ww.sciencedirect.com

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 3 ( 2 0 1 3 ) 3 5 5e3 6 1

Available online at w

journal homepage: www.JournalofSurgicalResearch.com

The effect of hyperbaric oxygen on nitric oxide synthaseactivity and expression in ischemia-reperfusion injury

Richard C. Baynosa, MD,* Anna L. Naig, MD, Patrick S. Murphy, MD, Xin Hua Fang, MT,Linda L. Stephenson, MT (ASCP), Kayvan T. Khiabani, MD, Wei Z. Wang, MD,and William A. Zamboni, MD

Division of Plastic Surgery, Department of Surgery, University of Nevada School of Medicine, Las Vegas, Nevada

a r t i c l e i n f o

Article history:

Received 26 September 2012

Received in revised form

20 December 2012

Accepted 3 January 2013

Available online 1 February 2013

Keywords:

Hyperbaric oxygen

Nitric oxide synthase

Nitric oxide

Ischemia-reperfusion

Nitric oxide synthase activity

Presented at American College of SurgeonsVegas, NV, June 16-18, 2005.* Corresponding author. Division of Plastic Su

Healing Center, University Medical Center of671-2256; fax: (702) 671-2245.

E-mail address: rbaynosa@medicine.neva0022-4804/$ e see front matter ª 2013 Elsevhttp://dx.doi.org/10.1016/j.jss.2013.01.004

a b s t r a c t

Background: Hyperbaric oxygen (HBO) mitigates ischemia-reperfusion (IR) injury via a nitric

oxide mechanism that is nitric oxide synthase (NOS) dependent. The purpose of this study

was to investigate this NOS-dependent mechanism by examining isoform-specific, tissue-

specific, and time-specific upregulation of NOS mRNA, protein, and enzymatic activity.

Methods: Weraised a gracilis flap inWistar rats thatwere separated into early and late phases.

Treatment groups included nonischemic control, IR, HBO-treated ischemia-reperfusion

(IR-HBO), and nonischemic HBO control. We harvested tissue-specific samples from gracilis,

rectus femoris, aorta, and pulmonary tissues and processed them by reverse transcription

polymerase chain reaction and Western blot to determine upregulation of isoform-specific

NOS mRNA and protein. We also harvested tissue for NOS activity to investigate upregula-

tion of enzymatic activity. Data are presented as mean � standard error of the mean with

statistics performed by analysis of variance. P � 0.05 was considered significant.

Results: There was no increase in NOSmRNA in the early phase. In the late phase, there was

a significant increase in endothelial-derived NOS (eNOS) mRNA in IR-HBO compared with

IR in gracilis muscle (79.4 � 22.3 versus 36.1 � 4.5; P < 0.05) and pulmonary tissues

(91.0 � 31.2 versus 30.2 � 3.1; P < 0.01). There was a significant increase in the late-phase

eNOS pulmonary protein IR-HBO group compared with IR (235.5 � 46.8 versus 125.2 �14.7; P < 0.05). Early-phase NOS activity was significantly increased in IR-HBO compared

with IR in pulmonary tissue only (0.049 � 0.009 versus 0.023 � 0.003; P < 0.05).

Conclusions: The NOS-dependent effects of HBO on IR injury may result from a systemic

effect involving an early increase in eNOS enzymatic activity followed by a late-phase

increase in eNOS protein expression within the pulmonary tissues.

ª 2013 Elsevier Inc. All rights reserved.

1. Introduction of perfusion, such as in myocardial infarction, cerebral

Ischemia-reperfusion (IR) injury is a common pathophysio-

logic process that can affectmultiple tissues after interruption

, San Francisco, CA, Octo

rgery, University of NevadSouthern Nevada, 2040 W

da.edu (R.C. Baynosa).ier Inc. All rights reserved

ischemia, crush injuries, acute vascular insufficiencies,

transplantation, replantation after traumatic amputation, and

free tissue transfer. Hyperbaric oxygen (HBO) is a treatment

ber 16-20, 2005 and Undersea and Hyperbaric Medical Society, Las

a School of Medicine, Hyperbaric Medicine and Advanced Wound. Charleston Boulevard, Suite 302, Las Vegas, NV 89102. Tel.: (702)

.

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 3 ( 2 0 1 3 ) 3 5 5e3 6 1356

modality that has proven to be safe and effective for various

conditions including decompression sickness, osteor-

adionecrosis, and hypoxic wounds [1]. More recently, there

has been basic science and clinical evidence indicating

a beneficial effect of HBO in the treatment of myocardial

infarction and cerebral ischemia, as well as renal and skeletal

muscle ischemia [2e8]. The lack of a well-defined mechanism

of action, however, has prevented widespread acceptance of

HBO as a treatment for IR injury. Our research strives to help

further elucidate the mechanism of the beneficial effect of

HBO therapy on skeletal muscle IR injury.

Previous studies have demonstrated the deleterious effect

of IR injury on the microcirculation. In a morphologic analysis

of the skeletal muscle microcirculation after IR injury,

a significant increase in adherent neutrophils to the endo-

thelium of postcapillary venules was noted compared to the

microcirculation of nonischemic controls [9]. This increased

neutrophil adherence was reversed with HBO treatment

both during and immediately after the ischemic event. This

benefit of HBO on skeletal muscle IR injury has been shown in

other studies via significant reductions in skeletal muscle

edema and necrosis [6,10,11].

Multiple studies from various researchers have implicated

nitric oxide (NO) as an important regulator of cell surface

adhesion molecules via cytoskeletal alterations [12e15]. We

have confirmed the importance of NO and nitric oxide syn-

thase (NOS) in preventing skeletal muscle necrosis after IR

injury using an NOS substrate (L-arginine) and NOS inhibitor

(L-NAME) [16]. Our recent work further confirmed that the

HBO reduction of IR-induced neutrophil polarization of CD18

and adherence to intercellular adhesion molecule-1 is medi-

ated through an NO mechanism that is NOS dependent [17].

The importance of CD18 and intercellular adhesion molecule-

1 in mediating the protective effect of HBO on IR injury has

been corroborated in separate rat skeletal muscle experi-

ments by other researchers [18].

The purpose of this study was to investigate the

NOS-dependent mechanism of HBO in IR injury by examining

isoform-specific, tissue-specific, and time-specific upregula-

tion of NOS mRNA, protein, and enzymatic activity. Nitric

oxide synthase is able to produce NO via two separate path-

ways. Increased NOmay result from either an increase in NOS

protein expression or an increase in NOS enzymatic activity.

We proposed to measure NOS expression by evaluating early-

and late-phase mRNA transcription via reverse transcription

polymerase chain reaction (RT-PCR) as well as subsequent

NOS protein expression via Western blot. In addition, we

measured NOS activity in vivo by means of a radioisotope

assay. Because the pathway of protein expression involves

numerous steps to raise overall NOS protein concentration,

whereas increasing NOS enzymatic activity may occur

rapidly, we hypothesized that HBO treatment for IR

injury would result in an early increase in NOS activity and

a late-phase increase in NOS expression.

2. Methods

The University of Nevada Institutional Animal Care and Use

Committee approved all experimental procedures as well as

the animal model and associated animal care during the

described study.

2.1. Rat gracilis muscle model

We anesthetized male Wistar rats weighing 275 � 30 g with

pentobarbital (50 mg/kg, intraperitoneally) with supplemen-

tation (10 mg/kg) as required to maintain anesthesia during

the surgical period. We raised the gracilis muscle flap as we

previously described [9]. Briefly, the right thigh musculature

and femoral vasculature were exposed, and the gracilis

muscle was dissected free on its vascular pedicle using stan-

dard microsurgical technique. Clamping the femoral artery

and vein for 4 h induced global muscle ischemia, after which

time the clampwas removed to initiate the reperfusionperiod.

2.2. Treatment groups

We randomly assigned the animals to either early-phase or

late-phase study groups. The early-phase group was further

subdivided into one of four experimental groups: (1) non-

ischemic control (NIC); (2) IR, consisting of 4 h ischemia and 30

min reperfusion; (3) IR-HBO, of 4 h ischemia and 30 min

reperfusion with HBO treatment during the last 90 min of

ischemia; or (4) HBO control (NIC-HBO) with HBO treatment

during the last 90 min of mock ischemia. We included the last

group to ensure that HBO treatment in normal controls did not

affect NOS expression or activity in the early phase.

We subdivided the late-phase group into one of three

experimental groups: (1) NIC; (2) IR, consisting of 4 h ischemia

and 24 h reperfusion; or (3) IR-HBO, consisting of 4 h ischemia

and 24 h reperfusion with HBO treatment during the last

90 min of ischemia.

2.3. Hyperbaric oxygen treatment

Hyperbaric oxygen treatment consisted of placing the animals

in a research-grade HBO chamber (Model 1300; Sechrist

Industries, Inc., Anaheim, CA) with 100% oxygen at 2.5 ATA

during the last 90 min of ischemia or NIC-ischemia.

2.4. Determination of early- and late-phase NOSexpression by reverse transcriptase polymerase chainreaction

We divided Wistar rats randomized to the early-phase group

into treatment groups, as described above. For those stratified

to the early phase, we subjected the animals to their respec-

tive treatments. After 30min reperfusion ormock reperfusion,

we harvested samples from the experimental gracilis muscle,

contralateral rectus femoris muscle, abdominal aorta, and

pulmonary tissue for RT-PCR (n ¼ 8). We harvested samples

from four separate and unique tissues to examine whether

the changes were local or systemic. We examined the gracilis

tissue to assess whether mRNA transcription was elevated in

the muscle subjected to the IR injury. Conversely, we also

sampled from the contralateral nonischemic rectus femoris

muscle to examine whether the increase in NOS mRNA could

be seen in skeletal muscle not subjected to IR injury. We iso-

lated and processed the abdominal aorta as well, to determine

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 3 ( 2 0 1 3 ) 3 5 5e3 6 1 357

whether an increase in NOS could be isolated systemically.

Finally, we examined pulmonary tissue to assess the effect of

the tissue’s first being exposed to the inhaled HBO.

We cleaned and weighed the tissue samples. The fresh

tissue for RT-PCRwasplaced inRNAlater (Qiagen Inc, Valencia,

CA) and stored at �80�C until processing. We purified the

samples for RNA using the RNeasy Mini Kit (Qiagen). We

assessed RNA concentration using a spectrophotometer.

Approximately 250 ng total RNA was reverse-transcribed into

cDNA using the Qiagen Omniscript Reverse Transcriptase kit,

according to the manufacturer’s protocol. We performed PCR

using the Qiagen Hotstar Taq Polymerase protocol with

primers for induciblenitric oxide synthase (iNOS), endothelial-

derivedNOS (eNOS),andneuronalnitricoxidesynthase (nNOS)

(Invitrogen; Life Technologies, Grand Island, NY). We mixed a

SYBR Green PCR Mastermix (Invitrogen) with 0.1 mmol/L of

each primer and the cDNA template in 25-mL volumes. Ampli-

fied DNA was run on agarose gel and stained with ethidium

bromide. We conducted semiquantitative analysis of fluores-

cence intensity between treatment groups using the Typhoon

Variable Wavelength Imager (Amersham; GE Healthcare

Biosciences, Pittsburgh, PA); We measured intensity and

expressed it as a percentage of intensity of the housekeeping,

reference gene, glyceraldehyde 3-phosphate dehydrogenase.

We subjected late-phase animals to their respective treat-

ments and afterward closed the surgical site with skin staples.

We returned the rats to their cages and allowed them to

recover with food and drink ad libitum. After 24 h reperfusion

or mock reperfusion, we harvested similar samples from

the four different tissue sites as for the early group for RT-PCR

(n ¼ 5). The technique and protocols for RT-PCR of the late-

phase tissue samples were identical to those followed for

the early-phase group.

2.5. Western blot analysis to determine NOS proteinexpression

We prepared late-phase tissue samples for Western blot

analysis (n ¼ 10). We flash-froze tissue for Western blot

analysis in liquid nitrogen and stored it at �80�C until pro-

cessing. The tissue samples were homogenized and protein

was isolated and quantified using the Bradford Assay (Bio-

Rad, Hercules, CA). Total protein volume was normalized to

20 mg/sample. We performed the detection of eNOS protein

using standard Western blot techniques. Briefly, we ran the

normalized protein samples use sodium dodecyl sulfa-

teepolyacrylamide gel electrophoresis, transferred them to

the nitrocellulose membrane, and then probed them with

monoclonal anti-eNOS antibody (sc-653; Santa Cruz Biotech-

nology, Inc, Dallas, TX). We employed chemiluminescent

detection using the ECL Plus kit (Amersham). We conducted

semiquantitative analysis of chemiluminescent intensity

between treatment groups using the Typhoon Variable

Wavelength Imager. We measured intensity and expressed it

as a percentage of intensity of the nonischemic control.

2.6. Nitric oxide synthase activity assay

We subjected rats from the early-phase group to their

respective treatments as described above and separately

prepared them for the NOS activity assay with samples har-

vested from the gracilis muscle and pulmonary tissue (n ¼ 20).

Tissue for NOS Activity was weighed, flash-frozen in liquid

nitrogen, and stored at �80�C until assayed. We thawed the

frozen tissue on ice and homogenized it for NOS activity using

the Cayman Chemical (Ann Arbor, MI) NOS Activity Assay Kit.

We incubated homogenized tissue (60 min) with the 3H-L-

arginine and measured 3H-L-citrulline formed by the

biochemical conversion of 3H-L-arginine by NOS. Concurrent

citrulline assay reactions were performed with L-NAME (an

NOS inhibitor) and rat cerebellum extract to serve as a blank

and positive linear control, respectively, for this quantitative

assay. We quantified NOS activity in the eluate by counting in

a liquid scintillation counter and expressed it as counts per

minute (cpm). This assay measures constitutive NOS (cNOS)

and inducible NOS (iNOS). There is no differentiation between

eNOS and nNOS, the calcium-dependent isoforms.

Data are reported as mean � standard error of the mean.

We compared the tissue and treatment groups using analysis

of variance, and used appropriate post hoc analysis for

comparison between means. P � 0.05 was considered

significant.

3. Results

In the early experimental group (30 min reperfusion), there

were no statistically significant differences in mRNA tran-

scription between tissue types, including gracilis muscle,

rectus femoris muscle, aorta, and pulmonary tissue, and

between treatment groups, including NIC, IR, IR-HBO, and

NIC-HBO (data not shown).

Likewise, in the late-phase group sustaining 24 h reperfu-

sion, there were no statistically significant increases in iNOS

mRNA transcription between tissue types or between treat-

ment groups, although there was a significant decrease in

iNOS mRNA after HBO compared with NIC in the aorta (Fig. 1).

However, there was a 120% increase in late-phase eNOS

mRNA expression in the IR-HBOetreated gracilis muscle

compared with the IR-treated gracilis muscle (79.4 � 22.3

versus 36.1� 4.5; P< 0.05) (Fig. 2). More impressively, therewas

an over 200% increase in late-phase eNOS mRNA expression

in the IR-HBOetreated pulmonary tissue compared wite the

NIC and IR treated pulmonary tissues (91.0� 31.2 versus 30.0�7.8 and 30.2 � 3.1; P < 0.01) (Fig. 2).

Subsequent Western blot testing on the late-phase group

(24 h reperfusion) failed to demonstrate an increase in eNOS

protein expression in the IR-HBOetreated gracilis muscle

compared with the IR-treated group. Figure 3 shows a repre-

sentative Western blot of eNOS protein at 24 h reperfusion

from two tissues from three treatment groups. However,

there was an increase in expression of eNOS protein> 100% in

the IR-HBOetreated pulmonary tissue compared with the

IRetreated pulmonary tissue (235.5 � 46.8 versus 125.2 � 14.7;

P < 0.05) (Fig. 4).

Based on the results of our late-phase groups, we limited

testing of NOS activity in the early phase to the experimental

gracilis muscle and the pulmonary tissues. We failed to detect

an increase in NOS activity in the gracilis muscle for any

treatment groups. This was true in testing for both cNOS and

Fig. 1 e RibonucleicacidpurificationandRT-PCRof iNOSfrom

gracilis, rectus femoris, aorta, and pulmonary vasculature

after24hreperfusion infourgroups:NIC,NIC-HBO, IR,andIR-

HBO. All are expressed as a percentage of glyceraldehyde 3-

phosphatedehydrogenase. TheNICgroupwas set at 100%.N

[ 5/group. *P< 0.05 versusNIC. Pulm[ pulmonary

vasculature. (Color version of figure is available online.)

Fig. 3 e Representative Western blot of eNOS protein at 24

h reperfusion in gracilis muscle and the pulmonary

vasculature from three groups: NIC, IR, and IR-HBO.

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 3 ( 2 0 1 3 ) 3 5 5e3 6 1358

iNOS activity (Fig. 5). Likewise, we did not demonstrate an

increase in iNOS activity in the pulmonary tissue in any

treatment group. However, there was a statistically significant

increase in cNOS activity in the pulmonary tissue of the

IR-HBOetreated group compared with the IR-treated group

(0.049 � 0.009 versus 0.023 � 0.003; P < 0.05) (Fig. 6).

4. Discussion

Previous work has suggested that the beneficial effect of HBO

in IR injury involves the inhibition of neutrophil-endothelial

adhesion and depends on NOS [19e21]. Studies have also

shown that NO is a potent modulator of cellular adhesion

molecules via a cyclic guanosine monophosphateeassociated

pathway [12]. These findings have been confirmed by our lab,

which has demonstrated that the HBO effect on IR-induced

neutrophil CD18 polarization and adhesion occurs by a NO

mechanism that is NOS-dependent [17,22]. The purpose of

this study was to investigate this NOS-dependent mechanism

Fig. 2 e Ribonucleic acid purification and RT-PCR of eNOS

from gracilis, rectus femoris, aorta, and pulmonary

vasculature after 24 h reperfusion in four groups: NIC,

NIC-HBO, IR, and IR-HBO.All are expressedasapercentageof

glyceraldehyde 3-phosphate dehydrogenase. The NIC group

was set at 100%.N[ 5/group. *P< 0.05 versus IR; **P< 0.05

versusNIC and IR. (Color version of figure is available online.)

of HBO in IR injury by examining isoform-specific, tissue-

specific, and time-specific upregulation of NOSmRNA, protein

and enzymatic activity.

Nitric oxide synthases are a family of enzymes that cata-

lyze the production of NO from L-arginine. Nitric oxide syn-

thase may be available as three different isoforms. The

constitutive isoforms include the eNOS and nNOS and are

calcium-dependent. The inducible form is referred to as iNOS

and is calcium-independent. Each of these isoforms has the

potential to increase the production of NO by one of two

pathways. Increased NOmay result from either an increase in

NOS protein expression or an increase in NOS enzymatic

activity. Recent literature has suggested that the impaired

vascular dysfunction associated with IR injury may result

from alterations and decreases in the bioavailability of eNOS,

leading to endothelial dysfunction [23,24]. Numerous studies

evaluating the treatment of IR injury via preconditioning,

postconditioning, or various chemical agents in tissue types,

including cardiac, renal, hepatic, and cerebral tissue, have

implicated increased eNOS activation and/or expression as

a key mechanism in protection against IR injury [25e37].

Our study suggests that the increased availability of NO

that facilitates the beneficial effect of HBOmay result from an

early increase in NOS enzymatic activity followed by a later

increase in eNOS protein expression. In the first phase of our

study, we isolated samples from different tissue sites during

the early phase of reperfusion and processed these for mRNA.

Samples were harvested from four separate and unique

tissues to examine whether the changes were local or

systemic. Interestingly, there was no upregulation of NOS

transcription in any of the tissues during the early time phase.

In the late phase, there was a significant decrease in iNOS

Fig. 4 e Endothelial NOS protein expression by Western

blot in gracilis muscle and the pulmonary vasculature after

24 h reperfusion. All are expressed as a percentage of NIC.

The NIC group was set at 100%. N [ 10/group. *P < 0.05

versus IR. (Color version of figure is available online.)

Fig. 5 e Constitutive NOS activity after 30 min reperfusion

in four groups: NIC, IR, IR-HBO, and NIC-HBO. All are

expressed as percent conversion per milligram tissue wet

weight. N [ 20/group. *P < 0.05 versus IR. (Color version of

figure is available online.)

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 3 ( 2 0 1 3 ) 3 5 5e3 6 1 359

mRNA in the HBO group compared with the NIC group.

Although it has been suggested that iNOS induction may

cause detrimental effects after IR, and others have linked

decreased iNOS expression to improvement in IR injury, its

role has been called into question and there is debate

regarding its deleterious or beneficial effects [32,34,38,39]. The

results are further complicated by the lack of any increase in

iNOS mRNA in any of the IR groups compared with NIC, and

no differences in iNOS activity in the early phase. These

results suggest that no conclusions can bemade regarding the

effect of iNOS in the beneficialmechanismof HBO on IR injury,

but further investigations may be warranted. However, there

was a statistically significant increase in eNOS transcription in

the IR-HBOetreated gracilis muscle as well as the pulmonary

tissue in the late phase. Further analysis using Western blot

techniques to evaluate protein expression, however, demon-

strated only a statistically significant increase in eNOS protein

expression in the pulmonary tissue and not the locally treated

skeletal muscle. Although it is possible that a statistically

significant increase in eNOS protein of the gracilis muscle

might be shown at an earlier or later time point, or perhaps

even through increasing the number of study animals,

it seems clear that after the first 24 h of reperfusion, the

Fig. 6 e Inducible NOS activity after 30 min reperfusion in

four groups: NIC, IR, IR-HBO, and NIC-HBO. All are

expressed as percent conversion per milligram tissue wet

weight. N [ 20/group. There were no significant

differences. (Color version of figure is available online.)

increased protein expression of eNOS could result from

a systemic response of the pulmonary tissue’s reaction

to HBO.

Evidence for a systemic pathway for the benefit of HBO-

treated IR injury is further enhanced by our results on NOS

activity testing in the early phase of reperfusion. There

was only a statistically significant increase in cNOS activity

that was seen in the systemic pulmonary tissue of the IR-

HBOetreated animal comparedwith the IR-treated pulmonary

tissue. No increases were demonstrated in the local gracilis

muscle or in the inducible form of NOS for either tissue group.

These results support the theory that the constitutive,

endothelial-type NOS is responsible for the beneficial effects

of HBO therapy in IR injury via a systemic response.

Previous studies in a rat skeletal muscle model of IR injury

treated with HBO have also suggested that the improved flap

survival was a systemic and not a local effect. This study used

a metal-coated Mylar bag to prevent oxygen diffusion locally

into the ischemic skeletal muscle flap, and demonstrated that

there was still a significant reduction in the percentage of flap

necrosis compared with IR controls that was comparable to

the HBO group without the Mylar bag [40]. This has been

further supported in the clinical realm with the use of multi-

place HBO chambers. In these facilities, the chamber is not

compressed with 100% oxygen as in a monoplace chamber,

but undergoes pressurization to 2.5 ATA with compressed air

(21% O2), and the patient is given 100% oxygen to breath via

a mask or oxygen hood. In these situations, the IR-injured

tissue is not directly exposed to the compressed oxygen

externally, but rather internally from the systemic circulation

and breathing of 100% oxygen at elevated pressure [41]. This

type of treatment has been demonstrated to be clinically

effective via multiple various clinical trials and is approved by

the Undersea and Hyperbaric Medical Society, whereas the

use of topical oxygen or HBO localized to specific tissues has

been shown to be ineffective [42].

Previous in vitro studies have also demonstrated an

increase in eNOS mRNA and protein expression after expo-

sure to HBO therapy [21]. In vivo and in vitro studies on fetal

pulmonary endothelial cells have demonstrated an increase

in eNOS mRNA and protein expression as well as eNOS

activity with increased oxygen concentrations at 1 ATA

[43,44]. Our study is the first to our knowledge not only to

demonstrate the upregulation of eNOS mRNA and protein in

an in vivo model of IR injury treated with HBO, but also to

localize this upregulation to a specific tissue type. Other

studies have also suggested that the increased oxidative stress

from HBO therapy and the resultant formation of reactive

oxygen and nitrogen species have critical roles as signaling

molecules in downstream transduction cascades [45,46]. We

suspect that the increased oxidative stress experienced by

pulmonary endothelial cells during HBO treatmentmay be the

catalyst for the upregulation of eNOS activity and subsequent

eNOS mRNA and protein expression within the pulmonary

tissue. The resultant reactive nitrogen species have a role in

the downstream protective effects on IR-injured tissues.

We have shown that the beneficial effects from HBO

treatment in IR injury that are NOS-dependent may rely on an

early increase in eNOS activity followed by a late-phase

upregulation of eNOS protein from endothelial cells within

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 3 ( 2 0 1 3 ) 3 5 5e3 6 1360

the pulmonary tissues. These findings suggest a possible

mechanism by which HBO-induced hyperoxia and oxidative

stress provide its paradoxical benefit to IR-injured tissues.

Further work is needed to delineate the link between the

systemic upregulation of eNOS from pulmonary endothelial

cells to the site of action in the IR-injured tissues.

r e f e r e n c e s

[1] Moon RE, Feldmeier JJ, Warriner RA. Decompressionsickness. In: Gesell LB, editor. Hyperbaric oxygen therapyindications. 12th ed. Durham, NC: Undersea and HyperbaricMedical Society; 2008. 51e6, 145e68, 67e83.

[2] Yogaratnam JZ, Laden G, Madden LA, et al. Hyperbaricoxygen: a new drug in myocardial revascularization andprotection? Cardiovasc Revasc Med 2006;7:146.

[3] Sterling DL, Thornton JD, Swafford A, et al. Hyperbaricoxygen limits infarct size in ischemic rabbit myocardiumin vivo. Circulation 1993;88:1931.

[4] Cabigas BP, Su J, Hutchins W, et al. Hyperoxic andhyperbaric-induced cardioprotection: role of nitric oxidesynthase 3. Cardiovasc Res 2006;72:143.

[5] Helms AK, Whelan HT, Torbey MT. Hyperbaric oxygentherapy of cerebral ischemia. Cerebrovasc Dis 2005;20:417.

[6] Strauss MB, Hargens AR, Gershuni DH, et al. Reduction ofskeletal muscle necrosis using intermittent hyperbaricoxygen in a model of compartment syndrome. J Bone JointSurg Am 1983;65:656.

[7] Garcia-Covarrubias L, McSwain NE, Van Meter K, Bell RM.Adjuvant hyperbaric oxygen therapy in the management ofcrush injury and traumatic ischemia: an evidence-basedapproach. Am Surg 2005;71:144.

[8] Bouachour G, Cronier P, Gouello JP, et al. Hyperbaric oxygentherapy in the management of crush injuries: a randomizeddouble-blind placebo-controlled clinical trial. J Trauma 1996;41:333.

[9] Zamboni WA, Roth AC, Russell RC, et al. Morphologicanalysis of the microcirculation during reperfusion ofischemic skeletal muscle and the effect of hyperbaricoxygen. Plast Reconstr Surg 1993;91:1110.

[10] Nylander G, Lewis D, Nordstrom H, Larson J. Reduction ofpost-ischemic edema with hyperbaric oxygen. Plast ReconstrSurg 1985;76:596.

[11] Skyhar MJ, Hargens AR, Strauss MB, et al. Hyperbaric oxygenreduces edema and necrosis of skeletal muscle incompartment syndromes associated with hemorrhagichypotension. J Bone Joint Surg Am 1986;68:1218.

[12] Banick PD, Chen Q, Xu YA, Thom SR. Nitric oxide inhibitsneutrophil beta 2 integrin function by inhibiting membrane-associated cyclic GMP synthesis. J Cell Physiol 1997;172:12.

[13] Ke X. Nitric oxide regulates actin reorganization throughcGMP and Ca(2þ)/calmodulin in RAW 264.7 cells. BiochemBiophys Acta 2001;10:1387.

[14] Loitto VM, Nilsson H, Sundqvist T, Magnusson KE. Nitricoxide induces dose-dependent CA(2þ) transients and causestemporal morphological hyperpolarization in humanneutrophils. J Cell Physiol 2000;182:402.

[15] Ingram AJ, James L, Cai L, et al. NO inhibits stretch-inducedMAPK activity by cytoskeletal disruption. J Biol Chem 2000;275:40301.

[16] Meldrum DG, Stephenson LL, Zamboni WA. Effects of L-NAME and L-arginine on ischemia-reperfusion injury in ratskeletal muscle. Plast Reconstr Surg 1999;103:935.

[17] Jones SR, Carpin KM,Woodward SM, et al. Hyperbaric oxygeninhibits ischemia-reperfusion-induced neutrophil CD18

polarization by a nitric oxide mechanism. Plast ReconstrSurg 2010;126:403.

[18] Hong JP, Kwon H, Chung YK, Jung SH. The effect ofhyperbaric oxygen on ischemia-reperfusion injury: anexperimental study in a rat musculocutaneous flap.Ann Plast Surg 2003;51:478.

[19] Chen Q, Banick PD, Thom SR. Functional inhibition of ratpolymorphonuclear leukocyte B2 integrins by hyperbaricoxygen is associated with impaired cGMP synthesis.J Pharmacol Exp Ther 1996;276:929.

[20] Thom SR, Mendiguren I, Hardy K, et al. Inhibition of humanneutrophil beta2-integrin-dependent adherence byhyperbaric O2. Am J Physiol 1997;272:C770.

[21] Buras JA, Stahl GL, Svoboda KK, Reenstra WR. Hyperbaricoxygen downregulates ICAM-1 expression induced byhypoxia and hypoglycemia: the role of NOS. Am J Physiol CellPhysiol 2000;278:C292.

[22] Khiabani KT, Bellister SA, Skaggs SS, et al. Reperfusion-induced neutrophil CD18 polarization: effect of hyperbaricoxygen. J Surg Res 2008;150:11.

[23] Kietadisorn R, Juni RP, Moens AL. Tackling endothelialdysfunction by modulating NOS uncoupling: new insightsinto its pathogenesis and therapeutic possibilities. Am JPhysiol Endocrinol Metab 2012;302:E481.

[24] Yang D, Xie P, Liu Z. Ischemia/reperfusion-induced MKP-3impairs endothelial NO formation via inactivation of ERK1/2pathway. PLoS One 2012;7:e42076.

[25] Yang CL, Talukder MA, Varadharaj S, Velayutham M,Zweier JL. Early ischaemic preconditioning requires Akt- andPKA-mediated activation of eNOS via serine1176phosphorylation. Cardiovasc Res 2013;97:33.

[26] Liu H, Liu X, Wei X, et al. Losartan, an angiotensin II type Ireceptor blocker, ameliorates cerebral ischemia-reperfusioninjury via PI3K/Akt-mediated eNOS phosphorylation. BrainRes Bull 2012;89:65.

[27] Ajamieh H, Farrell G, Wong HJ, et al. Atorvastatin protectsobese mice against hepatic ischemia-reperfusion injury byToll-like receptor-4 suppression and endothelial nitric oxidesynthase activation. J Gastroenterol Hepatol 2012;27:1353.

[28] Li XD, Yang YJ, Geng YJ, et al. Phosphorylation of endothelialNOS contributes to simvastatin protection againstmyocardial no-reflow and infarction in reperfused swinehearts: partially via the PKA signaling pathway.Acta Pharmacol Sin 2012;33:879.

[29] Li XD, Cheng YT, Yang YJ, et al. PKA-mediated eNOSphosphorylation in the protection of ischemicpreconditioning against no-reflow. Microvasc Res 2012;84:44.

[30] Abu-Amara M, Yang SY, Seifalian A, Davidson B, Fuller B.The nitric oxide pathwaydevidence and mechanisms forprotection against liver ischaemia reperfusion injury.Live Int 2012;32:531.

[31] Peng B, Guo QL, He ZJ, et al. Remote ischemicpostconditioning protects the brain from global cerebralischemia/reperfusion injury by up-regulating endothelialnitric oxide synthase through the PI3K/Akt pathway. BrainRes 2012;1445:92.

[32] Heeba GH, El-Hanafy AA. Nebivolol regulates eNOS and iNOSexpressions and alleviates oxidative stress in cerebralischemia/reperfusion injury in rats. Life Sci 2012;90:388.

[33] Mahfoudh-Boussaid A, Zaouali MA, Hadj-Ayed K, et al.Ischemic preconditioning reduces endoplasmic reticulumstress and upregulates hypoxia inducible factor-1a inischemic kidney: the role of nitric oxide. J Biomed Sci 2012;19:7.

[34] Betz B, Schneider R, Kress T, Schick MA, Wanner C,Sauvant C. Rosiglitazone affects nitric oxide synthasesand improves renal outcome in a rat model of severeischemia/reperfusion injury. PPAR Res 2012;2012:219319.

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 3 ( 2 0 1 3 ) 3 5 5e3 6 1 361

[35] Abu-Amara M, Yang SY, Quaglia A, et al. Role of endothelialnitric oxide synthase in remote ischemic preconditioning ofthe mouse liver. Liver Transpl 2011;17:610.

[36] Xia R, Zhao B, Wu Y, et al. Ginsenoside Rb1 preconditioningenhances eNOS expression and attenuates myocardialischemia/reperfusion injury in diabetic rats. J BiomedBiotechnol 2011;2011:767930.

[37] Posa A, Pavo N, Hemetsberger R, et al. Protective effect ofischaemic preconditioning on ischaemia/reperfusion-induced microvascular obstruction determined by on-linemeasurements of coronary pressure and blood flow in pigs.Thromb Haemost 2010;103:450.

[38] Darra E, Rungatscher A, Carcereri de Prati A, et al. Dualmodulation of nitric oxide production in the heart duringischaemia/reperfusion and inflammation. Thromb Haemost2010;104:200.

[39] Jones SP, Bolli R. The ubiquitous role of nitric oxide incardioprotection. J Mol Cell Cardiol 2006;40:16.

[40] Zamboni WA, Roth AC, Russell RC, et al. The effect of acutehyperbaric oxygen therapy on axial pattern skin flap survival

when administered during and after total ischemia.J Reconstr Microsurg 1989;5:343.

[41] Kindwall EP. The multiplace chamber. In: Kindwall EP,Whelan HT, editors. Hyperbaric medicine practice. 3rd ed.Flagstaff, AZ: Best Publishing Company; 2008. p. 191e208.

[42] Gesell LB, editor. Hyperbaric oxygen therapy indications.12th ed. Durham, NC: Undersea and Hyperbaric MedicalSociety; 2008. p. 191e208.

[43] Black SM, Johengen MJ, Ma ZD, et al. Ventilation andoxygenation induce endothelial nitric oxide synthase geneexpression in the lungs of fetal lambs. J Clin Invest 1997;100:1448.

[44] North AJ, Lau KS, Brannon TS, et al. Oxygen upregulatesnitric oxide synthase gene expression in ovine fetalpulmonary artery endothelial cells. Am J Physiol 1996;270:L643.

[45] Thom SR. Oxidative stress is fundamental to hyperbaricoxygen therapy. J Appl Physiol 1997;82:1424.

[46] Thom SR. Hyperbaric oxygen: its mechanisms and efficacy.Plast Reconstr Surg 2011;127:131S.