animals : four beagle dogs ( 2 males and 2 females , 2 
 years old , weighing 9.9 to 11.3 kg ) were included in this study .
the dogs had 
 no evidence of disease based on their histories and a clinical examination in which a blood 
 sample had been taken as part of a routine health check .
they were accustomed to being 
 restrained , having blood samples drawn and taking medications .
they were housed in individual cages , in 
 which the temperature was maintained at 23  1c and kept under a 12:12-hr light / dark cycle . 

the dogs were fed twice a day , at 10:00 and 16:00 , and water was available freely .
the 
 protocols in this study were approved by the animal care and use committee of osaka 
 prefecture university .
blood sampling catheter placement : in order to precisely obtain blood 
 samples , we placed catheters in the carotid arteries and jugular veins of the dogs .
seven 
 days before the start of the study protocol , all dogs were preanesthetized with 0.05 mg / kg 
 atropine sulfate hydrate subcutaneously , 0.25 mg / kg diazepam intravenously and 0.1 mg / kg 
 butorphanol tartrate intravenously .
the dogs were then injected with propofol intravenously 
 and anesthetized with isoflurane at a 2.0% concentration in expiration via an endotracheal 
 tube .
the depth of anesthesia was monitored according to the aaha anesthesia guidelines for 
 dogs and cats , and an adequate anesthesia level 
 was maintained for the operations .
all dogs were injected with 30 mg / kg cefazolin 
 subcutaneously as an antibiotic during the operations .
the catheter ( arrow central venous 
 catheterization set , single lumen , 18 ga  8  , 20 cm , teleflex medical japan 
 ltd . ,
tokyo , japan ) was placed in the left carotid artery and jugular vein according to a 
 general method .
the artery was incised transversely 
 with a number 11 scalpel blade and then dilated with a blunt instrument .
a catheter is 
 passed into the artery to the level of common carotid artery .
the proximal thread was 
 tightened around the catheter and then tied to the catheter to secure it .
the catheters were 
 flushed with saline containing 4.0 u / ml heparin sodium every 5 hr at night and during the day until they 
 were removed to prevent of embolism .
all dogs 
 were prescribed an antibiotic , 25 mg / kg cephalexin , to be taken orally twice a day from 
 catheter placement to 7 days after catheter removal . during the 7 
 days from catheter placement to the start of the study , the dogs were acclimated to the 
 catheters , the hyperbaric chamber and other apparatus used for this study .
study protocol : seven days after catheter placement , we studied the 4 dogs 
 in a crossover trial .
we did all studies during the morning to exclude the effects of daily 
 fluctuation .
( 1 ) when the dogs were quiet , we collected blood samples and measured 
 parameters .
( 2 ) next , the dogs ran on a treadmill ( corpo motor walker cp4000 , s. n. t. co. , 
 ltd . , kanazawa , japan ) at 2.0 m / sec for 10 min to induce physiological stress .
then , the dogs were placed 
 randomly ( 3 ) in a normal cage for 30 min ( non l - hbot ) or ( 4 ) in a hyperbaric chamber with 
 30% oxygen at 1.3 ata for 30 min ( l - hbot ) . after these treatments , we 
 collected blood samples and measured parameters again .
all dogs underwent treatment sets 
 consisting of ( 1 ) , ( 2 ) and ( 3 ) and ( 1 ) , ( 2 ) and ( 4 ) randomly . briefly
, a dog underwent one 
 treatment set and then underwent the other set 7 days later ( fig .
seven days after catheter placement , we studied the 4 dogs in a 
 crossover trial ( 1 ) when the dogs were quiet , ( 2 ) after running , ( 3 ) after non l - hbot 
 and ( 4 ) after l - hbot .
briefly , a dog underwent one study protocol and then underwent 
 the other 7 days later . ) .
all dogs were subjected to general blood tests at every trial to exclude anemia or 
 infections .
seven days after catheter placement , we studied the 4 dogs in a 
 crossover trial ( 1 ) when the dogs were quiet , ( 2 ) after running , ( 3 ) after non l - hbot 
 and ( 4 ) after l - hbot .
briefly , a dog underwent one study protocol and then underwent 
 the other 7 days later . hyperbaric oxygen chamber : we used a hyperbaric oxygen chamber for animals 
 ( o2 support-01 , liveaid , co. , ltd . , kanazawa , japan ) .
the chamber was clear and 
 had sufficient space for the dogs to walk around ( 870 mm length  780 mm width  790 mm 
 height ) .
air was instilled into the chamber through an air compressor , which condensed the 
 oxygen in the air to a 30% concentration using the pressure swing adsorption method .
this 
 method uses zeolite as an absorbent to absorb nitrogen and condense the oxygen in the air . 

the chamber was pressurized and maintained at approximately 1.3 ata . to depressurize the 
 chamber
, we cut off the power of the pressure device and exhausted the pressurized air . 

blood gas monitoring : pao2 , sao2 , ph and 
 paco2 were measured with a blood gas monitor ( i - stat 300f , fuso pharmaceutical 
 industries ltd . ,
approximately 0.5 ml arterial blood was 
 collected from the catheter and used for measuring pao2 and sao2 . 

cao2 was calculated using the following equation : cao2 
 ( ml / dl)=sao2 ( % ) /100
 hemoglobin 
 concentration in arterial blood ( g / dl )  1.39 + 0.0031  pao2 
 ( mmhg).the hemoglobin concentration was measured by using arterial blood 
 with a hemacytometer for animals ( poch-100iv diff , sysmex corporation , kobe , japan ) .
gpx activity : approximately 2.0 ml venous blood collected 
 via a catheter was placed into a tube containing sodium heparin and centrifuged at 1,500  
 g for 10 min at 4.0c . the plasma layer and buffy coat were removed to 
 obtain erythrocytes .
the erythrocytes were lysed in 4 volumes of ice - cold high - performance 
 liquid chromatography ( hplc ) grade water and centrifuged at 10,000  g for 
 15 min at 4.0c because hemoglobin absorbs significantly at 340 nm , and thus erythrocyte 
 lysates must be diluted before assaying .
the 
 supernatant was collected and frozen until analyzed for gpx activity using a commercial gpx 
 assay kit ( gpx assay kit , cayman chemical co. , ann arbor , mi , u.s.a . ) .
this kit measures gpx 
 activity using a coupled reaction with glutathione reductase ( gr ) .
oxidized glutathione 
 ( gssg ) , produced upon reduction of hydroperoxide by gpx , is recycled to its reduced state by 
 gr and nicotinamide adenine dinucleotide phosphate ( nadph).r - o - o - h + 2 
 glutathione   r - o - h + gssg + 
 h2ogssg + nadph + h 2 glutathione + nadp the oxidation of nadph to nadp is accompanied by a decrease in absorbance at 
 340 nm . under conditions in which the gpx activity is rate limiting , the rate of decrease in 
 the 340 is directly proportional to the gpx activity in the sample .
the gpx 
 activity assay range of this kit was between 50344 nmol / min / ml .
power spectral analysis of heart rate variability : lf / hf power values and 
 rri reflect autonomic nervous activity [ 1 , 7 , 40 ] .
a small 
 electrocardiograph ( digital quick corder qr2500 , fukuda m - e kogyo co. , ltd . ,
tokyo , japan ) , 
 suitable for small animals was used for electrocardiogram ( ecg ) recording .
electrodes were 
 taped to the chests of the dogs , and the dogs wore a vest with the electrocardiograph in its 
 pocket .
the data were collected every 24 hr , and 
 lf / hf and rri were calculated for every 5 min using an hs1000 lite holter analysis system 
 ( fukuda m - e kogyo co. , ltd . ) .
lf and hf power analysis was performed by power spectral analysis using the fast fourier 
 transform method .
briefly , 100 sec blocks of the date were resampled at 1.28 samples / sec and 
 subjected to a hamming window .
if there was a 
 run of arrhythmia or an artifact longer than 1 beat in length , the partial block was 
 discarded , and a new block was started as the first of the 100 sec blocks .
lf power was defined as the energy in the 
 power spectrum between 0.04 and 0.15 hz .
hf power was defined as the energy in the power 
 spectrum between 0.15 and 0.40 hz .
lf / hf was defined as the ratio of lf power to hf 
 power .
a linearly interpolated beat 
 was substituted to exclude intervals of ectopy or artifacts less than or equal to 2 rri 
 .
we used the following time points for the dogs : ( 1 ) quiet , a 5 min time point when the dogs 
 were in their cages before the start of the study ; ( 2 ) running , 5 min after running ; ( 3 ) non 
 l - hbot , 5 min after non l - hbot ; and ( 4 ) l - hbot , 5 min after l - hbot .
measurement of stress - related hormones : all stress - related hormone levels 
 were measured by an external facility ( japan clinical laboratories inc . ,
of 
 this , approximately 2.0 ml was placed into a tube with serum separating 
 medium and centrifuged at 1,000  g for 10 min at 4.0c .
the serum layer 
 was removed and frozen at 80c until analyzed for cortisol levels using an 
 electrochemiluminescence immunoassay .
briefly , samples were added to beads coated with an 
 anti - cortisol antibody , and then an antiprothrombotic antibody was added that labeled the 
 ruthenium complex .
this ruthenium complex emitted light as a result of an electrochemical 
 change . in this way , we could measure the level of cortisol in the samples .
the assay range 
 of cortisol was from 0.1 to 400 g / ml . the remaining 5.0 ml of collected blood was injected into a tube 
 containing sodium edta and centrifuged at 1,000  g for 10 min at 4.0c . 

the plasma layer was removed and frozen at 80c until analyzed for adrenaline and 
 noradrenaline levels using hplc .
briefly , the test material in the liquid mobile phase , 
 which was in contact with the stationary phase , was isolated by the gap of the affinity to 
 both phases .
the fraction isolated with the test material was identified and quantitatively 
 determined by its chromatogram acquired with a detector .
the assay ranges of both adrenaline 
 and noradrenaline were 6 to 10pg / ml . sc : sc was measured using a measuring instrument for dogs ( ps - imp001 , 
 liveaid co. , ltd . ) .
electrodes ( echorode iii , fukuda denshi co. , ltd . , tokyo , japan ) were 
 attached to the metacarpal pads , which contain sweat glands , and a pulse of 3.0 volts was 
 applied .
when sympathetic nervous activity increases , sweat glands produce more sweat , and 
 skin impedance decreases , resulting in increased sc .
the results were analyzed by parametric methods , 
 using statcel 3 ( oms publishing , tokorozawa , japan ) , and mean and standard deviation ( sd ) 
 values were reported .
differences for ( 1 ) quiet , ( 2 ) running , ( 3 ) non l - hbot and ( 4 ) l - hbot 
 in each of the measurement indicators were tested by analyses of variance and dunnett s 
 tests .
appearance of the dogs : each dog walked around and smelled the chamber for 
 the first 5 to 10 min of l - hbot or non l - hbot .
none of 
 the dogs showed any symptoms , such as seizures , salivation or vomiting , after l - hbot .
blood gas parameters : the hemoglobin concentration of the 4 dogs was 17.10 
  1.3 g / dl , almost within the reference range of 12.0 to 18.0 
 g / dl . none of the hemoglobin concentrations were significantly changed 
 after l - hbot ( the results are not shown ) . there were no significant differences in any blood 
 gas parameters among the dogs when they were quiet , after running , after non l - hbot or after 
 l - hbot .
pao2 and sao2 values were within the reference ranges 
 ( pao2 , 80.0 mmhg ; sao2 , 95 to 100% ) [ 11 , 51 ] .
the cao2 value 
 was within the range calculated by the equation stated above with the pao2 and 
 sao2 reference range ( 16.0 to 25.0 ml / dl ) . 

although ph was low ( when the dogs were quiet ) or almost normal ( after the running and 
 l - hbot conditions ) , hypocapnia was observed , except for after non l - hbot ( the reference 
 ranges : ph , 7.35 to 7.45 ; paco2 , 33.0 to 45.0 mmhg ) ( table 1table 1.blood gas parameters for the 4 treatmentstreatmentpao2(mmhg)sao2(%)cao2(ml / dl)phpaco2(mmhg)(1)86.0  4.394.0  4.221.9  0.77.25  0.2326.7  8.6(2)132  55.999.0  1.423.2  0.87.39  0.0524.9  8.9(3)91.5  16.396.5  0.726.5  0.17.35  0.0533.9  4.3(4)90.0  7.197.5  0.722.7  0.17.40  0.0023.6  5.1the
mean  sd values for pao2 , sao2 , cao2 , ph and 
 paco2 of arterial blood of the 4 dogs ( 1 ) when they were quiet , ( 2 ) after 
 running , ( 3 ) after non l - hbot and ( 4 ) after l - hbot . ) .
the mean  sd values for pao2 , sao2 , cao2 , ph and 
 paco2 of arterial blood of the 4 dogs ( 1 ) when they were quiet , ( 2 ) after 
 running , ( 3 ) after non l - hbot and ( 4 ) after l - hbot .
gpx activity : the gpx activity in erythrocytes of each dog when they were 
 quiet was 25.0  10.6 nmol / min / ml .
after running , it was 29.0  10.5 
 nmol / min / ml ; after non l - hbot , it was 37.2  17.6 
 nmol / min / ml ; and after l - hbot , it was 62.2  6.8 
 nmol / min / ml .
the mean gpx activity after l - hbot was about twice as high 
 as that when the dogs were quiet ( p<0.01 ) ( fig .
gpx activity in erythrocytes of the 4 dogs when 
 they were quiet , after running , after non l - hbot and after l - hbot .
the bars indicate 
 mean values , and the lines indicate the sd . * * significant difference vs quiet 
 ( p<0.01 ) . ) .
the gpx activity after running tended to decrease compared with that when the dogs 
 were quiet , but this was not statistically significant .
some of the gpx activity values were 
 out of the assay range , because lower values were measured in the erythrocyte samples due to 
 the interference of the absorbance of hemoglobin , as indicated in the resources provided 
 with the assay kit and a previous report .
gpx activity in erythrocytes of the 4 dogs when 
 they were quiet , after running , after non l - hbot and after l - hbot .
the bars indicate 
 mean values , and the lines indicate the sd . * * significant difference vs quiet 
 ( p<0.01 ) .
power spectral analysis of heart rate variability : one male dog was 
 excluded from the analysis , because he had removed the vest that contained the 
 electrocardiograph .
therefore , we analyzed the data of 3 dogs . the lf / hf value of each dog 
 when they were quiet was 0.69  0.32 .
after running , it was 0.87  0.43 ; after non l - hbot , 
 it was 0.58  0.18 ; and after l - hbot , it was 0.58  0.29 .
after running , it was 27.9  21.9 msec ; after non l - hbot , it was 26.5  
 13.8 msec ; and after l - hbot , it was 25.2  12.8 msec .
there were no significant differences 
 in the mean lf / hf and rri between the quiet , after running , after non l - hbot and after 
 l - hbot conditions ( fig . 3fig .
power 
 spectral analysis of heart rate variability of the 4 dogs when they were quiet , after 
 running , after non l - hbot and after l - hbot .
the bars 
 indicate mean values , and the lines indicate the sd . ) .
power 
 spectral analysis of heart rate variability of the 4 dogs when they were quiet , after 
 running , after non l - hbot and after l - hbot .
stress - related hormone levels : the cortisol level of each dog when they 
 were quiet was 7.1  1.9 g / dl . after running , it was 8.3 
  3.3 g / dl ; after non l - hbot , it was 3.9  2.1 
 g / dl ; and after l - hbot , it was 3.0  1.1 
 g / dl .
the adrenaline level when the dogs were quiet was 
 30.0  21.8 ng / ml . after running ,
it was 16.5  12.3 
 ng / ml ; after non l - hbot , it was 16.8  12.3 
 ng / ml ; and after l - hbot , it was 15.3  10.7 
 ng / ml . the noradrenaline level when the dogs were quiet 
 was 36.0  19.8 ng / ml .
after running , it was 29.8  25.7 
 ng / ml ; after non l - hbot , it was 27.3  21.8 
 ng / ml ; and after l - hbot , it was 25.0  13.8 
 ng / ml . the cortisol level after l - hbot was significantly 
 lower than that when the dogs were quiet ( p<0.05 ) .
there were no 
 significant differences in the mean adrenaline or noradrenaline among the treatments ( fig .
stress - related hormone levels in 
 blood of the 4 dogs when they were quiet , after running , after non l - hbot and after 
 l - hbot .
stress - related hormone levels in 
 blood of the 4 dogs when they were quiet , after running , after non l - hbot and after 
 l - hbot .
sc : the sc of each dog when they were quiet was 12.6  6.5% .
after 
 running , it was 47.8  40.8% ; after non l - hbot , it was 53.2  35.7% ; and after l - hbot , it 
 was 53.2  35.7% .
the mean sc was significantly higher after running 
 ( p<0.01 ) and after non l - hbot ( p<0.01 ) than when the 
 dogs were quiet ( fig . 5fig .
sc of the 4 dogs when they were quiet , after running , after 
 non l - hbot and after l - hbot .
there was no significant difference in mean sc between when the dogs were quiet and 
 after l - hbot .
sc of the 4 dogs when they were quiet , after running , after 
 non l - hbot and after l - hbot .
in the present study , blood gas parameters were not significantly changed after exercise or 
 l - hbot . a study that included healthy labrador retrievers indicated that immediately after 
 10 min of repeatedly retrieving a soft plastic tube thrown approximately 40 to 50 yards on 
 land , the pao2 of the dogs was significantly increased to 140.3  17.8 mmhg 
 .
that report suggested that hyperventilation 
 was induced response to increased oxygen demand after strenuous exercise and caused an 
 increase in pao2 . on the other hand
, a study with obese dogs indicated that the 
 blood oxygen saturation based on pulse oximetry of the dogs after walking for 6 min at their 
 own pace decreased significantly compared with that of dogs that participated in a weight 
 loss program .
as compared with these previous 
 studies , the exercise load might not have been large enough to change the blood gas 
 parameters in the present study .
the ph level increases , and paco2 decreases in 
 hyperventilation [ 9 , 28 , 30 ] . in the present study , although the 
 ph level and paco2 were low especially when the dogs were quiet , these two 
 parameters did not significantly change after l - hbot . from these results ,
l - hbot may be 
 useful in improving the oxygenation dynamics without inducing hyperventilation .
h - hbot sometimes induces hyperoxia , resulting in central nervous or pulmonary oxygen 
 toxicity .
a previous study showed that newborn 
 dogs that received h - hbot with 100% oxygen pressurized at 5.0 ata for about 40 min 
 experienced seizures .
this very high oxygen 
 concentration and pressure induces the oxidation of mitochondrial nicotinamide adenine 
 dinucleotide , which results in seizures . in the present study ,
we did not see seizures or 
 other neurological symptoms , and although the pao2 after l - hbot was lower than 
 the value we had expected , the dogs did not show hypoxia or any pulmonary oxygen toxicity 
 symptoms , such as chest pain or dry cough .
so , we considered that l - hbot may not induce 
 central nervous or pulmonary oxygen toxicity .
low pao2 after l - hbot may be caused 
 by problems related to measurement ; the sampling position or delay of measurements may 
 influence the values .
an important finding of the present study was that gpx activity increased after l - hbot 
 compared with after running in the dogs .
this result confirmed that the l - hbot could 
 increase gpx activity and reduce ros generated by stressful events . in a previous study in 
 rats , gpx activity increased in lung tissue and erythrocytes up to 30 min after h - hbot with 
 100% oxygen pressurized at 3.0 ata .
moreover , 
 another previous study recognized that gpx activity was higher in erythrocytes than in other 
 tissues in rats .
although only a few in 
 vivo studies have investigated gpx activities in dogs under hbot , we have shown 
 that gpx activity in erythrocytes increased at 30 min after l - hbot with 30% oxygen 
 pressurized at 1.3 ata in dogs . on the other hand , prolonged hbot ( 7 to 15 hbot sessions , 
 one session / day ) may induce increased levels of ros in the blood [ 6 , 34 ] .
it is thought that 
 activation of the redox - sensitive transcription factor , nuclear factor erythroid 2-related 
 factor 2 ( nrf2 ) , may play a pivotal role in the cellular defense against oxidative stress 
 via transcriptional upregulation of phase ii defense enzymes and antioxidant stress 
 proteins , such as gpx ; however , there have been only a few reports on this .
we considered from our results that the generation of 
 low levels of ros following a small increase in the supply of oxygen may activate nrf2 and 
 result in increased gpx activity .
further studies are required to determine the relationship 
 between l - hbot and the generation of ros , nrf2 and gpx .
we have previously shown that sc reflects the sympathetic nervous activity in dogs . in the present study , sc and the cortisol level 
 decreased after l - hbot compared with after running in the dogs , indicating that l - hbot may 
 control the sympathetic nervous activity in dogs . in professional divers ,
the heart rate and 
 lf / hf values decreased during h - hbot with 100% oxygen at 2.5 ata for 60 min .
this result suggests that h - hbot may control 
 sympathetic nervous activity and increase parasympathetic nervous activity .
increased 
 peripheral vessel resistance as a result of h - hbot may increase vagal efferent discharge . 

this would result in increased parasympathetic nervous activity and a decreased lf / hf value . 

the reactions of sc and stress - related hormones or the heart rate were verified in previous 
 studies in human beings ; however , the results varied . for example , sc and adrenaline levels 
 were correlated with perioperative stress , but the heart rate was not .
another study showed that both stress - related hormones and the heart 
 rate did not significantly reflect the stress level .
our previous study confirmed similar reactions for sc and stress - related 
 hormones in dogs during the perioperative period .
there are only a few reports available on sc , stress - related hormones and heart 
 rate during hbot in humans or other animals , so 
 further experiments are required to verify these relationships .
we studied only a small numbers of dogs , and all of them 
 were beagles .
l - hbot should be applied with caution concerning the respiration state in some 
 kinds of dogs , especially brachycephalic dogs in which respiratory diseases often occur 
 . moreover , increased red blood cells , 
 hemoglobin and hematocrit are observed in sight hound dogs . the oxygen circulation in these dogs is considered to be different from that 
 of the dogs included in this study .
more studies of l - hbot with more blood gas parameter 
 samples and other kinds of dogs are needed to create more adequate protocols and clarify the 
 safety . in the present study
l - hbot has low 
 influences on blood gas parameters in dogs . on the other hand
, the increased gpx activity 
 after l - hbot may bring new insights regarding oxidative stress mechanisms .
l - hbot may also change the oxidative stress 
 mechanism and autonomic nervous activity and therefore control body homeostasis .
 recently , hyperbaric oxygen therapy with a lower pressure and oxygen concentration 
 ( l - hbot ) than previous methods has been used for dogs in japan ; however , the influences of 
 l - hbot on dogs have not been clarified . to verify the influences of l - hbot on 
 physiological mechanism in dogs 
 , we investigated blood gas parameters , glutathione 
 peroxidase ( gpx ) activity , heart rate variability , stress - related hormones and skin 
 conductance ( sc ) in 4 clinically normal beagle dogs with catheters in their carotid 
 arteries and jugular veins when they were quiet , after running , after receiving l - hbot 
 ( 30% oxygen concentration , 1.3 atmospheres absolute , 30 min ) or after not receiving 
 l - hbot . 
 the results showed there were no changes in blood gas parameters , heart rate 
 variability and catecholamine levels after l - hbot . 
 gpx activity was significantly higher , 
 and the sc and cortisol level were lower in dogs that received l - hbot than those when they 
 were quiet . 
 these results suggested that l - hbot may have a small influence on oxygenation 
 dynamics , activate antioxidant enzymes such as gpx , restrain autonomic nervous activity 
 and control the balance between oxidation and antioxidation inside the body . 
MATERIALS AND METHODS
RESULTS
DISCUSSION