The Analgesic Effect And Synaptic Mechanism Of Gastrodin In The Spinal Dorsal Horn

Chronic pain is a kind of chronic disease, which attacks recurrently and protracts refractorily. It highly impairs mankind’s physical and mental health as well as quality of life, along with enormous medical costs of chronic pain. Persistent chronic pain makes the patients’ relationships and social activities in danger, and gradually leads to aversive emotion, such as anxiety, depression and fear. These patients were so overwhelmed with chornic pain that hardly wished to live. In recent years, substantial progress has been made in the mechanisms of chronic pain. However, the mainstream analgesics which are currently used in clinic, such as non-steroidal anti-inflammatory drugs(NSAIDs) and opioids, cannot meet demands of the patients. So it is an important medical and social significance to investigate the essence and pathogenesis of chronic pain in order to develop some new analgesics.Superficial dorsal horn(SDH) that transimits pain-related nociceptive information to the brain plays a crucial role on integration and sensitization. Spinal dorsal horn neurons and their dendrites or axons interrelation develop important excitatory and inhibitory loops. Our previous studies have shown that synaptic plasticity in spinal cord lamina I neurons played an important role during the process of the development of chronic pain. Therefore, the spinal cord lamina I neurons are one of the potential targets for analgesic drugs.Gastrodin(GAS) is one of the main bioactive constituent of traditional Chinese herbs “Tian ma”, which possesses anti-epileptic, anti-convulsant, sedative, analgesic, and neuroprotective effects. Amounting evidence has accumulated that GAS can significantly relieve the intractable chronic pain, such as trigeminal neuralgia, migraine, painful diabetic neuropathy, vascular headache in recent years. Previous studies have shown that GAS plays a significant inhibitory effect on the hyperexcitability of dorsal root ganglia(DRG) neurons under pathological states. Futher researches have found that the mechanisms may be through regulating the function of voltage-gated sodium channels, voltage-gated potassium channels and acid-sensing ion channels. However, very little is known whether GAS exerts analgesia in chronic inflammatory pain and which cellular and molecular mechanisms underlie this action. Therefore, it is important to study and explore the effect of GAS on chronic pain and its central analgesic mechanism, which will hopefully provide the scientific basis and theoretical guidance for the clinical application.Part 1: To expore the effect of GAS on spontaneous pain and hyperalgesia in inflammatory painObjective: To observe the analgesic effect of GAS on the mice of inflammatory pain. Methods: The pathological pain models were induced by unilateral subcutaneous(s.c.) of bee venom or Complete Freund’s Adjuvant(CFA) into the plantar surface of male adult mice hindpaws. Spontaneous pain was assessed immediately after injection of bee venom. Mechancial and thermal hyperalgesia as well as mechanical allodynia was assessed at different time points after CFA inflammation.Results: Different concentrations of GAS(50, 100, 200 mg/kg) or saline were intraperitoneally administered. As compared to vehicle, GAS dramatically attenuated spontaneous pain induced by bee venom and the development of mechanical allodynia and hyperalgesia as well as thermal hyperalgesia from CFA-inflammed mice in a dose-dependent manner(n = 5-8 mice per group, P < 0.05, one-way ANOVA). Moreover, the analgesic effect of GAS was naloxone-resistant, indicative of independence on μ opioid receptors. And no tolerance was seen after long-term administration of GAS. As compared to vehicle group, intrathecal GAS(10 m M, i.t.) resulted in a significant attenuation of mechanical allodynia, mechanical hyperalgesia and thermal hyperalgesia(n = 6 mice per group, P < 0.05, one-way ANOVA). However, both the basal nociception and motor coordination were not altered by GAS(200 mg/kg, i.p.)(n = 5-8 mice per group, P > 0.05, one-way ANOVA).Part 2: To explore the effect of GAS on spinal c-Fos expression induced by inflammationObjective: To obtain an overview of spinal cord that contributes to the analgesia induced by GAS, we sought a functional marker that permits assessment of populated neuronal activity to exert the second experiment.Methods: The bee venom-induced inflammatory pain models were set up with the above methods. Two hours later, the lumbar enlargement was removed after paraformaldehyde perfusion and fixation, then post-fixed, dehydrated, frozen sectioned and dryed by immunohistochemical ABC method.Results: The results showed bee venom injection produced a significant upregulation of c-Fos expression in the spinal dorsal horn of L4-L5 segments. Dense c-Fos was seen in the superficial(lamina I-II) and deep(lamina IV-V) lamina where the nociceptive primary afferents mainly terminates. As compared to vehicle, systemic delivery of GAS(50, 100, 200 mg/kg, i.p.) dose-dependently depressed c-Fos expression in both superficial and deep lamina of spinal dorsal horn(P < 0.05, one-way ANOVA). Moreover, the superficial lamina was inhibited by GAS more remarkably than deep lamina.Part 3: To explore the effect of GAS on excitatory synaptic transmission and excitability of SDH lamina I neuronsObjective: To explore the mechanisms by which GAS alleviates inflammatory pain, we further evaluated the effect of GAS on excitability synaptic transmission and excitability of neurons.Methods: The postnatal 14-18 d mice were injected with CFA into the plantar surface of hindpaw. At 24 h after CFA, transverse spinal slices with dorsal roots attached were obtained and whole cell patch clamp recordings of SDH lamina I neurons were performed. To observe the effect of GAS(300 μM) by bath application on excitatory synaptic transmission and hyperexcitability of neurons in chronic pain mice, the fiber-evoked excitatory postsynaptic currents(e EPSCs), miniature excitatory postsynaptic currents(m EPSCs), C-fiber-evoked EPSCs(C-e EPSCs), paired-pulse ratio(PPR) of C-e EPSCs as well as the active and passive membrane properties of SDH lamina I neurons were recorded.Results:(1) Compared with normal mice, input-output(I-O) curve of C-e EPSCs was shifted leftwards and upwards significantly after CFA inflammation. These results suggest that synaptic transmission of primary afferent synapse underwent a plastic potentiation in the spinal cord after peripheral inflammation. In 66.7%(10 out of 15) of spinal lamina I neurons examined, GAS significantly depressed the peak amplitude of C-e EPSCs recorded from CFA-inflamed mice(n = 10 neurons / 7 mice, P < 0.05, paired-t test). Quantitative analysis revealed an averaged inhibition of 29.18 ± 3.23% of C-e EPSCs by GAS.(2) Compared with normal mice, the frequency of m EPSCs were significantly increased from CFA-inflamed mice. Perfusing GAS significantly inhibited m EPSCs frequency without affecting the amplitude of m EPSCs, and the inhibition rate was 47.5 ± 7.66%(n = 7 neurons / 4 mice, P < 0.05, paired-t test). Moreover, GAS led to a remarkable change of PPR from C-e EPSCs in chronic inflammatory pain mice. Both m EPSCs and PPR analysis strongly support that the analgesic of GAS via presynaptic mechanisms.(3) Given strong inhibition of primary afferent synaptic potentiation by GAS in inflamed mice, we further tested the effect of GAS on the membrane properties and excitability of postsynaptic lamina I neurons, the output of primary afferent synapse.Compared with normal mice, following CFA inflammation, spinal lamina I neurons exhibited augmented excitabiliity under injection current condition, which manifested as increased action potential(AP) frequency, shortened AP half-width, greatened AP max-rise slope, lowered AP threshold and reduced rheobase. The passive membrane properties(Cm, Rm, RMP) of spinal lamina I neurons had no significant change(n = 16 neurons / 10 inflamed mice vs n = 16 neurons / 5 control mice, P < 0.05, one-way ANOVA). GAS could reverse the excitability of spinal lamina I neurons recorded from chronic inflammatory pain mice(n = 10 neurons / 8 inflamed mice vs n = 11 neurons / 5 control mice, P < 0.05, paired-t test). For example, AP frequency, AP half-width, AP max-rise slope, AP spike threshold and rheobase were normalized by GAS. Moreover, we found 28%(7/25) of spinal cord horn lamina I neurons from CFA inflammatory pain mice generated spontaneous firing under no injection current condition. GAS produced marked reduction on the frequency which the average inhibition rate was 33.04 ± 7.67%( n = 7 neurons / 4 mice, paired-t test) but not the amplitude of spontaneous firing.(4) What was different with the significant inhibition of inflammatory mice was that GAS had no effect on the peak amplitude of C-e EPSCs, the frequency and amplitude of m EPSCs, the change of PPR and the active and passive membrane properties of SDH lamina I neurons from normal mice.Conclusions:1, GAS(i.p.) dramatically attenuated spontaneous pain, allodynia and hyperalgesia from inflammed mice. GAS(i.t.) exerted an obvious analgesia at the spinal level. Both the basal nociception and motor coordination was not altered by GAS(i.p.). The above results revealed that GAS selectively inhibited pathological pain and had no significant effect on physiological pain, so it was a good analgesic.2, GAS(i.p.) dose-dependently depressed c-Fos expression upregulation of spinal dorsal horn from inflamed mice, indicating that GAS significantly inhibited the activity of spinal cord dorsal horn neurons induced by nociceptive stimulation.3, Superfusing GAS significantly inhibited synaptic potentiation of SDH lamina I neurons occurred in inflammatory pain states. GAS significantly inhibited m EPSCs frequency and resulted in a remarkable change of PPR in chronic inflammatory pain mice. The results strongly support that the inhibition of synaptic potentiation by GAS comes about via presynaptic mechanisms.4, GAS could reverse the excitability of spinal lamina I neurons recorded from chronic inflammatory pain mice. GAS remarkably inhibited the frequency of spontaneous firing. The above results suggest that GAS may inhibit spinal synaptic potentiation of presynaptic and further leading to reduction of postsynaptic hyperexcitability of spinal lamina I neurons.5, GAS had no effect on the peak amplitude of C-e EPSCs, the frequency and amplitude of m EPSCs, the change of PPR and the active and passive membrane properties of spinal cord lamina I neurons from normal mice. These results that GAS selectively inhibits pathological pain, without affecting normal physiological pain.