| Background:Dexmedetomidine（Dex）,as a highly selective a2 adrenergic receptor agonist,can produce a sedative effect similar to normal sleep,arousal,and has sedative,analgesic,anxiolytic,and stress-protective effects.Dex can reduce the body temperature and blood pressure within a safe range;it can inhibit the excitability of neurons in the locus coeruleus（LC）nucleus to reduce the release and oxidative phosphorylation of norepinephrine at the axon terminal,reducing the activity of Oxygen;it can also optimize the effects of cardiopulmonary status and cardiovascular system.At present,the drug has been widely used in clinical practice.It has been previously reported that administration of Dex（100 μg/kg）reduces the level of arginine vasopressin（AVP）in the blood.In addition,AVP and oxytocin（OXT）are structurally homologous peptide hormones synthesized by the hypothalamus,suggesting that the OXT and AVP systems interact at the hypothalamic level.However,whether Dex can directly act on brain-related neurons（such as AVP,OXT neurons）has not been reported yet.Objective:To explore the co-labeling of c-Fos protein expression and related brain regions of AVP and OXT neurons in mouse brain after administration of Dex（100 μg/kg）Methods:1.Methods of administration and grouping of miceC57 mice of 8-10 weeks were taken,and the administration method was intraperitoneal injection（ip）,and the injection dose:0.1 ml/10 g.The control group was given normal saline（Saline group）,and the experimental group was given gradient Dex 50,100,200 μg/kg（D1～3 groups）.According to the experimental category,it is divided into:（1）:D2 and Saline immunofluorescence c-Fos protein expression group;（2）:D2 and Saline immunofluorescence c-Fos protein co-labeling group with AVP/OXT neurons;（3）:D1～3 and Saline to mice 0.5,0.5-1,1-2,2-3,3-4 hours（h）（T1～5）period of drinking water and food intake measurement group;（4）:D2～3 and Saline on mice drinking water measurement group at 0.5,0.5-1,1-2,2-3 hours（h）（T1～4）;（5）:D3 and Saline on mouse blood and kidney function electrolyte detection group.2.The brain of mice was taken.After administration of group（1）and（2）,the mice waited in the home cage for 120 minutes,and were anesthetized with sodium pentobarbital,followed by 0.01 M phosphate buffer（PBS）,4%paraformaldehyde（PFA）was used to fix the brain by cardiac perfusion,the skull was removed and the brain was dehydrated in sucrose solution for three days.3.Frozen section of the whole mouse brain.First,the dehydrated and intact brain was placed under the brain trough apparatus and cut into two parts along the coronal plane.After the cut,the smooth surface was used as the base,embedded with OCT embedding medium,and placed on the ice base of the cryostat.After freezing,the whole brain was sliced with a thickness of 40 μm and placed in a 12-well plate containing 0.01 MPBS solution in sequence.4.Immunofluorescence.In the experiment（1）,the excised brain slices were incubated with goat antirabbit-derived c-Fos single antibody.The excised brain slices in experiment（2）were incubated with goat anti-mouse-derived c-Fos antibody and goat anti-rabbit-derived AVP/OXT neuron antibody for double staining.In this study,single-stained c-Fos was imaged using an Olympus（VS120-S6-W）slide scanner;c-Fos and AVP/OXT neurons double-stained were imaged using Zeiss LSM900 confocal.5.Measurement of drinking water and food intake of miceThe mice in the group（3）were fasted from water and food for 24 hours before and 6 hours after the drug administration.They ate normally during the measurement,and the water and food intake was measured.The mice in the group（4）were fasted from water and food for 24 hours before administration and 6 hours after administration.During the measurement,they continued to fast,only water was given,and the amount of water was measured.The experimental drinking water used a custom-made ball-type drinking bottle.After each stage of drinking water,together with the drinking water bottle,it was weighed under a precision balance.Food intake is measured by weighing under a precision balance along with the trough.Results:1.Immunofluorescence c-Fos protein expression in D2 group and Saline group.The results showed that compared with the Saline group,the D2 group had higher thalamotomy in the supraoptic nucleus（SON）,ventrolateral preoptic nucleus（VLPO）,subfornical organ（SFO）,solitary tract（SOL）,and area postrema（AP）;c-Fos in the locus coeruleus（LC）,lateral hypothalamic area（LH）,laterodorsal tegmental nucleus（LDT）,parabigeminal nucleus（PB）,ventral tuberomammillary nucleus（TMN）and motor cortex M1/M2 protein expression was significantly decreased（P<0.05）;there was no difference in c-Fos protein expression in parathalamic nucleus（PVN）and suprachiasmatic nucleus（SCN）.（P>0.05）.2.The water intake and food intake of D1～3 groups and Saline group.The results showed:（1）Compared with the Saline group,the water intake of the mice in the D1 group decreased during the period from T1 to 5,but the difference was not statistically significant（P>0.05）;（2）Compared with the Saline group,the water intake of the D2 group decreased significantly during the T1 and T2 periods（P<0.05）,and there was no difference between the T3 and 5 periods（P>0.05）;（3）Compared with Saline group,the water intake of D3 group decreased significantly in T1 period（P<0.01）,there was no difference in T2-5 period（P>0.05）.Compared with the Saline group,the food intake of the D1-3 groups decreased,but the difference was not statistically significant（P>0.05）.3.The effect of continuous fasting on water intake in D2～3 and Saline groups.The results showed:（1）Compared with the Saline group,the water intake of the D2 group was significantly decreased in the T1～3 period（P<0.05）,and there was a decreasing trend in the T4 period,but there was no statistical significance（P>0.05）;（2）with the compared with the Saline group,the water intake in the D3 group during T1～4 was significantly decreased（P<0.05）.4.D2 and Saline group co-labeled AVP,OXT neurons and c-Fos protein in PVN,SON and SCN brain regions.The results showed:（1）Compared with the Saline group,the co-labeling of AVP neurons and c-Fos in the PVN brain region of the D2 group was significantly decreased（P<0.05）;there was no difference in the SON and SCN brain regions（P>0.05）.（2）Compared with the Saline group,the PVN,OXT neurons and c-Fos protein in the SON brain region of the D2 group were co-labeled,and the results showed no difference（P>0.05）.5.Blood and kidney function and electrolyte indexes of mice in D3 and Saline groups.Compared with the Saline group,there was no difference in the indicators in the D3 group（P>0.05）.Conclusion:（1）For the first time,immunofluorescence co-labeling experiments showed that Dex can inhibit the expression of AVP neurons in the PVN brain region at the central level.（2）The increased expression of c-Fos protein in the SON brain region did not colabel with OXT neurons.（3）Giving Dex led the mice to be in a state of anesthesia and sedation.Compared with the Saline group,the energy consumption was lower,and there was no more urge to drink and eat,so the amount of water and food decreased.（4）After selecting mice treated with Dex at a dose much higher than the clinical dose,there was no abnormality in the renal function and other indicators of the mice,indicating that the drug has less nephrotoxicity.In conclusion,this study provides theoretical help for the clinical safety of Dex drug use provides a systematic analysis of the neural network of brain targets,and provides data support for subsequent research. |
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