Estrogen affects neuropathic pain through upregulating N-methyl-D-aspartate acid receptor 1 expression in the dorsal root ganglion of rats

Abstract

Estrogen affects the generation and transmission of neuropathic pain, but the specific regulatory mechanism is still unclear. Activation of the N-methyl-D-aspartate acid receptor 1 (NMDAR1) plays an important role in the production and maintenance of hyperalgesia and allodynia. The present study was conducted to determine whether a relationship exists between estrogen and NMDAR1 in peripheral nerve pain. A chronic sciatic nerve constriction injury model of chronic neuropathic pain was established in rats. These rats were then subcutaneously injected with 17β-estradiol, the NMDAR1 antagonist D(-)-2-amino-5-phosphonopentanoic acid (AP-5), or both once daily for 15 days. Compared with injured drug naïve rats, rats with chronic sciatic nerve injury that were administered estradiol showed a lower paw withdrawal mechanical threshold and a shorter paw withdrawal thermal latency, indicating increased sensitivity to mechanical and thermal pain. Estrogen administration was also associated with increased expression of NMDAR1 immunoreactivity (as assessed by immunohistochemistry) and protein (as determined by western blot assay) in spinal dorsal root ganglia. This 17β-estradiol-induced increase in NMDAR1 expression was blocked by co-administration with AP-5, whereas AP-5 alone did not affect NMDAR1 expression. These results suggest that 17β-estradiol administration significantly reduced mechanical and thermal pain thresholds in rats with chronic constriction of the sciatic nerve, and that the mechanism for this increased sensitivity may be related to the upregulation of NMDAR1 expression in dorsal root ganglia.

Keywords: 17β-estradiol; N-methyl-D-aspartic acid receptor 1; chronic constriction injury; dorsal root ganglion; estrogen; immunoreactivity; nerve regeneration; neural regeneration; neuropathic pain; D(-)-2-amino-5-phosphonopentanoic acid; pain; peripheral nerve injury; sciatic nerve; spinal cord; western blot assay.

Figure 1

Effects of estrogen on the CCI-induced changes in PWMT and PWTL. PWMT (A) and PWTL (B) of rats in the five treatment groups at various times after nerve injury. Data are expressed as the mean ± SD from 10 rats in each group (one-way analysis of variance and Student-Newman-Keuls test). Data are calculated by using the results of three measurements. *P < 0.05, **P < 0.01, vs. sham-operated group; #P < 0.05, vs. CCI group. Sham-operated group: Exposed sciatic nerve without ligation. Estrogen group: CCI surgery + 2 μL/day 17β-estradiol injected subcutaneously. AP-5 group: CCI surgery + 100 nmol/day AP-5 injected subcutaneously. Estrogen + AP-5 group: CCI model + 2 μL/day 17β-estradiol + 100 nmol/day AP-5 injected subcutaneously. T0–T4: 1 day before surgery and 1, 3, 7, 14 days after surgery, respectively. CCI: Chronic constriction injury; AP-5: D(-)-2-amino-5-phosphonopentanoic acid; PWMT: paw withdrawal mechanical threshold; PWTL: paw withdrawal thermal latency.

Figure 2

Estrogen effects on NMDAR1 immunoreactivity as assessed by immunohistochemistry in rat lumbar spine L_4–6 dorsal root ganglia 14 days after CCI surgery. (A–E) NMDAR1 immunoreactivity in L_4–6 dorsal root ganglia derived from the sham-operated, CCI, estrogen, AP-5, and estrogen + AP-5 groups, respectively (immunohistochemical staining, light microscope, × 200). Scale bars: 50 μm. Arrows show NMDAR1 immunoreactive cells. (F) Quantification (integrated optical density; IOD) of NMDAR1 immunoreactive cells. Data are expressed as the mean ± SD from five rats in each group (one-way analysis of variance and Student-Newman-Keuls test). *P < 0.05, vs. sham-operated group; #P < 0.05, vs. with CCI group. See Figure 1 legend for group definitions. NMDAR1: N-methyl-D-aspartic acid receptor 1; CCI: chronic constriction injury; AP-5: D(-)-2-amino-5-phosphonopentanoic acid. I: Sham-operated; II: CCI; III: Estrogen; IV: AP-5: IV: Estrogen + AP-5.

Figure 3

Estrogen effects on NMDAR1 protein expression as assessed by western blot assay in lumbar spine L_4–6 dorsal root ganglia of rats with CCI. (A) NMDAR1 protein expression in rat L_4–6 spinal dorsal root ganglia examined 14 days after nerve injury. (B) Quantification (absorbance ratio) of NMDAR1 protein expression in each group. Data are expressed as the mean ± SD from five rats in each group (one-way analysis of variance and Student-Newman-Keuls test). *P < 0.05, vs. sham-operated group; #P < 0.05. vs. CCI group. See Figure 1 legend for group definitions. NMDAR1: N-methyl-D-aspartic acid receptor 1; CCI: chronic constriction injury; AP-5, D(-)-2-amino-5-phosphonopentanoic acid. I: Sham-operated; II: CCI; III: estrogen; IV: AP-5: IV: estrogen + AP-5.