| Objective:Based on cortical EEG recording and analysing, to investigate sleep patterns of homozygous Period3 gene knockout mice. Here, on this basis, we examine the effects and mechanisms of albizia flower (flower of Albizia Julibrissin Durazz.) on sleep patterns changing of homozygous Period3 gene knockout mice.Methods: Part 1. Breeding of homozygous Period3 gene knockout mice and their offsprings genetic identificationThe heterozygous Period3 knockout mice (Period3+/-) and wild-type mice (WT) under C57BL/6J genetic background were raised by genotype separately,4 mice of each genotype (2 males and 2 females). Mice at age of 8 week were mated with the same genotype for breeding among themselves. Animals were housed in light-tight, ventilated compartment in a temperature-and humi-dity-controlled condition, food and water were available ad libitum, and 12h light-dark cycle. The fourth generation offsprings were genotyped at weaning by genomic DNA extracted, PCR reaction and agarose gel electrophoresis from tail biopsies (about 0.3-0.5 mm of tail tip). The F4 homozygous Periods gene knockout and WT mice which genotyped by PCR were breeding paired among themselves by same genotype and used to replace F1 generations. Then the fifth generation mice were made certain of genetic stability by collected tail tips at weaning to conduct genetic identification PCR again. Part 2. Preliminary research of sleep pattern of homozygous Period3 gene knockout mice and Per3 mechanism 1. Study of sleep structure of homozygous Period3 gene knockout miceMale homozygous Period3 gene knockout mice were taken as mutant group (Per3-/-), wild type (WT) mice as control group,8 mice in each group. Mice were proceeded EEG/EMG electrodes implantation after 7 days adaptation under experimental environ-ment conditions. EEG/EMG data recording for consecutive 5 days (120 hours) started after mice were allowed 7 days for recovery. The EEG and EMG recordings were determined in 10-s epochs to wake state, NREM sleep and REM sleep by using SleepSign 2.0, and to drew the hypnogram by Excel 2010. 2. The study of sleep-wake phase in homozygous Period3 gene knockout miceMale homozygous Period3 gene knockout mice were taken as mutant group (Per3-/-), wild type (WT) mice as control group,8 mice in each group. Mice were proceeded EEG/EMG electrodes implantation after 7 days adaptation under experimental environ-ment conditions. EEG/EMG data recording for consecutive 5 days (120 hours) started after mice were allowed 7 days for recovery. The EEG and EMG signals were determined in 10-s epochs to wake state, NREM sleep and REM sleep by using SleepSign 2.0, to calculate duration of each stage (wakefulness, NREMS, REMS). Time spent of wakefulness and each phase of sleep were calculated to percentage per hour, percent time spent per hour of wakefulness and each phase of sleep from 5 days were average to one day data. Average percentage of wakefulness and each sleep stages in 12h period (light-dark). Sleep bouts were expressed as number of total sleep per hour, per 12h (light-dark periods), from 5 days were average to number of total sleep per hour in 24h period and total number of sleep over light-dark periods. By SleepSign 2.0, to analyze δ power in NREM sleep, and calculated every hour NREM sleep δ activity by normalized to average 24h NREMS δ activity, and expressed as% δ activity in NREM sleep per hour. 3. Study of sleep time in homozygous Period3 gene knockout miceMale homozygous Period3 gene knockout mice were taken as mutant group (Per3-/-), wild type (WT) mice as control group,8 mice in each group. Mice were proceeded EEG/EMG electrodes implantation after 7 days adaptation under experimental environ-ment conditions. EEG/EMG data recording for consecutive 5 days (120 hours) started after mice were allowed 7 days for recovery. The EEG and EMG signals were determined in 10-s epochs to wake state, NREM sleep and REM sleep by using SleepSign 2.0. Percent time spent per hour of NREMS/TS and REMS/TS, and percent time spent over 12 hour (light-dark) period of NREMS/TS and REM/TS were calculated.4. Preliminary study of sleep pattern changing mechanisms in homozygous Period3 gene knockout miceSixteen of each homozygous Period3 gene knockout mice and WT mice were randomly divided into 4 groups by genotypes separately,4 mice in each group. The mice were adapted under experimental environment conditions for 7 days, then theirs hypothalamus were sampling every 6 hour (start at 7:00 am) on eighth day. The mice hypothalamic AVP and DBP genes expression were assessed by RNA extraction from whole hypotha-lamus and qRT-PCR.Part 3. Preliminary study of effects and mechanisms of albizia flower water decoction on sleep pattern of homozygous Period3 gene knockout mice1. Effects of albizia flower water decoction on sleep structure of homozygous Period3 gene knockout miceSixteen male homozygous Period3 gene knockout mice were randomly divided into 2 groups as treatment and blank control group after EEG/EMG electrodes surgical implantation and recovery for 7 days,8 mice in each group. Treatment group were consecutive 7 days administered with albizia flower water decoction 0.1mL/10g (lOg/kg) by gavage, and with the same volume of distilled water for blank control groups. On 7th day of treatment, the mice were started EEG/EMG recording together with medicine or water administration upon group at 7:00 am, EEG/EMG signals recording for consecutive 3 days (72 hours). The EEG and EMG signals were determined in 10-s epochs to wake state, NREM sleep and REM sleep by using SleepSign 2.0, and to drew the hypnogram by Excel 2010.2. Effects of albizia flower water decoction on sleep-wake phase of homozygous Period3 gene knockout miceSixteen male homozygous Period3 gene knockout mice were randomly divided into 2 groups as treatment and blank control group after EEG/EMG electrodes surgical implantation and recovery for 7 days,8 mice in each group. Treatment group were consecutive 7 days administered with albizia flower water decoction 0.1mL/10g (10g/kg) by gavage, and with the same volume of distilled water for blank control groups. On 7th day of treatment, the mice were started EEG/EMG recording together with medicine or water administration upon group at 7:00 am, EEG/EMG signals recording for consecutive 3 days (72 hours). The EEG and EMG signals were determined in 10-s epochs by using SleepSign 2.0, to calculate duration of each stage (wakefulness, NREMS, REMS). Time spent of wakefulness and each phase of sleep were calculated to percentage per hour, percent time spent per hour of wakefulness and each phase of sleep from 3 days were average to one day data. Average percentage of wakefulness and each sleep stages in 12h period (light-dark). Sleep bouts were expressed as number of total sleep per hour, per 12h (light-dark periods), from 3 days were average to number of total sleep per hour in 24h period and total number of sleep over light-dark periods. By SleepSign 2.0, to analyze 8 power in NREM sleep, and every hour NREM sleep δ activity by normalized to average 24h NREMS 8 activity were calculated, and expressed as% δ activity in NREM sleep per hour.3. Effects of albizia flower water decoction on sleep time in homozygous Period3 gene knockout miceSixteen male homozygous Period3 gene knockout mice were randomly divided into 2 groups as treatment and blank control group after EEG/EMG electrodes surgical implantation and recovery for 7 days,8 mice in each group. Treatment group were consecutive 7 days administered with albizia flower water decoction 0.1mL/10g (10g/kg) by gavage, and with the same volume of distilled water for blank control groups. On 7th day of treatment, the mice were started EEG/EMG recording together with medicine or water administration upon group at 7:00 am, EEG/EMG signals recording for consecutive 3 days (72 hours). The EEG and EMG signals were determined in 10-s epochs by using SleepSign 2.0. Percent time spent per hour of NREMS/TS and REMS/TS, and percent time spent over 12 hour (light-dark) period of NREMS/TS and REM/TS were calculated.4. Preliminary study the mechanisms of albizia flower water decoction on sleep pattern changing in homozygous Period3 gene knockout miceSixteen male homozygous Period3 gene knockout mice were randomly divided into treatment group and blank control group,8 mice of each. The mice were adapted under experimental environ-ment conditions for 7 days. Treatment group were cones-cutive 7 days administered with albizia flower water decoction 0.1mL/10g (10g/kg) by gavage at 7:00 am on every day. and with the same volume of distilled water for blank control groups. On 7th day, mice were suddenly decapitation and remove whole brain after 2h of treatment, and then on ice plate to sampling the hypothalamus, and kept in -80℃ refrigerator until next process. Finally, A VP and DBP genes expression were assessed by RNA extraction from whole hypothalamus and qRT-PCR.Results:Part 1. Breeding of homozygous Period3 gene knockout mice and theirs offspring genetic identificationBy PCR genotyping of our breeding homozygous Period3 gene knockout mice (Per3-/-) and homozygous WT mice (Per3+/+) indicated that the animals were have genotypic stability. The observation during raising, two genotypes mice were normally in phenotype, litters size, growth, and survival rate.Part 2. Preliminary research of sleep pattern of homozygousPeriod3 gene knockout mice and Per3 mechanism1. Study of sleep structure of homozygous Period3 gene knockout miceThe sleep-wake circadian and sleep structure of homozygous Period3 gene knockout mice were similar with WT mice. From hypnogram could be observed that differences of sleep-wake state between genotypes, but the most of data could not observe the obvious distinction between genotypes.2. The study of sleep-wake phase in homozygous Period3 gene knockout miceThe percentage of time spent in each stage of homozygousPeriod3 gene knockout mice, at first hour (ZTO) on every day, that were significantly more wakefulness and less NREM sleep compared with WT mice. The homozygous Period3 gene knockout mice had delayed to fall asleep. Most of times during light period, homozygous Period3 gene knockout mice had not significantly more REM sleep. Results of 5 days wake-sleep parameters average to one day were similar to results of 5 days. The percentage of sleep phase could be observed that homozygous Period3 gene knockout mice were not significantly more awake, less sleep time and NREM sleep, more REM sleep during light period compared with WT mice. At first hour after light-on (ZTO), homozygous Period3 gene knockout mice had significantly less sleep fragments and less NREM 8 activity compared with WT. The homozygous Period3 gene knockout mice showed not significantly increased sleep times and NREM 8 activity during early night.3. Study of sleep time in homozygous Period3 gene knockout miceThe homozygous Period3 gene knockout mice were no obvious difference in overall sleep phase, time spent in sleep, and percentage of sleep proportion when compared with WT mice. Homozygous Period3 gene knockout mice showed significantly less NREMS/TS and more REMS/TS at some time points during light period compared with WT mice. During early night period, homozygous Period3 gene knockout mice had trend to increase in NREMS/TS and REMS/TS, but they were significant difference in REM sleep at some time points compared with WT. The most obvious of the sleep results was significantly decreased in NREMS/TS. The REM sleep proportion of homozygous Period3 gene knockout mice was trend to increase but not significant.4. Preliminary study of sleep pattern changing mechanisms in homozygous Period3 gene knockout miceThe AVP gene expression of homozygous Period3 gene knockout mice hypothalamus showed trend to higher than WT mice, at ZT12 and ZT18 had significant difference between genotypes (P<0.05). The AVP gene expression of homozygous Period3 gene knockout mice also exhibited no significant delayed circadian phase of expression. The pattern of DBP gene expression of homozygous Period3 gene knockout mice was similar to WT mice, DBP expression levels was significantly higher than WT mice at ZT18 (P<0.05), and circadian expression of DBP gene had no obvious phase advance tendency.Part 3. Preliminary study of effects and mechanisms of albizia flower water decoction on sleep pattern of homozygous Period3 gene knockout mice1. Effects of albizia flower water decoction on sleep structure of homozygous Period3 gene knockout miceAccording to consecutive 10 second epoch analysis and labeled to wakefulness, NREM and REM sleep states, then depicted to hypnogram. On hypnogram we could observed that some distinctions of sleep-wake circadian and sleep structure between treatment and control groups, these showed increased in wakefulness and decreased in REM sleep of some mice.2. Effects of albizia flower water decoction on sleep-wake phase of homozygous Period3 gene knockout miceAt the early light period, treatment group mice shown decreased percentage of time spent in wakefulness, increased percentage of time spent in NREM sleep but percentage of time spent in REM sleep had no changing compared with control group. The results of consecutive 3 days indicated that albizia flower water decoction may have improve sleep circadian effects on homozygous Period3 gene knockout mice. The treatment group mice exhibited more NREM sleep proportion of light segment compared with control group, but NREM 8 activity of light segment was no significant difference. Treatment group mice were had less awake time, more NREM sleep time and more number of sleep bouts compared with control group, but not changed NREM 8 activity. The dark period on third day result showed that treatment group mice were more awake time than first day, and no significant difference between groups.3. Effects of albizia flower water decoction on sleep time in homozygous Period3 gene knockout miceWhen compared to blank control group mice, treatment group mice were more NREMS/TS per hour and less REMS/TS per hour, but most of time pointes had no significant difference. At ZTO, treatment group mice were significantly increased in NREMS/TS ratio compared with blank control group. At early of night period, treatment group showed increased in NREMS/TS ratio and REMS/TS ratio but no significant difference when compared to control group.Treatment group mice had significant more NREMS/TS proportion on second day light period, significant less REMS/TS proportion on first day light period than blank control group. In addition, average 3 days data to one day result showed more NREMS/TS ratio during light period of treatment group, had significant when compared to control group.4. Preliminary study the mechanisms of albizia flower water decoction on sleep pattern changing in homozygous Period3 gene knockout miceHypothalamic DBP gene expression levels of treatment group were significantly higher than control group mice. The AVP gene expression levels in hypothalamus were not significantly higher in treatment group mice than control group mice.Conclusions:1. At first hour in light period, homozygous Period3 gene knockout mice exhibited more awake time, less NREM sleep time and NREM δ activity, and delayed to fall asleep. During light period, they were more awake and REM sleep, but less NREM sleep.2. The Period3 gene functional mechanisms may be associated with the interaction between the circadian clock and the motivational drive of behavioral activity in response to light-dark cycle, and related to AVP gene regulation by Per3 gene in hypothalamus.3. Albizia flower water decoction affected to sleep pattern of homozygous Period3 gene knockout mice. These represented that, during early light period, albizia flower water decoction was reduced time spent in wakefulness together with increased time spent in NREM sleep, made the mice fall asleep earlier. Albizia flower water decoction reduced time spent in wakefulness over 12-h light-dark period, as well as increased time spent in NREM sleep and daytime NREMS/TS ratio.4. Albizia flower water decoction could be able to increase DBP gene expression levels. This was a possible mechanism of albizia flower water decoction to altered sleep pattern of homozygous Period3 gene knockout mice. |
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