melatonin , the main product of the pineal gland , is found in high concentrations in other body fluids and tissues [ 1 , 2 ] and possesses anti - inflammatory and antioxidant actions [ 36 ] .
we have evaluated the prophylactic   as well as the therapeutic   effect of melatonin in heatstroke rats under general anesthesia and showed the therapeutic effects of melatonin on heatstroke - induced multiple organ dysfunction syndrome . according to a more recent review , the ischemic and oxidative damage to the hypothalamus during heatstroke may cause multiple organ dysfunction or failure through hypothalamic - pituitary - adrenal ( hpa ) axis mechanisms .
studies are warranted to know whether the heatstroke - induced brain ( or hypothalamic ) inflammation and damage , thermoregulatory deficits , and multiple organ dysfunction can be affected by melatonin therapy in an unanesthetized and unrestrained mouse model [ 1012 ] . to deal with the hypothesis
, we assessed the temporal profiles of cellular markers of ischemia ( e.g. , glutamate and lactate / pyruvate ratio ) , damage ( e.g. , glycerol ) , inflammation ( e.g. , tumor necrosis factor - alpha ( tnf- ) , interleukin-1 ( il-1 ) , il-10 , and myeloperoxidase ( mpo ) activity ) , and oxidative damage ( e.g. , prooxidant enzymes ( e.g. , lipid peroxidation and glutathione oxidation ) , anti - oxidant defenses ( e.g. , glutathione peroxidase ( gpx ) , and glutathione reductase ( gr ) , oxidant radicals , nitric oxide metabolites ( nox ) , and dihydroxybenzoic acid ( dhba ) ) in the hypothalamus that occurred after heat regimen in mice treated with or without melatonin therapy . in addition , the influence of melatonin therapy on the heatstroke - induced thermoregulatory deficits as well as increased plasma levels of multiple organ dysfunction or failure [ 1012 ] was assessed .
present studies were performed in male icr mice ( 2937  g ) , whose stock originated from the institute of cancer research of the national institutes of health in the usa .
they were purchased from the national animal center ( taipei , taiwan ) and kept under a 12-hour light - dark cycle at controlled temperature ( 21  2c ) with free access to food and tap water .
institute of cancer research male mice 8 to 10 weeks old were exposed to whole body heating ( wbh ; 41.2c ; relative humidity 50%55% ; 1  h ) in an environment - controlled chamber [ 1012 ] .
the heat - stressed mice were returned to the normal room temperature ( 26c ) after the end of the heat exposure .
mice that survived to day 4 of heat treatment were considered survivors , and the data were used for analysis of the results .
core temperatures were measured every 1  min with a copper constant thermocouple inserted into the rectum and connected to a thermometer ( hr1300 ; yokogawa , tokyo , japan ) . before the start of thermal experiments ,
mice were housed at an ambient temperature ( 26c ) below the neutral zone for this species .
after 1-hour heating period , animals were properly fed and hydrated . in separate experiments ,
4  h post - wbh , animals were sacrificed and their brains and blood were obtained for biochemical verification [ 1012 ] .
three hundred mice were randomly divided into 3 major groups : ( a ) nonheated mice treated with vehicle solution  ( n = 60 ) : these groups of animals were s.c . injected with one dose of 0.2  ml of 0.25% ethanol - saline   immediately post - wbh ; ( b ) heated mice treated with vehicle solution  ( n = 60 ) ; and ( c ) heated mice treated with melatonin ( n - acetyl-5-methoxytryptamine ) ( 0.2  mg / kg , 1  mg / kg , or 5  mg / kg )   immediately post - wbh  ( n = 180 ) . in experiment  1 , effects of heat exposure on body core temperature and % survival of different groups of mice were assessed  ( n = 60 ) . in experiment  2 ,
effects of heat exposure on cellular ischemia and damage markers in brain ( or hypothalamus ) of different groups of mice were measured  ( n = 60 ) . in experiment  3 ,
effects of heat exposure on inflammatory mediators in brain ( or hypothalamus ) of different groups of mice were measured  ( n = 60 ) . in experiment  4 ,
effects of heat exposure on oxidative stress markers in brain ( or hypothalamus ) of different groups of mice were measured  ( n = 60 ) . in experiment  5 ,
effects of heat exposure on serum levels of multiple organ dysfunction indicators , acth , and corticosterone of different groups were measured  ( n = 60 ) .
hypothalamic samples were homogenized in 0.05  m phosphate buffer at ph7.0 and then centrifuged at 4000  g for 20  min at 4c .
the supernatants were used for the determination of cellular levels of glutamate , lactate - to - pyruvate ratio , glycerol , nitric oxide , and hydroxyl radicals . the dialysis probe ( 4  nm in length c cma/12 ; carnegie medicine , stockholm , sweden )
was put into the supernatants to obtain the dialysates . the nitric oxide ( nox )
concentration in the dialysates of hypothalamus was measured with the eicom eno-20  nox  analysis system ( eicom , kyoto ) . in the eicom eno-20  nox  analysis system , after the  no2  and  no3  in the sample have been separated by the column , the  no2  reacts in the acidic solution with the primary aromatic amine to produce an azo compound . following this , the addition of aromatic amines to the azo compound results in a coupling that produces a diazo compound , and the absorbance rate of the red color in this compound
lactate - to - pyruvate ratio , and glycerol , the dialysates were injected onto a cma600 microdialysis analyzer ( carnegie medicine , stockholm , sweden ) .
the concentrations of hydroxyl radicals were measured by a modified procedure based on the hydroxylation of sodium salicylates by hydroxyl radicals , leading to the production of 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid .
lipid peroxidation was assessed by measuring the levels of malondialdehyde ( mda ) with 2-thiobarbituric acid ( tba ) to form a chromophore absorbing at 532  nm .
about 0.1  g of tissue was homogenized with 1.5  ml of 0.1  m phosphate buffer at ph3.5 .
the reaction mixture ( 0.2  ml of sample , 1.5  ml of 20% acetic acid , 0.2  ml of 8.1% sodium dodecyl sulfate , and 1.5  ml of aqueous solution of 0.8% tba , up to 4  ml with distilled water ) was heated to 95c for 1  h , and then 5  ml of n - butanol and pyridine ( 15  :  1  vol / vol ) was added .
the mixture was vortexed vigorously and centrifuged at 1500  g for 10  min , and the absorbance of the organic phase was measured at 532  nm .
the values were expressed as nanomoles of tba - reactive substances ( mda equivalent ) per milligram of protein .
tissues were homogenized in 5% 5-sulfosalicylic acid ( 1  :  10  wt / vol ) at 0c , and the supernatants were used for analysis of total and oxidized glutathione .
total glutathione ( reduced - form glutathione ( gsh ) + oxidized - form glutathione ( gssg ) ) was analyzed according to the tietze method , and gssg was determined as described by griffith .
the recycling assay for total glutathione is oxidized by 5,5-dithiobis ( 2 acid ) ( dtnb ) to give gssg with stoichiometric formation of 5-thio-2-nitrobenzoic acid .
gssg is reduced to gsh by the action of the highly specific glutathione reductase ( gr ) and nicotinamide adenine dinucleotide phosphate ( reduced form ; nadph ) . the rate of 5-thio-2-nitrobenzoic acid formation is followed at 412  nm and is proportional to the sum of gsh and gssg present .
tissues were homogenized in 0.05  m phosphate buffer , ph7.0 and then centrifuged at 4000  g for 20  min at 4c .
the gpx and gr activities were performed with a commercial gpx assay kit ( sigma , usa ) and a gp assay kit ( sigma , usa ) , respectively .
one unit of gpx and gr activity was defined as the amount of sample required to oxidize 1  mmol of nadph per minute based on the molecular absorbance of 6.22  10 for nadph .
mpo activity , an indicator of polymorphonuclear leukocyte accumulation , was determined in the hypothalamus as described previously   at 4 hours after heat stress .
mpo activity was defined as the quantity of enzyme degrading 1  mol of peroxide / min at 37c and was expressed in milliunits / gram of wet tissue .
the homogenates were incubated on ice for 30  min and then centrifuged ( 15,000  g , 30  min , 4c ) twice . the concentrations of these cytokines in the supernatants were determined by commercially available elisa kits ( r & d systems , minneapolis , mn , usa ) according to the manufacturer 's instructions .
optical densities were read on a plate reader set at 450  nm for these cytokines .
the concentrations of these cytokines in the samples were calculated from the standard curve multiplied by the dilution factor and were expressed as pg / g .
creatinine , blood urea nitrogen ( bun ) , alanine aminotransferase ( alt ) , aspartate aminotransferase ( ast ) , and alkaline phosphatase ( alp ) were estimated in blood samples collected 4 hours after the start of heat stress or the equivalent time point for the nonheated animals .
the serum levels of creatinine , bun , alt , ast , and alp were determined by spectrophotometry ( hitachi 7600 , tokyo , japan ) .
plasma corticosterone and acth were assessed using corticosterone double antibody ria kit ( mp biomedicals , solon , oh , usa ) and acth ( rat , mouse)-ria kit ( phoenix pharmaceuticals , burlingame , ca , usa ) , respectively .
all values in the tables and text are expressed as mean  s.e.m . of n observations , where n represents the number of animals studied .
statistical evaluation was performed by using analysis of variance ( anova ) followed by a multiple - comparison test ( scheffe 's test ) .
a  p  value of less than 0.05 was considered to be statistically significant .
as summarized in table 1 , the body core temperature values of heated mice were significantly lower than those of nonheated mice kept at a normal ambient temperature ( 26c ) ( 33.2  0.2c and 37.2  0.3c , resp . )
additionally , the fraction survival of heated mice was significantly lower than those of nonheated mice ( 1/12 and 12/12 , resp . )
the heat - induced hypothermia and lethality were significantly reduced by melatonin therapy ( table 1 and figure 1 ) . 

table 2 summarizes the effects of heat exposure on cellular levels of glutamate , lactate - to - pyruvate ratio , glycerol , nitrite , and dhba in the hypothalamus in different groups of mice . as compared with nonheated mice , the heated mice had higher levels of glutamate , lactate - to - pyruvate ratio , glycerol , nitrite , and dhba in the hypothalamus ( p < 0.05 ) .
the heat - induced increased levels of glutamate , lactate - to - pyruvate ratio , glycerol , nitrite , and dhba in the hypothalamus were all significantly and dose - dependently reduced by melatonin ( 0.25.0  mg / kg ) . as shown in table 3 , hypothalamic levels of tnf- , il-1 , and mpo
again , heat - induced overproduction of tnf- , il-1 , and mpo was significantly and dose - dependently reduced by melatonin therapy ( 0.25.0  mg / kg ) ( p < 0.05 ) .
in contrast , hypothalamic levels of il-10 were significantly and dose - dependently increased by melatonin treatment in heated mice . as shown in table 4 ,
the hypothalamic levels of both mda and gssg / gsh in heated mice were significantly higher than those of nonheated mice .
in contrast , the hypothalamic levels of gp , gr of heated mice were significantly higher than those of nonheated mice ( p < 0.05 ) .
the heat - induced increased levels of mda and gssg / gsh as well as the decreased levels of gp , gr in the hypothalamus were significantly and dose - dependently reduced by melatonin therapy ( 0.25.0  mg / kg ) ( p < 0.05 ) .
the plasma levels of bun , creatinine , alt , ast , and ap in heated mice were all significantly higher than those of nonheated mice ( table 5 ) .
the heat - induced increased plasma levels of these parameters were all significantly and dose - dependently reduced by melatonin treatment ( 0.25.0  mg / kg ) ( p < 0.05 ) . as shown in table 6 ,
the plasma levels of both acth and corticosterone in heated mice were significantly higher than those of nonheated controls ( p < 0.05 ) .
the heat - induced increased plasma levels of both acth and corticosterone were all significantly enhanced by melatonin therapy ( p < 0.05 ; table 6 ) .
according to chatterjee et al . [ 10 , 11 ] , heat - treated mice display body core temperatures of > 40c immediately after the termination of wbh and profound hypothermia at + 4 , + 6 , and + 20  h after .
we further demonstrate that heat - induced hypothermia in mice can be significantly and dose - dependently prevented by melatonin therapy .
however , the contention is not consistent with the findings of leon et al . , who reported on mice exposed to an ambient temperature of ~39.5c until a maximal core temperature of 42.7c was attained . during their recovery ,
the mice had hypothermia ( 29.3c ) and , after 24  h of recovery , a fever - like elevation ( 37.8c ) .
the hypothalamus is believed to be involved in regulating homeostasis , motivation , and emotional behavior ; these functions are mediated through hypothalamic control of autonomic and endocrine activity .
the hypothalamus allows the output of pituitary hormones to response to changes in the autonomic nervous system activity and to the needs of temperature regulation , water balance , and energy requirements .
the hypothalamo - pituitary - adrenocortical ( hpa ) axis is also mobilized , as suggested by the increase in c - fos - positive cells   and c - fos mrna content   in the hypothalamic paraventricular nucleus , as well as the increase in blood adrenocorticotrophic - hormone ( acth ) and corticosterone concentrations [ 27 , 28 ] . decreased heat tolerance has been associated with hpa axis impairment .
more than half a century ago , thermal injury to the thermoregulatory centers of the hypothalamus was hypothesized to be the primary mechanism of mortality .
indeed , according to a more recent review , ischemic and oxidative damage to the hypothalamus may be responsible for heatstroke .
severe heat stress also decreases mean arterial pressure , increases intracranial pressure , and decreases cerebral perfusion pressure , all of which lead to cerebral ischemia and hypoxia . compared with normothermic controls , rodents with heatstroke have higher values of cellular ischemia ( e.g. , glutamate and lactate - to - pyruvate ratio ) and damage ( e.g. , glycerol ) markers , prooxidant enzymes ( e.g. , lipid peroxidation and glutathione oxidation markers ) , proinflammatory cytokines ( e.g. , interleukin-1 and tumor necrosis factor- ) , inducible nitric oxide - synthase - dependent nitric oxide and an indicator for the accumulation of polymorphonuclear leukocytes ( e.g. , myeloperoxidase activity ) as well as neuronal damage ( e.g. , apoptosis and necrosis ) in the hypothalamus after heat stroke .
hypothalamic values of antioxidant defenses ( e.g. , glutathione peroxidase and glutathione reductase ) , however , are lower .
melatonin therapy , in addition to attenuating heat - induced hypothermia ( thermoregulatory deficits ) , significantly attenuates heat - induced inflammatory , ischemic and oxidative damage to the hypothalamus .
furthermore , our present results demonstrated that melatonin therapy significantly enhanced hpa axis mechanisms ( as reflected by increased plasma levels of both acth and corticosterone in response to heat stress ) , led to reduction of multiple organ dysfunction or failure ( as reflected by decreased plasma levels of bun , creatinine , alt , ast , and alp ) , and resulted in attenuation of lethality in heatstroke mice .
, melatonin improved the clinical outcome of the septic newborns as judged by measurement of sepsis - related serum parameters via reducing the serum levels of lipid peroxidation products .
a major brain metabolite of melatonin acts as a potent nitric oxide scavenger , inhibitor , and/or downregulator of neuronal and inducible nitric oxide synthase and as a mitochondrial metabolism modulator .
melatonin protected against mitochondrial reactive oxygen species - mediated apoptosis in astrocytes   and isoproterenol - induced myocardial injury in the rat via its antioxidative mechanism .
melatonin also may attenuate peritonitis - induced lethality in conscious rats by exerting its antioxidant effect .
these observations prompted us to think that these multiple mitochondrial layers of protection provided by melatonin may be crucial for future therapeutic prevention and treatment of heatstroke .
systemic inflammatory response syndrome is characterized by increased serum levels of tnf- , icam-1 , e - selectin , il-1 , and il-6 [ 3740 ] .
increased levels of these systemic inflammatory response syndrome molecules have also been shown in patients   or rats .
in addition , the increased serum levels of these molecules during heatstroke in an anesthetized rat model could be reduced by melatonin treatment .
the present results further showed that the increased levels of some of these molecules in the hypothalamus in a mice could also be reduced by melatonin .
additionally , melatonin therapy increased the hypothalamic levels of il-10 in heatstroke , which was believed to be anti - inflammatory cytokine .
these results indicate that melatonin may reduce heat - induced activated inflammation by reducing the levels of these systemic inflammatory response molecules in the hypothalamus .
it is known that melatonin intensifies the expression of protective heat shock proteins   and that it enhances heat shock protein 27 expression   as well as having anti - inflammatory properties and antioxidant properties .
in summary , we report here that when untreated mice underwent heat stress , they displayed thermoregulatory deficit ( e.g. , animals display hypothermia during room temperature exposure ) , brain ( or hypothalamic ) inflammation , ischemia , and oxidative damage , hpa axis impairment , multiple organ dysfunction or failure , and lethality . melatonin therapy may improve outcomes of heatstroke in mice by reducing brain inflammation and oxidative damage , and multiple organ dysfunction .
 we report here that when untreated mice underwent heat stress , they displayed thermoregulatory deficit ( e.g. , animals display hypothermia during room temperature exposure ) , brain ( or hypothalamic ) inflammation , ischemia , oxidative damage , hypothalamic - pituitary - adrenal axis impairment ( e.g. , decreased plasma levels of both adrenocorticotrophic hormone and corticosterone during heat stress ) , multiple organ dysfunction or failure , and lethality . 
 melatonin therapy significantly reduced the thermoregulatory deficit , brain inflammation , ischemia , oxidative damage , hypothalamic - pituitary - adrenal axis impairment , multiple organ dysfunction , and lethality caused by heat stroke . 
 our data indicate that melatonin may improve outcomes of heat stroke by reducing brain inflammation , oxidative damage , and multiple organ dysfunction . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion