Altered expression and uptake activity of spinal glutamate transporters after nerve injury contribute to the pathogenesis of neuropathic pain in rats

Abstract

The central glutamatergic system has been implicated in the pathogenesis of neuropathic pain, and a highly active central glutamate transporter (GT) system regulates the uptake of endogenous glutamate. Here we demonstrate that both the expression and uptake activity of spinal GTs changed after chronic constriction nerve injury (CCI) and contributed to neuropathic pain behaviors in rats. CCI induced an initial GT upregulation up to at least postoperative day 5 primarily within the ipsilateral spinal cord dorsal horn, which was followed by a GT downregulation when examined on postoperative days 7 and 14 by Western blot and immunohistochemistry. Intrathecal administration of the tyrosine kinase receptor inhibitor K252a and the mitogen-activated protein kinase inhibitor PD98059 for postoperative days 1-4 reduced and nearly abolished the initial GT upregulation in CCI rats, respectively. Prevention of the CCI-induced GT upregulation by PD98059 resulted in exacerbated thermal hyperalgesia and mechanical allodynia reversible by the noncompetitive NMDA receptor antagonist MK-801, indicating that the initial GT upregulation hampered the development of neuropathic pain behaviors. Moreover, CCI significantly reduced glutamate uptake activity of spinal GTs when examined on postoperative day 5, which was prevented by riluzole (a positive GT activity regulator) given intrathecally twice a day for postoperative days 1-4. Consistently, riluzole attenuated and gradually reversed neuropathic pain behaviors when the 4 d riluzole treatment was given for postoperative days 1-4 and 5-8, respectively. These results indicate that changes in the expression and glutamate uptake activity of spinal GTs may play a critical role in both the induction and maintenance of neuropathic pain after nerve injury via the regulation of regional glutamate homeostasis, a new mechanism relevant to the pathogenesis of neuropathic pain.

Fig. 1.

Time course of GT changes after CCI. CCI induced a biphasic change in the expression of EAAC1, GLAST, and GLT-1 within the spinal cord dorsal horn ipsilateral to CCI. There was an initial increase in GT expression when rats were examined on postoperative days 1 and 4, which was followed by a downregulation of all three GTs when rats were examined on postoperative days 7 and 14. Top, Western blot bands from representative CCI and sham rats showing postoperative changes in protein levels of EAAC1 (69 kDa), GLAST (67 kDa), and GLT-1 (67 kDa) within the ipsilateral spinal cord dorsal horn. β-Actin is a loading control. Bottom, Statistical analysis of relative density changes of Western blots between CCI and sham rats. The data are expressed as the percentage of changes in CCI rats as compared with sham controls.CCI-D1 to CCI-D14 refers to CCI rats on postoperative days 1–14. *p < 0.05 as compared with the sham control.

Fig. 2.

Topographic distribution of GT changes within the ipsilateral spinal cord dorsal horn. The photo micrographs taken from representative CCI and sham rats show an increase in EAAC1, GLAST, and GLT-1 in CCI rats (B, E, H) on postoperative day 4 within the ipsilateral spinal cord dorsal horn as compared with those of the sham control (A, D, G). All three GTs were downregulated when examined on postoperative day 14 (C, F, I). Note that EAAC1, GLAST, and GLT-1 are located primarily within the superficial laminas of the spinal cord dorsal horn. Scale bar, 100 μm.

Fig. 3.

Modulation of GT expression by the Trk receptor inhibitor K252a or the MAPK inhibitor PD98059. The upregulation of EAAC1, GLAST, and GLT-1, respectively, was reduced and nearly abolished by intrathecal treatment with K252a (0.5 μg) and PD98059 (1 μg) given twice daily between postoperative days 1 and 4 beginning immediately after nerve ligation. Top, Western blots from representative CCI and sham rats with samples taken on postoperative day 3 (the C+P3 group) or day 5 (the remaining groups). Bottom, Statistical data showing GT expression in CCI rats treated with K252a (C+K), PD98059-day 3 (C+P3), PD98059-day 5 (C+P5), or vehicle (C+V) and in sham rats treated with vehicle (SHAM), K252a (S+K), or PD98059 (S+P). *p < 0.05 and **p < 0.01 as compared with the sham control; ⁺p < 0.05 as compared with the C+V group. Note that the C+V group shows the bands from vehicle-treated CCI rats on day 5, because the percentage of change of GT expression in vehicle-treated CCI rats did not differ significantly between postoperative days 3 and 5 (see Results for details).

Fig. 4.

Modulation of thermal hyperalgesia and mechanical allodynia by PD98059. Thermal hyperalgesia (A) and mechanical allodynia (B) were exacerbated in CCI rats treated with PD98059 (1 μg, i.t.) twice daily between postoperative days 1 and 4 beginning immediately after the operation. This exacerbation was manifested as a shortened onset of and an enhanced degree of thermal hyperalgesia and mechanical allodynia in those CCI rats. K252a did not increase neuropathic pain behaviors significantly. *p < 0.05 and **p < 0.01 as compared with sham control;⁺p < 0.05 as compared with CCI rats treated with vehicle. Diff. score, Difference scores (contralateral minus ipsilateral withdrawal latency).

Fig. 5.

MK-801 reversed thermal hyperalgesia and mechanical allodynia potentiated by PD98059. A single intrathecal administration of 10 nmol of MK-801 on postoperative day 5 reversed thermal hyperalgesia (top) and mechanical allodynia (bottom) in CCI rats treated with PD98059 (1 μg, i.t.) when they were examined at 30 min after the MK-801 treatment. The same dose of MK-801 also reversed thermal hyperalgesia and mechanical allodynia in CCI rats treated with vehicle or K252a. MK-801 alone did not change the baseline response to thermal or mechanical stimulation. *p < 0.05 and **p < 0.01 as compared with the corresponding baseline level. Pre-MKand After-MK refer to nociceptive responses before and at 30 min after the MK-801 treatment on postoperative day 5, respectively. For definition of Diff. score, see Figure4.

Fig. 6.

Changes in spinal glutamate uptake activity in CCI rats and its modulation by riluzole. CCI induced a significant reduction of glutamate uptake activity in the samples taken from the ipsilateral L₄–L₅ spinal cord dorsal horn as compared with that of sham rats when examined on postoperative day 5. When they were examined on postoperative day 5, glutamate uptake activity in CCI rats was enhanced by intrathecal treatment with 10 μg riluzole (CCI+RIL), but not vehicle (CCI+VEH), given twice daily for postoperative days 1–4 beginning immediately after the operation. Note that the 4 d riluzole treatment also moderately enhanced the glutamate uptake activity of the contralateral spinal cord dorsal horn in CCI rats and of bilateral spinal cord dorsal horn in sham rats (SHAM+RIL). *p < 0.05 as compared with the SHAM+VEH group.

Fig. 7.

Effects of riluzole on the induction of thermal hyperalgesia and mechanical allodynia after CCI. Intraperitoneal treatment with 1 or 4 mg/kg riluzole (RIL-1 orRIL-4) twice daily for postoperative days 1–4 beginning immediately after the operation significantly reduced the development of thermal hyperalgesia (A) and mechanical allodynia (B). Note that the effects of riluzole on thermal hyperalgesia and mechanical allodynia lasted at least several days after its discontinuation on postoperative day 5, indicating a significant role of regulating GT uptake activity in the development of neuropathic pain behaviors in CCI rats. The effect of riluzole on thermal hyperalgesia was dose-dependent. *p < 0.05 and **p < 0.01 as compared with CCI rats treated with vehicle (CCI+VEH); ⁺p < 0.05 as compared with the low dose (1 mg/kg) riluzole group. ForDiff. score, see Figure 4.

Fig. 8.

Effects of riluzole on the maintenance of thermal hyperalgesia and mechanical allodynia after CCI. Intraperitoneal treatment with 1 or 4 mg/kg riluzole (RIL-1 orRIL-4) twice daily for 4 d beginning on postoperative day 5 gradually reversed thermal hyperalgesia (A) and mechanical allodynia (B) when rats were examined on postoperative days 8 and 10. *p < 0.05 and **p < 0.01 as compared with CCI rats treated with vehicle (CCI+VEH); ⁺p < 0.05 as compared with the low dose (1 mg/kg) riluzole group.Arrows indicate the beginning of the riluzole treatment on postoperative day 5. For Diff. score, see Figure4.