| Objective:Normal circadian rhythms,such as normal sleep rhythm,contribute to the recovery of physical strength and energy.In clinic,circadian rhythm disorders often occur in patients after operation,mostly manifested as sleep deprivation and sleep disorders.This may lead to dysfunction of neuroendocrine and immune systems,which is associated with perioperative adverse cardiovascular events and cognitive dysfunction resulting in delayed recovery and increased perioperative mortality.The circadian rhythm of organisms is controlled by biological clock.The highest level of biological clock is located in suprachiasmatic nucleus(SCN)of hypothalamus.The clock signal of SCN is produced by molecular oscillation of clock gene expression in neurocytes.In the absence of external factors,its expression rhythm is fixed,known as endogenous biological clock.Affected by the "zeitgeber",its rhythm will be modulated to form an exogenous biological clock.The biological clock signal is transmitted to the peripheral region through nervous and endocrine pathways to regulate the rhythm of life.Recently,animal studies have showed that general anesthetics could change the expression of the clock gene period 2(per2)in animals,and also could change the rhythm of animal behavior.These results suggested that circadian rhythm disorders after general anesthesia be related to the effect of general anesthetics on the expression of clock genes.So,whether the alterd biological clock by anesthetics can be corrected in some way? A few studies found that light could activate the retinohypothalamic tract through acting on specific retinal ganglion cells in the eye,and then activate the N-methyl-d-aspartate glutamate(NMDA)receptors on the ventral neurons in SCN to trigger intracellular signal transduction pathway and induce transcription of per2 gene.This experiment was to observethe the change in expression of per2 in mice after long-term anesthesia,and to explore whether light could correct this change.The result might provide new ideas for the prevention and treatment of circadian rhythm disorders in patients after general anesthesia.Method:This experiment was divided into two parts:The first part: 114 C57BL/6 mice were cultured in a light-dark cycle of 12 hours:12 hours(light 8:00-20:00,darkness 20:00-8:00,LD)for 3 weeks.The daytime light intensity was 450 lux.Afterwards,all mice were cultured in continuous darkness(darkness 8:00-20:00,darkness 20:00-8:00,DD)for 3 days.At 8:00 on the fourth day of DD environment(DD4),the mice were randomly divided into three groups,namely the control group(group C,n = 42),the anesthesia group(group A,n = 36)and the 8-hour light group after anesthesia(group A + L,n = 36).The subsequent experiments were carried out in DD environment except for light treatment.In the group C,six mice were randomly selected every four hours from DD4 8:00 and killed to obtain SCN.In the group A,mice inhaled sevoflurane between DD4 8:00 and 12:00,and then were selected at random(n=6)and sacrificed every 4 hours from 12:00 to obtain SCN.In the group A+L,light treatment was administered between DD4 12:00 and 20:00 immediately after anesthesia as described in the group A.The sampling time points in the group A+L was the same as those in the group A.The expression of per2 in SCN was detected by RT-PCR.The second part: 168 C57BL/6 mice were cultured in the same light-dark period as described in the first part.At DD4 8:00,mice were randomly divided into four groups,namely the control group(group C,n = 42),the anesthesia group(group A,n = 42),the 8-hour light group after anesthesia(group A + L,n = 42),and the 12-hour light group after anesthesia(group A + EL,n = 42).Subsequent experiments were conducted in DD environment except for light treatment.Sevoflurane was inhaled between DD4 8:00 and 12:00 in the group A,group A+L and group A+EL.After anesthesia,mice in the group A+L and group A+EL were treated with light for 8 hours(DD4 12:00-20:00)or 12 hours(DD4 12:00-24:00)respectively.From DD5 8:00,6 mice in each group were randomly selected and killed every 4 hours to obtain SCN.The expression of per2 in SCN was detected by RT-PCR.Result:Part ?:1.Intra-group comparison: per2 expression in the group C increased from 12:00 with reaching its peak at 16:00,and then declined to reaching its bottom at 24:00.The pattern of per2 expression showed obvious rhythm,and its value at the peak or the bottom was significantly different from the baseline value(P < 0.01).The expression of per2 in the group A and group A+L showed a decrease at 12:00 compared with the baseline value(P< 0.01),but there were no significant changes at other time points compared with the baseline value(P > 0.05).However,the expression of per2 gene at 8:00 on DD5 was higher than that at 4:00 on DD5 in the group A+L(P < 0.05).2.Inter-group comparison: The values of per2 expression in the group A and group A+L at 12:00,16:00,and 20:00 were lower than those in the group C(P < 0.01),and were higher at 24:00 than those in the group C(P < 0.01).At 8:00 on DD5 the expression of per2 in the group A was lower than those in the group C and group A+L(P < 0.01).Part ?:1.Intra-group comparison: The pattern of per2 expression in the group C from DD5 8:00 to DD6 8:00 was identical with that in Part I.There was no significant difference in the expression of per2 at each time point in the group A compared to the baseline value(P > 0.05).However,the patterns of per2 expression in the A + L group and A + EL group all showed an obvious ryhthm with the peak value at 16:00 and the bottom value at 24:00 on DD5,which were significantly different from the baseline values respectively(P < 0.01 or P < 0.05).2.Inter-group comparison: The values of per2 expression in the group A and A+L were all lower than those in the group C at 8:00,12:00,and 16:00 on DD5,and were higher than that in the group C at 24:00 on DD5(P < 0.01).In the contrast,the expression of per2 in the group A + EL was in consistence with that in the group C at the same time point on DD5(P >0.05).At 16:00 on DD5 the expression of per2 in the group A+L was higher than that in the group A,but lower than that in the group A+EL(P < 0.01).At 24:00 on DD5 the expression of per2 in the group A+L was lower than that in the group A,but higher than that in the group A+EL(P < 0.05).Conclusion:1.The per2 gene was expressed rhythmically in SCN of normal mice,with obvious peak and valley.2.After sevoflurane anesthesia,the expression of per2 in SCN lost its rhythm with nither peak nor valley.Eight-hour light treatment immediately after anesthesia could not recover the altered ryhthem of per2 expression on the first day after anesthesia.3.On the next day after sevoflurane anesthesia,the rhythm of per2 expression in SCN did not recover.Eight-hour light treatment immediately after anesthesia could partly correct the rhythmic expression of per2 on the next day,but the peak and valley levels were still far from normal.Extending the time of light treatment to 12 hours after anesthesia could correct the rhythm and amplitude of per2 expression to almost normal level on the next day. |
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