRG from TLR4 mismatch (MM)-ODN-treated rats (−2.32 ± 3.6%, unpaired Student's t-test, n = 3, P > 0.05). The calculated molecular weight of TLR4 is 96 kDa (according to UniProtKB database entry Q9QX05). The difference between the calculated and apparent molecular weights may be due to the glycosylation of TLR4. β-Actin, which was used as a loading control, has a calculated molecular weight of ∼42 kDa (according to UniProtKB database entry P60771). The full-length gels and blots are included in Supplementary Fig. 3. (C) Male rats were treated intrathecally with AS-ODN or MM-ODN (both 120 μg in 20 μl/day) against TLR4 mRNA once a day for 4 days consecutively. On Day 0 (9 days after the last intrathecal ODN injection), rats received oxaliplatin [2 mg/kg, intravenously (i.v.)], and the mechanical nociceptive threshold was evaluated before the first intrathecal administration of ODN (Day −12) and then from Days 0 to 28. In the TLR4 AS-ODN-treated group, oxaliplatin did not develop hyperalgesia until Day 28. Two-way repeated-measures ANOVA, Time × TLR4 AS-ODN interaction, F(6,60) = 35.66, P < 0.0001; TLR4 AS-ODN treatment, F(1,10) = 881.1, P < 0.0001; Bonferroni's multiple post hoc comparisons test, ****P < 0.0001 (TLR4 MM-ODN versus TLR4 AS-ODN). n = 6 paws for each group. All data are mean ± SEM. CIPN = chemotherapy-induced peripheral neuropathy.