| Background General anesthesia can be described as a pharmacologic state involving amnesia,sedation,immobility,analgesia,and unconsciousness.According to the report of World Health Organization,there were 234 million operations in the world in 2007,most of which were performed under general anesthesia.At present,the number of anesthesia cases per year in China has reached 60 million,ranking first in the world.Though general anesthesia is widely required each day for surgical and medical procedures,the mechanisms by which how general anesthetics induce hypnosis and how to recover consciousness from general anesthesia are still unsolved questions in natural science.Anesthesia induction and emergence are somewhat similar to natural sleep and arousal.Currently,majority of study major focus on the mechanisms of general anesthesia by intervening one cerebral nucleus or one type of neurons.For instance,Optogenetic activation of dopamine neurons in the ventral tegmental area(VTA)can induce behavioral arousal response during isoflurane general anesthesia in mice.Lesion of cholinergic neurons in basal forebrain(BF)can accelerate induction and delay emergence from intravenous and Inhalation general anesthesia in rats,etc.At present,few studies have explored the mechanism of general anesthesia focused on the neural circuit of natural sleep awakening.Monochromatic blue light(MBL),with the wavelength between 400 nm and 490 nm,has both beneficial and harmful effects on living organisms.High energy short wave blue light between 415 nm and 455 nm is the most harmful,which causes photochemical retinal damage.The blue light with the band of 455 – 490 nm are involved in the regulation of biological rhythm,sleep and wakefulness,cognition,mood and memory,which is most beneficial.Light wavelength between 460 nm and 480 nm is absorbed by intrinsically photosensitive retinal ganglion cells(ip RGCs),engaged in regulation of sleep.In animal study,blue light(470 nm)enhances behavioural arousal via M1 ip RGCs.In human study,2-h evening exposure to MBL(460 nm),reduces slow wave activity(SWA: 0.75 – 4.5 Hz)in the first sleep cycle,while increase SWA during the third sleep cycle,and shorten rapid eye movement sleep(REMS)duration during these two sleep cycles.Though recent evidence suggests that general anesthesia shares some common neuronal mechanisms with sleep-wake,modulation of certain nuclei or neurotransmitters that regulate sleep-wake may affect the systemic effects of general anesthesia.General anesthetic can also act on the sleeping nervous system,propofol anesthesia is a sleep-like state,whose electroencephalogram(EEG)waves similar to non-rapid eye movement sleep(NREMS).However,whether MBL impacts on general anesthesia remains unknown.In mammals,the suprachiasmatic nucleus(SCN)in the hypothalamus is the main central nervous system circadian pacemaker,which regulates the 24-h biological rhythm including sleep-wake.As one of the most potent environmental stimuli,light has image-and nonimage-forming(NIF)visual functions in mammals.Light affects NIF functions directly or indirectly through melanopsin expressed on ip RGCs.ip RGCs project to the SCN and play a role in different aspects of physiology and behavior,including entrainment of circadian rhythms,suppression of pineal melatonin release,regulation of pupillary light reflex,altering sleep and wake,and affecting mood and cognition.Studies have confirmed that regulation of sleep-wake behavior by SCN involves both the autonomic nervous system and the central nervous system.Latest research shows that blue light increases the expression of Fos m RNA of SCN in mice,resulting in elevated corticosterone via autonomic innervation of the adrenal, thus increasing the arousal behavior of mice.SCN,act as an information integration hub for light regulating sleep-wake,sends both monosynaptic and multisynaptic connections to hypothalamic nuclei implicated in sleep and wake.Studies have confirmed that SCN has a dense projection to the dorsomedial hypothalamus(DMH),then DMH sends inhibitory GABAergic projections to the VLPO,and excitatory glutamatergic projections to the the LH,additionally,DMH also projects to the LC that sends excitatory outputs to the wakepromoting neurones in the sleep/arousal network,and the cerebral cortex.Pilorz et al.showed that blue light had an arouse-promoting effect on C57BL/6 mice by activating the SCN,while green light showed the opposite effect,namely sleep-promoting effect,by activating the VLPO.Therefore,it is unclear that whether MBL influences sevoflurane anesthesia via SCN and related nuclei downstream of the SCN.To investigate the effect of MBL on sevoflurane general anesthesia and underlying mechanisms,we test the time to loss of righting reflex(LORR)and recovery of the righting reflex(RORR)to explore the effect of MBL on induction and emergence of sevoflurane general anesthesia.Then,we record cortical electroencephalogram(EEG)and electromyogram(EMG)to monitor burst-suppression under sevoflurane anesthesia.The burst-suppression ratio(BSR)and EEG power are calculated to assess the effect of MBL on sevoflurane general anesthesia.We employ immunofluorescent staining and local field potential(LFP)recording to measure the SCN activity under sevoflurane anesthesia.We perform locally electrolytic ablated bilateral SCN neurons in mice to further confirm that SCN plays a key role in arousal-promoting effect of MBL under sevoflurane anesthesia.Finally,we observe expression of c-Fos in the SCN typical projection areas after sevoflurane anesthesia with application of MBL to explore the possible mechanism of SCN downstream.Methods and Results 1.Effect of monochromatic blue light on sevoflurane anesthesia induction and emergence Methods: Male C57BL/6J(C57)mice aged 10-12 weeks old weighing 22-26 g were used in the present study,all experiments were conducted during the daytime(8:00am-5:00pm).a)Effect of monochromatic blue light on sevoflurane anesthesia induction.Before sevoflurane anesthesia,mice were exposed to room light in cylindrical glass anesthesia chambers,when 1 MAC sevoflurane started,same illumination intensity(800 lux)of polychromatic white light(PWL)or monochromatic blue light(MBL)(460 nm)was administrated on acrylic glass chambers surface devoid of any other source of light,respectively.Then,the time to LORR in PWL or MBL was recorded.b)Effect of monochromatic blue light on sevoflurane anesthesia emergence.Mice were maintained under 1 MAC sevoflurane anesthesia 30 min and exposed to room light in cylindrical glass anesthesia chambers,when sevoflurane stoped,same illumination intensity(800 lux)of PWL or MBL(460 nm)was administrated on acrylic glass chambers surface devoid of any other source of light,respectively.Then,the time to RORR in MBL and PWL was recorded.c)Effect of monochromatic blue light on EEG when recovery from sevoflurane anesthesia.mice were maintained under 1 MAC sevoflurane anesthesia 30 min and exposed to room light in cylindrical glass anesthesia chambers,when sevoflurane stoped,same illumination intensity(800 lux)of PWL or MBL(460 nm)was administrated on acrylic glass chambers surface devoid of any other source of light,respectively.From the beginning of anesthesia,the EEG and EMG were recorded in the whole process until the mice showed RORR and body movements.Results: a)There was no significant difference in the time of LORR between PWL group(181.1 s ± 10.60)and MBL group(176.0 s ± 7.976)during sevoflurane anesthesia induction.b)time of RORR in MBL group(38.75 s ± 5.291)was significantly shorter than that in PWL group(133.1 s ± 13.53)(p < 0.001)during sevoflurane anesthesia emergence. c)EEG analysis results showed that MBL made a quick transition from an anesthesiaEEG state to a wake-EEG state during sevoflurane emergence period.Spectral analysis of EEG data at 100 s after sevoflurane stop showed that MBL decreased relative EEG delta power(p < 0.01)and increased spindle,beta,low gamma and high gamma power(p < 0.05),while theta and alpha band power remained unchanged compared to PWL group during sevoflurane emergence period.2.Effect of monochromatic blue light on burst-suppression induced by sevoflurane anesthesia Methods: Male C57BL/6J(C57)mice aged 10-12 weeks old weighing 22-26 g were used in the present study,all experiments were conducted during the daytime(8:00am-5:00pm).After mice were maintained under 2.5% sevoflurane anesthesia 30 min with exposed to room light in cylindrical glass anesthesia chambers,continually anesthetized mice were administrated with same illumination intensity(800 lux)of PWL or MBL(460 nm)for 30 min on acrylic glass chambers surface devoid of any other source of light,respectively.From the beginning of anesthesia,the EEG and EMG were recorded in the whole process until end of PWL or MBL.Results: a)During continuous inhalation of 2.5% sevoflurane,mice exposed to MBL showed a signifcant 31% decrease(p < 0.05)in BSR during MBL-post first 10 min and a 42% decrease(p < 0.01)during MBL-post second 10 min and a 55% decrease(p < 0.01)during MBL-post last 10 min when compared to the MBL-pre 10 min.However,PWL-exposed animals showed no signifcant difference in BSR compared to PWL-pre.b)EEG analysis results showed that MBL changed the burst suppression pattern to more burst occurrence during continuous inhalation of 2.5% sevoflurane.A spectrogram computed from the same animal showed that the EEG power after MBL administration was higher than MBL-pre.However,there was no change in EEG amplitude or frequency and power between PWL-pre and PWL-post.The combined power spectral densities computed from 120 s before and after MBL administration showed that power at all frequency bands except for spindle band was signifcantly increased after MBL administration(p < 0.05).Additionally,PWL-exposed animals showed no statistically signifcant difference in EEG power compared to PWL-pre.3.Monochromatic blue light plays a role in promoting arousal under sevoflurane anesthesia by activating the SCN Methods: Male C57BL/6J(C57)mice aged 10-12 weeks old weighing 22-26 g were used in the present study,all experiments were conducted during the daytime(8:00am-5:00pm).a)Effect of monochromatic blue light on the expression of c-Fos in SCN neurons under sevoflurane anesthesia.After mice were maintained under 2.5% sevoflurane anesthesia 30 min with exposed to room light in cylindrical glass anesthesia chambers,continually anesthetized mice were administrated with same illumination intensity(800 lux)of PWL or MBL(460 nm)for 30 min on acrylic glass chambers surface devoid of any other source of light,respectively.After sevoflurane anesthesia,mice were immediately deeply anesthetized with 1% sodium pentobarbital(100mg/kg,i.p),and then the heart was perfused with polyformaldehyde.The activation of SCN neurons was detected by immunofluorescence.b)Effect of monochromatic blue light on SCN local field potential(LFP)under sevoflurane anesthesia.After mice were maintained under 2.5% sevoflurane anesthesia 30 min with exposed to room light in cylindrical glass anesthesia chambers,continually anesthetized mice were administrated with same illumination intensity(800 lux)of PWL or MBL(460 nm)for 30 min on acrylic glass chambers surface devoid of any other source of light,respectively.From the beginning of anesthesia,the EEG,EMG and LFP were recorded in the whole process until end of PWL or MBL.c)SCN bilateral electrical lesions.40 male C57 mice were randomly divided into sham group and SCN-lesioned(SCNx)group,with 20 mice in each group.Sham group:the sham lesion mice underwent the same operation,but no current was passed through the electrode.SCNx group: bilateral electrolytic lesions of the SCN were made by using an isolated pulse stimulator(model 2100,A-M Systems,USA)to generate a current pulse of 2.5 m A for 20 sec.d)Effect of monochromatic blue light on sevoflurane anesthesia induction after SCN lesions.14 mice in the sham group were randomly divided into PWL group and MBL group,with 7 mice in each group.14 mice in the SCNx group were randomly divided into PWL group and MBL group,with 7 mice in each group.Method of measuring time to LORR in each group was same as the first part.e)Effect of monochromatic blue light on sevoflurane anesthesia emergence after SCN lesions.16 mice in the sham group were randomly divided into PWL group and MBL group,with 8 mice in each group.16 mice in the SCNx group were randomly divided into PWL group and MBL group,with 8 mice in each group.Method of measuring time to RORR in each group was same as the first part.f)Effect of monochromatic blue light on EEG when recovery from sevoflurane anesthesia after SCN lesions.12 mice in the sham group were randomly divided into PWL group and MBL group,with 6 mice in each group.12 mice in the SCNx group were randomly divided into PWL group and MBL group,with 6 mice in each group.Method of EEG monitoring for each group was same as the first part.g)Effect of monochromatic blue light on burst-suppression induced by sevoflurane anesthesia after SCN lesions.16 mice in the sham group were randomly divided into PWL group and MBL group,with 8 mice in each group.16 mice in the SCNx group were randomly divided into PWL group and MBL group,with 8 mice in each group.Method of EEG monitoring for each group was same as the second part.Results: a)Immunofluorescence results showed that MBL group under sevoflurane anesthesia elicited a significant increase in c-Fos expression in the SCN compared to PWL group(p < 0.0001).b)The results of LFP showed that the total power of SCN LFP after MBL exposure was significantly increased compared with that before MBL exposure(p < 0.05),while the difference in total power of SCN LFP before and after PWL exposure was not statistically significant.c)The results of Nissl staining showed that SCN in sham group was intact,evenly and symmetrically distributed on both sides of the third ventricle.SCN in SCNx group was ablated by direct current,the original SCN was vacuolated,indicating that SCN was completely lesioned.d)Behavioral results showed that there was no significant difference in the time of LORR between Sham+MBL group(171.3s ± 11.72)and Sham+PWL group(186.1s ±11.59).There was also no significant difference in the time of LORR between SCNx+MBL group(154.6 s ± 8.78)and SCNx+PWL group(158.4 s ± 9.604).e)Behavioral results showed that MBL promoted emergence from sevoflurane anesthesia in Sham group mice.Time of RORR in Sham+MBL group(60.13 s ± 13.38)was significantly shorter than that in Sham+PWL group(147.9s ± 16.27)(p < 0.001).However,MBL had no effect on sevoflurane anesthesia emergence in SCNx group,and there was no significant difference in the time of RORR between SCNx+MBL group(147.6s ± 12.71)and SCNx+PWL group(156.8s ± 12.32).f)EEG analysis results showed that compared with Sham+PWL group,a quick transition from an anesthesia-EEG state to a wake-EEG state was found in Sham+MBL group.In the SCNx group,MBL showed no such change,which was consistent with the effect of PWL on cortical EEG.Spectral analysis of EEG data at 100 s after sevoflurane stop showed that compared with Sham+PWL group,Sham+MBL group decreased relative delta power(p < 0.0001),increased theta power and beta power,low gamma and high gamma power,and shifted peak power from delta to theta(p < 0.05),while the relative power of alpha and spindle remained unchanged.However,there was no significant difference in EEG power between SCNx+MBL group and SCNx+PWL group.g)During continuous inhalation of 2.5% sevoflurane,Sham+MBL group showed a signifcant 21% decrease(p < 0.01)in BSR during MBL-post first 10 min and a 39% decrease(p < 0.01)during MBL-post second 10 min and a 56% decrease(p < 0.001)during MBL-post third 10 min when compared to the MBL-pre 10 min.While Sham+PWL group showed no signifcant difference in BSR between PWL-post and PWL-pre.In SCNx group,lesion of SCN neurons reversed the decrease of BSR during continuous inhalation of 2.5% sevoflurane induced by MBL.SCNx+MBL group showed no signifcant difference in BSR between MBL-post and MBL-pre.And lesion of SCN neurons did not affect the BSR between group PWL-pre and group PWL-post.h)EEG analysis results showed that,in the sham group,MBL changed the burst suppression pattern to more burst occurrence during continuous inhalation of 2.5% sevoflurane.A spectrogram computed from the same animal showed that the EEG power after MBL administration was higher than MBL-pre.However,there was no change in EEG amplitude or frequency and power between PWL-pre and PWL-post.Consistent with the previous EEG results above,Sham+MBL group showed that compared with MBL-pre,power at all frequency bands were signifcantly increased after MBL exposure(p < 0.01).While Sham+PWL group showed no statistically signifcant difference in EEG power between PWL-post and PWL-pre.However,in the SCNx group,MBL did not drive EEG change in EEG trace,EEG power spectrogram and group power spectral densities.SCNx+MBL group showed no signifcant difference in EEG power between MBL-post and MBL-pre.Additionally,SCNx+PWL group showed no signifcant difference in EEG power between PWL-post and PWL-pre. 4.Activation of the arousal nuclei downstream of the SCN is involved in the arousal effect of monochromatic blue light in sevoflurane anesthesia Methods: Male C57BL/6J(C57)mice aged 10-12 weeks old weighing 22-26 g were used in the present study,all experiments were conducted during the daytime(8:00am-5:00pm).After mice were maintained under 2.5% sevoflurane anesthesia 30 min with exposed to room light in cylindrical glass anesthesia chambers,continually anesthetized mice were administrated with same illumination intensity(800 lux)of PWL or MBL(460 nm)for 30 min on acrylic glass chambers surface devoid of any other source of light,respectively.After sevoflurane anesthesia,mice were immediately deeply anesthetized with 1% sodium pentobarbital(100mg/kg,i.p),and then the heart was perfused with polyformaldehyde.The activation of neurons in relevant brain regions downstream of SCN were detected by immunofluorescence.Results: Immunofluorescence results showed that there was no significant difference in VLPO c-Fos expression between MBL group and PWL group.Additionally,there was no significant difference in the merge of TH and c-Fos expression in LC between MBL group and PWL group.However,MBL group under sevoflurane anesthesia elicited a significant increase in c-Fos expression in the LH compared to PWL group(p < 0.0001),and the PFC c-Fos expression in MBL group also was significantly increased(p < 0.0001).5.Statistical analysis Data were represented as mean ± standard error of the mean(SEM)and analyzed using the software Graph Pad Prism 6.Group comparisons in repeated measures design(BSR)were tested with repeated measures(RM)one-way ANOVA and the Tukey's multiple comparisons test was applied to multiple hypothesis testing.Group differences in c-Fos numbers,behavior results(time to induction and time to emergence),and normalized EEG power during emergence period were compared using independent samples unpaired Student's t-tests with Welch's correction.After SCN lesioned,group comparisons in behavior results(time to induction and time to emergence),and normalized EEG power during emergence period were analyzed by ordinary one-way ANOVA,and a Tukey's multiple comparisons test was used.Differences in LFP power and EEG power during burst suppression period between the preand post-events were analyzed using paired Student's t-tests.In all cases,a p < 0.05 was considered statistically significant.Conclusions 1.Monochromatic blue light could accelerate emergence from sevoflurane anesthesia,but had no effect on induction.2.Monochromatic blue light could increase cortical EEG activity and decrease BSR during continuous inhalation of 2.5% sevoflurane.3.Monochromatic blue light plays a role in promoting arousal under sevoflurane anesthesia by activating the SCN and the related arousal nuclei downstream of the SCN.Significance There are currently no clinically approved interventions that could be used routinely to promote emergence from general anesthesia.As an innovative non-pharmacologic approach in preclinical study,MBL can accelerate emergence from sevoflurane anesthesia and induce cortical arousal during sevoflurane emergence period,on the other hand,MBL can decrease the depth of anesthesia during continuous deep sevoflurane anesthesia and increase cortical EEG activity.Additionally,in laboratory animal research and pilot human feasibility trial,bright blue light influences immunity,enhances bacterial clearance,attenuates inflammation and reduces oxidative stress.Zhou et al found that pharmacogenetic activation of mouse orexin neurons facilitates emergence from isoflurane anesthesia.In our study,we find MBL can activate LH neurons,and LH are mainly orexin neurons,thus we infer it may be the mechanism of MBL promoting arousal.Activation of orexin neurons has been reported to have analgesic effects.A study performed by Ibrahim et al also reported that blue light can resulte in thermal analgesia in naive rats.All in all,in future clinical practice,application of MBL in sevoflurane anesthesia would facilitate anesthesia emergence,pain and inflammation control,and further research to confirm this hypothesis is worthy to do. |
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