| The most common complication of herpes zoster (HZ) is postherpetic neuralgia (PHN) which has been defined as severe pain occurring 1 month after rash onset and persisting for more than 3 months. PHN is classed as a chronic pain syndrome which is characterized by all features of neuropathic pain, such as allodynia (normally nonpainful stimuli become painful), hyperalgesia (increased sensitivity to normally painful stimuli) and spontaneous pain. PHN is often prolonged, increases in incidence with age and responds poorly to current analgesics such as anticonvulsants, tricyclic antidepressants, opioids and non-steroidal anti-inflammatory drugs (NSAIDS). PHN can result in impaired sleep, emotional distress and depression which patients may suffer for years. Taken together, these manifestations of PHN has a major impact on life quality of HZ patients. However, there is still no ideal explanation on the neural mechanisms underlying PHN.According to classic pain research, the pain pathway has been assumed to be composed entirely of neurons. However, recent studies have shown that spinal cord glia and proinflammatory cytokine are identified to be strongly involved in the creation and maintenance of diverse exaggerated neuropathic pain states. After inflammation or nerve injury, spinal astrocytes can be activated and release cytokines that modulate neuronal activity and synaptic strength. Several recent studies showed that IL-1βsignaling may facilitate NMDAR activation (NMDAR activation is mainly manifested by NR1 subunit phosphorylation) in neurons to strengthen painful signal transmission. However, it remains unknown whether spinal glial cells are activated under the chronic pain state of PHN and whether they contribute to PHN.Investigation of the neural mechanisms underlying PHN has been facilitated by the introduction of an in vivo rat model of varicella zoster virus (VZV) persistent infection. Recent studies indicated that this rat model was a useful model for studying PHN, and this model could mimic the chronic pain states that occur in HZ patients. Therefore, we investigated the role of spinal cord glia in the pathophysiology of PHN by using this PHN model. In this study, it was found that:①Compared with naive rats and mock infected rats, the paw withdrawal threshold of VZV infected rats was significantly decreased. However, valaciclovir (antiviral agent) treatment had no effect on the development of mechanical allodynia (decreased paw withdrawal threshold). These results indicated that VZV infection contributed to mechanical allodynia which is resistant to antiviral therapy.②Immunohistochemistry indicated that compared with naive rats and mock infected rats, GFAP staining was significantly increased in the spinal cord of VZV infected rats. This enhanced staining concentrated in superficial dorsal horn. Using Western blot, we detected that GFAP expression was significantly increased in VZV infected rats. Furthermore, the expression level of GFAP was found to be significantly correlated to the paw withdrawal threshold in VZV infected group (P < 0.001, r = 0.868). Real-time reverse transcription polymerase chain reaction (RT-PCR) showed that GFAP mRNA expression was significantly increased in VZV infected rats compared to the control group. In all the mice, OX42 expression in spinal cord was unchanged through the period tested. These results indicated that spinal astrocyte but not microglia was activated in the state of PHN, which may be important for the regulation of spinal nociception.③We injected LAA or minocycline intrathecally and observed their effects on the induction and maintenance of mechanical allodynia in VZV infected rats. The astrocytic specific toxin LAA dose-dependently attenuated the allodynia. However, the microglial specific inhibitor minocycline did not influence mechanical allodynia. Most importantly, the combination of LAA with tricyclic antidepressants, antiepileptics or morphine showed synergistic analgesic effects on mechanical allodynia in VZV infected rats. Combined administration obviously relieved mechanical allodynia in a quick and stable manner. These results indicated that spinal astrocytic activation but not microglial activation surely contributed to allodynia. Combining LAA with widely used analgesics might be more effective and lead to less side effect.④Compared with naive rats and mock infected rats, the responsiveness of spinal WDR neurons was significantly enhanced in VZV infected rats, which indicated that spinal central sensitization occurred in VZV infected rat. We injected LAA or minocycline intrathecally and observed their effects on increased responsiveness of WDR neurons in VZV infected rats. The astrocytic specific toxin LAA significantly attenuated the increased responsiveness. However, the microglial specific inhibitor minocycline did not have any effect. These results indicated that spinal astrocytic activation contributed to central sensitization of spinal dorsal horn neurons.⑤Compared with naive rats and mock infected rats, inducible nitric oxide synthase (iNOS) expression was significantly increased in dorsal root ganglion and spinal cord of VZV infected rats. Furthermore, intrathecal treatment with L-NIL (inhibitor of iNOS) or PTIO (scavenger of NO) significantly reduced GFAP overexpression in VZV infected rats. These results indicated that spinal astrocytic activation depended on activation of iNOS and excessive production of NO.⑥Western blot analysis showed that spinal IL-1βexpression was significantly increased in VZV infected rats. We injected pentoxifylline (cytokine inhibitor) or IL-1ra (interleukin-1 receptor antagonist) intrathecally and observed their effects on mechanical allodynia in VZV infected rats. Both pentoxifylline and IL-1ra could significantly attenuated the allodynia. The time course of IL-1βexpression was similar to that of GFAP expression. Double immunofluorescent staining of spinal cord showed that IL-1β-immunoreactivity was only localized in GFAP-immunopositive cells but not in NeuN-immunopositive cells or OX42-immunopositive cells. In VZV infected rats, intrathecally administered LAA could significantly down-regulate IL-1βoverexpression. These results indicated that spinal astrocytes dramatically increased the expression of IL-1βwhich was related to allodynia.⑦Western blot analysis showed that phosphorylated NR1 subunit of NMDA receptor (P-NR1) expression in spinal cord was significantly increased in VZV infected rats. We injected AP5 (NMDA receptor antagonist) or MK-801 (non-competitive NMDA receptor antagonist) intrathecally and observed their effects on mechanical allodynia in VZV infected rats. Both AP5 and MK-801 could significantly attenuated the allodynia. The time course of P-NR1 expression was similar to that of IL-1βexpression or GFAP expression. Double immunofluorescent staining of spinal cord showed that P-NR1-immunoreactivity and IL-1RI-immunoreactivity were only localized in NeuN-immunopositive cells, and P-NR1-immunoreactivity and IL-1RI-immunoreactivity were totally double-labeled. In VZV infected rats, intrathecally administered LAA, pentoxifylline or IL-1ra could significantly down-regulate P-NR1 overexpression. These results indicated that IL-1βinduced neuronal NMDA receptor phosphorylation which finally contributed to mechanical allodynia.⑧Whole cell patch-clamp recordings from substantia gelatinosa neurons of spinal cord slices were performed in VZV infected rats. Both LAA and IL-1βneutralizing antibody could reduce the frequency, but not the amplitude of miniature excitatory postsynaptic currents (mEPSCs). Dorsal root stimulation resulted in evoked excitatory postsynaptic currents (eEPSCs) that were dose-dependently decreased in amplitude by LAA. The inhibitory effect of LAA could be facilitated by IL-1βneutralizing antibody. These results indicated that IL-1βreleased from astrocyte could bind to presynaptic or postsynaptic autoreceptor to mediate excitatory effect.In summary, the present study was the first time to provide evidence that spinal astrocytic activation contributed to mechanical allodynia in VZV-infected rats. The neural mechanism in astrocyte incudced allodynia may be that spinal activated astrocytes dramatically increased the expression of IL-1βwhich may induce NMDA receptor phosphorylation in spinal dorsal horn neurons to enhance neuronal activity and pain transmission. These findings suggest that spinal astrocytic inhibition may hold a therapeutic promise in the treatment of postherpetic neuralgia. |
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