Alteration Of Spinal Glutamate And Transporters In Arthritis Rats With Morphine Tolerance

Opioids have been regarded as the most effective analgesics for management ofacute and chronic pain,however,opioid tolerance occurs following chronic drugexposure which limits their usefulness.Studies on mechanism of opioid tolerancehave been developing generally and various models of opioid tolerance areconstructed to provide the basis for such studies.Recent reports indicate thatglutamatergic system is involved in the development of opioid tolerance and plays animportant role in neural plasticity of opioid tolerance.The glutamatergic systemconsists of excitatory amino acids,excitatory amino acid receptors and glutamatetransporters.For mammalians,spinal cord is the lower centre of somatic and visceralsensation and motion,and spinal dorsal horn is an important location where afferentinformation of nociception is introduced,processed and integrated.It is reported thatreleasable pools of EAA are found in opioid-sensitive primary afferent neurons inspinal cord,and generous glutamate receptors in spinal dorsal horn.In the dissertation,a novel model of arthritis-chronic morphine tolerant rats is constructed.Based on thismodel,the effect of glutamate and transporters leading to neural plastic change in thespinal cord of morphine tolerant rats is explored.The results of this study areexpected to provide important information for future treatment and studies associatedwith morphine tolerance.Part one:Construction of arthritis-chronic morphine tolerant rat model andimplication of glutamate in spinal nerve plasticity change of ratsObjective:To construct the rat model of arthritis-chronic morphine tolerance,discussthe implication of glutamate in spinal nerve plasticity change of rats witharthritis-chronic morphine tolerance.Methods:Healthy male SD rats,300±20g,wereimplanted intrathecal catheters and randomized into 6 groups.Amongst,the rats infour groups were made into model of adjuvant-induced arthritis and wereadministered intrathecally saline,morphine 10μg,morphine 20μg,morphine 10μg+naloxone 10μg,respectively.The rats in the other two groups without arthritis wereadministered intrathecally saline and morphine 20μg,respectively.The drugs wereadministered twice daily for 7 days.Mechanical withdrawal threshold (MWT) and thermal paw withdrawal latency (PWL) of rats was examined to evaluate theirbehavior.Hyperalgesia was concerned as the sign of genesis of morphine tolerance.The glutamate concentration in spinal cord tissue of rats was measured.MK-801 andexogenous glutamate was administered intrathecally concomitant with morphinerespectively,MWT and PWL was examined.Results:Mechanical and thermalhyperalgesia examination evinced that repeated intrathecal infusion of morphine forconsecutive 7 days induced tolerance.The spinal glutamate concentration furtherelevated in arthritis-chronic morphine tolerant rats on top of increasing in arthritis rats.The increasing of glutamate concentration was time-dependent as administration ofdrug.MK-801 and exogenous glutamate blokcked or enhanced hyperalgesia inducedby chronic morphine respectively.Conclusion:The rat model of arthritis-chronicmorphine tolerance is constructed successfully by repeatedly administering morphineintrathecally in the presence of painful arthritis,which develops morphine tolerancein the background of treating inflammatory pain.There is association between thehyperalgesia induced by chronic morphine tolerance and elevated spinal glutamateconcentration,excessive stimulation of glutamate receptor as well as the excitabilityof nervous system.Part two:The effect of spinal glutamate transporters on chronic morphinetoleranceObjective:To observe the change of expression of glutamate transporters in spinaldorsal horn of arthritis-chronic morphine tolerant rats and effect of glutamatetransporter inhibitor and promoter on behavior and pain modulators,explore theeffect of glutamate transporters on morphine tolerance mechanism.Methods:Immunohistochemical stain,western blot and RT-PCR were used to measure theexpression of glutamate transporters in spinal cord of each group of rats divided asmentioned in Part one.MWT and PWL was examined for rats which wereintrathecally infused Riluzole and PDC.The expression of OFQ and BDNF mRNA ofspinal cord was measured by RT-PCR.Results:The expression of protein and mRNAof GLAST and EAAC1 in spinal dorsal horn of arthritis-chronic morphine tolerantrats was downregulated.Riluzole and PDC attenuated or enhanced hyperalgesia inopioid tolerance respectively,and regulated the expression of pain modulators such as BDNF and OFQ.Conclusion:The downregulation of glutamate transporters resultsin elevation of extracellular glutamate concentration,which plays an important role inmechanism of hyperalgesia induced by morphine tolerance.Regulation of activity orexpression of glutamate transporters promises to become a new strategy for treatingmorphine tolerance induced hyperalgesia.Part three:The effect of glutamate and transporters on nerve cellular apoptosisof spinal dorsal horn induced by chronic morphine toleranceObjective:To elucidate the implication of glutamate introducing nerve cellularapoptosis of spinal dorsal horn in chronic morphine tolerance.Methods:TUNELstaining was used to observe the distribution of apoptotic cells in spinal dorsal hom ofarthritis-chronic morphine tolerant rats.The expression of Bax,caspase-3 and Bcl-2in spinal cord were determined by western blot.Results:Chronic administration ofmorphine induced nerve cellular apoptosis of spinal dorsal horn.The expression ofBax and caspase-3 in spinal cord was upregulated,and that of Bcl-2 wasdownregulated.Riluzole and MK-801 attenuated apoptosis in spinal dorsal horninduced by chronic opioids while PDC and exogenous glutamate enhanced apoptosis.Conclusion:Spinal neural apoptosis may be the basis of chronic opioid tolerancedeveloping.The neurotoxicity of glutamate contributes to nerve cellular apoptosis ofmorphine tolerance.Riluzole and PDC relieves or enhances hyperalgesia in opioidtolerance by inhibiting or enhancing neural apoptosis in spinal dorsal horn induced bychronic opioid.In conclusion,arthritis rat with chronic morphine tolerance is a novel chronicmorphine tolerant model which is formed in the backdrop of treatment ofinflammatory pain with morphine.The present study was on the basis of this modeland detected the important effect of glutamate and its transporters on mechanism ofchronic morphine tolerance.The results enlighten us to treat the hyperalgesia inducedby morphine tolerance through regulating the expression and activity of glutamatetransporters.Moreover,it promises to provide a new strategy for treatment andresearch associated with morphine tolerance.