ORIGINAL PAPER

The effect of hyperbaric oxygen treatmenton aspiration pneumonia

Sevtap Hekimoglu Sahin • Mehmet Kanter • Suleyman Ayvaz •

Alkin Colak • Burhan Aksu • Ahmet Guzel • Umit Nusret Basaran •

Mustafa Erboga • Ali Ozcan

Received: 13 May 2011 / Accepted: 24 May 2011 / Published online: 8 June 2011

� Springer Science+Business Media B.V. 2011

Abstract We have studied whether hyperbaric oxygen

(HBO) prevents different pulmonary aspiration materials-

induced lung injury in rats. The experiments were designed

in 60 Sprague-Dawley rats, ranging in weight from 250 to

300 g, randomly allotted into one of six groups (n = 10):

saline control, Biosorb Energy Plus (BIO), hydrochloric

acid (HCl), saline ? HBO treated, BIO ? HBO treated,

and HCl ? HBO treated. Saline, BIO, HCl were injected

into the lungs in a volume of 2 ml/kg. A total of seven

HBO sessions were performed at 2,4 atm 100% oxygen for

90 min at 6-h intervals. Seven days later, rats were sacri-

ficed, and both lungs in all groups were examined bio-

chemically and histopathologically. Our findings show that

HBO inhibits the inflammatory response reducing signifi-

cantly (P \ 0.05) peribronchial inflammatory cell infiltra-

tion, alveolar septal infiltration, alveolar edema, alveolar

exudate, alveolar histiocytes, interstitial fibrosis, granu-

loma, and necrosis formation in different pulmonary aspi-

ration models. Pulmonar aspiration significantly increased

the tissue HP content, malondialdehyde (MDA) levels and

decreased (P \ 0.05) the antioxidant enzyme (SOD, GSH-

Px) activities. HBO treatment significantly (P \ 0.05)

decreased the elevated tissue HP content, and MDA levels

and prevented inhibition of SOD, and GSH-Px (P \ 0.05)

enzymes in the tissues. Furthermore, there is a significant

reduction in the activity of inducible nitric oxide synthase,

TUNEL and arise in the expression of surfactant protein D

in lung tissue of different pulmonary aspiration models

with HBO therapy. It was concluded that HBO treatment

might be beneficial in lung injury, therefore, shows

potential for clinical use.

Keywords Pulmonary aspiration � iNOS � Surfactant

protein D � TUNEL � Hyperbaric oxygen

Introduction

Tracheobronchial aspiration can be defined as the inhala-

tion of oropharyngeal or gastric contents into the respira-

tory tract (Marik 2001). Although aspiration from either

source is important, the greatest concern in critically ill

tube-fed patients is from tracheobronchial aspiration of

gastric contents. The extent to which aspiration of gastric

contents occurs is difficult to determine, primarily because

most clinical studies have relied on flawed detection

methods (Winterbauer et al. 1981; Elpern et al. 1986; Potts

et al. 1993; Kinsey et al. 1994; Metheny et al. 2002, 2005).

S. H. Sahin � A. Colak

Department of Anesthesiology and Reanimation, Faculty

of Medicine, Trakya University, 22030 Edirne, Turkey

M. Kanter (&) � M. Erboga

Department of Histology and Embryology, Faculty of Medicine,

Trakya University, 22030 Edirne, Turkey

e-mail: mkanter65@yahoo.com

S. Ayvaz � B. Aksu � U. N. Basaran

Department of Pediatric Surgery, Faculty of Medicine, Trakya

University, 22030 Edirne, Turkey

A. Guzel

Department of Pediatrics, Faculty of Medicine,

19 Mayis University, 22030 Samsun, Turkey

A. Ozcan

Department of Biochemistry, Private Central Hospital,

Kozyatagi, Istanbul, Turkey

123

J Mol Hist (2011) 42:301–310

DOI 10.1007/s10735-011-9334-6

Different animal models have been developed to

investigate the mechanisms, characteristics, and patho-

physiology of lung injury (Leth-Larsen et al. 2003). Even

though the mechanism of lung injury is not accurately

comprehended there is evidence for the implication of

oxidative damage by reactive oxygen species (ROS) and

reactive nitrogen species (RNS), which are increasingly

regarded as key substances modulating the pulmonary

vascular endothelial damage that characterized acute

respiratory distress syndrome (ARDS). The resulting vas-

cular endothelial damage is accountable for the principal

clinical manifestations of ARDS (Bhatia and Moochhala

2004; Tasaka et al. 2008; Metnitz et al. 1999). There is

convincing evidence that ROS play a major role in medi-

ating injury to the endothelial barrier of the lung in the

presence of endotoxin or sepsis. ROS are capable of

reacting with cellular lipids, proteins, and nucleic acids

leading to changes in the structure and function of alveolar

cells. This condition is supported by several animal studies.

In these studies, antioxidant therapy has been beneficial in

the protection and the treatment of lung injury (Bhatia and

Moochhala 2004; Tasaka et al. 2008; Metnitz et al. 1999).

Surfactant protein D (SP-D) is a member of the collectin

family of proteins, which play important roles in innate

host defense of the lung and regulation of surfactant

homeostasis and is synthesized in alveolar type II cells and

Clara cells of lungs (Leth-Larsen et al. 2003). Alveolar cell

damage leads to decreased and impaired synthesis, secre-

tion, function, and composition of SP-D in acute lung

injury (Cheng et al. 2003; Herbein and Wright 2001; Guzel

et al. 2008). Therewithal, little knowledge presents about

the histopathological benefits of antioxidants on the

development of different gastric content induced lung

injury (Guzel et al. 2008).

Hyperbaric oxygen therapy (HBO) is defined as a

treatment in which a patient is intermittently exposed to

100% oxygen while the treatment chamber is pressurized

to a pressure above sea level ([1 ATA, 760 mmHg) (Gill

and Bell 2004). HBO therapy has been used in a number of

medical conditions with a proven efficacy in a limited

number of disorders (Gill and Bell 2004; Tibbles and

Edelsberg 1996; Hills 1999). HBO has been used as a

treatment modality in compromised tissue oxygenation,

such as refractory osteomyelitis, skin flaps, necrotizing

fasciitis and wound healing. There is increasing evidence

that HBO has beneficial effects on many more disease

including inflammatory and infectious processes (Slotman

1998). Despite these effects, it is also known that HBO

may be harmful to lung and brain tissue by increasing the

number of ROS (Harabin et al. 1990; Jamieson 1991).

The aim of the present study was to investigate the

effects of HBO treatment on lung injury due to different

aspiration materials in rats.

Materials and methods

The Ethical Committee of Trakya University approved all

animal procedures and the experimental protocol. Efforts

were made to minimize animal suffering and reduce the

number of animals used in experimental groups.

Hyperbaric oxygen treatment

Rats from saline ? HBO treated, BIO ? HBO treated, and

HCl ? HBO treated groups were placed in an experimental

hyperbaric chamber in which pure oxygen was adminis-

tered at a 2,4 atm 100% oxygen for 90 min at 6-h intervals

for a total of seven HBO sessions.

Animals

The experiments were performed in 60 Sprague-Dawley

rats, ranging in weight from 250 to 300 g. Rats were pro-

vided by the Experimental Research Center of the Medical

Faculty of Trakya University. The rats were kept in a

windowless animal quarter where temperature (22 ± 2�C)

and illumination were automatically controlled (light on at

7 a.m. and off at 9 p.m. 14 h light/10 h dark cycle).

Humidity ranged from 50 to 55%. Animals were given free

access to diet and water until the night before the experi-

ment, when they were fasted.

Experimental protocol

Sixty animals were included in each of the following six

groups (n = 10): saline control, enteral formula (Biosorb

Energy Plus (BIO), Nutricia, Zoetermeer, The Nether-

lands), Hydrochloric acid (HCl 0.1N, pH 1.25), sali-

ne ? HBO treated, BIO ? HBO treated, and HCl ? HBO

treated. The rats were anesthetized with ketamine hydro-

chloride (100 mg/kg) and xylazine (10 mg/kg) intraperi-

toneally, and allowed to breathe spontaneously throughout

the entire experimental protocol. The animals were placed

in a supine position with the extremities pulled caudally to

facilitate exposure of the trachea. The trachea was exposed

through an anterior neck incision and a direct puncture with

a 24-gauge needle on a 1-ml tuberculin syringe was per-

formed two to four tracheal rings below the larynx. Saline,

BIO, HCl were injected into the lungs in a volume of 2 ml/

kg. After instillation of saline, BIO, and HCl, the tuberculin

syringe was removed, and the neck incision was repaired

with a 6-0 Ethilon suture. Animals were observed until they

recovered from anesthesia. After surgical procedure, a total

of seven HBO sessions were performed at 2,4 atm 100%

oxygen for 90 min at 6-h intervals. Seven days later, all

rats were killed with intraperitoneal injection of ketamine

hydrochloride; the trachea and both lungs were removed.

302 J Mol Hist (2011) 42:301–310

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Histopathological procedures

Portions of right lung (anterior lobe, median lobe, posterior

lobe, and post caval lobe) and left lung (upper left lobe, and

lower left lobe) were individually immersed in 10% neu-

tral-buffered formalin, dehydrated in alcohol, embedded in

paraffin, and then cut into 5-lm-thick cross-sections

through the middle of the lobe so that each section included

hilum to periphery. Sections were placed on slides, depa-

raffinized, and stained with hematoxylin and eosin (H&E)

using standard procedures. Six slides were analyzed in a

standardized fashion. Each lung lobe sections were divided

equally for histopathological and biochemical investiga-

tions. Each slide was examined and evaluated in random

order under blindfold conditions for immunostaining by a

histologist and stained with H&E for histopathologic

assessment under a standard light microscopy by a

pathologist. The slides were examined for the presence of

peribronchial inflammatory cell infiltration (PICI), alveolar

septal infiltration (ASI), alveolar edema (AED), alveolar

exudate (AEX), alveolar histiocytes (AHI), interstitial

fibrosis (IF), granuloma (GRA), and necrosis (NEC) for-

mation. These changes were scored according to the four

point scale used by Takil et al. (2003). A histopathological

assessment was performed in at least randomly selected

eight microscopic high-power fields from each lung lobe.

The final score determined in each category for each

individual animal was the mean of the scores from the

sections of the lungs examined.

TUNEL assay

The TUNEL method, which detects fragmentation of DNA

in the nucleus during apoptotic cell death in situ, was

employed using an apoptosis detection kit (TdT-FragelTM

DNA Fragmentation Detection Kit, Cat. No. QIA33, Cal-

biochem, USA). All reagents listed below are from the kit

and were prepared following the manufacturer’s instruc-

tions. Five-lm-thick pulmonary sections were deparaffi-

nized in xylene and rehydrated through a graded ethanol

series as described previously. They were then incubated

with 20 mg/ml proteinase K for 20 min and rinsed in TBS.

Endogenous peroxidase activity was inhibited by incuba-

tion with 3% hydrogen peroxide. Sections were then

incubated with equilibration buffer for 10–30 min and then

TdT-enzyme, in a humidified atmosphere at 37�C, for

90 min. They were subsequently put into pre-warmed

working strength stop/wash buffer at room temperature for

10 min and incubated with blocking buffer for 30 min.

Each step was separated by thorough washes in TBS.

Labelling was revealed using DAB, counter staining was

performed using Methyl green, and sections were dehy-

drated, cleared and mounted.

Immunohistochemical procedures

Immunohistochemical reactions were performed according

to the ABC technique described by Hsu et al. (1981). The

procedure involved the following steps: (1) endogenous

peroxidase activity was inhibited by 3% H2O2 in distilled

water for 30 min, (2) the sections were washed in distilled

water for 10 min, (3) non-specific binding of antibodies

was blocked by incubation with normal goat serum (DAKO

X 0907, Carpinteria, CA) with PBS, diluted 1:4, (4) the

sections were incubated with specific rabbit polyclonal anti

inducible nitric oxide synthase (iNOS) antibody (Cat. #

RB-1605-P, Neomarkers, USA) and specific rabbit poly-

clonal anti-surfactant protein D (SP-D) antibody (Cat. #

AB3434, Chemicon, USA), diluted 1:50 for 1 h, and then

at room temperature, (5) the sections were washed in PBS

3 9 3 min, (6) the sections were incubated with biotinyl-

ated anti-mouse IgG (DAKO LSAB 2 Kit), (7) the sections

were washed in PBS 3 9 3 min, (8) the sections were

incubated with ABC complex (DAKO LSAB 2 Kit), (9) the

sections were washed in PBS 3 9 3 min, (10) peroxidase

was detected with an aminoethylcarbazole substrate kit

(AEC kit; Zymed Laboratories), (11) the sections were

washed in tap water for 10 min and then dehydrated, (12)

the nuclei were stained with hematoxylin, and (13) the

sections were mounted in DAKO paramount.

The positive staining of TUNEL, iNOS and SP-D cell

numbers were scored in a semiquantitative manner in order

to determine the differences between the control group and

the experimental groups in the distribution patterns of

intensity of immunolabeling of lung tissue. The numbers of

the positive staining were recorded as weak (±), mild (?),

moderate (??), strong (???) and very strong (????).

This analysis was performed in at least randomly

selected eight microscopic high-power fields from each

lung lobe section, in two sections from each animal at

4009 magnification. The final score determined in each

category for each individual animal was the average of the

scores from the sections of the lungs examined.

Biochemical procedures

At the end of the experiment, rats in all groups were

starved overnight for 12 h. After killing, the harvested lung

tissues samples were quickly washed in cold saline and

stored at -70�C.

Measurement of tissue hydroxyproline

Total hydroxyproline (HP) content of the lung tissue

samples was measured as an assessment of lung collagen

content. A spectrophotometric assay was used to quantify

lung HP (Kivirikko et al. 1967). Briefly, the lung was

J Mol Hist (2011) 42:301–310 303

123

removed from the -70�C freezer and homogenized in 5%

trichloroacetic acid (1:9 wt/vol). The homogenized samples

were centrifuged for 10 min at 4,000g, and the pellet was

washed twice with distilled water and then hydrolyzed for

16 h at 100�C in hydrochloric acid (6N HCl). The HP level

was expressed as micrograms per milligram wet lung

tissue.

Measurement of tissue malondialdehyde level

Lung tissue samples were frozen at -70�C and irrigated

well with a solution of sodium chloride (NaCl) (0.9%). By

admixing it with potassium chloride (KCl) (1.5%),

homogenization at a ratio of 1:10 was achieved. The

DIA99000 Homogenizer (Heidolph Instruments, Ger-

many) was used to homogenize the tissue samples. The

lipid peroxide level in the centrifuged tissue homogenates

was measured according to the method described by

Ohkawa et al. (1979). The reaction product was assayed

spectrophotometrically (Shimadzu UV-1700, Japan) at

532 nm. The lipid peroxide level was expressed as the

nanomole (nmol) of malondialdehyde (MDA) per milli-

gram of lung tissue protein. Protein levels were measured

according to the method described by Lowry et al. (1951).

Measurement of tissue superoxide dismutase activity

Superoxide dismutase (SOD) activity was determined

according to the method of Sun et al. (1988). This method

is based on the inhibition of nitroblue tetrazolium (NBT)

reduction by the XO system as a superoxide generator.

Activity was assessed in the ethanol phase of the lysate

after 1.0 ml ethanol/chloroform mixture (5/3, v/v) was

added to the same volume of sample and centrifuged. One

unit of SOD was defined as the enzyme amount causing

50% inhibition in the NBT reduction rate. SOD activity

was also expressed as units per milligram protein.

Glutathione peroxidase activity

Glutathione peroxidase (GSH-Px) activity was measured

by the method of Paglia and Valentine (1967). The enzy-

matic reaction was initiated in a tube containing the fol-

lowing items: NADPH, reduced glutathione (GSH),

sodium azide, and glutathione reductase by addition of

H2O2 and the change in absorbance at 340 nm was moni-

tored by a spectrophotometer. Activity was given in units

per gram protein. All samples were assayed in duplicate.

Statistical analysis

All statistical analyses were completed using SPSS statis-

tical software (SPSS for Windows, version 11.0). The data

of biochemical findings were presented in mean (±) stan-

dard deviation (SD) and dual comparisons between groups

exhibiting significant values were evaluated with Kruskal–

Wallis test followed by Mann–Whitney U test. The degrees

of histopathological severity were analyzed with one-way

analysis of variance and Tukey test for detection of signif-

icant differences between groups. These differences were

considered significant when probability was less than 0.05.

Results

Histopathological findings

Histopathological results of study groups are presented in

Fig. 1 and Table 1. Histopathological parameters including

PICI, ASI, AED, AEX, AHI, IF, GRA, and NEC decreased

in all groups treated with HBO compared to untreated

groups. There was a statistical difference between BIO and

saline control groups (P \ 0.01). PICI, ASI, AED, AEX,

AHI, IF, GRA, and NEC were found significantly higher in

HCl group compared to BIO (P \ 0.001) and saline control

groups (P \ 0.0001). Although IF, GRA, and NEC were

not observed in saline control and saline ? HBO groups,

HBO treatment significantly decreased (P \ 0.05) PICI,

ASI, AED, AEX, AHI, IF, GRA, and NEC in untreated

groups (Table 1).

Immunohistochemical findings

The number of alveolar type II cells positive for SP-D was

lower in the BIO and the HCl groups than in the saline

control group as a semi-quantitatively (Table 2). HBO

treatment significantly increased the number and degree of

SP-D reactivity in alveolar type II cells (Table 3; Fig. 2).

The number of alveolar cells positive for iNOS was semi-

quantitatively higher in the BIO and HCl groups than the

saline control group. HBO treatment significantly

decreased the number of iNOS immunopositive cells

(Table 3; Fig. 2).

TUNEL findings

The number of alveolar cells positive for TUNEL was

semi-quantitatively higher in BIO and HCl groups than

saline control group. Treatment of HBO markedly reduced

the reactivity and the number of TUNEL positive cells

(Table 3; Fig. 3).

Biochemical findings

The values of the tissue HP content, MDA levels, SOD and

GSH-Px activities, and statistical differences of these

304 J Mol Hist (2011) 42:301–310

123

measurements are shown in Table 4. Pulmonary aspiration

significantly increased the tissue HP content, MDA levels

and decreased (P \ 0.05) the antioxidant enzyme (SOD,

GSH-Px) activities (Table 4). HBO treatment significantly

(P \ 0.05) decreased the elevated tissue HP content, and

MDA levels and increased of SOD, and GSH-Px

(P \ 0.05) enzymes in the tissues (Table 4).

Discussion

Aspiration of gastric contents is a recognized risk factor for

ventilator-associated pneumonia in critically ill patients

(Torres et al. 1992; Cook et al. 1998; Drakulovic et al.

1999; Yavagal et al. 2000). However, more information is

needed to determine the extent to which the frequency of

tracheobronchial aspiration of gastric contents predisposes

to pneumonia and other adverse outcomes. An analysis to

determine the occurrence of aspiration as a risk factor for

pneumonia and as a determinant of clinical outcomes in

comparison with other factors has been reported (Metheny

et al. 2006).

In a study, the assessment of the histopathological

examination was based on the presence of acute-phase

result, peribronchial inflammatory cell infiltration; suba-

cute phase results, alveolar septal infiltration, alveolar

Fig. 1 Light microscopy of lung tissues in different groups. H&E:

a showing normal lung tissue morphology; b diffuse hemorrhagic

area (asterisk) is showed in the necrotic lung tissue; c rat lung

showing peribronchial inflammatory cell infiltration (asterisk), d rat

lung showing alveolar septal infiltration (asterisk), alveolar edema

(thin arrow), alveolar exudate (thick arrow) and multivacuolated

alveolar macrophages forming clusters in alveolar spaces (arrow-head) e rat lung showing granuloma in necrotic lung tissue (among

arrowhead). A alveol, B bronchiole (H&E, scale bar 50 lm)

J Mol Hist (2011) 42:301–310 305

123

edema, alveolar exudate, alveolar macrophages; chronic

phase results, interstitial fibrosis, granuloma, abscess, and

necrosis formation (Wright 1998). A standard diagnostic

finding in lipoid pneumonia is the observation of macro-

phages (Ahrens et al. 1999), which help in the diagnosis of

chronic pulmonary aspiration (Bauer and Lyrene 1999;

Umuroglu et al. 2006). These findings suggested that

multiple pulmonary aspirations of the enteral solutions

studied resulted in chronic, rather than acute or subacute,

histopathological changes.

Table 1 The 4-point scale used for histopathologic assessment

0 1 2 3

Peribronchial inflammatory cell

infiltration

No Prominent germinal centers of

lymphoid follicules

Infiltration between lymphoid

follicules

Confluent band like form

Alveolar septal infiltration No Minimal Moderate Severe, impending of lumen

Alveolar edema No Focal In multiple alveoli Widespread, Involving lobules

Alveolar exudate No Focal In multiple alveoli Prominent, widesp

Alveolar histiocytes No Scattered in a few alveoli Forming clusters in alveolar

spaces

Filling the alveolar spaces

Interstitial fibrosis No Focal, minimal Focal, prominent fibrous

thickening

Widespread, prominent fibrous

thickening

Granuloma No Rare, micro Focal, well formed Confluent, large

Necrosis No Focal, few necrotic cells Multifocal, small areas Larger areas

Table 2 Histopathological assessment of pulmonary tissue for each group

Saline Saline ? HBO BIO BIO ? HBO HCL HCL ? HBO

PICI 1.30 ± 0.15 0.60 ± 0.26a 2.10 ± 0.10b 1.30 ± 0.2a 3.00 ± 0.0c 1.80 ± 0.29a

ASI 0.50 ± 0.16 0.30 ± 0.21a 1.50 ± 0.16b 0.90 ± 0.23a 2.90 ± 0.10c 1.50 ± 0.16a

AED 0.20 ± 0.13 0.10 ± 0.10a 0.80 ± 0.13b 0.40 ± 0.13a 1.70 ± 0.15c 0.90 ± 0.23a

AEX 0.20 ± 0.13 0.10 ± 0.10a 0.90 ± 0.17b 0.50 ± 0.14a 1.80 ± 0.29c 0.70 ± 0.26a

AHI 0.30 ± 0.15 0.20 ± 0.13a 0.90 ± 0.10b 0.60 ± 0.16a 2.10 ± 0.10c 1.10 ± 0.10a

IF 0 ± 0 0 ± 0 0.60 ± 0.16b 0.30 ± 0.21a 2.20 ± 0.29c 1.12 ± 0.13a

GRA 0 ± 0 0 ± 0 0.20 ± 0.13b 0.10 ± 0.10a 1.80 ± 0.29c 0.90 ± 0.17a

NEC 0 ± 0 0 ± 0 0.30 ± 0.15b 0.10 ± 0.10a 2.30 ± 0.21c 1.40 ± 0.16a

Saline aspiration of saline, control group, Saline ? HBO aspiration of saline treated with HBO, BIO aspiration of BIO, BIO ? HBO aspiration of

BIO treated with HBO, HCL aspiration of HCL, HCL ? HBO aspiration of HCL treated with HBO groups (n: 10 pulmonary for each group),

PICI peribronchial inflammatory cell infiltration, ASI alveolar septal infiltration, AED alveolar edema, AEX alveolar exudate, AHI alveolar

histiocytes, IF interstitial fibrosis, GRA granuloma, NEC necrosis

Tukey test was used for statistical analysis. Values are expressed as means ± SD, n = 10 for each groupa P \ 0.05 compared to untreated groupsb P \ 0.01 compared to saline control groupc P \ 0.001 compared to BIO group; P \ 0.0001 compared to saline control group

Table 3 Semiquantitative comparison of the positive cell numbers for TUNEL, iNOS and surfactant protein D in lung tissues for each group

Saline Saline ? HBO BIO BIO ? HBO HCl HCl ? HBO

TUNEL ? ± ?? ? ???? ???

iNOS ? ± ?? ? ???? ???

Surfactant protein D ??? ???? ?? ??? ± ?

Saline aspiration of saline, control group, Saline ? HBO aspiration of saline treated with HBO, BIO aspiration of BIO, BIO ? HBO aspiration of

BIO treated with HBO, HCL aspiration of HCL, HCL ? HBO aspiration of HCL treated with HBO groups (n: 10 pulmonary for each group)

The numbers of the positive staining was recorded as absence (-), a few (±), few (?), medium (??), high (???) and very high (????)

306 J Mol Hist (2011) 42:301–310

123

The aspiration of lipoid material following the acci-

dental ingestion of enteral formulas is the most common

cause of lipoid pneumonia and the severity of lipoid

material aspiration induced-lung injury may depend on the

lipid content (Shepherd et al. 1995; Takil et al. 2003;

Garzon Lorenzo et al. 2008). The mechanism of lipoid

material in lung tissue after aspiration is not completely

known. Most authors have showed that vegetable oils are

found in BIO which do not dissolve in lung tissue and act

like a foreign body, whereas animal fats are hydrolyzed

into free fatty acids, induce acute or chronic inflammation

with NEC. However, the presence of histiocytes with lipid

content after lipid aspiration is the Standard diagnostic

histopathological finding in lipoid pneumonia in children

(Takil et al. 2003; Ahrens et al. 1999). As a clinic, it rep-

resents cough, increasing dyspnea and chest pain, together

with alveolar infiltrates in the radiography and the previous

accidental intake of a lipid substance and vomiting

(Metheny et al. 2006). According to one study, the pul-

monary histopathologic effects of aspiration of Impact

were more severe PICI (greater than aspiration of Biosorb

and Pulmocare), abundant AHI, and AED in comparison

with aspiration of saline, even though Impact had the

lowest lipid content of all studied formulas (Takil et al.

Fig. 2 Immunohistochemical expression of iNOS and SP-D in the

lung tissue. iNOS: a slightly positive iNOS immunoreactivity in

normal lung tissue; b strongly positive iNOS immunoreactivity in

necrotic lung tissue; c moderately positive iNOS immunoreactivity in

HBO treated lung tissue. SP-D: a strong SP-D immunostaining was

present in alveolar type II cells of normal lung tissue; b weak SP-D

expression in degenerative alveolar type II cells of necrotic lung

tissue (asterisk); c increase positive SP-D immunoreactivity in

alveolar type II cells of HBO treated lung tissue. A alveol, B bron-

chiole, arrows: positive reactivity (immunoperoxidase, haematoxylin

counterstain, scale bar 50 lm)

J Mol Hist (2011) 42:301–310 307

123

2003). In addition, some authors believe that the high lipid

content formula might not be a risk factor for lipid aspi-

ration (Takil et al. 2003). Our histopathological findings

are consistent with their results concerning the effects of

different content lipid formulas aspiration.

In our study, histopathological parameters including

PICI, ASI, AED, AEX, AHI, IF, GRA, and NEC decreased

in all groups treated with HBO compared to untreated

groups. There was a statistical difference between BIO and

saline control groups. PICI, ASI, AED, AEX, AHI, IF,

GRA, and NEC were found significantly higher in HCl

group compared to BIO and saline control groups.

Although IF, GRA, and NEC were not observed in saline

control and saline ? HBO groups, HBO treatment

significantly decreased PICI, ASI, AED, AEX, AHI, IF,

GRA, and NEC in untreated groups Our findings are con-

sistent with the results of Takil et al. (2003), Guzel et al.

(2008, 2009), and Kanter (2009) concerning the effects of

aspiration of different lipid formulas.

The role of nitric oxide (NO) is controversial in

inflammatory reactions that lead to injury, with evidence

for pro-inflammatory as well as anti-inflammatory effects

(Bogdan 2001). There is little knowledge concerning the

pharmacological benefits of NOS inhibitors on the devel-

opment of acid-induced lung injury. In previous studies,

aspiration of gastric components led to activation of mac-

rophages and the release of pro-inflammatory cytokines

such as NO. In these animal aspiration models selective

Fig. 3 Representative lung tissue photographs of TUNEL investiga-

tion a a few TUNEL positive cells in normal lung tissue; b many

TUNEL positive cells in necrotic lung tissue c moderately TUNEL

positive cells in lung tissue of HBO treated group. A alveol, arrows:

TUNEL positive cells (TUNEL staining, scale bar 50 lm)

Table 4 Tissue HP, SOD, GSH-Px, and MDA levels in groups

Saline Saline ?HBO BIO BIO ? HBO HCL HCL ? HBO

HP (lg/g tissue) 2,965.8 ± 422 2,662.2 ± 123a 6,733.7 ± 483b 664.3 ± 708a 8,683.2 ± 511c 845.3 ± 452a

MDA (nmol/g tissue) 54.7 ± 8.1 45.3 ± 7.16a 107.9 ± 10.32b 96.2 ± 8.02a 127.1 ± 11.91c 114.8 ± 12.06a

SOD (U/g tissue) 254.2 ± 25.2 311.9 ± 39.5a 189.3 ± 19.4b 229.1 ± 22.3a 162.4 ± 17.4c 204.4 ± 20.3a

GSH-Px (nmol/gr tissue) 91.7 ± 11.03 119.5 ± 21.72a 75.1 ± 11.54b 91.2 ± 10.99a 55.7 ± 7.77c 70.1 ± 9.23a

Kruskal–Wallis test was used for statistical analysis. Values are expressed as means ± SD, n = 10 for each groupa P \ 0.05 compared to untreated groupsb P \ 0.01 compared to saline control groupc P \ 0.001 compared to saline control group

308 J Mol Hist (2011) 42:301–310

123

iNOS inhibition prevented lung injury and a significant

increase in the metabolism of NO, nitrite and nitrate was

also observed in bronchoalveolar lavage fluid of acid-

instilled rats or dogs (Lee et al. 1998; Kudoh et al. 2001;

Miyakawa et al. 2002; Speyer et al. 2003; Harkin et al.

2004). Our data indicate that there is a significant increase

in iNOS activity in lung tissue of different aspiration

materials such as lipid content formula BIO and especially

HCl.

Acute lung injury is a frequent cause of morbidity and

mortality following pulmonary or systemic infections.

Injury to the alveolar epithelial barrier is a major deter-

minant of the extent and severity of acute clinical lung

injury (Bachofen and Weibel 1982; Cheng et al. 2003). SP-

D is a member of the collectin family of proteins, which

play important roles in innate host defense of the lung and

regulation of surfactant homeostasis (Botas et al. 1998;

Crouch 1998; Ikegami et al. 2007). SP-D is synthesized in

alveolar type II cells and Clara cells of rat lungs. In acute

lung injury, there is decreased and impaired synthesis,

secretion, function and composition of SP-D (Herbein and

Wright 2001; Cheng et al. 2003). There are only a few

reports of SP-D in lung injury due to gastric materials. This

study was undertaken to analyze the immunohistochemical

expression of SP-D in lung injury due to different aspira-

tion materials. Pursuant to our results, SP-D was weakly

expressed in lung injury our non-treated study groups.

Furthermore, our data indicate that there is a significant a

rise in the localization of SP-D in lung tissue of different

pulmonary aspiration models with HBO therapy.

Most of organs may be protected from the damaging

effects of the ROS by enzymatic and non-enzymatic anti-

oxidant defence. These include enzymes like SOD, cata-

lase, and GSH-Px (Bhatia and Moochhala 2004; Tasaka

et al. 2008; Metnitz et al. 1999). MDA is a good indicator

of free radical activity and its altitude presents increased

lipid peroxidation (Aksu et al. 2007). However, alveolar

macrophages can manufacture potent ROS such as super-

oxide radicals and consequently peroxynitrite which can be

produced by the reaction of NO with superoxide radicals

and exhibits a highly oxidative species (Bhatia and

Moochhala 2004; Tasaka et al. 2008; Metnitz et al. 1999).

ROS play major role in mediating damage to the endo-

thelial barrier of the lung in the sepsis. These species are

produced by activated alveolar macrophages, stimulated

endothelial cells, and damaged structure, and function of

alveolar cells (Bhatia and Moochhala 2004; Tasaka et al.

2008). In addition, several animal models have been

developed to investigate the mechanisms, characteristics,

and pathophysiology of aspiration lung injury and some

proinflammatory cytokines have been implicated in the

development of lung injury (Kudoh et al. 2001). A few

studies have shown that some inflammatory factors

associated with lung injury, such as neutrophilmediated

ROS and proinflammatory cytokines (Downey et al. 1999;

Davidson et al. 1999; Folkesson et al. 1995). In our study,

HBO which has been found to possess anti-inflammatory,

anticancer, and other treatment activities diminished HP

accumulation and MDA levels and prevented inhibition of

SOD, and GSH-Px enzymes in the tissues due to acid

instillation.

In conclusion, our results indicate that HBO exhibits an

obvious protective effect to oxidative alveolar cell damage

by increasing SP-D secretion and reduction of TUNEL and

iNOS activity. These findings support the use of HBO as a

potential therapeutic agent in acute lung injury.

Acknowledgments This study was supported as Project TUBAB

2010-179 by Trakya University Research Center, Edirne, Turkey.

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