| BackgroundSleep occupies one third of the human lifetime. A good and adequate sleep is essential to the human health which can help to eliminate fatigue,enhance immunity and so on.He who lacks of sleep for a long time is more likely to have health problem.Your immune function may decline although you just reduce several hours of your sleep time.Five days without sleep can endanger peoples's lives.A good sleep is so important to huam health。But with the aging population increasing, human face the unprecedented expansion pressure which lead to a high incidence of sleep disorders. Sleep disorders has become a comman problem threating haman health and social life.Insomnia is most common symptoms of sleep disorders by far. According to epidemiological survey in our country showed that:the incidence of insomnia is 26.27%,and female was significantly higher than men.Additional,the older also more likely to suffer from insomnia,the morbidity rate increas with the age.In fact,chronic diseases is also an important risk factor for insomnia.It estimated that 75%-90% insomnia patients complicted with chronic diseases which we call "comorbid insomnia".So many diseases is complicted with sleep disorders or main symptoms is sleep disorders,such as chronic skin diseases, rheumatoid arthritis, nervous system diseases such as cerebrovascular disease, peripheral nerve diseases, degenerative diseases,with their chief complaint of insomnia is more and more common, especially in older patients.Chronic cerebral ischemia is one of very common ischemia cerebrovascular disorders which is caused by arteriostenosis or arterial occlusion of cerebral perfusion.The main clinical manifestations involvs dizziness, headache, insomnia, numbness, memory impairment, depression, etc.All above symtoms recurrently occur,but no focal neurological was seen. According to statistics,2/3 of the elderly population has chronic cerebral insufficiency, so it is a common diseases among them. Clinical reports showed that many of patients with vertebral artery insufficiency compained with sleep disorders,especially in patients over the age of 45 years.The insomnia they face was mostly associated with chronic cerebral circulation insufficiency caused by cerebral arteriosclerosis and cervical disease.The etiology of chronic cerebral ischemia is complex and the course is not yet fully clear now.Modern medicine is lack of efficient drugs,no mention to treat this comorbid insomnia.They just use sedative hypnotics, antidepressants, antihistamines and so on to settle this comorbid insomnia, which can not do favorable to them but bring side effect and addiction.In Traditional Chinese Medicine, there is no the term "CCCI",but "vertigo", "headache", "insomnia", "forgetfullness", and other diseases are related to the disease.A lots of ancient physicians considered that deficiency syndromes and the blood stasis both lead to vertigo.The lesion focus on brain,and the fundamental cause is deficiency.But because all kinds factors can lead to the disease as its long course,deficiency and sthenia are shown at the same time. Our research group showed that improve the station of chronic cerebral circulation insufficiency can be effective in treating sleep disorders after a long-term clinical practice.Based on the long-term clinical experience,Professor Zang Kuntang has develop an experience prescription Wulongdan which is very effective in treating cerebral ischemia diseases such as Cerebral infarction, cerebral embolism, transient ischemic attack, stroke sequelae and recovery, cerebral arteriosclerosis, cerebral atrophy, high blood pressure dizziness, headache, cervical spondylosis (vertebral-basilar artery insufficiency), head injury or surgery complications and so on.Addtional,the Wulongdan also can effective ameliorate this comorbid insomnia. This all owe to Chinese herbal compound playing its multi-component,multi-target therapy.However,there has no mechanism study on the comorbid insomnia compicated with chronic cerebral hypoperfusion,also the mechanism of how the traditional medicine do the effective.Purpose and significance:Sleep deprivation (SD) is a artificial technology by reducing the normal amount of sleep to cause sleep disorders. Most researchers use the technology as a tool to uncover the function of sleep and mechanism of sleep disorders.In our study, we copy the chronic cerebral ischemia and sleep deprivation model then obseve changes of some related targets in chronic ischemia rats after sleep deprivation in order to explore the may mechanisms of sleep disorders in rats with chronic cerebral ischemia and further reveal the mechanism of Wulongdan efficacy. It also provide experimental basis for clinical application.Methods:1.group and model groupingAfter the purchase,the rats were breeded a whole week before experiment.The male rats model of chronic cerebral ischemia was induced by domestic improved method of permanent bilateral common carotid artery ligation.The sham-operated animal underwent the same surgical procedure without ligation of common carotid arteries.Nothing to the bland group.3 weeks after modeling,66 rats were survival.The survival rats were randomly allocated into 9 experimental group: control group, sham-operated group, the model group, blank and SD12h group, sham-operated and SD12h group, model and SD12h group, blank and SD3d group, sham-operated and SD3d group, the model and SD3d group. And the SD groups rats adapt to the small platform 1 hour a day for 1 weeks and then given sleep deprivation.2.the replication of sleep deprived rats modelUsing the "Sleep deprivation automatic recorder "a utility model patents made by our research team to replicated the sleep deprivation rats model(Patent No.:ZL 2009 2 0059193.3). The method we using to copy the model is the small platform aroud water environment (flower pots technique). Specific operations are:made a mouse box with 40×40×50cm big in the middle of which there is a platform with diameter of 4.5cm and height of 10.0cm,.The platform surrounding is filed with water, about 8.0cm high.The probe of the sleep deprivation automatic recorder penetration the platform from the bottom of the column to 2.0cm under the top.(Note: This sleep deprivation automatic recorder can record the number the rats falling into the water during the period of sleep deprivation.That is no other sleep deprivation experiment other can match.)3.test of effects of chronic cerebral ischemia on sleep deprivation rats' behavior:The sleep deprivation automatic recorder can record the number of rats fell into the water and the time staying into the water each time.4.The changes of orexin mRNA expression on the hypothalamic in chronic cerebral ischemia and REM sleep deprivation ratsIntraperitonealanesthesia by 10% chloral hydrate with 3.5ml/100g and removed the brain quickly, placing in 4℃refrigerator for 5 minutes. When it frozen to slightly harder, we strip the hypothalamus, placing in vials and throwing in liquid nitrogen,then stored in -80℃refrigerator.Extraction the total RNA and do the fluorescence quantitative PCR experiments.5.The effect of orexin immunoreactive neurons on hypothalamic in chronic cerebral ischemia and REM sleep deprivation ratsIntraperitonealanestied by 10% chloral hydrate with 4.0ml/100g leaded the rats into deep anesthesia station.Then made a sternotomy along the ventral midline and expose the heart.Then with 16-gauge needle puncture through the left ventricle to the ascending aorta. With a large curved needle clamp stay fixed the heart then broke the right atrial appendage using scissors.In the first quick rinse with PBS solution until a colorless supernatant liquid flow, following reperfusion with 4% paraformaldehyde and 0.1mol/L phosphate buffer (pH7.4). Hypothalamic dissection in 10% neutral formaldehyde fixative fixed. Follow the rules for dehydration, transparency, embedded in paraffin, sliced, do the immunohistochemistry staining experiment.6.Statisties analysisStatisties software SPSS 13.0 was used to analyze the experimental data.The expermental data were indicated by mean±standard deviation (X±s).Repeated measures variance analysis, the single-factor analysis of variance (One-way ANOVA) and independent-sample T test were used to analyze the data.LSD method was use in multiple comparisons between groups when the variance is homogeneous and Dunntt's T3 when the variance is not homogenous.Selecta=0.05 as inspection standard.Results:l.Test of effects of chronic cerebral ischemia on sleep deprivation rats' behavior:Data were analyzed by two-way analysis of variance with repeated measures and One-way ANOVA.And the data showed that:there was interaction between the time point and the groups(F=13.769,P=0.000),main effect among groups(F=22.952, P=0.000)and main effects among the time points(F=228.232, P=0.000)。The number of fell into the water among the SD3d groups have no statistical significance on the first day.But on both the second day and third day the model and SD3d group rats have fewer times of fell into the water,compared to the control and SD3d group and sham-operated and SD3d group(P<0.01).So as total times.2.The changes of orexin mRNA expression on the hypothalamic in chronic cerebral ischemia and REM sleep deprivation ratsThe data of prepro-orexin mRNA expression on the hypothalamus among groups was analyzed by One-way ANOVA indicated that there was significance statistical difference among them(F=103.932, P=0.000).The comparison between groups we use the Dunnett's T3 method.Campared to the sham-opreated group,the model and SD12h group and SD3d groups rats showed a significant increase of the prepro-orexin mRNA expression on the hypothalamus. Compared to the control group,the model group decresed the prepro-orexin mRNA expression on the hypothalamus,95%CI is(0.10,1.29) when the model and SD12h group,blank and SD3d group,the model and SD3d group increased the expression,95%CI are (-6.46,-1.05) (-12.63,-0.77) (-8.93,-0.07). The model and SD12h group increase the prepro-orexin mRNA expression compared to the sham-operated and SD12h group(P<0.01). Although there is no statistical significance among the SD3d groups,the model and SD3d group have a trend of rise compared to the other two groups.3.The effect of orexin immunoreactive neurons on hypothalamic in chronic cerebral ischemia and REM sleep deprivation ratsThe data was analyzed by One-way ANOVA and showed that there was significance statistical difference among them(F=110.765, P=0.000).The comparison between groups we use the LSD method.Compared to the control group,the model and SD12h,the blank and SD3d group,the sham-opeated and SD3d group,the model and SD3d group were all increased the number and the mean area of orexin immunoreactive neurons on hypothalamic. The difference was statistically significant (P<0.01).Conclusion:1.The ability of chronic cerebral ischemia rats to cope with the short-term stress is paralled to the normal group.But for the sustained stress,the chronic cerebral ischemia rats seen to more sensitive and maintain a high level of arousal.They stand on the platform longer and less times fell into the water during the SD.2. Compared to the control group,the model group decreased the prepro-orexin mRNA expression on the hypothalamic,which maybe the protective response of the organism. Campared to the sham-opreated group,the model and SD12h group and SD3d groups rats showed a significant increase of the prepro-orexin mRNA expression on the hypothalamus(P<0.01). The outcome is consistent with previous reseach.The model and SD12h group,the blank and SD3d group,the model and SD3d group increased the expression,compared to the control group. After -sleep deprivation 12h or 3d,the model rats increased the prepro-orexin mRNA expression compared to the sham-operated and SD group leading to a conclusion that the model itself is the reason for the expression increase when balanced of the sleep deprivation stimulation.3.The total number and the mean area of the orexin immunoreactive cells on the hypothalamic is not increase after a short period of sleep deprivation stimulation.SD3d can make the total number and the mean area of hypothalamic orexin immunoreactive cells increase. But the model groups were more vulnerable to the impact of sleep deprivation stimuli, compared with the control group.The model and SD12h group increased the expression of orexin neurons, compared with the blank and SD12h group.So as the SD3d groups.The difference was statistically significant (P<0.01). That is saying, the model group is more easy to active orexin neurons and make the expression increase than the control group. |
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