| SubjectiveThe tuberomammillary nucleus(TMN)and the ventrolateral preoptic nucleus(VLPO)are be considered as the awakening and the sleep-promoting center respectively.They are restriction and balance each other to mediate and control the sleep-awakening cycle.With the development of the research,the newly discovered functions in the central nervous system of microglia,as an important immune function in the central nervous system cells,are constantly being discovered.Whether microglia is functional in sleep-conscious regulation is rarely reported.Sleep deprivation is often used as an important research method in study sleep arousal mechanism.We adapted the sleep deprivation mode,to explore the role of microglia in sleep-wake steady-state regulation,by using tetracycline antibiotics,minocycline,which have been proved to inhibit the activation of microglia.Methods1.Animal groups:C57BL/6 mice were used in two parts according to the overall design of the experiment:1.1 multiple direction sleep tracking and analysis:To observe whether the minocycline had influence on normal sleep mice.The mice were divided into normal saline with time of day sleep group(NS-TODS group)and intraperitoneal injection of minocycline with time of day sleep group(mino-TODS group).To observe whether minocycline affect on sleep deprivation mice,mice were divided into NS-TODS group,normal saline with sleep-deprived group(NS-SD group)and intraperitoneal injection of minocycline with sleep-deprivation group(mino-SD group).1.2.Detecting the fluorescence expression of microglia in the sleep-wake relative brain region of mice:to detect the expression of iba-1 in the VLPO,TMN and superchiasmatic nucleus(SCN)of the mice after experienced intraperitoneal injection of normal saline and minocycline for seven days.NS-SD group and mino-SD group were carried out sleep deprivation for five hours,the mice of the NS-TODS group were only injected saline saline with time of day sleep,then the brain tissues were anesthetized and perfused operation.2.Animal surgeryMice were fixed on the mouse brain stereotactic adapter after treated with pentobarbital intraperitoneal anesthesia.Then cutting off their head hair and disinfecting the skin with alcohol and had it opened.The subcutaneous tissue was separated and washed with physiological saline so as to expose the skull completely.Drilling the skull with the skull drill in places that at the front of the anterior fontanel for 1.0 mm,1.5 mm next to the posterior fontanel and at the front of the after fontanel for 1.0 mm and 1.5 mm next to the after fontanel respectively,but not break the dura mater,and burying the brain electrode into them,with two EMG The electrodes inserted into the neck muscles on both sides.To fix the electrodes on the skull with dental cement,and then performing intraperitoneal injection of penicillin,then suturing the wound,putting the mouse into the box of the restoring cage in lateral position,to ensure adequate food and water,and observing the recovery of mice each day.3.PolysomnographyThe mice were put into the recovery cage for 7 days after operation.On the 8th day,the mouse head electrode was connected with the multi-channel caster by the recording cable.After three days of adaptation,the official documenting starts.Keep the cage out of light from 8:00-20:00.The record started from 8:00 am(noted as ZTO)and to record the electroencephalogram(EMG)activity of the mice with the software known as Acqknowlege 4.2.The mice were free to eat and drink water during the recording,no restriction on its activities.Each record all started at 8:00 and lasted for 24 hours.4.Sleep state data statistics standardsEEG data were divided into statistics of each four seconds,and the sleep awakening cycle was divided into three types:wakefulness(W),which is characterized by low amplitude EEG and apparent electromyography;Non-rapid eye movement(NREM),which is characterized by a significant decrease in EMG and slow wave low amplitude EEG;Rapid eye movement(REM),which is characterized by a significant decrease in EEG and electromyography.5.Sleep deprivation methodMice were placed in plexig lass drums(22 cm in diameter and 38 cm in height)from 8:00 to 13:00 when sleep deprivation was performed.Mice were touched on hips with hairbrush every 15 seconds,so that they were forced to remain awake,in order to achieve the purpose of sleep deprivation.6.Frozen brain tissue preparationAfter anesthetized,the mice will experience the intraperitoneally fix by using left ventricular intubation.Plunging the knife on the mucronate cartilage,cut the chest to fully expose he heart capsule,cutting the pericardium with the eye scissors to expose the entire heart,using the hemostatic clamp to clamp the inferior vena cava,and then insert the infusion needle in the left ventricular,cutting the right ear with no haste.Perfusion was divided into two parts:first rapid infusion in 4 ℃ saline,stopping it until the liquid out of the body is clear;then performing the infusion of fixed solution 4%paraformaldehyde(prepared with PBS buffer).The speed of infusion is quick at first and then slow,it took about 20-30 minutes to finish.Removing the tissue(the more complete mouse brain)after the body stiffed,and the perfusion was finished.The brain tissue was placed in the 4%paraformaldehyde(PBS buffer)for 24 hours and then dehydrated in PBS containing 10%,20%and 30%sucrose,respectively.The frozen sections can be made only when the tissue was sink on the bottom of the container.7.Frozen sectionsThe brain tissue embedded in the embedding agent was quickly frozen and subjected to brain slices.The slices were 30 μm in thickness and the brain slices containing the VLPO,TMN and SCN brain regions were placed in a 4 C refrigerator according to mouse brain atlas.8.Detection of VLPO,TMN and SCN in microglia immunofluorescence assay The cells containing VLPO,TMN and SCN were treated with 0.01mol/L PBS and 0.3%Triton X-100 and then incubated with primary antibody and secondary antibody respectively.Primary antibody is iba-1,microglia/macrophage-specific protein antibody;secondary antibody is Alexa fluor488,green fluorescent marker.9.Quantitative analysis of the diameter and optical density of the microglia Under the help of high magnification,VLPO,SCN and TMN regional image were,collected.Using the ImageJ-win32/fiji analysis software to process the images,and the mean fluorescence density value of AOI(area of interest),average cell diameter,cell number to reflect the protein expression level.10.Statistical processing Use the SPSS 17.0 software for analysis,use mean ± standard error to represent measurement data,use t test in cross-group comparison.Results(1)There was no statistical meaning of differences between the NS-TODS group and the mino-TODS group,the awakening(W),the rapid eye movement(REM)sleep and the non-rapid eye movement(NREM)whose sleep time show circadian change.(2)Compared with NS-TODS,NS-SD group showed significant increase in sleep time,mainly in the increase of NREM(P<0.05).(3)Compared with NS-SD group,the total amount of awakening was increased in the mino-SD group(P<0.05),but there was no significant change in the ZT6-11 period.(4)Expression of microglia in VLPO,SCN and TMN brain regions.Compared with blank control with PBS replacing primary antibody,iba-1 was positively expressed in VLPO,SCN and TMN brain regions,showing green staining and cell is easy to be identified,with clear structure,tentacles.(5)The expression of microglia in the VLPO region.The statistics showed that the number of microglia in the VLPO region of NS-SD group was smaller than that of NS-TODS group,the difference has no statistically significance compared with NS-SD group(P<0.01),and the diameter of cells was longer(P<0.05),and the number of microglia cells in min-SD group was larger.(6)The expression of microglia in TMN region.Compared with NS-TODS,the diameter of microglia in TMN area increased significantly(P<0.01),and the fluorescence intensity of the NS-SD group was increased(P<0.05).After 5 hours of sleep deprivation in mino-SD group,the volume of microglia decreased,but the difference has no statistically significance.(7)The expression of microglia in SCN region.It showed that there was no significant difference in the expression of microglia in NS-TODS group and NS-SD group min-SD group.Conclusion The Microglia in the VLPO and TMN of the brain may be involved in the regulation of sleep-wake steady state. |
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