the state of lowered metabolism during hibernation in mammals is a showcase of cell preservation strategies for muscle , bone , and the circulatory and innate immune systems .
hibernating mammals tolerate extremes in organ perfusion , oxygen saturation , temperature , immobilization , and calorie intake - which in combination would be lethal to humans .
hibernators may therefore act as reverse translational models for human health and disease . in most hibernating mammals ,
torpor phases are interrupted by euthermic arousal phases ( reviewed in : 1 ) . during torpor , metabolism is severely depressed but hibernation also involves the inhibition of thermogenesis , leading to a considerable decrease in body temperature ( tb ) .
the induction of torpor begins with lowering of the metabolic rate , followed by hypothermia as the tb drifts downward ( reviewed in : 1 - 3 ) .
various degrees of cold torpor have been observed over a phylogenetically wide range of mammals and is most drastic in bats and rodents .
the champion of all torpid mammals is the hibernating arctic ground squirrel ( urocitellus parryii , previously called spermophilus parryii ) , which exhibits body temperatures as low as -2.9c 4 . in a recent paper by bouma et al .
although there are few data from this field , one of the most striking phenomena is the reduced number of circulating leukocytes found in all hibernating mammals studies so far .
5 , finding leukopenia in hibernating mammals , were all conducted on small species , including the european hamster ( cricetus cricetus ) , european hedgehog ( erinaceus europeaus ) , european ground squirrel ( spermophilus citellus ) , arctic ground squirrel ( urocitellus parryii ) , and the thirteen - lined ground squirrel ( ictidomys tridecemlineatus , previously called spermophilus tridecemlineatus ) ; all species in which tb drops drastically during hibernation .
the authors 5 raised the question of whether the reduced number of circulating leukocytes in mammalian hibernators is due to torpor per se or secondarily to the low tb .
most recently , the same authors showed that , at least in small species ( syrian and djungarian hamsters ) , tb during hibernation indeed controls leukopenia 6 .
the definition of hibernation as a temporary physiological state , characterized by a controlled lowering of the metabolic rate , as well as a dramatic drop in tb 1 , does not strictly apply to the brown bear .
instead , the bear has been classified as a hibernator based on the length of the torpid period ( 5 - 7 months in scandinavia ) 7 - 9 . however , in a recent study on black bears ( ursus americanus ) , it was shown that bears are true hibernators ; involving processes of both metabolic and thermal regulation 10 . whereas the bear 's body temperature remains relatively stable during hibernation with a less dramatic drop in comparison to other hibernators ( from 37c in summer to 33 - 30c in winter )
10 , 11 , the absolute metabolic rate in the bear is substantially reduced by 75% , and it takes several weeks after emergence for metabolic rate to return to normal levels 10 .
the slight tb lowering in hibernating black bears is uncoupled from metabolic suppression 10 , and , most recently , it was demonstrated that the expression of a number of genes involved in regulating the metabolism is adjusted during winter hibernation 12 . by this
, the bear offers a unique opportunity to further elucidate whether immune depression during hibernation is primarily caused by a lowered tb or might be intrinsic to torpor per se . in the present study , we examined immune cell counts in peripheral blood of 13 free - ranging scandinavian brown bears during hibernation and again during the active period in summer .
the results were compared to data from the literature available on different species on immune function during hibernation .
blood samples were collected from 13 free - ranging brown bears , ( 7 females , 6 males ; age 2 ( n=4 ) , 3 ( n=8 ) and 4 ( n=1 ) years old ) , during hibernation ( february / march 2010 or 2011 ) and again from the same bears during their active period in summer ( june 2010 or 2011 ) .
the bears were immobilized in the den during february / march with combination of tiletamine - zolazepam , medetomidine and ketamine and from a helicopter during june by darting with a combination of tiletamine - zolazepam and medetomidine 13 , 14 .
, bod , norway ) , with a manufacturer reported accuracy of 0.1c , was used .
all bears were clinically examined by a wildlife veterinarian , and all animals were apparently healthy with no signs of infection .
none of the bears were pregnant , and since bears do not have menstruation or any other bleeding during the ovulatory cycle , we did not exclude female bears .
the study was approved by the swedish ethical committee on animal research ( c212/9 ) .
white blood cell ( wbc ) count and the number of different leukocytes were determined at the accredited clinical chemical laboratory at rebro university hospital , sweden , by a fully automated hematology analyzer ( xe-5000 , sysmex corporation , kobe , japan ) .
blood cells were differentiated by the instruments using standardized  wbc differential analysis settings  by applying a combination of forward scatter , side scatter and fluorescence of nucleic acid material using highly specific polymethine dyes .
the leukocyte differential counts were confirmed manually , by the same lab technician for all samples , with a microscope at the haematology and chemistry laboratory , university animal hospital , swedish university of agricultural sciences , uppsala , sweden .
a web of science search was performed to find studies on hibernators in which both white blood cell counts and tb data were available .
this search identified ten studies , including 9 different species6 , 16 - 22 ; european hamster ( cricetus cricetus ) , arctic ground squirrel ( urocitellus parryii ) , woodchuck ( marmota monax ) , 13-lined ground squirrels ( ictidomys tridecemlineatus ) , syrian hamster ( mesocricetus auratus ) , european ground squirrel ( spermophilus cirellus ) , northern leopard frog ( rana pipiens ) , painted turtle ( chrysemys picta ) and grass snake ( natrix natrix ) . for the later study ,
as the grass snake is an ectotherm , we made a search on meteorological websites .
the average temperature of grass snakes , corresponding to the month the study was conducted , was assumed as tb . including the present study , 10 species were included in the comparative analysis .
the effects of hibernation on innate immune cells were analyzed by using mixed linear models with time as the repeated measure ( hibernation vs. active ) , and bears and year of capture as random effects .
further analyses were performed by adjusting on sex , age and body masses to check for possible confounding factors on cell counts . the relationship between leukopenia ( expressed as a percentage of the active period ) and the drop in tb during hibernation
two - sided p - values are reported and significance was set to < 0.05 ( spps version 16 ) .
blood samples were collected from 13 free - ranging brown bears , ( 7 females , 6 males ; age 2 ( n=4 ) , 3 ( n=8 ) and 4 ( n=1 ) years old ) , during hibernation ( february / march 2010 or 2011 ) and again from the same bears during their active period in summer ( june 2010 or 2011 ) .
the bears were immobilized in the den during february / march with combination of tiletamine - zolazepam , medetomidine and ketamine and from a helicopter during june by darting with a combination of tiletamine - zolazepam and medetomidine 13 , 14 .
, bod , norway ) , with a manufacturer reported accuracy of 0.1c , was used .
all bears were clinically examined by a wildlife veterinarian , and all animals were apparently healthy with no signs of infection .
none of the bears were pregnant , and since bears do not have menstruation or any other bleeding during the ovulatory cycle , we did not exclude female bears .
the study was approved by the swedish ethical committee on animal research ( c212/9 ) .
white blood cell ( wbc ) count and the number of different leukocytes were determined at the accredited clinical chemical laboratory at rebro university hospital , sweden , by a fully automated hematology analyzer ( xe-5000 , sysmex corporation , kobe , japan ) .
blood cells were differentiated by the instruments using standardized  wbc differential analysis settings  by applying a combination of forward scatter , side scatter and fluorescence of nucleic acid material using highly specific polymethine dyes .
the leukocyte differential counts were confirmed manually , by the same lab technician for all samples , with a microscope at the haematology and chemistry laboratory , university animal hospital , swedish university of agricultural sciences , uppsala , sweden .
a web of science search was performed to find studies on hibernators in which both white blood cell counts and tb data were available .
this search identified ten studies , including 9 different species6 , 16 - 22 ; european hamster ( cricetus cricetus ) , arctic ground squirrel ( urocitellus parryii ) , woodchuck ( marmota monax ) , 13-lined ground squirrels ( ictidomys tridecemlineatus ) , syrian hamster ( mesocricetus auratus ) , european ground squirrel ( spermophilus cirellus ) , northern leopard frog ( rana pipiens ) , painted turtle ( chrysemys picta ) and grass snake ( natrix natrix ) .
for the later study , no data on tb was given in the reference 22 .
as the grass snake is an ectotherm , we made a search on meteorological websites .
the average temperature of grass snakes , corresponding to the month the study was conducted , was assumed as tb . including the present study , 10 species were included in the comparative analysis .
the effects of hibernation on innate immune cells were analyzed by using mixed linear models with time as the repeated measure ( hibernation vs. active ) , and bears and year of capture as random effects .
further analyses were performed by adjusting on sex , age and body masses to check for possible confounding factors on cell counts .
the relationship between leukopenia ( expressed as a percentage of the active period ) and the drop in tb during hibernation was checked by simple regression analysis .
two - sided p - values are reported and significance was set to < 0.05 ( spps version 16 ) .
blood samples were collected from all 13 bears during hibernation and again in summer . during winter , the rectal temperature , recorded with a digital thermometer , was 33.5  1.1 ( 32.2 - 35.4 ) c and during summer 39.6  0.8 ( 38.8 - 40.9 ) c ( figure 1a ) .
rectal temperatures seen during summer captures were typical for bears immobilized during the active period 23 .
bears had significantly lower counts of peripheral blood leukocytes during hibernation compared to the active period ( 4.5  1.1 vs. 7.4  2.4 x10/l , p = 0.001 ; figure 1b ) .
these data are in agreement with a previous study showing lower leukocyte counts in captive brown bears during denning compared to non - hibernating animals 11 . in addition
, we found that the bears had significantly fewer neutrophils ( 3.0  0.7 vs. 5.7  2.4 x10/l , p = 0.001 ; figure 1c ) and monocytes ( 0.35  0.08 vs. 0.61  0.4 , p = 0.039 ; figure 1d ) during hibernation .
these findings are consistent with the results for small hibernating animals 5 . on the other hand
, we could not detect any change in lymphocyte counts between hibernation and active periods ( 1.1  0.4 vs. 1.1  0.4 x10/l , p = 0.599 ; data not shown ) .
in one of the bears , wbc was 21.9 x10/l during hibernation , which is 4 - 5 times higher compared to the other bears ( figure 1 ) .
neutrophils , monocytes and lymphocytes were slightly elevated ( 4.3 , 1.1 and 2.0 x 10/l , respectively ) compared to the other bears ( figure 1 ) . however , the number of eosinophils was extremely high in this bear compared to the other bears ( 14.9 vs. 0.22  0.11 x10/l ) .
c - reactive protein ( crp ) was not elevated ( 0.085 vs. 0.046  0.04 mg /
the leukocyte count of this bear was similar to the other bears when examined in the summer .
still , based on the haematology analyses , this bear was excluded from the study .
our data support the view that the innate immune system is suppressed during hibernation . as discussed by bouma et al . 5
, this could affect the defence towards infections , as illustrated by the massive death of hibernating bats caused by a fungus 24 that has an optimal growth temperature of 12.5 - 15.8c but grows well at 3 - 6c 25 .
interestingly , it has been suggested that one reason why animals arouse periodically from torpor is to reactivate a dormant immune system to combat pathogens that enter the body during the torpor bouts 26 .
although arousals in bears show very different patterns as compared to small - bodied hibernators 10 , further investigation is required to find out whether or not hibernating bears exhibit increased cell counts upon arousal from torpor .
in contrast to the extreme decrease in peripheral blood lymphocytes in small hibernating animals 5 , 6 , our data suggest that cells of the adaptive immune system are not affected in bears during hibernation
. however , given that many acquired immune responses are stimulated by innate cells , there may still be an effect on the functioning of this immunological aspect . during arousal bouts in small hibernating animals
, there is a rapid ( within a few hours ) reappearance of retained lymphocytes probably from the gut and spleen to the blood 6 , 27 , 28 .
hibernating black bears do not show spontaneous arousals to normothermic levels of tb 10 , as do small hibernators .
instead , they change position on a daily basis and display multiday cycles of tb between 30 - 36c 10 .
still , bears can be aroused relatively easily , and one could speculate that the short awaken period before we anesthetized the bears could have influenced the number of lymphocytes . however , this period of time was very short ( approx .
15 min ) , and , in addition , the number of circulation lymphocytes during arousal found in small hibernators is still only half of the number found in active animals during summer 29 , suggesting that this was not a major factor influencing our results .
another explanation of depressed neutrophils and monocytes during torpor could be that longer - lived lymphocytes would remain in circulation longer than the short - lived neutrophils .
on the other hand , this explanation requires a decreased rate of granulopoiesis or a shorter life span of granulocytes .
if bear leukocytes are sequestered in peripheral tissue during hibernation as found in small hibernators 6 , 27 , 28 , this phenomenon might explain why monocytes are depressed with torpor due to their ability to leave the circulation .
also , further studies should investigate the hibernating effect of lymphocytes in sub - groups to see if there are different patterns between , for instance , b- and t - lymphocytes , and if this could explain the numbers of lymphocytes found in hibernating vs. active bears .
although the length of time and body temperature history during hibernation could play a role in determining the leukocyte profile at the particular time of sampling , this is unlikely based on work done in ground squirrels 19 that found that the numbers of leukocytes decreased by 90% within 24 hours of torpor and remained unchanged during the remainder of the torpor - period .
although this indicates that the length of time hibernating prior to sampling may not have an impact on the results , further studies including the temperature history ( using biologging techniques ) would be necessary to determine if these results in ground squirrels apply to the brown bear .
our study thus supports the concept that hibernation has significant effects on the immune system . whether or not the hibernation - induced leukopenia is a general phenomenon of hibernation , independently of body mass , resulting from a direct body temperature effect , is an important question from an evolutionary point of view .
in small hibernators , the number of circulating white blood cells drops dramatically when the body temperature decreases to 5c , and it was thus recently argued that the hibernation induced immune depression is a temperature - dependent mechanism 6 .
even more recently , metabolic suppression in bears during hibernation was suggested to be independent of tb 10 ; indicating that processes of hibernation may occur in a temperature - independent matter .
the direct corollary is to question whether the hibernation - induced leukopenia is independent of tb when body size is large or whether the temperature effect is a general feature of hibernation .
the between - species - comparison we performed shows that , across taxa , the depression of the immune function appears indeed as a direct function of the temperature reached during torpor ( figure 2 ) .
the intercept is slightly off zero , indicating that we can not rule out the contribution of other mechanisms .
however , given the strength of the relationship with tb explaining 83% of the relationship , it is likely that any alternative mechanisms are of modest importance , but further studies on large animals , such as different bear species , are needed .
taken together , our results suggest that the drop in tb , reached during the torpor bout , is the main drive of the process of leukopenia observed during hibernation and this may be a general mechanism in hibernators . we think that similar approaches are needed to investigate the relationship between metabolic depression and hypothermia .
indeed , we find it hard to explain how such an independency is possible given the first law of thermodynamics .
we rather think that further studies are clearly needed to look at regional / organ body temperature regulations . in conclusion
the present study supports the hypothesis that the reduced number of white blood cells during hibernation is a temperature - dependent phenomenon conserved across evolution .
future studies are needed to understand the mechanisms regulating the innate immune system during hibernation .
. one explanation could be that the suppression in immune function , as a function of body temperature , is proportional to the decrease in virulence of any pathogens present in the hibernator . whether this is due to energy benefits of a reduced immune function 6 or other mechanisms
, our findings of a hibernation - associated drop in white blood cells in brown bears could inspire research in human pathophysiological states and increase the understanding of hibernation physiology in general and aspects of ecological immunology .
high numbers of neutrophils are associated with disorders , including ischemic heart disease 30 , complications of metabolic syndrome 31 and hypertension 32 .
patients with coronary artery disease display dysregulated neutrophils 33 , 34 , and activated neutrophils play a role in morbid obesity 35 , rheumatoid arthritis 36 and bronchial asthma 37 .
if the regulatory mechanisms behind white blood cell lowering in bears could be worked out , this might have therapeutic potential .
understanding the mechanisms of specific physiological alterations involved in hibernation is of relevance for pharmacologically induced suspended animation - a therapeutic option in patients with acute severe cerebral or cardiovascular disease 6 .
acquisition of data : sahdo , evans , blanc , frbert . analysis and interpretation of data : sahdo , evans , arnemo , blanc , frbert , srndahl .
preparation of manuscript : sahdo , evans , arnemo , blanc , frbert , srndahl .
all authors were involved in revising the manuscript for important intellectual content and approved the final version .
 background : hibernation involves periods of severely depressed metabolism ( torpor ) and decreases in body temperature ( tb ) . small arctic mammals ( < 5 kg ) , in which tb generally drop drastically , display leukopenia during hibernation . 
 this raised the question of whether the decreased leukocyte counts in mammalian hibernators is due to torpor per se or is secondary to low tb . 
 the present study examined immune cell counts in brown bears ( ursus arctos ) , where torpor is only associated with shallow decreases in tb . 
 the results were compared across hibernator species for which immune and tb data were available.methods and results : the white blood cell counts were determined by flow cytometry in 13 bears captured in the field both during summer and winter over 2 years time . 
 tb dropped from 39.60.8 to 33.51.1c during hibernation . 
 blood neutrophils and monocytes were lower during hibernation than during the active period ( 47% , p= 0.001 ; 43% , p=0.039 , respectively ) , whereas no change in lymphocyte counts was detected ( p=0.599 ) . 
 further , combining our data and those from 10 studies on 9 hibernating species suggested that the decline in tb explained the decrease in innate immune cells ( r2=0.83 , p<0.0001).conclusions : bears have fewer innate immune cells in circulation during hibernation , which may represent a suppressed innate immune system . across species comparison 
 suggests that , both in small and large hibernators , tb is the main driver of immune function regulation during winter dormancy . 
 the lack of a difference in lymphocyte counts in this context requires further investigations . 
Introduction
Materials & Methods
Study subjects
Routine blood cell count
Comparative approach
Statistical analysis
Results & Discussion
Author Contributions