DA conceived and designed the article and coordinated the manuscript preparation. GD contributed the chapter devoted to "Eye, microgravity, and vision" and rewrote extensively several sections; EC wrote the chapter "Countermeasures for space from bed rest studies." CP contributed to the chapter "Weightlessness and fluid redistribution as stress factors in space." All other chapters were contributed by MG, IB, and DA. All authors read, discussed, edited, and approved the whole work.

#### ACKNOWLEDGMENTS

DA was recipient of contracts ILSRA-2009-1026 from the European Space Agency and 2013-066-R.0 (SFEF) from the Italian Space Agency. EC was the recipient of contracts 2013-032- R.0 (AEQUABED) and 2013-033-R.0 (QT-BED) from the Italian Space Agency. The authors are truly grateful to Antonio L'Abbate and Flavio Coceani for critical reading of the manuscript.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Demontis, Germani, Caiani, Barravecchia, Passino and Angeloni. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Fluoxetine Protection in Decompression Sickness in Mice is Enhanced by Blocking TREK-1 Potassium Channel with the "spadin" Antidepressant

Nicolas Vallée1 \*, Kate Lambrechts 1, 2, Sébastien De Maistre3 , Perrine Royal 1 , Jean Mazella4 , Marc Borsotto4 , Catherine Heurteaux 4 , Jacques Abraini 1, 5, 6 , Jean-Jacques Risso1 and Jean-Eric Blatteau1

1 Institut de Recherche Biomédicale des Armées, Equipe Résidante de Recherche Subaquatique Opérationnelle, Toulon, France, 2 UFR STAPS, Laboratoire Motricité Humaine Education Sport Santé, Université du Sud Toulon Var, La Garde, France, 3 Hôpital d'Instruction des Armées, Service de Médecine Hyperbare et Expertise Plongée, Toulon, France, 4 Centre National de la Recherche Scientifique and Université de Nice Sophia Antipolis, Institut de Pharmacologie Moléculaire et Cellulaire, UMR 7275, Valbonne, France, 5 Département d'Anesthésiologie, Université Laval, Québec, QC, Canada, 6 Faculté de Médecine, Université de Caen Normandie, Caen, France

#### Edited by:

Antonio L'Abbate, Scuola Superiore Sant'Anna, Italy

#### Reviewed by:

Angelo Gemignani, Consiglio Nazionale delle Ricerche, Italy Paul Kenneth Witting, The University of Sydney, Australia

> \*Correspondence: Nicolas Vallée<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Integrative Physiology, a section of the journal Frontiers in Physiology

Received: 27 August 2015 Accepted: 29 January 2016 Published: 16 February 2016

#### Citation:

Vallée N, Lambrechts K, De Maistre S, Royal P, Mazella J, Borsotto M, Heurteaux C, Abraini J, Risso J-J and Blatteau J-E (2016) Fluoxetine Protection in Decompression Sickness in Mice is Enhanced by Blocking TREK-1 Potassium Channel with the "spadin" Antidepressant. Front. Physiol. 7:42. doi: 10.3389/fphys.2016.00042 In mice, disseminated coagulation, inflammation, and ischemia induce neurological damage that can lead to death. These symptoms result from circulating bubbles generated by a pathogenic decompression. Acute fluoxetine treatment or the presence of the TREK-1 potassium channel increases the survival rate when mice are subjected to an experimental dive/decompression protocol. This is a paradox because fluoxetine is a blocker of TREK-1 channels. First, we studied the effects of an acute dose of fluoxetine (50 mg/kg) in wild-type (WT) and TREK-1 deficient mice (knockout homozygous KO and heterozygous HET). Then, we combined the same fluoxetine treatment with a 5-day treatment protocol with spadin, in order to specifically block TREK-1 activity (KO-like mice). KO and KO-like mice were regarded as antidepressed models. In total, 167 mice (45 WTcont 46 WTflux 30 HETflux and 46 KOflux) constituting the flux-pool and 113 supplementary mice (27 KO-like 24 WTflux2 24 KO-likeflux 21 WTcont2 17 WTno dive) constituting the spad-pool were included in this study. Only 7% of KO-TREK-1 treated with fluoxetine (KOflux) and 4% of mice treated with both spadin and fluoxetine (KO-likeflux) died from decompression sickness (DCS) symptoms. These values are much lower than those of WT control (62%) or KO-like mice (41%). After the decompression protocol, mice showed significant consumption of their circulating platelets and leukocytes. Spadin antidepressed mice were more likely to exhibit DCS. Nevertheless, mice which had both blocked TREK-1 channels and fluoxetine treatment were better protected against DCS. We conclude that the protective effect of such an acute dose of fluoxetine is enhanced when TREK-1 is inhibited. We confirmed that antidepressed models may have worse DCS outcomes, but concomitant fluoxetine treatment not only decreased DCS severity but increased the survival rate.

Keywords: bubble, decompression sickness, diapedesis, kcnk2, TREK-1, diving, depression, capillary leak

### INTRODUCTION

In this study, fluoxetine and the new antidepressant spadin were used to find a treatment strategy against decompression sickness.

Circulating bubbles cause cell damage (Vallee et al., 2013), prothrombotic phenomena, ischemia, and diapedesis (Dutka et al., 1989; Zamboni et al., 1989, 1992, 1993; Dal Palu and Zamboni, 1990; Helps and Gorman, 1991). This inflammation can spread systemically and may degenerate into a vicious cycle, ending in multiple organ failure (Jacey et al., 1976; DeGirolami and Zivin, 1982; Ersson et al., 1998). Spinal cord and brain neurological damage underlie the most serious symptoms of decompression sickness (DCS; Gempp et al., 2008). These symptoms result from circulating bubbles generated by a pathogenic decompression, following a dive for example. Even after standard treatment with hyperbaric oxygen, 20–30% of victims suffer from sequelae after a neurological DCS (Blatteau et al., 2011). We aimed to establish a potential treatment strategy using an animal model of DCS.

Fluoxetine, the active compound in the antidepressant Prozac™, prevents the reuptake of serotonin (5 hydroxytryptamine, 5-HT) by inhibiting serotonin transporters (SERT) located in neurons, platelets (Lesch et al., 1993), and leukocytes (Faraj et al., 1994; Lima and Urbina, 2002; Yang et al., 2007). SERT increases the concentration of circulating serotonin (Brenner et al., 2007). When used in a single high dose, fluoxetine is also believed to mediate neuroprotection (Pariente et al., 2001; Chollet et al., 2011; Taguchi et al., 2012) by inhibiting NMDA-R (Vizi et al., 2013), regulating inflammatory effects (Kubera et al., 2001; Jin et al., 2009; Lim et al., 2009) and algesia (Kostadinov et al., 2014). We have previously demonstrated that WT mice treated with fluoxetine are more resistant to DCS and that fluoxetine inhibits the inflammatory process by reducing the level of circulating IL-6, a pro-inflammatory cytokine (Blatteau et al., 2012).

TREK-1, the product of the kcnk2 gene, regulates cell excitability and prevents neuron death by inhibiting NMDAdependent glutamatergic excitotoxicity induced by ischemia (Franks and Honore, 2004; Heurteaux et al., 2004; Buckler and Honore, 2005; Honore, 2007; Dedman et al., 2009). The mechanosensitive TREK-1 channel (Franks and Honore, 2004) is activated by a mechanical deformation of the cellular membrane, for example by a deformation induced by air depression (Heurteaux et al., 2004). Additionally, in mice, decompressioninduced desaturation also activates TREK-1. Consequently, KO ("KO" is used in the mean of "TREK-1−/− mice") mice are more sensitive to DCS than WT (TREK-1+/+) mice (Vallee et al., 2012). TREK channel activity is also under the control of several different mechanisms acting either on channel trafficking and surface density or directly on gating properties (Noel et al., 2011). Furthermore, KO mice display a depression-resistant phenotype, similar to chronic fluoxetine-treated mice (Heurteaux et al., 2006).

We have shown that the opening of TREK-1 channels is protective in DCS, suggesting that TREK-1 channel activity limits ischemia-induced glutamatergic toxicity (Vallee et al., 2012). The TREK-1 channel is directly inhibited by fluoxetine (Heurteaux et al., 2006; Bogdan et al., 2011; Sandoz et al., 2011) and by spadin, a new antidepressant that internalizes the channel after a 5-days treatment and consequently abolish channel activity (Mazella et al., 2010; Moha Ou Maati et al., 2011), so the question arises as to whether acute fluoxetine treatment could be significantly more efficient when the TREK-1 channel is impaired: we suggest if less fluoxetine can link to TREK-1, the fluoxetine anti-inflammatory effect should be enhanced in a dose-dependent manner, although the neuroprotection afford by the TREK-1 activity could be loss. Conversely, we wondered whether mice pre-treated with an anti-depressant drug, that impairs TREK-1 activity, would be sensitized to DCS.

In this study, we wanted to obtain a better understanding of the protection afforded by fluoxetine in our DCS model. We aimed to block the fluoxetine binding on TREK-1, and therefore to promote other fluoxetine pathways, resulting in a better efficiency for the inhibition of the NMDA-R or the regulation of interleukin releases for example (**Figure 2**).

Before the exposure to the pathogenic decompression, we gave an acute dose of fluoxetine on wild-type (WT: TREK-1+/+) mice and on mice to whom TREK-1 disappeared from the membrane surface. We therefore used TREK-1 knockout (KO: TREK-1 −/−) and heterozygous (HET: TREK-1+/−) mice, or 5 days of treatment with spadin resulting in KO-like (TREK-1−/−like) mice, to inhibit TREK-1 channel activity. KO and KO-like mice were considered the antidepressed models. We obviously did not use chronic fluoxetine (more than 21 days are necessary) on WT mice to obtain an antidepressive state, followed by an acute dose of fluoxetine; while of interest, this would not allow us to reach a conclusion.

### MATERIALS AND METHODS

#### Animals and Ethical Statement

All procedures involving experimental animals were in line with European Union rules (Directive 2010/63/EU) and French law (Decree 2013/118). The ethics committee of the Institut de Recherche Biomédicale des Armées approved this study. According to our animal care committee, a scoring system inspired by the Swiss veterinary guidelines was implemented to ensure the welfare of animals. A dedicated observer scored (from 0 to 3) the stress or pain relating to some criteria for each animal, and then completed a sheet (Supplementary Data). Pain of degree 3 (very painful) in one case or a total score of 12 in the table were the ethical endpoints. On this sheet, the most commonly found were: vocalizing, aggression or withdrawn behavior, reduction in exploratory behavior, licking, closed eyes, tears, bubbles in the eyes, high respiratory rate, runny nose, fur bristling, labored breathing, convulsions, paralysis, difficulty moving, and problems with the fore or rear limbs (classified as motor disorders). In this study, no score reached 12 and there was no need to cull the animal based on these criteria. Actually, mice displaying degree 3 convulsions died very rapidly. At the end of the experiment, mice were anesthetized first with halothane (5% in oxygen, Halothane, Belamont, France) in order to gain time and to minimize stress, and then with an intraperitoneal injection of a mixture of 16 mg/kg xylazine (Rompum R 2%, Bayer Pharma) and 100 mg/kg ketamine (Imalgène R 1000, Laboratoire Rhône). Our investigator (NV) is associated with agreement number 83.6 delivered by the Health and Safety Directorate of our department, as stated in the French rules R.214-93, R-214- 99, and R.214-102. Mice were housed in an accredited animal care facility. Mice kept were in group cages both during rest and during the experiments and maintained on a regular day (6:00 a.m.–6:00 p.m.)/night (12 h) cycle. Food (AO3, UAR) and water were provided ad libitum and the temperature was kept at 22 ± 1 ◦C.

In TREK-1−/− mice, the gene encoding the TREK-1 channel was knocked out by Cre-Lox recombination (Heurteaux et al., 2004). The comparator animals were analogous C57Bl/6 mice (Charles River Laboratory, Arbresle, France). In order to preclude phenotypic variation between the different strains (Sato et al., 2006), crosses were made every 11 generations. Wildtype (WT, TREK-1+/+) heterozygous (HET, TREK-1+/−) and knockout (KO, TREK-1−/−) mice were produced for this study. To avoid fluctuations due to female hormonal cycles, only males were used in this study.

#### Flux-Pool

In total, 167 mice (6–9 week-old) constituting the flux-pool were exposed to compressed air to induce DCS. The mice were randomly divided into four groups and numbered (**Figure 1**): 46 for the wild-type group treated with fluoxetine (WTflux), 30 for the heterozygous group treated with fluoxetine (HETflux), 46 for the knockout group treated with fluoxetine (KOflux), and 45 wild-type controls (WTcont).

Fluoxetine (50 mg/kg) was administered by gavage to experimental animals as an oral solution (Prozac™ 20 mg/5 ml, oral solution bottle of fluoxetine hydrochloride, Lilly Laboratories, France) 18 h before hyperbaric exposure, while the wild type control group (WTcont) received a similar saccharine fluoxetine-free solution (7.4 g/kg). This high dose of fluoxetine was determined on the basis of previous results from a mouse model of ischemia (Jin et al., 2009; Blatteau et al., 2012; Taguchi et al., 2012).

#### Spad-Pool

In total, 113 mice (strain C57Black/6, 6–9 weeks of age, Harlan laboratories, Gannat, France) constituted the spad-pool and were used to replicate the experiment performed with the flux-pool, but substituting TREK-1 KO mice with spadin-induced KO mice (KO-like mice). To generate KO-like mice, mice were treated for 5 days at 9:00 a.m. with intraperitoneal injections of spadin (100µg/Kg) in a bolus of 100µL of NaCl 0.9%. The mixture for the acute fluoxetine treatment (50 mg/kg; 1.5 mg per mouse) was obtained from 20 mg capsules (fluoxetine hydrochloride Prozac™, Lilly Laboratories, France) diluted in 300µL of a solution of NaCl 9% and gum arabic 5%. It was injected intraperitoneally 18 h before the end of the hyperbaric exposure (p.m. 5:00).

The first group (WTcont2, n = 21), for control, received the equivalent 5 days of treatment (100µL per day of NaCl 0.9%, i.p.) plus the vehicle (300µL of NaCl 0.9% with gum arabic 5%) corresponding to the acute dose of fluoxetine. A second group (KO-like = WTspadin 5d, n = 27) received a daily dose of spadin for 5 days plus the vehicle of the acute fluoxetine treatment. A third group (WTflux2, n = 24) received an acute i.p. injection of fluoxetine plus the analogous 5 days-treatment without spadin. A fourth group (KO-likeflux = WTflux+spadin 5d, n = 24) received 5 days of treatment with spadin and the acute dose of fluoxetine. A fifth group (WTno dive, n = 17) not submitted to the hyperbaric protocol received the same injections as WTcont2 mice. Batches were mixed for the hyperbaric protocol.

#### Hyperbaric Procedure

Hyperbaric Exposure Began at 12:30 p.m.

Each mouse was weighed 30 min before the dive. Samples of 20 mice (10 per cage) from the flux-pool or batches of 6–8 mice from the spad-pool were subjected to the hyperbaric protocol in a 200-l tank fitted with three observation ports. The mice were free to move around the cage.

The compression protocol involved two ramps of pressure increase, first at 0.1 atm/min up to 1 atm, followed by 1 atm/min up to 9 atm; 9 atm corresponds to the pressure where animals were kept for 45 min before decompression. The decompression rate was 60 atm/min up to the surface. Compression and decompression were automatically controlled by a computer linked to an analog/digital converter (NIUSB-6211, National Instrument, USA) with two solenoid valves (Belino LR24A-SR, Switzerland) and a pressure transmitter (Pressure Transmitter 8314, Burket Fluid Control System, Germany). The software was programmed on a DasyLab (DasyLab National Instrument, USA) by our engineer. The software also controlled the temperature and oxygen rate. Compressed air was generated using a diving compressor (Mini Verticus III, Bauer Comp, Germany) coupled to a 100-l tank at 300 bars. The oxygen analyzer was based on a MicroFuel electrochemical cell (G18007 Teledyne Electronic Technologies/Analytical Instruments, USA). The temperature inside the tank was monitored using a platinum-resistance temperature probe (Pt 100, Eurotherm, France).

Water vapor and CO2 produced by the animals were captured with soda lime (<300 ppm captured by the soda lime) and seccagel (relative humidity: 40–60%). Gases were mixed by an electric fan. The day-night cycle was respected throughout.

#### Behavioral and Clinical Observations

At the end of the decompression time, mice were observed for 30 min. All signs were recorded together with their time of onset: labored breathing, convulsions or death. Paralysis, difficulty moving and problems with the fore or rear limbs were classified as motor deficits.

The grip test, a motor/sensory test adapted from Hall et al. (Hall, 1985), was used to quantify forelimb involvement 15 and 30 min after the end of decompression. The mouse was placed in the middle of a 60 cm-long cord suspended at a height of 40 cm hanging from its fore paws and each performance was timed with a stop-watch, over a test duration of 30 s. Mice which escaped by climbing up and then walking along the cord corresponded to the highest score of 30 s. Mice which failed at least one test were considered as symptomatic (Grip−). The results of this behavioral test were used to define DCS and distinguished the following groups: dead (fatal DCS), mice that failed at least one grip test (Grip−) and mice that passed the grip test (Grip+).

#### Anesthesia and Sacrifice

All mice were anaesthetized 30 min after surfacing (after grip tests) first with halothane (5% in oxygen, Halothane, Belamont, France) in order to gain time and to minimize stress, and then by intraperitoneal injection of a mixture of 16 mg/kg xylazine (Rompum R 2%, Bayer Pharma) and 100 mg/kg ketamine (Imalgène R 1000, Laboratoire Rhône). Mice in the spad-pool were kept for heart blood sampling for biochemistry and then sacrificed by injecting pentobarbital (200 mg/kg ip, Sanofi Santé, France).

#### Blood Tests

Blood tests were carried out using an automatic analyzer (ABCvet, SCIL Animal care company, France) on samples taken before the dive and again 30 min afterwards. The second test values were corrected according to the hematocrit variation. Leukocytes, erythrocytes, hematocrit, platelets and Mean Platelet Volume (MPV) were analyzed in 20µl samples taken from the tip of the tail and diluted in the same volume of 2 mM EDTA (Sigma, France).

 Hemolytic samples were rejected.

#### Genotyping

For first part of the study (flux-pool), DNA for PCR was extracted from cells from the tip of the tail (5 mm) after overnight digestion at 56◦C with protease K (200µg/ml) (Promega, Charbonnière, France) freshly added in a buffer solution containing 100 mM Tris (pH 8.5), 200 mM NaCl, 5 mM EDTA, and 0.2% SDS. The protease K was then heat-inactivated (95◦C for 5–10 min). The lysate was diluted 20-fold in ultrapure water before amplification.

PCR was carried out on 5µl of lysate added to 20µl of the reaction mixture. For negative controls, water was substituted for the lysate. The reaction mixture contained a pair of primers (10 pM/µl) [1–2] or [1–3] with an amplification mixture (GoTaq R Green Master Mix 2X, Promega France). DNA primers (MWG-Opéron Biotech, France) corresponding to the loci of interest in the kcnk2 gene (Primers #1 [5′ GGT GCC AGG TAT GAA TAG AG 3′ ]; Primers #2 [5′ TTC TGA GCA GCA GAC TTG G 3′ ]; Primers #3 [5′ GTG TGA CTG GGA ATA AGA GG 3′ ]) were used with the following thermocycler (MultiGene Gradient, Labnet International, USA) settings: initialization step 94◦C/3 min>> [denaturation step 94◦C/25 s >> annealing step 61◦C/25 s >> elongation step 72◦C/35 s] for 35 cycles.

Amplified DNA sequences were resolved by electrophoresis (Biorad Generator, Powerpac 200; 90 V 45 min) on buffered 1.2% Tris acetate EDTA agar gels supplemented with BET for UV detection (Geneflash, Syngene Bioimaging). PCR-detected bands at both 680 bp [1–2] and 1870 bp [1–3] characterize the homozygous wild-type (WT, TREK-1+/+), and a single band at 650 bp [1–3] characterizes the homozygous knock-out (KO, TREK-1−/−). A pair of bands characterizes heterozygous mice (HET, TREK-1−/+).

### Statistical Analyses

Individual blood cell count data were calculated as the percentage change from baseline (the measurement before hyperbaric exposure). Numerical data points are expressed as mean and standard deviation. A contingency table was used for independence and association tests coupled with the χ 2 significance test. Different groups were compared using the Mann-Whitney (MW) test and matched comparisons within groups were analyzed using the Wilcoxon (W) test. Multiple comparisons were performed using the Kruskal-Wallis test followed by the Bonferroni-Dunn post-hoc test. The significance threshold was 95% with an α-risk of 5%.

### RESULTS

Flux-pool: 167 mice (91 WT, 30 HET, and 46 KO) were subjected to the hyperbaric protocol to induce DCS. In these groups, mouse weights were similar [weightWTcont: mean = 24.0 ± 2.3 g (range: 19.4–29.3 g); weightWTflux: mean = 24.2 ± 0.6 g (range: 23.7–25.1 g); weightHETflux: mean = 24.5 ± 2.4 g (range: 20.4– 29.4 g); weightKOflux: mean = 24.4 ± 2.1 g (range: 20.8–30.6 g)] (KWWTcont/WTflux/HETflux/KOflux: n = 45/46/30/46, α = 0.05, p = 0.657).

Spad-pool: 113 mice were used in this pool. Spadin was used to generate TREK-1 KO-like mice. Again, mouse weights were similar [weight KOlike: mean = 27.4 ± 2.2 g (range: 23.0–31.5 g); weight WTflux: mean = 28.2 ± 2.7 g (range: 23.7–34.4 g); weight KO-like flux: mean = 28.7 ± 2.0 g (range: 25.1–33.0 g) weight WTcont2: mean = 27.2 ± 2.1 g (range: 23.1– 31.3 g); weight WTno dive: mean = 27.2 ± 1.9 g (range: 24.9– 32.6 g)] (KWKO-like/WTflux,/KO-like flux/WTcont2/WTno dive, n = 27/24/24/21/17, p = 0.121). These weights were significantly higher than those of the flux-pool (KWAllgroups, n = 27/24/24/21/17/45/46/30/46, p < 0.0001), which may increase susceptibility to DCS.

Hence, in order to avoid confusion, results of both pools, i.e., flux and spad, are presented separately.

### Clinical Observations

Compared to WT mice, the KO or KO-like mice showed no abnormal behavioral or phenotypic signs. All groups displayed a similar range of DCS symptoms (**Figure 1**).

Most of the mice were prostrate, suggesting that they were in physiological distress. Symptoms of DCS or death occurred after returning to the surface. Mice died from convulsions (on the outer edge of the radar map, **Figure 1**) and/or respiratory distress. Mice essentially displayed neurological symptoms with varying degrees of severity with motor and locomotor impairments (paraplegia, paraparesis) and, in some cases, convulsions. We also observed fewer convulsions and slightly more labored breathing in KO-likeflux (**Figure 1B** Radar chart). The opposite trend was found for WTcont2 (Contingency table, χ 2 KO-like/WTflux2/KO-like flux/WTcont2/WTno dive = 63.175 vs. 21.026, p < 0.0001).

In the flux-pool, DCS symptoms generally occurred at 5.8 ± 3.5 min on average after the end of the dive. When mice succumbed to DCS, it occurred rapidly, i.e., 6.1 ± 3.1 min on average. There was no significant difference for the onset of first symptoms or death latency, regardless of the group (KWWTcont/WTflux/HETflux/KOflux: p = 0.399, p = 0.390, respectively).

In the spad-pool, first DCS symptoms generally occurred at 6.2 ± 3.4 min on average after the end of the dive. When mice succumbed from DCS, it occurred more rapidly, at 2.1 ± 4.5 min on average. There was no significant difference in the onset of first symptoms or death, regardless of the treatment (KW KO-like/WTflux2/KO-like flux/WTcont2/WTno dive: p = 0.620, p = 0.550, respectively).

## Lethal DCS, Grip−, Grip+ Status

KOflux and KO-likeflux mice were less susceptible to the hyperbaric protocol than other groups. Fewer KO-like mice succeeded in the grip tests (**Figure 1** histograms).

Analysis of the different clinical status of DCS showed a significant difference between the WTcont group and WTflux, HETflux, and KOflux groups (KWWTcont/WTflux/HETflux/KOflux: n = 45/46/30/46, α = 0.05, p < 0.0001) (**Figure 1**; **Table 1**). Only 7% of KOflux mice died as a consequence of DCS. Additionally, KOflux mice presented a better success rate in both their grip tests (Contingency table, χ 2 WTcont/WTflux/HETflux/KOflux = 39.194 vs. 16.919, n = 45/46/30/46, α = 0.05, p < 0.0001).

In the spad-pool, analysis of the different DCS statuses showed a significant difference between the different groups (KW KO-like/WTflux2/KO-like flux/WTcont2/WTno dive: p < 0.0001). Interestingly, a significant difference appeared between the KOlike group and the KO-likeflux group. The main difference corresponded to the number of animals that died from DCS, i.e., 41 and 4% for the KO-like group and KO-likeflux group, respectively (**Figure 1**; **Table 1**). Surprisingly, the number of mice that died was lower in the KO-likeflux group than in the WTflux2 group, i.e., 4 and 17% respectively (**Figure 1**; **Table 1**). The success rate in both grip tests (Grip+) was lower for the KO-like group (**Figure 1**; **Table 1**; Contingency table, χ 2 KO-like/WTflux2/KO-like flux/WTcont2/WTno dive = 14.496 vs. 15.507, p = 0.070).

### Mice that Failed the Grip Test (Grip−)

Survivors underwent two grip tests (**Figure 1**; **Table 1**). While results of grip tests were different according to the treatment group, no treatment seems to better promote recovery or worsen the physical state of mice once symptoms become manifest.

#### TABLE 1 | Clinical status after a dive.

Survivors underwent two grip tests. Grip tests, i.e., motor/sensory tests, were used to quantify forelimb involvement 15 and 30 min after the end of decompression. Mice which failed at least one test were considered to be symptomatic (Grip−). The results of this behavioral test were used to define DCS and distinguish the following groups: dead (Lethal DCS), mice that failed at least one grip test (Grip−) and mice that passed both grip tests (Grip+).

We previously suggested that the absence of TREK-1 channel activity could limit recovery after DCS (Vallee et al., 2012), but our present data are not in accord with such a hypothesis. For example, a large number of KOflux mice passed both grip tests, but without an improvement between both tests, whereas KOlikeflux mice not only passed both grip tests, but improved their scores between test 1 and test 2.

In more detail and with regard to the timed performance in the grip tests, WTflux mice improved their mean time spent suspended from the cord (WWTflux: n = 6, α = 0.05, p = 0.034) in the second test carried out 15 min later (16.8 ± 2.1s vs. maximal time 30.0 ± 0.0 s). In the spad-pool study, better performances were observed in the second grip tests for the KO-like population (WKO-like: p = 0.014, 4.1 ± 5.8 s vs. 16.0 ± 11.2 s) and the KO-likeflux population (WKO-like flux: p = 0.014, 11.5 ± 8.9 s vs. 22.2 ± 11.1 s).

Significant differences were observed between populations in both grip tests (first test, KWWTcont/WTflux/HETflux/KOflux : n = 7/6/7/7, α = 0.05, p = 0.048 and second test, KW WTcont/WTflux/KOflux: n = 7/6/7/7, α = 0.05, p = 0.039): the WTcont group did not perform as well as mice treated with fluoxetine (first test: MW WTcont/KOflux: n = 7/7, p = 0.054, MW WTcont/WTflux: n = 7/6, p = 0.002; second test: MW WTcont/WTflux: n = 7/6, p < 0.0001). WTflux mice performed better in the second grip test than HETflux mice (MWHETflux/WTflux: n = 7/7, p < 0.009). With regard to timed performance in the grip tests of the spad-pool, no significant differences were observed between populations, either in the first (KW: p = 0.190) or second grip test (KW: p = 0.453), or in their delta performance (KW: p = 0.193).

## Full Blood Counts

#### Erythrocyte Counts

To a small extent, a decrease in red cell counts, usually though diapedesis, can be observed after a dive and can be attributed to the decompression protocol (**Table 2**). Overall, we cannot link this decrease to an effect of treatment or to mouse genotype.

In the flux-pool, differences in erythrocyte counts could be attributed to the treatment before the dive (KW: p < 0.0001): WTcont mice displayed lower erythrocyte counts than both WTflux (p < 0.001) and KOflux (p < 0.003) mice. HETflux mice also presented lower erythrocyte counts than KOflux mice (p = 0.01). Globally, erythrocyte counts decreased on average after the dive, to a small extent (W: p = 0.003, mean = −0.4 ± 7.9), with lower counts in Grip− mice (KW p = 0.017). These differences can therefore be related to treatments given to the mice (KW p < 0.0001: WTcont and WTflux presented lower erythrocyte counts than KOflux), but there were no variations in their consumption rate (KW p = 0.382) after the dive. This suggests that the red cell count decrease was due to the decompression, even when basal levels were different according to treatment.

In the spad-pool, no effect on erythrocyte counts could be attributed to the sole effects of treatments before the dive (KW: p = 0.999), or afterwards (KW: p = 0.392), or in its variation between before and after the dive (KW: p = 0.172). Following the dive, a significant decrease in erythrocyte counts (W p < 0.001, mean = −8.6 ± 10.1) was observed after the decompression protocol in all survivors, but the variation was not linked to the clinical state after the dive (Grip+ vs. Grip−: MW p = 0.299: Grip+: mean = −7.3 ± 9.4%; Grip−: mean = −10.2 ± 11.0%).

#### Platelet Counts

Globally, platelet loss occurs following a dive that can be linked to the physical state of mice. Platelet consumption seemed to be more related to the clinical state of the mice than to their treatment or their genetic status (**Table 2**). Platelet consumption increased with the severity of DCS. This drop in platelet counts was attributed to clotting activity following exposure of the collagen under bubble-damaged endothelial cells in blood vessels (Persson et al., 1978; Haller et al., 1987; Thorsen et al., 1987; Nossum et al., 1999) or to direct interactions between bubbles and platelets (Hallenbeck et al., 1973; Warren et al., 1973; Giry et al., 1977).

TABLE 2 | Variation (%) in erythrocyte, leukocyte, and platelet counts before and after decompression.

The column on the right shows the averaged count carried out for all survivors (Grip+ and Grip−). Grip− mice were considered to be symptomatic. Grip+ mice were considered to be asymptomatic. \*Denotes a significant difference within all the survivors between pre- and post-decompression counts. #Denotes a significant difference between Grip+ and Grip− mice.

In more detail, and with regards to the first part of the study, some differences in platelet counts could be attributed to the treatments before the dive (KW: p < 0.001): WTcont displayed lower platelet counts than both HETflux (p = 0.004) and KOflux (p < 0.001). WTflux also presented lower platelet counts than HETflux (p = 0.004) and KOflux (p < 0.001).

Following the dive, no significant decrease in platelet counts (mean = +4.5 ± 33.5%) was recorded when all survivors were considered (W p = 0.728). Nonetheless, Grip− mice tended to exhibit higher consumption of platelets than Grip+ mice (KW, p = 0.005; Grip+: mean = +9.2 ± 33.1%; Grip−: mean = −13.6 ± 29.2%). No link could be established between platelet consumption and treatments (KW, p = 0.076). This suggests that platelet consumption, attributed to clotting activity, is mainly due to the clinical state induced by decompression rather than to treatment or genotype.

In the spad-pool, the absence of a difference in platelet counts could be attributed to the treatment effects before the dive (KW: p = 0.209), and levels before the dive had no consequences on the clinical state after the dive (KW: p = 0.735).

Thirty minutes after the dive ended, a significant decrease in platelet counts (−21.2 ± 26.5%) was recorded in all survivors (MW: p < 0.0001). Platelet consumption was independent of the nature of the treatment (KW: p = 0.132). Grip+ mice had more circulating platelets (KW, p = 0.042) and tended to have a lower consumption (in proportion) of their platelets than Grip− mice (KW, p = 0.069; Grip+: mean = −26.1 ± 23.8%; Grip−: mean = −41.2 ± 29.3%). Actually, Grip− platelets had a greater volume than that of Grip+ mice (p = 0.0014), confirming that old platelets (with a lower normal volume) had been used in the aggregation process.

#### Leukocyte Counts

Globally, there was a loss of leukocyte following the dive (**Table 2**) that was usually attributed to diapedesis (Dutka et al., 1989; Zamboni et al., 1989, 1992, 1993; Dal Palu and Zamboni, 1990; Helps and Gorman, 1991). Neither treatment seemed to influence leukocyte movement greatly, or the clinical state.

In the flux-pool, differences in leukocyte counts could be attributed to the treatments before the dive (KW: p = 0.001), as WTcont displayed lower leukocyte counts than both HETflux (p = 0.010) and KOflux (p < 0.005). These differences influenced their clinical state after the dive (KW: p = 0.004). Therefore, mice that died after the dive-decompression protocol (Lethal DCS mice) had lower leukocyte counts before the dive than Grip+ mice (4.3 ± 1.2 vs. 5.3 ± 1.6 × 2.103 /µl).

After the dive, in all survivors, leukocyte counts decreased by 2.4 ± 7.9% (W: p = 0.003). These differences in counts or variation between the different groups were not significant with regard to genotype or treatment (KW: p = 0.129 and p = 0.342), or clinical status after the dive, i.e., Grip+, Grip−, or Lethal (KW p = 0.870 and p = 0.416).

In the spad-pool, differences in leukocyte counts could be attributed to the 5 days of spadin treatment (KW, p = 0.008). KO-like mice displayed higher leukocyte counts than KO-likeflux mice (p = 0.016), WTflux2 mice (p = 0.007) or WTno dive mice (a trend, p = 0.096). Differences observed before the dive possibly influenced the clinical state after the dive (KW: p = 0.022). Here again, Lethal DCS mice had higher leukocyte counts than Grip+ mice (6.3 ± 1.7 vs. 5.0 ± 2.0×02.103 /µl, p = 0.032). Nonetheless, this is the opposite result to what has just been proposed for the flux-pool study, what must invalidate the hypothesis.

Following the dive, a significant decrease in leukocyte counts (W: p < 0.0001, mean = −27.4 ± 62.8%) was recorded in all survivors. Nonetheless, there was no difference in the consumption of leukocytes between Grip+ and Grip− mice (KW: p = 0.8930: Grip+: mean = −29.0 ± 51.7%; Grip−: mean = −25.4 ± 74.7%). Leukocyte recruitment was independent of the nature the treatment (KW: p = 0.905).

Finally, fluoxetine administered orally before the dive tended to increase the number of circulating leukocytes, compared to control mice. When injected i.p., fluoxetine reduced leukocyte counts. In fact, intraperitoneal injection of fluoxetine induces irritation and leukocyte adhesion in the abdominal cavity (Herr et al., 2014). This effect was not counteracted by the 5 days of spadin treatment, while the latter, when given alone i.p., tended to increase leukocyte counts. Finally, however, this prestimulation was equal in all groups for the spad-pool and controlled by the administration of the vehicle in the same way as fluoxetine administration. These paradoxical effects on leukocyte counts before the dive did not influence the (high) survival rate of the KOflux and the KO-likeflux mice.

As already described (Pontier et al., 2008; Blatteau et al., 2012; Vallee et al., 2012), the leukocyte counts dropped after decompression in all survivors, and leukocyte recruitment was independent of the type of treatment.

#### Blood Biochemistry

Following the dive, blood biochemistry was assessed on samples from heart punctures. Compared to WTno dive, sodium (KW: p = 0.007; post-hoc p = 0.019 p = 0.01), potassium (KW: p = 0.022; post-hoc p = 0.006 p = 0.004), globulin (KW: p = 0.01; post-hoc p = 0.008 p = 0.001), and transaminase (KW: p = 0.001; post-hoc p = 0.002 p < 0.0001) were found at increased levels in mice subjected to the hyperbaric protocol, regardless of the clinical state (Grip+ or Grip−) (**Table 3**). Grip− mice had higher levels of creatinine kinase (KW p = 0.062; post-hoc p = 0.019). The BUN level was lower in the Grip+ groupthan in the WTno-dive group (KW p = 0.041; post-hoc p = 0.016). The albumin level was lower in the Grip+ and the Grip− groups than in the WTno-dive group (KW: p < 0.001; posthoc p = 0.002 p < 0.0001). Triglyceride levels were higher in Grip− than in Grip+ mice (KW p = 0.005; post-hoc p = 0.01). Overall, the dive induced a hemoconcentration regarding sodium potassium and AST levels. This could be due to a capillary leak consider low albumin levels (in contrast to high globulin levels). Cell destructions and/or liver shouting could also be engaged, especially when taking into account high triglycerides levels then recorded in Grip−. Increase in creatinine kinase also suggested muscle or heart straining when DCS was stated.

Blood sample analyses from two mice, punctured just before they died (Lethal DCS) showed (i) higher levels of sodium (KW p = 0.007; post-hoc p = 0.001), potassium (KW p = 0.022; posthoc p = 0.028) or creatinine kinase (KW p = 0.062; post-hoc p =

0.064) in comparison with WTno dive mice, (ii) a higher sodium level in comparison with both the Grip− and Grip+ groups (KW p = 0.007; post-hoc p = 0.006 and p = 0.031 respectively), (iii) a higher lactate level (KW p = 0.016; post-hoc p = 0.010) and triglyceride concentration (KW p = 0.005; post-hoc p = 0.005) when compared to the Grip+ group, (iv) higher bilirubin levels than those of WTno dive, Grip+ and Grip− mice (KW p = 0.004; post-hoc p = 0.002 p < 0.001 p = 0.002, respectively), (v) unchanged glycemia, calcium, cholesterol, creatinine, and total protein levels vs. the other groups.

When looking at the effect of the treatments per se on blood biochemistry after the dive (**Table 4**), we pointed out that mice treated with fluoxetine, such as KO-likeflux and WTflux2 and corresponding to the best survival rates, had higher levels of potassium (hemoconcentration, cell destruction), AST (aspartate aminotransferase, liver function) and globulin (hemoconcentration), and lower levels of albumin (an indicator of capillary leak) and lactate (high levels indicate anaerobic) than those of the WTno-dive group.

KO-like mice, receiving spadin for 5 days, displayed the worst survival rate. In comparison with the WTno-dive group, they expressed very low levels of bilirubin, a toxic and hydrophobic

breakdown product of red blood cells that is bound to and carried by albumin. They also had very high levels of lactate (anaerobic metabolism and possible mitochondria dysfunction), sodium and triglycerides; the origin of these high triacylglycerol levels in their blood could be due to adipocyte release, pancreatitis, autoimmune disease or nephritic syndrome.

#### DISCUSSION

In this study, fluoxetine should be regarded as an antiinflammatory drug, considering that we opted for an acute, high dose of 50 mg/kg; the antidepressant effect of fluoxetine requires long-term treatment (28 days at 20 mg/kg) to induce synaptogenesis, and should not be considered in this study. In contrast, 5 days of spadin treatment was sufficient to internalize TREK-1 channels and consequently to exert antidepressant activity (Mazella et al., 2010; Moha Ou Maati et al., 2012).

#### Prevention of Decompression Sickness

The decompression protocol used in this study is comparable with that used in previous studies on mice of a similar weight (Berghage et al., 1979; Blatteau et al., 2012; Vallee et al., 2012). Mice in the spad-pool were heavier, which may have increased their susceptibility to DCS. Nonetheless, the protocols used in this study induced neurological DCS with motor and locomotor impairments and convulsions, suggesting damage to the spinal cord or brain.

The increase in AST (aspartate aminotransferase) may be due to fluoxetine hepatotoxicity, as fluoxetine is extensively metabolized in the liver to norfluoxetine and this is a well-known side effect (Inkielewicz-Stepniak et al., 2014). Fluoxetine may also induce moderate lymphocyte infiltration within the portal tracts and ballooning degeneration of hepatocytes (Kwak et al., 2000). The low mortality associated with fluoxetine treatment led us to conclude that increased AST levels may have a minor effect on DCS outcomes. Nonetheless and except for Lethal DCS mice, we also observed increased concentrations of sodium and potassium ions and globulins, after the dive. These variations may be explained at least in part by hemoconcentration (increased hematocrit in the spad-pool) or by hepatocyte damage due to the presence of bubbles in the liver, as previously observed in rats (L'Abbate et al., 2010). Arguing in favor of liver dysfunction, the albumin and BUN (both synthetized by the liver) concentrations were low, but these lower concentrations could also been explained by damage to the glomerulus, as occurs in nephritic syndrome (Blann and Ahmed, 2014), or by capillary leak that may lead to edema (Clarkson et al., 1960). This albumin loss demonstrated that fluoxetine did not prevent capillary leak per se, a symptom of severe DCS. Finally, the blood biochemistry results indicate that damage induced by bubbles may have induced capillary leak, cell destructions and liver dysfunction. However, other blood components were not affected by the dive. Consequently, additional data will be necessary to validate or invalidate the liver dysfunction hypothesis. Dramatically, multivisceral dysfunction could be supported by changes in analyte concentrations in Lethal DCS mice, such as high levels of creatinine kinase or lactate.

This protocol also decreased erythrocyte, leukocyte and platelet counts, suggesting diapedesis, inflammation and clotting activity, as well as ischemia, the usual symptoms described for DCS. Platelet dysfunction has been described for fluoxetine (Lesch et al., 1993; Brenner et al., 2007), but this effect did not influence platelet recruitment or DCS outcomes in our study. On the other hand, TREK-1 channels are not known to affect clotting function directly, although they are activated by platelet activating factor (Maingret et al., 1999). Under these conditions, TREK-1 channels likely played a minor role in DCS outcomes.

The main result from our work is that mice with impaired TREK-1 channel function and treated with fluoxetine are more resistant to the consequences of decompression than WT mice treated with fluoxetine, and far more resistant than untreated KO or KO-like mice. Our data suggest that the presence of the TREK-1 channel may mitigate the global benefit of fluoxetine in DCSrelated ischemia and inflammation. The second important result from our work is that 5 days of spadin antidepressant treatment, thereby blocking TREK-1 channel activity specifically, increased DCS susceptibility. However, when the same 5 days of spadin treatment was administered to mice treated with fluoxetine, the protective effect of fluoxetine was increased. These data indicate that the main protective effect of fluoxetine is mediated by a cellular pathway independent of TREK-1 channel activity.

#### Loss of Protective Activity of TREK-1

The high mortality rate of KO-like mice confirmed that the neuroprotection induced by the TREK-1 channel in WT was prevented by spadin (5 days of treatment). The importance of the TREK-1 channel in neuroprotection is well-documented for different physio-pathological processes, including hypoxia (Moha Ou Maati et al., 2011), glutamatergic excitotoxicity and neuronal death (Heurteaux et al., 2004). According to our previous and present studies (Vallee et al., 2012), mice that do not express the TREK-1 channel (KO-like or KO) are more likely to develop neurological symptoms after DCS damage to the central nervous system. This could be attributed to the stimulation of NMDA receptor activity, which exacerbated glutamate excitotoxicity, making the brain blood barrier (BBB) more permeable to leukocytes (Bittner et al., 2014). These KO mice are known to display an inflammatory phenotype and accelerated leukocyte trafficking across the BBB (Bittner et al., 2014).

This study focused on fluoxetine, which inhibited TREK-1 channel activity (Kennard et al., 2005; Sandoz et al., 2011) and thus reduced its neuroprotective effects. However, in the present work and in previous experiments on fluoxetine and DCS (Blatteau et al., 2012), we found that WT mice treated with fluoxetine were more resistant to DCS than untreated mice. These observations suggest that the positive antiinflammatory effects of fluoxetine outweigh fluoxetine-induced TREK-1-inhibition.

#### Enhanced Protective Activity of Fluoxetine on TREK-1−/−(like) Mice

KO and KO-like mice treated with fluoxetine appeared to be the most resistant to decompression. It was recently shown that fluoxetine selectively inhibits glutamate-N2B NMDA receptors and then induces neuroprotection (Vizi et al., 2013). This property of fluoxetine could partly substitute for the loss of TREK-1-mediated neuroprotection. Another hypothesis can also be drawn. In the absence of TREK-1 channels at the cell membrane, more fluoxetine may have been available for binding to other fluoxetine targets, thereby increasing its protective effects. This hypothesis is suggested by the dose dependence of DCS in KOflux HETflux and WTflux mice treated with fluoxetine. We previously attributed the main protective effect of fluoxetine in DCS to its anti-inflammatory properties from the observed decrease in IL-6 (Blatteau et al., 2012). Decreased IL-6 secretion was also described in TREK-1-deficient cells (Schwingshackl et al., 2012) and this could superimpose on the effect of fluoxetine to further reduce inflammation in TREK-1 deficient mice. It could constitute an efficient response to TREK-1−/−-induced BBB damage (Bittner et al., 2013, 2014) or more hypothetically, an opportunity to cross the BBB more readily and more efficiently (**Figure 2**). Finally, fluoxetine is also known to prevent BBB disruption (Lee et al., 2014) thanks to its anti-inflammatory activity coupled with its ability to block NMDA receptors and attenuate glutamatergic excitotoxicity. Further studies will be required to confirm this hypothesis.

### Should SSRI Treatment be a Contraindication for Diving?

Spadin was described as a new concept of antidepressant and it was shown that 4 days of treatment with spadin corresponds to 21–28 days of treatment with classical SSRIs (Mazella et al., 2010; Moha Ou Maati et al., 2012; Borsotto et al., 2015; Devader et al., 2015; Veyssiere et al., 2015). Spadin treatment induces TREK-1 internalization, resulting in the disappearance of the channel

from the plasma membrane (Mazella et al., 2010). KO mice are known for developing a depression-resistant phenotype and also for being more sensitive to ischemia (Heurteaux et al., 2006). Both KO and KO-like TREK-1 mice were more likely to succumb to DCS after dive. Therefore, we legitimately wonder whether SSRIs, in their usual indication against depression in humans, could represent a risk factor in diving, or more generally in risky sports that may induce ischemia and inflammation.

## Fluoxetine, TREK-1 and Clinical Status: Weight

TREK-1 is widely expressed in central and peripheral tissues. It is highlyexpressedin thenervoussystem,digestivesystem,endocrine system and reproductive system, as well as in the muscular system. In the CNS, TREK-1 is expressed in both astrocytes and neurons. It should be kept in mind that fluoxetine, like spadin, could spread throughout whole body and thus could induce many interactions not anticipated in this study, even if the symptoms are mainly nervous. Moreover, it could be useful to include in further studies immuno-histological technics to confirm the infiltrating leukocyte population in the brain injury. The signaling pathways should also be of interest to assess cell viability.

## CONCLUSION

We previously found that acute fluoxetine treatment affords the best protection against DCS when the TREK-1 potassium channel is impaired. We suggest that this drop in mortality may be due to decreased ischemia-induced glutamatergic toxicity, which could be due to the blockage of NMDA receptors by fluoxetine, and/or to an enhancement of the anti-inflammatory effect of fluoxetine. Following this study, exact mechanism remains to be elucidated. We also found that antidepressed mice, i.e., KO or spadin treated, showed increased susceptibility to DCS compared to WT mice. It remains to be determined whether antidepressants could be contraindicated in humans participating in risky activities such as diving that may induce ischemia, even though we found an efficient but paradoxical solution to DCS by delivering fluoxetine.

## AUTHOR CONTRIBUTIONS

JB and NV: conception and design of research; JB, SM, KL, PR and NV: performed experiments; JB, KL, and NV: analyzed data; JB, KL, SM, JM, MB, CH, JA, JR, and NV: interpreted results of experiments; NV: prepared figures; JB: drafted manuscript; NV: edited and revised manuscript; JB, KL, SM, JM, MB, CH, JA, JR, and NV: approved final version of manuscript.

#### FUNDING

The work should be attributed to the "Institut de Recherches Biomédicales des Armées" laboratories. It is supported by Grant No. PDH-1-SMO-2-722 from the Délégation Générale pour

#### REFERENCES

l'Armement of the French Army, Paris, France, and by a special Grant from "La fondation des gueules Cassées," Paris, France.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fphys. 2016.00042

potassium channel, TREK-1, by fluoxetine and its metabolite norfluoxetine. Br. J. Pharmacol. 144, 821–829. doi: 10.1038/sj.bjp.0706068

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The Reviewer AG declares that, despite sharing a common affiliation with the Guest Associate Editor, the review process was carried out objectively and no conflict of interest exists.

Copyright © 2016 Vallée, Lambrechts, De Maistre, Royal, Mazella, Borsotto, Heurteaux, Abraini, Risso and Blatteau. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Strong Ion Regulatory Abilities Enable the Crab Xenograpsus testudinatus to Inhabit Highly Acidified Marine Vent Systems

Marian Y. Hu1, 2, Ying-Jey Guh3 , Yi-Ta Shao4 , Pou-Long Kuan5 , Guan-Lin Chen5 , Jay-Ron Lee1 , Ming-Shiou Jeng6 and Yung-Che Tseng5 \*

*1 Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan, 2 Institute of Physiology, Christian-Albrechts University Kiel, Kiel, Germany, 3 Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, 4 Institute of Marine Biology, National Taiwan Ocean University, Keelung, Taiwan, 5 Department of Life Science, National Taiwan Normal University, Taipei, Taiwan, 6 Biodiversity Research Center, Academia Sinica, Taipei, Taiwan*

#### Edited by:

*Antonio L'Abbate, Scuola Superiore Sant'Anna, Italy*

#### Reviewed by:

*Nia M. Whiteley, Bangor University, UK Michael B. Morris, The University of Sydney, Australia*

> \*Correspondence: *Yung-Che Tseng<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Integrative Physiology, a section of the journal Frontiers in Physiology*

Received: *01 August 2015* Accepted: *11 January 2016* Published: *01 February 2016*

#### Citation:

*Hu MY, Guh Y-J, Shao Y-T, Kuan P-L, Chen G-L, Lee J-R, Jeng M-S and Tseng Y-C (2016) Strong Ion Regulatory Abilities Enable the Crab Xenograpsus testudinatus to Inhabit Highly Acidified Marine Vent Systems. Front. Physiol. 7:14. doi: 10.3389/fphys.2016.00014* Hydrothermal vent organisms have evolved physiological adaptations to cope with extreme abiotic conditions including temperature and pH. To date, acid-base regulatory abilities of vent organisms are poorly investigated, although this physiological feature is essential for survival in low pH environments. We report the acid-base regulatory mechanisms of a hydrothermal vent crab, *Xenograpsus testudinatus,* endemic to highly acidic shallow-water vent habitats with average environment pH-values ranging between 5.4 and 6.6. Within a few hours, *X. testudinatus* restores extracellular pH (pHe) in response to environmental acidification of pH 6.5 (1.78 kPa pCO2) accompanied by an increase in blood HCO− levels from 8.8 ± 0.3 to 31 ± 6 mM. Branchial Na+/K+-ATPase 3 (NKA) and V-type H+-ATPase (VHA), the major ion pumps involved in branchial acid-base regulation, showed dynamic increases in response to acidified conditions on the mRNA, protein and activity level. Immunohistochemical analyses demonstrate the presence of NKA in basolateral membranes, whereas the VHA is predominantly localized in cytoplasmic vesicles of branchial epithelial- and pillar-cells. *X. testudinatus* is closely related to other strong osmo-regulating brachyurans, which is also reflected in the phylogeny of the NKA. Accordingly, our results suggest that the evolution of strong ion regulatory abilities in brachyuran crabs that allowed the occupation of ecological niches in euryhaline, freshwater, and terrestrial habitats are probably also linked to substantial acid-base regulatory abilities. This physiological trait allowed *X. testudinatus* to successfully inhabit one of the world's most acidic marine environments.

Keywords: hydrothermal vent, V-type H+-ATPase, Na+/K+-ATPase, hypercapnia, invertebrate physiology, gill, crustacean

### INTRODUCTION

Deep sea hydrothermal vent systems support ecosystems with an enormous biomass, and reveal a rich biodiversity ranging from microbes to vertebrates (Tunnicliffe, 1992). To survive in these extreme habitats, vent associated organisms show a range of morphological and physiological adaptations to cope with challenging environmental conditions including temperature, metallic sulfides, anoxia, hypercapnia, and low pH (Goffredi et al., 1997; Ramirez-Llodra et al., 2007). Highly acidified conditions due to the release of HCl and CO2 are a characteristic of most seafloor vent systems including the shallow-water hydrothermal vent system of Kueishan Island (24◦ 50′N, 121◦ 57′E), off the coast of Taiwan (Han et al., 2014). This shallow water hydrothermal vent system has been described as one of the most acidic vents in the world, discharging water with a high content of elemental sulfur particles, having temperatures ranging between 76 and 116◦C and a minimum pH of 1.52 (Chen et al., 2005; **Figure 1A**). The gas composition released by the underwater volcano is mainly CO2 (<92%; Han et al., 2014). Even in the surrounding areas with depths between 2 and 14 m, the seawater is highly acidic ranging from pH 6.6 to 5.4 (Han et al., 2014). This challenging hydrothermal vent habitat is inhabited by Xenograpsus testudinatus, a crab species that is endemic to shallow-water (<200 m) vent systems (Ng et al., 2000). X. testudinatus is the only metazoan species found in the direct surroundings of the vents, and individuals congregate in large numbers in vent crevices with an average of 364 individuals per m2 (**Figure 1B**). These crabs have evolved a unique feeding behavior by feeding on dead zooplankton killed by the toxic vent discharges (Jeng et al., 2004). During slack water conditions, when there are no currents, the crabs swarm out of their crevices (**Figures 1C,D**) to rapidly feed on this "marine snow" of dead zooplankton (Jeng et al., 2004).

Crustaceans are probably one of the most successful invertebrate group that occupy ecological niches in marine systems ranging from polar regions (Frederich et al., 2001)

FIGURE 1 | Xenograpsus testudinats crabs in the hydrothermal vent habitat at Kueishan island located off Taiwan's east coast. Underwater photographs of the hydrothermal vent system at Kueishan island, showing an active chimney discharging acidic, CO2 rich water (note the *X. testudinatus* crab in the direct vicinity of the vent opening) (A). High densities of *X. testudinatus* crabs can be found in the direct surrounding of the vents hiding in sulfur-rich crevices (B). Whilst feeding on zooplankton killed by the toxic discharges of the vents crabs swarm out of their shelters, and are directly exposed to highly acidic waters (C,D). All images were obtained and authorized from the co-author Dr. Ming-Shiou Jeng.

to hydrothermal vents (Joel and Haney, 2005; Dittel et al., 2008) and have radiated from seawater to freshwater and even into terrestrial habitats (Schubart et al., 1997). While those found in stenohaline marine habitats are more likely to be weak osmo-regulators or even osmo-conformers crustaceans from euryhaline or intertidal habitats are moderate to strong osmo-regulators, a physiological feature that is beneficial for adaptation to habitats with fluctuating salinities (Charmantier and Charmantier-Daures, 2001; Henry et al., 2012). The gills are the major organ involved in extracellular ion homeostasis, equipped with an efficient ion regulatory machinery that shows evolutionary conserved features comparable to the ion regulatory epithelia in cephalopods and fish (Henry et al., 2012). Similar to the situation in most vertebrate and invertebrate systems, the ubiquitous Na+/K+-ATPase localized in basolateral membranes creates the electro-chemical gradient that is used by secondary active transporters such as apical Na+/H+-exchangers or anion exchangers that can mediate ionic and pH homeostasis in crustaceans (Cameron, 1978; Heisler, 1986; Pörtner et al., 1991; Gutowska et al., 2010; Henry et al., 2012). The V-type H+-ATPase (VHA) has also been described in gill epithelia of crustaceans, demonstrating a cytosolic or apical localization of this enzyme (Weihrauch et al., 2001; Tsai and Lin, 2007). The VHA has been shown to be involved in the acidification of intracellular organelles, and in the secretion of protons across the plasma membrane of specialized cells located in ion-regulatory epithelia (Weihrauch et al., 2002; Tresguerres et al., 2006; Hwang, 2009; Hu et al., 2014). Based on the structure of the VHA with the catalytic (V1 complex) site facing the cytoplasm, the VHA is restricted to transport protons out of the cytoplasm (Beyenbach, 2006).

In euryhaline crabs changes in environmental salinity were demonstrated to directly affect the extracellular acid-base status of these animals (Truchot, 1981, 1992). The fact that a reduction in environmental salinity induces a metabolic alkalosis while an increase in salinity leads to a metabolic acidosis demonstrated that osmo-regulation and the maintenance of acidbase homeostasis are directly linked (Whiteley et al., 2001). This connection between osmo and acid-base regulation may explain why most osmo-regulating crustaceans are relatively tolerant to environmental acid-base disturbances (Henry and Cameron, 1982; Spicer et al., 2007).

X. testudinatus is a brachyuran crab species that has a phylogenetic position close to other crab species that are characterized as strong osmo-regulators including the euryhaline crab Eriocheir sinensis and crabs of the genus Hemigrapsus spp. (Hicks, 1973; Bedford and Leader, 1977; Onken and Graszynski, 1989; Ki et al., 2009). The potential link between acid-base and osmo-regulation in many euryhaline decapod crustaceans prompted us to formulate the hypothesis that X. testudinatus utilizes conserved ion pumps to regulate extracellular pH. To test this hypothesis we exposed X. testudinatus to CO2 induced seawater acidification (pH 6.5), and monitored changes in extracellular acid-base status as well as expression, protein concentrations and activities of the branchial Na+/K+-ATPase and V-type H+-ATPase over a time course of 48 h. These results will demonstrate that evolution of strong osmo-regulatory

abilities in this phylogenetic group may represent a requisite that is accompanied by substantial acid-base regulatory abilities that allowed X. testudinatus to inhabit one of the most acidic marine habitats.

### MATERIALS AND METHODS

#### Acidification Experiments

X. testudinatus with carapace width ranging between 3 to 4 cm were obtained by SCUBA divers from Kueishan Island, Taiwan (ROC) in July 2013. Crabs were collected from depth ranging between 5 and 10 m and held in recirculating natural seawater systems (500 l total volume, nitrification filter, salinity 31–32h, temperature 28◦C, constant 12 h dark:12 h light cycle) at the Institute of Cellular and Organismic Biology, Academia Sinica. Animals were fed ad libitum twice per day with tilapia meat. CO2 perturbation experiments were carried our using a total of 48 animals that were distributed into six 20 l tanks (8 animals per tank). The six tanks, with three replicate tanks for each pH treatment were connected to a flow through system providing filtered (0.2µm), natural seawater. Water was exchanged at a flow rate of ∼3 l h−1 to guarantee high water quality inside the test aquaria. The pH (8.0 and 6.5) in the experimental tanks was continuously adjusted by the addition of the appropriate gas mixtures using a continuous pH-stat system (pH controller, MACRO) that controlled the addition of pure CO2 into the seawater. The experimental tanks were additionally aerated with air (O2 saturation > 90%) to assure sufficient seawater pO2 during the experiment. Specific seawater physicochemical conditions for the different incubations are shown in Table S1. Animals were not fed during the 48 h experimental period in order to minimize physiological artifacts caused by feeding activity. pHSW was measured using a WTW 340i meter equipped with a WTW SenTix 81 electrode that was calibrated daily with Tris and AMP buffers at a salinity of 31 to monitor the experiment. Alkalinity in seawater samples was determined spectrophotometrically according to Sarazin et al. (1999). The carbonate system of seawater was calculated from AT and pHSW using the software CO2SYS (Lewis and Wallace, 1998), including the dissociation constants of Mehrbach et al. (1973) as refitted by Dickson and Millero (1987). Along the experimental duration of 48 h, extracellular acid-base parameters were measured, and gill tissue samples were collected for gene expression, protein, and enzyme analyses. Tissue and hemolymph sampling was carried out at six time points (0, 1, 4, 12, 24, and 48 h) along the time course of 48 h. At each sampling time point, one to two animals were sampled from the three experimental replicate tanks, leading to a biological replication of n = 4. Animals were anesthetized by cooling on ice and were killed by an incision of the frontal region of the carapace. The carapace was removed to access the branchial chamber where the gills are located. X. testudinatus has six gill pairs (Figures S2A,B), of which the anterior-most gill 1 (G1), is minute (length 1–2 mm); G6 is the posterior-most gill with a total length of ∼10 mm. G1–G3 are designated as anterior gills, while G4–6 are considered posterior gills. G3–G6 are similar in size while G2 is smaller (∼5 mm long). The gill formula of X. testudinatus is: G1,G2 podobranch, G3 and G4 arthrobranchs with a common insertion point, G5 and G6 arthrobranchs with a common insertion point. Gills from the left side were sampled for gene expression studies while those from the right side were sampled for protein and enzyme analyses. Pooled samples of posterior gills (4,5,6) were used for gene expression, activity and protein analyses. The experimental protocols for the present study were approved by the National Taiwan Normal University Institutional Animal Care and Utilization Committee (approval no.: 101005).

### Extracellular Acid-Base Status

Hemolymph samples were taken from the coxa using a gas-tight Hamilton syringe. Determination of pHe in venous hemolymph was performed in 100µl samples using a microelectrode (WTW Mic-D) and a WTW 340 pH meter (precision ± 0.01 units) that was calibrated with Radiometer precision buffers 7 and 10 (S11M44, S11 M007). Measurements were performed inside a temperature controlled water bath adjusted to 28◦C. Due to low hemolymph sample volumes for total dissolved inorganic carbon (CT) determinations (∼100µl) samples were diluted 1:1 with de-ionized water prior to measurements. After hemolymph sampling, the dilution and measurement of samples was carried out within less than 30 s to guarantee negligible changes in the carbonate system of hemolymph samples. CT was determined in duplicates (100µl each) via a Corning 965 carbon dioxide analyzer (precision ± 0.1 mmol l−1 ; Olympic Analytical Service, England) that was calibrated using a fresh dilution series of 40, 20, 10, 5, and 2.5 mM bicarbonate in distilled water to generate a sodium bicarbonate standard curve. Carbonate system speciation (i.e., pCO2, [HCO− 3 ]) of hemolymph samples of X. testudinatus was calculated from extracellular pH (pHe) and CT using the Henderson–Hasselbalch equation with dissociation constants and solubility coefficients as previously described for the shore crab Carcinus maenas (Truchot, 1976).

### Immunohistochemistry and Western Blot Analyses

For immunohistochemistry gill tissues from control animals were fixed and mounted to slides as previously described (Hu et al., 2014). The primary antibodies, a mouse monoclonal antibody α5, raised against the avian α subunit of the Na+/K+-ATPase (Hybridoma Bank) and a polyclonal antibody raised against part the subunit A region (SYSKYTRALDEFYDK) of the molluscan V-type-H+-ATPase (VHA; for more detail see Hu et al., 2013) were diluted in PBS (1:100) and placed in droplets of 200µl onto the sections, and incubated over night at 4◦C inside a wet chamber. Sections were then washed (3 × 5 min) with PBS and incubated for 1 h with the secondary antibody, anti-mouse Alexa Fluor 488, or anti-rabbit Alexa Fluor 568 (Invitrogen) (dilution 1:250). After rinses in PBS (3 × 5 min), sections were examined and photographed using a fluorescence microscope (Zeiss imager A1) equipped with an appropriate filter set. Negative controls were performed several times for every antibody by omitting the primary antibody.

Immunoblotting was essentially performed as previously described (Hu et al., 2014) using 15µL of gill crude extracts. Proteins were fractionated by SDS-PAGE on 10% polyacrylamide gels, and transferred to PVDF membranes (Millipore), using a tank blotting system (Bio-Rad). Blots were exposed to the primary antibody (see previous section) diluted 1:250–500 and incubated at 4◦C overnight. After washing with PBS-T (phosphate buffered saline containing 0.1% Tween20), blots were incubated for 2 h with horseradish conjugated goat antirabbit IgG antibody (diluted 1:1000–2000, at room temperature; Amersham Pharmacia Biotech). Protein signals were visualized using the enhanced chemiluminescence system (ECL, Amersham Pharmacia Biotech) and recorded using Biospectrum 600 imaging system (UVP, Upland, CA, USA). Signal intensities were calculated using the free software "Image J" (e.g., Schneider et al., 2012).

#### Enzyme Activity

ATPase activity was measured in crude extracts of the three posterior gills (4,5,6). The measurement is based on a coupled enzyme assay containing pyruvate kinase (PK) and lactate dehydrogenase (LDH) as previously described (Hu et al., 2014). Crude extracts were obtained by quickly homogenizing the tissue samples using a pestle followed by complete homogenization in a tissue lyzer (Qiagen) in five volumes of ice-cold imidazole buffer (Hu et al., 2014). After centrifugation for 10 min at 1000 g and 4◦C, cell debris was removed and the supernatant was used as a crude extract. The reaction was started by adding 1.5µl of the sample homogenate to the reaction buffer. The coupled to The hydrolysis of ATP reflected by the oxidation of NADH was measured photometrically at 30◦C in a temperature controlled plate reader (Molecular Device, Spectra Max, M5), over a period of 15 min, with the decrease of extinction being measured at λ = 339 nm. Addition of 2µl ouabain (5 mM final concentration) or bafilomycin (Bafilomycin A1, Sigma-Aldrich) (1µM final concentration) to the assay was used to determine the fraction of Na+/K+-ATPase or H+-ATPase activity from the total ATPase (TA) activity. The concentrations of inhibitors used in this enzyme assay are sufficient to fully inhibit the NKA (Morris et al., 1997) and VHA (Dröse and Altendorf, 1997), respectively. Six measurement replicates were performed for each sample (three with inhibitor dissolved in DMSO and three with DMSO). Enzyme activities were calculated by using the extinction coefficient for NADH of ε = 6.31 mM−1 ·cm−1 and given as micromoles of ATP consumed per gram tissue fresh mass (gFM) per hour.

### Preparation of mRNA

Separated gills (1–6) were homogenized in Trizol reagent (Invitrogen, Carlsbad, CA, USA) using a Tissue lyser (Quiagen). Chloroform was added to the Trizol homogenates, and total RNA was extracted from the aqueous phase and purified by addition of isopropanol. Genomic DNA contaminations were removed by DNase I (Promega, Madison, WI, USA) treatment. The mRNA for the RT-PCR was obtained using a QuickPrep Micro mRNA Purification Kit (Amersham Pharmacia, Piscataway, NJ, USA) according to the supplier protocol. Extracted mRNA concentrations were determined by spectrophotometry (ND-2000, NanoDrop Technol, Wilmington, DE), and the integrity

### Cloning of xtNKA Fragment

Fragments of the X. testudinatus Na+/K+-ATPase (NKA) and V-Type H+-ATPase (VHA) genes were amplified from gill tissue by means of reverse transcription followed by PCR (RT–PCR) using primers based on highly conserved regions of the NKA and VHA from the green shore crab C. maenas. Reverse transcription was performed as previously described (Hu et al., 2013) and the primer pair 5′ -CAGTCACTTCATCCACATCA-3′ and 5′ - CACATCTCCAATAGCCAGTT-3′ resulted in a 540 bp fragment of the NKA. PCR fragments were separated by electrophoresis in 1.5% agarose gels. Extraction, purification, and cloning of the PCR fragments from the gel was accomplished as previously described (Hu et al., 2013). Plasmids were sequenced and sequence analysis was performed using the BLASTx program (NCBI, http://blast.ncbi.nlm.nih.gov/Blast.cgi).

### Real-Time Quantitative PCR (qPCR)

The mRNA expressions of selected candidate genes were measured by qPCR using the Roche LightCycler R 480 System (Roche Applied Science, Mannheim, Germany). Primers for the Na+/K+-ATPase, V-type H+-ATPase and the reference gene arginine kinase were designed using Primer Premier software (vers. 5.0; PREMIER Biosoft International, Palo Alto, CA) and are provided in Table S2. PCR reactions were performed as previously described (Hu et al., 2013, 2014) and PCR products were subjected to a melting-curve analysis. Primer efficiencies were >96% and control reactions were performed using nuclease-free water to determine background levels. Additionally, DNAse I treated RNA samples served as a control, demonstrating that no PCR product was obtained, and thus the success of the DNase I treatment. The standard curve of each gene was in a linear range with arginine kinase (AK) that served as reference gene. The expression of this reference gene has been demonstrated to be stable in the green shore crab C. maenas during CO2 treatments (Fehsenfeld et al., 2011).

#### Statistical Analyses

Statistical analyses were performed using Sigma Stat 3.0 (Systat) software. Statistical differences between pH treatments within one time point were analyzed using a Student's t-test. The significance levels were set to p < 0.05∗ and p < 0.01∗∗ .

### RESULTS

#### Extracellular Acid-Base Status

Mean extracellular pH (pHe) measured in hemolymph samples along the time series of 48 h ranged between pH 7.50 ± 0.02 to 7.59 ± 0.01 in control animals (**Figure 2A**; Figure S1). In response to acidified conditions of pH 6.5, pHe dropped (p < 0.05) by ∼0.25 pH units compared to control animals after 1 h. pHe was partially restored after 4 h and remained stable at levels of ∼0.1 pH units below control pHe. Mean blood HCO− 3 levels were found to range from 6.4 ± 0.5 to 8.2 ± 1.1 mM in

between pH treatments (\**p* < 0.05 and \*\**p* < 0.001). Bars represent mean ± SE (*n* = 4).

control animals along the incubation period of 48 h (**Figure 2B**). In response to acidified conditions of pH 6.5, hemolymph HCO− 3 levels progressively increased from 8.8 ± 0.3 to 31 ± 6 mM during the 48 h incubation period and significant differences (p < 0.05 and 0.01) to control animals were observed after 4 h incubation until the end of the experiment. Hemolymph pCO2 levels ranged from 0.41 to 0.55 kPa in control animals along the incubation period of 48 h (**Figure 2C**). In response to acidified conditions, hemolymph pCO2 levels increased to peak values of 2.7 ± 0.3 kPa (**Figure 2C**).

### Regulation of Branchial Na+/K+-ATPase and V-Type H+-ATPase upon Low pH Exposure

Comparisons of routine mRNA levels of NKA and VHA did not reveal any significant differences between anterior (1–3) and posterior gills (4–6; Figure S2). In response to acidified conditions, mRNA levels of the NKA and VHA measured in the posterior gills (4,5,6) increased rapidly within 1 h by 2.3 and 11.4-fold, respectively (**Figures 3A,B**). While NKA mRNA levels returned to control levels within the acute acclimation phase of 4 h, VHA mRNA levels rapidly increased upon low pH exposure for 1 h and then decreased back to control levels within 24 h (**Figures 3A,B**). The increase in mRNA concentrations is paralleled by an increase in NKA and VHA enzyme activities (**Figures 3C,D**). Compared to control conditions where maximum NKA enzyme activities ranged from 335 ± 60 to 456 ± 9µmolATP h −1 g −1 FM, branchial NKA activities in crabs exposed to acidified conditions increased within 1 h to a maximum of 719 ± 115µmolATP h −1 g −1 FM. Along the period of 24 h, NKA activities decreased back to control levels (**Figure 3C**). Branchial VHA enzyme activities increased in response to acidified conditions with an activity peak after 12 h reaching 1.7-fold increased enzyme activities compared to control animals (**Figure 3D**). After 24 h of low pH exposure, branchial VHA activities returned back to control levels and remained slightly above control levels for the experimental period of 48 h. When comparing relative changes of NKA mRNA and activity levels along the entire experimental period a slight shift by ∼3 h in peak NKA activities can be found in comparison to mRNA levels. Moreover, while NKA mRNA levels returned to control conditions after 4 h, NKA enzyme activities remain elevated by 40% to 30% along the experimental duration of 48 h (**Figure 3E**). A more pronounced shift between peak mRNA and enzyme activity levels of ∼9 h has been observed for the branchial VHA (**Figure 3F**). After this acute phase, VHA enzyme activities decreased back to control levels after 48 h exposure to acidified conditions.

### Localization of Na+/K+-ATPase and V-Type H+-ATPase in Gill Epithelia

Immunohistochemical analyses demonstrate the sub-cellular localization of Na+/K+-ATPase and V-type H+-ATPase in gill epithelia of posterior gills from control animals (**Figure 4A**). Using double staining, high concentrations of Na+/K+-ATPase were detected in basolateral membranes of the entire gill epithelium as well as in pillar cells spanning between the two epithelial layers (**Figure 4A**). V-type H+-ATPase was predominantly located in the cytoplasm of epithelial- and pillarcells. In contrast to the distribution of NKA in cells of the entire gill lamella, VHA immunoreactivity is only observed in single cells, predominantly pillar cells. Negative controls performed by omitting the primary antibody, did not show any signal in posterior gill lamellae, supporting the specificity of the primary antibodies used (Figure S3). Western blot analyses demonstrate specific immunoreactivity of antibodies with proteins including Na+/K+-ATPase (≈110 kDa) and V-type H+-ATPase (≈70 kDa; **Figure 4B**). Western blot analyses of posterior gills of pH 8.0 and 6.5 treated crabs demonstrates increased NKA as well as VHA protein concentrations in low pH treated animals during the acute low pH acclimation phase (**Figure 4C**). NKA protein concentrations in the acute acclimation phase (12 h) are 1.4-fold higher whereas VHA protein concentrations of posterior gills are increased by 3-fold in low pH treated animals compared to pH 8.0 acclimated animals.

 In these taxa, buffering of extracellular pH (pHe) is associated with an increase in blood HCO− 3 levels, which is a conserved and efficient mechanism to counter respiratory acidosis (Heisler, 1986; Pörtner et al., 1991; Gutowska et al., 2010; Henry et al., 2012). The present work demonstrates powerful extracellular acid-base regulatory abilities of X. testudinatus that are beneficial for this species to inhabit highly acidic hydrothermal vent habitats over long time scales. The hyperbolic increase in blood HCO− 3 levels of X. testudinatus in response to acidified conditions is in general accordance with findings for other strong acidbase regulators (Heisler, 1986). For example, most teleosts can rapidly and fully compensate pHe during moderate to strong hypercapnia, accompanied by an increase in blood [HCO3 −] in excess of 20 to 30 mM (Larsen et al., 1997; Perry et al., 2010). While cephalopods were characterized to have moderate acid-base regulatory abilities (Gutowska et al., 2010; Hu et al., 2014), most brachyuran crabs were described to be moderate to strong acid-base regulators (Cameron, 1978; Truchot, 1984; Pane and Barry, 2007; Spicer et al., 2007). Detailed investigations of extracellular acid-base parameters during hypercapnic exposure in crustaceans are restricted to a few species, including the European shore crab, C. maenas (Truchot, 1984), the blue crab, Callinectes sapidus (Cameron, 1978), Cancer magister, Chionoecetes tanneri (Pane and Barry, 2007), and the velvet swimming crab, Necora puber (Spicer et al., 2007). All species showed an initial depression of extracellular pH over the course of 4 h, which was partially restored over the next 24–48 h through an active accumulation of hemolymph HCO− 3 . Such a marked

acidosis in the acute acclimation phase (0–12 h) was not observed in X. testudinatus exposed to pH 6.5 (1.78 kPa pCO2), a level greatly in excess of those experimental conditions under which C. maenas (0.57 kPa CO2; ≈pH 7.2), C. sapidus (1.0 kPa CO2; pH 7.08), C. magister and C. tanneri (1.0 kPa CO2; pH 7.08) were examined. As gills were demonstrated to be the major site for ion and acid-base regulation in decapod crustaceans, (Henry et al., 2012) the following paragraph focuses on the branchial mechanisms that mediate extracellular acid-base balance in the hydrothermal vent crab X. testudinatus.

### Branchial Acid-Base Regulatory Mechanisms

Immunohistochemical analyses demonstrate the presence of Na+/K+-ATPase and V-type H+-ATPase in branchial epithelia of the hydrothermal vent crab X. testudinatus. These primary active ion-transporters are key players for intra- and extra-cellular regulation of ion and pH homeostasis in all animals (Emery et al., 1998; Tresguerres et al., 2005; Colina et al., 2007; Horng et al., 2009). The ubiquitous NKA located in basolateral membranes creates an electrochemical gradient that fuels secondary active transporters. For example, blocking of NKA in perfused gills of the crab Neohelice granulata inhibited HCO− 3 secretion and H+ reabsorption indicating a central role in fueling secondary active transport mechanisms relevant for acid-base regulation (Tresguerres et al., 2008).

The sub cellular localization of VHA in branchial epithelia of marine organisms seems to be less conserved compared to the NKA. For example, in most teleosts, the VHA is located in apical membranes where it is believed to mediate the direct secretion

of protons (Horng et al., 2007; Tsai and Lin, 2007; Hwang et al., 2011). However, in marine species like the teleost, Oryzias latipes (Lin et al., 2012) and the squid Sepioteuthis lessoniana the VHA is located in basolateral membranes of ion-regulatory epithelia where it is involved in acid-base regulatory processes as well (Hu et al., 2013). In most crustaceans, the VHA has been demonstrated to be localized in apical membranes and/or in the cytoplasm of branchial epithelial cells (Weihrauch et al., 2001; Tsai and Lin, 2007). The strong cytosolic but weak apical abundance of the VHA in branchial cells of X. testudinatus is in accordance with observations made on other marine and intertidal brachyuran crabs including Ocypodid crabs, Uca laceta, and Macrophthalmus spp. as well as the brachyuran crabs, Hemigrapsus sanguineus, H. penicillatus, Perisesarma bidens, and Chiromantes dehaani (Tsai and Lin, 2007). The cytoplasmic localization of the VHA has been hypothesized to be involved in the trapping of NH+ 4 within acidified vesicles and subsequent exocytosis across the apical membrane (Weihrauch et al., 2002). As secretion of NH+ 4 also results in a net export of protons, it is likely that excretion of nitrogenous waste products and acid-base regulation are linked processes.

Gene expression analyses and enzyme activity measurements in the present study indicate that branchial NKA and VHA are important players of acid-base regulation that mediate bicarbonate accumulation as well as H+ secretion in this hydrothermal vent species. In fish, cephalopods and crustaceans, the compensation of an extracellular acidosis by an accumulation of HCO− 3 is always associated with a significant net export of protons (Heisler, 1984, 1986; Cameron, 1986). As HCO− 3 formation through the hydration of CO2 is always associated with the generation of H+, H+ export pathways represent an essential feature in animals that compensate an extracellular acidosis. These observations are in line with the results of the present study, demonstrating that environmental acidification stimulates expression levels of branchial VHA that is involved in the secretion of proton equivalents. Moreover, basolateral NKA has been hypothesized to energize HCO− 3 transport in crustacean branchial epithelia (Tresguerres et al., 2008). In this context a Na+/HCO− 3 co-transporter (NBC) located in basolateral membranes has been proposed for the branchial epithelium of Neohelice granulate based on pharmacological observations (Tresguerres et al., 2008). Other studies suggested that basolateral anion exchangers are involved in the HCO− 3 re-absorption in crustacean gills (Freire et al., 2008; Harms et al., 2014). Although the existence, function and cellular localization of HCO− 3 transporters from the SLC 4 family are not confirmed for X. testudinatus, this work strongly suggests that active HCO− 3 re-absorption significantly contributes to

extracellular pH homeostasis during environmental acidification. Active HCO− 3 transport mechanism can be considered essential for the HCO− 3 buffering since dissolution of the carapace has been demonstrated to represent only a minor contribution to extracellular HCO− 3 accumulation in crustaceans (Cameron, 1985). According to this information, we propose a first model for acid-base regulatory mechanisms in gill epithelia of the hydrothermal vent crab, X. testudinatus (**Figure 4D**). The NKA energizes HCO− 3 uptake via a currently not identified basolateral HCO− 3 transporter and cytosolic carbonic anhydrase (CA). Based on a study by Weihrauch et al. (2002) we hypothesize that also in X. testudinatus cytoplasmic VHA is involved in countering an acidosis by pumping H+ ions into vesicles that exocytose protons or proton equivalents (e.g., NH+ 4 ) across the apical membrane. Accordingly, during vesicle fusion at the apical membrane a certain fraction of the cellular VHA must be temporarily transferred to the apical plasma membrane as well. In order to support this hypothesis of vesicular proton excretion in this species future studies will compare the subcellular distribution of VHA-rich vesicles between control and low pH acclimated animals.

Based on the findings of the present work this model suggests that NKA seems to be involved in acid-base compensatory mechanisms by fueling additional secondary active ion-transporters. The electroneutral transport of acidbase equivalents (e.g., HCO− 3 and H+) via anion and cation exchangers is directly connected to the transport of counter ions (e.g., Na+ and Cl−) that supports the link between acid-base and osmoregulation found in marine euryhaline decapod crustaceans (Truchot, 1981; Whiteley et al., 2001). Accordingly it is very likely that X. testudinatus utilizes Na+- and Cl−-dependent acid-base regulatory pathways via Na+/H+-exchangers (NHEs), and maybe also NBCs or anion exchangers that are fueled by the NKA. Using X. testudinatus as a model for a strong acid-base regulating crustacean further studies regarding the functional expression, enzyme abundance and subcellular localization of these secondary active transporters will provide important insights to the mechanistic connection between acid-base and osmotic regulation in decapod crustaceans.

Interestingly, the increase of the NKA and VHA on the mRNA level is only visible in the acute low pH acclimation phase, whereas enzyme activities increase with a short delay and stay elevated until the time point of 24 h. This observation reflects the time gap between mRNA expression and translation into the functional protein, which is in general accordance with findings in other eukaryotes as well as prokaryotes (Marsh et al., 2000; Glanemann et al., 2003). Although NKA and VHA enzyme activities return back to control levels after 24 h, blood HCO− 3 levels remain increased after 48 h in the low pH treatment. This suggests, that initial accumulation of HCO− 3 and the associated secretion of H+ are reflected in the up regulation of NKA and VHA mRNA and activity levels. However, after 24 h the pHe compensation process is completed and routine NKA and VHA activity levels are probably sufficient to maintain increased hemolymph HCO− 3 levels to protect pHe homeostasis. This energy saving strategy of stabilizing hemolymph pH over long exposure times can be regarded as a key adaptation of X. testudinatus to occupy an ecological niche in a highly acidified hydrothermal vent habitat.

### CONCLUSION

Strong acid-base regulatory abilities of X. testudinatus can be regarded an essential feature of this species to successfully inhabit a highly acidic hydrothermal vent environment. Functional and histological results of the present study demonstrated that extracellular acid-base regulatory mechanisms in X. testudinatus rely on a conserved set of ion pumps that are also found in other brachyuran crabs. In accordance to phylogenetic analyses based on mitochondrial genomes (Ki et al., 2009) and morphological systematics (Ng et al., 2000), amino acid sequence comparisons of the NKA demonstrate that X. testudinatus is closely related to other varunid and grapsid species including E. sinensis and Pachygrapsus marmoratus which are characterized as powerful osmo-regulators (Gross, 1961; Onken, 1999; **Figure 5**). NKA homologies also confirm molecular systematics demonstrating that deep-sea hydrothermal vent crabs of the genus Bythograea spp. (Mateos et al., 2012) constitute an own phylogenetic group within the crustacea (**Figure 5**). Despite having weak osmoregulating abilities, Bythograea thermydron (Martinez et al., 2001) may be able to increase ATPase activities to increase H+ efflux rates to protect from strong pH fluctuations that are a characteristic of deep-sea vent habitats (Von Damm, 1995; Tivey, 2004). However, additional studies addressing the mechanistic basis of extracellular pH regulation in crustaceans from deepsea vent systems will be needed to support this hypothesis. Such comparative studies between deep-sea and shallow-water hydrothermal vent crabs will provide a basis for new and exciting research regarding the evolution of pH regulatory systems in crustaceans that have the ability to occupy ecological niches in highly acidic hydrothermal vent habitats.

### AUTHOR CONTRIBUTIONS

MH and YT designed and conducted experiment, analyzed the data, and compiled the main manuscript. MH conducted immunohistochemical experiments and evaluate enzyme activities. YS and MJ collected and acquired the animals and photos from open filed. YG and YT carried out the molecular cloning and expression studies. PK, GC, and JL conducted CO2 perturbation experiments, physiological measurements, and sample preparation. All authors reviewed and approved the manuscript.

### ACKNOWLEDGMENTS

This study was financially supported by the grants to YT. YT from the Ministry of Science and Technology, Taiwan, Republic of China (MOST 104-2321-B-003-001), Alexander von Humbold/National Science Council (Taiwan) grant (NSC 102- 2911-I-001-002-2), and a Cluster of Excellence "The future ocean" grant (CP1409) awarded to MH. We gratefully thank Mr. H. T. Lee (assistant in marine station of Institute of Cellular and Organismic Biology, Academia Sinica) for his assistance to maintain experimental systems.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fphys. 2016.00014

## REFERENCES

Harms, L., Frickenhaus, S., Schiffer, M., Mark, F. C., Storch, D., Held, C., et al. (2014). Gene expression profiling in gills of the great spider crab Hyas araneus in response to ocean acidification and warming. BMC Genomics 15:789. doi: 10.1186/1471-2164-15-789

Heisler, N. (1984). Acid-Base Regulation in Fishes. Amsterdam: Academic Press.

family Bythograeidae: a comparison of three Bayesian species tree methods. PLoS ONE 7:e32066. doi: 10.1371/journal.pone.0032066

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Hu, Guh, Shao, Kuan, Chen, Lee, Jeng and Tseng. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Double Knockdown of PHD1 and Keap1 Attenuated Hypoxia-Induced Injuries in Hepatocytes

Jing Liu, Yiping Li, Lei Liu, Zhi Wang, Chuanbing Shi, Zhengyuan Cheng, Xiaoyi Zhang, Fengan Ding and Ping Sheng Chen\*

*Department of Pathology and Pathophysiology, School of Medicine, Southeast University, Nanjing, China*

Background and Aims: Hypoxia and oxidative stress contribute toward liver fibrosis. In this experiment, we used small hairpin RNA (shRNA) to interfere with the intracellular oxygen sensor—prolyl hydroxylase 1 (PHD1) and the intracellular oxidative stress sensor—kelch-like ECH associated protein 1 (Keap1) in the hypoxic hepatocytes in order to investigate the function of PHD1and Keap1.

Methods: We first established the CCl4-induced liver fibrosis model, subsequently, the levels of the PHD1, hypoxia-inducible factor-1α (HIF-1α), hypoxia-inducible factor-2α (HIF-2α), Keap1, and nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) were detected in liver tissues. Simultaneously, AML12 cells co-transfected with PHD1 and Keap1shRNAs were constructed *in vitro*, then the intracellular oxidative stress, the proportion of cells undergoing apoptosis, and cell viability were measured. The expression of pro-fibrogenic molecules were analyzed via quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The level of alpha-1 type I collagen (COL1A1) was determined using an enzyme-linked immunosorbent assay (ELISA). Finally, serum-free "conditioned medium" (CM) from the supernatant of hypoxic AML12 hepatocytes was used to culture rat hepatic stellate cells (HSC-T6), and the levels

## of fibrosis-related molecules, apoptosis, and cell proliferation were determined. Results: The marker of hypoxia—HIF-1α and HIF-2α in the livers with fibrosis were upregulated, however, the increase in PHD1 expression was not statistically significant in comparison to the control group. Sign of oxidative stress—Keap1 was increased, while the expression of Nrf2, one of the Keap1 main downstream molecules, was reduced in the hepatocytes. And *in vitro*, the double-knockdown of PHD1 and Keap1 in AML12 hepatocytes presented with decreased hypoxia-induced oxidative stress and

apoptosis, furthermore, these hypoxic AML12 cells showed the increased cell viability and the doweregulated expression of pro-fibrogenic molecules. In addition, HSC-T6 cells cultured in the hypoxic double-knockdown CM demonstrated the downregulation of fibrosis-related molecules, diminished cell proliferation, and enhanced apoptosis.

Conclusions: Our study demonstrated that double-knockdown of PHD1 and Keap1 attenuated hypoxia and oxidative stress induced injury in the hepatocytes, and subsequently inhibited HSC activation, which offers a novel therapeutic strategy in the prophylaxis and treatment of liver fibrosis.

Keywords: liver fibrosis, hepatocytes, PHD1, Keap1, hypoxia, oxidative stress

#### Edited by:

*Maria Giovanna Trivella, Consiglio Nazionale Delle Ricerche (CNR), Italy*

#### Reviewed by:

*Katja Breitkopf-Heinlein, Heidelberg University, Germany Antonella Cecchettini, University of Pisa, Italy*

\*Correspondence:

*Ping Sheng Chen<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Integrative Physiology, a section of the journal Frontiers in Physiology*

Received: *22 October 2016* Accepted: *21 April 2017* Published: *10 May 2017*

#### Citation:

*Liu J, Li Y, Liu L, Wang Z, Shi C, Cheng Z, Zhang X, Ding F and Chen PS (2017) Double Knockdown of PHD1 and Keap1 Attenuated Hypoxia-Induced Injuries in Hepatocytes. Front. Physiol. 8:291. doi: 10.3389/fphys.2017.00291*

### INTRODUCTION

Liver fibrosis is a pathological process caused by numerous chronic liver damages—such as viral hepatitis, alchoholic hepatitis, and drug induced hepatotoxicity (Begriche et al., 2011). As a highly active metabolic organ, the liver is particularly prone to hypoxic environments and the damages caused by it (Nakanishi et al., 1995). A convincing body of evidence suggest that hypoxia plays an important role in the pathogenesis of liver fibrosis (Cannito et al., 2014).

Cellular hypoxia leads to activation of hypoxia-inducible factors (HIF), which is crucial for the survival of an asphyxiated/ischemic hepatocyte. In normoxia, HIF-1α and HIF-2α are first hydroxylated by prolyl hydroxylase (PHD) and then degraded by proteasomes (Kamura et al., 2000). Whereas during hypoxic condition, HIF-1α and HIF-2α accumulate and get translocated to the nuclei, thereby activating the genes responsible for limiting hypoxia-induced injury and cell death. Recently, Nimker et al. reported that the pharmaceutical inhibition of PHDs with ethyl 3, 4-dihydroxy benzoate (EDHB) protects myoblasts against hypoxia-induced oxidative damage by upregulating HIFs (Nimker et al., 2015). However, non-specific inhibition of PHDs also induces the activation of HIFs but at the cost of adverse effects such as steatosis (Minamishima et al., 2009; Rankin et al., 2009). PHD has three isoforms—PHD1, PHD2, and PHD3, each of these has numerous functions. Selective loss of PHD1, but not those of PHD2 or PHD3 could induce hypoxia tolerance in the skeletal muscle and liver cells via reprogramming of basal oxygen metabolism without inducing angiogenesis and erythrocytosis (Aragones et al., 2008; Schneider et al., 2010). Therefore, the specific inhibition of PHD1 could serve as a potential therapeutic strategy against hypoxia during liver fibrosis.

In recent years, many studies have demonstrated that oxidative stress also contributes to the pathogenesis of liver fibrosis (Ghatak et al., 2011; Mormone et al., 2012; Yang et al., 2013).The nuclear factor-erythroid 2-related factor 2 (Nrf2) has been suggested to be involved in this process. Under normal condition, Nrf2 exists in the cytoplasm where it binds to kelchlike ECH associated protein 1 (Keap1), thereby hastening the Nrf2 ubiquitination and degradation (Kang et al., 2004). During oxidative stress, Nrf2 evades Keap1 and translocates to the nucleus, where it activates antioxidant genes. Chen et al. has also demonstrated that glycyrrhetinic acid could ameliorate chronic liver fibrosis via upregulation of Nrf2 (Chen et al., 2013). Additonally, Tanaka et al. also reported that Nrf2-null mice had an increased susceptibility to liver injury (Tanaka et al., 2012). Keap1 serves to negatively regulate Nrf2 (Miyata et al., 2011), for example, Keap1-knockdown mice are less vulnerable to oxidative liver injuries during obstructive cholestasis mediated by enhanced expression of Nrf2 (Okada et al., 2009). Thus, Keap1 may be an efficient therapeutic target for relieving oxidative stress injury during liver fibrosis.

It has been reported that many cell types are involved in the pathogenesis of liver fibrosis, activated hepatic stellate cells (HSCs) play a central role in liver fibrogenesis. It is worth noting that injuried hepatocytes are considered to be the primary activator of HSCs (Nieto et al., 2002), and hepatocytes are the major parenchymal cells that account for more than 70% of all liver cells (Bogdanos et al., 2013). Furthermore, hepatocytes are highly susceptible to hypoxic injury and the drugs induced toxicity, therefore we chose hepatocytes as a therapeutic target in an attempt to ameliorate liver fibrosis. Hypoxic hepatocytes are known to generate excessive amount of reactive oxygen species (ROS) which exert oxidative stress on the liver. Moreover, such oxidative stress can further exacerbate the hypoxia in the hepatocytes, and the subsequent re-oxygenation following hypoxia leads to additional oxidative stress (Miyata et al., 2011). Therefore, the ideal therapy for liver fibrosis could be the alleviation of hypoxia along with the oxidative stress, however, no such related studies have been done so far.

RNA interference (RNAi) is a method that uses a small complementary double-stranded RNA (dsRNA) molecule to silence a target gene (Inoue et al., 2006). Considering the short half-life and high cost of current small interfering RNA (siRNA) vectors, they are not suitable for many in vivo studies. In contrast, short hairpin RNA (shRNA) is widely used due to its enhanced stability and transfer efficiency. Accordingly, for the present study we used shRNAs to simultaneously knockdown the expressions of PHD1 and Keap1 in the hepatocytes with the aim of exploring therapeutic target for liver fibrosis.

### MATERIALS AND METHODS

#### Animals and Treatment

Six-week-old male Sprague-Dawley rats (250 ± 30 g) were purchased from the Animal Center of Yangzhou University (Yangzhou, China). All animals were housed in the animal experimental center of Southeast University under constant temperature (22◦C) and humidity (55 ± 5%) in a controlled room with a 12–12 h light-dark cycle, where the diet and water were available ad libitum. The rats were acclimatized under these conditions for at least 1 week prior to experiments. All animals were randomly divided into two groups: a treatment group (n = 10) which received 1.5 ml/kg body weight carbon tetrachloride (CCl4; 40% CCl4 in olive oil) via intraperitoneal injection twice a week for eight weeks and a normal control group (n = 10) which received a saline injection in parallel. This study was carried out in accordance with the recommendations of the European Council Directive of the 24th November 1986 (86/609/EEC). All procedures were approved by the Animal Research Ethics Committee at the Medical School of the Southeast University (Nanjing, China).

#### Histology and Immunohistochemistry

Excised livers were fixed in 10% neutral-buffered formalin, embedded in paraffin, and cut into sections of 5 µm thickness. Slides were then deparaffinized with dimethylbenzene, dehydrated with graded ethanol, and stained with hematoxylin and eosin (H&E) and Masson dyes to evaluate the degree of liver fibrosis.

For immunohistochemical probing, liver slides were initially boiled in a pressure cooker containing a citric acid buffer (pH 6.0) to retrieve antigens. Then the slides were blocked with 5% BSA and incubated with primary antibodies (**Table 1**) overnight at 4◦C, and with a goat anti-rabbit biotinylated secondary antibody for 20 min at 37◦C. Immunolabels were detected with 3, 3-Diaminobenzidine (DAB), after which the nuclei were counterstained with hematoxylin. Slides were inspected under a fluorescence microscope at 200× magnification. For quantification analysis, the percentage of positive area for immunohistochemistry was determined using ImageJ software.

#### Cell Cultures and Hypoxia Treatment

AML12 (alpha mouse liver 12 cells) and HSC-T6 (rat hepatic stellate cells) were purchased from the Shanghai Institute of Biochemistry and Cell Biology. Cells were cultured in

TABLE 1 | The antibody for immunohistochemistry and western blot.

#### TABLE 2 | Primer sequences for quantitative PCR.

DMEM-F12 (Hyclone, USA) supplemented with 10% heatinactivated fetal bovine serum (Bioind, Israel), and were maintained in a humidified atmosphere with 5% CO2 at 37◦C. For hypoxic exposure, AML12 cells were incubated in a trigas incubator (Thermo, America) of 1% O2, 5% CO2, and 94% N2. Serum-free "conditioned medium" (CM) was obtained from the supernatant of AML12 cells to eliminate the influence of serum cytokines. HSC-T6 cells were cultured in CM and the cells cultured in DMEM were used as control.

### RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction

Total RNA was extracted from cells with Trizol reagent (TaKaRa, Japan). Then, 1 µg RNA was added to a 20 µl reaction volume for cDNA reverse transcription using the Prime ScriptTM RT reagent Kit with gDNA Eraser (TaKaRa, Japan), and quantitative real-time polymerase chain reaction (qRT-PCR) was performed using the BR R Premix Ex TaqTM (TaKaRa, Japan) in a Step One Plus real-time PCR system (Applied Biosystems, USA). Gene expression was quantified according to the 2−11Ct method. All PCR primers are listed in **Table 2**.

#### Protein Extraction and Western Blot

Cells were lysed with RIPA buffer (Beyotime, China) containing a Protease Inhibitor Cocktail (Roche, Germany) and then centrifuged at 12,000 × g for 15 min at 4◦C. Equal amounts of protein were separated via gel electrophoresis and transferred to a PVDF membrane (Millipore Corp, USA). Membranes were blocked with 5% skimmed milk for 1 h at 37◦C and incubated with primary antibodies (**Table 1**) overnight at 4◦C and then with corresponding secondary antibodies for 1 h at 37◦C. The blotting signal was visualized using enhanced chemiluminescence reagent (HaiGene, China). Final protein levels were normalized to that of GAPDH.

percentage of positive area were quantified (C). The scale bar represents 200µm. \**p* < 0.05, \*\**p* < 0.01 vs. control.

### Malondialdehyde (MDA), Reduced Glutathione (GSH) Levels, and Lactate Dehydrogenase (LDH) Activity Assay

MDA and GSH levels were detected in total cell lysates via commercial assay kits (Jian Cheng Bioengineering Institute, China). All levels are expressed as µmol/g protein. For detection of LDH activity, supernatants were collected and analyzed with the LDH assay kit (Jian Cheng Bioengineering Institute, China). LDH activity is expressed as U/L.

### Enzyme-Linked Immunosorbent Assay for Collagen Secretion

The level of alpha-1 type I collagen (COL1A1) in the supernatants of AML12 cells was determined using a commercial enzymelinked immunosorbent assay (ELISA) kit (Hengyuan Biology, China) according to the manufacturer's protocol. The optical density was measured at a wavelength of 450 nm.

### Cell Viability Assay

Cellular viability was measured using the cell counting kit-8 (CCK-8) assay (Dojindo, Japan) following the manufacturer's protocol. Absorbance (OD value) was measured at 450 nm using an enzyme-linked immunosorbent assay reader (Thermo Fisher Scientific).

### Apoptosis Assay

Cells undergoing apoptosis were stained with the Annexin-V/PI stain (Ebscience, America). For cells that were transfected with plasmids with green and red fluorescence, PI was replaced by DAPI to label apoptosis. A Flow cytometer (Becton Dickinson, USA) was used to quantify apoptotic cells.

### Statistical Analysis

Results are expressed as mean ± SEM. One-way ANOVA and the Student's t-test were used to evaluate any variations in outcomes between different groups. A p-value < 0.05 was considered as statistically significant.

### RESULTS

### Hypoxia and Oxidative Stress in the Rats with Liver Fibrosis

To understand the microenvironment in the fibrotic liver tissues, we established CCl4-induced liver fibrosis rat model. H&E staining showed that CCl4 exposure resulted in severe damage of the liver cells, including hepatocytes necrosis, inflammatory cells infiltration, steatosis, and fibrosis connective tissues proliferation. However, the control group presented normal histological morphology (**Figure 1A**). Masson's trichrome staining imparts a blue color to collagen against the red background of other structures. According to the microscopic examination, CCl4 treatment resulted in an increased collagen deposition around the portal area and formation of septal fibrosis (**Figure 1A**). Furthermore, immunohistochemical staining demonstrated an increased expression of α-smooth muscle actin (α-SMA, a marker of HSC activation, which plays a critical role in liver fibrogenesis) in fibrous septae (**Figure 1A**). These results indicated that the CCl4-induced liver fibrosis was conducted successfully. Sitmultaneously, we found the upregulation of HIF-1α and HIF-2α (markers of hypoxia), and Keap1 (an intracellular oxidative stress sensor) in hepatocytes in response to CCl4 treatment (**Figures 1B,C**). However, Nrf2, one of the main downstream molecules of Keap1, was downregulated, while PHD1 showed no obvious change (**Figures 1B,C**).

### Effects of PHD1shRNA on HIF Levels in the Hypoxic AML12 Cells

To clarify the function of PHD1 during hypoxia-induced liver injuries, PHD1shRNA was applied to transfect the AML12 cells, then the cells were exposed to hypoxia for 0, 6, 12, 24, and 48 h, the expression of PHD1, HIF-1α and HIF-2α mRNAs and proteins were measured. As shown in **Figure 2A**, PHD1mRNA expression was reduced in hypoxic shNC (negative control) cells compared to that in normoxic shNC cells. Introduction of PHD1shRNA inhibited 60–80% of PHD1 mRNA expression, achieving optimum gene silencing at 12 h of hypoxia. However, PHD1 protein level was invariably reduced by ∼50% regardless of the duration of hypoxia (**Figure 2B**). Under normoxic condition, the expression of HIF-1α and HIF-2α mRNAs (**Figure 2A**) and proteins (**Figure 2B**) were in low expression, and upon exposed to hypoxia, these expression were upregulated. Furthermore, PHD1 silencing further increased the expression of HIF-2α but not that of HIF-1α at both the mRNA and protein levels.

### Effects of Keap1shRNA on Nrf2 Level in the Hypoxic AML12 Cells

To clarify the effect of Keap1shRNA, the AML12 cells were transfected with Keap1shRNA and were administrated with hypoxia for 0, 6, 12, 24, and 48 h, then the expression of Keap1 and Nrf2 at the mRNA and protein levels was determined. An 84% reduction in Keap1 mRNA (**Figure 3A**) and 50% reduction in Keap1 protein (**Figure 3B**) levels were observed at 12 h of hypoxia in the cells silenced with Keap1shRNA. As expected, Nrf2 mRNA and protein levels were increased in the hypoxic shNC cells compared to that in the normoxic shNC cells, and the expression of Nrf2 mRNA and protein were further increased in hypoxic cells treated with Keap1shRNA, which peaked at 12 h of hypoxia (**Figures 3A,B**).

### Effects of PHD1 and Keap1shRNAs on Oxidative Stress in the Hypoxic AML12 Cells

shNC under normoxia. N, normoxia; H, hypoxia.

Since hypoxia led to an increase in oxidative stress, we next studied how molecular markers of oxidative stress—MDA and GSH (important lipid peroxidation and endogenous antioxidant marker, respectively) were regulated. The AML12 hepatocytes were co-transfected with PHD1 and Keap1shRNAs, and then were cultured for 12 h under hypoxia. When cells exposed to hypoxia, a dramatic enhancement of MDA was observed, but the treatment of single knockdown of PHD1 or Keap1 suppressed the increase, furthermore, the double knockdown group was more effective in reducing the hypoxia-induced MDA elevation (**Figure 4A**). However, no significant differences in the levels of MDA and GSH between gene knockdown cells and control cells during normoxia (**Figure 4A**), The intracellular GSH concentration presented the inverse correlation with the MDA level (**Figure 4A**).

### Effects of PHD1 and Keap1shRNAs on AML12 Cells Viability during Hypoxia

To understand the effects of PHD1 and Keap1 molecules on cell biological behavior, the Annexin V-APC/DAPI double staining was used to analyze cell apoptosis. The results were presented as the sum of the percentage of early apoptotic

cells and late apoptotic cells. In **Figures 4B,C**, during hypoxia, the percentage of apoptotic cells in the single PHD1shRNA (6.49 ± 0.50%) or Keap1shRNA (5.86 ± 0.45%) treatment group were notably lower than in the hypoxic control group (10.14 ± 0.79%). Moreover, the apoptotic rate was further reduced in the double-knockdown group (4.63 ± 0.36%). A similar trend among treatment and control groups were also observed during normoxia.

Next, the cell viability was examined using the CCK-8 assay. The data in **Figure 4D** indicated that hypoxia evidently reduced cell viability, however, co-treatment with PHD1 and Keap1 shRNAs led to an increase in cell viability compared to single shRNA transfection. Cellular membrane integrity was analyzed by detecting LDH release. As shown in **Figure 4E**, LDH release in different hypoxia groups fluctuated with a similar tendency as MDA levels. During normoxia, we found no significant difference in LDH levels between the gene silencing and normoxic control groups. In hypoxia, PHD1shRNA or Keap1shRNA treatment group led to a decrease in LDH release, furthermore, the LDH level of the co-transfected group was lower than that of the single groups.

### Effects of PHD1 and Keap1shRNAs on the Expression of Pro-fibrogenic Molecules in the Hypoxic AML12 Cells

To clarify the function of PHD1 and Keap1 during fibrogenesis, the expression of some pro-fibrogenic molecules including Liu et al. Inhibitting Hepatocytes Hypoxia and OS

COL1A1, transforming growth factor-β1 (TGF-β1), vascular endothelial growth factor-A (VEGF-A), and insulin-like growth factor 1 (IGF-1) was evaluated by qRT-PCR and western blot in the hypoxic AML12 cells. Upon exposure to hypoxia, mRNAs (**Figure 5A**) as well as proteins (**Figures 5B,C**) levels of COL1A1, TGF-β1, VEGF, and IGF-1 were upregulated, but the changes in TGF-β1 and IGF-1 proteins were not statistically significant.

During hypoxia, single knockdown of PHD1 or Keap1 induced the downregulation of mRNAs (**Figure 5A**) and proteins (**Figures 5B,C**) levels of COL1A1, TGF- β1, VEGF-A, and IGF-1 compared to the control. More interestingly, the decrease was further enhanced when PHD1 and Keap1shRNAs were simultaneously introduced. To know whether the release of COL1A1 levels were affected by PHD1 and Keap1shRNAs, an ELISA assay was performed using the AML12 cells culture supernatants. The results showed that the levels of COL1A1 in the supernatants had the same trend of reduction as those in the cell lysates (**Figure 5D**).

### Effects of PHD1 and Keap1shRNAs-Treated Hypoxic AML12 Cells on the Expression of Fibrosis-Related Molecules in HSCs

Considering the complex intrahepatic microenvironment, especially the complicated relationship between hepatocytes and HSCs, HSC-T6 cells were cultured in double-knockdown CM to better simulate the in vivo local environment, and then the corresponding fibrosis-related molecules were examined at the mRNAs and proteins levels. During hypoxia CM, COL1A1, α-SMA, TGF-β1, VEGF-A, and IGF-1 mRNAs (**Figure 6A**) and proteins (**Figure 6B**) in hypoxic single-knockdown CM were lower than those in the hypoxic control CM, and were further decreased in double-knockdown CM. However, no significant difference in these mRNAs and proteins expression was observed between the PHD1 or Keap1shNC CM and the control CM group.

### Effects of PHD1 and Keap1shRNAs-Treated Hypoxic AML12 Cells on Apoptosis and Proliferation of in HSCs

Since double-knockdown CM deregulated the expression of fibrosis-related molecules in HSCs, we next detected how the apoptosis of HSCs was regulated using Annexin V-APC/PI double staining. Apoptosis was increased in hypoxic single-knockdown CM, moreover, the apoptotic rate was further enhanced in both normoxic and hypoxic double-knockdown CM (**Figures 7A–C**). However, there was no significant difference between the knockdown

FIGURE 5 | Co-transfection of PHD1 and Keap1shRNAs inhibited expressions of pro-fibrogenic molecules during hypoxia. AML12 cells were co-transfected with PHD1 and Keap1 shRNAs, and then exposed to hypoxia for 12 h. (A) mRNAs and (B,C) proteins levels of COL1A1, TGF-β1, VEGF-A, and IGF-1 were detected by qRT-PCR and western blot. (D) COL1A1 level was measured by ELISA.

CM groups and control CM group in normoxic CM. In addition, **Figure 7D** showed that cells cultured in CM enhanced cell viability compared with cells in DMEM, and the effect was enhanced in hypoxic CM. However, the cell proliferation of the HSC-T6 cells was reduced when cultured in single-transfected CM and was further weakened in the co-transfected CM.

### DISCUSSION

In this study, we first successfully established a model of liver fibrosis induced by CCl4, which is a suitable model for exploring the underlying mechanisms involved in liver fibrosis (Brattin et al., 1985; Lee and Friedman, 2011). By analyzing the liver tissues from model animals with fibrosis, we observed that the

molecules related to hypoxia (HIF-1α, HIF-2α) and oxidative stress (Keap1) were increased in hepatocytes, which provided a valid theoretical basis for the in vitro research.

Next, to elucidate the underlying mechanisms we chose hepatocytes as the primary target. To simulate the hypoxia in vitro, we used 1% oxygen (normoxia with ∼20% oxygen) in the incubators similar to Fingas' study (Fingas et al., 2011). In the hepatocytes, we observed that hypoxia only leads to a decrease in PHD1mRNA but not that of its protein expression, which implied that hypoxia controls the expression of PHD1 only at the transcript level. PHD1 is not only sensitive to oxygen concentration but also vulnerable to ROS generation (Kaelin and Ratcliffe, 2008). Our results also showed that knockdown of PHD1 increased the expression of both HIF-2α mRNA and protein in the hepatocytes, but had no effect on HIF-1α level. These findings were consistent with the previous studies reporting HIF-2α as a major downstream mediator of PHD1 during hypoxia tolerance (Aragones et al., 2008; Schneider et al., 2010) and HIF-1α may be regulated by the PHD2 pathway (Takeda et al., 2007). Furthermore, we found that Keap1shRNA could efficiently suppress Keap1 expression and enhance Nrf2 expression, which suggest that Keap1 negatively regulates Nrf2. Therefore, knockdown of Keap1 is vital for promoting Nrf2-mediated cytoprotection.

Until now, most anti-fibrotic approach researches are focused on either alleviating hypoxia or oxidative stress alone, however, a single treatment have showed an unsatisfactory result with a more side-effects (Halliwell, 2013; Kim and Yang, 2015; Biswas, 2016). Considering the interrelation between hypoxia and oxidative stress, in this experiment we used shRNAs to simultaneously knockdown the expressions of both PHD1 and Keap1 so that one sided dominance of either factor was eliminated. Our data suggest that optimal interference of PHD1 or Keap1 was achieved at 12 h in hypoxic condition, therefore the co-transfected cells were cultured in 1% hypoxia up to this time point. We found that loss of PHD1 or Keap1 reduced oxidative stress by activating GSH and by subsequent inhibition of MDA. Schneider et al. reported that knockdown of PHD1 in the hepatocytes led to reduced oxidative stress (Schneider et al., 2010), and inhibition of Keap1 in the hepatocytes improved the resistance against oxidative stress by upregulating Nrf2 (Miyata et al., 2011), which are also inline with our findings and further supported our data. Importantly, our results indicated that

combined interference of PHD1 and Keap1 had a synergistic effect on the inhibition of oxidative stress.

Previous studies have shown that apoptosis was involved in the pathogenesis of hepatic fibrosis (Hamdy and El-Demerdash, 2012; Klein et al., 2012). In the study, we observed that apoptosis was decreased in the PHD1-knockdown hypoxic hepatocytes, and the anti-apoptotic effect observed was independent of the HIF signal pathway, which may be associated with the activation of NF-κB (Fitzpatrick et al., 2016). Meanwhile, Hamdy et al. reported that Nrf2 inhibited apoptosis via activation of the antiapoptotic Bcl-2 protein (Hamdy and El-Demerdash, 2012), and we also demonstrated that knockdown of Keap1 could inhibit the apoptosis of hepatocytes. Furthermore, combined knockdown of PHD1 and Keap1 led to stronger anti-apoptotic effects and improved viability of hepatocytes during hypoxia. Indeed, the hypoxia tolerance in the PHD1-knockdown hepatocytes is mainly as the result of the subsequent decrease in glucose oxidation and oxygen consumption.(Aragones et al., 2008; Schneider et al., 2010).

It is well known that cytokines play central roles in liver fibrosis by regulating inflammatary mediators toward various injuries (Marra, 2002). VEGF contributes to the process of liver fibrosis by promoting angiogenesis and activating HSCs (Yoshiji et al., 2003), TGF-β1 plays a critical role in inducing the epithelial-mesenchymal transition of hepatocytes and thereby promoting collagen synthesis (Gressner and Weiskirchen, 2006; Kaimori et al., 2007), and IGF-1 also stimulates collagen synthesis and HSCs proliferation (Scharf et al., 1998). Ma et al. proposed that both HIF-1α and HIF-2α acted as positive regulators of VEGF (Ma et al., 2014), moreover the changes in TGF-β1 expression was in HIF dependent manner (Qu et al., 2015). However, our data indicated that the levels of the pro-fibrogenic molecules—COL1A1, TGF-β1, VEGF-A, and IGF-1 were downregulated in PHD1 knockdown cells during hypoxia, and this may be because TGF-β1 and VEGF-A may also be under regulation of ROS besides HIF (Kuroki et al., 1996; Jobling et al., 2006). In addition, we found that the knockdown of Keap1 could inhibit the expression of pro-fibrogenic molecules. Furthermore, concomitant knockdown of both PHD1 and Keap1 synergistically reduced the expression of pro-fibrogenic molecules, thereby inhibiting the progression of fibrosis, however, the specific mechanism is still unclear.

Hypoxic hepatocytes are capable of promoting the expression of TGF-β1 and VEGF-A, which further activates HSCs (Qu et al., 2015). In addition, hepatocytes undergoing apoptosis and oxidative stress which in turn activates HSCs (Zhan et al., 2006; Subhadip et al., 2011), Therefore, considering the complex intrahepatic microenvironment and the interaction between hepatocytes and HSCs (Ayako et al., 2004), we used CM from the supernatant of hypoxic hepatocytes to culture HSCs. Our data showed that double-knockdown CM from hepatocytes could suppress the HSCs activation and prevent the progression of fibrosis. Alternatively, prompting apoptosis of activated HSCs may also be an alternative approach to resolve fibrosis (Iredale, 2001). It is worthly noted that double-knockdown CM could induce an increased apoptosis of HSCs, thereby inhibiting their proliferation.

The possible mechanism of hypoxia-induced hepatocytes injury are illustrated in **Figure 8**. Hypoxia is the initiator of this process, and can induce oxidative stress, which decreases the cellular acitivity, enhances apoptosis, promotes expression of pro-fibrogenic molecules, and hence activates HSC. In our study, we have demonstrated that single-knockdown of PHD1 or Keap1 could alleviate hepatocellular hypoxic injury by upregulating the expression of HIF-2α and Nrf2 respectively, which then inhibits the HSC activation (**Figure 8**), moreover, double-knockdown of PHD1 and Keap1 could synergistically enhance this effect. Collectively, these findings will provide new

#### REFERENCES

targets for developing new effective clinical drugs to treat liver fibrosis. However, further studies are needed to confirm our hypothesis in vivo.

### AUTHOR CONTRIBUTIONS

Conceived and designed the experiments: JL, YL, and PC; Performed the experiments: JL, LL, ZW, CS, ZC, XZ, and FD; Analyzed the data and wrote the paper: JL and YL; Revised the paper: YL and PC.

#### FUNDING

This work was financially supported by the National Nature Science Foundation of China (No 81370868), the Fundamental Research Funds for the Central Universities and the Scientific Research Innovation Program for Graduate Students of Jiangsu Province, China (No KYLX16\_0298), and (No KYZZ15\_0062).

growth factor expression in vitro and in vivo. J. Clin. Invest. 98, 1667–1675. doi: 10.1172/JCI118962

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Liu, Li, Liu, Wang, Shi, Cheng, Zhang, Ding and Chen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Neurosensory and Cognitive Modifications in Europe's Toughest RandoRaid Competition: the Transpyrénéa Extreme Study

Alessandro Tonacci 1 \*, Simona Mrakic-Sposta2 , Kristian Ujka1 , Francesco Sansone1 , Alice Ferrisi 3 , Guido Giardini 4 , Raffaele Conte1 and Lorenza Pratali 1 \*

1 Institute of Clinical Physiology, National Research Council (IFC-CNR), Pisa, Italy, 2 Institute of Bioimaging and Molecular Physiology, National Research Council, Segrate, Italy, 3 Department of Psychology, University of Torino, Torino, Italy, 4 Mountain Medicine Center, Valle d'Aosta Regional Hospital Umberto Parini, Aosta, Italy

Introduction: Given the wide proliferation of ultra-long endurance races, it is important to understand the physiological response of the athletes to improve their safety. We evaluated the cognitive and neurosensory effects on ultra-endurance athletes during the Transpyrénéa (866 Km, 65,000 m positive slope), held on the French Pyrenees.

#### Edited by:

Enrico Capobianco, University of Miami, USA

#### Reviewed by:

Jie Liu, Tangdu Hospital, Fourth Military Medical University, China Rimoldi Stefano, University Hospital Bern, Switzerland

\*Correspondence:

Alessandro Tonacci<EMAIL_ADDRESS>Lorenza Pratali<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Integrative Physiology, a section of the journal Frontiers in Physiology

Received: 28 November 2016 Accepted: 20 March 2017 Published: 04 April 2017

#### Citation:

Tonacci A, Mrakic-Sposta S, Ujka K, Sansone F, Ferrisi A, Giardini G, Conte R and Pratali L (2017) Neurosensory and Cognitive Modifications in Europe's Toughest RandoRaid Competition: the Transpyrénéa Extreme Study. Front. Physiol. 8:201. doi: 10.3389/fphys.2017.00201 Materials and Methods: 40 athletes were enrolled (age 43.8 ± 8.8 years; 36 males). Olfactory and cognitive tests were performed before the race (T0, n = 40), at 166 kms (T1, n = 28), at 418 kms (T2, n = 20), and after the race (T3, 866 kms, n = 13). The effect of dehydration and sleep deprivation on cognitive features were also studied.