Research Article | Open Access
Pretreatment of Small-for-Size Grafts In Vivo by γ-Aminobutyric Acid Receptor Regulation against Oxidative Stress-Induced Injury in Rat Split Orthotopic Liver Transplantation
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 γ-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.
Oxygen is required for cell survival. 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) [1–3]. 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 [1–3]. 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 [1–3]. These antioxidants ensure a defense against ROS/RNS-induced OS .
The predominant inhibitory neurotransmitter in the brain is γ-aminobutyric acid (GABA), and almost all researchers have focused on GABA or the regulation of GABA receptor (GABAR) in the brain [4–8]. 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]). Liver contains GABA and its transporter , and hepatic GABAR has been also detected . 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]. Currently, strategic procedures are required to improve liver tolerance against CIWR injury. 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 cannot 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. Therefore, SFSGs result in a higher mortality and morbidity after LDLT or SOLT. 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]. Currently, the 40%-SFSG is a critical issue .
Our laboratory has focused on the effect of GABAR regulation on liver damage by using rodent models [26–28]. 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.
2. Materials and Methods
Lewis rats () were purchased from Harlan Laboratories, Inc. (Indianapolis, IN, USA). Graft donors and recipients were 8–12-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. A19609). 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.
2.2. Intravenous Injection of a GABAR Agonist
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. Louis, MO, USA) was used . Four hours before graft harvest, the donor rat intravenously received 1.0 mL of GABAR agonist into the penile vein.
2.3. SOLT with 40%-SFSG and Postoperative Care
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 4°C 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.
2.4. Study Design
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 μg)/g body weight, 1.0 mL, i.v.) and SOLT with 40%-SFSG.
First, a survival study was performed ( in each group). Cell signalings involving cell proliferation, differentiation, and apoptosis were investigated from the early postoperative period [18, 31–33], and subsequently, progressive necrosis was observed [18, 31–33]. Serum, plasma, and liver samples for histopathological/immunohistological assessment and western blotting analyses were then collected 6 h after SOLT ( in each group).
2.5. Biochemical Assay and Coagulation Profile
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).
2.6. Histopathological and Immunohistological Assessments
Liver tissue was fixed in 10% neutral-buffered formalin, embedded in paraffin, and sliced into 4 μm 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. Caspase-3-positive nuclei were stained brown, and negative nuclei were counterstained 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 mm2 was determined.
2.7. Western Blotting Analysis
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 4°C. 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 4–20% Tris-glycine gels and transferred onto 0.45 μm nitrocellulose membranes. The membranes were then blocked with 5% nonfat milk made up in a Tris-buffered saline solution. After blocking, the membranes were incubated at 4°C overnight with the primary antibody. 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. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a control. Signals were quantified by using ImageQuant 5.0 software (Molecular Dynamics, Sunnyvale, CA, USA).
2.8. Statistical Analysis
The results are presented as mean ± standard deviation. The Student’s -test was used for the comparison of unpaired continuous variables between groups. Survival curves were constructed by the Kaplan-Meier method (log-rank test). Statistical calculations were performed using SPSS Software Version 16.0 (SPSS Inc., Chicago, IL, USA). A value <0.05 was considered statistically significant.
3.1. Survival Curves
Survival curves in each group are shown in Figure 1(a). SOLT with a 40%-SFSG clearly showed poorer survival than laparotomy (), and graft pretreatment by GABAR agonist prolonged survival after SOLT ().
3.2. Parenchymal Damage in Grafts
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 ( versus points, ) and between SOLT with saline and SOLT with GABAR agonist ( versus points; ) (Figure 1(e)).
3.3. Biochemical and Coagulation Profiles
There were significant differences in serum AST levels between laparotomy and SOLT with saline ( versus U/L; ) and between SOLT with saline and SOLT with GABAR agonist ( versus U/L; ) (Figure 2(a)).
There were significant differences in serum ALT levels between laparotomy and SOLT with saline ( versus U/L; ) and between SOLT with saline and SOLT with GABAR agonist ( versus U/L; ) (Figure 2(b)).
There were significant differences in serum T-Bil levels between laparotomy and SOLT with saline ( versus mg/dL; ) and between SOLT with saline and SOLT with GABAR agonist ( versus mg/dL; ) (Figure 2(c)).
There were significant differences in PT-INR between laparotomy and SOLT with saline ( versus ; ) and between SOLT with saline and SOLT with GABAR agonist ( versus ; ) (Figure 2(d)).
3.4. Apoptotic Induction
TUNEL immunostaining in each group is shown in Figures 3(a)–3(c). The ratio of TUNEL-positive nuclei was significantly different between laparotomy and SOLT with saline ( versus ; ) and between SOLT with saline and SOLT with GABAR agonist ( versus ; ) (Figure 3(d)).
Caspase 3 immunostaining in each group is shown in Figures 4(a)–4(c). The ratio of caspase 3-positive nuclei was significantly different between laparotomy and SOLT with saline ( versus ; ) and between SOLT with saline and SOLT with GABAR agonist ( versus ; ) (Figure 4(d)).
Actual intensities of 4-HNE are shown in Figure 5(a). Normalized 4-HNE showed significant differences between laparotomy and SOLT with saline ( versus ; ) and between SOLT with saline and SOLT with GABAR agonist ( versus ; ) (Figure 5(b)).
3.6. Response to and Repair of DNA Damage
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 ( versus ; ) and between SOLT with saline and SOLT with GABAR agonist ( versus ; ) (Figure 6(b)).
Normalized H2AX showed significant differences between laparotomy and SOLT with saline ( versus ; ) and between SOLT with saline and SOLT with GABAR agonist ( versus ; ) (Figure 6(c)).
3.7. Promotion of Cell Survival
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 ( versus ; ) and between SOLT with saline and SOLT with GABAR agonist ( versus ; ) (Figure 7(b)).
Normalized Akt showed significant differences between laparotomy and SOLT with saline ( versus ; ) and between SOLT with saline and SOLT with GABAR agonist ( versus ; ) (Figure 7(c)).
3.8. Activities of Antioxidant Enzymes
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 ( versus ; ) but not between SOLT with saline and SOLT with GABAR agonist ( versus ; ) (Figure 8(b)).
Normalized SOD 2 showed significant differences between laparotomy and SOLT with saline ( versus ; ) but not between SOLT with saline and SOLT with GABAR agonist ( versus ; ) (Figure 8(c)).
Normalized catalase showed no significant differences between laparotomy and SOLT with saline ( versus ; ) and between SOLT with saline and SOLT with GABAR agonist ( versus ; ) (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, 36–38]. 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 [1–3], 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 [1–3], 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]. Our results of 4-HNE showed that OS occurred after SOLT. 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 [1–3]. 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 . This DNA damage-inducible kinase activates histone H2AX . 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, 43–45]. 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 [47–50]. 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 [5–7]. 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).
Any pretreatment in a living donor violates ethical policy and spoils donor regulations. 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.
Conflict of Interests
All authors have no financial conflict of interests.
This work was partially supported by Grants to J. H. Nguyen from the Deason Foundation, Sandra and Eugene Davenport, Mayo Clinic CD CRT-II, and from the National Institutes of Health (R01NS051646-01A2) and a Grant to T. Hori from the Uehara Memorial Foundation (no. 200940051, Tokyo, Japan). The authors are grateful to Dickson W. Dennis, Monica Castanedes-Casey, Virginia R. Phillips, Linda G. Rousseau, and Melissa E. Murray (Department of Neuroscience, Mayo Clinic Florida, Jacksonville, FL, USA) for their diagnostic and technical support in the histopathological and immunohistological assessments.
- D. A. Castroviejo, L. C. Lopez, G. Escames, A. Lopez, J. A. Garcia, and R. J. Reiter, “Melatonin-mitochondria interplay in health and disease,” Current Topics in Medicinal Chemistry, vol. 11, pp. 221–240, 2011.
- N. Ghosh, R. Ghosh, and S. C. Mandal, “Antioxidant protection: a promising therapeutic intervention in neurodegenerative disease,” Free Radical Research, vol. 45, no. 8, pp. 888–905, 2011.
- B. Turan, “Role of antioxidants in redox regulation of diabetic cardiovascular complications,” Current Pharmaceutical Biotechnology, vol. 11, no. 8, pp. 819–836, 2010.
- M. E. Pamenter, D. W. Hogg, J. Ormond, D. S. Shin, M. A. Woodin, and L. T. Buck, “Endogenous GAB and GAB receptor-mediated electrical suppression is critical to neuronal anoxia tolerance,” Proceedings of the National Academy of Sciences of the United States of America, vol. 108, no. 27, pp. 11274–11279, 2011.
- M. Andäng, J. Hjerling-Leffler, A. Moliner et al., “Histone H2AX-dependent GAB receptor regulation of stem cell proliferation,” Nature, vol. 451, no. 7177, pp. 460–464, 2008.
- J. M. Ferreira, A. L. Burnett, and G. A. Rameau, “Activity-dependent regulation of surface glucose transporter-3,” The Journal of Neuroscience, vol. 31, no. 6, pp. 1991–1999, 2011.
- Y. Hirooka, T. Kishi, K. Sakai, A. Takeshita, and K. Sunagawa, “Imbalance of central nitric oxide and reactive oxygen species in the regulation of sympathetic activity and neural mechanisms of hypertension,” American Journal of Physiology—Regulatory Integrative and Comparative Physiology, vol. 300, no. 4, pp. R818–R826, 2011.
- S. Kurt, J. M. Crook, F. W. Ohl, H. Scheich, and H. Schulze, “Differential effects of iontophoretic in vivo application of the GABA A-antagonists bicuculline and gabazine in sensory cortex,” Hearing Research, vol. 212, no. 1-2, pp. 224–235, 2006.
- Y. Gong, M. Zhang, L. Cui, and G. Y. Minuk, “Sequence and chromosomal assignment of a human novel cDNA: similarity to gamma-aminobutyric acid transporter,” Canadian Journal of Physiology and Pharmacology, vol. 79, no. 12, pp. 977–984, 2001.
- T. Norikura, A. Kojima-Yuasa, D. O. Kennedy, and I. Matsui-Yuasa, “Protective effect of gamma-aminobutyric acid (GABA) against cytotoxicity of ethanol in isolated rat hepatocytes involves modulations in cellular polyamine levels,” Amino Acids, vol. 32, no. 3, pp. 419–423, 2007.
- R. N. Fernando, B. Eleuteri, S. Abdelhady, A. Nussenzweig, M. Andäng, and P. Ernfors, “Cell cycle restriction by histone H2AX limits proliferation of adult neural stem cells,” Proceedings of the National Academy of Sciences of the United States of America, vol. 108, no. 14, pp. 5837–5842, 2011.
- C. E. Irarrazabal, J. C. Liu, M. S. Burg, and J. D. Ferraris, “ATM, a DNA damage-inducible kinase, contributes to activation by high NaCl of the transcription factor TonEBP/OREBP,” Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 23, pp. 8809–8814, 2004.
- J. Xu, C. Li, X. Yin, and G. Zhang, “Additive neuroprotection of GABA A and GABA B receptor agonists in cerebral ischemic injury via PI-3K/Akt pathway inhibiting the ASK1-JNK cascade,” Neuropharmacology, vol. 54, no. 7, pp. 1029–1040, 2008.
- J. E. Lee, J. S. Kang, Y. Ki et al., “Akt/GSK3β signaling is involved in fipronil-induced apoptotic cell death of human neuroblastoma SH-SY5Y cells,” Toxicology Letters, vol. 202, no. 2, pp. 133–141, 2011.
- S. Tejada, C. Roca, A. Sureda, R. V. Rial, A. Gamundí, and S. Esteban, “Antioxidant response analysis in the brain after pilocarpine treatments,” Brain Research Bulletin, vol. 69, no. 5, pp. 587–592, 2006.
- J. Zhou, Y. Li, G. Yan et al., “Protective role of taurine against morphine-induced neurotoxicity in C6 cells via inhibition of oxidative stress,” Neurotoxicity Research, vol. 20, no. 4, pp. 334–342, 2011.
- M. P. Biju, S. Pyroja, N. V. Rajeshkumar, and C. S. Paulose, “Hepatic GAB receptor functional regulation during rat liver cell proliferation,” Hepatology Research, vol. 21, no. 2, pp. 136–146, 2001.
- J. Lemasters, H. Bunzendahl, and R. Thurman, “Preservation of the liver,” in Transplantation of the Liver, W. Maddrey and M. Sorrell, Eds., pp. 297–321, Appleton & Lange, East Norwalk, Conn, USA, 2nd edition, 1995.
- H. B. Abdennebi, M. A. Zaoualí, I. Alfany-Fernandez, D. Tabka, and J. Roselló-Catafau, “How to protect liver graft with nitric oxide,” World Journal of Gastroenterology, vol. 17, no. 24, pp. 2879–2889, 2011.
- Y. Ogura, T. Hori, W. M. El Moghazy et al., “Portal pressure <15 mm Hg is a key for successful adult living donor liver transplantation utilizing smaller grafts than before,” Liver Transplantation, vol. 16, no. 6, pp. 718–728, 2010.
- F. Wang, K. Pan, S. Chu et al., “Preoperative estimation of the liver graft weight in adult right lobe living donor liver transplantation using maximal portal vein diameters,” Liver Transplantation, vol. 17, no. 4, pp. 373–380, 2011.
- S. He, C. Atkinson, F. Qiao, K. Cianflone, X. Chen, and S. Tomlinson, “A complement-dependent balance between hepatic ischemia/reperfusion injury and liver regeneration in mice,” Journal of Clinical Investigation, vol. 119, no. 8, pp. 2304–2316, 2009.
- J. Wu, Q. Tang, J. Shen et al., “Comparative proteome profile during the early period of small-for-size liver transplantation in rats revealed the protective role of Prdx5,” Journal of Hepatology, vol. 53, no. 1, pp. 73–83, 2010.
- T. Hori, S. Uemoto, L. B. Gardner, L. Sibulesky, Y. Ogura, and J. H. Nguyen, “Left-sided grafts for living-donor liver transplantation and split grafts for deceased-donor liver transplantation: their impact on long-term survival,” Clinics and Research in Hepatology and Gastroenterology, vol. 36, no. 1, pp. 47–52, 2012.
- R. W. Busuttil and J. A. Goss, “Split liver transplantation,” Annals of Surgery, vol. 229, no. 3, pp. 313–321, 1999.
- T. Hori, L. B. Gardner, T. Hata et al., “Pretreatment of liver grafts in vivo by γ-aminobutyric acid receptor regulation reduces cold ischemia/warm reperfusion injury in rat,” Annals of Transplantation, vol. 18, pp. 299–313, 2013.
- L. B. Gardner, T. Hori, F. Chen et al., “Effect of specific activation of γ-aminobutyric acid receptor in vivo on oxidative stress-induced damage after extended hepatectomy,” Hepatology Research, vol. 42, pp. 1131–1140, 2012.
- T. Hori, L. B. Gardner, F. Chen et al., “Liver graft pretreated in vivo or ex vivo by γ-aminobutyric acid receptor regulation,” Journal of Surgical Research, vol. 182, pp. 166–175, 2013.
- T. Hori, J. H. Nguyen, X. Zhao et al., “Comprehensive and innovative techniques for liver transplantation in rats: a surgical guide,” World Journal of Gastroenterology, vol. 16, no. 25, pp. 3120–3132, 2010.
- T. Hori, S. Uemoto, X. Zhao et al., “Surgical guide including innovative techniques for orthotopic liver transplantation in the rat: key techniques and pitfalls in whole and split liver grafts,” Annals of Gastroenterology, vol. 23, no. 4, pp. 270–295, 2010.
- S. Padrissa-Altés, M. A. Zaouali, R. Franco-Gou et al., “Matrix metalloproteinase 2 in reduced-size liver transplantation: beyond the matrix,” American Journal of Transplantation, vol. 10, no. 5, pp. 1167–1177, 2010.
- Z. Y. Ma, J. M. Qian, X. H. Rui et al., “Inhibition of matrix metalloproteinase-9 attenuates acute small-for-size liver graft injury in rats,” American Journal of Transplantation, vol. 10, no. 4, pp. 784–795, 2010.
- V. Defamie, M. Laurens, D. Patrono et al., “Matrix metalloproteinase inhibition protects rat livers from prolonged cold ischemia-warm reperfusion injury,” Hepatology, vol. 47, no. 1, pp. 177–185, 2008.
- T. Iida, S. Yagi, K. Taniguchi et al., “Significance of CT attenuation value in liver grafts following right lobe living-donor liver transplantation,” American Journal of Transplantation, vol. 5, no. 5, pp. 1076–1084, 2005.
- T. Hori, T. Iida, S. Yagi et al., “K1CGvalue, a reliable real-time estimator of graft function, accurately predicts outcomes in adult living-donor liver transplantation,” Liver Transplantation, vol. 12, no. 4, pp. 605–613, 2006.
- H. Rehman, H. D. Connor, V. K. Ramshesh et al., “Ischemic preconditioning prevents free radical production and mitochondrial depolarization in small-for-size rat liver grafts,” Transplantation, vol. 85, no. 9, pp. 1322–1331, 2008.
- Z. Zhong, H. D. Connor, M. Froh et al., “Free radical-dependent dysfunction of small-for-size rat liver grafts: prevention by plant polyphenols,” Gastroenterology, vol. 129, no. 2, pp. 652–664, 2005.
- K. Man, S. Fan, C. Lo et al., “Graft injury in relation to graft size in right lobe live donor liver transplantation: a study of hepatic sinusoidal injury in correlation with portal hemodynamics and intragraft gene expression,” Annals of Surgery, vol. 237, no. 2, pp. 256–264, 2003.
- Y. C. Awasthi, R. Sharma, A. Sharma et al., “Self-regulatory role of 4-hydroxynonenal in signaling for stress-induced programmed cell death,” Free Radical Biology and Medicine, vol. 45, no. 2, pp. 111–118, 2008.
- G. Voulgaridou, I. Anestopoulos, R. Franco, M. I. Panayiotidis, and A. Pappa, “DNA damage induced by endogenous aldehydes: current state of knowledge,” Mutation Research, vol. 711, no. 1-2, pp. 13–27, 2011.
- J. Kim and P. K. Y. Wong, “Loss of ATM impairs proliferation of neural stem cells through oxidative stress-mediated p38 MAPK signaling,” Stem Cells, vol. 27, no. 8, pp. 1987–1998, 2009.
- J. Yuan, R. Adamski, and J. Chen, “Focus on histone variant H2AX: to be or not to be,” FEBS Letters, vol. 584, no. 17, pp. 3717–3724, 2010.
- E. P. Rogakou, D. R. Pilch, A. H. Orr, V. S. Ivanova, and W. M. Bonner, “DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139,” The Journal of Biological Chemistry, vol. 273, no. 10, pp. 5858–5868, 1998.
- S. Burma, B. P. Chen, M. Murphy, A. Kurimasa, and D. J. Chen, “ATM phosphorylates histone H2AX in response to DNA Double-strand breaks,” The Journal of Biological Chemistry, vol. 276, no. 45, pp. 42462–42467, 2001.
- E. P. Rogakou, C. Boon, C. Redon, and W. M. Bonner, “Megabase chromatin domains involved in DNA double-strand breaks in vivo,” Journal of Cell Biology, vol. 146, no. 5, pp. 905–916, 1999.
- S. Maruyama, R. Shibata, K. Ohashi et al., “Adiponectin ameliorates doxorubicin-induced cardiotoxicity through Akt protein-dependent mechanism,” The Journal of Biological Chemistry, vol. 286, no. 37, pp. 32790–32800, 2011.
- Y. Sánchez, D. Amrán, E. de Blas, and P. Aller, “Regulation of genistein-induced differentiation in human acute myeloid leukaemia cells (HL60, NB4). Protein kinase modulation and reactive oxygen species generation,” Biochemical Pharmacology, vol. 77, no. 3, pp. 384–396, 2009.
- T. F. Franke, D. R. Kaplan, and L. C. Cantley, “PI3K: downstream AKTion blocks apoptosis,” Cell, vol. 88, no. 4, pp. 435–437, 1997.
- B. M. Burgering and P. J. Coffer, “Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction,” Nature, vol. 376, no. 6541, pp. 599–602, 1995.
- T. F. Franke, S.-I. Yang, T. O. Chan et al., “The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase,” Cell, vol. 81, no. 5, pp. 727–736, 1995.
- D. Kim and J. Chung, “Akt: versatile mediator of cell survival and beyond,” Journal of Biochemistry and Molecular Biology, vol. 35, no. 1, pp. 106–115, 2002.
- S. R. Datta, H. Dudek, T. Xu et al., “Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery,” Cell, vol. 91, no. 2, pp. 231–241, 1997.
- A. Khalil, R. N. Morgan, B. R. Adams et al., “ATM-dependent ERK signaling via AKT in response to DNA double-strand breaks,” Cell Cycle, vol. 10, no. 3, pp. 481–491, 2011.
- C. Dal Ponte, E. Alchera, A. Follenzi et al., “Pharmacological postconditioning protects against hepatic ischemia/reperfusion injury,” Liver Transplantation, vol. 17, no. 4, pp. 474–482, 2011.
- S. Haga, M. Ozaki, H. Inoue et al., “The survival pathways phosphatidylinositol-3 kinase (PI3-K)/phosphoinositide-dependent protein kinase 1 (PDK1)/Akt modulate liver regeneration through hepatocyte size rather than proliferation,” Hepatology, vol. 49, no. 1, pp. 204–214, 2009.
- D. S. A. Majid and L. Kopkan, “Nitric oxide and superoxide interactions in the kidney and their implication in the development of salt-sensitive hypertension,” Clinical and Experimental Pharmacology and Physiology, vol. 34, no. 9, pp. 946–952, 2007.
- G. Wright, V. Reichenbecher, T. Green, G. L. Wright, and S. Wang, “Paraquat inhibits the processing of human manganese-dependent superoxide dismutase by Sf-9 insect cell mitochondria,” Experimental Cell Research, vol. 234, no. 1, pp. 78–84, 1997.
- N. Maulik and D. K. Das, “Emerging potential of thioredoxin and thioredoxin interacting proteins in various disease conditions,” Biochimica et Biophysica Acta, vol. 1780, pp. 1368–1382, 2008.
- T. Cesetti, T. Fila, K. Obernier et al., “GAB receptor signaling induces osmotic swelling and cell cycle activation of neonatal prominin+precursors,” Stem Cells, vol. 29, no. 2, pp. 307–319, 2011.
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