animals and housing conditions : we used male c57bl/6j mice at 8 weeks of age from clea japan , 
 inc . the animals were 
 housed in stable groups of 3 or 4 each , in polycarbonate cages with autoclaved bedding ( aspen chips , tapvei 
 ltd . , kortteinen , finland ) . each cage had tap water in a polycarbonate bottle , and the mice were supplied with 
 sterilized food ( clea japan , inc . ) . the ambient temperature was controlled by a central temperature management 
 system and was maintained from 20 to 26c . mice were maintained at a 
 12:12-hr light : dark cycle ( light on from 7 a.m. to 7 p.m. ) . all of the animal procedures were conducted in 
 accordance with the approved guidelines of vet-300 ( 2014 ) , vet-700 ( 2014 ) and car-100 ( 2014 ) of the 
 institutional animal care and use committee at chugai pharmaceutical co. , ltd . the mmb combination in this study 
 contained medetomidine ( domitol , nippon zenyaku kogyo co. , ltd . , fukushima , japan ) , midazolam ( dormicum , 
 astellas pharma inc . , tokyo , japan ) and butorphanol ( vetorphale , meiji seika pharma co. , ltd . , atipamezole ( antisedan , nippon zenyaku kogyo co. , ltd . ) was used as a medetomidine antagonist . the other 
 anesthetics assessed included isoflurane ( escain , mylan co. , ltd . , tokyo , japan ) , pentobarbital sodium 
 ( somnopentyl , kyouritu co. , ltd . , tokyo , japan ) , and a mixture of ketamine ( ketalar , daiichi sankyo co. , ltd . , 
 tokyo , japan ) and xylazine ( celactal , bayer yakuhin co. , ltd . , all drugs were diluted by saline 
 ( otsuka normal saline , otsuka pharmaceutical factory , inc . , tokushima , japan ) . and preparation of the drugs : all drugs were prepared according to table 1table 1.list of drugs used in this studygroup namedrugproduct concentrationagent doseadministration routecontrolsalinesubcutaneousiso sa ( + ) salinesubcutaneousisoflurane2%4 l / mininhalationatipamezole*5.0 mg / ml1.8 mg / kgsubcutaneousiso sa ( )isoflurane2%4 l / mininhalationmmb lowmedetomidine1.0 mg / ml0.45 mg / kgsubcutaneousmidazolam5.0 mg / ml6.0 mg / kgbutorphanol5.0 mg / ml7.5 mg / kgatipamezole*5.0 mg / ml0.9 mg / kgmmb highmedetomidine1.0 mg / ml0.9 mg / kgsubcutaneousmidazolam5.0 mg / ml12.0 mg / kgbutorphanol5.0 mg / ml15.0 mg / kgatipamezole*5.0 mg / ml1.8 mg / kgpentpentobarbital sodium64.8 mg / ml48.6 mg / kgintraperitonealk / xketamine50 mg / ml75 mg / kgintraperitonealxylazine20 mg / ml10 mg / kgmedmedetomidine1.0 mg / ml0.9 mg / kgsubcutaneousmidmidazolam5.0 mg / ml12.0 mg / kgbutbutorphanol5.0 mg / ml15.0 mg / kgatipamezole * is only administrated in experiment 3 . and were diluted in saline . preparation of mmb was conducted in accordance with previous reports 
 [ 3 , 12 , 18 , 37 ] . each mmb dose was determined by 
 pre - examination that evaluated mmb singly administrated in mice ; mmb low dose was the minimum dose that enabled 
 surgery , and the mmb high dose was the maximum single dose that did not result in death . the dosage of other drugs followed the guidelines of the institutional animal care and use 
 committee at chugai pharmaceutical co. , ltd . evaluation of the depth of anesthesia : three parameters were used to assess the depth and 
 duration of anesthesia , namely , the righting reflex , abdominal region pinch reflex and pedal withdrawal reflex 
 in the hind limbs . recovery of the righting reflex was defined as the time when the animals began to roll over . 
 for the pedal withdrawal reflex , the interdigital web of limbs on both sides was lightly pinched and pulled 
 using atraumatic forceps [ 17 , 19 ] . for the abdominal region pinch reflex , a 1 cm region of the abdominal skin was lightly pinched using hooked 
 forceps . the depth of 
 anesthesia was evaluated at 10 , 30 and 60 min after commencement of the experiment . experimental design : animals were divided into the following experimental groups : iso group , 
 mmb low group , mmb high group , pent group , k / x group , medetomidine ( med ) group , midazolam ( mid ) group and 
 butorphanol ( but ) group ( table 1 ) . in addition , the iso group was 
 divided into the saline administered group ( iso sa ( + ) ) and not administered group ( iso sa (  ) ) , to confirm the 
 effect of saline administration . a control group was also included , in which a necropsy was performed under 
 isoflurane anesthesia 1 hr after saline administration . experiment 1 : blood sample for biochemical measurements was collected from abdominal portion 
 of vena cava under isoflurane anesthesia in all groups , other than the mmb high group that maintained an 
 adequate depth of surgical anesthesia . necropsies were performed on mice to confirm the influence on organ of 
 mmb in the control group ( n=3 ) , iso sa ( + ) group ( n=6 ) , iso sa (  ) group ( n=6 ) , pent group ( n=6 ) , k / x group , 
 ( n=6 ) , mmb low group ( n=7 ) and mmb high group ( n=19 ) 1 hr after the experiment was commenced . blood biochemical 
 levels ( blood sugar ( gluc ) , inorganic phosphorus ( ip ) , calcium ( ca ) , lactate dehydrogenase ( ld ) , creatine kinase 
 ( ck ) , sodium ( na ) , potassium ( k ) and chlorine ( cl ) ) at the time of necropsy were compared between groups . experiment 2 : necropsies were performed on control group ( n=3 ) , iso sa (  ) group ( n=6 ) , mmb 
 high group ( n=12 ) , pent group ( n=6 ) , k / x group ( n=6 ) , med group ( n=6 ) , mid group ( n=6 ) and but group ( n=6 ) 
 mice 1 hr after commencement of the experiment . experiment 3 : changes in biochemical parameters in the iso sa ( + ) ( n=18 ) , mmb low ( n=19 ) and 
 mmb high groups ( n=27 ) over time were compared in each group . in addition , 1 hr after commencement of 
 anesthesia , mice in all the groups were given atipamezole ( iso sa ( + ) and mmb high ; 1.8 mg / kg , mmb low ; 0.9 
 mg / kg ) for recovery from anesthesia ( table 1 ) . then , necropsies were 
 performed in 6 7 animals in each group at 1 , 4 and 24 hr after commencement of the experiment . blood biochemical level measurements : after blood collection , the blood was poured into a tube 
 containing heparin lithium and centrifuged at 1,900 g and 15c for 10 min , and the resultant plasma was 
 collected . plasma was analyzed for blood biochemical levels by chemical automatic analyzer tba-120fr ( toshiba 
 medical systems , co. , ltd . the ck isoenzyme was measured using a ck isoenzyme kit ( titan gel 
 ck ( qg ) ) with automatic electrophoretic device , epalyzer 2 ( helena laboratories , co. , ltd . , beaumont , tx , 
 u.s.a . ) . data analysis : all results are expressed as mean  standard deviation . all data were analyzed 
 by parametric dunnett s multiple comparison tests with sas software ver.5.0 ( sas institute inc . , cary , nc , 
 u.s.a . ) . in the mmb low group , surgical level anesthesia was achieved in 50% of all mice at 10 min and 60 min . in pent 
 and k / x groups , all mice recovered from anesthesia within 1 hr after administration of the anesthetics . hence , 
 necropsies were performed in all the groups immediately after isoflurane anesthesia , other than the mmb high 
 group that maintained an adequate depth of surgical anesthesia . in the med , mid and but groups , an adequate 
 surgical depth of anesthesia was not achieved . in addition , death due to anesthetic overdosage did not occur in 
 any of the mice in this study . changes in blood biochemical levels 1 hr after start of the experiment : in experiment 1 , gluc , 
 ip and k levels increased 1 hr after commencement of the experiment in the k / x ( p<0.0001 ) , 
 mmb low ( p<0.0001 ) and mmb high groups ( p<0.0001 ) and beyond the 
 reference range ( table 2table 2.blood biochemical levels 1 hr after commencement of the experiment in male miceitemcontrol(n=3)iso sa ( + ) ( n=6)iso sa (  ) ( n=6)pent ( n=6)k / x ( n=6)mmb low ( n=7)mmb high ( n=19)gluc ( mg / dl)276.314.8289.822.5356.055.5251.831.2653.547.1721.455.2714.865.4ip ( mg / dl)6.90.35.10.47.81.18.10.610.40.710.50.410.50.5ca ( mg / dl)9.10.39.60.29.60.19.40.39.00.19.10.29.10.2ld ( u / l)228.718.9252.883.4187.358.8214.862.9203.250.3268.382.5278.6108.4ck ( u / l)271.081.7197.534.568.515.7224.063.2188.344.1348.663.8604.4150.7na ( mmol / l)147.70.6145.00.9145.31.0150.31.2144.81.3144.11.3145.72.2k ( mmol / l)4.40.24.60.25.50.85.40.26.10.46.60.46.50.6cl ( mmol / l)116.31.5113.01.5110.20.8115.51.4110.33.1109.31.8109.81.9the value is mean  standard deviation . the statistics processing uses the parametric dunnett s multiple 
 comparison tests . a ) p<0.05 vs. control , b ) p<0.01 vs. 
 control . ) . in addition , ck increased in the mmb high group 
 ( p=0.0058 ) . on the other hand , ld levels did not change in any of the groups and within the 
 reference range ( table 2 ) . 
 in the iso sa ( + ) , iso sa (  ) and pent groups , blood biochemical levels did not change significantly or were 
 within the reference range ( table 2 ) . the value is mean  standard deviation . the statistics processing uses the parametric dunnett s multiple 
 comparison tests . change in ck isoenzyme levels 1 hr after commencement of the experiment : in experiment 2 , ck 
 in the mmb high ( p=0.0036 ) and mid groups ( p=0.027 ) increased and in the med 
 group ( p=0.0503 ) had a tendency to increase at 1 hr after start of the experiment . skeletal 
 muscle type isoenzyme of ck ( ck - mm ) also had a tendency to increase in the mmb high group 
 ( p=0.0993 ) . in addition , the brain - type isoenzyme of ck ( ck - bb ) increased in the med group 
 ( p=0.0003 ) , while ck - mm increased in the mid group ( p=0.0184 ) ( table 3table 3.ck and ck isoenzyme levels in plasma 1 hr after anesthetic drug administration in male miceitemcontrol ( n=3)iso sa (  ) ( n=6)mmb high ( n=12)pent ( n=6)k / x ( n=6)med ( n=6)mid ( n=6)but ( n=6)ck ( u / l)271.081.768.515.7572.9138.3224.063.2188.344.1584.7296.9616.0297.3240.2134.0ck - mm ( u / l)190.071.028.014.5394.2111.2114.639.084.825.2306.0243.8484.1232.9149.6124.8ck - bb ( u / l)77.19.037.310.5157.945.2106.127.992.419.9259.0130.3125.268.086.022.3ck - mb ( u / l)3.92.03.21.120.816.03.30.911.25.619.819.26.83.14.62.0the value is mean  standard deviation . the statistics processing uses the parametric dunnett s multiple 
 comparison tests . ck - mm : skeletal muscle type isoenzyme of ck , ck - bb : brain type isoenzyme of ck , ck - mb : myocardial type 
 isoenzyme of ck . ) . the value is mean  standard deviation . the statistics processing uses the parametric dunnett s multiple 
 comparison tests . ck - mm : skeletal muscle type isoenzyme of ck , ck - bb : brain type isoenzyme of ck , ck - mb : myocardial type 
 isoenzyme of ck . changes in blood biochemical levels 1 , 4 and 24 hr after mmb administration : in experiment 3 , 
 the changes in blood biochemical parameters seen in experiment 1 returned to the levels seen in the iso sa ( + ) 
 group at 24 hr after anesthetic administration ( table 4table 4.changes in blood biochemical parameters 1 , 4 and 24 hr after anesthetic 
 administration in male miceitemiso sa ( + ) mmb lowmmb high1 hr ( n=6)4 hr ( n=6)24 hr ( n=6)1 hr ( n=7)4 hr ( n=6)24 hr ( n=6)1 hr ( n=13)4 hr ( n=7)24 hr ( n=7)gluc ( mg / dl)289.822.5258.330.0290.227.9721.455.2473.866.2284.328.7724.974.1453.491.6214.333.3ip ( mg / dl)5.10.47.81.68.10.410.50.48.40.97.30.510.50.58.61.07.90.8ca ( mg / dl)9.60.29.40.19.80.39.10.29.40.29.50.29.10.29.40.29.50.1ld ( u / l)252.883.4327.854.8194.592.2268.382.5340.794.7140.520.4269.7128.4346.774.2136.943.0ck ( u / l)197.534.5364.2209.3145.0136.0348.663.8437.759.778.027.3577.7170.9826.7206.0130.4128.5na ( mmol / l)145.00.9149.31.0147.30.8144.11.3145.52.4148.50.5145.72.5148.92.6150.31.0k ( mmol / l)4.60.23.80.23.60.46.60.44.40.23.40.26.60.64.20.33.90.3cl ( mmol / l)113.01.5115.71.6112.81.5109.31.8110.83.8115.21.5110.22.1114.62.4116.71.7the value is mean  standard deviation . the statistics processing uses the parametric dunnett s multiple 
 comparison tests . a ) p<0.05 vs. iso sa ( + ) , b ) p<0.01 vs. iso sa 
 ( + ) . ) ( supplementary table 1 ) . the levels 
 of gluc ( p<0.0001 ) , ip ( p<0.0001 ) and k ( p<0.0001 ) 
 increased at 1 hr after mmb administration in the mmb low and high groups . all the blood biochemical parameters , except gluc , returned to the level 
 in the iso sa ( + ) group 4 hr after mmb administration . there were some statistically significant changes in 
 other blood biochemical items ( alp , tcho , tg , tp , un , ca , na and cl ) as well . however , these changes were not 
 considered to be important ( supplementary table 2 ) , 
 because they were unrelated to the dose of anesthetic administered or the passage of time , and they were within 
 the reference range . an additional experiment proved that there were 
 no gender related differences in the results ( supplementary tables 3 and 4 ) . the value is mean  standard deviation . the statistics processing uses the parametric dunnett s multiple 
 comparison tests . a ) p<0.05 vs. iso sa ( + ) , b ) p<0.01 vs. iso sa 
 ( + ) . in this study , iso sa ( + ) group and iso sa (  ) group of the comparison group of the anesthesia were 
 anesthetized with isoflurane ; the isoflurane is frequently used during surgery , because of its easy control of 
 anesthesia depth and its relative safety compared to injection anesthesia . a previous report indicated that isoflurane increases gluc levels by decreasing insulin secretion 
 [ 7 , 11 , 40 , 41 ] and the surgical stress itself conduces to the 
 hyperglycemia . so , the gluc level in the control , iso sa ( + ) and iso 
 sa (  ) groups might have been influenced by the surgery stress . the reason for the elevated gluc and k levels in 
 the mmb low , mmb high and k / x groups is inhibition of insulin secretion of the pancreatic -cells and closing 
 of the atp - dependent potassium ion channels of the cell membrane by 2-adrenoreceptor agonists ; medetomidine and 
 xylazine [ 2,3,4 , 6 , 11 , 13 , 18 , 23 , 36 , 41 ] 
 ( table 2 ) . since pentobarbital does not affect insulin secretion , 
 gluc levels in the pent group become the level in the control group ( table 
 2 ) [ 5 , 16 , 27 , 40 ] . further , atipamezole has been 
 reported to have 2-adrenoreceptor blocking effects . in the mmb high 
 group , the blood level of gluc and ck remained high at 3 hr after atipamezole administration ( table 4 ) . this suggests the possibility that the dose of atipamezole 
 used in this study was not enough to reverse the effects of mmb . according to a previous report , it requires a 
 long time for the gluc level to decrease when the dose of atipamezole on medetomidine is not enough . all blood biochemical levels finally became the level in the iso sa ( + ) 
 group or returned to reference levels at 24 hr after administration of the anesthetic agents ( table 4 ) . the blood levels of gluc , ip and k in the k / x group were increased , as in the mmb administration group . the k 
 / x combination contains xylazine , which stimulates on the 2-adrenoceptors , similar to that of medetomidine 
 [ 3 , 4 , 13 , 18 ] ( table 
 2 ) . however , ck levels in the k / x group did not increase . and , it is thought that the effect of mmb 
 administration was beyond the influence of the s.c . administration . because , the ck level of the mmb high group 
 was higher than the control group by s.c . muscle twitching is reported to be caused by 
 2-adrenoceptor stimulants , the degree of muscle twitching varying according to the route of drug administration 
 . hence , the difference in ck levels between the k / x and mmb 
 groups is probably related to the difference in the method of administration of the drugs . ck levels increased 
 in both the med and mid groups . therefore , because of the cumulative effect of both medetomidine and midazolam , 
 ck levels in the mmb group probably increased to a greater extent than in the k / x group ( table 3 ) . to clarify which drug in the mmb combination caused the increase in ck , ck isoenzymes in each group were 
 examined . we observed increases in ck levels in the mmb high , med and mid groups ( table 3 ) . in the mid group , ck - mm levels were elevated , similar to what was seen in 
 the mmb high group . ck - bb levels in the med group were also clearly high in comparison with the control group . this indicates that the rise in ck level in the mmb high group was due to the combined influence of both 
 medetomidine and midazolam . in addition , it appears that most of the changes in blood biochemistry , except for 
 the change in ck levels , were mainly due to medetomidine ( supplementary table 1 ) . in this study , administration of mmb in mice caused a temporary rise in the levels of blood biochemical 
 parameters . this , however , did not seem to adversely affect the health of the mouse . our results highlight the 
 fact that when mmb is used to anesthetize animals for experiments , the effects of the anesthetic agents on blood 
 biochemistry should be considered during experimental data analysis . in addition , use of mmb in pathological 
 animal models , such as diabetes models , requires caution , because gluc levels increase greatly with mmb . our study suggests that the anesthetic and analgesic agents that are usually used for animal experiments may 
 cause changes in the blood biochemical parameters that are commonly measured as experimental data . hence , it is 
 important to be aware of the effects of the drugs before their use .
currently , from the viewpoint of animal welfare , anesthesia or analgesia is required during experimental 
 procedures in animals that are likely to cause pain . a part of these anesthetics 
 have been reported to 
 influence a blood biochemical level . 
 it is important for us to understand the effect of the anesthetic on 
 blood biochemistry when we choose the anesthetic agent to be used in experiments . in this study 
 , we examined 
 the blood biochemical changes in mice after administration of a new mixture of three anesthetic agents 
 medetomidine / midazolam / butorphanol ( mmb ) . 
 we subcutaneously administered two dose combinations of mmb 
 ( 0.45 / 6 / 7.5 and 0.9 / 12 / 15 mg / kg ) in mice , followed by administration of atipamezole , for reversal of 
 anesthetic effects , after 1 hr . thereafter , blood biochemistry was assessed at 1 , 4 and 24 hr after mmb 
 administration . 
 we observed that mmb administration caused a transient increase in blood sugar , inorganic 
 phosphorus , potassium and creatine kinase levels . these , however , returned to the reference range 24 hr after 
 mmb administration . in conclusion 
 , mmb changes the levels of some blood biochemical parameters , but not to an 
 extent that would threaten health . 
 however , when using laboratory animals , this effect of mmb may influence 
 the experimental results , depending on the experimental content . hence , the choice of anesthetic agents used 
 in laboratory animals should be based on detailed knowledge of their pharmacological effects .