| About 2 million people suffer from spontaneous intracerebral hemorrhage(ICH)every year in the world,which has the characteristics of high mortality and disability rate,and the current treatment measures are not effective.Therefore,it is of great scientific significance and clinical value to find new strategies and targets for the treatment of cerebral hemorrhage.The so-called chemical genetics technology(designer receptors exclusively activated by designer drugs,DREADDs)is a new discipline that uses genetic methods,chemical small molecules as tools to solve biological problems,or through interference and regulation of normal physiological processes to understand the function of egg white matter,and uses chemical tools to explore and study life processes.In this paper,we will use adeno-associated virus transfection to make mouse motor cortex express hM3D(Gq),combined with intraperitoneal injection clozapine-N-oxide(CNO)specifically activated glutamatergic neurons in the motor cortex,and explored the role and possible mechanism of glutamatergic neurons in the recovery process of cerebral hemorrhage in mice.The research of this paper is as follows:First of all,this paper will be able to target the glutamatergic neurons of the adeno-associated virus vector,the use of stereotactic technology to build a chemical genetic mouse model.After feeding for 3 weeks,clozapine-N-oxide was injected intraperitoneally to regulate the activity of glutamate neurons in the animal cortex.Some model animals were perfused and collected for immunofluorescence detection to verify the expression of DREADDs vector neurons,and the expression of c-Fos gene activated by identification cells of model animals was detected by immunohistochemistry to verify the chemmogenetic activation of glutamatergic neurons.Then,the animal model of cerebral hemorrhage was established by stereotactic injection of striatum of tail artery blood,and the stability and success rate of the model were evaluated.The results showed that stereotaxic injection of adeno-associated virus hm3d mCherry into the brain clozapine-N-oxide combined with intraperitoneal injection can activate glutamatergic neurons in cortex chemically and genetically,and cause glutamatergic neurons to be excited.At the same time,the mouse model of intracerebral hemorrhage established by our group through directional striatum injection of tail artery blood has the characteristics of high model success rate,high survival rate of mice,relatively fixed hematoma volume and small group difference,which can be well used for To carry out research on cerebral hemorrhage.Furthermore,for the mice with cerebral hemorrhage induced by DREADDs,we evaluated the neural function by Longa neural deficit scale,and evaluated the recovery of motor sensory disfunction by grip test,hanging test and rotate beam test.Morris water maze test was used to evaluate the recovery of cognitive disfunction in mice.The results showed that the activation of glutamatergic neurons in the motor cortex by chemogenetics could promote the recovery of the whole neural state,motor sensory function,coordination and exercise endurance after cerebral hemorrhage,and promote the recovery of the damage of spatial learning and memory function in mice caused by cerebral hemorrhage.In order to further explore the chemical genetics technology to activate glutamatergic neurons in the cortex to improve the motor and cognitive functions of mice with cerebral hemorrhage,this paper explored the internal mechanism of glutamatergic neurons activation to improve the recovery of neural function by detecting the mitochondrial function and cell proliferation of hippocampal dentate gyrus(DG).Firstly,the neuron single cell suspension of model animal was prepared,the mitochondrial membrane potential was detected by flow cytometry,the level of mitochondrial ATP and the content of 8-OHdG,a biomarker of DNA oxidative damage,were detected by ELISA,and the effect of glutamatergic neurons on mitochondrial function was revealed;then,through the assay of 5-bromodeoxyuridine(BrdU)and immunohistochemistry,the proliferation of hippocampal dentate gyrus(DG)cells was detected,and the intrinsic mechanism of cortical glutamatergic neurons activation promoting cognitive function in mice with cerebral hemorrhage was clarified..The results showed that the activation of glutamatergic neurons in the motor cortex could improve mitochondrial membrane potential,ATP activity,DNA oxidative damage,mitochondrial function,and promote the functional recovery after cerebral hemorrhage in mice.According to the detection of BrdU markers in the newborn neurons in the dentate gyrus of the hippocampus,it was found that BrdU+cells in the DG area of the hippocampus were significantly increased,indicating that the glutamate in the cortex was significantly increased After the activation of neurons,the proliferation of neurons in this area was enhanced,which promoted the recovery of cognitive function in mice after cerebral hemorrhage.To sum up,in this paper,we use chemical genetics technology to activate glutamatergic neurons in the superficial layer of the cortex,through their synaptic connections and fiber projections,to significantly improve the mitochondrial function of neurons after cerebral hemorrhage and the proliferation of hippocampal DG cells,which is conducive to the improvement of motor and cognitive dysfunction.This study suggests that the activation of glutamatergic neurons in the superficial cortex may be a potential new way to accurately intervene the neurologic dysfunction after cerebral hemorrhage,and provide new means or strategies for the future drug development and clinical precise treatment of white matter damage and cognitive dysfunction caused by cerebral hemorrhage.Part 1 The Establishment of Wild-type and hM3Dq-mCherry Stable Expression Mouse Model of Cerebral Hemorrhage and the Verification of its Biological Characteristics1.Objective:In this part,we will construct the animal model of glutamatergic neurons in motor cortex by chemical genetics technology,and carry out the biological verification of adeno-associated virus infection and activation of neurons.2.Method(1)This study was divided into four groups:sham operation group(sham),cerebral hemorrhage model group(ICH),pAAV2/9-CaMK ? ?-hM3Dq mCherry+ICH group(hM3Dq),pAAV2/9-CaMK ? ?-mCherry+ICH group(mCherry).Three weeks before the establishment of ICH mice model,the experimental virus pAAV2/9-CaMK ? ?-hM3Dq-mCherry(hM3Dq group)and the control virus pAAV2/9-CaMK ? ?-mCherry(mCherry group)were injected into the M1 area of bilateral motor cortex of mice according to different groups.A total of 220 adult mice were randomly divided into 55 animals in each group.In addition,24 suckling mice(15-17 days old)were injected with pAAV2/9-CaMK ? ?-hM3Dq-m cherry to identify the stable transfection of motor cortex chemical heritage.The ICH mice model was established 3 weeks after virus injection,and CNO(7 p.m.)was injected intraperitoneally for 21 days.(2)After 3 weeks of AAV injection,the aorta of mice was perfused,and the brain tissue of mice was collected to make brain slices.The fluorescence expression of mCherry in the motor cortex of mice was observed by fluorescence microscope,and the expression of CaMK ? ? positive glutamatergic neurons was detected by immunofluorescence staining.(3)One week after the continuous intraperitoneal injection of CNO,the aorta was perfused and sampled,and the expression of c-fos in the motor cortex of suckling rats was detected by immunohistochemistry to verify the chemical genetic activation of glutamatergic neurons in the motor cortex.3.Result(1)Virus expression:after 4 weeks of virus injection,the fluorescence microscope showed that the M1 area of motor cortex showed red fluorescence,indicating that the virus was expressed in motor cortex.Further immunofluorescence staining showed that both CaMK? ? and mCherry fluorescent protein were expressed in glutamatergic neurons in M1 area of motor cortex,indicating that pAAV2/9-CAMK ? ?-hM3Dq-mCherry transfected glutamatergic neurons and expressed stably.(2)Immunohistochemical detection of early gene c-fos:One week after the intraperitoneal injection of CNO,perfusion and sampling were carried out.Immunohistochemistry showed that the expression level of c-fos was significantly increased after the application of CNO,which indicated that CNO could regulate the activity of glutamatergic neurons and cause the excitability of glutamatergic neurons to increase.4.Conclusion:Brain stereotaxic injection of chemical genetic tools pAAV2/9-CAMK ? ?-hM3Dq-mCherry and pAAV2/9-CAMK ? ?-mCherry combined with intraperitoneal injection of CNO can activate glutamatergic neurons in motor cortex and regulate the excitability of glutamatergic neurons.Part 2 Effects of glutamatergic neurons in motor cortex stimulated by Chemogenetics on motor and cognitive functions in mice with cerebral hemorrhageI Establishment of animal model of cerebral hemorrhage by stereotactic target injection of striatum with autogenous tail artery blood1.Objective:to establish a mouse model of cerebral hemorrhage and evaluate its stability and success rate.2.Method(1)The model of cerebral hemorrhage was established by stereotactic injection:C57BL/6 mice weighing 25-30g(as before).All mice were anesthetized with 5%isoflurane,intubated and maintained with 2-3%isoflurane.The ICH model of mice was established by stereotactic striatum injection of tail artery blood.The patients were fasted 8 hours before operation and water was forbidden 4 hours before operation.After anesthesia,they were fixed on the stereotactic operating table of small animals in prone position(512600,Stoelting,USA).After shaving the hair on the surface of skull,disinfecting with Iodophor and deioding with 75%alcohol,cut off the scalp(in order to ensure the later recovery,the operation window should be as small as possible),the periosteal peeler should peel off the periosteum,expose the anterior fontanelle and the coronal suture,dry with a small amount of 3%hydrogen peroxide,and cut off the residual damaged mucosa tissue.The height of the anterior fontanelle and the posterior fontanelle should be adjusted with the stereo positioning instrument to make them in the same level.According to the anatomical map of mouse brain,the anterior fontanelle was used as the origin to carry out three-dimensional stereotaxic localization(AP=0.00 mm,ML=+2.2 mm,DV=-2.5 mm).After positioning,use a dental drill to drill through the skull.Warm the tail with 40? warm water,and sterilize it with ethanol after congestion,2cm from the tail end Cut the tail of the mouse,take 50ul of non anticoagulant with a micro syringe,fix the syringe on the stereotaxic instrument,inject the tail artery slowly(15ul/5min at a constant rate)through the automatic micro injection pump,wait for 5min after the injection,withdraw the needle by 2.0mm,stop the needle for 4min,and exit the syringe slowly.During the period of stopping the needle,the cut of the tail of the rat was bound with alcohol cotton ball.After the operation,the bone holes of the skull were sealed with bone wax,disinfected with Iodophor,and the scalp was sutured with erythromycin ointment to prevent local infection.There was no stereotactic injection of the caudal artery into the striate body in the sham group.(2)Evaluation of animal models of cerebral hemorrhage:two hours and 24 hours after the operation,the neural deficit of each experimental group was evaluated by Longa scale.Except group.If the weight loss of mice is more than 20%and the neurological function score,further experiments will be excluded.3.Results(1)Survival rate:after 2 hours of operation,the survival rate of sham group,ICH model group,hm3dq group and mcurry group was 100%,98.18%,94.55%and 96.36%,respectively.After 24 hours of operation,the survival rate of sham group,ICH group,hm3dq group and mcurry group were 100%,96.36%,92.73%and 94.55%,respectively.(2)Two hours after the operation,76%of the mice in ICH group scored 1-3,77%of the mice in hM3Dq group scored 1-3,76%of the mice in mCherry group scored 1-3,and 76%of the mice in ICH group scored 24 hours after the operation 66%of the mice scored 1-3,65%of the mice scored 1-3 in the hM3Dq group and 63%of the mice scored 1-3 in the mCherry group.4.Conclusion:The model of intracerebral hemorrhage in mice established by stereotactic striatum injection of tail artery blood has the characteristics of high success rate,high survival rate,relatively fixed hematoma volume and small intra group difference.It is a good model for the research of intracerebral hemorrhage.II Effects of glutamatergic neurons in motor cortex stimulated by Chemogenetics on motor and cognitive functions in mice with cerebral hemorrhage1.Objective:In this part,we will build a model of cerebral hemorrhage based on the activation of glutamatergic neurons in the cortex,and further explore the effects of chemical genetics on the motor and cognitive functions of mice with cerebral hemorrhage.2.Method:In this part,the experimental groups are as follows.clozapine-N-oxide(CNO)was injected intraperitoneally(3mg/kg)continuously in each experimental group one week after the establishment of cerebral hemorrhage model.On the 7th and 14th day of continuous intraperitoneal injection of CNO,the neurofunctional assessment of motor and cognition was conducted within 6 hours of the last intraperitoneal injection of CNO.Longa neural deficit scale was used to evaluate the overall neural function of the mice,rod rotation test,grip test and suspension test were used to evaluate the motor function of the mice,Morris water maze test was used to evaluate the cognitive function of the mice.3.Result(1)Overall neurological deficit and motor function recovery:the results of longa neurological deficit scale showed that the neurological deficit scores of hm3dq,ICH,and mCherry groups were significantly higher than those of sham group(P=0.031,P ?0.0001,P=0.0002),ICH group and mCherry group were higher than those of hm3dq group(P=0.0086),There was no significant difference between ICH group and mCherry group(P? 0.05).The results of rotating bar test showed that from the 7th day of CNO intervention,the movement distance and speed of hm3dq group were significantly higher than those of mCherry group and ICH group(P ?0.05),but lower than the score of sham group(P ?0.05)(Table 2.4 and figure 2.5).Suspension test showed that the scores of hm3dq group,ICH group and mCherry group were significantly lower than those of sham group(P ?0.0001),ICH group and mCherry group were lower than those of hM3Dq group(P ?0.001),and there was no significant difference between ICH group and mCherry group(P?0.05)(2)Improvement of memory function:Morris water maze experiment showed that escape latency of hM3Dq group was lower than that of control groups(P ?0.05),the swimming speed was faster than that of the control group(P ?0.05).In the exploration experiment,when the platform was removed,there was a significant difference in the time spent in the target quadrant between the hM3Dq group and the ICH and mCherry groups(P=0.0123,P=0.0112),there was no significant difference between the two control groups(P? 0.05);in the contraposition training,the number of crossing platform positions in the hM3Dq group was significantly increased compared with the ICH and mCherry control groups(P=0.0341,P=0.0177),and there was no significant difference between the control groups(P=0.6433)4.Conclusion:The activation of glutamatergic neurons in the motor cortex by chemogenetics can promote the recovery of the overall neural state,motor function,coordination and endurance after cerebral hemorrhage,and then promote the recovery of spatial learning and memory damage in mice caused by cerebral hemorrhage.Part 3 The Mitochondrial Mechanism of Chemogenetics Activating Glutamatergic Neurons to Promote the Recovery of Neurological Function in Mice after Cerebral Hemorrhage1.Objective:To investigate the effect of activating glutamatergic neurons in motor cortex by chemical genetics on mitochondrial function in mice with cerebral hemorrhage.2.Method(1)Flow cytometry was used to detect mitochondrial membrane potential:the experimental animals were killed after cervical dislocation,followed by operation in the ice box.Soak 75%alcohol for 1-3 minutes,then put it into the super clean working table,and take the brain aseptically.The brain of the model side was cut up with small scissors,then the tissue was put into a centrifuge tube,and the 0.25%pancreas EDTA compound digestive solution was added to digest at 37? for 20-30 min.during digestion,the brain was vibrated and blown intermittently.Using precooling neurobase Gamma The special medium for neurons is used to stop digestion.After the pipette is gently blown repeatedly and evenly,the supernatant is put into 400 mesh cell sieve for filtration,and a container is placed under it to collect cell suspension.After the pipette is blown evenly,it is centrifuged(1000rpm,5min),poured out the supernatant,rinsed repeatedly for 1-2 times,and then precooled neurobase is added Gamma The single cell suspension of neuron was made from the special medium for neuron.Then,count the cells and adjust the cell concentration.Four parallel samples were set up in each group,and TMRM mitochondrial staining solution(30 nm)was added to the cell culture box.The cells were incubated at 37? in dark for 30 minutes.After pre cooling PBS cleaning,flow cytometry was used to detect.The average value of fluorescence intensity of 1×106 neurons was taken as the measured value of each group.Its mean fluorescence intensity(MFI)represents the state of MMP in neurons.The excitation wavelength and emission wavelength of TMRM are 561nm and 581nm,respectively,which are analyzed by cell quest pro.(2)Detection of mitochondrial ATP level:the animals were killed by dislocating the cervical vertebrae,soaked in 75%alcohol for 1-3 minutes in the ice box,and then placed on the super clean working table.The brain was taken out by aseptic operation,and a small piece of brain tissue with a weight of about 50-100mg was cut from the side of the model.The protein was removed by 10 kDa MWCO ultrafiltration centrifuge tube after 10 times lapping every 10 mg brain tissue in 100 uL ATP Asay buffer.The ATP assay kit(sigma,mak1901kt)reagent was dissolved in an ice bath,then 10 ml(10 nmole/ml)of ATP standard solution was diluted with 90 mL of water to generate 1 mM(1 nmole/ml)of ATP standard solution.Add 0,2,4,6,8 and 10 uL of 1 mM ATP standard solution to 96 well plate to generate 0(blank),2,4,6,8 and 10 nmol/L standard solution;add ATP assay buffer solution to each well to make the volume reach 50 uL.According to the product manual,configure the reaction mixture of ATP Asay buffer,ATP probe,ATP converter and developer mix according to the fixed proportion,sample in 96 hole plate,50ul for each hole.Omitting ATP converter in sample blank to correct the background in the sample.In order to ensure that the reading is within the linear range of the standard curve,3 multiple holes are taken for each sample.A suitable reaction mixture of 50 uL was added to each pore.Use a horizontal shaker or pipette to mix thoroughly.The absorbance was measured at 570nm(A570)after incubation at room temperature for 30 minutes.Prepare a standard curve,where the ordinate represents the RLU value and the abscissa represents the ATP concentration.(3)Detection of 8-hydroxydeoxyguanosine(8-OHdG),the oxidized product of mitochondrial DNA:the animal cervical vertebrae were dislocated and killed.The animals were immersed in 75%alcohol for 1-3 minutes in an ice box and then placed on a super clean working table.The brain was taken out by aseptic operation.The brain tissue with a weight of about 50-100mg on the side of the model was cut and frozen in liquid nitrogen.5mL of homogenate buffer(0.1 M PBS,pH 7.4,including 1 mM EDTA)was added to every gram of tissue to homogenize the sample with ultrasonic instrument,centrifuged at 1000 x g for 10 min,and purified the supernatant with DNA Extraction Kit(Abcam,kit0103).DNA was digested by nuclease P1(sigma Aldrich,n8630-1v1).1 mM Tris adjusted the pH value to 7.5-8.5,every 100 ug one unit of alkaline phosphatase(sigma,p7640)was added to the DNA,which was incubated at 37? for 30 min,boiled for 10 min,and placed on ice for standby.The 8-OHdG ELISA kit(Abcam,ab201734)was rewarming to room temperature(20-25?),and the storage solution of wash buffer(1:10)and 8-hydroxy-2-deoxyguanosine:HRP combined with monoclonal antibody(1:100)was diluted with ultrapure water to prepare the working solution.Prepare the standard solution according to the ELISA guidelines until the final concentration is 0(blank),0.94,1.875,3.75,7.5,15,30,60 ng/ml,then use the multi-channel pipette to add 50ul of standard solution and sample solution to 96 well plate,and add 50uL 8-OHdG preparation solution(except for the blank hole),add 50uL of standard,sample diluent and antibody diluent(the total volume of each hole is 100uL)into the blank hole,mix the 96 hole plate on the back cover and incubate at room temperature for 1 hour.Fully wash 96 pore plates with 300uL of lx washing buffer solution for 4 times,then add 100uL of TMB substrate to each pore and cover the second pore plate cover,add 100uL of termination buffer solution after incubation for 30min,and read the absorbance value with the wavelength of 450nm by the enzyme analyzer.After preparing the standard curve,the 8-OHdG values of each experimental group were calculated and analyzed statistically.3.Result(1)Activation of cortical glutamatergic neurons improved mitochondrial membrane potential:The results of flow cytometry showed that the ratio of neurons in ICH group was lower than sham group(P<0.0001),and that in hM3Dq group was higher than ICH group(P<0.0005);There was no significant difference in the ratio of cells with high fluorescence intensity between mCherry group and ICH group(P>0.05).It is suggested that the activation of glutamatergic neurons in motor cortex can reduce the damage of mitochondrial membrane potential caused by cerebral hemorrhage.(2)Activation of cortical glutamatergic neurons upregulated cellular ATP level:It was found that the ATP content in the tissues around hematoma in ICH group was significantly lower than that in sham group(P<0.0001)after the glutamatergic neurons in motor cortex were excited by chemical genetics technology,indicating that the ATP production of cells was damaged by cerebral hemorrhage.At the same time,there was significant difference between the hM3Dq group and the ICH group and the mCherry group(P<0.0001).The results clearly indicate that the stimulation of glutamatergic neurons by chemical genetics can up regulate ATP content in cells,and the excitation of glutamatergic neurons is closely coupled with energy regulation.(3)Glutamatergic neuron activation inhibits DNA oxidative damage:The results showed that 8-OHdG in ICH group The content of 8-OHdG in brain tissue of hM3Dq group was significantly lower than that of ICH group(P=0.0032),indicating that the oxidative damage caused by cerebral hemorrhage increased significantly(P=0.0012)There was no significant difference in the content of 8-OHdG in the brain between the mCherry group and the ICH group(P? 0.05).These results indicate that activation of glutamatergic neurons in motor cortex can reduce free radical production and DNA oxidative damage after cerebral hemorrhage.4.ConclusionMitochondria,known as the "energy factory" of cells,provide most of the ATP energy source for cells.The function of mitochondria is directly dependent on the normal mitochondrial membrane potential.Under the condition of normal cell activity,the mitochondrial membrane potential is high,which can provide the cell with the energy needed for various physiological activities.When cells tend to apoptosis,the most obvious characteristic is the decrease of mitochondrial membrane potential.It can be said that the decrease of mitochondrial membrane potential is a marker event in the early stage of apoptosis.According to the changes of ATP level,mitochondrial membrane potential,DNA oxidative damage product 8-OHdG and other indicators in the tissues around the focus of cerebral hemorrhage under different excitability conditions of glutamatergic neurons in the motor cortex,we can confirm that the activation of glutamatergic neurons in the motor cortex can maintain the normal function of mitochondria.In conclusion,the activation of glutamatergic neurons in the motor cortex can maintain the normal mitochondrial function around the hematoma,so as to improve the motor and cognitive functions of mice after cerebral hemorrhage.Part 4 Mechanism of the Proliferation of Dentate Gyrus Cells in the Hippocampus of Mice with Cerebral Hemorrhage by Chemogenetics Activating Glutamatergic Neurons in M11.Objective:To investigate the mechanism of chemogenetics technology activating glutamatergic neurons in motor cortex to promote cognitive function improvement in mice after cerebral hemorrhage.2.Method:Proliferation detection of hippocampal dentate gyrus(DG area):the mice in each group were intraperitoneally injected with BrdU solution(dose:50mg/kg)21 days after CNO injection,twice a day,12 hours apart,for 2 days.After 7 days of administration,ketamine and xylazine were injected into abdominal cavity for anesthesia(ketamine 100mg/kg,xylazine 25mg/kg).After corneal reflex and muscle tension disappeared,4?precooling 4%paraformaldehyde solution(50mL)was given 0.9%Pre rinse with normal saline,then 250ml 4%paraformaldehyde was fixed before,fast first and then slow)the brain tissue was perfused and fixed through the ascending aorta of the left ventricle.The mouse brain was taken out completely in the ice box,and the hippocampus was preserved.The continuous coronal section was made on the vibration microtome.The thickness of the brain slice was 40 ? M.one piece was selected from each 5 pieces.12 pieces of each sample were collected and placed in PBS for BrdU immunohistochemical detection.The slices were placed in a water bath of 65? in 50%formamide solution for 2 h,PBS(pH=7.4)washes tablets,incubate in 2 M hydrochloric acid at 37? for 30 min,0.1M Wash the tablet with boric acid for 10 min,and incubate it with 30%H2O2 at room temperature for half an hour after PBS washing the tablets,incubate the serum at room temperature for 60min,add BrdU primary antibody(1:1000)after the blood washing tablets are discarded,incubate at 4? for 24h,discard primary antibody and PBS washing tablets,add biotin labeled secondary antibody(dilution:1:100),incubate at room temperature for 30min,incubate the PBS washing tablets at room temperature for half an hour with ABC reagent,develop the color with DAB reagent after PBS washing tablets,rinse the slices with ddH2O after the staining is satisfactory,and stop the staining.The stained sections were spread to the glass slides and dried,then the neutral gum wrap and nail polish were fixed.3.Result:The results showed that the number of BrdU+cells in hippocampal DG area of ICH group was lower than that of sham group,the difference was statistically significant(P ?0.05),indicating that cerebral hemorrhage damaged the proliferation ability of hippocampal DG area;the number of BrdU+cells in hippocampal DG area of hm3dq group was significantly higher than that of ICH group,and there was significant difference(P ?0.01),indicating that the activation of glutamatergic neurons in motor cortex promoted the neurogenesis of hippocampal DG area There was no significant difference in the number of BrdU+cells between ICH group and mcurry group(P?0.05).Based on the above results,it can be concluded that the activation of glutamatergic neurons in Ml area of motor cortex may promote the proliferation of DG cells in hippocampus.4.Conclusion:The activation of glutamatergic neurons in motor cortex by chemical genetics can promote the proliferation of neurons in DG area of hippocampus,and improve the cognitive function of mice with cerebral hemorrhage. |
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