The Mechanisms Of Locus Coeruleus Mediates The Analgesic And Sedative Effects Of Dexmedetomidine

ObjectiveDexmedetomidine（DEX）developed at the end of the 20th century is a novelα₂adrenergic receptor agonist with a highα₂ receptor selectivity,about 8-10 times that of clonidine.Due to its central anti-sympathetic properties,it not only has certain analgesic and anxiolytic effects,but also produces a sedative effect similar to natural sleep.Locus coeruleus（LC）,referred to as the blue spot,is located in the anterior and posterior pons of the pons,close to the end of the inferior temporal sac of the fourth ventricle,and is the site where the noradrenergic neurons are concentrated in the brain.As a brain area associated with pain and arousal,we suspect that the analgesic and sedative effects of DEX may be associated with LC.This study aims to study the functional mechanism of DEX on normal mice by detecting intraperitoneal injection of DEX combined intrathecal injection of various blockers to observe changes of mechanical pain and heat-tail pain in mice and electrophysiology.Methods1.Assessment of mechanical withdrawal thresholdThe mice were habituated in the transparent acrylic chambers on a metal mesh floor for30 minutes before the experiment.During the measurement,the von Frey-type monofilaments were delivered to vertically stimulate the plantar surface of the hind paws of the habituated mice.The interval time between different filaments was 3seconds.If any phenomenon such as rapid lifting,shaking or lameness occurred when the mice were stimulated,we have reasons to believe it is a positive reaction.The method of up-down can be used to calculate and analyze mechanical withdrawl threshold of the mice.2.Assessment of the tail-flick latencyIn the tail immersion experiment,firstly,the body of the mouse was wrapped with a soft towel gently,then,the lower 5 cm section of tail of mice was immersed in the Thermostatic Water Bath in which temperature of water was maintained at 50±2°C.In order to prevent the mouse tail to get a burn,each test interval should be greater than10 seconds.The latency for tail withdrawal from the water was recorded as the reaction time which is the indicators of the pain thershold.3.Intrathecal administrationMicrosyringe was inserted between L4 and L5 segment,and the flick or formation of an‘S’shape by the tail was considered as a successful injection.Drugs were administrated with a total volume of 10μl at a total time of 30 s.At the same time,the animals with dyskinesia were removed.4.Assessment of sedationThe individual mice were placed in a clean cage and allowed to move freely.After 30min of adaptation,their behaviors were observed and recorded scores according to their behaviors.The scoring criteria depended on Chuck’s sedation rating scale.5.Immumohistochemical stainingThe brain slices were rinsed twice,and the solution was blocked at room temperature for 1 h.The blocking solution was discarded,and the primary antibody was added at 4°C overnight,then the primary antibody was rinsed 3 times and then the secondary antibody was added.After incubation for 2 h at room temperature,the secondary antibody was discarded and rinsed.After 3 times,the brain piece was attached to a glass slide and the film was taken.6.Electrode productionThe electrodes consisted of 16 individually insulated nichrome wires（35μm internal diameter,impedance 300-900 Kohm,Stablohm 675）.The 16 microwire arrays were arranged in a 4′4′4′4 pattern（approximately 200μm spacing between lines）.Soldered to an 18-pin connector（Mil-Max）,the welds were sealed with AB glue,and the naked nichrome wire was protected with polyethylene glycol 2000.7.Electrode implantationAnesthetized the animal with continuous isoflurane during the operation（induction 3%,1.5%was maintained）and placed the mouse in a stereotaxic frame.Chlortetracycline ophthalmic ointment was applied to protect the eyes of the mice,shaved the head skin with small scissors and cleaned the scalp with a cotton swab.Cutted the scalp along the midline with scissors and pushed it aside,exposed the skull.Removed the periosteum with a cotton swab dipped in 70%alcohol and dried the skull.Adjust the mouse skull to a position that meets the positioning criteria.Unilateral holes were drilled above the targeted brain locations,two additional holes were drilled,and screws were implanted to secure electrode array implants.Multi-wire electrodes were unilaterally implanted in the targeted brain regions with the following coordinates（AP-5.20 mm,ML-0.92 mm,DV-3.58 mm）.In order to mark the location of the implant,a small amount of Neuro-DiI dye was applied to the tip of the electrode array before implantation.3 M Vetbang tissue glue was painted to the skull and screws of skull surface.The frontal and parietal bone of the skull were puted with dental cement to fix the head post to the skull.After the dental cement became hard,the mouse was removed from the stereotaxic frame,placed on the heat pad in a cage until it awakes,and returned to a housing cage.8.The electrophysiological record in vivoNeuronal signal recordings were performed in a quiet room with a shielded box and started at the 7th day after the electrode was implanted in mouse.In order to reduce electrode shedded,the mice were lightly anesthetized with isoflurane before recording and the electrodes connected to a recording system were quickly.Then the mice were placed in a recording box using a multi-channel data acquisition system in free moving for 30 min to adapt the environment.After the mice injected EDX by intraperitoneal injection and naloxon and yohimbine by intrathecal injection,electrophysiological signals of neurons in LC were recorded using a multi-channel data acquisition system.The sampling frequency of recording was 500 Hz and the recordings were sustained for40 minutes.9.Analysis of in vivo electrophysiological dataThe Offline Sorter software was used to remove the distinct mussy waves from the normal wave in order to screen out high-quality single-cell aggregation waves.The selected data were analyzed by NeuroExplorer software to obtain the graph and chart of the firing frequency of the neurons in LC when intraperitoneal injection of DEX from low to high dose.10.Statistical AnalysisSPSS 17.0 was used to statistical process.All data were expressed as the mean?±?standard error of the mean（SEM）.Two independent samples t test was used for comparison between two groups,and one-way analysis of variance was applied for comparison between multiple groups.GraphPad Prism 5.0 software was used to draw statistical graphs.Differences were considered to be significant with p<0.05.Results1.The results of mechanical withdrawal threshold measurements and sedation scores show that dexmedetomidine in low dose（0-14μg/kg）intraperitoneal injected could increase the thresholds of mechanical and thermal pain in mice.However,DEX in high dose（16-20μg/kg）could obviously make mice sedate.Both the sedative and analgesic effects of DEX were dose-dependent.2.Multichannel electrophysiological recordings in vivo showed that the discharge frequencies of LC neurons were increased by DEX in low dose,while were decreased by DEX in high dose.3.Immunohistochemistry experiments showed that after intraperitoneal injection of DEX 20μg/kg LC noradrenergic neuron activity was significantly decreased compared with 6μg/kg in the-5.34 and-5.40 points from Bregma.4.Yohimbine intrathecal injected,an alpha 2 receptor antagonist,significantly inhibited the analgesic effect of DEX in low dose,but did not affect the sedative effect of DEX in high dose.5.Naloxone intrathecal injected,a morphine receptor antagonist,did not affect the analgesic effect of DEX.ConclusionDexmedetomidine exerts analgesic effects by activating the descending inhibition system of LC,and the spinal alpha 2 receptor involves in analgesic effects of DEX.Nevertheless,the sedative effects of DEX are not mediated by the descending inhibition system from LC to spinal cord.