Modification of Preservative Fluids with Antioxidants

Modification of Preservative Fluids with Antioxidants: Comparison

Please note this is a comparison between Version 1 by Aneta Ostróżka-Cieślik and Version 2 by Sirius Huang.

Transplantation is currently the only effective treatment for patients with end-stage liver failure. Many advanced studies have been conducted to improve the efficiency of organ preservation techniques. Modifying the composition of the preservation fluids currently used may improve graft function and increase the likelihood of transplantation success. The modified fluid is expected to extend the period of safe liver storage in the peri-transplantation period and to increase the pool of organs for transplantation with livers from marginal donors.

liver transplantation
antioxidants
organ preservation solution

1. Introduction

Liver injury due to ischemia and reperfusion is a significant problem in the peri-transplantation period. Warm ischemia results in damage to hepatocytes through the activation of Kupffer cells and pro-inflammatory cytokines. Cold IRI (ischemia–reperfusion injury) results in the dysfunction of hepatic sinusoidal endothelial cells and impaired microcirculation ^[1][2][3][4][1,2,3,4]. Endothelin-1 increase and nitric oxide decrease cause vasoconstriction [5]. Oxidative phosphorylation in mitochondria is inhibited. ATP stores are depleted and, consequently, the activity of the cell’s active transport system and membrane potential decreases. The level of Ca²⁺ ions in the cell increases, which affects the activation of cell-damaging enzymes such as phospholipases, endonucleases, proteases, and ATP-ase. The rate of anaerobic glycolysis increases. Mitochondria become swollen and highly permeable channels are formed in their inner membrane. Cytochrome c is released into the cytoplasm, which can direct the cell into the apoptosis pathway. The cell membrane and elements of the cytoskeleton are damaged ^[3][6][3,6].

Oxidative stress generates the production of ATP metabolites, accompanied by a sharp increase in the production of superoxide anion radical (O₂^•−), hydrogen peroxide (H₂O₂), and hydroxyl radicals (-OH). These initiate circulatory disturbances and a cascade of inflammatory reactions. Mitochondrial membrane morphology and permeability are altered. Damage to the mitochondrial respiratory chain leads to inhibition of oxidative phosphorylation and disruption of energy metabolism. Free oxygen radicals formed in mitochondria cause damage to DNA and organelle proteins and peroxidation of membrane lipids, consequently leading to cell death by apoptosis or necrosis ^[7][8][7,8].

Recent years have seen an increase in research into developing organ perfusion and preservation techniques to minimize ischemia-related graft damage and improve marginal donor utilization rates. The optimization of organ preservation techniques, i.e., static cold storage (SCS; 0–4 °C), hypothermic machine perfusion (HMP, 0–4 °C), subnormothermic machine perfusion (SNMP; 20–30 °C), normothermic machine perfusion (NMP; 32–37 °C), and the introduction of novel substances into preservation fluid compositions, can significantly improve organ function before transplantation. A relatively new generation of organ preservation technology is NMP, which allows blood flow in the organ to be reconstructed and its vital functions to be assessed outside the human body. Transplant rejection rates using this method are 50% lower compared to static cold storage [9].

Modifying the composition of the preservation fluids currently used may improve graft function and increase the likelihood of transplantation success. The modified fluid is expected to extend the period of safe liver storage in the peri-transplantation period and to increase the pool of organs for transplantation with livers from marginal donors.

2. Strategies Based on Modifications of Preservative Solutions with Antioxidants

Transplant fluids provide the environment in which organs are stored during the peri-transplantation period. They extend the ischemic period of the graft and prevent the development of damage during this time. Preserving the optimal vital functions of the organ improves its function in the subsequent postoperative period. The development of an optimal preservative fluid is an important element of transplant success. The substances contained in it should have a multidirectional effect, including anti-inflammatory and cytoprotective effects, which will increase the effectiveness of the transplants performed. Commercially available preservative fluids have been discussed in detail by me in previous publications ^{[10][11][12][13]}[10,11,12,13]. Studies indicate that UW (University of Wisconsin), HTK (histidine–tryptophan–ketoglutarate), and IGL-1 (Institut Georges Lopez-1) are the most commonly used in liver transplantation [14]. Antioxidants are important components of organ perfusion and preservation fluids. An analysis of the available literature (Table 1 and Table 2) indicates that the challenge is to develop a preservative fluid formulation for effective liver preservation in which the antioxidant is compatibility with the other ingredients, demonstrates efficacy and safety of use, and does not complicate the manufacturing process.

Table 1.

Studies on the effectiveness of supplementing preservative fluids with antioxidants. Basic research.

Author, Year of Publication	Antioxidant	Species	Preservation Solution Modification /Cold Ischemia	Outcome Measures, (Intervention, I/Control, C)	Antioxidant Dose	Effects of Antioxidant
Species	Preservation Solution Modification		/Cold Ischemia	Outcome Measures, (Intervention, I/Control, C)	Antioxidant Dose	Effects of Antioxidant
Enzymatic antioxidant
Enzymatic antioxidant
Lauschke et al., 2003 [15]	Taurine SOD
Hide et al.,	Isolated perfused rat liver model	2014 [54]	rMnSOD	human liver samples, LSEC, liver grafts from healthy and steatotic ratsUW 24 h; 4 °C; SCS with VSOP	Celsior 16 h, 4 °C; SCSI1: UW + SOD I2: UW + TAU C: UW	SOD: 600 U/mL; Taurine: 0.5 mg/mL	↓ lipid peroxidation;	I: Celsior + rMnSOD C: no SCS↓ vascular resistance; ↓ LDH, GLDH; ↑ bile production
rMnSOD: 0,15 µM	↓ oxidative stress;		↑ NO;	Minor et al., 1995 [16]	Taurine	Wistar rats	UW 24 h; 4 °C; SCS	I: UW + TAU C1: UW	1 mM/L	improve hepatic circulation; enhance viability of the liver upon reperfusion
Non-enzymatic antioxidants							Non-enzymatic antioxidants
Low-molecular-weight antioxidant
Low-molecular-weight antioxidant
Aliakbarian et al., 2017 [55]	N-Acetylcysteine	Human	UW 4 °C; SCS	I: UW + NAC C: UW	2 g	addition of NAC does not decrease the rate of ischemia–reperfusion injury	Bardallo et al., 2022 [17]	PEG35, Glutathione	Zücker rats	IGL 24 h; 4 °C; SCS	I1: IGL + GSH I2: IGL + PEG35 + GSH C: IGL	PEG35: 1 g/L, 5 g/L GSH: 3 mM/L, 9 mM/L	IGL + PEG35 (5 g/L) + GSH (9 mM/L): maintained ATP production; ↓ succinate accumulation; ↑ expression of the OXPHOS complexes, UCP2, PINK-1, Nrf2, and HO-1; protected against lipid and protein oxidation; increased the GSH/GSSG ratio; ↓ inflammasome NLRP3 expression; protecting mitochondrial integrity
Quintana et al., 2001 [18]	S-Nitrosoglutathione	Isolated perfused rat liver model	UW 48 h; 4 °C; SCS	I: UW + GSNO C: UW	100 µM	the hepatic morphology was conserved showing little vacuolation; avoiding hepatic injury post cold preservation/reperfusion
Quintana et al., 2002 [19]	S-Nitrosoglutathione	Isolated perfused rat liver model	UW 48 h; 0 °C; SCS	I: UW + GSNO C: UW	50 µM, 100 µM, 250 µM, 500 µM	100 µM: prevented the ischemia/reperfusion injuries; ↓ LDH; improved bile production; partially reduced endothelial cell damage
Quintana et al., 2004 [20]	S-Nitrosoglutathione	Isolated perfused rat liver model	UW 48 h; 0 °C; SCS	I: UW + GSNO C: UW	500 μM 100 μM	500 μM: interstitial edema after normothermic reperfusion; 100 μM: damages on mast cells were avoided
Minor et al., 2000 [21]	N-Acetylcysteine, SOD	Isolated perfused rat liver model	UW 24 h; 4 °C; SCS VSOP	I1: UW + SOD I2: UW + NAC C: UW	SOD: 600 U/mL; NAC: 20 mM	prevented an increase in free radical-mediated lipid peroxidation; NAC counteracted the phosphorylation of Iκb
Srinivasan et al., 2014 [22]	N-Acetylcysteine	Lewis rat	HTK 1 h, 3 h, 24 h, 168 h; 5 °C; SCS	12 h, 16 h, 20 h; I: HTK + NAC C: HTK	20 mM	4 °C; SCS	I: UW + PTX C: UW↓ PVP; ↓ ALT; improved microcirculation; diminished histologic graft damage; ↓ lipid peroxidation; ↑ total antioxidant capacity
1 g/L	↓ TNF-α, MDA;		↓ ALT, AST;		↑ ATP;	100% 1-week survival; improved microcirculation	Scherer de Fraga et al., 2010 [23]	S-Nitroso-N-Acetylcysteine	Wistar rats	UW 2 h, 4 h, 6 h; 4 °C; SCS	I: UW + SNAC C: UW	200 nM	↓ AST ↓ Liver injury
Alix et al., 2020 [59]	M101	Pig allogeneic liver orthotopic transplantation	UW 9 h, 4 °C; SCS	I: UW + M101 C: UW	1 g/L	improved liver graft oxygenation during SCS; livers cold stored with UWSCS + M101 did not reach the oxygenation level achieved with machine perfusion	Kerkweg et al., 2002 [24]	Deferoxamine	Hepatocytes from male Wistar rats, rat liver endothelial cells	UW 24 h; 4 °C; SCS	I: UW + DFX C: UW	10 mM	↓ lipid peroxidation; ↓ apoptosis
Jain et al., 2008 [25]	N-Acetylcysteine, Trolox C, Deferoxamine	Isolated perfused rat liver model	UW 4 °C; HMP	I: UW + Gly + NAC + TRX-C + DFX C: UW	NAC: 5 mM TRX-C: 0.2 mM DFX: 0.25 mM	↓ LDH, ALT; ↑ bile production; improved mitochondrial function; improved liver microcirculation; intact hepatocytes
Vreugdenhil et al., 1997 [26]	Deferoxamine, Trolox C, Dithiothreitol	Hepatocytes from Sprague-Dawley rats; isolated perfused rat liver model	UW 24 h, 48 h; 4 °C; SCS	I1: UW + DFX I2: UW + TRX I3: UW + DTT C: UW	DFX: 2.5 mM, 5 mM, 10 mM TRX: 3 mM, 5 mM, 10 mM DTT: 5 mM, 10 mM, 20 mM	poor distribution of antioxidants in isolated rat liver; only DFX was effective when added to the UW: suppressed oxidative stress only in isolated hepatocytes stored in cold storage; ↓ LDH
Wu et al., 2009 [27]	LK 614	Isolated perfused rat liver model	HTK 24 h; 4 °C; SCS	I: HTK + LK 614 C: HTK	20 μM	↓ LDH during reperfusion; increased bile secretion; better preserved hepatic microcirculation
Stegemann et al., 2010 [28]	Deferoxamine, LK 614	Wistar rats	HTK 18 h; 4 °C; HMP	I: HTK-N + LK 614 + DFX C1: HTK C2:HTK-N	DFX: 25 μM LK 614: 2.5 μM, 7.5 μM	↓ ALT, LDH; DFX: 25μM and LK 614: 2.5μM improved metabolic activity, reduced cleavage of caspase 9 and apoptotic index
Mitochondria-targeted antioxidants
Cherkashina et al., 2011 [29]	SkQ1	Rat	Sucrose–saline 24 h; 4 °C; SCS	I: Sucrose–saline + SkQ1 C: Sucrose–saline	1 μM	↓ hepatic injury and oxidative stress; ↑ liver and mitochondrial function
Wieland et al., 1995 [30]	Idebenone (QSA-10)	Rat liver microsomal model	UW, HTK 4 °C; SCS	I1: UW + QSA-10 I2: UW + Q-10 I3: HTK + QSA-10 I4: HTK + Q-10 C1: UW C2: HTK	QSA-10: 0.1 µM/L, 20 µM/L Q-10: 20 µM/L, 100 µM/L	protection against lipid peroxidation (HTK + QSA-10: 0.1 µM/L); prevented protein damage (HTK + QSA-10: 20 µM/L and UW + QSA-10: 20 µM/L); Q-10: 20 µM/ partial protection in UW; QSA-10 have the potential to increase the efficacy of organ preservation
Kondo et al., 2013 [31]	Phosphoenolpyruvate	Mouse liver (ex vivo)	UW, PBS 24 h, 48 h, 72 h, 4 °C; SCS	I1: UW + PEP I2: PBS + PEP C1: UW C2: PBS	PEP: 1 mM, 10 mM, 100 mM (PBS) PEP: 100 mM (UW)	↓ oxidative stress; attenuate ATP depletion; prevents increases in biochemical parameters
Polyphenols
Johnston et al., 1999 [32]	Curcumin	Sprague-Dawley rat livers (ex vivo)	UW, EC 24 h; 4 °C; oxygenated perfusion	I1: EC+ CUR C1: EC C2: UW	100 μM	curcumin-enhanced EC solution was equivalent to the UW solution
Chen et al., 2006 [33]	Curcumin	Sprague-Dawley rat livers	UW, EC, PBS 24 h, 36 h, 48 h; 4 °C; SCS	I1: UW + CUR I2: EC+ CUR I3: PBS+ CUR C1: UW C2: EC C3: PBS	25–200 μM	curcumin at 100 μM concentration had the optimal preservation characteristics; ↑ portal flow rates and bile production; ↓ ALT, AST, LDH; improves the quality of organs
McNally et al., 2006 [34]	Curcumin	Human hepatocytes	UW 16 h, 24 h, 48 h, 72 h; 4 °C; SCS	I: UW + CUR C: UW	10 μM	induces HO-1; maximum protection cells between 16 and 24 h of CS
Kato et al., 2020 [35]	Quercetin	Rat (isolated hepatocytes and whole liver)	UW 24 h; 4 °C; SCS	I: UW + QE C: UW	QE: 0.33; 33.1 µM/L Suc: 0.1 M/L	optimal dose of QE: 33,1 µM/L ↓ ALT mild vascular degradation; improvement of histological changes
Ligeret et al., 2008 [36]	Silibinin	Isolated perfused rat liver model	UW 24 h; 4 °C; SCS	I: UW + SB C: UW	100 μM	↑ ATP, RCR ↓ oxidative stress
Chiu et al., 1999 [37]	Magnolol	Rat	UW, Ringer’s lactate 24 h, 48 h; 96 h, 4 °C; SCS	I1: UW + MAG I2: Ringer’s lactate + MAG C1: UW C2: Ringer’s lactate	10–6 M/L	↓ lipid peroxidation
Bioactive metabolites from marine algae
Gdara et al., 2018 [38]	Phycocyanin	Rat	KH 12 h, 24 h; 4 °C; SCS	I: KH + Pc C:KH	0.1; 0.2 mg ml	⁻¹	g	⁻¹	of liver	optimal dose of Pc: 0.1 mg ml	⁻¹	g	⁻¹	; ↓ ALT, AST, ALP; ↓ MDA; ↓ GST, GPx
Slim et al., 2020 [39]	Fucoidan	Isolated perfused rat liver model	IGL-1 24 h, 4 °C; SCS	I1: IGL-1 + FUC C1: IGL-1 C2: Ringer’s lactate	10 mg/L, 50 mg/L, 100 mg/L, 250 mg/L	optimal dose of FUC: 100 mg/L; ↓ ALT, AST; ↑ phosphorylation of AMPK, AKT protein kinase, and GSK-3β; reduction in apoptosis (caspase 3); reduction of mitochondrial damage; reduction oxidative stress; reduction of ER stress markers; ↓ ERK1/2 and p38 MAPKs phosphorylation
Vitamins and vitamin-like substances
Homozygous obese and lean Zücker rats
IGL-1

24 h, 4 °C; SCS
I: IGL-1 + TMZ
	C: IGL-1	10	⁻⁵	M/L	↓ ALT, AST (especially in steatotic livers);	↑ SIRT1 protein levels; ↓ HMGB1 protein level; ↓ TNF-α release; increasing the tolerance of steatotic liver graft against cold IRI
Pantazi et al., 2015 [50]	Trimetazidine	Rat orthotopic liver transplantation	IGL-1 8 h, 4 °C; SCS	I: IGL-1 + TMZ C: IGL-1	10	⁻⁶	M/L	↓ ALT, GLDH; Reduction of oxidative stress; reduction of mitochondrial damage; enhanced SIRT1 protein expression
Kozaki et al., 1995 [51]	Pentoxifylline	Isolated perfused rat liver model	UW 4 h, 24 h; 0–4 °C; SCS	I: UW + PTX C: UW	25 mg PTX/kg body weight	the Kupffer cells produced significantly less O	₂⁻	and TNF-α
Arnault et al., 2003 [
Mitochondria-targeted antioxidants
Aghdaie et al., 2019 [56]	α-Lipoic acid (ALA) ursodeoxycholic acid (UDCA)	Isolated human hepatocytes derived from livers of deceased donors	UW 24 h; 4 °C; SCS	I1: UW + α-Lipoic acid I2: UW + ursodeoxycholic acid C: UW	ALA: 5 mM/L UDCA: 5 mM/L	does not increase the number of viable hepatocytes
Polyphenols
Otani et al., 2023 [57]	Quercetin	Isolated pig livers	UW 6 h, 4 °C; SCS	I: UW+ QE C:UW	QE: 33.1 µM/L Suc: 0.1 M/L	↓ ALT, AST, LDH; improvement of histological changes; prevent tissue edema
Drugs							Bae et al., 2014 [40]	α-Tocopherol	Wistar rats after cardiac death	Vasosol 4 °C; HMP	I: Vasosol + α-TCP C1: Vasosol C2: KPS-1	5.4 × 10	⁻²	mM	↓ ALT; ↓ inflammatory cytokines (IL-6, TNF-α, MCP-1); ↓ caspase 3/7 expression in the circulation
Tolba et al., 2003 [41]	L-carnitine	Isolated perfused rat liver model	HTK 24 h, 4 °C; SCS	I: HTK + L-CAR C: HTK	5 mM	↓ ALT, GLDH; improved the hepatic energy metabolism; preserved integrity of the mitochondria and the endoplasmic reticulum
Coskun et al., 2007 [42]	L-carnitine	Wistar Albino rat	UW 2 h, 24 h, 36 h, 48 h; 4 °C; SCS	I: UW + L-CAR C: UW	5 mM/L	↓ ALT, ACP; ↓ MDA
52]	Pentoxifylline	Isolated perfused Wistar rat liver model	UW 18 h, 24 h; 4 °C; SCS
Qing et al., 2006 [58]	Pentoxifylline	Simple porcine orthotopic liver transplantation	Drugs
UW	Ben et al., 2010 [43]	Carvedilol	Isolated Zücker rat liver (steatotic and non-steatotic)	UW 24 h; 4 °C; SCS	I: UW + CVD C: UW	10	⁻⁵	M/L	reduced hepatic injury, and improved hepatic functionality in both liver types
Ben et al., 2006 [44]	Trimetazidine	Zücker rats (steatotic and non-steatotic)	UW 24 h; 4 °C; SCS	I: UW + TMZ C: UW	10	⁻⁶	M/L	protects against mitochondrial damage; preserves more ATP; decreases oxidative stress higher bile production
Ben et al., 2007 [45]	Trimetazidine, aminoimidazole-4-carboxamide ribonucleoside	Zücker rats (steatotic and non-steatotic)	UW 24 h; 4 °C; SCS	I1: UW + TMZ I2: UW + AICAR C: UW	TMZ: 10	⁻⁸	M/L 10	⁻⁶	M/L, 10	⁻⁴	M/L AICAR: 10 μM/L, 20 μM/L, 40 μM/L, 80 μM/L TMZ + AICAR: 10	⁻⁶	M/L + 20 μM/L; 10	⁻⁶	M/L + 40 μM/L;	↓ AST (TMZ) TMZ improved bile production; increase in cNOS via increasing AMPK; TMZ and AICAR protected, with a similar degree of effectiveness, against cold I/R injury in steatotic and non-steatotic livers; not necessary to combine AICAR and TMZ
Zaouali et al., 2010 [46]	Trimetazidine	Zücker rats	IGL-1 24 h; 4 °C; SCS	I: IGL-1 + TMZ C: IGL-1	10	⁻⁶	M/L	↓ AST, ALT; higher bile production; increased NO production; HIF-1α accumulation; increased HO-1 expression
Zaouali et al., 2013 [47]	Trimetazidine melatonin	Zücker rats (steatotic)	UW, IGL-1 24 h, 4 °C; SCS	I2: IGL-1 + TMZ +MEL C1: IGL-1 C2: UW	TMZ: 10	⁻³	µM MEL: 100 µM	↑ liver autophagy; ↓ GRP78, pPERK, and CHOP after reperfusion; improved steatotic liver preservation through AMPK activation; synergism of action MEL and TMZ
Zaouali et al., 2017 [48]	Trimetazidine	Sprague-Dawley rats	UW, IGL-1 8 h, 4 °C; SCS	I1: IGL-1 + TMZ C: IGL-1 C: UW	10	⁻⁶	M/L	↓ ALT, GLDH, MDA; protects the mitochondria; inhibited of GSK3β and VDAC phosphorylation; reduced apoptosis; decreased ER stress
Zaouali et al., 2017 [49]	Trimetazidine	I: UW + PTX		C: UW	30 mM (during cold storage) 3 mM (at reperfusion);	improve microcirculation in the liver; decrease in vascular resistance at reperfusion of 18 h and 24 h; decreased number of foci of peliosis after an 18 h preservation; ↓ LDH, AST, ALT after 24 h cold ischemia time
Asong-Fontem et al., 2021 [53]	M101	Zücker rats	IGL-1 24 h, 4 °C, SCS 2 h, 37 °C, NR	I: IGL-1 + M101 C: IGL-1	1 g/L	↓ AST, ALT, Lactate, GLDH; ↓ MDA; higher production of NO2-NO3; less inflammation (HMGB1)

Table 2.

Studies on the effectiveness of supplementing preservative fluids with antioxidants. Preclinical and clinical studies.

Author, Year of Publication	Antioxidant

Abbreviations: ACP, acid phosphatase; AICAR, aminoimidazole-4-carboxamide ribonucleoside; ALA, α-Lipoic acid; ALT, alanine aminotransferase; AMI, amifostine; AMPK, AMP-activated protein kinase; AraA, adenine9-b-D-arabinofuranoside; AST, aspartate transferase; ATP, adenosine triphosphate; CHOP, C/EBP homologous protein; cNOS, constitutive nitric oxide synthase; CUR, curcumin; CVD, carvedilol; DFX, Deferoxamine; DTT, Dithiothreitol; EC, Euro-Collins solution; ER, endoplasmic reticulum; ERK1/2, extracellular signal-regulated protein kinase; FUC, Fucoidan; GLDH, glutamate dehydrogenase; Gly, glycine; GRP78, glucose-regulated protein 78; GSH, reduced glutathione; GSK-3β, glycogen synthase kinase 3 beta; GSNO, S-Nitrosoglutathione; GSSG, oxidized glutathione; HMGB1, High-mobility Group Box 1; HMP, hypothermic machine perfusion; HO-1, heme oxygenase 1; HOPE, hypothermic oxygen perfusion; HTK, histidine–tryptophan–ketoglutarate; IL-6, interleukin 6; Iκb, inhibitor of nuclear factor kappa; KH, Krebs–Henseleit; KPS-1, kidney machine perfusion solution; L-CAR, L-carnitine; LDH, lactate dehydrogenase; LSEC, liver sinusoidal endothelial cell; M101, Hemo2Life^®; MAG, magnolol; MCP-1, monocyte chemotactic protein 1;MEL, melatonin; NAC, N-Acetylcysteine; NLRP3, pyrin domain-containing protein 3; NR, normothermic reperfusion; Nrf2, nuclear factor-erythroid 2-related factor 2; OXPHOS, oxidative phosphorylation complexes; p38 MAPK, mitogen-activated protein kinase; PBS, phosphate buffer saline; PEP, phosphoenolpyruvate; PINK1, PTEN-induced kinase 1; pPERK, protein kinase R-like endoplasmic reticulum kinase; PTX, Pentoxifylline; PVP, portal venous pressure; QE, quercetin; QSA-10, idebenone; RCR, respiratory control ratio; RL, Ringer Lactate; SB, silibinin; SCS, static cold storage; SIRT1, Sirtuin 1; p-p38, phosphor-p38; SkQ1, 10-(6′-plastoquinonyl)decyltriphenylphosphonium; SNAC, S-nitroso-N-acetylcysteine; Suc, sucrose; TAU, taurine; TMZ; trimetazidine; TNF-α, tumor necrosis factor α; TRX-C, Trolox-C; UCP2, uncoupling protein 2; UDCA, ursodeoxycholic acid; UW, University of Wisconsin solution; VDAC, voltage-dependent anion channel; VSOP, venous systemic oxygen persufflation; α-TCP, α-Tocopherol; ↑ increase ↓ decrease.