Effects Of Minocycline On The Orofacial Inflammatory Pain And P38MAPK Pathway

The orofacial region is one of the most densely innervated (by the trigeminal nerve) areas of the body, which focuses some of the most common pains. However, the mechanisms underlying these pains are still poorly understood, partly due to the relative scarcity of investigations devoted to the face and the mouth, compared to the rest of the body. P38MAPK represents a group of enzymes in the MAPK family that phosphorylated and activated by a variety of physical, chemical and biological stimuli. Accumulating evidences suggested that p38MPAK played an important role in neural plasticity through regulating cell transcription, protein synthesis and receptor expression. Recent work suggested that activation of p38 mitogen-activated protein kinase (p38) in dorsal root ganglia and spinal cord played a critical role in nerve injury and inflammation-induced spinal pain processing. Inhibition of this kinase prevents hypersensitivity induced by both peripheral inflammation and nerve injury. Minocycline is a semi-synthetic second-generation tetracycline that penetrates well into the central nervous system (CNS) via blood-brain barrier. In addition to its actions as an antibiotic, minocycline has neuroprotective and anti-inflammatory effects in the CNS. Recently, it has been demonstrated that the inhibition of microglial activation by minocycline attenuates the development of pain hypersensitivity in rat models of neuropathic pain. This finding raises the possibility that minocycline may behave as a potential analgesic for other pain model types. Using the orofacial formalin test, we evaluated the potential role of p38 mitogen-activated protein kinase (MAPK) in the spinal trigeminal nucleus in the orofacial inflammatory pain and investigated whether minocycline had effects on orofacial inflammatory pain. Object:l.To investigate the changes of c-fos, GFAP and OX-42 expression in Vc in formalin-induced orofacial pain rat to explore the mechanism of central sensitization.2. To evaluate the potential role of p38 mitogen-activated protein kinase (MAPK) in the orofacial inflammatory pain. 3. To investigate the effects of minocycline on orofacial inflammatory pain and p38 MAPK path way.Materials and methods:1. Male SD rats received subcutaneous injection of 2.5% formalin 50μl in the left vibrissa pad to establish the inflammatory pain model. The expressions of c-fos, GFAP and OX-42 were detected at specific time point by immunohistochemical and immunofluorescence staining.2. At each time point of 10,20,30,40 minutes after formalin injection, p38 MAPK activity in Vc were examined by means of western blot analysis; SB203580, a p38 MAPK inhibitor was inserted into the rat's cisterna magna 20 minutes prior to the formalin injection, then the behavioral changes and the expression of c-fos in Vc were tested.3.1 hour before formalin injection,60 mg/kg, 90mg/kg minocycline intraperitonially or 20 minutes before formalin injection,60μg,120μg minocycline intracisterna magna injected respectively, then the behavioral changes and the expression of p-p38,c-fos and OX-42 in Vc were tested.Results:1. Fos-immunoreactivity (Fos-IR) neurons were presented at 30 minutes, peaked at 120 minutes and remained at a higher level by 360 min after injection. Meanwhile, the expression of GFAP-IR was observed higher at 30 minutes, peaked at 60 minutes, then declined and returned to normal level by 240 minutes after injection. Fos-IR and GPAP-IR showed similar distribution. The expression of OX-42-IR started on day 1 and peaked at day 7, then declined at day 14 post-injection.2. P38 MAPK was constitutively expressed in Vc and p38 MAPK was activated following formalin injection. The level of phosphorylated p38 expression was increased 10 minutes after the formalin injection, peaked at 20 minutes, and declined at 30 minutes. Intracisterna magna pretreatment of p38 MAPK inhibitor-SB203580 resulted in potent attenuation of phase II of pain behavior but not phaseⅠ. Meanwhile, the expression of c-fos was also been inhibited.3. Formalin induced orofacial pain behavioral responses included two Phases, and intraperitonial or intracisterna magna preadministration of minocycline significantly suppressed phase II with little effects on phase I. Intraperitonial or intracisterna magna preadministration of minocycline produced significant suppression on the activation of p38MAPK, c-fos and OX-42 in Vc.Conclusion1. Subcutaneous injection of formalin into the vibrissa pad could produced two phases nociceptive behavioral responses in rat, which is a suitable model for orofacial inflammatory pain research.2. The results suggested that the neurons and glia (astrocytes and microglia) in the Vc might jointly participate in pain modulation in CNS and glia (astrocytes and microglia) and regulate neuron's function in formalin-induced orofacial pain model.3. Activation of p38 mitogen-activated protein kinase played a major role in the development of orofacial inflammatory pain and it was verified by the experimental result that p38 MAPK inhibitor-SB203580 inhibited the fonnalin-induced orofacial pain.4. Preadministration of minocycline produced a potent antinociception in formalin-induced orofacial pain model and this effect was mediated by inhibition of activation of p38MAPK and microgia in Vc.