Sexual dimorphism in the nociceptive effects of hyaluronan

Abstract

Intradermal administration of low-molecular-weight hyaluronan (LMWH) in the hind paw induced dose-dependent (0.1, 1, or 10 µg) mechanical hyperalgesia of similar magnitude in male and female rats. However, the duration of LMWH hyperalgesia was greater in females. This sexual dimorphism was eliminated by bilateral ovariectomy and by intrathecal administration of an oligodeoxynucleotide (ODN) antisense to the G-protein-coupled estrogen receptor (GPR30) mRNA in females, indicating estrogen dependence. To assess the receptors at which LMWH acts to induce hyperalgesia, LMWH was administered to groups of male and female rats that had been pretreated with ODN antisense (or mismatch) to the mRNA for 1 of 3 hyaluronan receptors, cluster of differentiation 44 (CD44), toll-like receptor 4, or receptor for hyaluronan-mediated motility (RHAMM). Although LMWH-induced hyperalgesia was attenuated in both male and female rats pretreated with ODN antisense for CD44 and toll-like receptor 4 mRNA, RHAMM antisense pretreatment only attenuated LMWH-induced hyperalgesia in males. Oligodeoxynucleotide antisense for RHAMM, however, attenuated LMWH-induced hyperalgesia in female rats treated with ODN antisense to GPR30, as well as in ovariectomized females. Low-molecular-weight hyaluronan-induced hyperalgesia was significantly attenuated by pretreatment with high-molecular-weight hyaluronan (HMWH) in male, but not in female rats. After gonadectomy or treatment with ODN antisense to GPR30 expression in females, HMWH produced similar attenuation of LMWH-induced hyperalgesia to that seen in males. These experiments identify nociceptors at which LMWH acts to produce mechanical hyperalgesia, establishes estrogen dependence in the role of RHAMM in female rats, and establishes estrogen dependence in the inhibition of LMWH-induced hyperalgesia by HMWH.

Figure 1.. Dose- and time-dependence of LMWH-induced hyperalgesia.

Different groups of male (A) and female (B) rats received an intradermal injection of one of three doses of LMWH (0.1, 1, 10 μg/5 μL), on the dorsum of the hind paw. Mechanical nociceptive threshold was then measured 5, 10, 15, 20 and 30 min after the injection of LMWH. Measurement of mechanical nociceptive threshold showed that LMWH produced robust mechanical hyperalgesia in male (A. Significant effect of Time F_4,60=50.19, p<0.0001, Dose F_2,15=12.28, p=0.0007 and their Interaction F_8,60=2.908, p=0.0083. Two-way ANOVA followed Holm-Šídák multiple comparisons test showed significant differences at 10 mins for 0.1 vs. 1 μg **p=0.0081; at 15 mins for 0.1 vs. 1 μg ***p=0.0004; and 0.1 vs. 10 μg, ***p=0.0004; and at 30 mins for 0.1 vs. 10 μg ***p=0.0002), and female (B. Significant effect of Time F_4,72=68.75, p<0.0001, Dose F_2,18=25.94, p<0.0001, but not their Interaction F_8,72=1.641, P=1.641. Two-way ANOVA followed Holm-Šídák multiple comparison test showed that significant differences at 5 min for 1 vs. 10 μg *p=0.0184; at 10 mins for 0.1 vs. 10 μg ***p=0.0007, at 15 mins for 0.1 vs. 10 μg ***p=0.0001 and 1 vs. 10 μg **p=0.0045; and at 20 and 30 mins for 0.1 vs. 10 μg **p=0.0016 and **p=0.0022, respectively) rats. Sex differences. Repeated measures ANOVA showed that at a dose of 0.1 μg of LMWH while there was a significant effect of Time (F_4,44=28.63, p<0.0001), there was no significant effect of Sex (F_1,11=0.9382, p=0.3536) or Sex X Time Interaction (F_4,44=1.441, p=0.2365); at 1 μg, there was a significant effect of Time (F_4,44=46.26, p<0.0001), and Sex (F_1,11=5.522 p=0.0385) but no significant Interaction (F_4,44=0.4066, p=0.8029); and at 10 μg, there was a significant effect of Time (F_4,32=32.38, p<0.0001), but no significant effect of Sex (F_1,8=1.849, p=0.2017) or their Interaction (F_4,32=5.515, p=0.0017). n=6 for each Male group, n=7 for each Female group. Over the first 30 min there was no significant difference between males and females, in the magnitude of hyperalgesia, at any dose. C. Time course of LMWH-induced hyperalgesia. Different groups of male, female, orchiectomized male, and ovariectomized female rats received an intradermal injection of LMWH (1 μg/5 μL), on the dorsum of the hind paw. Mechanical nociceptive threshold was evaluated before and then 5, 10, 15, 20 and 30 min, and 1, 2, 4, 8 and 24 h after intradermal LMWH. While LMWH hyperalgesia was no longer present in males, 2 h after administration, it was still present in intact females 4h after administration (F_(5,25)=19.38, *p<0.0250, when the female-group was compared with male-, ovariectomized female- and orchiectomized male- group respectively 1 h after intradermal LMWH; ****p<0.0001, when the female-group was compared with male- and orchiectomized male-group and ***p=0.0002, when the ovariectomized female-group was compared with orchiectomized male-group 2 h after intradermal LMWH; ****p<0.0001, when the female-group was compared with male-, ovariectomized female- and orchiectomized male-group and 4 h after intradermal LMWH; two-way repeated-measures ANOVA followed by Holm-Šídák multiple comparison test) n=6 per group.

Figure 2.. Role of CD44 in LMWH-induced hyperalgesia in male and female rats.

Male and female rats received intrathecal injections of antisense (120 μg/20 μL) or mismatch (120 μg/20 μL) ODN against CD44 mRNA, once a day for three consecutive days. On the fourth day, 24 h after the last intrathecal administration of ODNs, LMWH (1 μg/5 μL) was injected intradermally, on the dorsum of the hind paw, and mechanical nociceptive threshold evaluated 5, 10, 15, 20, 25, and 30 min later. An additional group of male and female rats received intrathecal ODN treatment against CD44 mRNA, LMWH injected intradermally, and mechanical nociceptive threshold evaluated 1, 2, 4, 8 and 24 h after LMWH. A. Males: On the fourth day, at which time the mechanical nociceptive threshold was not significantly different from mismatch controls (t₍₅₎=0.0000; p>0.9999; for antisense ODN male group and t₍₅₎=1.000; p=0.3632 to mismatch ODN male group, when the mechanical nociceptive threshold is compared before the 1^st and approximately 24 h after the 3^rd intrathecal injection of ODNs; paired Student’s t test), LMWH-induced hyperalgesia was significantly attenuated 15, 20 and 30 min and 1 h after its injection, in the group of male rats treated with CD44 antisense ODN, compared to male rats treated with CD44 mismatch ODN (F_(1,10)=20.31, *p=0.0373, **p=0.0042, ****p<0.0001 and ***p=0.0002; when the LMWH-induced hyperalgesia is compared between CD44 antisense ODN- and mismatch ODN-treated groups 15, 20, 30 min and 1 h after intradermal LMWH, respectively; two-way repeated-measures ANOVA followed by Holm-Šídák multiple comparison test). n=6 per group. B. Females: In female rats, at a time at which the mechanical nociceptive threshold was not significantly different from mismatch controls (t₍₅₎=2.236; p=0.0756; for antisense ODN female group and t₍₅₎=0.0000; p>0.9999 to mismatch ODN female group; paired Student’s t test) rats treated with CD44 antisense ODN or mismatch ODN, LMWH-induced hyperalgesia was significantly attenuated, 10, 15, 20, 30 min and 1, 2 and 4 h later, in the CD44 antisense ODN treated group (F_(1,10)=48.63, *p=0.0210, when the LMWH-induced hyperalgesia is compared between CD44 antisense ODN- and CD44 mismatch ODN treated groups 10 and 15 min after intradermal LMWH; **p=0.0047, when the LMWH-induced hyperalgesia is compared between CD44 antisense ODN- and CD44 mismatch ODN-treated groups 20 and 30 min after intradermal LMWH,****p<0.0001; when the LMWH-induced hyperalgesia is compared between CD44 antisense ODN- and CD44 mismatch ODN-treated groups 1, 2 and 4 h after intradermal LMWH; two-way repeated-measures ANOVA followed by Holm-Šídák multiple comparison test). n=6 per group.

Figure 3.. Role of TLR4 in LMWH-induced hyperalgesia.

Male and female rats received intrathecal injections of antisense (120 μg/20 μL) or mismatch (120 μg/20 μL) ODN for TLR4 mRNA, once a day for three consecutive days. On the fourth day, 24 h after the last intrathecal administration of ODN, LMWH (1 μg/5 μL) was injected intradermally, on the dorsum of the hind paw, and mechanical nociceptive threshold evaluated 5, 10, 15, 20, 25, and 30 min after injection of LMWH. An additional group of male and female rats received the same intrathecal ODN treatment against TLR4 mRNA and LMWH intradermally, and mechanical nociceptive threshold evaluated 1, 2, 4, 8 and 24 h after LMWH. A. Males: In male rats treated with TLR4 ODN, pre-ODN baseline mechanical nociceptive threshold was not significantly different from threshold in mismatch controls (t₍₅₎=1.865; p=0.1212; for antisense ODN male group and t₍₅₎=1.000; p=0.3632 to mismatch ODN male group; paired Student’s t test), hyperalgesia induced by i.d. LMWH was significantly attenuated at the 15–30 min time points, in male rats treated with TLR4 antisense ODN (F_(1,10)=2.103, **p=0.0030, when the LMWH-induced hyperalgesia is compared between TLR4 antisense ODN- and TLR4 mismatch ODN-treated groups 15 min after intradermal LMWH; *p=0.0154, when the LMWH-induced hyperalgesia is compared between TLR4 antisense ODN- and TLR4 mismatch ODN-treated groups 20 and 30 min after intradermal LMWH, respectively; two-way repeated-measures ANOVA followed by Holm-Šídák multiple comparison test). n=6 per group. B. Females: In female rats, treated with antisense ODN for TLR4 mRNA, pre-ODN baseline mechanical nociceptive threshold was not significantly different from the threshold in mismatch controls (t₍₅₎=0.8771; p=0.4206; to antisense ODN female group and t₍₅₎=1.234; p=0.2722 to mismatch ODN female group; paired Student’s t test). In the TLR4 antisense ODN-treated group, LMWH-induced hyperalgesia was also significantly attenuated 15–25 min later (F_(1,10)=48.63, **p=0.0068, when the LMWH-induced hyperalgesia is compared between TLR4 antisense ODN- and TLR4 mismatch ODN-treated groups 15 min after intradermal LMWH; *p=0.0135, when the LMWH-induced hyperalgesia is compared between TLR4 antisense ODN- and TLR4 mismatch ODN-treated groups 20 after intradermal LMWH; ***p=0.0003, when the LMWH-induced hyperalgesia is compared between TLR4 antisense ODN- and TLR4 mismatch ODN-treated group 30 after intradermal LMWH; **p=0.0070; when the LMWH-induced hyperalgesia is compared between TLR4 antisense ODN- and TLR4 mismatch ODN-treated groups 1 and 2 h after intradermal LMWH; ***p=0.0001; when the LMWH-induced hyperalgesia is compared between TLR4 antisense ODN- and TLR4 mismatch ODN-treated groups 4 h after intradermal LMWH; two-way repeated-measures ANOVA followed by Holm-Šídák multiple comparison test). n=6 per group.

Figure 4.. Role of RHAMM in LMWH-induced hyperalgesia.

Male and female rats received intrathecal injections of antisense (120 μg/20 μL) or mismatch (120 μg/20 μL) ODN against RHAMM mRNA, once a day for three consecutive days. On the fourth day, after the last intrathecal administration of ODN, LMWH (1 μg/5 μL) was injected intradermally, on the dorsum of the hind paw, and mechanical nociceptive threshold evaluated 5, 10, 15, 20, 25, and 30 min later. An additional group of male and female rats received the same intrathecal ODN treatment against RHAMM mRNA, LMWH injected intradermally, and mechanical nociceptive threshold evaluated 1, 2, 4, 8 and 24 h after LMWH. A. Males: While pre-ODN baseline mechanical nociceptive threshold was not significantly different in male rats treated with antisense or mismatch ODN for RHAMM mRNA (t₍₅₎=2.445; p=0.0583; for the antisense ODN male group and t₍₅₎=1.732; p=0.1438 for the mismatch ODN male group; paired Student’s t test), in male rats treated with RHAMM antisense ODN, the hyperalgesia induced by intradermal LMWH was significantly attenuated when measured 20–25 min later (F_(1,10)=4.270, **p=0.0042, when the LMWH-induced hyperalgesia is compared between RHAMM antisense ODN- and RHAMM mismatch ODN-treated groups 20 min after intradermal LMWH; *p=0.0488, when the LMWH-induced hyperalgesia is compared between RHAMM antisense ODN- and RHAMM mismatch ODN-treated groups 30 min after intradermal LMWH, respectively; two-way repeated-measures ANOVA followed by Holm-Šídák multiple comparison test). n=6 per group. B. Females: In female rats treated with antisense or mismatch ODN for RHAMM mRNA, pre-ODN baseline mechanical nociceptive thresholds were not significantly different (t₍₅₎=1.291; p=0.2532; for the antisense ODN female group and t₍₅₎=0.5222; p=0.6238 for the mismatch ODN female group; paired Student’s t test). In groups treated with RHAMM antisense or mismatch ODN, LMWH-induced hyperalgesia was present at all time points (F_(1,10)=0.6706, p=0.4319; two-way repeated-measures ANOVA followed by Holm-Šídák multiple comparison test). Our findings support the suggestion that in both male and female rats LMWH-induced hyperalgesia is dependent on CD44 and TLR4. However, the hyperalgesia induced by LMWH is dependent on RHAMM in male rats, but not females. n=6 paws per group.

Figure 5.. Sex difference in LMWH-induced hyperalgesia.

A. Ovariectomy. Groups of female rats underwent ovariectomy, and one group of rats also received 17β-estradiol replacement, 14 days prior to intrathecal injection of antisense (120 μg/20 μL) or mismatch (120 μg/20 μL) ODN for RHAMM mRNA, once a day for three consecutive days. On the fourth day, after the last intrathecal administration of ODN, LMWH (1 μg/5 μL) was injected intradermally, on the dorsum of the hind paw, and mechanical nociceptive threshold evaluated 5, 10, 15, 20, and 30 min later. The magnitude of the mechanical hyperalgesia in ovariectomized female rats was attenuated in those treated with RHAMM antisense ODN, compared to those treated with its mismatch ODN, while ovariectomized females with 17β-estradiol replacement showed a similar response to the gonad intact female group. (F_(3,20)=17.93, *p=0.0182, when the LMWH-induced hyperalgesia is compared between RHAMM antisense ODN- and RHAMM mismatch ODN-, 17β-estradiol plus RHAMM antisense ODN- and 17β-estradiol plus RHAMM mismatch ODN-treated groups 15 min after intradermal LMWH; ***p=0.0008, when the LMWH-induced hyperalgesia is compared between RHAMM antisense ODN- and RHAMM mismatch ODN-, 17β-estradiol plus RHAMM antisense ODN- and 17β-estradiol plus RHAMM mismatch ODN-treated groups 20 min after intradermal LMWH; **p=0.0024, when the LMWH-induced hyperalgesia is compared between RHAMM antisense ODN- and RHAMM mismatch ODN-, 17β-estradiol plus RHAMM antisense ODN- and 17β-estradiol plus RHAMM mismatch ODN-treated groups 30 min after intradermal LMWH; two-way repeated-measures ANOVA followed by Holm-Šídák multiple comparison test). n=6 paws per group. B. GPR30 ODN-treatment. Female rats were treated daily with spinal intrathecal injections of antisense or mismatch ODN (120 μg/20 μl) to GPR30 and RHAMM mRNA, for 3 consecutive days. On the fourth day, LMWH (1 μg/5 μL) was injected intradermally, on the dorsum of the hind paw, and the magnitude of mechanical nociceptive threshold, evaluated over 30 min. LMWH-induced hyperalgesia was significantly attenuated in rats that received antisense to both GPR30 and to RHAMM mRNA (F_(3,20)=6.789, **p=0.0094, when the LMWH-induced hyperalgesia is compared between GPR30 antisense ODN + RHAMM antisense ODN- and others-treated groups 20 and 30 min after intradermal LMWH; two-way repeated-measures ANOVA followed by Holm-Šídák multiple comparison test). Antisense ODN to GPR30 alone did not affect the magnitude of LMWH-induced hyperalgesia (data not shown). n=6 paws per group.

Figure 6.. Attenuation of LMWH hyperalgesia by HMWH.

Male and female rats received HMWH (1 μg/5 μL) or vehicle (5 μL) intradermally, on the dorsum of the hind paw, 10 min after LMWH (1 μg/5 μL) was injected in the same site. Mechanical nociceptive threshold was evaluated over the 30 min after LMWH administration. A. Male: HMWH significantly attenuated LMWH-induced hyperalgesia in males (F_(1,10)=33.54, *p=0.0103, when the LMWH-induced hyperalgesia is compared between HMWH- and vehicle-treated groups 10 min after intradermal LMWH; **p=0.0040, when the LMWH-induced hyperalgesia is compared between HMWH- and vehicle-treated groups 15 and 30 min after intradermal LMWH; ***p=0.0001, when the LMWH-induced hyperalgesia is compared between HMWH- and vehicle-treated groups 20 min after intradermal LMWH; two-way repeated-measures ANOVA followed by Holm-Šídák multiple comparison test). n=6 paws per group. B. Female: HMWH did not significantly attenuate LMWH-induced hyperalgesia in females (F_(1,11)=0.6400, p=0.440, when the LMWH-induced hyperalgesia is compared between HMWH- and vehicle-treated groups after intradermal LMWH; two-way repeated-measures ANOVA followed by Holm-Šídák multiple comparison test). n=6 paws per group.

Figure 7.. HMWH attenuates LMWH hyperalgesia in ovariectomized female rats and in female rats treated with ODN antisense to GPR30 mRNA.

A. Ovariectomy. Female rats underwent ovariectomy 14 days prior to administration of HMWH (1 μg/5 μL) or vehicle (5 μL), injected intradermally on the dorsum of the hind paw, 10 min after LMWH (1 μg/5 μL) was injected in the same site. Magnitude of mechanical hyperalgesia, evaluated over 30 min after intradermal LMWH, in ovariectomized females was attenuated by HMWH (in contrast to intact females (see Figure 2)) (F_(1,10)=33.37, **p=0.0073, when the LMWH-induced hyperalgesia is compared between HMWH- and vehicle-treated groups 10 min after intradermal LMWH; *p=0.0143, when the LMWH-induced hyperalgesia is compared between HMWH- and vehicle-treated groups 15 min after intradermal LMWH; **p=0.0018, when the LMWH-induced hyperalgesia is compared between HMWH- and vehicle-treated groups 20 min after intradermal LMWH; ***p=0.0003, when the LMWH-induced hyperalgesia is compared between HMWH- and vehicle-treated groups 30 min after intradermal LMWH; two-way repeated-measures ANOVA followed by Holm-Šídák multiple comparison test). n=6 paws per group. B. GPR30 ODN-treated. Female rats were treated daily with spinal intrathecal injections of antisense or mismatch ODN (120 μg/20 μL) to GPR30 mRNA, for 3 consecutive days. On the fourth day, HMWH (1 μg/5 μL) was injected intradermally, on the dorsum of the hind paw, and 10 min later, LMWH (1 μg/5 μL) was injected in the same site. The magnitude of mechanical hyperalgesia, evaluated over 30 min after intradermal LMWH, was significantly attenuated in rats treated with antisense, compared to mismatch, GPR30 ODN (F_(1,10)=33.59, **p=0.0071, when the LMWH-induced hyperalgesia is compared between GPR30 antisense ODN- and GPR30 mismatch ODN treated groups 10 and 30 min after intradermal LMWH; *p=0.0142, when the LMWH-induced hyperalgesia is compared between GPR30 antisense ODN and GPR30 mismatch ODN treated groups 15 min after intradermal LMWH; ****p<0.0001, when the LMWH-induced hyperalgesia is compared between GPR30 antisense ODN- and GPR30 mismatch ODN-treated groups 20 min after intradermal LMWH; two-way repeated-measures ANOVA followed by Holm-Šídák multiple comparison test). n=6 paws per group. C. Ovariectomy plus 17β-estradiol. A group of female rats that underwent ovariectomy received 17β-estradiol replacement, 14 days prior to administration of HMWH (1 μg/5 μL) or vehicle (5 μL), injected intradermally on the dorsum of the hind paw, 10 min after LMWH (1 μg/5 μL) was injected in the same site. The magnitude of mechanical hyperalgesia was evaluated over 30 min after intradermal LMWH. Ovariectomized females with 17β-estradiol replacement show similar response to gonad intact female group, HMWH did not attenuate LMWH hyperalgesia (F_(1,10)=1.471, p=0.2531; two-way repeated-measures ANOVA followed by Holm-Šídák multiple comparison test). n=6 paws per group.