however , oxygen also poses a potential hazard via reactive oxygen species ( ros ) and reactive nitrogen species ( rns ) , with biological and functional alterations of lipids , proteins , and deoxyribonucleic acid ( dna ) [ 13 ] .
therefore , ros / rns have been initially considered as harmful products of the normal aerobic metabolism .
the control of ros / rns production plays physiological roles , especially , in regulating cell signaling to involve cell proliferation , differentiation , and apoptosis [ 13 ] .
oxidative stress ( os ) mediated by free radicals is defined as an imbalance between the production of ros / rns and the antioxidant capacity of the cell [ 13 ] .
the predominant inhibitory neurotransmitter in the brain is -aminobutyric acid ( gaba ) , and almost all researchers have focused on gaba or the regulation of gaba receptor ( gabar ) in the brain [ 48 ] .
currently , gaba is considered to be a multifunctional molecule with various physiological effects throughout the body [ 9 , 10 ] . in the brain , many researchers have found that the regulation of gabar has preventive effects against os - induced damage [ 5 , 7 , 8 ] .
these results in the brain were mainly explained via specific pathways against os ( i.e. , inhibition of the response to dna damage [ 5 , 11 , 12 ] and promotion of cell survival [ 13 , 14 ] or the free radical scavenging system [ 15 , 16 ] ) .
however , in the liver , the effects of gabar regulation have not been reported .
orthotopic liver transplantation ( olt ) is an accepted therapy for children and adults with end - stage liver disease , and it currently provides long - term survival and quality lifestyle .
however , cold ischemia during organ storage and subsequent reperfusion severely damage the transplanted liver . during cold ischemic preservation ,
parenchymal cells swell and bleb , and then kupffer and endothelial cells trigger ros / rns production after warm reperfusion .
this cold ischemia / warm reperfusion ( ciwr ) injury is still a major cause of morbidity and mortality after olt due to primary graft dysfunction or a nonfunctioning graft .
reperfusion not only triggers the liver regeneration cascade but also causes fatal damage in the liver graft due to os [ 18 , 19 ] .
proactive strategies through pharmacological pretreatment to limit graft damage from ciwr injury have the advantage of excellent graft function after olt .
a small - for - size graft ( sfsg ) is also an issue in deceased - donor liver transplantation ( ddlt ) and living - donor liver transplantation ( ldlt ) .
an sfsg is defined as a ratio of graft weight against standard liver volume < 40% [ 20 , 21 ] . an inevitable insufficiency of graft size can not be avoided in ldlt or split orthotopic liver transplantation ( solt ) for ddlt .
shear stress not only triggers the liver regeneration cascade but also causes fatal damage in the sfsg by os [ 22 , 23 ] .
an sfsg in ldlt or solt is accompanied by ciwr injury , as well as shear stress with portal hypertension .
the choice of a left - side graft is preferred from the viewpoint of greater donor safety and expanded donor candidates in ldlt [ 20 , 24 ] .
guaranteed solt with successful outcomes resolves a donor shortage in ddlt [ 24 , 25 ] .
our laboratory has focused on the effect of gabar regulation on liver damage by using rodent models [ 2628 ] .
we failed to show beneficial effects in gabar regulation ex vivo and in gabar regulation by a specific antagonist [ 27 , 28 ] .
however , gabar regulation in vivo by a specific agonist showed a subtle reduction in liver damage in a murine hepatectomy model involving shear stress with portal hypertension   and in a rat orthotopic liver transplantation model with a whole - liver graft involving ciwr injury .
proactive strategies through pharmacological pretreatment to limit graft damage from ciwr injury and shear stress with portal hypertension have advantages for overcoming a current issue . as a final goal of gabar regulation in the liver
, we investigated the strategic potential of graft pretreatment in vivo by a gabar agonist in the rat solt model with a 40%-sfsg , and we examined the possible pathways involved .
graft donors and recipients were 812-week - old rats ( approximately 250  g ) .
the experimental protocols were approved by the ethical committee of our institution ( mayo clinic , institutional animal care and use committee , no .
rats were cared for in accordance with the institutional guidelines for animal welfare based on the national institutes of health guide for the care and use of laboratory animals . a dose of 43.56  nmol / g body weight of gabar agonist ( gabaa receptor agonist , muscimol , 114.10  g / mol ; 70015 , fluka , sigma - aldrich co. , st .
four hours before graft harvest , the donor rat intravenously received 1.0  ml of gabar agonist into the penile vein .  
comprehensive details of the surgical procedures for rat solt and postoperative care in our institution have been previously described [ 29 , 30 ] .
briefly , the syngeneic graft had a cold ischemic time of 2  h at 4c in normal ringer 's solution .
the liver graft was washed twice by 10  ml of normal ringer 's solution , immediately after the graft harvest and before graft implantation .
the 40%-sfsg was made by the left median and lateral segments at the back table [ 29 , 30 ] . to avoid any irrelevant signaling ,
the hepatic artery was reconstructed by ultramicrosurgery in this study [ 29 , 30 ] .
each rat was housed separately after surgery , and body temperature was maintained by a heating pad .
postoperative observation was performed every 30  min until 6  h after solt , and 1.0  ml of warm lactate ringer 's solution was routinely administered every 1  h until 6  h after solt . in this model , we previously demonstrated the importance of a shortened anhepatic phase and exclusion of unreliable samples based on autopsy findings [ 29 , 30 ] . in this study ,
the anhepatic phase was maintained within 20  min in each solt , and no surgical complications were observed in each case at autopsy .
recipient rats were divided into three groups according to donor treatments and the recipient 's surgery as follows : ( i ) saline ( normal saline , 1.0  ml , i.v . ) and laparotomy , ( ii ) saline ( normal saline , 1.0  ml , i.v . ) and solt with 40%-sfsg , and ( iii ) gabar agonist ( muscimol , 43.56  nmol ( 4.98  g)/g body weight , 1.0  ml , i.v . ) and solt with 40%-sfsg .
first , a survival study was performed ( n = 10 in each group ) .
cell signalings involving cell proliferation , differentiation , and apoptosis were investigated from the early postoperative period [ 18 , 3133 ] , and subsequently , progressive necrosis was observed [ 18 , 3133 ] .
serum , plasma , and liver samples for histopathological / immunohistological assessment and western blotting analyses were then collected 6  h after solt ( n = 5 in each group ) .
aspartate aminotransferase ( ast ) , alanine aminotransferase ( alt ) , and total bilirubin ( t - bil ) levels , and the international normalized ratio of prothrombin time ( pt - inr ) were measured .
serum ast , alt , and t - bil levels were assessed ( sgot , sgpt , and total bilirubin reagent , respectively , biotron , hemet , ca , usa ) .
the pt - inr in plasma was measured by the i - stat system ( abbott , princeton , nj , usa ) .
liver tissue was fixed in 10% neutral - buffered formalin , embedded in paraffin , and sliced into 4  m sections .
morphological characteristics and graft injury scores were assessed after hematoxylin - eosin ( he ) staining .
the graft damage score ( points ) has previously been described elsewhere [ 30 , 34 , 35 ] .
scores were counted in 10 fields ( 100 ) in each slide , and then these scores were averaged .
induction of apoptosis was assessed by immunostaining of terminal deoxynucleotidyl transferase - mediated deoxyuridine triphosphate nick - end labeling ( tunel ) ( apoptag peroxidase in situ apoptosis detection kit , s7100 , chemicon international , inc . ,
billerica , ma , usa ) and cysteine aspartic acid protease ( caspase ) 3 ( cleaved caspase-3 ( asp175 ) antibody , 9661s , cell signaling technology , inc . , danvers , ma , usa ) .
tunel - positive nuclei were stained brown , and negative nuclei were counterstained light blue .
slides were scanned with an automated high - throughput scanning system ( scanscope xt , aperio technologies , inc . , vista , ca , usa ) .
to quantify the immunohistological findings , positively stained nuclei were counted by aperio imagescope software ( aperio technologies , inc . ) .
all nuclei were classified into four color intensity levels , and the higher two levels were considered as positive .
the ratio of positively stained nuclei to all nuclei was calculated , and the mean ratio per mm was determined .
the primary antibodies for 4-hydroxynonenal ( 4-hne ) ( 4 hydroxynonenal antibody , ab46545 , abcam , cambridge , ma , usa ) , ataxia - telangiectasia mutated kinase ( atm ) ( phospho - atm / atr substrate rabbit mab , cell signaling technology ) , phosphorylated histone h2ax ( phospho - histone h2ax antibody , 2577 , cell signaling technology ) , phosphatidylinositol-3 kinase ( pi3k ) ( phospho - pi3k p85/p55 antibody , 4228 , cell signaling technology ) , akt ( phospho - akt rabbit mab , 4058 , cell signaling technology ) , superoxide dismutase ( sod ) 1 ( cu / zn superoxide dismutase , ls - b2907 , lifespan biosciences , seattle , wa , usa ) , sod 2 ( mn superoxide dismutase , ls - c62194 , lifespan biosciences ) , and catalase ( catalase , ls - b2554 , lifespan biosciences ) were used .
liver samples were collected , homogenized , and centrifuged at high speed for 10  min at 4c .
the supernatant was then collected and used for bicinchoninic acid protein determination ( bca protein assay reagent , thermo fisher scientific , rockford , il , usa ) and western blot analysis .
forty micrograms of protein were run on 420% tris - glycine gels and transferred onto 0.45  m nitrocellulose membranes .
the membranes were then blocked with 5% nonfat milk made up in a tris - buffered saline solution . after blocking ,
the next day , the membranes were washed three times for 10  min with tris - buffered saline solution and then incubated with the peroxidase - conjugated secondary antibody for 1  h , with shaking at room temperature .
after incubation , the membranes were once again washed three times for 10  min with tris - buffered saline solution and then developed using chemiluminescence .
signals were quantified by using imagequant 5.0 software ( molecular dynamics , sunnyvale , ca , usa ) .
the student 's t - test was used for the comparison of unpaired continuous variables between groups .
survival curves were constructed by the kaplan - meier method ( log - rank test ) .
solt with a 40%-sfsg clearly showed poorer survival than laparotomy ( p < 0.0001 ) , and graft pretreatment by gabar agonist prolonged survival after solt ( p = 0.0369 ) .
inflammatory cell infiltration , vacuolization , hepatocyte ballooning , and necrosis were confirmed after solt with a 40%-sfsg .
actual histopathological findings in h - e staining are shown in each group in figures 1(b)1(d ) .  
there were significant differences between laparotomy and solt with saline ( 0.0  0.0 versus 5.8  1.1 points , p < 0.0001 ) and between solt with saline and solt with gabar agonist ( 5.8  1.1 versus 4.1  1.0 points ; p = 0.0280 ) ( figure 1(e ) ) .
there were significant differences in serum ast levels between laparotomy and solt with saline ( 45.4  10.3 versus 387.4  36.8 u / l ; p < 0.0001 ) and between solt with saline and solt with gabar agonist ( 387.4  36.8 versus 296.0  32.3
there were significant differences in serum alt levels between laparotomy and solt with saline ( 54.2  9.2 versus 354.2  32.1  u / l ; p < 0.0001 ) and between solt with saline and solt with gabar agonist ( 354.2  32.1 versus 272.4  31.3 
there were significant differences in serum t - bil levels between laparotomy and solt with saline ( 0.28  0.04 versus 1.37  0.29  mg / dl ; p < 0.0001 ) and between solt with saline and solt with gabar agonist ( 1.37  0.29 versus 1.02  0.15  mg / dl ; p = 0.0453 ) ( figure 2(c ) ) .
there were significant differences in pt - inr between laparotomy and solt with saline ( 0.99  0.04 versus 1.22  0.06 ; p = 0.0001 ) and between solt with saline and solt with gabar agonist ( 1.22  0.06 versus 1.13  0.06 ; p = 0.0456 ) ( figure 2(d ) ) .
the ratio of tunel - positive nuclei was significantly different between laparotomy and solt with saline ( 0.001  0.002 versus 0.166  0.052 ; p < 0.0001 ) and between solt with saline and solt with gabar agonist ( 0.166  0.052 versus 0.092  0.038 ; p = 0.0324 ) ( figure 3(d ) ) .
the ratio of caspase 3-positive nuclei was significantly different between laparotomy and solt with saline ( 0.0001  0.0001 versus 0.115  0.019 ; p < 0.0001 ) and between solt with saline and solt with gabar agonist ( 0.115  0.019 versus 0.080  0.024 ; p = 0.0347 ) ( figure 4(d ) ) .
normalized 4-hne showed significant differences between laparotomy and solt with saline ( 1.00  0.06 versus 1.38    0.22 ; p = 0.0068 ) and between solt with saline and solt with gabar agonist ( 1.38    0.22 versus 1.05    0.15 ; p = 0.0276 ) ( figure 5(b ) ) .
actual intensities of atm and h2ax in each group are shown in figure 6(a ) .
normalized atm showed significant differences between laparotomy and solt with saline ( 1.00  0.11 versus 1.21  0.11 ; p = 0.0131 ) and between solt with saline and solt with gabar agonist ( 1.21  0.11 versus 0.90  0.28 ; p = 0.0477 ) ( figure 6(b ) ) .   
normalized h2ax showed significant differences between laparotomy and solt with saline ( 1.00  0.10 versus 2.59  0.66 ; p = 0.0007 ) and between solt with saline and solt with gabar agonist ( 2.59  0.66 versus 0.83  0.25 ; p = 0.0005 ) ( figure 6(c ) )
. actual intensities of pi3k and akt in each group are shown in figure 7(a ) .
normalized pi3k showed significant differences between laparotomy and solt with saline ( 1.00  0.11 versus 0.59  0.27 ; p = 0.0139 ) and between solt with saline and solt with gabar agonist ( 0.59  0.27 versus 0.92  0.13 ; p = 0.0443 ) ( figure 7(b ) ) .  
normalized akt showed significant differences between laparotomy and solt with saline ( 1.00  0.12 versus 0.34  0.24 ;
p = 0.0006 ) and between solt with saline and solt with gabar agonist ( 0.34  0.24 versus 1.11  0.22 ; p = 0.0007 ) ( figure 7(c ) ) . actual intensities of sod 1 , sod 2 , and catalase in each group are shown in figure 8(a ) .
normalized sod 1 showed significant differences between laparotomy and solt with saline ( 1.00  0.10 versus 0.81  0.16 ; p = 0.0445 ) but not between solt with saline and solt with gabar agonist ( 0.81  0.16 versus 0.82  0.12 ; p = 0.8248 ) ( figure 8(b ) ) .
normalized sod 2 showed significant differences between laparotomy and solt with saline ( 1.00  0.13 versus 0.79  0.14 ; p = 0.0361 ) but not between solt with saline and solt with gabar agonist ( 0.79  0.14 versus 0.84  0.15 ; p = 0.5765 ) ( figure 8(c ) ) .
normalized catalase showed no significant differences between laparotomy and solt with saline ( 1.00  0.14 versus 0.95  0.14 ; p = 0.6904 ) and between solt with saline and solt with gabar agonist ( 0.95  0.14 versus 0.96  0.26 ; p = 0.9764 ) ( figure 8(d ) ) .
  based on the current situation in the clinical field , the 40%-sfsg needs to be investigated in detail because successful solt overcomes a donor shortage in ddlt , and the shift to a left - lobe graft provides donor safety in ldlt [ 20 , 24 , 30 ] .
however , the 40%-sfsg is prone to ischemia / reperfusion injury and shear stress with portal hypertension , and therefore , the os - induced damage after solt is more fatal [ 18 , 3638 ] . in our study ,
a survival study , biochemical assays , and histopathological assessment showed that the 40%-sfsg received the liver injury enough .
os causes dna damage and subsequent apoptosis [ 13 ] , and in our study , immunohistochemistry showed that solt induced apoptosis in the 40%-sfsg .
ros / rns can attack and damage a variety of critical biological molecules [ 13 ] , and the products of lipid peroxidation reliably and rapidly reflect sensitive and specific signals due to os occurring in vivo [ 39 , 40 ] . the fatty aldehyde 4-hne is an end product of lipoperoxidation [ 39 , 40 ] .
therefore , os after solt with a 40%-sfsg resulted in apoptotic induction and subsequent necrosis .
os mediated by free radicals is defined as an imbalance between the production of ros / rns and antioxidant capacity [ 13 ] .
ros / rns have been suggested as a major contributing factor for dna damage in the progression of os . as a sensor of dna damage responses
, the protein kinase atm can be initiated through rapid intermolecular autophosphorylation induced by dna damage [ 12 , 41 ] ; it phosphorylates various proteins , and subsequently amplifies the responses to dna damage .
h2ax is required for cell cycle arrest and dna repair following double - stranded dna breaks [ 5 , 42 ] .
dna damage results in the rapid phosphorylation of h2ax by atm at sites of dna damage [ 5 , 4345 ] .
our study showed that this response to and repair of dna damage via atm / h2ax was clearly triggered after solt with a 40%-sfsg and that this cascade is a possible pathway in the process of os - induced injury after solt with sfsg .
our preliminary data in the rat olt model with whole - liver grafts ( i.e. , a model for only ciwr injury ) suggested that gabar regulation by a specific agonist showed differences in atm / h2ax .
we consider that gabar regulation may have a beneficial effect against ciwr injury via the atm / h2ax pathway in the liver . from the viewpoint of the production of ros
/ rns in the process of os , akt also plays a critical role in controlling apoptosis [ 41 , 46 , 47 ] and promotes cell survival [ 4750 ] .
apoptotic machinery is inhibited by the activation of akt [ 46 , 51 , 52 ] .
akt is a component of the antiapoptotic process related to the activation of pi3k , and pi3k is upstream from akt [ 47 , 53 ] .
the cell survival pathway via pi3k / akt is also considered as an important signaling pathway to control apoptotic induction in the liver [ 54 , 55 ] .
our study showed that this promotion of cell survival via pi3k / akt was disturbed after solt with a 40%-sfsg and that this cascade could be one of the possible pathways in the process of os - induced injury after solt with sfsg . our preliminary data in the murine hepatectomy model ( i.e. , a model for only shear stress with portal hypertension ) suggested that gabar regulation by a specific agonist showed differences in pi3k / akt .
therefore , we consider that gabar regulation may have a beneficial effect against shear stress with portal hypertension via the pi3k / akt pathway in the liver . from the viewpoint of antioxidant defenses , free radical scavenging enzymes , such as sod and catalase , also play an important role in reducing dna damage and subsequent apoptosis [ 2 , 3 , 56 ] .
normal cells are able to defend themselves against os through this scavenging system [ 3 , 56 ] .
our study showed a decrease in sod 1 and sod 2 levels after solt with a 40%-sfsg , although we initially expected that antioxidant enzymes would increase .
our results appear to be consistent with a previous opinion that os impairs mitochondrial importing and processing of sod .
however , another possible explanation for our results may be that this scavenging system failed , and some reactive molecules evaded the detoxification process and damaged potential targets because of drastic damage after solt with a 40%-sfsg , even though these scavenging enzymes can handle large amounts of ros / rns .  
our results of the survival study , histopathological assessment , and biochemical assays showed that pretreatment for sfsg by gabar regulation in vivo affected graft damage after solt .
moreover , immunohistochemistry showed that this pretreatment reduced apoptotic induction after solt . in the field of brain research ,
the effect of gabar regulation on the prevention of os has been reported [ 57 ] .
although gaba was initially thought to be confined to the central nervous system , gaba is currently considered to be a multifunctional molecule with various physiological effects throughout the body [ 9 , 10 ] .
although the liver contains gaba and hepatic gabar [ 10 , 17 ] , the effects of gabar regulation in the liver are unknown .
our study suggests that gabar regulation may have a strategic potential for 40%-sfsgs as a pharmacological pretreatment for reducing os - induced damage after solt , although solt with a 40%-sfsg involves fatal os due to dual damage ( i.e. , ciwr injury and shear stress with portal hypertension ) .
whether gabar regulation ex vivo ( i.e. , a procedure during organ storage ) is more suitable for ldlt is unknown .
although our results showed the strategic potential of gabar regulation in vivo as a pretreatment for liver grafts , we failed to confirm a positive effect of gabar regulation ex vivo .
therefore , some innovations are still required for clinical application . in previous reports on the brain ,
many investigators have suggested that gabar regulation by a specific agonist or antagonist affects the response to reduce os - induced injury [ 5 , 7 , 8 ] .
their preventive effects in the brain have been mainly explained via specific pathways against os ( i.e. , inhibition of the response to dna damage [ 5 , 11 , 12 ] and promotion of cell survival [ 13 , 14 ] or the free radical scavenging system [ 15 , 16 ] ) .
many previous investigators have suggested that gabar regulation in the brain has certain effects on the response to and repair of dna damage via the atm / h2ax pathway in vivo and in vitro in the process of os [ 5 , 11 , 12 ] .
our study showed that the regulation of hepatic gabar also appeared to reduce os - induced dna damage via the atm / h2ax pathway as well as to have effects in the brain . with regard to the effects of gabar regulation on os in the brain
, the pi3k / akt pathway promotes cell survival against dna damage [ 5 , 13 , 14 , 46 , 59 ] .
our study showed that regulation of hepatic gabar appeared to promote cell survival via the pi3k / akt pathway against os - induced dna damage as well as to have effects in the brain .
however , antioxidant enzymes reduce os - induced damage . from the viewpoint of this scavenging system
, some researchers have shown that gabar regulation in the brain has preventive effects against os - induced damage via antioxidant enzymes [ 15 , 16 ] .
although sod 2 plays an important role in preventing dna damage in the sfsg , our results suggested that the effects of the regulation of hepatic gabar against os did not depend on this scavenging system .
overall , we speculate that the regulation of hepatic gabar has a preventive effect against os , by reducing dna damage via the atm / h2ax pathway and by promoting cell survival via the pi3k / akt pathway
. however , antioxidant enzymes might be important for gabar regulation in the brain [ 15 , 16 ] .
in conclusion , regulation of gabar by a specific agonist in vivo works well in the liver , as well as the brain .
even though ciwr injury and shear stress with portal hypertension affect 40%-sfsgs after solt and results in fatal os , graft pretreatment in vivo by gabar regulation clearly improves graft damage after solt .
this strategy may be advantageous for overcoming current issues in the ddlt and ldlt fields .
the effects of gabar regulation on graft damage after solt with a 40%-sfsg appear to prevent os by reducing dna damage via the atm / h2ax pathway and by promoting cell survival via the pi3k / akt pathway .
 
 background . graft pretreatment to limit postoperative damage has the advantage of overcoming a current issue in liver transplantation ( lt ) . 
 the strategic potential of graft pretreatment in vivo by a specific agonist for -aminobutyric acid receptor ( gabar ) was investigated in the rat lt model with a small - for - size graft ( sfsg ) . 
 methods . 
 recipient rats were divided into three groups according to donor treatments and recipient surgeries : ( i ) saline and laparotomy , ( ii ) saline and split orthotopic liver transplantation ( solt ) with 40%-sfsg , and ( iii ) gabar agonist and solt with 40%-sfsg . 
 survival was evaluated . 
 blood and liver samples were collected 6  h after surgery . 
 immunohistological assessment for apoptotic induction and western blotting for 4-hydroxynonenal , ataxia - telangiectasia mutated kinase ( atm ) , histone h2ax , phosphatidylinositol-3 kinase ( pi3k ) , akt , and free radical scavenging enzymes were performed . 
 results . 
 pretreatment by gabar showed improvement in survival , histopathological assessment , and biochemical tests . 
 apoptotic induction and oxidative stress were observed after solt with an sfsg , and this damage was limited by gabar regulation . 
 gabar regulation appeared to reduce dna damage via the atm / h2ax pathway and to promote cell survival via the pi3k / akt pathway . 
 conclusions . 
 pretreatment in vivo by gabar regulation improves graft damage after solt with an sfsg . 
 this strategy may be advantageous in lt . 