plasma and serum samples are generally acceptable for the detection of various 
 physiologically active substances ; however , drawing blood can induce stress in animals . in 
 the instance of collecting repeated blood samples from mice , using the saphenous and lateral 
 tail vein
these sites yield a 5% volume of circulating blood from the 
 saphenous veins and 0.10.15 ml from lateral tail vein . however , the small volume of blood sampled limits 
 detection of substances by any assay .
blood sampling by decapitation or cardiac puncture is 
 employed as a collection method to obtain whole blood , gathering approximately 1 
 ml of whole blood and 0.5 ml of plasma .
this volume is 
 adequate to measure multiple substances using an immunoassay . in this case ,
a large number 
 of mice are required , due to the sacrifice of animals at each time point when exhibiting 
 responses to experimental treatments .
in addition to this problem , it is impossible to 
 investigate multiple time points , before and after responses to the treatment in the same 
 mouse . alternatively ,
non - invasive samples ( including feces , urine and saliva ) can be used 
 for the assays . in particular , many studies discuss salivary biomarkers in responses to 
 stress ( for reviews [ 10 , 21 ] ) .
stress responsive biomarkers , including glucocorticoid , -amylase 
 and chromogranin a , have demonstrated some correlations between salivary and plasma 
 concentration in human and animals [ 19 , 21 ] . however , there are few reports evaluating saliva for 
 these biomarkers in response to stress using small experimental animals , particularly 
 mice .
human saliva can be easily collected using absorbent materials , such as cotton balls , 
 placed in the mouth . in contrast , it is more difficult to obtain mouse saliva as mice 
 swallow their saliva while they are awake ; thus , the mice need an anesthetic for saliva 
 collection [ 3 , 11 ] .
in addition to anesthesia , pilocarpine ( muscarinic receptor agonist ) is also 
 needed as a salivator [ 11 , 30 ] , since salivary volume is expected to be low without pilocarpine . 

however , anesthesia itself may induce a stress response [ 1 , 4 ] in mice during saliva collection . in order to clarify
whether the salivary corticosterone is available to evaluate stress 
 response in mice , first we confirmed the transfer of glucocorticoid from the blood to the 
 saliva after administration of exogenous cortisol .
secondly , we compared corticosterone 
 levels between plasma and saliva in response to restraint stress , and finally , we 
 investigated the adequate recovery , at set intervals after anesthesia , for saliva collection 
 between pre - stress and post - stress in mice .
animals : adult male icr mice were purchased from kiwa laboratory animals 
 co. , ltd .
all mice were allowed 
 ad libitum access to a commercial diet ( ef ; oriental yeast co. , ltd . , 
 tokyo , japan ) and tap water .
mice were maintained under controlled conditions : temperature 
 ( 2325c ) , relative humidity ( 4060% ) and light ( 12 hr , 07:0019:00 hr ) .
nine- to 
 thirteen - week - old animals were used in each experiment ; their body weights before the 
 experiment were 42.0  0.3 g
mice were treated according to the 
 provisions for animal welfare of the nippon veterinary and life science university , which 
 follows the guidelines for animal experimentation issued by the japanese association for 
 laboratory animal science .
drug preparation : all agents were injected intraperitoneally ( ip ) at an 
 injection volume of 0.1 ml/10 g body weight / mouse . a mixture of three drugs 
 ( medetomidine , midazolam and butorphanol ) acting as the anesthetic agents , were used 
 throughout the present study .
the injection of atipamezole ( an antagonist of medetomidine ) 
 allowed mice to undergo a rapid recovery from anesthesia [ 6 , 25 ] .
the mixed anesthetic agents were 
 prepared as a three - drug mix , modifying the dose 
 slightly dose according to naganuma et al . . the preparation involved using sterilized saline ( 0.9% ) : 0.3 mg / kg of 
 medetomidine hydrochloride ( domitor ; nippon zenyaku kogyo co. , ltd . , fukushima , 
 japan ) , 6.0 mg / kg of midazolam ( dormicum ; astellas pharma inc . , tokyo , japan ) 
 and 7.5 mg / kg of butorphanol tartrate ( vetorphale ; meiji seika pharma co. , ltd . , 
 tokyo , japan )
. pilocarpine hydrochloride ( nacalai tesque , inc . , kyoto , japan ) , as a 
 salivator to enhance salivary secretion , and atipamezole hydrochloride 
 ( antisedan ; nippon zenyaku kogyo co. , ltd . ) , as an antagonist of medetomidine , 
 were dissolved in saline and treated at a dosage of 0.5 and 0.3 mg / kg , respectively . 

cortisol ( hydrocortisone ; nacalai tesque , inc . ) was dissolved in sesame oil ( nacalai tesque , 
 inc . ) and treated at a dosage of 2.0 mg / kg .
saliva collection procedure : mice were treated with the mixed 
 anesthetic agents , and 10 min later , they were additionally treated with pilocarpine ( 0.5 
 mg / kg ip ) .
saliva was collected for 40 min by absorption onto a cotton roll ( of 
 approximately 5 mm in diameter , 10 mm in length ) and placed in the oral cavity , exchanging 
 the cotton occasionally .
after the end of the saliva collection , atipamezole was injected 
 into mice to allow recovery from the anesthetic . during anesthesia and saliva collection , 
 the body temperature of the mice was maintained at 38c by using a heating plate . to extract 
 saliva from the cotton rolls ,
they were transferred to 1.5 ml centrifuge 
 tube with a hole ( approximately 1.5 mm in diameter ) at the bottom onto empty the tube .
effect of cortisol administration on cortisol levels in plasma and saliva : 
 mice were treated simultaneously with the mixed anesthetic agents and 2.0 mg / kg ip of 
 cortisol or by vehicle injection , and 10 min later , they were additionally treated with 
 pilocarpine .
after the end of the saliva collection , mice were euthanized by decapitation 
 under anesthesia , and trunk blood was collected in heparinized sampling tubes .
blood samples 
 were centrifuged to obtain the plasma for the determination of cortisol levels by enzyme 
 immunoassay ( eia ) .
comparison of corticosterone levels before and after the restraint stress in plasma 
 without anesthesia : the restraint stress group was immobilized for 60 min by a 
 restrainer that was made of transparent acrylic , forming a cylinder ( icn-3 ; inner diameter , 
 36 mm ; length 100 mm ; icm co. , ltd . ,
blood samples were treated as described above , and 
 the plasma was obtained for the determination of corticosterone levels by eia .
comparison of corticosterone levels before and after the restraint stress in plasma 
 and saliva under anesthesia : the restraint stress group was immobilized for 60 
 min by the restrainer and then treated with the mixed anesthetic agents immediately after 
 release from the restraint . at the end of the 40 min saliva collection period
, mice were 
 euthanized by decapitation for blood sample collection . on the other hand
, the intact 
 control group without restraint stress was euthanized by decapitation after the saliva 
 collection under anesthesia .
investigation of the adequate recovery period from anesthesia for saliva 
 collection : to investigate the adequate recovery period from anesthesia for 
 saliva collection between pre - stress and post - stress in the same individual , three different 
 recovery times were examined . for the collection of saliva samples in the pre - stress 
 condition , the male mice without restraint stress were treated with the mixed anesthetic 
 agents , and saliva was collected for 40 min under anesthesia .
after 1 , 3 and 7 days since 
 the first saliva collection , the same individual male mice were immobilized for 60 min by 
 the restrainer and treated with the mixed anesthetic agents immediately after release from 
 the restraint .
saliva was collected for 40 min , and atipamezole was injected into mice for 
 recovery from anesthesia at the end of the saliva collection .
enzyme immunoassay and statistical analysis : plasma samples were extracted 
 by diethyl ether , according to the method described by kanesaka et al . 
 
the levels of cortisol and corticosterone in plasma and saliva were measured by competitive 
 enzyme immunoassay , using anti - cortisol-3-cmo - bsa igg ( fka 404-e ; cosmo bio co. , ltd . , 
 tokyo , japan ) and anti - corticosterone-3-cmo - bsa igg ( fka 420-e ; cosmo bio co. , ltd . ) as the 
 primary antibody .
cortisol-3-cmo - hrp ( fka 403 ; cosmo bio co. , ltd . ) and 
 corticosterone-3-cmo - hrp ( fka 419 ; cosmo bio co. , ltd . ) were used as the labeled steroid . 

the intra- and inter - assay coefficients of variation were 4.5 and 4.4% for cortisol , and 7.0 
 and 14.0% for corticosterone , respectively .
t - test or paired t - test was performed using jstat 
 software , version 10.0 for windows ( http://toukeijstat.web.fc2.com/ ) , for the data analysis 
 of plasma and salivary corticosterone levels .
the differences were considered significant at 
 probabilities of less than 5% ( p<0.05 ) .
effect of cortisol administration on cortisol levels in plasma and saliva : 
 the results are shown in fig . 1.fig .
1.effect of cortisol injection ( 2.0 mg / kg ip ) on plasma and salivary cortisol levels in 
 adult male icr mice .
nd denotes  not detectable. the cortisol levels in the treatment group administered with 2.0 mg / kg ip of cortisol 
 were 633.1  35.9 ng / ml in the plasma and 75.6  4.4 
 ng / ml in the saliva .
salivary cortisol was not detected 
 in the group treated by vehicle injection , while plasma cortisol was detected by eia ; 
 however , it was only represented by very low levels .
the total salivary cortisol levels , 
 from 10 to 50 min after the treatment of cortisol , were approximately 12% of the plasma 
 cortisol levels at the time point of 50 min after the treatment .
effect of cortisol injection ( 2.0 mg / kg ip ) on plasma and salivary cortisol levels in 
 adult male icr mice .
nd denotes  not detectable. comparison of corticosterone levels before and after the restraint stress in plasma 
 without anesthesia : figure 2afig .
2.effects of restraint stress ( 60 min ) on corticosterone levels in adult male icr mice 
 treated with or without anesthesia .
the restraint group was euthanized by decapitation 
 immediately after the restraint stress ( 60 min ) without anesthesia ( a ) .
the intact and 
 restraint groups were euthanized by decapitation after saliva collection , and saliva 
 was collected for 40 min following 10 min anesthesia induction ( b ) .
the open bars show 
 intact control ( non - stress ) group , and the hatched bars show restraint stress groups . 

statistically significant corticosterone levels were determined by student s 
 t - test ( * * * p<0.001 ) .
indicates the corticosterone levels of plasma obtained immediately after 60 min 
 restraint stress without anesthesia .
the levels of the restraint group were significantly 
 higher than the levels of the intact group ( 403.0  16.5 vs. 97.9  12.1 
 ng / ml , p<0.001 ) .
effects of restraint stress ( 60 min ) on corticosterone levels in adult male icr mice 
 treated with or without anesthesia .
the restraint group was euthanized by decapitation 
 immediately after the restraint stress ( 60 min ) without anesthesia ( a ) .
the intact and 
 restraint groups were euthanized by decapitation after saliva collection , and saliva 
 was collected for 40 min following 10 min anesthesia induction ( b ) .
the open bars show 
 intact control ( non - stress ) group , and the hatched bars show restraint stress groups . 

statistically significant corticosterone levels were determined by student s 
 t - test ( * * * p<0.001 ) .
comparison of corticosterone levels before and after the restraint stress in plasma 
 and saliva under anesthesia : figure 2b 
 indicates the corticosterone levels of plasma and saliva under anesthesia .
the plasma was 
 obtained after the end of the 40 min saliva collection following restraint stress .
the 
 plasma corticosterone levels of the restraint group were significantly higher than the 
 levels of the intact group ( 258.5  11.3 vs. 170.4  6.0 
 ng / ml , p<0.001 ) .
the salivary 
 corticosterone levels of the restraint group were also significantly higher than the levels 
 of the intact group ( 19.7  1.8 vs. 6.3  1.3 ng / ml , 
 p<0.001 ) .
investigation of the adequate recovery period from anesthesia for saliva 
 collection : the results are shown in fig . 
 3.fig .
3.examination of adequate recovery period from anesthesia for saliva sampling in stress 
 experiment using adult male icr mice tested as individuals .
on 1 , 3 and 7 days ( day 1 , 
 day 3 and day 7 , respectively ) after the prior saliva collections ( intact control 
 groups ) , saliva was collected again following restraint stress ( 60 min ) by the 
 restrainer .
the open bars show intact control groups , and the hatched bars show restraint 
 stress groups .
statistically significant corticosterone levels were determined by 
 student s paired t - test ( * p<0.05 , 
 * * * p<0.001 ) .
the salivary corticosterone levels of the 1- , 3- and 7-day recovery group ( day 1 , day 
 3 and day 7 ) treated with restraint stress were significantly higher than the levels of the 
 respective intact control .
the numerical results were as follows : day 1 , 11.3  0.9 vs. 8.1 
  1.2 ng / ml , p=0.0429 ; day 3 , 11.7  1.7 
 vs. 4.4  0.8 ng / ml , p=0.0165 ; and day 7 , 
 14.1  1.5 vs. 5.4  0.5 ng / ml , 
 p<0.001 .
the ratio of salivary corticosterone levels from the recovery groups 
 with restraint stress was compared with the levels of each intact control group .
the 7-day 
 recovery group showed a significant increase in salivary corticosterone levels compared to 
 the intact control group , p=0.0036 . on the other hand ,
1- and 3-day 
 recovery groups did not show a significant increase in levels when compared with each intact 
 control group , p=0.1247 and p=0.0610 ( data not shown as 
 the figure ) .
in addition to this comparison , the increase of salivary corticosterone induced 
 by restraint stress , was significantly suppressed ( p<0.01 ; one - way anova 
 followed by tukey s test ) in 1-day or 3-day recovery ( repeated anesthesia ) group ( fig .
the increased corticosterone secretion was 
 not suppressed in the 7-day recovery ( repeated anesthesia ) group ( data not shown ) .
examination of adequate recovery period from anesthesia for saliva sampling in stress 
 experiment using adult male icr mice tested as individuals . on 1 , 3 and 7 days ( day 1 , 
 day 3 and day 7 ,
respectively ) after the prior saliva collections ( intact control 
 groups ) , saliva was collected again following restraint stress ( 60 min ) by the 
 restrainer .
the open bars show intact control groups , and the hatched bars show restraint 
 stress groups .
statistically significant corticosterone levels were determined by 
 student s paired t - test ( * p<0.05 , 
 * * * p<0.001 ) .
in this paper , cortisol was detected in both the plasma and saliva as a result of the ip 
 administration of cortisol . on the other hand ,
cortisol was not detected in the saliva , but 
 was detected in the plasma at very low levels , after treatment with vehicle ( fig .
rodents , including mice , possess 
 corticosterone as a glucocorticoid and do not retain endogenous cortisol . in the present 
 study ,
the anti - cortisol showing 2% cross - reactivity with corticosterone was used for eia . 

the reason why cortisol was detected in vehicle control group , might be accounted for by the 
 cross - reactivity of anti - cortisol .
cortisol unconjugated with protein in the blood 
 circulation has been reported to pass easily through the acinar cells on the secretory 
 end - piece of the salivary glands .
the present 
 data showed that the origin of cortisol  detected in mouse saliva  is exogenous corticosteroid 
 and that the cortisol was considered to spread to the saliva from the blood via the salivary 
 glands , suggesting that glucocorticoids in saliva may reflect the levels present in the 
 blood circulation .
the result of the present study indicated that the restraint stress significantly increased 
 plasma and salivary corticosterone levels ( fig . 

2 ) , and our observations agreed with previous studies , reporting plasma 
 corticosterone levels in mice and rats treated with restraint stress [ 9 , 22 , 28 , 29 ] . in humans ,
saliva , in 
 addition to blood samples , is readily available for the measurement of cortisol in various 
 stress - related studies [ 8 , 13 , 26 ] .
however , there is no 
 scientific literature mentioning salivary corticosterone levels in mice treated with stress , 
 with only one study reporting salivary corticosterone in rats treated with isolation stress 
 .
the present report indicates that mouse 
 salivary corticosterone is detected and the level is significantly increased following 60 
 min restraint stress .
these results suggest that salivary corticosterone levels reflect 
 changes in plasma corticosterone levels , caused by induced restraint stress in mice . in the experiments investigating the adequate recovery periods from anesthesia , three 
 different time periods (
restraint stress significantly increased 
 salivary corticosterone levels in all three groups ( fig . 

3 ) ; even so , the statistical evidence of corticosterone increase is stronger in a 
 7-day recovery group ( p<0.001 ) than the others 
 ( p<0.05 ) . when comparing the ratio of corticosterone levels of intact 
 control group , restraint stress increased the levels significantly in 7-day recovery group , 
 but did not significantly increase the levels in 1- and 3-day recovery groups ( data not 
 shown as the figure ) .
repeated restraint stress after 7 days , increased the levels of plasma 
 corticosterone . in rats ,
a study shows an increase of heterogeneous nuclear rna of 
 corticotrophin - releasing hormone in the paraventricular nucleus 
equivalent to levels in 
 single acute stress  while the others experiencing repeated restraint stress ( daily , 
 alternate days and at 3 day intervals ) increased , but to lower equivalent levels of single 
 acute stress .
moreover , rat serum corticosterone 
 levels elevated by repeated nicotine treatment as a chemical stress , decreased to baseline 
 levels after 7 days withdrawal .
nicotine 
 elevated the rat plasma adrenocorticotropic hormone levels   in addition to corticosterone , suggesting that this treatment would induce 
 chemical stress in animals .
these previous studies also indicate that the recovery period 
 from physical or chemical stress is estimated at one week .
additionally , the previous 
 studies showing that the diethyl ether anesthesia increased the plasma corticosterone levels 
 in rats [ 7 , 27 ] , indicate that the anesthesia itself may induce a stress response . to evaluate 
 corticosterone secretion in response to restraint stress using saliva samples
, it could be 
 possible to set a 1-day recovery period from anesthesia , however , 7-day recovery periods 
 would be preferable for using the same individual mice .
when using small experimental animals , such as mice and rats , few studies have examined the 
 evaluation of salivary corticosterone as a stress biomarker , while numerous studies have 
 examined salivary cortisol for stress research in humans [ 8 , 13 , 26 ] .
the results of present study may contribute to stress - related research using 
 saliva samples of mice .
saliva collection is a less invasive procedure compared to blood 
 sampling and allows for repeat sampling in the same individual mouse .
moreover , the 
 anesthetic mixture of the three drugs ( medetomidine , midazolam and butorphanol )   may make it easier for repeated salvia collection . 

thus , it is easy to 
 investigate the time course of a response to any stimulus using saliva samples from mice 
 using the same individual . in the laboratory animal science field ,
russell and burch proposed a practical principle of 
 three rs ( reduction , refinement and replacement ) in 1959 .
reduction is defined as obtaining equivalent levels of information from fewer 
 animals in scientific investigation or to obtain maximum information without rise in 
 discomfort level from a small number of animals maintained long term .
refinement is also defined as performing appropriate experimental 
 procedures to improve animal well - being and minimize or avoid discomfort .
our proposal techniques in the present study , using 
 saliva sampling to measure levels of corticosterone in it , would make it possible to 
 compliment refinement in addition to reduction practices , since the saliva collection is low 
 invasive technique and is performed under anesthetized condition . in conclusion , we suggest that salivary corticosterone levels reflect plasma corticosterone 
 levels , and thus , it will be a useful and less - invasive biomarker of physical stress in 
 mice
. moreover , to evaluate restraint stress by the corticosterone level in saliva collected 
 from mice under anesthesia , an adequate recovery period of one week is preferable .
the 
 present study may contribute to the concepts of reduction and refinement as part of the 
 three rs , in small animal experiments .
 saliva as a sampling method is a low invasive technique for the detection of 
 physiologically active substances , as opposed to sampling the plasma or serum . in this 
 study 
 , we obtained glucocorticoids transferred from the blood to the saliva from mice 
 treated with 2.0 mg / kg via an intraperitoneal injection of cortisol . 
 next , to evaluate the 
 effect of restraint stress using mouse saliva  collected under anesthesia by mixed 
 anesthetic agents  we measured plasma and salivary corticosterone levels at 60 min after 
 restraint stress . moreover , to evaluate salivary corticosterone response to stress in the 
 same individual mouse , an adequate recovery period ( 1 , 3 and 7 days ) after anesthesia was 
 examined . 
 the results demonstrate that exogenous cortisol was detected in the saliva and 
 the plasma , in mice treated with cortisol . 
 restraint stress significantly increased 
 corticosterone levels in both the plasma and saliva ( p<0.001 ) . 
 
 monitoring the results of individual mice showed that restraint stress significantly 
 increased salivary corticosterone levels in all three groups ( 1- , 3- and 7-day recovery ) . 
 
 however , the statistical evidence of corticosterone increase is stronger in the 7-day 
 recovery group ( p<0.001 ) than in the others 
 ( p<0.05 ) . 
 these results suggest that the corticosterone levels in 
 saliva reflect its levels in the plasma , and salivary corticosterone is a useful , 
 less - invasive biomarker of physical stress in mice . 
 the present study may contribute to 
 concepts of reduction and refinement of the three rs in small animal experiments . 