Intrathecal leptin inhibits expression of the P2X2/3 receptors and alleviates neuropathic pain induced by chronic constriction sciatic nerve injury

Abstract

Background: Leptin, an adipocytokine produced mainly by white adipose tissue, has a broad role in the regulation of neuronal functions. Accumulating evidence has revealed that leptin plays an important role in influencing neuropathic pain, shown recently by the finding that chronic administration of leptin induced thermal hyperalgesia and mechanical allodynia in naïve rats. Chronic constriction sciatic nerve injury (CCI) is a well characterized model used for studying neuropathic pain. The present study was designed to investigate whether leptin plays a role in neuropathic pain in rats induced by CCI by examining particular pain behaviors.

Results: After sciatic nerve injury in rats, endogenous levels of leptin and leptin receptor (OB-Rb) were increased in a time dependent manner within the ipsilateral dorsal root ganglion (DRG). Intrathecal administration of leptin once daily for 6 days, beginning 7 days after CCI, alleviated neuropathic pain and decreased the expression of IL-6, TNFα, and the P2X2 and P2X3 receptors. Attenuation of endogenous OB-Rb in the DRG by intrathecal administration of OB-Rb antisense oligonucleotides did not change thermal hyperalgesia or mechanical allodynia induced by CCI.

Conclusions: Our findings suggest that exogenous leptin can alleviate the chronic neuropathic pain caused by CCI. The leptin effect may be mediated by attenuated expression of IL-6, TNFα, and the P2X2 and P2X3 receptors in the DRG of CCI rats.

Figure 1

Leptin and OB-Rb were upregulated in a time dependent manner in L4-6 DRG of CCI rats. (A) The expression of leptin mRNA and OB-Rb mRNA was increased in the DRG of CCI rats. Leptin and OB-Rb mRNAs were detected by RT-PCR at days 1, 7, 14 and 21 after CCI. (B) Leptin and OB-Rb protein were increased in the DRG of CCI rats. Leptin and OB-Rb protein were detected by Western Blot analysis at days of 1,7,14 and 21 after CCI. Data are shown as mean ± S.E.M, n = 6. ^*P < 0.05, ^#P < 0.01 and ^@P < 0.001 vs sham group.

Figure 2

Leptin alleviates the neuropathic pain. The thermal withdrawl latency TWL (A) and mechanical withdrawl threshold MWT (B) were measured after intrathecal administration of leptin. Leptin was intrathecally delivered once daily for 6 days beginning 7 days after CCI, as indicated by arrows. The TWL and MWT were tested 2 h after each administration of leptin. Data are shown as mean ± S.E.M, n = 5. ^*P < 0.05, ^#P < 0.01 and ^@P < 0.001 vs CCI + Vehicle group.

Figure 3

Leptin attenuates the expression of IL-6, TNFα, and the P2X₂ and P2X₃ receptors in L4-6 DRG of CCI rats. (A) Leptin decreased the expression of P2X₂, P2X₃ receptors, IL-6 and TNFα mRNAs in a dose dependant manner. Leptin was intrathecally delivered once daily for 6 days beginning 7 days after CCI. The rats were sacrificed 24 h after the last leptin administration. The expression of P2X₂ and P2X₃ receptors mRNAs, and IL-6 and TNFα mRNAs were detected using RT-PCR. (B) Leptin decreased the expression of P2X₂, P2X₃, IL-6 and TNFα protein in a dose dependant manner. Leptin was delivered intrathecally once daily for 6 days beginning 7 days after CCI. The rats were sacrificed 24 hour after the last leptin administration. The expression of P2X₂, P2X₃, IL-6 and TNFα protein was detected by Western Blot. Data are shown as mean ± S.E.M, n = 6. ^*P < 0.05, ^#P < 0.01 and ^@P < 0.001 vs CCI + Vehicle group.

Figure 4

Leptin administration decreases the immunoreactivities of P2X₂ and P2X₃ receptors in L4-6 DRG of CCI rats. Leptin administration decreased the immunoreactivity of P2X₂(A) and P2X₃(B) receptors within the DRG of CCI rats. P2X₂ and P2X₃ receptors immunoreactivity (brown) was detected in DRG neurons of sham, CCI + vehicle, CCI + Leptin (10 μg), CCI + Leptin (50 μg) and CCI + Leptin (200 μg) treatments. Leptin was intrathecally delivered once daily for 6 days beginning 7 days after CCI. The rats were sacrificed 24 h after the last leptin administration. Arrows indicate P2X₂ and P2X₃ receptor positive neurons. Scale bar is 20 μm. Data are shown as mean ± S.E.M, n = 5. ^*P < 0.05, and ^#P < 0.01 vs CCI + Vehicle group.

Figure 5

Intrathecal administration of OB-Rb antisense oligonucleotides decreased OB-Rb mRNA and protein expression. (A) Intrathecal OB-Rb antisense oligonucleotides (120 μg/kg and 240 μg/kg) significantly decreased the expression of OB-Rb mRNA. (B) Intrathecal OB-Rb antisense oligonucleotides (120 μg/kg and 240 μg/kg) significantly decreased the expression of OB-Rb protein. The drugs were delivered intrathecally once daily for 6 days beginning on day 7 after CCI. The rats were sacrificed 24 h after of the last leptin administration. Data are shown as mean ± S.E.M, n = 6. ^*P < 0.05, and ^#P < 0.01 vs CCI + Vehicle group.

Figure 6

Effects of intrathecal OB-Rb antisense oligonucleotides on the pain behavior induced in CCI rats. The thermal withdrawl latency TWL (A) and mechanical withdrawl threshold MWT (B) were measured after intrathecal administration of antisense nucleotides directed against the leptin receptor. The AS oligonucleotides were delivered intrathecally once daily for 6 days beginning on day 7 after CCI, as indicated by arrows. The TWL and MWT were tested 2 h after each administration of leptin. Data are shown as mean ± S.E.M, n = 6.