| α2-adrenoceptor(α2-AR)agonist is one of the major class of drug used in animals for its analgesic and sedative effects.The sedative and analgesic effects of these drugs in ruminants(such as cattle and sheep)are far superior to other animals,and their dosage is less than 1/10 of other animals.Different effects of α2-AR agonists(sedative,analgesic and side effects)are related to different types and characteristics of α2-AR subtype.According to their amino acid sequence and affinity with drug ligand,α2-AR subtypes can be divided into α2A α2B α2C and α2D.Studies have found that pigs,horses,dogs,cats and other animals have α2A,α2B and α2C subtypes,while ruminants have special α2D subtypes in addition to α2B and α2C homologous to other domestic animals.Whether the better analgesic effect of α2-AR agonists in ruminants is related to α2D subtypes is still unclear,and the distribution and functional characteristics of these subtypes in ruminants are also lacking in research.The purpose of this study was to explore the distribution of α2-AR subtypes in the central and peripheral regions of goats and their role in analgesia.In order to study the analgesic properties of different subtypes of α2-AR and their effects on physiological indicators,we use healthy adult goat as animal model in this study.Thirty-six male goats were randomly divided into six groups,which are:normal saline(NS)group,non-specific globulin(Ig G)group,yohimbine hydrochloride(Yoh)group,α2B adrenergic receptor(ADRA2B)antibody group,α2C adrenergic receptor(ADRA2C)antibody group,and α2D adrenergic receptor(ADRA2D)antibody group There are 6 goats in each group,and intrathecal cannulation and lateral ventricle cannulation are performed on all goats.Six groups of goats were injected intrathecal(intracerebroventricular)with 100 μL of specific antibody(ADRA2B,ADRA2 C or ADRA2 D antibody,30 μg/kg),Ig G(30 μg/kg),NS or α2-AR non-selective inhibitor Yoh 3 μg/kg.Specific antibodies are used to block the binding of α2-AR agonists to specific subtype receptors.Goats were injected intramuscularly with the α2-AR agonist xylazine hydrochloride(Xyl)0.05 mg/kg 30 minutes after the antibody/dug was injected.During the whole experiment,the changes of pain threshold and physiological indexes of goats were observed.Intrathecal(lateral ventricle)injection experiment results show that compared with the Ig G group,the hypothermic effect of Xyl can be inhibited by the injection ofα2-AR subtype specific antibody into the lateral ventricle,and the injection of ADRA2 C specific antibody has the best inhibitory effect on the hypothermic effect of Xyl.Compared with the Ig G group,the analgesic effect of Xyl can be inhibited by intrathecal(lateral ventricle)injection of ADRA2 D specific antibody,and the analgesic effect of Xyl cannot be injected with ADRA2 B and ADRA2 C specific antibody inhibition.It is suggested that the analgesic effect mediated by α2-AR in goats may be mainly mediated by ADRA2 D,and ADRA2 C may play an important role in the regulation of body temperature mediated by α2-AR.In order to explore the relationship between the distribution of different subtypes of goat α2-AR in the body and their different physiological effects,real-time fluorescent quantitative PCR and Western blot were used to determine the distribution of α2-AR subtypes,including analgesia-related nucleus and regions: caudate putamen nucleus(Cpu),nucleus accumbents(Acb),paraventricular thalamus(PVT),hypothalamic paraventricular nucleus(PVN),arcuate nucleus(Arc),amygdaloid nucleus(Amy),ventromedial hypothalamic nucleus(VMH),hippocampus(Hip),thalamic nucleus submedius(Sm),habenular nucleus(Hb),parafascicular nucleus(Pf),substantia nigra(SN),periaqueductal gray(PAG),parabrachial nucleus(PBN),locus coeruleus(LC),nucleus raphe magnus(NRM),gigantocellular reticular nucleus(GRN),nucleus of solitary tract and L4-L6 spinal dorsal horn(SCDH),and peripheral tissues: myocardium,hepar,spleen,lung,kidney,aorta,superior vena cava,inferior vena cava,arterioles and venules.Real-time fluorescent quantitative PCR experiment results show that compared with other nuclei and regions and tissues involved in the experiment: ADRA2 B mRNA is expressed in higher amounts in the liver,spleen,aorta,superior vena cava,arterioles and venules,and the expression of ADRA2 B mRNA in the analgesia-related nuclei and regions is relatively low;the expression of ADRA2 C mRNA is relatively high in the Cpu,Acb and PVT;the expression of ADRA2 D mRNA is relatively low in Cpu,Acb,PVT,myocardium,hepar,kidney and lung.Comparing the relative expression of ADRA2 B,ADRA2C and ADRA2 D in different analgesia-related nuclei and regions and peripheral tissues,it is found that the expression level of ADRA2 D mRNA is higher than the expression levels of ADRA2 B and ADRA2 C mRNA in PVN,VMH,Sm,Hb,Pf,SN,PAG,PBN,LC,NRM,GRN,SCDH,kidney,aorta,inferior vena cava and arterioles,and ADRA2 C mRNA expression is higher than ADRA2 B and ADRA2 D mRNA in Cpu,Acb,PVN,Acb,Amy,Hip,NTS,spleen,lung,superior vena cava and venules,ADRA2 B mRNA expression is higher than ADRA2 C and ADRA2 D mRNA in myocardium and liver.Western blot results showed that the relative trends of protein expression of ADRA2 B,ADRA2C and ADRA2 D in various analgesia-related nuclei and regions and peripheral tissues were roughly in line with the relative trends of mRNA.ADRA2B,ADRA2 C and ADRA2 D are not uniformly expressed in the CNS and peripheral regions,and may be involved in the regulation of different physiological effects.The analgesic effect mediated by α2-AR is mainly mediated by ADRA2 D,which is widely distributed in analgesic-related nuclei and regions.The negative regulation of goat body temperature by α2-AR can be reversed by α2-AR subtypes specific antibody.This study helps to clarify the analgesic regulation mechanism ofα2-AR agonists,and also lays the foundation for the search and development of α2-AR subtype agonists with high specificity,strong analgesic effect and low side effects. |
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