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Mechanical ventilation (MV) can provoke oxidative stress and an inflammatory response, and subsequently cause ventilator-induced lung injury (VILI), a major cause of mortality and morbidity of patients in the intensive care unit. Inhaled hydrogen can act as an antioxidant and may be useful as a novel therapeutic gas. We hypothesized that, owing to its antioxidant and anti-inflammatory properties, inhaled hydrogen therapy could ameliorate VILI. VILI was generated in male C57BL6 mice by performing a tracheostomy and placing the mice on a mechanical ventilator (tidal volume of 30 ml/kg without positive end-expiratory pressure, FiO(2) 0.21). The mice were randomly assigned to treatment groups and subjected to VILI with delivery of either 2% nitrogen or 2% hydrogen in air. Sham animals were given same gas treatments for two hours (n = 8 for each group). The effects of VILI induced by less invasive and longer exposure to MV (tidal volume of 10 ml/kg, 5 hours, FiO(2) 0.21) were also investigated (n = 6 for each group). Lung injury score, wet/dry ratio, arterial oxygen tension, oxidative injury, and expression of pro-inflammatory mediators and apoptotic genes were assessed at the endpoint of two hours using the high-tidal volume protocol. Gas exchange and apoptosis were assessed at the endpoint of five hours using the low-tidal volume protocol. Ventilation (30 ml/kg) with 2% nitrogen in air for 2 hours resulted in deterioration of lung function, increased lung edema, and infiltration of inflammatory cells. In contrast, ventilation with 2% hydrogen in air significantly ameliorated these acute lung injuries. Hydrogen treatment significantly inhibited upregulation of the mRNAs for pro-inflammatory mediators and induced antiapoptotic genes. In the lungs treated with hydrogen, there was less malondialdehyde compared with lungs treated with nitrogen. Similarly, longer exposure to mechanical ventilation within lower tidal volume (10 mg/kg, five hours) caused lung injury including bronchial epithelial apoptosis. Hydrogen improved gas exchange and reduced VILI-induced apoptosis. Inhaled hydrogen gas effectively reduced VILI-associated inflammatory responses, at both a local and systemic level, via its antioxidant, anti-inflammatory and antiapoptotic effects.
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Hydrogen inhalation ameliorates ventilator-induced lung injury
Short Running Head: Hydrogen Mitigates Lung Injury
Chien-Sheng Huang1,2, Tomohiro Kawamura1,3, Sungsoo Lee1,4, Naobumi Tochigi5, Norihisa
Shigemura1, Bettina M Buchholz6, John D Kloke7, Timothy R Billiar8, Yoshiya Toyoda1,
Atsunori Nakao1,3,8
1 Department of Cardiothoracic Surgery, University of Pittsburgh Medical Center, Pittsburgh,
Pennsylvania, USA
2 Division of Thoracic Surgery, Department of Surgery, Taipei-Veterans General Hospital and
National Yang-Ming University School of Medicine, Taipei, Taiwan
3Thomas E Starzl Transplantation Institute, University of Pittsburgh Medical Center,
Pittsburgh, Pennsylvania, USA
4 Department of Thoracic and Cardiovascular Surgery, Ajou University School of Medicine,
Youngtong-gu, Suwon, South Korea
5Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh,
Pennsylvania, USA
6Department of Medicine, Division of Gastroenterology and Hepatology, University of
Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA
7Center for Research on Health Care Data Center, University of Pittsburgh, Pittsburgh,
Pennsylvania, USA
8Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania,
USA
Corresponding author
Atsunori Nakao e-mail: anakao@imap.pitt.edu
Word count: 3511
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Chien-Sheng Huang (huangc@upmc.edu)
Tomohiro Kawamura (kawamurat2@upmc.edu)
Sungsoo Lee (lees3@upmc.edu)
Naobumi Tochigi (tochigin@upmc.edu)
Norihisa Shigemura (shigemuran@upmc.edu)
Bettina B Buchholz (bmb60@pitt.edu)
John D Kloke (klokejd@upmc.edu)
Timothy R Billiar (billiartr@upmc.edu)
Yoshiya Toyoda (toyoday@upmc.edu)
Atsunori Nakao (anakao@imap.pitt.edu)
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Abstract: 333 (limitation: 350)
Introduction: Mechanical ventilation (MV) can provoke oxidative stress and an inflammatory
response, and subsequently cause ventilator-induced lung injury (VILI), a major cause of
mortality and morbidity of patients in the intensive care unit. Inhaled hydrogen can act as an
antioxidant and may be useful as a novel therapeutic gas. We hypothesized that inhaled
hydrogen therapy could ameliorate VILI due to its antioxidant and anti-inflammatory
properties.
Methods: VILI was generated in male C57BL6 mice by performing a tracheostomy and
placing the mice on a mechanical ventilator (tidal volume of 30 ml/kg without positive end-
expiratory pressure, FiO2 0.21). The mice were randomly assigned to treatment groups and
subjected to VILI with delivery of either 2% nitrogen or 2% hydrogen in air. Sham animals
were given same gas treatments for two hours (n=8 for each group). The effects of VILI
induced by less invasive and longer exposure to MV (tidal volume of 10 ml/kg, 5 hours, FiO2
0.21) were also investigated (n=6 for each group). Lung injury score, wet/dry ratio, arterial
oxygen tension, oxidative injury, and expression of pro-inflammatory mediators and
apoptotic genes were assessed at the endpoint of two hours using the high-tidal volume
protocol. Gas exchange and apoptosis were assessed at the endpoint of five hours using the
low-tidal volume protocol.
Results: Ventilation (30 ml/kg) with 2% nitrogen in air for 2 hours resulted in deterioration of
lung function, increased lung edema, and infiltration of inflammatory cells. In contrast,
ventilation with 2% hydrogen in air significantly ameliorated these acute lung injuries.
Hydrogen treatment significantly inhibited upregulation of the mRNAs for pro-inflammatory
mediators and induced antiapoptotic genes. In the lungs treated with hydrogen, there was
less malondialdehyde compared with lungs treated with nitrogen. Similarly, longer exposure
to mechanical ventilation within lower tidal volume (10 mg/kg, five hours) caused lung injury
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including bronchial epithelial apoptosis. Hydrogen improved gas exchange and reduced
VILI-induced apoptosis.
Conclusions: Inhaled hydrogen gas effectively reduced VILI-associated inflammatory
responses, at both a local and systemic level, via its antioxidant, anti-inflammatory and
antiapoptotic effects.
Key words: mechanical ventilation, inflammation, ventilator-induced lung injury, hydrogen,
antioxidant, apoptosis
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Introduction
Although ventilatory support is often required in the intensive care unit (ICU) for the
treatment of critically ill patients with respiratory failure, including acute respiratory distress
syndrome (ARDS), pneumonia, septic shock, trauma, aspiration of vomit and chemical
inhalation, mechanical ventilation (MV) itself can induce lung injury and worsen pre-existing
lung injury depending on the setting and the length of ventilation [1, 2]. This condition has
been recognized as ventilator-induced lung injury (VILI). Despite recent progress in reducing
the time on MV (e.g. earlier weaning and extubation) and improving safety of MV (e.g. lung
protective ventilation with lower tidal volume), VILI remains a major concern in the ICU and
can lead to remote organ dysfunction and multiple organ failure [3].
Multifactorial etiologies of VILI, from either direct or indirect injury to the lung, are
postulated [4]. MV with high tidal volumes and pressure can lead to increased alveolar-
capillary permeability accompanied by the release of pro-inflammatory mediators by the lung
cells in response to mechanical stretch. These stimuli trigger detachment of endothelial cells
from the basement membrane and synthesis of extracellular matrix components [5, 6].
Injurious MV also promotes alveolar coagulopathy and fibrin deposition within the airways
[7]. In addition, generation of reactive oxygen species (ROS) during VILI causes direct
cellular injury and triggers ROS-sensitive, aberrant activation of cellular mechanisms leading
severe inflammation, resulting in rapid transcription of pro-inflammatory cytokines and
chemokines [8, 9].
Adjunctive therapy with inhaled therapeutic medical gas is promising and might be
reasonable for lung disease as it would be an easily delivered and straightforward
therapeutic option [10]. Hydrogen, recently discovered to be a novel therapeutic medical gas
in a variety of biomedical fields, has potent antioxidant and anti-inflammatory efficacies by
eliminating toxic ROS [11-14]. However; to our knowledge, hydrogen therapy has not been
tested in the VILI setting. Although hydrogen is highly flammable, it is safe in concentrations
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of <4.6% when mixed with air and at concentrations of <4.1 % when mixed with oxygen [15].
In this study, we investigated the hypothesis that inhaled hydrogen therapy could ameliorate
VILI due to its antioxidant and anti-inflammatory properties.
Methods
Animals
Male wild type C57BL6 mice (10-12 weeks old, 25-30 gram) were purchased from the
Jackson Laboratory (Bar Harbor, ME). All animals were maintained in laminar flow cages in
a specific pathogen-free facility at the University of Pittsburgh. The experimental protocol
was approved by the Institutional Animal Care and Use Committee of the University of
Pittsburgh and all experiments were performed in adherence to the National Institute of
Health guidelines for the use of laboratory animals.
Lung injury model
Mice were anesthetized by intraperitoneal injection of 85 mg/kg ketamine and 15 mg/kg
xylazine. Then, under the sterile conditions, a tracheostomy was performed with a 20-gauge
angiocatheter and sutured in place. Mice were placed in a supine position on a warming
device and then connected to a ventilator (Harvard Apparatus Co., Holliston, MA, USA) on
volume-control mode at a constant inspiratory flow. MV was initiated with a tidal volume of
30 ml/kg or 10 ml/kg without an end expiratory pressure at a respiratory rate of 120
breaths/minute [16, 17]. Mean arterial blood pressure was continuously monitored via
catheterization of a femoral artery using a blood pressure monitor (Cardiomax-III, Columbus,
OH). Mice received intravenous injection of 0.05 ml/hour saline, as well as intraperitoneal
ketamine and xylazine to maintain the blood pressure at 75-80 mmHg. At the end of the
experiment, the animal was euthanized with 150 mg/kg ketamine intraperitoneally.
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Experimental design
Mice were randomly assigned to 1 of 4 experimental groups: MV (tidal volume 30 ml/kg,
FiO2 0.21, 2 hours) with 2% nitrogen in air (Praxair, Danbury, CT), MV with 2% hydrogen in
air (Praxair), sham controls exposed to 2% nitrogen, or sham controls exposed to 2%
hydrogen (n=8 for each group). The concentration of 2% hydrogen was determined, on the
basis of previous observations, as an optimal and safe concentration [11, 18]. Mice under
ventilation received the therapeutic (2% hydrogen) or control (2% nitrogen) gases via the
tracheal tube. Animals in the sham groups were given therapeutic gases for 2 hours using a
gas chamber [19] and underwent anesthesia only prior to sacrifice and procurement of
tissue. While under anesthesia, the control mice received hydrogen or nitrogen through a
face mask by spontaneous respiration. In separate experiments, mice were subjected to MV
with lower tidal volume for a longer period (10 ml/kg, FiO2 0.21, 5 hours) with 2% nitrogen or
hydrogen in air (n=6 for each group). At sampling, after the right lung was isolated and tied
off with a microclamp at the right bronchus, the left lung was used for bronchoalveolar
lavage (BAL). The right lower lobe was used for wet/dry ratio measurement, the right middle
lobe was used for histologic examination and the other portions of the right lung were
immediately snap frozen in liquid nitrogen for further experiments, including gene expression
analyses.
Bronchoalveolar lavage
The left lung was used for BAL via slow intratracheal injection of three sequential 0.5 ml
aliquots of sterile normal NaCl. Cell pellets obtained by centrifuging BAL samples at 1500
rpm for 5 minutes at 4°C were resuspended in 1 ml of PBS. The cell viability was determined
via trypan blue exclusion assay. In brief, 10 µl of cells were mixed with 10 µl of 0.4% trypan
blue and loaded onto a hemocytometer. Protein concentration in the bronchoalveolar lavage
fluid (BALF) was measured with bovine IgG as a standard as previously described [20].
Histopathological, immunohistochemistry and TUNEL staining
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For histological evaluation, the right middle lobes of the lungs were fixed in 10% formalin,
embedded in paraffin, sectioned to 6 µm, in thickness, and stained with hematoxylin and
eosin. The slides were blindly reviewed by one of the authors (NT) without knowledge of
experimental groups (n=6 for each group). Acute lung injury was scored according to the
following four items: alveolar congestion, hemorrhage, infiltration or aggregation of
neutrophils in the airspace or the vessel wall, and thickness of the alveolar wall/ hyaline
membrane formation [21]. For analysis of macrophage infiltration, formalin-fixed, paraffin-
embedded mouse lung sections (4 µm) were deparaffinized and underwent antigen
unmasking with an appropriate buffer. After protein blocking (Dako Cytomation Carpinteria,
CA) for 15 min, rat anti-mouse F4/80 primary antibody (clone: CI:A3-1, AbD Serotec, NC)
was applied and incubated for overnight at 4˚C. After blocking endogenous peroxidase,
biotinylated goat anti-rat secondary antibodies (Jackson ImmunoResearch, West Grove, PA)
were applied followed by ABC Elite reagent (Vector Laboratories, Burlingame, CA). Staining
was developed with AEC chromogen (Scytek Laboratories, Cache, UT) and the tissue was
counterstained with hematoxylin. The terminal deoxynucleotidyl transferase-mediated
deoxyuridine triphosphate nick-end labeling method (TUNEL) was used for identification of
bronchiolar cell apoptosis with the ApopTag Peroxidase Kit (Intergen Co., Purchase, NY).
TUNEL-positive bronchial epithelial cells in 5 random, high-power fields per section were
counted with the samples’ identities masked.
Arterial blood analysis
At the end of the experiment, arterial blood was obtained from the abdominal aorta. Blood
gas analyses and measurement of lactate concentration were performed using an iSTAT
handheld device (Abaxis, Union City, CA).
Wet-to-dry weight ratio
The right lower lobe was weighed immediately after collection and placed into a 60°
C oven to
dry for 2 days. The dried tissue was weighed to determine the wet-to-dry (W/D) weight ratio.
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Real-time RT-PCR & TNF-α
αα
α ELISA
Since one of the underlying mechanisms of VILI is the release of pro-inflammatory mediators
by the lung cells and airway epithelial cell apoptosis in response to mechanical stretch,
quantitative real-time RT-PCR for inflammatory mediators was conducted on RNA extracted
from the lung tissues. The mRNAs for early growth response (Egr)-1, chemokine (CC motif)
ligand 2 (CCL2), interleukin (IL)-1ß, TNFα, B-cell lymphoma-2 (Bcl-2), Bcl-xL (B-cell
lymphoma-extra large), Bax (B-cell lymphoma-2-associated X-protein) and ß-actin were
quantified in duplicate using SYBR Green two-step, real-time RT-PCR as previously
described [13]. The levels of serum TNF-α were detected by specific enzyme-linked
immunosorbent assay (ELISA) (Thermo scientific, Rockford, IL) according to the
manufacturer’s protocol.
Malondialdehyde measurement
Oxidative injury from VILI was determined by measuring the tissue concentration of
malondialdehyde (MDA), a marker of lipid peroxidation, using the MDA-586 kit
(Oxidresearch, Portland, OR) according to the manufacturer’s instructions.
Statistical Analysis
Results are presented as mean ± standard deviation (SD). The EZAnalyze add-in for
Microsoft Excel was used to perform ANOVA with an F-test and Bonferroni posthoc group
comparisons. After applying a log transformation to the data, Bartlett’s test of homogeneity
was not significant and comparison boxplots indicated that the assumption of constant
variance was not violated. Nonetheless, in cases of possible heterogeneity, analysis was
performed on the log-transformed data. Histopathological score was analyzed with a
Kruskal-Wallis test with post hoc Steel-Dwass test for group comparisons. A probability level
of p<0.05 was considered statistically significant.
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Results
Lung injury after MV with high tidal volume
To evaluate the magnitude of lung injury by MV, sequential blood gas analysis of arterial
blood from the mice exposed to 2% nitrogen via the ventilator was performed. Ventilator
support improved gas exchange for the first 1 hour; while inhibition of gas exchange was
seen in the animals at beginning of MV due to muscle relaxation by general anesthesia.
Subsequently, partial pressure of arterial oxygen (PaO2) gradually decreased with time in the
ventilated mice and the partial pressure of arterial carbon dioxide (PaCO2) increased,
suggesting that MV with high tidal volume provoked lung damage, related to alveolar
overdistension or volutrauma, in a time-dependent manner (Figure 1A). To determine
whether hydrogen inhalation affected hemodynamics, we monitored blood pressure and
heart rate under MV. One hour after starting MV, there was a significant decrease in mean
arterial pressure in the mice ventilated with 30 ml/kg of tidal volume. There was no
significant difference in hemodynamics between the VILI/N2 and VILI/H2 treatment groups
over the 2-hour period. Inspiratory pressure was continuously monitored. Peak inspiratory
pressure of the mice ventilated with 30 mL of tidal volume was 26.2 ± 0.7 cm H2O, which
remained at constant levels throughout the experiment regardless of exposure to nitrogen or
hydrogen.
Gas exchange during VILI
Although the effects of MV with either 2% nitrogen or 2% hydrogen in air using a tidal
volume of 30 ml/kg for 30 minutes on lung function were negligible, VILI was induced by 2
hours of ventilation with 2% N2 in air with a tidal volume of 30 ml/kg, as indicated by a
significant decrease in PaO2 and an increase in PaCO2. Ventilation with 2% hydrogen in air
exerted protective effects on the lungs and improved oxygenation of the arterial blood
(Figure 1B). There was no statistical difference in PaCO2 levels among the groups. The
blood pH did not differ between the 30 minute and 2 hour time points nor did it differ between
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the treatment groups (VILI/N2 30 min; blood pH 7.25 ± 0.05, VILI/H2 for 30 minutes; pH 7.28
± 0.06, VILI/N2 for 2 hours; pH 7.24 ± 0.04, and VILI/H2 for 2 hours; 7.25 ± 0.05).
VILI-induced pulmonary edema
MV exacerbated pulmonary inflammation and injury, as indicated by thickening of the
alveolar septum and infiltration of inflammatory cells, evident in histopathological
examination. In the presence of hydrogen, both edema and inflammatory cell infiltration were
reduced despite exposure to MV with a 30 ml/kg tidal volume (Figure 2A) and the lung injury
score was significantly improved with hydrogen inhalation (Table 1). Two hours of MV with
high tidal volume (2% N2 in air) significantly increased the lung W/D ratio compared with
lungs of sham mice. Ventilation with 2% hydrogen in air ameliorated ventilator-induced
edema, as indicated by a significantly decrease in lung W/D ratio as compared with
ventilation with 2% N2 in air (Figure 2B).
Lung lipid peroxidation
Although there were increased MDA-protein adducts in the lung ventilated with a high tidal
volume of 2% nitrogen in air, ventilation with 2% hydrogen reduced tissue MDA levels after 2
hours of MV with high tidal volume (Figure 2C).
Alveolar-capillary leak due to VILI
MV with 30 ml/kg tidal volume caused an acute exudative phase with alveolar-capillary leak
in conjunction with leukocyte extravasation and resulted in an increase in the total cell
number in the BALF. The effects of hydrogen on total cells or protein concentration in the
BALF were marginal (Figure 3A and B). While blood lactate levels increased in ventilated
mice receiving 2% nitrogen, they did not increase in mechanically ventilated mice receiving
2% hydrogen in air (Figure 3C).
Expression of inflammatory mediators and apoptosis-related genes
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VILI after ventilation with 2% nitrogen resulted in upregulation of mRNAs for Egr-1, TNFα, IL-
1ß and CCL2. Hydrogen administration significantly attenuated the upregulation of the
mRNAs for these inflammatory mediators (Figure 4). Hydrogen inhalation increased the
expression of antiapoptotic genes, such as Bcl-2 and Bcl-xL, and reduced VILI-induced
expression of the pro-apoptotic Bax gene (Figure 5). There was no significant difference in
mRNA expression for the housekeeping gene ß-actin among the groups (data not shown).
VILI with lower tidal volume
Finally, we analyzed whether hydrogen therapy could also attenuate VILI induced using a
less invasive protocol with longer exposure to MV of lower tidal volume. Inducing VILI in the
mice via a tidal volume of 10 ml/kg for 5 hours resulted in deterioration of gas exchange in
mice receiving 2% nitrogen with an associated increase in pulmonary edema. These lung
injuries were significantly attenuated by treatment with 2% hydrogen (Figure 6A, B).
Hydrogen treatment significantly reduced serum TNFα concentrations as compared with the
serum levels of TNFα in mice with VILI caused by ventilation with 2% nitrogen (Figure 6C).
In this VILI model with lower-tidal volume, hydrogen treatment significantly decreased
macrophage infiltration, as determined by F4/80 staining, as compared with lungs ventilated
with 2% nitrogen (Figure 7A&C). MV with 2% nitrogen also increased apoptotic cell death of
bronchial epithelial cells, as determined by TUNEL. Ventilation with 2% hydrogen
significantly reduced TUNEL-positive epithelial cells as compared with ventilation with 2%
nitrogen (Figure 7B&C).
Discussion
In this study, we demonstrated that administration of hydrogen gas mitigated VILI
and VILI-associated oxidative and inflammatory responses, as well as VILI-induced
apoptotic cell death of bronchial epithelial cells. To our knowledge, this is the first study to
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demonstrate that hydrogen gas significantly reduces VILI. Since VILI is a major concern with
intensive care, approaches to minimize VILI will advance critical care medicine and could
have substantial clinical impact.
Recently, the biological functions of therapeutic gases have received considerable
attention and hydrogen was identified as a physiologically relevant gaseous signaling
molecule, like other endogenously generated gases including nitric oxide, carbon monoxide,
and hydrogen sulfide [22-24]. Thus, hydrogen has been described as “the fourth signaling
gaseous molecule” [25].
Hydrogen has a high potential as a safe and potent therapeutic medical gas, as well
as several potential advantages as a therapeutic option for VILI. Inhalation therapy is a
straightforward approach to lung disease and can be administered by simply providing gas
for the patient to inhale. Hydrogen may be relatively easily incorporated into our current
interventional or surgical procedures without increasing their complexity. Inhaled hydrogen
gas has been safely utilized for treatment of decompression syndrome in divers [26],
suggesting that hydrogen can be safely administered to patients. Hydrogen is a stable
molecule and does not react with other therapeutic medical gases at room temperature,
thus, it may be administered as a combined gas with other therapeutic gases or inhaled
anesthesia agents [18]. Hydrogen does not alter nitric oxide (NO) levels [11]. Endogenous
NO signaling pathways are critical for modulating pulmonary vascular tone and
leukocyte/endothelial interactions; therefore, it may be beneficial to spare endogenous NO
[27]. Hydrogen treatment does not eliminate superoxide anion (O2-) or hydrogen peroxide
(H2O2) [11]. O2- and H2O2 have important functions in neutrophils and macrophages,
allowing phagocytosis. Hydrogen therapy may spare the innate immune system, which
would be beneficial because lung infection accompanies VILI in many cases [28].
Importantly, experimental studies have demonstrated the protective effects of
hydrogen for septic shock [29], brain injury [11], liver injury [12], ischemic heart disease [30],
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and paralytic ileus [13]. As all of these diseases frequently coincide with VILI in ICU
patients, inhaled hydrogen therapy by simply delivering the gas through the ventilator could
be a very promising adjunctive therapy in the ICU or operating room.
In our study, hydrogen inhalation ameliorated upregulation of the mRNAs for TNFα,
IL-1ß, Egr-1 and CCL2 after 2 hours of MV, which may explain the anti-inflammatory
mechanisms afforded by hydrogen in this VILI model. Egr-1 acts as a key pro-inflammatory
regulator in VILI. Hoetzel and colleagues demonstrated that Egr-1-deficient mice did not
sustain lung injury after ventilation, relative to wild-type mice [31]. The CC chemokine family
is essential for the leukocyte recruitment during inflammation. Mounting evidence suggests
that CCL2, one of CC chemokine family, is involved in numerous inflammation disorders of
the lung including VILI [32]. Pro-inflammatory cytokines, such as TNFα and IL-1ß, are
elevated and play pivotal roles during the pathogenesis of VILI [33].
In our model, the increase in W/D ratio due to MV was relatively mild, despite the
larger changes observed in lung function (gas exchange). The histopathology changes were
moderate. We are unsure of the reason for these discrepancies, though, perhaps W/D ratio
is a very sensitive method to detect lung edema and small difference of W/D ratio may
represent significant edema. The gravimetric measure of lung edema poses a considerable
technical challenge, including evaporative loss and regional heterogenesity. W/D can be
complicated by the inclusion of blood in the wet lung weight both from residual intravascular
blood and blood introduced into the lung interstitium via bleeding or injury [34]. In addition,
extravasated protein can contribute to total lung weight. Although there are mismatches in
magnitude of lung injury depending which parameter is evaluated, each evaluation is
scientifically sound and each indicates some degree of VILI that was ameliorated by
hydrogen treatment.
VILI has been shown to induce apoptosis of airway epithelial cells [35]. We
demonstrated, in the present study, that hydrogen could upregulate antiapoptotic genes,
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including Bcl-2 and Bcl-xL. Although our findings do not explain the all the mechanisms
underlying the protective effects of hydrogen, we postulate that the Bcl-2/Bcl-xL pathway
might be one of the key mechanisms. Additionally, cyclic stretch associated with high-tidal
volume MV generates ROS and redox imbalance in lung epithelial and endothelial cells [36].
The antioxidant properties of hydrogen to eliminate ROS may contribute to mitigation of VILI
in our model.
In our blood gas analyses, there were no significant differences in PaCO2 levels
between animals exposed to 2% nitrogen or 2% hydrogen, although MV for 2 hours
significantly increased PaCO2 levels compared with MV for 30 minutes. These findings
suggest that PaCO2 levels may not the best therapeutic indicator in our model, although
PaO2 levels are very sensitive indicators of the magnitude of VILI. Our ventilation protocol
was based on maintaining PaCO2 in a range of 25-35 mmHg during the first 30 minutes of
MV period. We acknowledge the limitations of using PaCO2 as a marker of VILI in our
model, including that PaCO2 as a marker would not be applicable for clinical use and may be
influenced by auto-positive end-expiratory pressure (PEEP), overdistension or atelectasis;
however, the changes in PaCO2 observed in our study were likely caused by VILI, and not
by a poorly-controlled MV setting.
PaCO2 levels can influence inflammation and edema of the lungs. Hypocapnia
increases microvascular permeability and impairs alveolar fluid reabsorption, which may
influence the pathogenesis of pulmonary edema [37]. Furthermore, hypocapnia is directly
injurious to lung parenchyma and worsens ischemia/reperfusion injury [38]. Therefore,
permissive hypercapnia has been proposed as a protective strategy during MV and
contributes to attenuation of lung inflammatory response and pulmonary edema [39-42] and
the potential exists for an independent protective/pathologic role of alterations in CO2 tension
in the context of VILI. However, the VILI model in this study was designed to be
normocapnic and our results demonstrated that higher PaCO2 levels were not associated
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with less lung inflammation or edema, suggesting no influence of PaCO2 levels in our VILI
model.
A more comprehensive understanding of the pharmacokinetics, biology, and toxicity
of hydrogen will certainly help us harness the protective potential of hydrogen gas prior to
clinical application. Virtually, all patients under ventilation receive oxygen and, in particular,
patients with pulmonary disease usually require high levels of oxygen. Although hydrogen
poses no risk of explosion in air and oxygen when present at concentrations of <4%, safety
is still a concern and the desired concentration of hydrogen must be legitimately monitored
and maintained with commercially available tools. In this study, no adverse events related to
hydrogen were observed. We acknowledge that this experimental model, like most animal
models, does not recapitulate all aspects of clinical VILI. However, it is a scientifically sound
study using a model appropriate to address the hypothesis. These findings will serve as a
springboard to further translational research. Although extensive studies on toxicity and
safety are needed, hydrogen treatment of ventilated patients may be clinically feasible and
would be easy to incorporate without alteration of interventional and surgical procedures.
Conclusions
This study demonstrated a novel anti-inflammatory, antioxidative and antiapoptotic
function of hydrogen that ameliorated mechanical ventilation-induced lung injury.
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Key messages
Hydrogen inhalation therapy at a safe concentration mitigated ventilator-induced lung
injury in mice.
Hydrogen inhalation demonstrated potent antioxidant and anti-inflammatory effects in
a mouse ventilator-induced lung injury model.
Hydrogen reduced ventilation-induced epithelial apoptosis by induction of
antiapoptotic genes.
Adjunctive therapy with inhaled therapeutic medical gas is promising and might be
reasonable for lung disease as it would be an easily delivered and straightforward
therapeutic option
Hydrogen treatment of ventilated patients may potentially yield a novel, clinically
feasible therapy that would be easy to incorporate without alteration of interventional
and surgical procedures
Abbreviations
BAL, bronchoalveolar lavage; BALF, bronchoalveolar lavage fluid; Bax, B-cell lymphoma-2-
associated X-protein; Bcl-2, B-cell lymphoma-2; Bcl-xL, B-cell lymphoma-extra large; CCL2,
chemokine (CC motif) ligand 2; Egr, early growth response; ELISA, enzyme-linked
immunosorbent assays; HPF, high-power field; IL, interleukin; MDA, malondialdehyde; MV,
mechanical ventilation; PaCO2, partial pressure of arterial carbon dioxide; PaO2, partial
pressure of arterial oxygen; TNF, tumor necrosis factor; TUNEL, terminal deoxynucleotidyl
transferase-mediated deoxyuridine triphosphate nick-end labeling; ROS, reactive oxygen
species; VILI, ventilator-induced lung injury; W/D, wet-to-dry.
Competing interests:
The authors report no competing interests. The authors alone are responsible for the content
and writing of the paper.
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Authors’ contribution:
CH initiated the experimental design, participated in animal study and drafted manuscript.
TK and SL carried out the molecular genetic studies and participated in the sequence
alignment. NS, TRB and YT participated in its design and coordination and helped to draft
the manuscript. NT contributed to histopathological analysis. BMB and JDK contributed to
statistical analysis. AN provided the working hypothesis, designed the study, participated in
the performance of the research and wrote the manuscript. All authors read and approved
the final manuscript.
Acknowledgement
We thank Lisa Chedwick, Kumiko Isse and John Brumsfield for their excellent technical
support
Source of Support: NIH Grants GM R37-44100 (to TRB) and HL102528-01 (to AN),
Research funds of Department of Cardiothoracic Surgery, University of Pittsburgh Medical
Center
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Figure Legends
Figure. 1
(A) Sequential blood gas analysis of mice exposed to MV with high tidal volume (30 ml/kg).
The impact of MV with 2% nitrogen in air on the lungs in time-dependent manner was
investigated. Animals showed deteriorated gas exchange before ventilation (0 hour) due to
respiratory insufficiency caused by general anesthesia. Although MV improved gas
exchange for the first hour, partial pressures of arterial oxygen (PaO2) were decreased with
time and PaCO2 increased with time. N=3-6 for each time point. (B) Blood gas analysis for
arterial blood after the end of 2 hours of MV with high tidal volume. There was improved
pulmonary function in mice exposed to VILI for 2 hours under 2% hydrogen, compared with
nitrogen controls. N=8 for each group, *p<0.05 versus VILI 30min/N2 and VILI 30min/H2,
#p<0.05 versus VILI 2 hrs/N2.
Figure. 2
(A) Lung sections with MV with high tidal volume (30 ml/kg) stained with hematoxylin and
eosin. Alveolar septal thickening and inflammatory cell infiltration were observed in the lung
with VILI (2% N2). Hydrogen administration markedly reduced these histopathological
changes. 400X magnification; representative images are shown. N=6 animals for each
experimental group. (B) W/D ratio of the lungs with MV with high tidal volume (30 ml/kg).
VILI for 2 hours was accompanied with an increase of W/D ratio; ventilation with 2%
hydrogen still induced lung edema but to a lesser extent compared to MV with 2% nitrogen
in air. N=6 for each group. (C) Tissue malondialdehyde (MDA) levels. MV with high tidal
volume (30 mg/kg) with 2% nitrogen in air increased tissue MDA levels. The
supplementation of hydrogen significantly lowered levels of tissue MDA, a marker of lipid
20
peroxidation. N=6 for each group, *p<0.05 versus sham/N2 and sham/H2; #p<0.05 versus
VILI/N2.
Figure. 3
(A) The number of infiltrating cells recovered in the BALF obtained from the lungs with
MV with higher tidal volume (30 ml/kg). Administration of hydrogen had no effect on
inflammatory cell accumulation in the BALF. N=6 for each group, #P<0.05 versus sham/N2
controls. (B) Protein concentration in BALF. VILI resulted in significant increases of protein
contained in BALF. Hydrogen did not reduce leaked protein. N=6 for each group. (C) Blood
lactate concentration. VILI by mechanical ventilation for 2 hours was associated with
hyperlactatemia. Mice subjected to ventilation with 2% hydrogen did not show significant
increase of blood lactate levels compared to those of sham controls. N=6 for each group,
*p<0.05 versus sham/N2 and sham/H2; #p<0.05 versus VILI/N2.
Figure. 4
Quantitative RT-PCR for inflammatory mediators and transcripts in lung tissues with MV with
higher tidal volume (30 ml/kg). The levels of mRNAs for Egr-1(A), TNFα (B), IL-1ß (C) and
CCL-2 (D) significantly increased after mechanical ventilation with either with 2% nitrogen in
air (VILI/N2) or 2% hydrogen in air (VILI/H2). However, mRNA expression was significantly
less in VILI/H2 group compared with the VILI/N2 group. All values are reported as %ß-actin
with normalization to ß-actin mRNA expression. N=8 for each group, *p<0.05 versus
sham/N2 and sham/H2; #p<0.05 versus VILI/N2.
Figure. 5
21
Quantitative RT-PCR for apoptosis-related genes in lung tissues with MV of higher tidal
volume (30 ml/kg). The levels of mRNAs for Bcl-2 (A) and Bcl-xL (B) significantly increased
after mechanical ventilation in the presence of 2% hydrogen in air (VILI/H2). On the other
hand, Bax (C) was significantly increased after MV with 2% nitrogen in air (VILI/N2) and was
not increased after MV with 2% hydrogen in air. All values are reported as %ß-actin with
normalization to ß-actin mRNA expression. N=6 for each group, *p<0.05 versus sham/N2
and sham/H2; #p<0.05 versus VILI/N2.
Figure.6
The effects of hydrogen on VILI-induced by MV with low tidal volume (TV) (10 ml/kg). (A)
Arterial PaO2 levels of mice ventilated with 2% hydrogen for 5 hours were significantly higher
than those of mice ventilated with 2% nitrogen. N=6 for each group. (B) Lung edema caused
by VILI was determined by measuring W/D ratio. Hydrogen inhalation therapy ameliorated
VILI-induced lung edema caused by mechanical ventilation with low tidal volume. N=6 for
each group. (C) Serum TNFα concentrations were measured after VILI with tidal volume of
10 ml/kg for 5 hours. Hydrogen treatment significantly reduced VILI-induced elevation of
serum TNFα. N=6, *p<0.05 versus sham/N2 and sham/H2; #p<0.05 versus VILI/N2
Figure. 7
The effects of hydrogen on VILI-associated histopathological changes by MV with low
tidal volume (10 ml/kg). (A) Representative images of lungs stained with F4/80 to visualize
macrophages. Left panel - There were scarce F4/80-positive cells in the lungs of sham
control mice. Middle panel - A marked increase in F4/80-postive cells was observed in the
lung with low-tidal volume ventilation. Right panel -Ventilation with 2% hydrogen significantly
reduced the number of macrophages. 600X magnification; representative images from 4
22
animals for each experimental group are shown. White arrowheads indicate F4/80-positive
cells. (B) Bronchial cell apoptosis was determined by TUNEL assay. Left panel - Few
apoptotic cells were seen in the bronchial epithelium of sham control mice. Middle panel -
There was an increase in the number of TUNEL-positive cells in the bronchial epithelium in
the lung with VILI in 2% nitrogen. Right panel - Hydrogen therapy decreased the number of
TUNEL-positive cells. 800X magnification; representative images from 4 animals for each
experimental group are shown. Black arrowheads indicate TUNEL-positive cells. (C) The
positive cells were counted in a blinded manner and expressed as the number per high-
power field (HPF). Histograms indicating the number of F4/80-positive cells/HPF and TUNEL
positive epithelial cells/HPF. N=4, *p<0.05 versus sham/N2 and sham/H2; #p<0.05 versus
VILI/N2.
23
Table 1. Lung injury scores after 2 hrs VILI (30 ml/kg)
Lung injury score
Group Treatment Alveolar
congestion
Hemorrhage Infiltration
of
neutrophils
Alveolar
wall
thickness
Total score
sham N2 0.25±0.50 0 0.50±0.58 0 0.75±0.50
sham H2 0.25±0.50 0 0.25±0.50 0 0.50±0.58
VILI N2 1.75±0.50 0 1.75±0.96 1.50±0.58 5.0±1.63
VILI H2 0.75±0.50 0 1.50±0.57 0.75±0.50 3.0±0.82*
Data are mean ± standard deviation (p<0.05 vs VILI/N2)
Acute lung injury was scored in each sample (n=6 for each group) according to the following
four items: alveolar congestion, hemorrhage, infiltration or aggregation of neutrophils in
airspace or the vessel wall, and thickness of the alveolar wall/hyaline membrane formation.
Each item was graded according to a five-point scale: 0; minimal (little) damage, 1; mild
damage, 2; moderate damage, 3; severe damage, and 4; maximal damage.
24
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*
#*
*
Figure 1
Figure 3
VILI
N2
VILI
H2
Sham
H2
Sham
N2
Lactate (mmol/L)
1.5
1.0
0.5
2.0
A B
VILI
N2
VILI
H2
Sham
H2
Sham
N2
10
20
30
5
25
BALF cell (x103)
15
0.3
0.2
VILI
N2
VILI
H2
Sham
H2
Sham
N2
0.1
C
BALF protein (mg/mL)
#
*
*
*
*
Figure 3
Egr-1
150
TNFα
200
IL-1βCCL2
*
100
200
200
VILI
N2
VILI
H2
Sham
H2
Sham
N2
VILI
N2
VILI
H2
Sham
H2
Sham
N2
150
100
100
150
150
100
50
250
50
50
Figure 4
50
%β-actin
*
%β-actin
VILI
N2
VILI
H2
Sham
H2
Sham
N2
VILI
N2
VILI
H2
Sham
H2
Sham
N2
*
*
A B
C D
#
##
#
**
*
Figure 4
Figure 5
100
80
60
80
300
60
40
200
100
40
20
Bcl-xL
BAX
20
% β-actin
VILI
N2
VILI
H2
Sham
H2
Sham
N2
VILI
N2
VILI
H2
Sham
H2
Sham
N2
% β-actin
VILI
N2
VILI
H2
Sham
H2
Sham
N2
*
Bcl-2
A B
C
*
*
#
#
#
Figure 5
Figure 6
B
VILI
low TV
H2
(5 hrs)
VILI
low TV
N2
(5 hrs)
60
40
20
80
PaO2 (mmHg)
0
4
6
2
W/D ratio
A
*
*
C
20
TNFα (pg/mL)
40
60
20
30
10
PaCO2 (mmHg)
40
VILI
low TV
H2
VILI
low TV
N2
sham
N2
*
80
VILI
low TV
N2
(30 min)
VILI
low TV
H2
(5 hrs)
VILI
low TV
N2
(5 hrs)
VILI
low TV
N2
(30 min)
VILI
low TV
H2
VILI
low TV
N2
sham
N2
#
#
#
*
**
Figure 6
A
5
15
20
10
F4/80 positive cells (/HPF)
*
C
10
TUNEL positive cells (/HPF)
20
30
VILI
low TV
H2
VILI
low TV
N2
sham
N2
*
40
VILI
low TV
H2
VILI
low TV
N2
sham
N2
Figure 7
B
25
#
#
*
*
Figure 7
Additional files provided with this submission:
Additional file 1: competinginterests - Huang et al -9110853384136199.doc,
202K
http://ccforum.com/imedia/1692180904879677/supp1.doc
... A study by Huang et al. has shown that hydrogen inhalation can attenuate VILI (Huang et al., 2010(Huang et al., , 2011. NFκB was activated after one hour in experimental mice that received 2 % hydrogen instead of nitrogen during ventilation and this was correlated with an increase in anti-apoptotic Bcl-2 protein levels and a decrease in pro-apoptotic Bax protein. ...
... As is the case in most articles with a focus on signaling, we have followed a general schema to describe how an initial stimulus; i.e. mechanical forces in MV, leads to the release of various molecules and how the interaction of these molecules with their receptors start a more or less linear set of events that lead to the final pathophysiological outcome. Intravenous iPSCs and iPSC-CM ( Li et al., 2013) Mouse, n = 10 per group A: VILI HMGB1 and PAI-1, MIP-2, malondialdehyde, glutathione, p-Akt in BALF p-Akt: D = B=C < A B: VILI + iPSCs C: VILI + iPSC-CM iPSCs and iPSC-CM attenuate VILI through suppressing PI3K/Akt D: VILI + LY294002 Inhaled Hydrogen (Huang et al., 2010(Huang et al., , 2011 Mouse, n = 6 per group A: VILI + N2 Lung injury score, Lung wet-to-dry weight ratio, TNF-α, Inhaled anesthetic isoflurane ( Faller et al., 2012) Mouse, n = 8 per group A: VILI lung damage, inflammation, and stress protein expression, IL-1β, MIP-2 in BALF ...
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... SARS-CoV-2 induced activation of p53 apoptosis signaling pathway in lymphocytes may play an important role in the development of lymphopenia of patients (Xiong et al., 2020). H 2 may exert antiapoptotic effects in peripheral blood lymphocytes, a benefit to COVID-19 (Sim et al., 2020); moreover, H 2 can also increase surfactant proteins to further prevent lung injury (Huang et al., 2010), which may be used for prevention and treatment in patients with COVID-19. As a landmark event, recent public reports by China's National Health Commission and the Chinese Center for Disease Control and Prevention, the Chinese Clinical Guidance for COVID-19 Pneumonia Diagnosis and Treatment (7th edition) issued by China National Health Commission recommended the inhalation of O 2 mixed with H 2 gas (33.3% O 2 and 66.6% H 2 ), bringing H 2 to the forefront of contemporary therapeutic medical gas research. ...
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Molecular hydrogen (H 2 ) is a colorless and odorless gas. Studies have shown that H 2 inhalation has the therapeutic effects in many animal studies and clinical trials, and its application is recommended in the novel coronavirus pneumonia treatment guidelines in China recently. H 2 has a relatively small molecular mass, which helps it quickly spread and penetrate cell membranes to exert a wide range of biological effects. It may play a role in the treatment and prevention of a variety of acute and chronic inflammatory diseases, such as acute pancreatitis, sepsis, respiratory disease, ischemia reperfusion injury diseases, autoimmunity diseases, etc.. H 2 is primarily administered via inhalation, drinking H 2 -rich water, or injection of H 2 saline. It may participate in the anti-inflammatory and antioxidant activity (mitochondrial energy metabolism), immune system regulation, and cell death (apoptosis, autophagy, and pyroptosis) through annihilating excess reactive oxygen species production and modulating nuclear transcription factor. However, the underlying mechanism of H 2 has not yet been fully revealed. Owing to its safety and potential efficacy, H 2 has a promising potential for clinical use against many diseases. This review will demonstrate the role of H 2 in antioxidative, anti-inflammatory, and antiapoptotic effects and its underlying mechanism, particularly in coronavirus disease-2019 (COVID-19), providing strategies for the medical application of H 2 for various diseases.
... In addition to inflammation, oxidative stress response also plays an important role [26,27] in VILI. During VILI or ARDS, the activated neutrophils convert oxygen into hydrogen peroxide and superoxide anions through NADPH oxidase [28,29]. ...
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