The aims of the present study were two-fold. First, we examined the effects of antiinflammatory cytokine and glial inhibitors on orofacial hyperalgesia following focal microinjection into the subregions of the spinal trigeminal complex. Second, we further established the role of the trigeminal Vi/Vc transition zone in the development of hyperalgesia associated with deep tissue injury. After systematic comparison, it is clear that injection of an antiinflammatory cytokine, IL-10, and two glial inhibitors, fluorocitrate and minocycline, into the Vi/Vc transition zone attenuated hyperalgesia after masseter muscle inflammation, but had no effect on hyperalgesia after cutaneous inflammation. On the other hand, injection of these agents into the caudal Vc attenuated hyperalgesia associated with both masseter and cutaneous inflammation. Western blot data showed that masseter inflammation induced an enhanced NMDA receptor phosphorylation and upregulation of GFAP in both Vi/Vc and Vc zones, while cutaneous inflammation only induced similar effects in the caudal Vc. These findings support the view that glial activation and cytokine activity are important factors in persistent pain development and the Vi/Vc transition zone provides additional processing for deep orofacial input.
Cytokines are a complex group of proteins able to exert pleiotropic effects on activity of a variety of cells. Together with a group of so-called proinflammatory cytokines including tumor necrosis factor (TNF), IL-1, IL-6 and IL-8, a variety of antiinflammatory cytokines including IL-10 and the IL-1 receptor antagonist (IL-1ra) are also produced during humoral and cell-mediated immune responses . Interleukin-10 is induced in the CNS in a number of disease conditions . Analysis of cytokine mRNA expression in the CNS of mice with experimental autoimmune encephalomyelitis reveals that IL-10 mRNA expression correlates with recovery .
Interleukin-10 acts on immune cells to suppress the release of IL-1 and TNF-α . Interleukin-1ra, an analogue of IL-1, antagonizes IL-1 activity by competing for IL-1 receptors. We have previously shown that IL-1ra attenuated inflammation-induced NMDA receptor phosphorylation and hyperalgesia [17, 57]. The present study further found that IL-10 produced antihyperalgesia and inhibition of IL-1β transcription in a rat model of orofacial inflammatory pain when injected into the identified brain stem sites involved in trigeminal pain processing. These results are consistent with the antinociceptive and neuroprotective effects of IL-10 at the spinal level and support the use of IL-10 as a therapeutic agent against persistent pain [34, 35, 58–60].
The use of two glial inhibitors in the present study produced attenuation of orofacial hyperalgesia. These observations are consistent with the literature [61–63]. Minocycline selectively inhibits microglia [47, 48]. Fluorocitrate has been shown to be relatively selective against astroglia [19, 64]. Our results support the current view that central glial activation plays a role in orofacial hyperalgesia [17, 19, 20] or persistent pain in general . It appears that the involvement of glia occurred at multiple levels since the injection of glial inhibitors into the two subregions of the spinal trigeminal complex was effective in attenuating hyperalgesia. Evidence suggests that glia are intimately involved in the control of neuronal activity [65, 67]. Glial TNF-α modulates synaptic strength in the brain [68, 69]. However, the functional consequence of glial activation is not fully understood. Models of peripheral nerve injury and neuropathic pain have been associated with a decrease in astrocytic glutamate transporter activity [70–73] and astroglia are apparently activated in response to peripheral nerve or tissue injury [17, 74]. Thus, there is a reciprocal relationship between the astrocytic activation state after nerve injury and astrocytic glutamate transporter GLT-1 expression. In primary astrocytic cultures that exhibit an activated phenotype, propentofylline, a glial modulator, induces glutamate transporter GLT-1 expression and glutamate uptake . Since astroglial glutamate transporters play an important role in maintaining an appropriate level of glutamate extracellular concentration, a reduction in GLT-1 glutamate transporter expression may lead to a build up of glutamate concentration in the synaptic cleft, resulting in neuronal hyperexcitability and behavioral hyperalgesia.
After masseter inflammation, IL-1β is selectively induced in astroglia in the Vi/Vc region , although previous studies have indicated that IL-1β is produced primarily in microglia . It is interesting that both glial inhibitors suppressed inflammation-induced IL-1β mRNA upregulation. These results suggest interaction between microglia and astroglia in the mechanisms of inflammatory hyperalgesia. Studies have suggested that microglial activation precedes activation of astrocytes [see , for a review]. Activation of TLR4 on microglial cells may lead to astroglial activation . Kawasaki et al.  showed that early microglial and later astroglial activation were associated with neuropathic pain. Recent studies suggest that IL-18, a member of the IL-1 cytokine family, is released from microglia and acts on the IL-18 receptor on astrocytes . The IL-18-mediated microglia-astroglia interaction potentiates neuropathic pain behavior in rats; and inhibition of IL-18 signaling pathways suppresses astroglial activity and nerve injury-induced allodynia . Taken together, these findings support the view that coordinated activation of microglia and astroglia contribute to the development and maintenance of persistent pain.
The present study showed that orofacial inflammatory hyperalgesia was attenuated by injection of an antiinflammatory cytokine and two glial inhibitors into the spinal trigeminal complex, associated with a reduction of IL-1β mRNAs. However, these drugs did not produce an effect on baseline responses before inflammation, suggesting that cytokine and glial activity do not contribute to pain processing under normal conditions. While these data support the involvement of both glia and inflammatory cytokines in persistent pain, the mechanisms of interactions between IL-10 and glia during the pain processing are only speculative. Interleukin-10 not only inhibits synthesis of proinflammatory cytokines by macrophages/microglia, it also induces anergy in brain-infiltrating T cells . The activation of astroglia may be suppressed by IL-10 indirectly through inhibition of cytokine production . We have shown recently that astroglial activation and IL-1β upregulation in the Vi/Vc transition zone contributes to trigeminal responses to deep orofacial tissue inflammation and hyperalgesia . The inhibition of IL-1β mRNA transcription by IL-10 and glial inhibitors would be consistent with their antihyperalgesic effect. Importantly, IL-10 suppresses p38 mitogen-activated protein kinase activation in immune cells , which may be a key interface between IL-10 and glial cells in producing antihyperalgesia [see ].
Through a systematic comparison of the effects of IL-10, fluorocitrate and minocycline on masseter and cutaneous hyperalgesia, we further demonstrate a selective involvement of the trigeminal Vi/Vc transition zone in response to deep orofacial tissue injury. Injection of these agents into the Vi/Vc transition zone only attenuated hyperalgesia after masseter inflammation without an effect on cutaneous hyperalgesia. In contrast, both masseter and cutaneous hyperalgesia were attenuated after injection of these agents into the caudal Vc, which is consistent with a role of laminated Vc in trigeminal pain processing. These results are in good agreement with our previous study where an NMDA receptor antagonist was injected . These observations are further supported by Western blot analysis that shows a selective enhancement of NMDA receptor phosphorylation and GFAP upregulation in the Vi/Vc transition zone after masseter inflammation. The findings strengthen the view that while both deep and cutaneous orofacial nociceptive input are processed in the laminated Vc, the trigeminal Vi/Vc transition zone is also involved in integrating responses to deep tissue injury .
One important feature of neuronal activation in the Vi/Vc transition zone is that a unilateral injury always produces bilateral activation, particularly in the ventral Vi/Vc [8, 10, 1, 13]. This is in sharp contrast to caudal laminated Vc where a predominantly unilateral neuronal activation is always observed . Interestingly, contralateral as well as ipsilateral hyperalgesia developed after a unilateral injection of CFA into the masseter. In contrast, unilateral cutaneous inflammation only produced hyperalgesia on the ipsilateral side. It appears that contralateral Vi/Vc activation underlies the development of contralateral hyperalgesia, which should be categorized as secondary hyperalgesia or pain referred to a site remote from the injury. We also observed for the first time that unilateral administration of IL-10, fluorocitrate or minocycline into the Vi/Vc transition zone was able to attenuate ipsilateral, as well as contralateral, hyperalgesia after masseter inflammation. These results suggest an indirect or polysynaptic circuitry from the ipsilateral to the opposite Vi/Vc. An important relay is likely in the rostral ventromedial medulla (RVM), the pivotal structure in descending pain modulation including inhibition and facilitation . Sugiyo et al.  have shown a reciprocal pain facilitatory circuitry between the RVM and Vi/Vc transition zone. Lesions of the RVM eliminate masseter hyperalgesia on both sides . Neuronal activation in the Vi/Vc is also modulated by input from the caudal Vc [14, 84–86]. However, injection of either one of the three drugs into the Vc only reduced ipsilateral hyperalgesia without an effect on contralateral hyperalgesia. This would suggest that, with regard to secondary hyperalgesia, facilitation from the RVM-Vi/Vc circuitry is sufficient and necessary.