Campolo et al. Journal of Neuroinflammation 2014, 11:196
http://www.jneuroinflammation.com/content/11/1/196

RESEARCH

JOURNAL OF
NEUROINFLAMMATIONOpen AccessHydrogen sulfide-releasing cyclooxygenase
inhibitor ATB-346 enhances motor function andreduces cortical lesion volume following
traumatic brain injury in miceMichela Campolo1†, Emanuela Esposito1†, Akbar Ahmad1, Rosanna Di Paola1, Irene Paterniti1, Marika Cordaro1,
Giuseppe Bruschetta1, John L Wallace2 and Salvatore Cuzzocrea1,3*

Abstract
Background: Traumatic brain injury (TBI) induces secondary injury mechanisms, including dynamic interplay
between ischemic, inflammatory and cytotoxic processes. We recently reported that administration of ATB-346(2-(6-methoxynapthalen- 2-yl)-propionic acid 4-thiocarbamoyl-phenyl ester), a hydrogen sulfide-releasing
cyclooxygenase inhibitor, showed marked beneficial effects in an animal model of spinal cord injury, significantly
enhancing recovery of motor function and reducing the secondary inflammation and tissue injury.
Methods: Here we evaluated the neuroprotective potential of ATB-346, a hydrogen sulfide-releasing derivative of
naproxen, using the controlled cortical impact (CCI) injury model in mice, one of the most common models of TBI.
Moreover, the aim of the present study was to carefully investigate molecular pathways and subtypes of glial cells
involved in the protective effect of ATB-346 on inflammatory reaction associated with an experimental model ofTBI. In these studies, TBI was induced in mice by CCI and mice were orally administered ATB-346, naproxen (both at
30 μmol/kg) or vehicle (dimethylsulfoxide:1% carboxymethylcellulose [5:95] suspension) one and six hours after
brain trauma and once daily for 10 days.
Results: Results revealed that ATB-346 attenuated TBI-induced brain edema, suppressed TBI-induced neural cell
death and improved neurological function. ATB-346 also significantly reduced the severity of inflammation andrestored neurotrophic factors that characterized the secondary events of TBI.
Conclusions: These data demonstrate that ATB-346 can be efficacious in a TBI animal model by reducing thesecondary inflammation and tissue injury. Therefore, ATB-346 could represent an interesting approach for the
management of secondary damage following CNS diseases, counteracting behavioral changes and inflammatory
process.
Keywords: Brain trauma, Hydrogen sulfide, Neurotrophic factor, Inflammation, Motor recovery, Infarct area, Infarct volume,
Nitrosative stress, Astrogliosis, Neuroprotection* Correspondence: salvator@unime.it†
Equal contributors
1
Department of Biological and Environmental Sciences, University of Messina,
Viale Ferdinando Stagno D’Alcontres, 31-98166 Messina, Italy
3
Manchester Biomedical Research Centre, Manchester Royal Infirmary, Schoolof Medicine, University of Manchester, 29 Grafton Street Manchester, M13
9WU Manchester, UK
Full list of author information is available at the end of the article© 2014 Campolo et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.

Campolo et al. Journal of Neuroinflammation 2014, 11:196
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Background
Traumatic brain injury (TBI) is a growing public healthconcern worldwide. There are over 1.35 million emergency
room visits and 275,000 hospitalizations for nonfatal TBI
each year in the United States, and approximately 40% of
these individuals suffer from long-term disability due totheir injury [1]. The pathophysiology of TBI can be divided
into primary and secondary brain injury [2]. Primary injury
results from the direct, physical brain trauma with tissue
distortion, shearing, vascular injury and cell destruction
probably related to rotational acceleration and deceleration
inertial forces. Secondary brain injury is related to destructive inflammation and biochemical changes. Secondary injury onsets within minutes of primary injury, may last forseveral days and contributes to final outcome [3]. Primaryand secondary brain injuries induce cerebral edema andbleeding. During secondary neuronal injury, healthy neurons around the injury site progressively degenerate, eventually leading to more serious clinical symptoms. Therefore,
secondary neuronal injury plays a key role in the severity of
insult and subsequent clinical prognosis.
Non-steroidal anti-inflammatory drugs (NSAIDs) areamong the most commonly used anti-inflammatory drugs,
but their use is associated with significant, sometimes lifethreatening, adverse effects, particularly in the gastrointestinal (GI) tract [4]. Along with nitric oxide (•NO) and carbon monoxide (CO), hydrogen sulfide (H2S) is regarded as
an important gasotransmitter and endogenous neuromodulator, drawing increasing attention in the literature.
Traditional neurotransmitters bind and activate membrane receptors, while gasotransmitters can freely diffuse
to adjacent cells and directly bind to their target proteins
to modify biological functions. Therefore, H2S is a physiologic gasotransmitter as important as •NO and CO. In thepast decade, increasing evidence shows that H2S plays
multiple roles in the CNS under physiological and pathological states. H2S is produced in various parts of the body
including the heart [5], the cardiovascular system [6] andthe central nervous system (CNS) [7]. With respect to the
CNS, H2S has been reported to exert neuroprotective andneuromodulatory effects [8,9]. Thus, H2S has recently
been exploited in the design of novel NSAID derivatives
that exhibit little, if any, side effects in the GI tract, despite
producing suppression of prostaglandin synthesis andreduction of inflammation at least as effectively as theparent NSAID [10]. Recently, beneficial effects of an H2Sreleasing derivative of naproxen have been shown in an
animal model of spinal cord injury (SCI), significantly enhancing recovery of motor function, possibly by reducing
the secondary inflammation and tissue injury that characterizes this model. The combination of inhibition of cyclooxygenase [11] and delivery of H2S may offer a promisingalternative to existing therapies for traumatic injury [12].
On the basis of these data, H2S could have an important

Page 2 of 15role in reducing inflammatory processes and tissue damage post-brain trauma. Therefore, in the current study weevaluated ATB-346, a novel H2S-releasing derivative of naproxen, for neuroprotective properties in experimental
murine TBI using controlled cortical impact injury (CCI),a model of focal brain injury. Moreover, the aim of thepresent study was to carefully investigate molecular pathways and subtypes of glial cells involved in the protective
effect of ATB-346 on inflammatory reaction associatedwith an experimental model of TBI. In particular, our attention shifts to post-injury recovery of motor function,
reduction of infarct area and of brain tissue inflammation
after TBI.

Methods
Animals

Male CD1 mice (25 to 30 g, Harlan, Milan, Italy), aged between 10 and 12 weeks, were used for all studies. Mice
were housed in individual cages (five per cage) and maintained under a 12:12 hour light/dark cycle at 21 ± 1°C and
50 ± 5% humidity. Standard laboratory diet and tap water
were available ad libitum. The study was approved by the
University of Messina Review Board for the care of animals. Animal care was in compliance with Italian regulations on protection of animals used for experimental andother scientific purposes (Ministerial Decree 16192) aswell as with the Council Regulation (EEC) (Official Journalof the European Union L 358/1 12/18/1986).
Controlled cortical impact experimental traumatic brain
injuryTBI was induced in mice by a controlled cortical impactor.
The mice were anesthetized under intraperitoneal ketamine and xylazine (2.6/0.16 mg/kg of body weight, respectively). A craniotomy was made in the right hemisphere,encompassing bregma and lambda, and between the sagittal suture and the coronal ridge, with a Micro motor handpiece and drill (UGO Basile SRL, Comerio Varese, Italy).
The resulting bone flap was removed and the craniotomy
enlarged further with cranial rongeurs (New Adalat Garh,
Roras Road, Pakistan). A cortical contusion was producedon the exposed cortex using the controlled impactor device Impact OneTM Stereotaxic impactor for CCI (Leica,
Milan, Italy). Briefly, the impacting shaft was extended,and the impact tip was centered and lowered over the craniotomy site until it touched the dura mater. Then, the rod
was retracted and the impact tip was advanced farther toproduce a brain injury of moderate severity for mice (tipdiameter: 4 mm; cortical contusion depth: 3 mm; impact
velocity: 1.5 m/sec). Immediately after injury, the skin incision was closed with nylon sutures, and 2% lidocaine jelly
was applied to the lesion site to minimize any possible
discomfort.

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Test drugs

ATB-346 (2-(6-methoxynapthalen-2-yl)-propionic acid 4thiocarbamoyl phenyl ester) is a derivative of naproxen,which includes a H2S-releasing moiety referred to hereafter
as ‘TBZ’ (4-hydroxythiobenzamide) [13]. ATB-346, TBZ
and naproxen were prepared freshly each day as suspensions in dimethylsulfoxide:1% carboxymethylcellulose (5:95).
Prior to beginning these experiments, a pilot study
was performed to confirm the equipotency of naproxen
and ATB-346 in suppressing cyclooxygenase at the dose
selected. At 30 μmol/kg (oral administration), naproxen
and ATB-346 equally suppressed gastric (prostaglandin
E2) PGE2 synthesis (by more than 90%) and whole blood
thromboxane synthesis ( by more than 95%). This levelof inhibition was evident within 15 minutes and persisted for at least 12 hours after drug administration.
TBZ had no effect on gastric PGE2 synthesis or whole
blood thromboxane synthesis.
Experimental groups

Mice were randomly allocated into one of five groups. In
the TBI + vehicle group, mice were subjected to TBI andreceived the vehicle for TBZ, naproxen and ATB-34(dimethylsulfoxide:1% carboxymethylcellulose) (orally), at
one and six hours after brain trauma (N = 20). The TBZgroup was the same as the TBI + vehicle group, but mice
were administered TBZ only (30 μmol/kg, orally), at one
and six hours after brain trauma (N = 20). The naproxen
group was the same as the TBI + vehicle group, but mice
were administered naproxen only (30 μmol/kg, orally), at
one and six hours after brain trauma (N = 20). The ATB346 group was the same as the TBI + vehicle group, but
mice were administered ATB-346 only (30 μmol/kg, orally), at one and six hours after brain trauma (N = 20). In
the sham + vehicle group mice were subjected to identical
surgical procedures, except for TBI, and were kept underanesthesia for the duration of the experiment (N = 20).
As described below, mice were sacrificed at 24 hours
after TBI in order to evaluate the following parameters:
2,3,5-triphenyltetrazolium chloride (TTC) staining (N =four out of 20 for each group) [14]; histology analysis(N = three out of 20 for each group) and Tumor necrosis
factor (TNF)α, Interleukin (IL)-1β, Glial fibrillary acidic
protein (GFAP) and Ionized calcium binding adaptor
molecule (Iba)1 immunofluorescence (N = three out of
20 for each group) [14]; Western blot analysis (N = fiveout of 20 for each group) and RT-PCR analysis for Glial
cell-Derived Neurotrophic Factor (GDNF), Nerve Growth
Factor (NGF) and Vascular Endothelial Growth Factor(VEGF) levels (N = five out of 20 for each group). In a
separate set of experiments, another 10 animals fromeach group were observed after TBI in order to evaluate
the behavioral testing. Several recent results illustrate
the importance of initiating therapeutic interventions asPage 3 of 15

soon as possible following TBI, preferably within fourhours post-injury, to achieve the best possible neuroprotective effect [15].
Behavioural testing

Mice with TBI display motor and cognitive deficits.
Thus, the present behavioural tests involved analyses ofmotor asymmetry (elevated biased swing test (EBST)
and rotarod test). Training for the rotarod test was initiated at one week. Before the CCI injury, whereas no
training was required for the EBST. The retard treadmill(Accuscan, Inc., Columbus, Ohio, United States) provided a motor balance and coordination assessment.
Data were generated by averaging the scores (total timespent on treadmill divided by five trials) for each animal
during training and testing days. Each animal was placedin a neutral position on a cylinder (3 cm and 1 cm diameter for rats and mice, respectively) then the rod was rotated with the speed accelerated linearly from 0 to 24
rpm within 60 seconds, and the time spent on the retard
was recorded automatically. The maximum score given
to an animal was fixed to 60. For training, animals were
given five trials each day and declared having reached
the criterion when they scored 60 in three consecutive
trials. For testing, animals were given three trials and theaverage score of these three trials was used as the individual rotarod score. The EBST provided a motor asymmetry parameter and involved handling the animal by its
tail and recording the direction of the biased bodyswings. The EBST consisted of 20 trials with the numberof swings ipsilateral and contralateral to the injured
hemisphere recorded and expressed in percentage to determine the biased swing activity.
Quantification of infarct volume

Mice were anesthetized with ketamine and decapitated.
Their brains were carefully removed. The brains were cut
into five coronal slices of 2-mm thickness. Slices were incubated in a 2% solution of TTC at 37°C for 30 minutes
and immersion fixed in 10% buffered formalin solution.
TTC stains the viable brain tissue red while infracted tissue remains unstained [16,17]. For quantification of
infracted area and volumes, the brain slices were photographed using a digital camera (HP Photosmart R707,
Milan, Italy) and then image analysis was performed on a
personal computer with an image analysis software program (using ImageJ for Windows (Institute of Mental
Health, Maryland, USA). To compensate for the effect ofbrain edema the corrected infarct volume was calculated
as previously described in detail [18]:Corrected infarct area ¼ left hemisphere area ‐ ðrighthemisphere area ‐ infarct areaÞ:Campolo et al. Journal of Neuroinflammation 2014, 11:196
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Coronal sections of 5-μm thickness were sectioned fromthe perilesional brain area of each animal and were evaluated by an experienced histopathologist. Damaged neurons were counted and the histopathologic changes of thegrey matter were scored on a six-point scale [19]: 0, nolesion observed; 1, grey matter contained one to five eosinophilic neurons; 2, grey matter contained five to 10 eosinophilic neurons; 3, grey matter contained more than 10
eosinophilic neurons; 4, small infarction (less than one
third of the grey matter area); 5, moderate infarction (onethird to one half of the grey matter area); 6, large infarction (more than half of the grey matter area). The scoresfrom all the sections of each brain were averaged to give a
final score for individual mice. All the histological studies
were performed in a blinded fashion.

that blots were loaded with equal amounts of protein lysates, they were also incubated in the presence of the antibody against β-actin or lamin A/C (1:5,000; Santa Cruz
Biotechnology, Heidelberg, Germany). The signals weredetected with enhanced a chemiluminescence detection
system reagent according to manufacturer’s instruction(Super Signal West Pico Chemiluminescent Substrate,
Pierce Thermo Scientific, Rockford, IL. USA). The relative
expression of the protein bands of IκBα (approximately 37kDa), NFκB (approximately 65 kDa), eNOS (approximately
140 kDa), iNOS (approximately 130 kDa) and COX-2 (approximately 72 kDa) were quantified by densitometry withGel Logic 200 PRO software (GE Healthcare, Milwaukee,
Wisconsin, USA) and standardized to β-actin and lamin
A/C levels. Images of blot signals were imported to analysis software Image Quant TL Software, version 2003 (GEHealthcare, Milwaukee, Wisconsin, USA). A preparationof commercially available molecular weight 10 to 250 kDa
was used to define molecular weight positions, and as
reference concentrations for each molecular weight.

Western blot analyses

Reverse transcription polymerase chain reaction

Cytosolic and nuclear extracts were prepared as previously described [20], with slight modifications. The ipsilateral hemisphere after injury from each mouse wassuspended in extraction Buffer A containing protease inhibitors, homogenized for two minutes, then centrifugedat 1,000 × g for 10 minutes at 4°C. Supernatants contained the cytosolic fraction. The pellets, containing
enriched nuclei, were resuspended in Buffer B containing 1% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl
pH 7.4, 1 mM, ethylene glycol tetraacetic acid (EGTA), 1
mM, ethylenediaminetetraacetic acid (EDTA), 0.2 mM
phenylmethanesulfonylfluoride (PMSF) and protease inhibitors. After centrifugation for 30 minutes at 15,000 ×
g at 4°C, the supernatants containing the nuclear protein
were stored at −80°C for further analysis. The levels of
inducible nitric oxide synthase (iNOS), cyclooxygenase(COX)-2, endothelial nitric oxide synthase (eNOS) andIκBα were quantified in cytosolic fractions. NFκBp65
was quantified in nuclear fractions from brain tissue collected 24 hours after TBI. The filters were probed withspecific Abs anti-iNOS (1:1,000; BD Biosciences, Milan,
Italy), anti-COX-2 (1:1,000; Cayman Chemicals, Tallinn
Estonia), anti-eNOS (1:1000; BD Biosciences, Milan,
Italy), anti-NFκBp65 (1:500; Santa Cruz Biotechnology,
Heidelberg, Germany) and anti-IκBα antibody (1:500;
Santa Cruz Biotechnology, Heidelberg, Germany) at 4°C
overnight in 1 × PBS, 5% (w/v), non-fat dried milk and0.1% Tween-20 (PMT). Membranes were incubated withperoxidase-conjugated bovine anti-mouse IgG secondary
antibody or peroxidase-conjugated goat anti-rabbit IgG(1:2,000; Jackson ImmunoResearch, West Grove, PA,USA) for one hour at room temperature. To ascertain

Total RNA, from contused brain tissue at the impact site
after injury, was extracted by a modified method [21],using TRIzol™ Reagent (Life Technologies, Milan, Italy) according to the manufacturer’s instructions. Reverse transcription was performed by a standard procedure using 2
μg of total RNA. After reverse transcription, 1 μl of reverse
transcriptase (RT) products were diluted in 24 μl of PCRmix, to give a final concentration of 50 U ml −1 of Taq
DNA polymerase (Life Technologies, Milan, Italy), 10 μmof 5′ and 3′ primers, 10 mM of each deoxynucleotide triphosphates (dNTP), 50 mM MgCl2 and 10 × NH4 buffer.
cDNAs underwent 30 cycles for GDNF, NGF, VEGF and
β-actin, each one performed at 94°C for one minute, melting temperature (Tm) °C for 45 seconds and 72°C for 55
seconds (Table 1). After this treatment 10 μl of RT-PCR
products were separated by 1.5% agarose gel electrophoresis in Tris/Borate/EDTA (TBE) 0.5 × (Tris-base 0.089 m,boric acid 0.089 m) containing 0.1 μg ml−1 of ethidium
bromide. Fragments of DNA were seen under ultraviolet
light. β-actin was used as an internal reference.

Values are given as mean ± SEM. The corrected total infarct volume was calculated by summing the infarct area
in each slice and multiplying it by slice thickness (2 mm).
Tissue processing and histology

Immunofluorescence

After deparaffinization and rehydration, detection of TNFα,
IL-1β, GFAP and Iba1 was carried out after boiling in 0.1
M citrate buffer for one minute. Non-specific adsorption
was minimized by incubating the section in 2% (volume/
volume (vol/vol)) normal goat serum in PBS for 20 minutes. Sections were incubated with mouse monoclonalanti-GFAP (1:100, vol/vol Santa Cruz Biotechnology(Heidelberg, Germany), or with polyclonal rabbit antiTNFα (1:100, vol/vol Santa Cruz Biotechnology, Heidelberg,
Germany), or with rabbit anti-IL-1β (1:100, vol/vol Santa

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Table 1 Specific primer sequences
Gene

Forward primer (5′-- > 3′)

Reverse primer (5′-- > 3′)

GDNF

TCA CTG ACT TGG GTT TGG
GCT ATTCA GAC GGC TGT TCT CAC
TCC TANGF

GCA TCG AGT GAC TTT GGAGCGTA CGC CGA TCA AAA ACG
CA

VEGF

TGG ATG TCT ACC AGC GAAGCACA AGG CTC ACA GTG ATTTTβ- actin CAT GAA GTG CGA CGT TGACACAC ATC TGC TGG AAG GTG
GACruz Biotechnology, Heidelberg, Germany) or withmouse monoclonal anti-Iba1 (1:100, vol/vol Santa Cruz
Biotechnology, Heidelberg, Germany) antibody in a humidified oxygen and nitrogen chamber for over night at
37°C. Sections were incubated with secondary antibody
Fluorescein isothiocyanate (FITC)-conjugated anti-mouse
Alexa Fluor-488 antibody (1:2,000 vol/vol Molecular
Probes, Monza, Italy) and with TEXAS RED-conjugatedanti-rabbit Alexa Fluor-594 antibody (1:1000 in PBS, vol/vol Molecular Probes, Monza, Italy) for one hour at 37°C.
For nuclear staining, 2 μg/ml 4′,6′-diamidino-2-phenylindole (DAPI; Hoechst, Frankfurt, Germany) in PBS wasadded. Sections were observed at 20× magnification usinga Leica DM2000 microscope (Leica, Milan, Italy). Opticalsections of fluorescence specimens were obtained using a
HeNe laser (543 nm), an ultraviolet laser (361 to 365 nm)
and an argon laser (458 nm) at a one-minute, two seconds
scanning speed with up to eight averages; 1.5 μm sections
were obtained using a pinhole of 250. Contrast and brightness were established by examining the most brightly
labeled pixels and applying settings that allowed clear
visualization of structural details, while keeping the highest pixel intensities close to 200. The same settings wereused for all images obtained from the other samples that
had been processed in parallel. Digital images were
cropped and figure montages prepared using Adobe
Photoshop 7.0 (Adobe Systems; Palo Alto, California,
United States).

Materials
ATB-346 (2-(6-methoxy-napthalen-2-yl)-propionic acid 4thiocarbamoyl-phenyl ester), sodium naproxen and TBZ(4-hydroxythiobenzamide) were provided by Antibe Therapeutics Inc. (Toronto, Canada). Unless otherwise stated,
all other compounds were obtained from Sigma-AldrichCompany Ltd. (Milan, Italy). All stock solutions were prepared in non-pyrogenic saline (0.9% NaCl; Baxter, Italy) or
10% dimethyl sulfoxide (DMSO).

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analyzed using two factor repeated measures analysis ofvariance (RM ANOVA, group × time). Infarct area, lesion
volume and densitometric analysis data were analyzed by
ANOVA followed by a Bonferroni post-hoc test for multiple
comparisons. Histological score and percentage total tissue
area were analyzed by Student’s t-test. For all comparisons,P <0.05 was considered to be significant. In the experiments
involving histology or immunohistochemistry, the figures
shown are representative of at least three experiments
performed on different experimental days on the tissue
sections collected from all the animals in each group.

Results
Effect of ATB-346 on IκBα degradation and NFκBp65
translocation

To investigate whether the cellular mechanism throughATB-346 could attenuate inflammatory processes weassessed Western blot analysis of the ipsilateral hemisphere after TBI, using an IκBα and an NFκB-specific antibodies. The results showed a basal expression of IκBα inthe brain from sham-mice (Figure 1A, see densitometry
analysis A1), while IκBα expression was significantly reduced in mice subject to TBI and TBZ administration, as
showed in Figure 1A and 1A1. Naproxen treatment
blunted the degradation of IκBα but ATB-346 was able tosignificantly restore IκBα degradation (Figure 1A, seedensitometry analysis A1). Moreover, p65 subunit translocation was increased after TBI and TBZ injection in the
nuclear brain homogenates, compared with sham-group.
ATB-346 administration significantly reduced the translocation of p65 in nuclei compared to the TBI group(Figure 1B, see densitometry analysis B1).
Effect of ATB-346 on iNOS and COX-2 expression

To determine the role of •NO produced during TBI, iNOS
expression was evaluated by Western blot analysis. A significant increase in iNOS expression was observed in thecontused area from mice subjected to TBI and TBZ administration (Figure 1C, see densitometry analysis C1). Consequently, naproxen reduced TBI-induced iNOS expression(Figure 1C, see densitometry analysis C1); on the other
hand, a significant decrease in iNOS expression was observed after ATB-346 treatment (Figure 1C see densitometry analysis C1). Similarly, COX-2 expression was induced
by TBI and TBZ administration compared to the shamgroup (Figure 1D, see densitometry analysis D1). Bothtreatments with naproxen and ATB-346 lowered COX-2
expression (Figure 1D, see densitometry analysis D1).

Statistical evaluation

Effect of ATB-346 on TNFα and IL-1β expression in astrocytes
after traumatic brain injury

Data are mean ± SEM. Data were analyzed using Graphpad
PRISM V (Graphpad Software Inc., La Jolla, California,
United States). Swing activity and time on platform wereTo analyse the activation of astrocytes and cytokines expression, contused brain tissue at the impact site after
injury was double-stained with antibodies against GFAPCampolo et al. Journal of Neuroinflammation 2014, 11:196
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Figure 1 Effects of ATB-346 on Nuclear factor κB (NFκB) pathway and pro-inflammatory enzymes. Degradation of IκBα was significantly blocked
by Naproxen and ATB-346 treatment (A). Moreover, ATB-346 treatment resulted in an inhibition of nuclear translocation of p65 (B). Translocation of NFκB isa critical step in the coupling of extracellular stimuli to the transcriptional activation of specific target genes. A significant increase in inducible nitric oxide
synthase (iNOS) and cyclooxygenase (COX)-2 (C and D, respectively) was observed in the injured area from TBI mice compared with the Sham mice.
ATB-346-treated mice had notably reduced expression of pro-inflammatory enzymes (C and D, respectively). Data show one representative blot from three
independent experiments with similar results. Mean ± SEM of four to five animals per group. One-way ANOVA, followed by Bonferroni’s multiple
comparison test. ***P <0.001 versus sham, ##P <0.01, ###P <0.001 versus TBI, °P <0.05 versus TBI + naproxen.

(green; Figures 2 and 3) and TNFα (red; Figure 2) or IL1β (red; Figure 3). Brain sections revealed increased
astrogliosis (GFAP+ cells) in TBI and TBZ panels. Moreover, a marked co-localization of TNFα in GFAP+ cells
was present after TBI and TBZ administration (merged,
Figure 2). TNFα and IL-1β expressions were significantly
reduced by ATB-346 treatment (TBI + ATB-346 panels;
Figures 2 and 3).

or IL-1β (red; Figure 5). Microglial cells (Iba1- + cells)
expressed TNFα and IL-1β in TBI and TBZ panels(Figures 4 and 5, respectively). There was an evident colocalization of TNFα and Iba1 in TBI and TBZ panels(merged, Figure 4). Considerable reductions in cytokines
expressions were evident in naproxen panels (Figures 4 and
5, respectively); however, ATB-346 reduced TNFα and IL1β expressions in microglia (Figures 4 and 5, respectively).

Effect of ATB-346 on TNFα and IL-1β expression in
microglia after traumatic brain injury

Effect of ATB-346 on mRNA levels of neurotrophic factors

To evaluate the microglia activation and its correlation
with cytokines expression, ipsilateral hemisphere to theinjury site were double-stained with antibodies against
Iba1 (green; Figures 4 and 5) and TNFα (red; Figure 4)

To test whether ATB-346 modulates the levels of theneurotrophic factors, we studied GDNF and NGF levels inbrain tissue using semi-quantitative RT–PCR analysis. A
significant decrease in GDNF (470 bp) and NGF (318 bp)
mRNA expression following TBI and TBZ administration

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Figure 2 Effects of ATB-346 on tumor necrosis factor (TNF)α expression in glial fibrillary acidic protein (GFAP) positive cells. Brain tissue
was double-stained with antibodies against GFAP, green) and TNFα (red). The red spots indicate the co-localizations (merged). Brain sections
revealed increased astrogliosis (GFAP+ cells) in TBI and TBZ panels. Considerable GFAP immunoreactivity was present in TBI and TBZ panels. TNFα
expression was significantly reduced by ATB-346 treatment (TBI + ATB-346 panels) compared to naproxen treatment (TBI + naproxen panels).
All images were digitalized at 600 dpi.

Figure 3 Effects of ATB-346 on interleukin (IL)-1β expression in glial fibrillary acidic protein (GFAP) positive cells. Brain tissue wasdouble-stained with antibodies against GFAP (green) and IL-1β (red). Brain sections revealed increased astrogliosis (GFAP+ cells) in TBI and TBZ
panels. Considerable GFAP immunoreactivity was present in TBI and TBZ panels. IL-1β expression was significantly reduced after ATB-346
treatment (TBI + ATB-346 panels) respect to naproxen treatment (TBI + naproxen panels). All images were digitalized at 600 dpi.

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Figure 4 Effects of ATB-346 on tumor necrosis factor (TNF)α in ionized calcium binding adaptor molecule (Iba)1 positive cells. Braintissue was double-stained with antibodies against Iba1 (green) and TNFα (red). Microglial cells (Iba1+ cells) expressed TNFα in TBI and TBZ panels.
A considerable reduction of cytokine expression was present in naproxen panels; however, ATB-346 reduced notably TNFα expressions (TBI + ATB-346
panels). The red spots indicate the co-localizations (merged). All images were digitalized at 600 dpi.

was evident. Moreover, ATB-346 significantly increasedmRNA levels of both neurotrophic factors examined(Figure 6A and B respectively, see densitometry analysis
A1 and B1 respectively).
Effect of ATB-346 on vascular components after traumaticbrain injury

To investigate whether ATB-346 could promotenormalization of the impaired neurovascular unit, weobserved VEGF level and eNOS expression. RT-PCR
showed a significant increase in VEGF (308 bp) mRNA
expression, and ATB-346 significantly increased its level(Figure 6C, see densitometry analysis C1). Moreover, byWestern blot analysis we observed a significantly
increase in eNOS expression in the TBI group, and
ATB-346 upregulated its expression (Figure 6D, seedensitometry analysis D1).
Infarct outcome in ATB-346-treated mice after traumaticbrain injury

A histological examination of brain sections at the level ofthe perilesional area, stained 24 hours after injury, revealed
significant damage in the TBI and TBI + TBZ groups, suchas prominent and thickened blood vessels, ischemicchanges and gliosis in the brain parenchyma (Figure 7B
and C respectively, see densitometry analysis F) compared
to sham mice (Figure 7A, see densitometry analysis F).

Naproxen treatment attenuated the development of inflammation at 24 hours after TBI; ATB-346 significantly
reduced the degree of brain injury (Figure 7D and E respectively, see densitometry analysis F).
Brain edema indicates pathology associated with endothelial cell activation and endothelial dysfunction. To
evaluate the effect of ATB-346 on brain edema and infarctions in the TBI and TBZ group, we performed TTC staining (Figure 8A). At 24 hours after TBI, ATB-346-treatedmice had a significantly smaller infarct area (Figure 8B)
and volume (Figure 8C).
Neurological deficits after ATB-346 administration

To investigate the relationship between neurological deficits in the setting of TBI we used two different tests: theEBST and the rotarod test, considered the most sensitive
vestibular motor test to assess motor function. The EBST
provided a motor asymmetry parameter and involved
handling the animal by its tail and recording the directionof the biased body swings. The EBST consisted of 20 trials
with the number of swings ipsilateral and contralateral tothe ischemic hemisphere recorded and expressed in percentage to determine the biased swing activity. Mice were
tested seven days after TBI for both behavioral tests. CCIinjured mice and TBZ-treated mice displayed a range ofimpairments in locomotor tasks as showed in Figure 8D
and E, respectively. Both groups of animals that received

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Figure 5 Effects of ATB-346 on interleukin (IL)-1β expression in ionized calcium binding adaptor molecule (Iba)1 positive cells. Braintissue was double-stained with antibodies against Iba1 (green) and IL-1β (red). Microglial cells (Iba1+ cells) expressed IL-1β in TBI and TBZ panels.
A considerable reduction of IL-1β expression was present in naproxen panels; at least, ATB-346 markedly reduced TNFα and IL-1β expressions(ATB-346 panels). The red spots indicate the co-localizations (merged). All images were digitalized at 600 dpi.

naproxen or ATB-346 were significantly less impaired in
the EBST and rotarod tests compared with the TBI group(Figure 8D and E, respectively).

Discussion
A number of animal models have been developed to induce
brain trauma. Of these the most commonly used areweight-drop injury, fluid percussion injury (FPI) and CCI.
The use of TBI models has resulted in an increased understanding of the pathophysiology of TBI, including changes
in molecular and cellular pathways and neurobehavioral
outcomes. CCI models utilize a pneumatic pistol to laterally
deform the exposed dura and provide controlled impact
and quantifiable biomechanical parameters. This model
produces graded, reproducible brain injury. Dependent on
the severity of injury, CCI results in an ipsilateral injury
with cortical contusion, hemorrhage and blood-brain barrier disruption. CCI injury reproduces changes reported inclinical head injuries such as cortical contusion, brain
edema, subarachnoid hemorrhage, elevated intracerebral
pressure, reduced cortical perfusion, decreased cerebral
blood flow and neuro-endocrine and metabolic changes[22]. The predominantly focal brain injury caused by CCI
makes this model to a useful tool for studying the pathophysiology of the secondary processes induced by focal
brain injury. However, there is a lack of brain stem deformation in this model and thus a low mortality rate.

In recent years, H2S has been recognized as a fundamental signalling molecule that plays important roles inexerting cytoprotective effects in the CNS, since it canprotect neurons and glia from oxidative stress [9,23].
H2S also exert many anti-inflammatory effects, including
inhibition of leukocyte-endothelial adherence, reductionof edema formation [24,25] and inhibition of NFκB activation [26]. H2S is produced endogenously via enzymatic
activity, non-enzymatic pathways (such as reduction of
thiol-containing molecules), and is also released fromintracellular sulfur stores (sulfane sulfur). Cystathionine
β-synthase (CBS) is believed to be the critical enzymethat produces H2S, resulting in the modulation of neurological function. H2S generated by cystathionine γ-lyase(CSE) was next discovered as an important modulator ofvasorelaxation in smooth muscle. They separately coordinate with L-cysteine to produce H2S, L-serine and ammonium. After the discovery of H2S as a potential
neurological and vasorelaxant signaling molecule, moretargets were expected to be found [27,28]. The enhanced
beneficial effects of ATB-346 over those of naproxen are
most likely attributable to the H2S release from theformer, and may be due to the neuroprotective and antiinflammatory properties of this gaseous mediator, acting
in a complimentary manner to the anti-inflammatory effects associated with inhibition of COX activity. Indeed,
the marked reduction of gastrointestinal toxicity ofCampolo et al. Journal of Neuroinflammation 2014, 11:196
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Figure 6 RT–PCR analysis for GDNF, NGF and VEGF (A, B and C, respectively). ATB-346 treatment significantly increased both GDNF and NGF
mRNA expression compared to TBI (A and B, respectively). ATB-346 determined an important increase in VEGF mRNA expression (C). β-actin wasused as an internal control. mRNA was extracted and reverse-transcribed as described in the Methods section. Similar results were obtained infour additional separate experiments. No bands were observed in the absence of cDNA. Western blot analysis showed that eNOS expression in
TBI mice was increased compared with sham mice (D), while ATB-346 upregulated its expression (D). A representative blot of homogenatesobtained from five animals per group is shown, and densitometry analysis of all animals is reported (A1 to D1). A P value of less than 0.05 wasconsidered significant. *P < 0.05, **P <0.01, ***P <0.001 versus sham, #P <0.05 versus TBI.

ATB-346 versus naproxen has been attributed to themucosal protective and anti-inflammatory effects (forexample, inhibition of leukocyte adherence to vascularendothelium) of the H2S released from this drug [29].
We observe a light beneficial effect of TBZ, the H2Sreleasing moiety of ATB-346, on several parameters. The
release of H2S from TBZ may not be as great as thatfrom an equimolar amount of ATB-346. Previous studies
have shown that TBZ alone releases very little H2S, butwhen covalently linked to another drug, such as an
NSAID, considerably more H2S is released [30].

Focal lesions to the brain display a characteristic
inflammatory response with infiltration of peripheral
immune cells after injury. These cells are believed to beimportant because they contain and release a multitudeof inflammatory mediators associated with increased tissue injury. Neutrophils peak approximately two days
post-TBI, and monocytes slightly later [31]. Leukocyte
homogenates from post-TBI patients display upregulation of iNOS, COX-2 and nicotinamide adenine dinucleotide phosphate-oxidase; all enzymes involved in
producing the damaging neutrophilic oxidative burst

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Figure 7 Histological examination of brain sections after 24 hours. Brain sections from TBI mice and TBZ-treated mice (B and C respectively,
see densitometry analysis F) demonstrated brain tissue injury and inflammatory cell infiltration. Naproxen treatment did not attenuate completely
the development of acute brain injury at one and six hours after TBI (D, see densitometry analysis F). On the contrary, ATB-346 treatment reduced
the degree of brain injury and the inflammatory cells infiltration (E, see densitometry analysis F). Figure is representative of at least three experiments
performed on different experimental days. ###P <0.001 versus TBI.

[32]. To confirm the pathological contributions to brain
inflammation, we have demonstrated here expression of
COX-2 and iNOS in the injured tissue after TBI, but
TBI-induced iNOS and COX-2 expression are significantly lower in injured brains from ATB-346-treatedmice.
Post-TBI there is increased infiltration of neutrophils,
astrocytosis, edema and both pro- and anti-inflammatory
cytokines release. The major pro-inflammatory cytokines
released are IL-1β, IL-6 and TNFα. The anti-inflammatory
cytokines are IL-10 and transforming growth factor beta.
We demonstrate that increased microglial and astrocyte
activation is present 24 hours after TBI. Moreover, immunofluorescence staining showed increased TNFα andIL-1β expression in astrocytes and microglia in TBI group.
ATB-346 treatment importantly reduced TNFα and IL-1β
expression in these glial cells. Apparently, ATB-346 mightenhance actual functional neuronal regeneration via inhibiting glial scar formation during TBI.
Neurotrophic factors have well-established roles insurvival, differentiation and function of CNS neurons.
Exogenous NGF, for example, plays a critical role inneuronal plasticity and regenerative potential, as well as
the inhibition of neural apoptosis after TBI [33,34]. A
study found brain-derived neurotrophic factor,(BDNF)
and neurotrophin-3 (NT-3) support the survival of injured CNS neurons in vitro and in vivo, induce neurite
outgrowth and increase the expression of key enzymes
for neurotransmitter synthesis [35]. GDNF is capable ofprotecting against hippocampal neuronal death [36],attenuating brain swelling and reducing the lesion volume [37] after TBI. Our results visibly showed restored
GDNF and NGF levels after ATB-346 treatment, maintaining their protective action.
VEGF, an angiogenic growth and survival factor for
endothelial cells that also exhibits neurotrophic and neuroprotective effects, has been implicated in neovascularization that precedes brain tissue repair and nerve
regeneration following brain injury and is required tore-establish metabolic support [38]. VEGF is upregulated
during many pathological events, and it is induced in
astrocytes located in and surrounding edematous tissue
following brain contusion [39]. Our study showed that
ATB-346 significantly increased VEGF levels. Thus, it
could be hypothesized that ATB-346-facilitated the increase in VEGF expression in the lesion area, resulting
in the secretion of VEGF from synthesizing cells and therestoration of the neurovascular unit.
•NO is a key regulator of cerebral circulation by its
contribution to basal vascular tone that in vasculature ismainly derived from eNOS. eNOS is predominantly
expressed by vessels endothelial cells and are also located in Purkinje cell bodies in the cerebellar cortex,
olfactory bulb, dentate nucleus in granular layer andhippocampal pyramidal cells and astrocytes surrounding
the cerebral blood vessels [40]. A recent paper showedthat eNOS is central after trauma for the maintenance ofblood flow in the injured cortex for at least 24 hours
after TBI; this is based on the observation that eNOS
knockout mice have lower cerebral blood flow at that

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Figure 8 Effect of ATB-346 on brain edema, infarction and locomotor activity. Representative TTC stained brain section (four out of the six
consecutive sections from cranial to caudate region) corresponding to largest infraction from each group (A). Brain sections (2 mm thick) were
stained with TTC at 24 hours after TBI and show significant difference after ATB-346 treatment in terms of area (B) and volume (C) of infarctions.
The figures are representative of at least three experiments performed on different experimental days. TBI determined a range of impairments inlocomotor tasks, as showed by the EBST (D) and the rotarod test (E), after seven days. Both groups of animals that received naproxen or ATB-346
were significantly less impaired in EBST and rotarod tests compared with the TBI group. ATB-346-treated mice displayed significant improvementin their behavioral performance as revealed by decreased biased swing activity in the EBST (D) and increased time on the rotating rod in therotarod test (E). Each data are expressed as mean ± SEM from N = four male CD mice for each group. A P value of less than 0.05 was consideredsignificant. ***P <0.001 versus sham, ###P <0.001 versus TBI.

time point compared to wild-type mice [41]. According
to this data, our results showed an evident increase ineNOS expression 24 hours after TBI. The increase ineNOS protein may represent either a protective or a reparative response, since it has been reported that eNOS isnecessary to counteract posttraumatic cerebral hypoperfusion at 24 hours after CCI-TBI in mice [42]. There issubstantial evidence that H2S upregulates •NO production in endothelial cells through the activation of eNOS,
inducing angiogenesis and improving functional outcome[43-45]. Therefore, ATB 346 upregulates its expression, increasing functional protein expression and augmentationof cerebral blood flow, also in the brain. Furthermore,treatment with ATB-346 results in a significant reduction
in inflammation and it is also accompanied by a detectablehistological improvement of TBI. As shown in our recent
paper, ATB-346 can markedly accelerate recovery of
motor function in mice subjected to SCI [12]. Here, in adifferent model of neurotrauma, we confirmed that ATB346 significantly improved the latency compared to thenaproxen group, indicated as a mediator of the mechanism to promote recovery and to enhance the repair mechanism. In the present study, post-TBI administration of
ATB-346 not only facilitated functional recovery, but also
reduced tissue damage within hours following injection.
The ameliorating effect of ATB-346 at tissue level was
further corroborated by its ability to reduce the extent ofneurodegeneration. The neuroprotective basis for theseactions seems to be dependent on the H2S-releasing moiety of ATB-346, as also stated in a recent paper [46].

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Figure 9 Schematic representation of the method outlined in this experiment and results obtained.

Moreover, the observation that neither naproxen nor TBZ
produce the same ‘restorative’ effect on neurotrophic factors as seen with ATB-346 suggests that both suppressionof COX and delivery of H2S is required to achieve the observed effect. The properties of this compound are summarized in Figure 9. In May 2014 Antibe Therapeutics hasannounced the submission of a clinical trial application to
Health Canada for ATB-346.

Conclusions
The need for developing new therapeutics for TBI treatment and the current lack of specific therapy for this indication underscore the importance of identification andcharacterization of novel neuroprotective compounds.
Released-H2S may account for the absence of deleterious
gastric effects, thus making of ATB-346 a potentially
useful therapeutic alternative to traditional naproxen forthe management of secondary damage following CNS

diseases, counteracting behavioral changes and inflammatory process.
Abbreviations
CCI: Controlled cortical impact; COX: Cyclooxygenase; EBST: Elevated Biased
Swing Test; TBZ: 4-hydroxythiobenzamide; DMSO: dimethyl sulfoxide;dNTP: deoxynucleotide triphosphates; EGTA: ethylene glycol tetraacetic acid;
EDTA: ethylenediaminetetraacetic acid; iNOS: inducible nitric oxide synthase;
NSAIDs: Non-steroidal anti-inflammatory drugs; IL-1β: interleukin 1β;
Iba1: ionized calcium binding adaptor molecule 1; TNFα: tumor necrosis
factor α; GFAP: glial fibrillary acidic protein; NFκB: Nuclear factor κB.

Competing interests
The authors declare that they have no competing interests.

Authors’ contributions
MC and EE performed experiments and prepared the manuscript. AA, RD, RS,
MC and GB performed experiments and the biochemical analysis. JLW and
SC planned the study, analyzed the results and prepared the manuscript. All
authors read and approved the final manuscript.

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Acknowledgements
The authors would like to thank Mrs Maria Antonietta Medici for her
excellent technical assistance during this study, Mr Francesco Soraci and Mr
Giuseppe Mancuso for their secretarial and administrative assistance andMiss Valentina Malvagni for her editorial assistance with the manuscript.
Author details
1
Department of Biological and Environmental Sciences, University of Messina,
Viale Ferdinando Stagno D’Alcontres, 31-98166 Messina, Italy. 2Inflammation
Research Network, University of Calgary, 3330 Hospital Drive NW, Calgary,Alberta T2N 4 N1, Canada. 3Manchester Biomedical Research Centre,
Manchester Royal Infirmary, School of Medicine, University of Manchester, 29
Grafton Street Manchester, M13 9WU Manchester, UK.
Received: 4 September 2014 Accepted: 7 November 2014

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Cite this article as: Campolo et al.: Hydrogen sulfide-releasing
cyclooxygenase inhibitor ATB-346 enhances motor function andreduces cortical lesion volume following traumatic brain injury in mice.
Journal of Neuroinflammation 2014 11:196.

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