Expression Of IL-33in The Nervous System And Its Role In Pain Modulation

Part Ⅰ Expression of IL-33in the nervous system of ratsObjective:Increasing evidences have shown that IL-33participates in a variety of nervous system diseases. However, little is known about the expression of IL-33in the nervous system in vivo. In this study, we aim to study the expression of IL-33in the nervous system.Methods:The expression of IL-33protein in the nervous system was determined by immunofluorescence staining.Results:In wild type rat, IL-33, mainly located in the nucleus, could be observed in the inner cell layer of the retina and the Bergmann glia in the cerebellum Purkinje cell layer, stain with astrocytes marker Glutamine synthetase, GS. In ganglionic layer of retina, trigeminal ganglion and dorsal root ganglion, IL-33was not only co-localized with the astrocytes marker GS, but also expressed in neurons within these tissues.Conclusions:IL-33is co-expressed with GS-positive cells in the retina and cerebellum and located both in GS-positive cells and neurons in retinal ganglionic layer, trigeminal ganglion and dorsal root ganglion. Part II The expression of IL-33in the dosal root ganglion and spinal cord after inflammationObjective:We determine the expression of IL-33and its relevant molecules in the dorsal root ganglia (DRG) and spinal cord after complete Freund’s adjuvant (CFA) inflammation.Methods:By using the CFA-induced inflammatory model, we determine the mRNA expression profiles of IL-33, ST2, ST2L, IL-lRAcP, IL-1and IL-1R1in rat Lumbar4-5DRG and the corresponding spinal cord using quantitative real-time PCR at different time points. The expression of IL-33protein was also determined by western blot and immunofluorescence staining in ipsilateral lumbar4-5DRG of inflammatory rats.Results:In CFA-induced inflammation model, the mRNA expression of IL-33, ST2, ST2L, IL-1in ipsilateral L4-5DRG was time-dependently increased. CFA increased IL-33, ST2, ST2L and IL-1at day1, reached to the peak by day3, but decreased by day7. The mRNA expression of these proteins returned to the baseline at day14and without significant changes on the contralateral side. The mRNA level of IL-1RAcP increased only at Day3and the level of IL-1Rl decreased at Day7. The protein expression of IL-33protein was significantly increased at Day7. Interestingly, IL-33mRNA was decreased on the bilateral spinal cords after CFA-induced inflammation.Conclusions:Under peripheral CFA-induced inflammation, ipsilateral DRG showed a time-dependent increase of IL-33/ST2/IL-1RAcP, while both sides of spinal cord showed a transitory decreasion of IL-33mRNA expression. Part Ⅲ Role of IL-33in pain modulationObjective:We focus on the roles of IL-33in pain modulation and investigate its underlying mechanisms.Methods:Immunofluorescence staining was used to determine the expression of IL-33and its receptor ST2in the acutely isolated rat Lumbar4-6DRG neurons. The whole cell patch-clamp recording method was used to explore the effects of recombinant IL-33(final concentration10ng/ml and100pg/ml) on the DRG neuron membrane excitability. Recombinant IL-33was injected intrathecally to determine the thermal withdrawal latency and50%paw withdrawal threshold.Results:1) Acutely isolated DRG neurons express not only IL-33, but the receptor ST2.2)10ng/ml application of IL-33significantly depolarized the rest membrane potentials and markedly reduced rheobase. In100pA,300pA and500pA of Ramp stimuli, the administration of IL-33showed a significant increase in the firing rate. And, IL-33application at the lower concentration (100pg/ml) significantly reduced rheobase, action potential threshold and duration.3) Intrathecal injection of IL-33showed a dose-dependent potential of lowering thermal withdrawal latency. Especially in0.1ug group, after injection of5h,7h,24h, the thermal withdrawal latency was significantly decreased comparing with the saline group at the corresponding time point. In contrast, the50%paw withdrawal threshold was not significantly changed when comparing with the saline group at the corresponding time points.Conclusions:Both IL-33and it receptors are expressed in DRG neurons, which indicated an autocrine and/or paracrine mechanism within DRGs. Bath application of IL-33enhances the excitability of DRG neurons, which may contribute to the thermal hypersensitivity.