| ObjectiveVentrolateral preopic (VLPO) is an important area involving in sleep regulation.Adenosine (AD) is known as an important neuro-hormonal factor which modulatessleep-wakefulness cycle. The concentrations of AD in VLPO during before sleepdeprivation, sleep deprivation and sleep recovery period of two groups were studiedwith brain stereotaxic, microdialysis, polysomngraphy, and high performance liquidchromatograph.Methods1. Animal model Adult male SD rats (280-320g and clean stage) were used in theexperiments. Under pentobarbital anesthesia (40mg/kg, i.p.), electroencephalogram(EEG) and electromyography (EMG) electrodes were implanted for polysomnographicrecordings, and a guide cannula was inserted into VLPO (AP：–0.36mm；L/R：1.30mm；H：7mm) for drug application or microdialysis in rats. The EEG electrodes and theguide cannula were fixed to the skull with dental cement. After7days for recovery in asoundproof recording room, each animal was connected to an EEG/EMG recordingcable and habituated for1day before polygraphic recording.2. Grouping The rats in the experiments were divided into two groups: control groupand sleep deprivation group.3. Microdialysis One week after surgery, a microdialysis probe was inserted throughthe guide cannula into the target structure and the tissue was allowed to stabilize for24h. Artificial cerebrospinal fluid (ACSF) was perfused for2h before sample collected,which was set at9:00-19:00. ACSF was perfused in control group and sleep deprivationgroup and the flow rate was at2μl/min. Samples of rat cerebrospinal fluid werecollected about30-minute interval and were immediately stored at80°C until assayed.4. Method of sleep deprivation of rats The rats were placed in plexiglass barrels (22cm,38cm high), and were gently touched on the rats hip from10:00to16:00every15s with a brush in order to cause sleep deprivation, while the rats in control groupwere not given any treatment. Samples of rat cerebrospinal fluid were collected starting from9:00a.m. In the sleep deprivation group, the period form9:00to10:00is beforesleep deprived (B-SD) period without any treatment, the period form10:00to16:00issleep deprivation (SD) period with a brush touched, and the period from16:00to19:00is recovery (REC) period.5. HPLC and AD analysis The concentrations of AD were determined usinghigh-performance liquid chromatography (HPLC) combined with UV detection. Themobile phase, consisting of10mM ammonium phosphate (pH6.0) and14%(v/v)methanol, was pumped at1ml/min at23oC. The chromatograms were recorded at254nm. The injection volume was10μl. The concentrations of sample were calculated asfollows: the calibration curve equation of the adenosine standard concentration wasproducted with peak area and the sample peak area would be substituted into thecalibration curve of the adenosine standard concentration regarded as the sampleconcentration.6. Polysomnography Polysomnography (PSG, including EEG and EMG) was startedat9:00and sustained for10h. According to the PSG results, the sleep-wakefulness wasdivided into three stages:(1) Wakefulness (W) period was characterized by highfrequency and low amplitude waves of EEG and relatively high tone EMG.(2)Non-rapid eye movement (NREM) period was characterized by low frequency, spindles,high amplitude and δ slow waves of EEG, and significantly decreased EMG tone.(3)Rapid eye movement (REM) was characterized by high frequency and low amplitudewaves of EEG, and lack of EMG tone except occasional muscle twitches.7. Histological identification After sleep recording, a unipolar stainless steelelectrode was inserted into VLPO through the guide cannula. Lesions were made bycurrents (2.5mA for10s) to the microdialysis or microinjection site through anelectrode. Only animals whose sites of lesion were located in the VLPO were used.8. Statistical analyses SPSS12.0software was used for statistical analyses. The datawere presented as x±S. Differences between the mean. The values were analyzedwith Student’s t-test, groups were compared by analysis of variance, and those at P <0.05were considered significant.Results1. The ratios of W, NREM sleep and REM sleep in control group accounted of totaltime are37.3±2.4%,59.1±1.8%, and3.6±0.7%, respectively. The ratios of W, NREM sleep and REM sleep in SD group accounted of total time are:35.6±3.6%,61.0±2.9%,and3.2±0.8%, respectively during B-SD period;95.8±1.2%,3.4±0.8%, and0.8±0.4%,respectively during SD peroid;18.9±4.4%,76.5±5.6%, and4.8±1.7%, respectively,during REC peroid.2. The ratios of W, NREM sleep and REM sleep accounted of total time during B-CDperiod have no significant difference compared with the control group (n=6, P>0.05).The ratio of W accounted of total time is enhanced by58.5%(n=6, P <0.01), the ratioof NREM sleep accounted of total time is decreased by55.7%(n=6, P <0.01), andthe ratio of REM sleep accounted of total time is no significant changed during sleepdeprivation period. The ratio of W accounted of total time is decreased by18.4%(n=6,P <0.05), NREM sleep accounted of total time is enhanced by17.4%(n=6, P <0.01),and REM sleep accounted of total time was shown no significant change during sleeprecovery period (n=6, P>0.05).3. The concentration of AD in VLPO during B-SD period of SD group is42.90±11.50nmol/L, which is no significant difference from that in control group,36.40±9.98nmol/L,(n=6, P>0.05). The relative concentration of AD in VLPO during SD periodof SD group is199.8±23.5%, which is significant difference from that of control group,142.0±7.0%,(n=6, P <0.05). Therein, the concentration of AD in VLPO in SDgroup had no significant change in the initial2h after sleep deprivation, and theconcentration of AD in VLPO increased significantly in the following4h after sleepdeprivation compared with the same period of control group. The relative concentrationof AD in VLPO during REC period of SD group is138.3±11.7%, which is significantdifference from that in control group,36.40±9.98nmol/L,(n=6, P>0.05). During theinitial3h sleep recovery period, the relative concentration of AD is significantlydecreased.ConclusionAD in VLPO participates in the sleep-wakefulness cycle regulation, might promotesleep. |
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