Metabolomic Study Of Cerebrospinal Fluid In Rats With Chronic Sleep Deprivation Based On Uplc/q-tof-ms

Objective:Based on UPLC/Q-TOF-MS,to investigate the changes of endogenous metabolites in cerebrospinal fluid of rats after chronic sleep deprivation and finding sleep factors.Method:1.Replicated chronic sleep deprivation rat modelTake 48 male rats as experimental animals,keep a week under experimental conditions to adapt to the environment,drink water and obtain food freely during the period.One week later,the rats were randomly divided into 6 groups.The rats were placed in a sleep deprivation apparatus in batches of 8 at a time and sleep deprivation was performed for 1,3,5,7 and 9 days respectively,drink water and obtain food freely during the period.Sleep deprivation speed is set to 5m/min,exercise 5 seconds,pause 5 seconds.The experimental animals in the blank control group were kept on a sleep deprivation apparatus at a speed of 0 m/min,and were allowed to freely drink water and obtain food.2.Based on UPLC/Q-TOF-MS,metabolomics study of cerebrospinal fluid of rats with chronic sleep deprivation.After chronic sleep deprivation,in order to avoid the regression of sleep deprivation,the rats are anesthetized by intraperitoneal injection of chloral hydrate immediately.Rats were fixed with a stereotaxic apparatus.The head feather was cut off,disinfected,and a sponge was placed on the abdomen to maintain the temperature.With reference to the stereotaxic map of the rat brain,needles were inserted at the muscle gap 3 mm below the occipital condyle.The needle insertion angle is parallel to the body,with the needle slope facing upwards and slowly moving forward to the cisterna magna,with a depth of approximately 0.5 cm.Take about 80 microliters of cerebrospinal fluid slowly,then quickly withdraw the needle.The cerebrospinal fluid was transferred into an EP tube,frozen in liquid nitrogen,and transferred to a-80?freezer.After all the samples were collected,the cerebrospinal fluid was treated centrally and analyzed by UPLC/Q-TOF-MS.Screen out endogenous metabolites and explore their changing trends and find sleep factors.Result:1.Replicated chronic sleep deprivation rat modelThe principle of sleep deprivation on this subject is to make use of the random movement of the platform on which the experimental animal stands to prevent the experimental animals from sleeping.Most of the sleep deprivation experiments used water platforms or electrical stimulation methods to cause harm to rats.The method of this subject has reduced the damage to experimental animals.This device is characterized by:running time and rest time can be carried out in a cyclic manner,achieving the purpose of chronic sleep deprivation,well mimicking people's lack of sleep,and successfully completing the replication of the chronic sleep deprivation animal model.2.Based on UPLC/Q-TOF-MS,metabolomics study of cerebrospinal fluid of rats with chronic sleep deprivation.2.1 In positive ion mode,based on UPLC/Q-TOF-MS,metabolomics study of cerebrospinal fluid of rats with chronic sleep deprivation.The grouping trends presented by the PLS-DA score graph are as follows:Samples in the blank group and chronic sleep deprivation for 1 day were concentrated in the fourth quadrant.Samples for chronic sleep deprivation for 3 days,5 days,and 7 days were concentrated in the first quadrant.Samples for chronic sleep deprivation for 9 days were concentrated in the first quadrant and it differs from other groups in the major components 1.After cross-validation,the accumulated R2Y decreased from 0.77 to 0.51 after the label was disrupted,and the cumulative Q2 decreased from 0.56 to-0.14.There was no overfitting phenomenon in the model.The grouping information in the model was credible,and it is necessary to carry out more in-depth analysis of different metabolites.The selected endogenous metabolites are:histidine cysteine,L-histidine,L-lysine,L-methionine,L-tryptophan,octanoyl L-carnitine,L-glutamine,L-camitine,acetyl L-carnitine,valeryl L-carnitine,L-carnitine and so on.There are a total of 25 endogenous metabolites.In positive ion mode,based on UPLC/Q-TOF-MS,the contents of L-methionine,L-carnitine and L-tryptophan in the cerebrospinal fluid of rats deprived by sleep showed a trend of increasing first and then decreasing.In positive ion mode,based on UPLC/Q-TOF-MS,The contents of L-histidine,L-glutamine,butyryl L-carnitine,tetradecane L-carnitine and phospholipids(0-16:0/0:0)in the cerebrospinal fluid of rats showed an overall increasing trend and there is a difference in the upward trend.In positive ion mode,based on UPLC/Q-TOF-MS,There was no significant change in the content of 1-linoleoylglycerophosphocholine and glutaryl-L-camitine in rat cerebrospinal fluid,which remained basically stable.In positive ion mode,based on UPLC/Q-TOF-MS,The changes in the levels of hexene-L-carnitine,methyl-pentyl-L-camitine,hexadecane-L-camitine and lysophospholipids in the cerebrospinal fluid of rats were as follows:decreased first and then increased and there is a difference in the upward trend.In positive ion mode,based on UPLC/Q-TOF-MS,The content of octanoyl-L-carnitine and valeryl-L-carnitine in rat cerebrospinal fluid was relatively stable,and it was significantly increased around the third day with significant difference.In positive ion mode,based on UPLC/Q-TOF-MS,The contents of L-carnitine,acetyl L-carnitine,undecane L-camitine,and lysophospholipids in the cerebrospinal fluid of rats were changed to increase first,then decrease,and then increase again and there is a difference in the upward trend.In positive ion mode,based on UPLC/Q-TOF-MS,The contents of histamine cysteine and L-lysine in the cerebrospinal fluid of rats did not change significantly during the first 5 days of the experiment,and histamine cysteine significantly decreased on the 7th day of the experiment and afterwards Rapidly rising,L-lysine rapidly decreased around the 7th day of the experiment.In positive ion mode,based on UPLC/Q-TOF-MS,The content of L-camitine in the cerebrospinal fluid of rats showed a wide range of changes,which rapidly increased from the first day to the seventh day of the experiment,and remained stable from the seventh day to the ninth day.In positive ion mode,based on UPLC/Q-TOF-MS,The content of stearoyl-L-camitine in the cerebrospinal fluid of rats showed a decreasing trend,and there was a short-term slight increase around the third day.In positive ion mode,based on UPLC/Q-TOF-MS,Among 25 endogenous metabolites,L-tryptophan,L-histidine and histidine cysteine are associated with sleep.2.2 In negative ion mode,based on UPLC/Q-TOF-MS,metabolomics study of cerebrospinal fluid of rats with chronic sleep deprivation.The grouping trends according to the PLS-DA score plots are as follows:Sample blank groups and 1 day samples,3 days and 5 days samples,and 7 days and 9 days samples were separated.After cross-validation,the accumulated R2Y decreased from 0.74 to 0.58 after the label was disrupted,and the cumulative Q2 decreased from 0.41 to-0.034.There was no overfitting phenomenon in the model.The grouping information in the model was credible,and it is necessary to carry out more in-depth analysis of different metabolites.The selected endogenous metabolites are:Citric acid,d-galactose,L-aspartic acid,L-glutamate,L-serine,L-tyrosine,succinic acid,(R)-3-hydroxy-hexadecanoic acid,phospholipids(0-16:0/2:0).There are 9 endogenous metabolites.In negative ion mode,based on UPLC/Q-TOF-MS,The approximate trends of changes in the content of d-galactose,L-aspartate,and L-glutamate in the cerebrospinal fluid of rats are as follows:first decrease,then increase,and then decrease.In negative ion mode,based on UPLC/Q-TOF-MS,The content of L-serine,citric acid,succinic acid and phospholipids in the cerebrospinal fluid of rats increased first and then decreased.In negative ion mode,based on UPLC/Q-TOF-MS,The content of L-tyrosine in the cerebrospinal fluid of rats showed a decreasing trend overall,with significant differences.In negative ion mode,based on UPLC/Q-TOF-MS,The content of(R)-3-hydroxy-hexadecanoic acid in the cerebrospinal fluid of rats was relatively stable,only significantly higher on the fifth day of the experiment.In negative ion mode,based on UPLC/Q-TOF-MS,Among 9 endogenous metabolites,L-aspartate,L-glutamate,L-serine,L-tyrosine are associated with sleep.Conclusion:1.Chronic sleep deprivation changes multiple endogenous metabolites in the cerebrospinal fluid of rats and their change trends are different.2.Through UPLC/Q-TOF-MS detection technology.In positive ion mode,there are 25 endogenous metabolites were detected in the rat cerebrospinal fluid.In negative ion mode,there are 9 endogenous metabolites were detected in the rat cerebrospinal fluid.3.Among these endogenous metabolites,L-tryptophan,L-histidine L-aspartate,L-glutamate,L-serine,L-tyrosine and histidine cysteine are associated with sleep.