Hyperbaric Oxygen Preconditioning Induces Tolerance against Oxidative Injury and Oxygen-Glucose Deprivation by Up-Regulating Heat Shock Protein 32 in Rat Spinal Neurons
et al. (2014) Hyperbaric Oxygen Preconditioning Induces Tolerance against Oxidative Injury and Oxygen-Glucose
Deprivation by Up-Regulating Heat Shock Protein 32 in Rat Spinal Neurons. PLoS ONE 9(1): e85967. doi:10.1371/journal.pone.0085967
Hyperbaric Oxygen Preconditioning Induces Tolerance against Oxidative Injury and Oxygen-Glucose Deprivation by Up-Regulating Heat Shock Protein 32 in Rat Spinal Neurons
Guoyang Huang 0
Jiajun Xu 0
Li Xu 0
Shifeng Wang 0
Runping Li 0
Kan Liu 0
Juan Zheng 0
Zhiyu Cai 0
Kun Zhang 0
Yuandeng Luo 0
Weigang Xu 0
Ken Arai, Massachusetts General Hospital/Harvard Medical School, United States of America
0 1 Department of Diving and Hyperbaric Medicine, Faculty of Naval Medicine, Second Military Medical University , Shanghai , People's Republic of China, 2 Naval Medical Institute , Shanghai , People's Republic of China
Objective: Hyperbaric oxygen (HBO) preconditioning (HBO-PC) has been testified to have protective effects on spinal cord injury (SCI). However, the mechanisms remain enigmatic. The present study aimed to explore the effects of HBO-PC on primary rat spinal neurons against oxidative injury and oxygen-glucose deprivation (OGD) and the relationship with heat shock proteins (HSPs). Methods: Primary rat spinal neurons after 7 days of culture were used in this study. HSPs were detected in rat spinal neurons following a single exposure to HBO at different time points by Western blot. Using lactate dehydrogenase release assay and cell counting kit-8 assay, the injuries induced by hydrogen peroxide (H2O2) insult or OGD were determined and compared among neurons treated with HBO-PC with or without HSP inhibitors. Results: The results of Western blot showed that HSP27, HSP70 and HSP90 have a slight but not significant increase in primary neurons following HBO exposure. However, HSP32 expression significantly increased and reached highest at 12 h following HBO exposure. HBO-PC significantly increased the cell viability and decreased the medium lactate dehydrogenase content in cultures treated with H2O2 or OGD. Pretreatment with zinc protoporphyrin IX, a specific inhibitor of HSP32, significantly blocked the protective effects of HBO-PC. Conclusions: These results suggest that HBO-PC could protect rat spinal neurons in vitro against oxidative injury and OGD mostly by up-regulating of HSP32 expression.
Funding: This work was supported by the National Natural Science Foundation of China, No. 81171873. The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
Spinal cord injury (SCI) is a serious health problem, which may
result from surgical operations on the spinal column or
thoracoabdominal aorta, or decompression sickness (DCS) associated with
sport or commercial diving. The reported rate of paraplegia or
paraparesis after thoracoabdominal aortic aneurysms surgery
varies from 3.8% to 16.7% [1,2] and the incidence of injured
divers with incomplete recovery from neurological DCS has been
reported between 20% and 30% . Under those high risk
conditions, effective preventive measures are in desperate need to
reduce the incidence of SCI.
Hyperbaric oxygen (HBO) therapy is the way that a person
breathes pure oxygen under a pressure greater than 1 atmosphere
absolute in a specially designed chamber . It is widely used in
the management of various diseases and conditions including
DCS, carbon monoxide (CO) poisoning, gas embolism and
neurologic diseases . As an effective therapy for SCI, HBO
has been reported to accelerate neurologic recovery by reversing
hypoxia, ameliorating mitochondrial dysfunction, arresting the
spread of hemorrhage, reducing edema, up-regulating the activity
of antioxidant enzymes and decreasing the production of
inflammatory factors . In addition, recent studies have found
that preconditioning with HBO could be an effective preventive
measure to alleviate SCI. In 2002, HBO preconditioning
(HBOPC) was first applied in experimental SCI with beneficial effects
. Later studies demonstrated that HBO-PC exerted its
neuroprotective effects by up-regulating the activity of catalase
and superoxide dismutase, promoting axonal growth and
suppressing mitochondrial apoptosis pathway . Our
previous work found that HBO-PC alleviated SCI by
upregulating of heat shock protein (HSP) 70 expression in a DCS
rat model . However, there is still short of evidence about the
direct effects of HBO-PC on spinal neurons in vitro and the
HSPs normally perform as molecular chaperones and are called
protein guardians because they play a crucial role in repairing
partially damaged proteins . In the setting of SCI, HSPs
induction has been shown to be beneficial, which can alleviate SCI
through its anti-inflammatory and anti-apoptotic actions [15,16].
The aim of the present study was to observe the protective
effects of HBO-PC on primary rat spinal neurons, and to explore
the possible roles of HSPs.
Materials and Methods
The experimental procedures were carried out in accordance
with the National Institutes of Health Guidelines on the Use of
Laboratory Animals and approved by the Institutional Animal
Care and Use Committee of the Second Military Medical
Primary culture of rat spinal neurons was done using the
method described by Jiang et al. with minor modifications .
Spinal neurons were obtained from embryonic day 1415
Sprague-Dawley rats. The spinal cords were rapidly dissected
from embryo and cut into 1 mm3 slices in a 35 mm diameter dish
at 4uC. The slices were then digested in pre-warmed 0.05% trypsin
(Invitrogen, USA) at 37uC for 18 min in a conical flask agitated by
hand every 5 min. After digestion, the supernatant was removed
and the remained trypsin was inactivated with Dulbeccos
modified Eagles medium (DMEM; Invitrogen) supplemented
with 20% heat-inactivated fetal bovine serum (Invitrogen) at room
temperature (RT). Afterwards, tissues were centrifuged for 5 min
at 1,500 rpm. The supernatant was removed and the tissues were
resuspended in DMEM containing 10% heat-inactivated fetal
bovine serum, 10% heat-inactivated horse serum (Invitrogen) and
triturated 1520 times with a fire-polished Pasteur pipette. After
the slices were let to settle, single cell suspensions were collected,
counted with a hemocytometer, and diluted to a density of
16106 cells/ml. The cells were plated onto poly-L-lysine-
(molecular weight, 30,00070,000; Sigma, USA) coated culture plates at
a density of 1.26106 cells/well on 6-well plates (for western blot)
or 36105 cells/well on 12-well plates (for lactate dehydrogenase
assays; LDH) or 16105 cells/well on coverslips (for
immunocytochemistry) or 0.56105 cells/well on 96-well plates (for cell
counting kit-8 assays; CCK-8), then the cultures were kept at
37uC in a 5% CO2 humidified incubator. Four hours later, the
medium was replaced with serum-free neurobasal medium
(Invitrogen) supplemented with 2% B27 supplement (Invitrogen),
100 U/ml penicillin, 100 mg/ml streptomycin, and 0.5 mM
glutamine (Invitrogen). 25 mM glutamate (Invitrogen) was added
during the first day in vitro. On the second day, 5 mM
cytosine-bD-arabinofuranoside (Sigma, USA) was added into the medium
for 24 h to inhibit non-neuronal cell division. After that, half
volume of culture media was replaced with fresh media every 3
days. The cultures consisted of more than 90% neurons as
identified by immunocytochemistry staining and those cultures
were utilized for the experiments described below at 7 days in
Identification of cultured cells
Rat spinal neurons were identified by immunocytochemistry
staining. Briefly, at 7 d in vitro, cultures on PLL-coated coverslips
were fixed with 4% paraformaldehyde (PFA) for 30 min, and
rinsed three times with 0.01 M phosphate-buffered saline (PBS)
and blocked with 5% BSA for 20 min at RT. The monoclonal
rabbit anti-rat primary antibody against b-tubulin III (Sigma) was
added to the cell slides and co-cultured with the cells in a
humidified chamber overnight at 4uC. After three washes with
PBS, cultures were incubated for 1 h at 37uC with the
HRPconjugated goat anti-rabbit antibody (Sigma). Then cultures were
stained with peroxidase substrate diaminobenzidine. 49,
6diamidino-2-phenylindole (DAPI; Sigma) was used to stain nuclei
of all cells, including neurons and glial cells. The purity of cultured
spinal neuron was expressed as the percentage of b-tubulin III
positive cells relative to the total number of DAPI labeled nuclei.
Hyperbaric oxygen preconditioning
HBO exposure was performed in a temperature and humidity
controlled hyperbaric incubator (OxyCure 3000, OxyHealH
Health Group, USA). The pressure-duration was 280
kPa60 min, which is frequently used in animal and cell study. The
compression and decompression were both carried out within
5 min. The chamber was flushed and compressed with pure
oxygen containing 1.79% CO2. Thus, at pressure of 280 kPa, the
partial pressure of CO2 reached 5 kPa to maintain a physiological
pH of the culture. All the pressures described in this text are
Determination of heat shock protein
HSP27, 32, 70 and 90 were determined at 0, 6, 12, 18, 24, and
30 h following HBO exposure by Western blot and verified by
Western blot. The neurons were harvested and lysed in
Radio-Immunoprecipitation Assay Lysis Buffer (Sigma). Protein
samples were electrophoresed on 8% SDS-polyacrylamide gels,
and transferred onto a polyvinyldifluoridine membrane (Millipore,
USA) and detected with rabbit monoclonal primary antibodies
directed against rat HSP27 (Cell Signaling Technology, USA),
HSP32 (Abcam, USA), HSP70 (GeneTex, USA), HSP90 (Cell
Signaling Technology) or b-actin (Abcam). Proteins were
visualized by using HRP-conjugated goat anti-rat IgG (Abcam) and the
intensity of each band was measured by using Kodak Digital
Science 1D Image Analysis System (Eastman Kodak, USA).
Immunofluorescence staining. The results of western blot
showed that only HSP32 was significantly expressed (see in
Results). To verify the expression, the cultures on PLL-coated
coverslips were fixed with 4% PFA for 30 min, blocked with 5%
BSA for 20 min at RT and incubated with monoclonal rabbit
antirat primary antibody against HSP32 (Abcam) overnight at 4uC
and then with the FITC-conjugated goat anti-rabbit IgG (Sigma)
for 1 h at 37uC, and mounted with Gel/Mount aqueous mounting
media containing DAPI. Then cultures were observed under a
Determination of the effects of HBO-PC on neurons
Groups and treatments. Neuron injuries were induced at
12 h after the HBO-PC using hydrogen peroxide (H2O2) or
oxygen-glucose deprivation (OGD). Cultures were randomly
divided into nine groups (n = 4, each with 6 parallel wells): (1)
Air, (2) HBO, (3) Air +H2O2, (4) Air +OGD, (5) HBO+H2O2, (6)
HBO+OGD, (7) HBO+H2O2+Zn-pp, (8) HBO+OGD+Zn-pp, (9)
Air +Zn-pp. For H2O2 mediated oxidative injury, H2O2 (Sigma)
was diluted into the culture medium at a final concentration of
200 mM and maintained for 4 h at 37uC. For OGD, the cells were
cultured with sugar-free Earles solution (in mmol/L: 121.7 NaCl,
0.8 MgSO4, 20.7 NaHCO3, 5.5 KHCO3, 1 NaH2PO4, 1.8 CaCl2,
0.01 glycine, and 10 HEPES, pH 7.4) in an anaerobic chamber
(Thermo Forma Scientific, USA) that was filled with an anoxic gas
mixture (5% CO2 and 95% N2) at 37uC for 2 h and for another
24 h under normal conditions. Control cells were incubated in the
same solution with glucose (4.5 g/L) under normal conditions.
Znic protoporphrin IX (Zn-pp; Sigma), a specific inhibitor of
HSP32, was dissolved in complete dimethyl sulfoxide (DMSO, the
final concentration in the culture medium was 0.15%), and added
into the cultures immediately before HBO treatment at a final
concentration of 3 mM. Group 8 and 9 were adopted to show
whether HBO or Zn-pp alone has effects on the determined
Injury parameter determination. To determine whether
the protocol of HBO-PC can protect rat spinal neurons from
H2O2-induced oxidative injury or OGD, we detected the cell
viability and the medium lactate dehydrogenase (LDH) content
after those injuries using cell counting kit-8 (CCK-8) assay
(Dojindo Laboratories, Japan) and LDH release assay (Nanjing
Jiancheng Bioengineering Institute, China), respectively. Medium
LDH content was measured 24 h after injuries following the
manufactures instruction. After injuries, the medium was replaced
with normal medium and CCK-8 solution was added to the cell
culture medium at a final concentration of 10 ml/100 ml, and
incubated for an additional 3.5 h under normal conditions, the
absorbance at 450 nm were measured.
All data are presented as mean 6 standard deviation (SD).
Statistical analysis was made using one-way ANOVA and was
performed with SPSS software (version 16.0). A P-value of less
than 0.05 was considered to be statistically significant.
Identification of spinal neurons
Immunocytochemistry staining of cultures at 7 d in vitro
showed that over 90% of the cells counterstained with DAPI
were immuno-positive for b-tubulin III, a marker of neurons,
which confirmed their neuronal identity (Figure 1).
HSP expression after HBO exposure
The results of Western blot showed that HSP27, HSP70 and
HSP90 had a slight and non-significant increase after HBO
exposure, while for HSP32, it increased and reached at a peak
level at 12 h after HBO exposure (P,0.01; Figure 2).
Immunofluorescence staining further verified that the expression of HSP32
significantly increased in neuronal cultures at 12 h after HBO-PC
(Figure 3). Therefore, the 12 h time point following HBO
exposure was selected to observe the effects of HBO-PC.
The effects of HBO-PC on spinal neurons against injuries
As shown in Figure 4, both H2O2 insult and OGD significantly
decreased the cell viability and increased the medium LDH
content compared with the normal control (P,0.01). HBO-PC
significantly increased the cell viabilities and decreased the
medium LDH contents (P,0.01). HBO has no effects on the
viability or LDH release of the neurons under normal conditions.
The role of HSP32 in HBO-PC on spinal neurons against
To explore the role of HSP32 in HBO-PC induced
neuroprotection, Zn-pp was added into the medium at the final
concentration of 3 mM immediately before HBO exposure.
Pretreatment with Zn-pp significantly offset the protective effects
induced by HBO (P,0.01; Figure 4). Zn-pp at 3 mM has no effects
on the viability or LDH release of the neurons under normal
SCI includes primary and secondary injury processes. Primary
injury is immediate and irreversible, which results in direct
damage to spinal cord tissues. The delayed secondary damage
defines a cascade of chemical and physiological events that are
initiated by an original insult including ischemia-reperfusion injury
(IRI), edema, inflammation, excitotoxicity and oxidative cell
damage [18,19]. Among those events, oxidative stress and IRI
are two pivot mechanisms . It has been proved that HBO-PC
is effective to increase the ability of spinal cord to counteract
oxidative stress and IRI insults in rats and rabbits [10,12,13].
In this study, we detected the protective effects of HBO-PC on
primary rat spinal neurons against oxidative injury induced by
H2O2 or OGD, a cell IRI model. The results showed that a single
episode of HBO exposure 12 h before significantly enhanced the
ability of spinal neurons to counteract the oxidative or OGD
insults, which is related to HSP32 expression.
As a safe and clinically viable therapy, HBO has been widely
used in the management of various diseases and conditions
including DCS, carbon monoxide poisoning, gas embolism and
neurologic diseases . In addition to elevating the partial pressure
of oxygen, HBO causes moderate oxidative stress and further
induces the expression of cell protective proteins, which can
enhance the cellular tolerance against harmful stimuli .
HSP family plays a crucial role in maintaining cell homeostasis
and survival against various harmful stimuli . The inducible
members including HSP 27, 32, and 70 are associated with
cellular protection . HSP90 is constitutive and regulates
numerous client proteins to counteract various injuries and in
some circumstances is also inducible [23,24]. HBO has been found
to enhance the expression of the above HSPs in liver, heart, brain,
kidney and some cell lines . Our previous work found that
HBO-PC significantly induced the expression of HSP70 in rat
spinal cord and lung, and contributed to the protection against
DCS injuries . A significant increasing of HSP70 expression
but not others was also observed in human umbilical vein
endothelial cells after a single exposure to HBO (our unpublished
data). However, in this study only HSP32 increased significantly
and reached a peak level at 12 h following a same profile of HBO
exposure. These results indicate that HBO induces different
subtype of HSPs in different organs or cells, in vivo or in vitro. The
underlying mechanisms deserve further study.
In this study, the protective effects of HBO on rat spinal neurons
were significantly blocked by Zn-pp, which can effectively compete
for iron protoporphyrin, the natural substrate of HSP32, and
inhibit enzyme activity . This and the increased level of
HSP32 expression following HBO exposure suggests that the
beneficial effects of HBO on rat spinal neurons against oxidative
injury and OGD were achieved by up-regulating the expression of
HSP32, also known as heme oxygenase-1, is one of the
ratelimiting enzymes in heme catabolism, which leads to the
generation of biliverdin, ferrous iron, and CO . Recent data
indicates that CO can exert anti-inflammatory and anti-apoptotic
effects by modulating the MAPK-signaling pathways or activating
soluble guanylate cyclase . Ferrous iron released from heme
can maintain cells with a low level of iron pools and enhance cell
antioxidant capacity by increasing the expression of ferritin and
iron ATPase pump . The beneficial roles of biliverdin and
bilirubin are to act as physiological antioxidants by efficiently
scavenging peroxyl radicals . The exact mechanism by which
HSP32 exerts its neuroprotective effects in vitro warrants further
A previous study has reported that HBO-PC protected primary
cultured mice spinal neurons against H2O2-induced oxidative
injury via up-regulating the expression of HSP32 . However,
in that study, the profile of HBO treatment used was 350 kPa,
98% O2 and 2% CO2 for 2 h. Except for the higher pressure of
O2 never used in real practice, the partial pressure of CO2 in the
hyperbaric mixture reached 7 kPa, which was significant higher
than in the sham pretreatment groups and control groups (2 kPa),
and is higher than that required to maintain the acid-base
equilibrium for the culture medium (5 kPa). Thus, the influence of
CO2 on the results cannot be ruled out.
In conclusion, this study revealed that a single HBO exposure
increased the ability of primary rat spinal neurons to counteract
oxidative or OGD injuries mostly by up-regulating the expression
of HSP32. Whether a double or multi-exposure further enhances
the expression of HSPs and simultaneously the protective effects
deserve further study. As a feasible and safe treatment modality,
HBO-PC may be a promising way to alleviate the possible SCI in
some scheduled operations, such as surgeries on thoracoabdominal
aorta and spinal column and diving practice.
Conceived and designed the experiments: WGX GYH JJX. Performed the
experiments: GYH LX KL JZ. Analyzed the data: WGX SFW ZYC KZ
YDL. Wrote the paper: WGX JJX GYH RPL.
Figure 4. Cell viability and LDH levels in culture medium after oxidative injury or OGD insult. HBO-PC significantly increased the viability
of neurons and decreased the medium LDH content. (A) H2O2 injury, (B) OGD insult. Pretreatment with Zn-pp (3 mM) significantly blocked the
protective effects. HBO or Zn-pp has no effects on the parameters under normal conditions. #, *, &: P,0.01.
1. Coselli JS , Bozinovski J , LeMaire SA ( 2007 ) Open surgical repair of 2286 thoracoabdominal aortic aneurysms . Ann Thorac Surg 83 : S862-864; discussion S890-862.
2. Verhoeven EL , Tielliu IF , Bos WT , Zeebregts CJ ( 2009 ) Present and future of branched stent grafts in thoraco-abdominal aortic aneurysm repair: a singlecentre experience . Eur J Vasc Endovasc Surg 38 : 155 - 161 .
3. Blatteau JE , Gempp E , Simon O , Coulange M , Delafosse B , et al. ( 2011 ) Prognostic factors of spinal cord decompression sickness in recreational diving: retrospective and multicentric analysis of 279 cases . Neurocrit Care 15 : 120 - 127 .
4. Grim PS , Gottlieb LJ , Boddie A , Batson E ( 1990 ) Hyperbaric oxygen therapy . JAMA 263 : 2216 - 2220 .
5. Al-Waili NS , CHT GJB , Beale J , Abdullah MS , Hamilton RB , et al. ( 2005 ) Hyperbaric oxygen in the treatment of patients with cerebral stroke, brain trauma, and neurologic disease . Adv Ther 22 : 659 - 678 .
6. Tai PA , Chang CK , Niu KC , Lin MT , Chiu WT , et al. ( 2010 ) Attenuating experimental spinal cord injury by hyperbaric oxygen: stimulating production of vasculoendothelial and glial cell line-derived neurotrophic growth factors and interleukin-10 . J Neurotrauma 27 : 1121 - 1127 .
7. Topuz K , Colak A , Cemil B , Kutlay M , Demircan MN , et al. ( 2010 ) Combined hyperbaric oxygen and hypothermia treatment on oxidative stress parameters after spinal cord injury: an experimental study . Arch Med Res 41 : 506 - 512 .
8. Al-Waili NS , Butler GJ , Beale J , Abdullah MS , Hamilton RW , et al. ( 2005 ) Hyperbaric oxygen in the treatment of patients with cerebral stroke, brain trauma, and neurologic disease . Adv Ther 22 : 659 - 678 .
9. Dong H , Xiong L , Zhu Z , Chen S , Hou L , et al. ( 2002 ) Preconditioning with hyperbaric oxygen and hyperoxia induces tolerance against spinal cord ischemia in rabbits . Anesthesiology 96 : 907 - 912 .
10. Nie H , Xiong L , Lao N , Chen S , Xu N , et al. ( 2006 ) Hyperbaric oxygen preconditioning induces tolerance against spinal cord ischemia by upregulation of antioxidant enzymes in rabbits . J Cereb Blood Flow Metab 26 : 666 - 674 .
11. Lu PG , Hu SL , Hu R , Wu N , Chen Z , et al. ( 2012 ) Functional recovery in rat spinal cord injury induced by hyperbaric oxygen preconditioning . Neurol Res 34 : 944 - 951 .
12. Wang L , Li W , Kang Z , Liu Y , Deng X , et al. ( 2009 ) Hyperbaric oxygen preconditioning attenuates early apoptosis after spinal cord ischemia in rats . J Neurotrauma 26 : 55 - 66 .
13. Ni XX , Ni M , Fan DF , Sun Q , Kang ZM , et al. ( 2013 ) Heat-shock protein 70 is involved in hyperbaric oxygen preconditioning on decompression sickness in rats . Exp Biol Med (Maywood) 238 : 12 - 22 .
14. Reddy SJ , La Marca F , Park P ( 2008 ) The role of heat shock proteins in spinal cord injury . Neurosurg Focus 25 : E4 .
15. Zhang Q , Hu W , Meng B , Tang T ( 2010 ) PPARgamma agonist rosiglitazone is neuroprotective after traumatic spinal cord injury via anti-inflammatory in adult rats . Neurol Res 32 : 852 - 859 .
16. Robinson MB , Tidwell JL , Gould T , Taylor AR , Newbern JM , et al. ( 2005 ) Extracellular heat shock protein 70: a critical component for motoneuron survival . J Neurosci 25 : 9735 - 9745 .
17. Jiang XY , Fu SL , Nie BM , Li Y , Lin L , et al. ( 2006 ) Methods for isolating highlyenriched embryonic spinal cord neurons: a comparison between enzymatic and mechanical dissociations . J Neurosci Methods 158 : 13 - 18 .
18. Juurlink BH , Paterson PG ( 1998 ) Review of oxidative stress in brain and spinal cord injury: suggestions for pharmacological and nutritional management strategies . J Spinal Cord Med 21 : 309 - 334 .
19. Norenberg MD , Smith J , Marcillo A ( 2004 ) The pathology of human spinal cord injury: defining the problems . J Neurotrauma 21 : 429 - 440 .
20. Thom SR ( 2009 ) Oxidative stress is fundamental to hyperbaric oxygen therapy . J Appl Physiol 106 : 988 - 995 .
21. Macario AJ , Conway de Macario E ( 2007 ) Molecular chaperones: multiple functions, pathologies, and potential applications . Front Biosci 12 : 2588 - 2600 .
22. Chow AM , Tang DW , Hanif A , Brown IR ( 2013 ) Induction of heat shock proteins in cerebral cortical cultures by celastrol . Cell Stress Chaperones 18 : 155 - 160 .
23. Latchman DS ( 2001 ) Heat shock proteins and cardiac protection . Cardiovasc Res 51 : 637 - 646 .
24. Redaelli CA , Wagner M , Kulli C , Tian YH , Kubulus D , et al. ( 2001 ) Hyperthermia-induced HSP expression correlates with improved rat renal isograft viability and survival in kidneys harvested from non-heart-beating donors . Transpl Int 14 : 351 - 360 .
25. Soejima Y , Ostrowski RP , Manaenko A , Fujii M , Tang J , et al. ( 2012 ) Hyperbaric oxygen preconditioning attenuates hyperglycemia enhanced hemorrhagic transformation after transient MCAO in rats . Med Gas Res 2 : 2 - 9 .
26. Liu Y , Sun XJ , Liu J , Kang ZM , Deng XM ( 2011 ) Heme oxygenase-1 could mediate the protective effects of hyperbaric oxygen preconditioning against hepatic ischemia-reperfusion injury in rats . Clin Exp Pharmacol Physiol 38 : 675 - 682 .
27. He X , Xu X , Fan M , Chen X , Sun X , et al. ( 2011 ) Preconditioning with hyperbaric oxygen induces tolerance against renal ischemia-reperfusion injury via increased expression of heme oxygenase-1 . J Surg Res 170 : e271 - 277 .
28. Shyu WC , Lin SZ , Saeki K , Kubosaki A , Matsumoto Y , et al. ( 2004 ) Hyperbaric oxygen enhances the expression of prion protein and heat shock protein 70 in a mouse neuroblastoma cell line . Cell Mol Neurobiol 24 : 257 - 268 .
29. Maines MD ( 1981 ) Zinc. protoporphyrin is a selective inhibitor of heme oxygenase activity in the neonatal rat . Biochim Biophys Acta 673 : 339 - 350 .
30. Maines MD ( 1997 ) The heme oxygenase system: a regulator of second messenger gases . Annu Rev Pharmacol Toxicol 37 : 517 - 554 .
31. Tsuchihashi S , Fondevila C , Kupiec-Weglinski JW ( 2004 ) Heme oxygenase system in ischemia and reperfusion injury . Ann Transplant 9 : 84 - 87 .
32. Baranano DE , Wolosker H , Bae BI , Barrow RK , Snyder SH , et al. ( 2000 ) A mammalian iron ATPase induced by iron . J Biol Chem 275 : 15166 - 15173 .
33. Stocker R , Yamamoto Y , McDonagh AF , Glazer AN , Ames BN ( 1987 ) Bilirubin is an antioxidant of possible physiological importance . Science 235 : 1043 - 1046 .
34. Li Q , Li J , Zhang L , Wang B , Xiong L ( 2007 ) Preconditioning with hyperbaric oxygen induces tolerance against oxidative injury via increased expression of heme oxygenase-1 in primary cultured spinal cord neurons . Life Sci 80 : 1087 - 1093 .