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. 2022 Sep 1;163(9):1728-1739.
doi: 10.1097/j.pain.0000000000002558. Epub 2021 Dec 6.

Second messengers mediating high-molecular-weight hyaluronan-induced antihyperalgesia in rats with chemotherapy-induced peripheral neuropathy

Ivan J M Bonet  1 , Larissa Staurengo-Ferrari  1 , Dionéia Araldi  1 , Paul G Green  2 , Jon D Levine  3
Affiliations

Second messengers mediating high-molecular-weight hyaluronan-induced antihyperalgesia in rats with chemotherapy-induced peripheral neuropathy

Ivan J M Bonet et al. Pain. .

Abstract

High-molecular-weight hyaluronan (HMWH) is an agonist at cluster of differentiation (CD)44, the cognate hyaluronan receptor, on nociceptors, where it acts to induce antihyperalgesia in preclinical models of inflammatory and neuropathic pain. In the present experiments, we studied the CD44 second messengers that mediate HMWH-induced attenuation of pain associated with oxaliplatin and paclitaxel chemotherapy-induced peripheral neuropathy (CIPN). While HMWH attenuated CIPN only in male rats, after ovariectomy or intrathecal administration of an oligodeoxynucleotide (ODN) antisense to G protein-coupled estrogen receptor (GPR30) mRNA, female rats were also sensitive to HMWH. Intrathecal administration of an ODN antisense to CD44 mRNA markedly attenuated HMWH-induced antihyperalgesia in male rats with CIPN induced by oxaliplatin or paclitaxel. Intradermal administration of inhibitors of CD44 second messengers, RhoA (member of the Rho family of GTPases), phospholipase C, and phosphatidylinositol (PI) 3-kinase gamma (PI3Kγ), attenuated HMWH-induced antihyperalgesia as does intrathecal administration of an ODN antisense to PI3Kγ. Our results demonstrated that HMWH induced antihyperalgesia in CIPN, mediated by its action at CD44 and downstream signaling by RhoA, phospholipase C, and PI3Kγ.

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Conflict of interest statement

Conflict of Interest: The authors declare no competing financial interests.

Figures

Figure 1.
Figure 1.. HMWH induces anti-hyperalgesia in male rats with oxaliplatin- and paclitaxel-induced CIPN
A. Male rats received oxaliplatin (2 mg/kg, i.v.) on day 0. On day 7 after oxaliplatin administration, HMWH was injected (1 μg/ 5 μL, i.d.) on the dorsum of the hind paw at the site of nociceptive threshold testing. Mechanical nociceptive threshold was evaluated before oxaliplatin and 30 min, and 7 days after its administration, and 30 min after HMWH. Oxaliplatin decreases mechanical nociceptive threshold (i.e., produces hyperalgesia) in male rats. Intradermal administration of HMWH attenuates the hyperalgesia induced by oxaliplatin, in male rats (F(3,30)= 18.37, ****p<0.0001, when CIPN, HMWH was compared to CIPN, vehicle-treated group; two-way ANOVA followed by Bonferroni’s post hoc comparisons test). B. Male rats received paclitaxel (1 mg/kg, i.p.), every other day for a total of 4 doses (days 0, 2, 4 and 6). On day 7, approximately 24 h after the last dose of paclitaxel, HMWH (1 μg/ 5 μL) was injected intradermally (i.d.) on the dorsum of one hind paw. Mechanical nociceptive threshold was evaluated before the 1st paclitaxel injection and 1, 3, 5 and 7 days after, and then 30 min after HMWH. Paclitaxel decreases mechanical nociceptive threshold (i.e., produces hyperalgesia) in male rats. Intradermal administration of HMWH attenuates the hyperalgesia induced by paclitaxel (F(5,50)= 8.60, ****p<0.0001, when CIPN, HMWH-treated group was compared to the CIPN, vehicle-treated group; two-way ANOVA followed by Bonferroni’s post hoc comparisons test). C. Female rats received oxaliplatin (2 mg/kg, i.v.) on day 0. On day 7 after oxaliplatin administration, HMWH was injected (1 μg/ 5 μL, i.d.) on the dorsum of the hind paw, at the site of nociceptive threshold testing. Mechanical nociceptive threshold was evaluated before oxaliplatin and 30 min, and 7 days after its administration, and then 30 min after HMWH. Oxaliplatin decreases mechanical nociceptive threshold (i.e., produces hyperalgesia) in male rats. Intradermal administration of HMWH attenuates the hyperalgesia induced by oxaliplatin, in male rats. In female rats, oxaliplatin also decreases mechanical nociceptive threshold. However, in females HMWH does not attenuate oxaliplatin-induced hyperalgesia (F(3,30)= 0.06, p=0.98, when CIPN treated with HMWH was compared to the CIPN vehicle-treated group; two-way ANOVA followed by Bonferroni’s post hoc comparisons test). n=6 per group. D. Female rats received paclitaxel (1 mg/kg, i.p.), every other day for a total of 4 doses (days 0, 2, 4 and 6). On day 7, approximately 24 h after the last dose of paclitaxel, HMWH (1 μg/ 5 μL) was injected intradermally (i.d.) on the dorsum of one hind paw. Mechanical nociceptive threshold was evaluated before the 1st paclitaxel injection and 1, 3, 5 and 7 days after, and then 30 min after HMWH. Administration of paclitaxel decreases mechanical nociceptive threshold in female rats. However, HMWH does not attenuate the hyperalgesia induced by paclitaxel in females (F(5,50)= 0.80, p=0.56, when CIPN, HMWH-treated group was compared to CIPN, vehicle-treated group; two-way ANOVA followed by Bonferroni’s post hoc comparisons test). Results in all figures are presented as mechanical nociceptive threshold in grams. n=6 per group.
Figure 2.
Figure 2.. HMWH-induced anti-hyperalgesia in females is sex hormone dependent
A. Female rats underwent ovariectomy 14 days prior to receiving oxaliplatin (2 mg/kg, i.v.), on day 0. On day 7, HMWH (1 μg/ 5 μL, i.d.) was injected on the dorsum of the hind paw. Mechanical nociceptive threshold was evaluated before oxaliplatin and 30 min, and 7 days after, and again 30 min after HMWH. Intradermal administration of HMWH attenuates the hyperalgesia induced by oxaliplatin in ovariectomized female rats (F(3,30)= 17.04, ****p<0.0001, when CIPN treated with HMWH was compared to the CIPN vehicle-treated group; two-way ANOVA followed by Bonferroni’s post hoc comparisons test). n= 6 per group. B. Another group of female rats underwent ovariectomy 14 days prior to receiving paclitaxel (1 mg/kg, i.p.), every other day for a total of 4 doses (days 0, 2, 4 and 6). Seven days later, rats were treated with HMWH (1 μg/ 5 μL, i.d.) on the dorsum of the hind paw. Mechanical nociceptive threshold was evaluated before paclitaxel, and on days 1, 3, 5 and 7 after, and then 30 min after HMWH. Intradermal administration of HMWH attenuates the hyperalgesia induced by paclitaxel in ovariectomized rats (F(5,50)= 9.61, ****p<0.0001, when CIPN treated with HMWH was compared to the CIPN vehicle-treated group; two-way ANOVA followed by Bonferroni’s post hoc comparisons test). n= 6 per group. C. Female rats received oxaliplatin (2 mg/kg, i.v.) on day 0. Four days later, they were treated with an oligodeoxynucleotide (ODN) antisense or mismatch (120 μg/ 20 μL, i.t.) for GPR30 mRNA, daily for 3 consecutive days. On day 7, approximately 24 h after the last dose of ODN, HMWH (1 μg/ 5 μL, i.d.) was injected on the dorsum of the hind paw. Mechanical nociceptive threshold was evaluated before oxaliplatin and 30 min, 4 and 7 days after its administration, and then 30 min after HMWH. Oxaliplatin similarly decreases mechanical nociceptive threshold in both GPR30 antisense- and mismatch-treated rats. Intradermal administration of HMWH attenuates the hyperalgesia induced by oxaliplatin in the GPR30 antisense-treated group (F(4,40)= 15.99, ****p<0.0001, when CIPN treated with HMWH was compared to the CIPN vehicle-treated group; two-way ANOVA followed by Bonferroni’s post hoc comparisons test). n= 6 per group. D. Female rats received paclitaxel, administered intraperitoneally (1 mg/kg, i.p.) every other day for a total of 4 doses (days 0, 2, 4 and 6). Four days after the 1st paclitaxel injection, rats were treated with antisense or mismatch ODN antisense for GPR30 mRNA (120 μg/ 20 μL, i.t.), daily for 3 consecutive days. On day 7, approximately 24 h after the last ODN dose and the last dose of paclitaxel, HMWH (1 μg/ 5 μL, i.d.) was injected on the dorsum of the hind paw. Mechanical nociceptive threshold was evaluated before paclitaxel, and on day 1, 3, 5 and 7 after its administration, and then 30 min after HMWH. In both GPR30 antisense- and mismatch-treated groups, paclitaxel decreases mechanical nociceptive threshold. In the group treated with ODN antisense to GPR30 mRNA, intradermal administration of HMWH attenuates the hyperalgesia induced by paclitaxel (F(5,50)= 8.75, ****p<0.0001, when CIPN treated with HMWH was compared to the CIPN vehicle-treated group; two-way ANOVA followed by Bonferroni’s post hoc comparisons test). n= 6 per group.
Figure 3.
Figure 3.. HMWH-induced anti-hyperalgesia is attenuated by ODN antisense to CD44 mRNA
A. Male rats received oxaliplatin (2 mg/kg, i.v.) or saline (i.v.) on day 0. Four days later, they were treated with an ODN antisense or mismatch for CD44 mRNA (120 μg/ 20 μL, i.t.), daily for 3 days. On day 7, approximately 24 h after the last intrathecal administration of ODN, HMWH was injected (1 μg/ 5 μL, i.d.). Mechanical nociceptive threshold was evaluated on day 0, and on day 4 and 7 after administration of oxaliplatin, and then 30 min after HMWH. Results are presented as mechanical nociceptive threshold in grams. Oxaliplatin decreased mechanical nociceptive threshold (i.e., produced hyperalgesia), measured 4 days after intravenous injection (F(7,35)= 97.99, ****p<0.0001; two-way ANOVA followed by Bonferroni’s post hoc comparisons test), in both CD44 antisense- and mismatch-treated groups. HMWH attenuates the hyperalgesia induced by oxaliplatin (F(7,35)= 97.99, ****p<0.0001; two-way ANOVA followed by Bonferroni’s post hoc comparisons test) only in the CD44 mismatch-treated group. n= 6 per group. B. Male rats received paclitaxel (1 mg/kg, i.p.), every other day for a total of 4 doses (days 0, 2, 4 and 6). Four days after the 1st paclitaxel injection, rats were treated with an oligodeoxynucleotide (ODN) antisense or mismatch (120 μg/ 20 μL, i.t.) for CD44 mRNA, daily for 3 consecutive days. On day 7, approximately 24 h after the last dose of ODN, and the last intraperitoneal injection of paclitaxel, HMWH was injected (1 μg/ 5 μL, i.d.). Mechanical nociceptive threshold was evaluated on day 0, and 4 and 7 days after oxaliplatin, and then 30 min after HMWH. Paclitaxel decreased mechanical nociceptive threshold (i.e., produced hyperalgesia), measured 4 days (F(7,35)= 52.53, ****p<0.0001; two-way ANOVA followed by Bonferroni’s post hoc comparisons test) after its first dose, in both CD44 antisense- and mismatch-treated groups. HMWH attenuates the hyperalgesia induced by paclitaxel in the CD44 mismatch-treated group (F(7,35)= 52.53, *p=0.0107; two-way ANOVA followed by Bonferroni’s post hoc comparisons test). n= 6 per group.
Figure 4.
Figure 4.. HMWH anti-hyperalgesia is attenuated by a RhoA inhibitor
A. Male rats received oxaliplatin (2 mg/kg, i.v.) or saline (i.v.) on day 0. On day 7, a ROK inhibitor (a component of RhoA signaling pathway) (Y27632, 1 μg/ 5 μL, i.d.) or vehicle (5 μL, i.d.) was injected. Ten minutes later, rats received an injection of HMWH (1 μg/5 μL, i.d.) or vehicle (5 μL, i.d.) and mechanical nociceptive threshold was evaluated on day 0 and 7 days after administration of oxaliplatin, and then again 30 min after intradermal HMWH or vehicle. Results are presented as mechanical nociceptive threshold, in grams. Oxaliplatin decreased mechanical nociceptive threshold measured 7 days after its administration (F(8,40)= 106.85, ****p<0.0001; two-way ANOVA followed by Bonferroni’s post hoc comparisons test). Anti-hyperalgesia induced by HMWH was attenuated by the ROK inhibitor (F(8,40)= 106.85, ****p<0.0001; two-way ANOVA followed by Bonferroni’s post hoc comparisons test). n= 6 per group. B. Paclitaxel was administered intraperitoneally (1 mg/kg, i.p.), in male rats, every other day for a total of 4 doses (days 0, 2, 4 and 6). On day 7, approximately 24 h after the last dose of paclitaxel, a RhoA inhibitor (Y27632, 1 μg/ 5 μL, i.d.) or vehicle (5 μL, i.d.) was injected. Ten minutes later, rats received an injection of HMWH (1 μg/5 μL, i.d.) or vehicle (5 μL, i.d.) and mechanical nociceptive threshold evaluated on days 0 and 7 after the 1st paclitaxel injection, and then again 30 min after HMWH or vehicle. Paclitaxel decreases mechanical nociceptive threshold on day 7 after its first dose (F(8,40)= 118.45, ****p<0.0001; two-way ANOVA followed by Bonferroni’s post hoc comparisons test). Intradermal administration of RhoA the inhibitor attenuates HMWH-induced anti-hyperalgesia (F(8,40)= 118.45, ****p=0.0010; two-way ANOVA followed by Bonferroni’s post hoc comparisons test). n= 6 per group.
Figure 5.
Figure 5.. HMWH anti-hyperalgesia is attenuated by a PLC inhibitor
A. Male rats received oxaliplatin (2 mg/kg, i.v.) or saline (i.v.) on day 0. On day 8, a PLC inhibitor (U73122, 1 μg/ 5 μL, i.d.) or vehicle (5 μL, i.d.) was injected. Ten minutes later, rats received an injection of HMWH (1 μg/5 μL, i.d.) or vehicle (5 μL, i.d.) and mechanical nociceptive threshold evaluated on day 0, and 7 days after oxaliplatin, and then again 30 min after HMWH or vehicle. Oxaliplatin decreased mechanical nociceptive threshold after administration on day 7 (F(8,40)= 104.79, ****p<0.0001; two-way ANOVA followed by Bonferroni’s post hoc comparisons test). Anti-hyperalgesia induced by HMWH was attenuated by the PLC inhibitor (F(8,40)= 104.79, ****p<0.0001; two-way ANOVA followed by Bonferroni’s post hoc comparisons test). n= 6 per group. B. Male rats received paclitaxel (1 mg/kg, i.p.), administered every other day for a total of 4 doses (on days 0, 2, 4 and 6). On day 7, approximately 24 h after the last dose of paclitaxel, a PLC inhibitor (U73122, 1 μg/ 5 μL, i.d.) or vehicle (5 μL, i.d.) was injected. Ten minutes later, rats received an injection of HMWH (1 μg/5 μL, i.d.) or vehicle (5 μL, i.d.). Mechanical nociceptive threshold was evaluated on day 0 and on day 7 after the 1st dose of paclitaxel, and then again 30 min after HMWH or vehicle. Paclitaxel induced mechanical hyperalgesia on day 7 (F(8,40)= 84.20, ****p<0.0001; two-way ANOVA followed by Bonferroni’s post hoc comparisons test). Intradermal administration of the PLC inhibitor also attenuated HMWH-induced anti-hyperalgesia (F(8,40)= 84.20, **p=0.0028; two-way ANOVA followed by Bonferroni’s post hoc comparisons test). n= 6 per group.
Figure 6.
Figure 6.. HMWH anti-hyperalgesia is attenuated by ODN antisense to PI3Kγ mRNA
A. Male rats received oxaliplatin (2 mg/kg, i.v.) or saline (i.v.) on day 0. Four days later they were treated with an oligodeoxynucleotide (ODN) antisense or mismatch (120 μg/ 20 μL, i.t.) for PI3Kγ mRNA, daily for 3 consecutive days. On day 7, approximately 24 h after the last dose of ODN, HMWH (1 μg/ 5 μL, i.d.) was injected. Mechanical nociceptive threshold was evaluated on days 0, 4 and 7 after administration of oxaliplatin and 30 min after HMWH. Oxaliplatin decreased mechanical nociceptive threshold (i.e., produced hyperalgesia), observed 4 days after intravenous injection in both PI3Kγ antisense- and mismatch-treated groups (F(7,35)= 103.47, ****p<0.0001; two-way ANOVA followed by Bonferroni’s post hoc comparisons test). Intradermal administration of HMWH does not attenuate the hyperalgesia induced by oxaliplatin in PI3Kγ antisense-treated rats (F(7,35)= 103.47, ****p<0.0001; two-way ANOVA followed by Bonferroni’s post hoc comparisons test). n= 6 per group. B. Male rats received paclitaxel (1 mg/kg, i.p.) every other day for a total of 4 doses (days 0, 2, 4 and 6). Four days after the 1st dose of paclitaxel, rats were treated with an oligodeoxynucleotide (ODN) antisense or mismatch (120 μg/ 20 μL, i.t.) against PI3Kγ mRNA, daily for 3 consecutive days. On day 7, approximately 24 h after the last injection of ODN, and paclitaxel, HMWH (1 μg/ 5 μL, i.d.) was injected. Mechanical nociceptive threshold was evaluated on day 0 and days 4 and 7 after administration of oxaliplatin, and again 30 min after HMWH. Paclitaxel decreases mechanical nociceptive threshold (i.e., produces hyperalgesia), measured on day 4 (F(7,35)= 54.74, ****p<0.0001; two-way ANOVA followed by Bonferroni’s post hoc comparisons test), in both PI3Kγ antisense- and mismatch-treated groups. However, HMWH does not attenuate the hyperalgesia induced by paclitaxel in PI3Kγ antisense-treated rats (F(7,35)= 54.74, ****p<0.0001; two-way ANOVA followed by Bonferroni’s post hoc comparisons test). n= 6 per group.
Figure 7.
Figure 7.. HMWH anti-hyperalgesia is attenuated by a PI3Kγ inhibitor
A. Male rats received oxaliplatin (2 mg/kg, i.v.) or saline (i.v.) on day 0. On day 7, a PI3Kγ inhibitor (AS605240, 1 μg/ 5 μL, i.d.) or vehicle (5 μL, i.d.) was injected. Ten minutes later, rats received an injection of HMWH (1 μg/5 μL, i.d.) or vehicle (5 μL, i.d.); mechanical nociceptive threshold was evaluated on days 0 and 7 after administration of oxaliplatin, and again 30 min after intradermal HMWH or vehicle. Oxaliplatin decreased mechanical nociceptive threshold on day 7 (F(8,40)= 61.33, ****p<0.0001; two-way ANOVA followed by Bonferroni’s post hoc comparisons test). HMWH-induced anti-hyperalgesia was attenuated in the rats treated with the PI3Kγ inhibitor (F(8,40)= 61.33, ****p<0.0001; two-way ANOVA followed by Bonferroni’s post hoc comparisons test). n= 6 per group. B. Male rats received paclitaxel (1 mg/kg, i.p.) every other day for a total of 4 doses (days 0, 2, 4 and 6). On day 7, approximately 24 h after the last dose of paclitaxel, a PI3Kγ inhibitor (AS605240, 1 μg/ 5 μL, i.d.) or vehicle (5 μL, i.d.) was injected. Ten minutes later, rats received an injection of HMWH (1 μg/5 μL, i.d.) or vehicle (5 μL, i.d.) and mechanical nociceptive threshold was evaluated on day 0 and day 7 after the 1st dose of paclitaxel, and then again 30 min after HMWH or vehicle. The PI3Kγ inhibitor attenuates HMWH anti-hyperalgesia (F(8,40)= 79.37, ****p<0.0001; two-way ANOVA followed by Bonferroni’s post hoc comparisons test). n= 6 per group.
Figure 8.
Figure 8.. HMWH-induced anti-hyperalgesia signaling pathway
In male and gonadectomized female rats, oxaliplatin and paclitaxel induces CIPN-hyperalgesia that is reversed by HMWH, which binds to CD44 to induce its clustering in cell membrane lipid rafts and initiate signaling in downstream second messenger pathways. After binding to CD44, HMWH can signal via RhoA and Rac1, which in turn, activate ROK and PKN, respectively, leading to phosphorylation of PLCε and PLCγ1, respectively. Binding of HMWH to CD44 also stimulates RhoA, which activates ROK to phosphorylate PLCε, increasing serine/threonine phosphorylation of the adaptor protein, Gab-1 and leading to activation of PI3Kγ. Abbreviations: CD44, cluster of differentiation 44 (hyaluronan receptor); Gab1, scaffold protein; HMWH, high molecular weight hyaluronan; PI3Kγ, phosphatidylinositol (PI) 3-kinase gamma; PKN, fatty acid-activated serine/threonine kinase; PLCε, phospholipase C epsilon; PLCγ1, phospholipase Cγ1; Rac1, Rho family of GTPases; RhoA, Rho family of GTPases; ROK, Rho-associated kinase.
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