Spinal microglial β-endorphin signaling mediates IL-10 and exenatide-induced inhibition of synaptic plasticity in neuropathic pain

Abstract

Aim: This study aimed to investigate the regulation of pain hypersensitivity induced by the spinal synaptic transmission mechanisms underlying interleukin (IL)-10 and glucagon-like peptide 1 receptor (GLP-1R) agonist exenatide-induced pain anti-hypersensitivity in neuropathic rats through spinal nerve ligations.

Methods: Neuropathic pain model was established by spinal nerve ligation of L5/L6 and verified by electrophysiological recording and immunofluorescence staining. Microglial expression of β-endorphin through autocrine IL-10- and exenatide-induced inhibition of glutamatergic transmission were performed by behavioral tests coupled with whole-cell recording of miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) through application of endogenous and exogenous IL-10 and β-endorphin.

Results: Intrathecal injections of IL-10, exenatide, and the μ-opioid receptor (MOR) agonists β-endorphin and DAMGO inhibited thermal hyperalgesia and mechanical allodynia in neuropathic rats. Whole-cell recordings of bath application of exenatide, IL-10, and β-endorphin showed similarly suppressed enhanced frequency and amplitude of the mEPSCs in the spinal dorsal horn neurons of laminae II, but did not reduce the frequency and amplitude of mIPSCs in neuropathic rats. The inhibitory effects of IL-10 and exenatide on pain hypersensitive behaviors and spinal synaptic plasticity were totally blocked by pretreatment of IL-10 antibody, β-endorphin antiserum, and MOR antagonist CTAP. In addition, the microglial metabolic inhibitor minocycline blocked the inhibitory effects of IL-10 and exenatide but not β-endorphin on spinal synaptic plasticity.

Conclusion: This suggests that spinal microglial expression of β-endorphin mediates IL-10- and exenatide-induced inhibition of glutamatergic transmission and pain hypersensitivity via presynaptic and postsynaptic MORs in spinal dorsal horn.

Keywords: IL-10; Neuropathic pain; exenatide; mEPSCs; mIPSCs; microglia; β-endorphin.

FIGURE 1

Mechanical allodynia and thermal hyperalgesia (A), increased neuronal c‐fos expression (B), regulation of astrocytic, microglial, and neuronal biomarkers GFAP, Iba‐1, and NeuN (C), enhanced excitatory synaptic transmission (D‐F), and reduced inhibitory transmission (G‐I) in the ipsilateral spinal dorsal horns of neuropathic rats. Neuropathic rats were induced by ligation of spinal nerves. Representative images and quantitative measurement of immunofluorescence staining of astrocytic, microglial, and neuronal biomarkers GFAP, Iba‐1, and NeuN and double immunofluorescence staining of c‐fos/NeuN in the ipsilateral spinal dorsal horns of lamina II of sham and neuropathic rats. Scale bar, 200 μm. Representative recording traces of mEPSCs and mIPSCs and cumulative distribution and statistical analyses were obtained from the lamina II spinal dorsal horn neurons. The data are presented as the means ± SEM (n = 4–8 animals). *p < 0.05, by unpaired and two‐tailed Student's t test or repeated‐measures two‐way ANOVA followed by Sidak's post‐tests

FIGURE 2

Schematic diagram showed the time procedures for spinal nerve surgery and drug administration (A). Blockade effects of the β‐endorphin antiserum and μ‐opioid receptor (MOR) antagonist CTAP on IL‐10 mechanical antiallodynia and thermal antihyperalgesia in the ipsilateral hind paws (B), inhibition of spinal excitatory synaptic transmission (C‐E), and effects on inhibitory synaptic transmission (F‐H) in neuropathic rats. Neuropathic rats were induced by ligation of spinal nerves. Representative mEPSCs and mIPSCs traces, cumulative distribution and statistical analyses were obtained from the lamina II spinal dorsal horn neurons. The data are presented as the means ±SEM (n = 5–6 animals). *p < 0.05, by one‐way or repeated‐measures two‐way ANOVA followed by Sidak's post‐tests)

FIGURE 3

Blockade effects of the IL‐10 antibody, β‐endorphin antiserum, and μ‐opioid receptor (MOR) antagonist CTAP on the GLP‐1 receptor agonist exenatide‐induced inhibition of mechanical allodynia and thermal hyperalgesia in the ipsilateral hind paws (A), inhibition of spinal excitatory synaptic transmission (B‐D), and effects on inhibitory synaptic transmission (E‐G) in neuropathic rats. Neuropathic rats were induced by ligation of spinal nerves. Representative mEPSCs and mIPSCs traces, cumulative distribution and statistical analyses were obtained from the lamina II spinal dorsal horn neurons. The data are presented as the means ±SEM (n = 5–6 animals). *p < 0.05, by one‐way or repeated‐measures two‐way ANOVA followed by Sidak's post‐tests

FIGURE 4

Blockade effects of the μ‐opioid receptor (MOR) antagonist CTAP on β‐endorphin and DAMGO‐induced mechanical antiallodynia and thermal antihyperalgesia in the ipsilateral hind paws (A), inhibition of spinal excitatory synaptic transmission (B‐D), and effects on inhibitory synaptic transmission (E‐G) in neuropathic rats. Neuropathic rats were induced by ligation of spinal nerves. Representative mEPSCs and mIPSCs traces, cumulative distribution and statistical analyses were obtained from the lamina II spinal dorsal horn neurons. Scale bars, 40 pA and 10 s. Representative double immunofluorescence staining of MORs with the presynaptic and postsynaptic biomarkers Bassoon and PSD‐95 in the spinal dorsal horns from three normal rats (H). Scale bar: 100 μm. The data are presented as the means ± SEM (n = 6 animals per group). *p < 0.05, by one‐way or repeated‐measures two‐way ANOVA followed by Sidak's post‐tests)

FIGURE 5

The effects of the microglial inhibitor minocycline on IL‐10‐, GLP‐1 receptor agonist exenatide‐, and β‐endorphin‐induced suppression of spinal excitatory synaptic transmission in neuropathic rats (A‐C). Neuropathic rats were induced by ligation of spinal nerves. Representative mEPSCs recording traces, cumulative distribution and statistical analyses were obtained from the lamina II spinal dorsal horn neuron. Scale bars, the data are presented as the means ± SEM. *p < 0.05, by one‐way ANOVA followed by Sidak's post‐tests

FIGURE 6

Schematic diagram showing the role of microglial expression of β‐endorphin in IL‐10‐ and specific GLP‐1 receptor agonist exenatide‐induced inhibition of spinal excitatory synaptic transmission and pain hypersensitivity in neuropathic pain. Following activation of GLP‐1 receptors, IL‐10 is released and then activates IL‐10 receptors via a microglial autocrine mechanism. Afterward, the β‐endorphin is released to microglial neuronal synapses and activates neuronal presynaptic and postsynaptic μ‐opioid receptors (MORs) to inhibit the enhanced glutamatergic transmission, leading to pain anti‐hypersensitivity