β2-Adrenoceptor Activation Modulates Skin Wound Healing Processes to Reduce Scarring

During wound healing, excessive inflammation, angiogenesis, and differentiated human dermal fibroblast (HDF ) function contribute to scarring, whereas hyperpigmentation negatively affects scar quality. Over 100 million patients heal with a scar every year. To investigate the role of the beta 2 adrenergic receptor (β2AR) in wound scarring, the ability of beta 2 adrenergic receptor agonist (β2ARag) to alter HDF differentiation and function, wound inflammation, angiogenesis, and wound scarring was explored in HDFs, zebrafish, chick chorioallantoic membrane assay (CAM), and a porcine skin wound model, respectively. Here we identify a β2AR-mediated mechanism for scar reduction. β2ARag significantly reduced HDF differentiation, via multiple cAMP and/or fibroblast growth factor 2 or basic FGF (FGF2)-dependent mechanisms, in the presence of transforming growth factor betaβ1, reduced contractile function, and inhibited mRNA expression of a number of profibrotic markers. β2ARag also reduced inflammation and angiogenesis in zebrafish and CAMs in vivo, respectively. In Red Duroc pig full-thickness wounds, β2ARag reduced both scar area and hyperpigmentation by almost 50% and significantly improved scar quality. Indeed, mechanisms delineated in vitro and in other in vivo models were evident in the β2ARag-treated porcine scars in vivo. Both macrophage infiltration and angiogenesis were initially decreased, whereas DF function was impaired in the β2ARag-treated porcine wound bed. These data collectively reveal the potential of β2ARag to improve skin scarring.


INTRODUCTION
Wound repair is complex, requiring the coordinated action of numerous cell types and physiological processes to facilitate healing. In contrast to the perfect regeneration in embryonic wounds (Redd et al., 2004), adult wounds heal with fibrotic scars (Shaw and Martin, 2009). Evolution has primed repair processes to heal quickly, but imperfectly (Stramer et al., 2007). Excessive inflammation (Stramer et al., 2007), angiogenesis (DiPietro, 2013), and DF function (Hinz, 2007) all contribute to scarring. Annually, 100 million patients, in the developed world, heal with scars after elective procedures, trauma, and burn injuries, causing serious cosmetic/functional problems, which are emotionally and physically debilitating, placing hefty financial burdens on Healthcare Systems (Bayat et al., 2003).
No previous study has addressed the role of b2AR in wound scarring. The b2ARag Salbutamol is a safe and widely used asthma medication (Boskabady and Saadatinejad, 2003). Here, the effect of b2ARag on DF differentiation and function, inflammation (zebrafish wounds) (Renshaw et al., 2006), and angiogenesis (chick chorioallantoic membrane assay (CAM)) (Ausprunk et al., 1974) was investigated. Furthermore, as porcine skin is anatomically similar to human skin (Montagna and Yun, 1964), the effect of Salbutamol on wound scarring was explored in the Red Duroc pig, a widely used model for evaluating human scar reduction interventions (Gallant-Behm et al., 2008).
Transforming growth factor beta (TGFb)1, a fibroblast differentiation promoter strongly upregulated in wounds (Hinz, 2007), increased the ratio of SMA-positive HDFs by 14.5-fold (Figure 1a). Regardless of whether the b2ARag was added 6 hours prior or subsequent to TGFb1 for a further 42 hours, similar, marked inhibition was observed, decreasing the ratio of SMA-positive HDFs by up to 95% (Figure 1a).
A b2ARag-mediated decrease in TGFb1 secretion could underpin its reduction in HDF SMA expression. TGFb1 was detected in the HDF supernatant, but there was no b2ARagmediated effect (Supplementary Figure S2a online). FGF2 can reduce myofibroblast function (Ishiguro et al., 2009;Tiede et al., 2009). b2ARag increased FGF2 secretion by 4.3-and 0.59-fold after 6 and 24 hours, respectively ( Figure 1b). Indeed, exogenous FGF2 alone decreased the ratio of SMApositive HDFs by 79% (Figure 1c). HDFs express the FGF2 receptor fibroblast growth factor receptor1 (Takenaka et al., 2002). Treatment with the fibroblast growth factor receptor1 inhibitor PD173074 (PD) (Miyake et al., 2010) increased the ratio of SMA-expressing HDFs by 2-fold ( Figure 1c). Meanwhile, in the presence of PD, b2ARag no longer reduced SMApositive HDF numbers, both in the presence and absence of TGFb1 b2ARs can couple to Ga s , increasing intracellular cAMP (Scott et al., 1999). A cAMP analog (sp-cAMP) alone decreased SMA-positive HDFs by 40% (Figure 1d, Supplementary Figure S2c online), whereas a cAMP analog that inhibits protein kinase A (rp-cAMP) (Dostmann et al., 1990) had no significant effect alone, but completely prevented the b2ARag-mediated reduction in SMAexpressing HDFs (Figure 1d, Supplementary Figure S2c online), suggesting that the mechanism was cAMP and protein kinase A dependent.
To investigate downstream signaling through mitogen-activated protein kinases (MAPK), experiments were performed in (1 ng ml À 1 ), FGF2 (10 ng ml À 1 ), PD173074 FGFR inhibitor (PD173074,50 nM), sp-cAMP/rp-cAMP (50 mM), alone or pretreated, as indicated. (a) HDFs were treated for 48 hours with TGFb1 alone or 6 hours pretreatment with TGFb1 or b2ARag before b2ARag or TGFb1 addition, respectively, for 42 hours. Ratios of smooth muscle a actin (SMA)-positive cells to total cells were normalized to control. Scale bar ¼ 100 mm. (b) HDF FGF2 secretion was ELISA analyzed 6/24 hours post treatment in the presence or absence of b2ARag. (c) HDFs were treated for 48 hours with FGF2, PD, or b2ARag, with/without 6 hours of PD pretreatment. (d) HDFs were treated for 2 hours with sp-cAMP, rp-cAMP, or b2ARag, with/without rp-cAMP pretreatment, 30 minutes before b2ARag for 2 hours. Data presented are means ± SEM; 4 independent experiments. NS, not significant.
the presence of selective inhibitors of MEK1/2 (U0126), p38 MAP kinase (SB202190), or JNK (JNK inhibitor VIII, which inhibits JNK1/2/3). Inhibiting the MAP kinases individually had no effect and did not prevent the b2ARag-mediated reduction in the number of SMA-expressing HDFs (Supplementary Figure S2d online).
To determine whether b2ARag altered the number of M/ supermature focal adhesion (SMFAs), the length of FAs at the HDF periphery was analyzed. b2ARag reduced both peripheral HDF MFA and SMFA numbers by 23 and 58%, respectively ( Figure 2a). PD was used to probe FGF2 involvement, and it had no effect alone and it did not prevent the b2ARagmediated reduction in M/SMFA numbers (Figure 2a). However, exogenous FGF2 also reduced HDF peripheral MFA and SMFA numbers by 29 and 63%, respectively, whereas PD completely prevented any decrease ( Figure 2b).
Similar to the SMA studies above, the use of rp-cAMP prevented the b2ARag-mediated reduction in M/SMFA numbers, demonstrating that the underpinning mechanism was cAMP dependent (Figure 2c To determine whether b2ARag altered HDF profibrotic capability, RT-PCR was performed for profibrotic genes (Hinz, 2007). Salbutamol and Formoterol decreased SMA, fibronectin (FN) EDA, type I collagenA1, TGFb1, and connective tissue growth factor (CCN2) gene expression by 24 to 49% and increased FGF2 gene expression by 1.2-and 2.1-fold, respectively, after 6 hours ( Figure 2d). No effects on TGFb2, TGFb3, COL1A2, PDGF-A, MMP2, or decorin gene expression were observed (results not shown). In addition, whereas TGFb1 decreased HDF b2AR gene expression by 73%, FGF2 increased b2AR expression by 1.5-fold

b2ARag reduces zebrafish inflammation and embryonic chick angiogenesis
Excessive wound inflammation contributes to scarring (Stramer et al., 2007). A zebrafish tail wound model was used to visualize neutrophil guidance to wounds in real time (Renshaw et al., 2006). b2ARag reduced neutrophil recruitment by 60% after 6 hours (Supplementary Figure S3a online). Although angiogenesis is essential for wound repair, reduced angiogenesis is linked with improved healing (DiPietro, 2013) and less angiogenesis occurs in nonscarring oral wounds (Szpaderska et al., 2005) and scarless fetal wounds (Wilgus et al., 2008). b2ARag significantly reduced angiogenesis in the chick chorioallantoic membrane assay (CAM) by 29% (Supplementary Figure S3b online).
b2ARag altered the open wound area within the first 14 days of wound healing but did not affect the re-epithelialization of fullthickness Red Duroc pig wounds The effect of b2ARag on wound scarring was tested in a porcine skin wound model. Similar to humans, Red Durocs heal wounds with excessive collagen fiber formation (Gallant-Behm and Hart, 2006), generating raised hyperpigmented scars (Gallant et al., 2004). Twenty full-thickness wounds were created along each back ( Figure 4a); pigs were randomized and treated daily with vehicle alone or containing 5 mM Salbutamol sulfate. Two wounds/animal were biopsied weekly initially (7/14/21/28/42 days post wounding), and all wounds were digitally photographed. Remaining scars were photographed and collected 56 days post wounding.
Calibrated digital wound pictures were used to determine the open wound area. b2ARag-treated open wound area was 11% smaller and appeared less red and swollen than controls, at 7 days post wounding ( Figure 3a). After 14 days, b2ARagtreated open wound area was 23% larger, but there was no significant difference in the open wound area after 28 days, when wounds were almost completely healed (Figure 3a). Re-epithelialization rates could not be assessed 7 days post wounding, but they were similar in both groups after 14 or 28 days and almost all wounds were completely re-epithelialized by 28 days post wounding (Figure 3b). b2ARag reduces scar area and improves the appearance of Red Duroc scars Scars were visible from day 28 onward, and scar area was measured from calibrated digital pictures in a double-blind manner. One scar (1-10, control/b2ARag, Figure 4a), closest to the average scar area at that position, is presented alongside a mask delineating scar area (Figure 4b). b2ARag reduced scar area by 34, 38, and 47%, 28, 42, and 56 days post wounding, respectively ( Figure 4c). Tension worsens scarring (Ogawa et al., 2011) and varies along the porcine back, with the highest tension most caudally (positions 9/10). Indeed, wounds in positions 9/10 had scars almost twice the size of wounds in the most cranial positions, 1/2 (Figure 4b and d).
Immunohistochemistry reveals a reduction in profibrotic mechanisms underpinning the b2ARag-mediated scar reduction Wound inflammation was investigated using both the macrophage a-naphtyl acetate esterase (a-NAE) stain and an anti-macrophage antibody, CD163, specific for the M2 alternatively activated/healer macrophages (Weisser et al., 2013). a-NAE staining revealed an 18% decrease in biopsy macrophage-infiltrated area, after 7 days, in b2ARag-treated wounds ( Figure 5a). In contrast, after 14 days, b2ARag treatment increased the macrophage-infiltrated area by 28% (Figure 5a). From 21 to 56 days post wounding, there was no difference in macrophage-infiltrated area between groups (Figure 5a). At 14 days post wounding, CD163-specific staining appeared similar to the a-NAE staining (Figure 5b).
Discrete blood vessels were detected by anti-von Willebrand factor (vWF) immunohistochemistry and counted within defined areas of biopsies/excised scars. After 7 days, the number of discrete vWF-stained structures decreased by 22% in b2ARag-treated biopsies (Figure 5c). Similar to the pattern observed with macrophage infiltration, b2ARag increased discrete vWF-stained structure density by 2-fold, after 14 days (Figure 5c). In contrast, after 21/28/42 days, discrete vWF-stained structure densities were lower in b2ARag-treated biopsies (Figure 5c). By 56 days post wounding, angiogenesis was markedly reduced in all wounds, and there was no difference in vWF-stained structure density between groups at the end of study (Figure 5d). Biopsies were stained with the proliferation marker Ki67, but no differences were observed between groups (results not shown).
Collagen deposition and fiber formation were analyzed by histology on cranial-caudal cross-sections of whole excised scars to observe the entire wound bed, 56 days post wounding. Masson's trichrome stain was used to detect the presence (green) and absence (red) of collagen fibrillar structures within the wound bed. In b2ARag-treated wounds, the area without collagen fibers was 1.5-fold larger than in control-treated wounds (Figure 5e), but there was no difference in COL1 staining between groups (Supplementary Figure S4a online).
Biopsies, from day 14 onward, were positively stained for SMA and FN EDA, markers of DF profibrotic phenotype (Hinz, 2007). The wound bed area positively stained for SMA and FN EDA was almost halved in b2ARag-treated scars (Figure 5f and g). However, there was no difference in COL3 (not shown) or CCN2 (downstream of TGFb1) staining between groups (Supplementary Figure S4b online). To investigate underpinning mechanisms for reducing fibroblast differentiation, FGF2 staining was performed on excised scars. FGF2 staining intensity was 2-fold higher in the b2ARag-treated wound bed (Figure 5h).

DISCUSSION
b2ARag restrained mechanisms that contribute to scarring, including HDF differentiation and function in vitro, and inflammation (zebrafish) and angiogenesis (chick chorioallantoic membrane assay (CAM)) in vivo. Moreover, in Red Duroc pigs, Salbutamol significantly reduced wound scarring by 47%, 56 days post wounding. Histology confirmed that b2ARag initially reduced inflammation and angiogenesis, whereas DF function was restrained in the wound dermis. Indeed, at the end of study, scar area and hyperpigmentation were reduced by almost half, and scar appearance was significantly improved in b2ARag-treated wounds compared with controls.
In vitro, b2ARag reduced HDF profibrotic nature by impairing their ability to sense tension within the wound (reduced peripheral MFA and SMFA numbers) and reduced profibrotic gene expression (SMA, FN EDA, TGFb1, CCN2, type I collagenA1), leading to a reduction in differentiation via cAMP and/or FGF2 dependent and MAPK-independent mechanisms, resulting in reduced contractile function (Figures 1 and 2, Supplementary Figures S1 and S2 online). Indeed, b2ARag increased HDF FGF2 mRNA expression and secretion in vitro (Figures 1b and 2d) and increased FGF2 staining intensity in excised scars (Figure 5h).
Recently, cAMP was highlighted as a regulator in tissue fibrosis (Insel et al., 2012) and FGF2, highly expressed in scarless fetal skin (Chen et al., 2006), reduced scarring in human acute incisional wounds (Ono et al., 2007). The b2ARmediated increases in cAMP and FGF2 were likely highly beneficial in steering wound healing toward regeneration and away from scarring in the porcine full-thickness wounds.
Here, b2ARag reduced inflammatory cell guidance to zebrafish wounds in vivo within hours post wounding (Supplementary Figure S3a online) and reduced the macrophage-infiltrated area in 7-day porcine wound biopsies, the earliest time point that biopsies could be taken from the porcine wound edge (Figure 5a). However, the macrophageinfiltrated area was actually 28% larger in b2ARag-treated wounds, 14 days post wounding, compared with controls, whereas there was no difference from day 21 onward (Figure 5a). The main macrophage phenotypes, characterized in murine models, are classically activated killer macrophages (M1), or alternatively activated healer macrophages (M2) (Weisser et al., 2013), although subsets of these phenotypes exist in wounds (Sindrilaru and Scharffetter-Kochanek, 2013). Murine M2 can be identified by CD163 expression, also identified on pig macrophage populations (Fairbairn et al., 2013). In the present study, the pattern of macrophage staining with a-NAE and CD163 was identical 14 days post wounding, suggesting that all biopsy macrophages expressed CD163 (Figure 5b). Therefore, either M2 alone is recruited to porcine wounds, perhaps contributing to the b2ARag-mediated scar reduction, or CD163 is not a phenotype-specific pig M2 marker. The effect of b2ARag on angiogenesis and macrophage infiltration appeared highly correlated (Figure 5a and b). M2 macrophages promote wound angiogenesis (Lucas et al., 2010), and macrophages are necessary to stimulate and fuse tip cells at the leading edge of angiogenic sprouts (Fantin et al., 2010). It is possible that the b2ARag-mediated increase in CD163-expressing macrophages, 14 days post wounding, facilitated the large increase in angiogenesis observed in the b2ARag-treated wounds at this time point.
b2ARag-treated and control wounds re-epithelialized at the same rate from day 14 onward (Figure 4b). Previously, an early bARag-mediated delay in murine skin wound re-epithelialization was observed (Pullar et al., 2006a;Sivamani et al., 2009). Perhaps we missed an early delay in this study. Rodent skin is untethered, however, and wounds heal primarily by wound contraction (Hayward and Robson, 1991); therefore, a bAR-mediated reduction in wound contraction (Pullar and Isseroff, 2005) (Figure 3a) could have underpinned this delay in murine models. In contrast, porcine and human skin wounds close primarily by wound re-epithelialization (Sullivan et al., 2001).
Histology confirmed that b2ARag had restrained DF differentiation in excised scars; the area of the dermis stained for SMA/FN EDA was significantly reduced, whereas FGF2 staining intensity was increased 2-fold (Figure 5f-h). In addition, after 14 days, b2ARag-treated wounds were larger, suggesting a reduction in wound contraction. Mechanisms delineated in vitro, underpinning the b2ARag-mediated reduction in DF differentiation/function, appeared operational in vivo  ( Figure 5f-h). Moreover, Masson's trichrome staining revealed a 1.5-fold increase in wound bed area without collagen fibers, but no significant difference in the collagen I staining, suggesting that b2ARag had not altered collagen I deposition in the neodermis but had delayed the incorporation of collagen I into fibers (Figure 5e, Supplementary Figure S4a online). Keloids and hypertrophic scars contain a large increase in collagen fibers and bundles (Tuan and Nichter, 1998); therefore, the b2ARag-mediated reduction in collagen fiber formation perhaps contributed to the reduced scar area and improved scar appearance.
b2ARag also reduced scar hyperpigmentation and improved sheen, height, texture, and pliability ( Figure 4). Indeed, knockdown of Adrb2a, a b2AR ortholog expressed in zebrafish brain and epidermis, induced significant hypopigmentation, revealing a functional b2AR role in pigmentation (Wang et al., 2009). Inflammation is essential for zebrafish wound hyperpigmentation (Levesque et al., 2013). Here, b2ARag reduced inflammation (Supplementary Figures S3a, Figure 5a and b online), which could have contributed to the reduced hyperpigmentation; future work will address this.
In conclusion, our work highlights the b2AR as a regulator of wound healing/scarring. There are currently no clinically tested or licensed interventions/pharmaceuticals available to reduce wound scarring/fibrosis or to improve scar hyperpigmentation. Topical Salbutamol significantly improved acute skin scarring in vivo and could have significant potential as a treatment. Future work will address the potential to improve hypertrophic scarring, keloid formation, and organ fibrosis.

Ethics statement
Scar study was performed under UK Home Office License (40/3535). Local ethics approval was obtained from The University of Nottingham ACUC.

Animals
Adult zebrafish were maintained in compliance with the Animals (Scientific Procedures) Act, 1986. Embryos were collected and raised in 28.5 1C egg water until the required developmental stage (Westerfield, 1994;Kimmel et al., 1995).
Ten female, 3-month-old Red Duroc pigs (30-35 kg) were purchased from a certified breeder. Animals were acclimatized the week before study commencement and were maintained on a standard diet.

ELISA
HDFs were plated on collagen I-coated (30 mgml À 1 , Invitrogen) 6well plates and incubated for 24 hours in fibroblast complete medium to 80% confluency, and then washed and serum-starved for 24 hours before incubation with SFM alone or SFM containing 10 mM Salbutamol for 6 or 24 hours. Supernatant growth factor levels were determined using human Duoset ELISA kits (R&D Systems).

Immunocytochemistry
SMA Immunocytochemistry: 8,500 HDFcm À 2 were plated on sterile coverslips for 24 hours in fibroblast complete medium, then serumstarved, overnight. Pretreatment was applied for 6 hours with either SFM alone or SFM containing Formoterol/Salbutamol (10 mM), TGFb1 (1 ng/ml), FGF2 (10 ng/ml), or PD173074 (50 nM), before adding TGFb1 (1 ng/ml) or Salbutamol (10 mM) for a further 42 hours, as per combinations stated in the figures. sp-cAMP or rp-cAMP (50 mM) were applied for 30 minutes before treatment with SFM alone or SFM containing 10 mM Salbutamol for 2 hours. Cells were fixed in ice-cold methanol for 10 minutes and blocked for 1 hour with 2% donkey serum/5% BSA.
Coverslips were mounted using ProLong Gold containing DAPI (Invitrogen). Image acquisition was performed on a Nikon Eclipse TE2000-E microscope using the Volocity software (Improvision, Perkin Elmer).
Analyses were performed on 20 separate random fields from duplicate samples, per condition. The mean ratio of SMA-positive HDFs/total HDFs was determined using Volocity, counting total DAPI-stained nuclei and SMA-positive HDF numbers, within defined fluorescence thresholds in blue and green channels, respectively. On 20 HDF per experiment (n ¼ 4), FAs were counted and measured manually on calibrated pictures using Volocity, then classified according to their length: immature (o4 mm), mature (4-8 mm) and supermature FAs (48 mm) (Dugina et al., 2001). Only peripheral FAs were measured.
Animals were placed in ventral recumbency, and their backs were shaved and washed with skin prep. Twenty 2 Â 2 cm 2 full-thickness wounds were created in 5 rows and 4 columns down the back, with a minimum of 2.5 cm between each wound (Figure 4a) (Gallant-Behm et al., 2008). Using a scalpel and metal guide, the skin, including subcutaneous fat, was removed exposing the underlying fascia. Wound corners and the edge centers were marked with ink tattoos and animals were randomized into two groups. A volume of 500 ml of Granugel (Convatec, Uxbridge, UK) diluted 2:1 in sterile water, alone (controls) or containing 5 mM Salbutamol sulfate (1.2 mgml À 1 Salbutamol), was freshly prepared and added to each wound after surgery and daily thereafter.
Wounds were covered with a new dressing daily (Steroplast, Manchester, UK) and custom jackets (Agenda Group, Hull, UK) for study duration.
On days 7,14,21,28,42 post wounding, animals were provided with analgesia/anesthesia, skin was shaved, each wound was digitally photographed, and four 6-mm punch biopsies (Miltex, Weymouth, UK) were taken from 2 wounds/animal, as shown in (Figure 4a). On day 56 post wounding, animals were provided with analgesia/ anesthesia, skin was shaved, and each wound was digitally photographed and assessed for scar appearance. Animals were then euthanized using a schedule 1 method, and the remaining ten scars/animal were harvested for histology.

Wound healing and scar formation assessment
Wound healing and scar area were analyzed from calibrated wound photographs in a double-blind manner, using Adobe Photoshop. Wound area was measured from day 7 to day 28, using the wound edges marked by tattoos. Re-epithelialized area was calculated at days 14/28 by measuring the non-re-epithelialized area of the wound, dividing it by the wound area at day 0, and multiplying it by 100, to obtain the percentage non-re-epithelialized area, which was then subtracted from 100 to give % re-epithelialization.
Visible scar area was measured from day 28 to day 56. Hyperpigmented, red, raised, or damaged skin at the wound site was included in the scar area. Scar assessment was performed at day 56 before animal euthanasia using a composite of the Manchester (Beausang et al., 1998) and Vancouver (Sullivan et al., 1990) scar scales with modifications for porcine wounds (Supplementary Table S1 online). The results are presented as mean values/scores þ / À SEM (n ¼ 10 for biopsies; n ¼ 50 for excised scars).
Brightfield pictures of scar sections were acquired on a Zeiss Axiovert 200 microscope with the Axiovision 4.8 software using multiacquisition stitching. Calibrated images were analyzed with Adobe Photoshop (Maidenhead, UK). Neo-collagen fiber deposition was determined by measuring collagen fiber negative areas (red), within the dermis (defined from H&E sections), in the Masson's trichrome staining. Neo-angiogenesis was determined by the number of discrete vWF-stained structures counted within a 0.25 mm 2 (biopsies) or 1 mm 2 (excised scars) area within the most densely stained area (biopsies) or wound bed directly below the epidermis (excised scars). Inflammation was assessed by the % macrophageinfiltrated area of the biopsy (NAE/anti-CD163 staining) or wound bed (excised scars). The wound bed on excised scar sections was defined from H&E-stained sections and demarcated on Figure 5d-h and Supplementary Figure S4a and b online with a black line.

Statistical analysis
Statistical significance was analyzed using the GraphPad Prism6 software (La Jolla, CA). Data distribution was first assessed using the D'Agostino & Pearson normality test. Continuous variables were analyzed with the two-tailed unpaired Student's t-test (Gaussian distributions), with a Welch's correction for unequal variances when necessary, or with the nonparametric Mann-Whitney test (non-Gaussian distributions). One or two categorical variables were analyzed by one-way or two-way ANOVA (Gaussian distributions), with the Bonferroni's post-test, or by the Kruskal-Wallis test (non-Gaussian distributions) with a Dunn's post-test. Values are presented as mean þ / À SEM, with statistical significance ascribed as *Po0.05/ **Po0.01/***Po0.001.