The Central Mechanism Of Homer1b/c Mediates Pain Nociception Induced By Complete Freund's Adjuvant And Chronic Constriction Injury Of The Sciatic Nerve In Rats

Chronic pain still a problem that continues to plague us,even after more than a decade of aggressive efforts at improvement.Persuasive epidemiologic evidence, mainly drawn from developed nations,has proven that chronic pain is a widespread public health issue.Despite the heterogeneity of study methods,surveys find that 15%-25%of adults suffer from chronic pain at any given time,a figure that increases to 50%in those older than 65 yr.Chronic pain is linked with a constellation of maladaptive physical,psychologic,family,and social consequences,and can be regarded as a disease entity per se.Inadequately treated pain has major physiological, psychological,economic,and social ramifications for patients,their families,and society.The majority of research into neuropathic pain mechanisms has concentrated on changes in the peripheral nerve or spinal cord after peripheral nerve injury and, therefore,most available evidence relates to changes in these parts of the nervous system.Nevertheless,it is important to recognize that alterations in the brain have also been demonstrated following peripheral nerve injury,but much less is known about the significance of these changes.It is well known that the excitatory amino acid glutamate is the major excitatory neurotransmitter released at the central terminals of primary afferent nociceptive neurones after noxious stimulation.Whilst glutamate acts at a number of post-synaptic receptors.Previous studies have demonstrated that postsynaptic protein, Homer 1b/c,a molecular scaffolding protein that binds and clusters mGluR receptors at neuronal synapses,plays an important role in the signaling process of GluRs. Moreover,a large body of evidence suggests that the ionotropic NMDA sub-type is one of the most intimately involved in both inflammation and nerve injury-induced central sensitization.Previous studies have demonstrated that postsynaptic protein-95(PSD-95),that binds and clusters NMDA receptors,plays an crucial role in the signaling process.Chronic monoarthritis(MA),induced by intraarticular injection of complete Freund's adjuvant(CFA) into the tibiotarsal joint,provides a localized and stable form of unilateral inflammation with animals displaying hyperalgesia and allodynia and is very suitable for studies of persistent(nociceptive) arthritic pain.Chronic MA is one of the most used animal models to study neuronal plasticity and chronic pain.Besides monoarthritis,chronic constriction inury(CCI) of the sciatic nerve is another one of most used experimental mononeuropathy model in chronic pain research.The current study,utilizing animal behavioral test,immunohistochemistry and co-immunoprecipitation,investigated the possible involvement of Homer 1b/c and PSD-95 in the maintenance of inflammatory pain induced by complete Freund's adjuvant(CFA),and neuropathic pain induced by chronic constriction inury(CCI) of the sciatic nerve in rats.The results are as follows:1.Exclusive expression of Homer1b/c protein in the spinal cord.Immunohistochemistry found that Homer1b/c immunoreactivity was localized mainly in laminaeⅠ-Ⅱand outer laminaⅢ.Under high magnification,Homer1b/c immunoreactive neuronal cell bodies were observed in the dorsal horn.Western blot confirmed the high level of Homer1b/c expression in the dorsal hom,and the undetectable level in the ventral hom.2.Increased expression of Homer1b/c following inflammatory pain induced by CFA.Injection of CFA into the tibio-tarsal joint of the rat produced a stable monoarthritic model.Injected ankle joint showed redness and swelling at day 1 after CFA injection,and maintained for 14 days or longer.Unilateral intra-articulation of CFA induced significantly increased expression of Homer1b/c in the ipsilateral spinal dorsal hom.While expression of PSD-95 is not affected by CFA-induced inflammatory pain,moreover,pain retrival of the inflammatory ankle significantly decreased expression of PSD-95 in the ipsilateral spinal dorsal horn.3.Increased interaction of PSD protein-95 and Homer1b/c following chronic painCo-immunoprecipitation of PSD protein-95 and Homer1b/c is increased significantly in the spinal dorsal horn and(anteior cingulate cortex,ACC) following CFA-induced inflammatory pain,but acute pain retrival of the inflammatory ankle significantly decreased co-immunoprecipitation of these two proteins in the ipsilateral spinal dorsal horn.4.Homer1b/c mediates neuropathic painUnilateral CCI induced significantly increased expression of Homer1b/c in the Ipsilateral spinal dorsal horn,and co-immunoprecipitation of PSD protein-95 and Homer1b/c is increased significantly in the contralateral ACC following CCI-induced neuropathic pain.This result suggested that Homer1b/c,both in ACC and spinal dorsal hom,plays role in the maintance of neupathic pain induced by chronic constriction inury of the sciatic nerve.5.Homer1b/c ASODN attenuated paw withdrawal responses to mechanical Stimuli Intrathecal injection of the the highest doses(10μg/10μl) of Homer1b/c antisense oligonucleotide(ASODN) every 24 h for 4 days,reduced expression of Homer1b/c protein in the spinal cord.Meanwhile,Homer1b/c ASODN attenuated CFA-induced mechanical hyperalgesia at day 3,4,5 and 6 after ODN injection.The lowest dose(2.5μg/10μl) of Homer1b/c AS ODN and missense ODN(10μg/10μl) had no effect.6.Homer1b/c ASODN attenuated paw withdrawal responses to thermal stimuliIntrathecal injection of a higher dose(5μg and 10μg/10μl) of Homer1b/c AS ODN every 24 h for 4 days,reduced CFA-induced thermal hyperalgesia significantly at day 3,4 after ODN injection.The low dose(2.5μg/10μl) of Homer1b/c AS ODN and Missense ODN(10μg/10μl) had no effect on paw withdrawal responses to thermal stimuli.7.Homer1b/c ASODN has no effect on acute painFollowing the intrathecal injection of antisense ODN(10μg/10μl) every 24 h for 4 days,no significant change of behavioral was found in paw-withdrawal threshold (PWT) to mechanical stimuli with von Frey filaments and paw-withdrawal latencies (PWL) to heat stimuli.Denotes knock down of Homer1b/c in the spinal has no effect on acute pain in intact animals.8.Homer1b/c mediates c-fos expression and CREB phosphorylationExpression of Fos protein and phosphorylated CREB(pCREB) in the spinal dorsal horn following retrival of inflammatory pain induced by CFA injection was reduced by intrathecal injection of Homer1b/c anti-sense ODN.This suggests expression of Fos protein and pCREB following retrival is,at least partly,mediated by Homer1b/c.In conclusion,the present study demonstrated that1.Homer1b/c in the spinal dorsal horn and ACC plays role in the maintance of CFA-induced inflammatory pain and neuropathic pain.2.Interaction of Homer1b/c and PSD-95 may be the possible mechanism underlying chronic pain.3.Homer1b/c mediates c-fos expression and CREB phosphorylation in the spinal dorsal horn following retrival of inflammatory nociception.