Central glucocorticoid receptors modulate the expression and function of spinal NMDA receptors after peripheral nerve injury

Abstract

Central glucocorticoid receptors (GRs) and NMDA receptors (NMDARs) have been shown to play a significant role in the mechanisms of neuropathic pain after peripheral nerve injury; however, how central GRs and NMDARs interact in this process remains unknown. Here we show that the expression and function of spinal NMDARs after peripheral nerve injury were modulated by central GRs. Chronic constriction nerve injury (CCI) in rats induced a time-dependent upregulation of NR1 and NR2 subunits of the NMDAR within the spinal cord dorsal horn ipsilateral to CCI. The upregulation of NMDARs was significantly diminished by intrathecal administration (twice daily for postoperative days 1-6) of either the GR antagonist RU38486 or an antisense oligonucleotide against GRs. Moreover, this CCI-induced expression of NMDARs was significantly attenuated in rats receiving intrathecal treatment with an interleukin-6 (IL-6) antiserum and in mice with protein kinase Cgamma (PKCgamma) knock-out. Because IL-6 and PKCgamma mediated the upregulation of central GRs after CCI as demonstrated previously, the results suggest that IL-6 and PKCgamma served as cellular mediators contributing to the GR-mediated expression of NMDARs after CCI. Functionally, nociceptive behaviors induced by NMDAR activation and CCI were reversed by a single intrathecal administration of the GR antagonist RU38486. Conversely, a single intrathecal injection with the noncompetitive NMDAR antagonist MK-801 reversed neuropathic pain behaviors exacerbated by the GR agonist dexamethasone in CCI rats. These data suggest that interactions between central GRs and NMDARs through genomic and nongenomic regulation may be an important mechanism critical to neuropathic pain behaviors in rats.

Figure 1.

a, b, RT-PCR (a) and statistical analysis (b) showed the time course of NR1 and NR2 expression after CCI within the ipsilateral (I) and contralateral (C) (NR1 only) spinal cord dorsal horn. c, d, Western blot (c) (100 kDa), as well as statistical analysis (d), also showed an increased NR1 expression within the ipsilateral spinal cord dorsal horn in the CCI rats. In a and b, ^*p < 0.05 and ^**p < 0.01 compared with sham rats. In this figure, the data from day 7 after sham operation were presented as sham control because there were no differences in the expression of NR1 or NR2 in sham rats across postoperative days 1, 3, 5, and 7 in our pilot experiments. For all figures, the number of animals per group is given in Results. Actin, β-Actin for loading control in all panels.

Figure 2.

a-c, Colocalization of GRs (a) and NR1 (b), as shown in the emerged image (c), was observed in the ipsilateral spinal cord dorsal horn of a representative CCI rat. The sample was taken at day 7 after CCI. DL, Dorsal lateral part of the dorsal horn. Scale bar, 60 μm. d, e, The GR antagonist RU38486 (2 μg, i.t.) given twice daily on postoperative days 1-6 attenuated the expression of NR1 (Western blot) within the ipsilateral spinal cord dorsal horn of CCI rats, when examined on postoperative day 7. ^*p < 0.05, compared with sham rats. S/V, Sham plus vehicle; C/V, CCI plus vehicle; C/RU, CCI plus RU38486. RU38486 alone did not change the baseline NR1 expression (data not shown).

Figure 3.

a, b, The expression of NR1 and NR2 (RT-PCR) was attenuated substantially in CCI rats treated with a GR antisense OND but not a sense or mixed-base OND. ^*p < 0.05 and ^**p < 0.01, compared with day 1 of each corresponding group. S1, S7; A1, A7; M1, M7: day 1 and day 7 from CCI rats treated with a GR sense (S), antisense (A), and mixed-base (M) OND, respectively. Sham, Sham control. c, d, GR expression (Western blot) (95 kDa) within the ipsilateral spinal cord dorsal horn was substantially attenuated in CCI rats treated with a GR antisense OND. ^*p < 0.05 and ^**p < 0.01, compared with CCI rats treated with vehicle (Veh). Actin, β-Actin (42kDa) for loading control. S1d-M7d: see legend for a and b.

Figure 4.

a, IL-6 antiserum (0.5 μg, i.t.) given once daily on postoperative days 1-6 attenuated the NR1 expression within the ipsilateral spinal cord dorsal horn in CCI rats, when examined on day 7. ^*p < 0.05 compared with both sham rats and CCI rats treated with the IL-6 antiserum. C/V, CCI plus vehicle; S/V, sham plus vehicle; C/IS, CCI plus IL-6 antiserum. b, Time course of NR1 and NR2 expression (RT-PCR) within the spinal cord dorsal horn of naive rats induced by IL-6 (33 ng) or a vehicle given intrathecally once daily for 6 consecutive days. c, NR1 expression was also observed using Western blot, and NR1 upregulation returned to baseline at 7 d after the discontinuation of IL-6 treatment (D14). ^*p < 0.05 and ^**p < 0.01 compared with the corresponding vehicle (Veh) treatment group. D1-D14 refers to the samples taken on days 1-14 of the experimental period.

Figure 5.

a,b, NR1 expression (RT-PCR) within the ipsilateral spinal cord dorsal horn after CCI was nearly prevented in PKCγ KO mice compared with the matched PKCγ WT mice. Con., Contralateral; Ipsi., ipsilateral.

Figure 6.

a, b, A single injection of the NMDAR agonist NMDA (5 μg, i.t.) induced both thermal hyperalgesia (a) and mechanical allodynia (b) in naive rats when examined at 30 min after the NMDA injection, which was prevented by pretreatment with 2 μg of RU38486 (RU) at 30 min before the NMDA injection. c, d, A single injection of RU38486 (2 μg, i.t.) on day 4 reversed both thermal hyperalgesia (c) and mechanical allodynia (d) induced by repeated treatment with 5 μg of NMDA (twice daily for 3 d), when examined at 30 min after the RU38486 injection. ^*p < 0.05 compared with the baseline in each corresponding group. FWL, Foot-with-drawal latency. Threshold force, Threshold bending force from von Frey filament stimulation.

Figure 7.

a, b, A single injection of RU38486 (2 μg, i.t.), but not vehicle, on postoperative day 7 reversed thermal hyperalgesia (a) and mechanical allodynia (b) in CCI rats when examined at 30 min after the RU38486 injection. ^*p < 0.05 compared with sham rats. C/RU, CCI plus RU38486; C/V, CCI plus vehicle. c, d, A single injection of the noncompetitive NMDAR antagonist MK-801 (10 nmol, i.t.) on day 7 reversed thermal hyperalgesia (c) and mechanical allodynia (d) exacerbated by the dexamethasone treatment (DEX) (4 μg, i.t.; twice daily for 6 d) in CCI rats, when examined at 30 min after the MK-801 injection. ^*p < 0.05 compared with sham rats. CCI, CCI rats; C/D/V, CCI plus DEX plus vehicle; C/D/MK, CCI plus DEX plus MK-801. FWL/Threshold force: see Figure 6 legend.