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Cardiovascular Research 2004 61(1):22-29; doi:10.1016/j.cardiores.2003.10.010
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Copyright © 2004, European Society of Cardiology

Gender differences in superoxide generation in microvessels of hypertensive rats: role of NAD(P)H-oxidase

Ana Paula V Dantas, Maria do Carmo P Franco, Michele M Silva-Antonialli, Rita C.A Tostes, Zuleica B Fortes, Dorothy Nigro and Maria Helena C Carvalho*

Laboratory of Hypertension, Department of Pharmacology, Institute of Biomedical Science, University of Sao Paulo, 05508-900, Av Prof Lineu Prestes, 1524, Room 217, Sao Paulo, SP, Brazil

* Corresponding author. Tel.: +55-11-3091-7433; fax: +55-11-3091-7237. mhcarval{at}icb.usp.br

Received 1 August 2003; revised 17 September 2003; accepted 8 October 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: This study is aimed to explore whether gender plays a role in the generation of nitric oxide (NO) and superoxide anion (O2–) in microvessels of hypertensive rats (SHR), as well as the potential mechanisms involved in these effects. Methods and results: NO generation in mesenteric arterioles was evaluated by measuring NO synthase (NOS) activity and protein expression. Oxidative stress was studied in vivo in mesenteric arterioles from male and female SHR by hydroethidine microfluorography. Although we did not observe any sex-related differences in NO generation, we found that hydroethitine oxidation is markedly increased (30.9±2.4%) in male compared to female (12.3±2.5%; p<0.05), demonstrating a gender difference in O2– production. The treatment of mesenteries with DPI (NAD(P)H-oxidase inhibitor) and treatment of SHR with losartan [Angiotensin-II type 1 (AT-1) receptor antagonist] markedly reduced O2– production in male, while produced a minor effect in female, suggesting that overexpression/activity of AT-1 receptor and NAD(P)H-oxidase contribute for the sexual dimorphism in superoxide generation. Immunoblot analyses provide evidences of overexpression of the NAD(P)H-oxidase components p22phox, gp91phox, p47phox and p67phox in arterioles from male in comparison to female. Losartan treatment inhibited the overexpression of these subunits in male, without affecting the responses in female. Conclusion: Taken together, our findings demonstrate that male SHR presents higher superoxide anion concentration under basal condition than does female. An AT-1-dependent overexpression of the NAD(P)H-oxidase components may account for the sexual dimorphism in oxidative stress, and may play an important role in the noted gender differences on incidence of cardiovascular disease.

KEYWORDS Oxygen radical; Nitric oxide; NAD(P)H-oxidase; Hypertension; Endothelial function; Angiotensin-II; Gender


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Compared to men, young adult women are relatively protected against the development of hypertension and its deleterious consequences in the cardiovascular system [1,2]. Studies using ambulatory blood pressure monitoring have shown that the sexual dimorphism in the incidence of elevated blood pressure becomes apparent prematurely during adolescence and persists throughout adulthood [3]. Moreover, the gender-associated differences in the development of hypertension have also been observed in various animal models, such as spontaneously hypertensive rats (SHR) [4], deoxycorticosterone-salt hypertensive rats [5], Dahl salt-sensitive rats [6] and New Zealand genetically hypertensive rats [7]. In these animals, males develop an earlier and more severe hypertension than do females.

Although the mechanisms underlying the sexual dimorphism in the control of blood pressure are uncertain, gender differences in nitric oxide (NO) release, related to a differential modulation of the endothelial NO synthase (eNOS) expression and/or activity, have been proposed. In fact, consensus from in vitro studies has demonstrated that NO-mediated endothelium-dependent relaxation is more pronounced in the vasculature from female in comparison to male [8–10]. Furthermore, experimental studies have shown that normotensive males have an inferior expression/activity of eNOS than do females [10,11]. Interestingly, contrary to data in normotensive animals, studies in hypertensive animals have documented that eNOS levels in male and female are similar or even elevated in male [12]. Therefore, it can be speculated that hypertensive females may have increased NO availability, not only by enhancing NO release, but above all by decreasing its degradation after synthesis.

It is currently known that the biological activity of NO may be modified by reactive oxygen species (ROS), such as superoxide anion (O2–). An increased O2– concentration in the vasculature results in rapid scavenging of NO, leading to increase of vascular tone and consequent elevation of blood pressure [13,14]. There are great evidences in recent years showing that rising in O2– levels plays an important role in the pathophysiology of hypertension [14,15]. In addition, few data from studies in normotensive animals have shown a sex-related difference in oxidative stress in the cardiovascular system [16,17]. However, whether or not the higher incidence and severity of hypertension observed in male may be due to excessive production of O2– remains poorly understood. The present study addresses this issue by assessing the in vivo O2– generation in mesenteric microvessels from male and female spontaneously hypertensive rats (SHR), as well as by exploring the potential mechanisms involved in the gender differences in O2– generation.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
The experiments were performed with age-matched (16–18 week old) male and female SHR obtained from the breeding stock at our own institute, housed according to institutional guidelines (constant room temperature, 12-h light/dark cycle, 60% humidity, standard rat chow and water ad libitum). Arterial blood pressure (BP) was measured in unanesthetized animals by an indirect tail-cuff method (pneumatic transducer, PowerLab 4/S, AD Instruments). All procedures used in this study were approved and performed in accordance with guidelines of the Ethics Committee of the Institute of Biomedical Science, University of Sao Paulo, conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.1 Intravital fluorescence microscopy
The in vivo generation of superoxide anion in mesenteric arteriolar wall was determined by hydroethidine microfluorography, essentially as described recently [18]. Briefly, the mesentery was exposed for microscopic observation in situ and continuously superfused (1.0 ml/min) with a Krebs–Henseleit bicarbonate-buffered solution, saturated with a 95% N2/5% CO2 gas mixture to minimize the production of oxygen free radicals. Special precautions were taken to avoid interruption of the suffusion solution on the tissue because even superficial drying causes rapid cell injury. Arterioles were classified according to their branching order beginning at the capillary level and reaching up to the arteriolar side. Single unbranched A2 arterioles (15–25 µm) were selected for this study. After an initial 30-min stabilization period, the preparation was then superfused for 60 min with a buffer solution containing hydroethidine (HE; 10.0 µmol/l, Polysciences). In the presence of oxidative stress, hydroethidine is transformed intracellularly into the fluorescent compound ethidium bromide (EB), which binds to DNA and can be detected by virtue of its red fluorescence. The number of nuclei labeled with ethidium bromide (EB-positive nuclei) along arterioles (NEB) was determined every 15 min after the onset of HE superfusion. At the end of the experiments, the tissue was superfused with absolute ethanol for 5 min followed by a superfusion with a 10% EB solution (v/v) to establish the total number of nuclei along the vessel wall (NT). The EB-positive number was counted (double-blind) and expressed as a percentage of EB-positive nuclei=(NEB/NT) x 100 (%).

In order to evaluate the source of superoxide generation, the mesenteries from male and female SHR were treated with diphenyleneiodonium (DPI 20 µmol/l, a NAD(P)H-oxidase inhibitor) or with oxypurinol (1 mmol/l, a xanthine-oxidase inhibitor). In all treatments, each drug was superfused separately during the 30-min stabilization period and maintained throughout the experiment. In another series of experiments, the animals were orally treated with the following drugs: diclofenac (a cyclooxygenase inhibitor, 1 mg/kg/15 days) or losartan [Angiotensin-II type 1 (AT-1) receptor antagonist, 15 mg/kg/15 days]. Because losartan, at this dose, causes a decrease in BP that may contribute to a decrease in superoxide generation [19], SHR were treated with verapamil (10 mg/kg/15 days), which does not interfere with pro-oxidative proteins but has an antihypertensive action.

2.2 Nitric oxide synthase (NOS) activity measurement
Total NOS activity was measured in supernatants from mesenteric arterioles as previously described [20]. Briefly, the NOS assay was performed by incubation (37 °C for 60 min) of 100 µg of protein extracted from mesenteric arterioles in a assay mixture containing Tris–HCl 50 mmol/l, tetrahydrobiopterin 6 µmol/l, FAD 2 µmol/l, FMN 2 µmol/l+NAD(P)H 10 mmol/l, L-arginine/L-[H3]arginine 100 mmol/l (5 µCi/ml), CaCl2 6 mmol/l, and calmodulin 0.1 µmol/l. Cation-exchange resin (200 µl) (Dowex, Na+ form, equilibrated with HEPES 50 mmol/l; pH 5.5) was added to each reaction mixture to remove the excess of L-[H3]arginine. The aliquots were placed in spin cups and centrifuged for 1 min at 12,000 x g. The supernatants were collected in vials with scintillation liquid and the radioactivity was quantified.

2.3 SDS-PAGE and immunoblotting
Arterioles from mesenteric bed were rapidly dissected in a dissecting dish containing iced-cold phosphate-buffered saline (PBS) plus a mixture of protease inhibitors (as described below), and then homogenized in lysis buffer (50 mM Tris, pH 7.4; 1 mM EDTA; 1% v/v NP-40; 150 mM NaCl; 0.25% sodium deoxycolate; a mixture of protease inhibitors—2 µg/ml leupeptin, antiapin, soybean trypsin inhibitor, lima trypsin inhibitor). After 30 min of incubation on ice, samples were centrifuged for 20 min at 14,000 x g at 4 °C to yield a solubilized preparation. Protein concentration was measured using the Bio-Rad protein assay (Bio-Rad, Richmond, CA). Equal quantities of protein from each sample were resolved by SDS-PAGE on 9% gels and electroblotted onto nitrocellulose. Protein expression was assayed by high-sensitivity immunoblots [21] probed with specific primary antibody as follow: 1:10,000 monoclonal anti eNOS (Transduction Laboratories); 1:500 polyclonal anti p22-phox (Santa Cruz Biotechnology); 1:500 polyclonal anti gp91-phox (Upstate Biotechnology); 1:500 polyclonal anti p47-phox (Upstate Biotechnology) and 1:500 polyclonal anti p67-phox (Upstate Biotechnology). Densitometric analyses of Western blots were performed using a ChemiImager 4000 (Alpha-Innotech). Loading and transfer of equal amounts of protein in each lane were verified by reprobing the membrane with a polyclonal anti-β-actin antibody (1:1000 dilution, Santa Cruz Biotechnology), followed by densitometry.

2.4 Statistical analysis
The results are shown as mean±S.E.M. Statistical analysis was performed using the nonparametric test Kruskal–Wallis for multiple comparisons and unpaired Student's t-test for comparison of a single observation between male and female groups. Values were considered statistically significant when p<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
In this study, we observed that the mean arterial pressure in male SHR was higher (178±1.9 mm Hg; n = 8; p<0.05) than in female SHR (155±2.3 mm Hg; n = 10). Treatment with losartan and verapamil significantly decreased BP in both male (losartan: 143±5.6; n = 6; verapamil: 137±1.4; n = 6) and female (losartan: 126±2.6; n = 6; verapamil: 123±1.4; n = 6). Diclofenac treatment had no effects on BP in male (182±2.5; n = 6) or female SHR (152±3.3; n = 6).

To first explore a possible sexual dimorphism on nitric oxide (NO) production, the abundance and activity of endothelial NO synthase (eNOS) were studied. As shown in Fig. 1, we found no sex differences in both protein expression (Fig. 1A and B) and activity (Fig. 1C) of eNOS, suggesting that the release of the endothelial derived NO is not altered by gender. Besides NO, reactive oxygen species (ROS) are known to be involved in the onset and development of cardiovascular disease. Therefore, the effect of gender difference on ROS generation was evaluated in vivo in mesenteric arterioles from SHR. As illustrated in Fig. 2A, hydroethidine fluorescence was markedly enhanced in arterioles of male SHR in comparison to female. Fig. 2B represents the time course for the relative number of EB-positive nuclei along the mesenteric arteriolar wall. Compared to female, the number of EB-positive nuclei in arterioles from male was significantly increased at 45 and 60 min after the onset of hydroethidine superfusion, suggesting that superoxide anion generation is markedly enhanced in male SHR.


Figure 1
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  		Fig. 1 eNOS activation and expression in male and female SHR. The upper panel (A) shows the results of immunoblots analyzed in mesenteric arterioles isolated from male and female SHR and probed with antibodies against eNOS or β-actin. The middle graph (B) shows the results of densitometric analyses from pooled data (n = 4). Values were normalized by the corresponding optical density for β-actin, used as internal control. The bottom panel (C) shows the results of an eNOS activity assay performed in mesenteric arterioles isolated from male (n = 6) and female (n = 6) SHR. Each data point represents the mean±S.E.M. Groups were compared with the use of unpaired Student's t-test.

 

Figure 2
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  		Fig. 2 Sexual dimorphism in oxidative stress. The upper panel (A) shows the representative images of hydroethidine microfluorography obtained in male (left) or female (right). Each panel shows both transillumination images and ethidium bromide fluorographs 60 min after onset of hydroethidine superfusion. The lower graph (B) shows time course of the EB-positive nuclei along mesenteric arterioles of male (n = 6) and female (n = 6) SHR. Each point represents the mean±S.E.M. Groups were compared with the use of unpaired Student's t-test. *p<0.05 compared to male.

 
The overproduction of superoxide anion observed in male was significantly attenuated by treatment with the NAD(P)H oxidase inhibitor DPI, but not with oxypurinol, a xanthine-oxidase inhibitor (Fig. 3A). On the other hand, DPI had a smaller but statistically significant effect on hydroethidine oxidation in microvessels from female (Fig. 3A). Comparable pattern of reduction in superoxide generation was seen in mesenteric arterioles from both male and female SHR chronically treated with losartan (Fig. 3B). Taken together, these findings may suggest that NAD(P)H-oxidase and AT-1-dependent pathway mediates the overproduction of superoxide anion generation in male SHR. The lack of any effect after diclofenac and verapamil treatment suggests that neither cyclooxygenase nor changes in blood pressure are associated with superoxide generation in this study (Fig. 3B).


Figure 3
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  		Fig. 3 Characterization of the source of superoxide generation in the mesenteric arterioles. This figure shows bar graphs of the percentage of EB-positive nuclei 60 min after onset of hydroethidine superfusion obtained in mesenteric arterioles of male and female SHR. Panel A shows data obtained from experiments performed in the absence (control) or presence of DPI (20 µmol/l, n = 4–6) or oxypurinol (1 mmol/l, n = 5–6). Each drug was superfused separately during the 30-min stabilization period and maintained throughout the experiment. In another series of experiments (data shown in panel B), the animals were chronically treated with losartan (15 mg/kg/15 days, n = 6); diclofenac (1 mg/kg/15 days, n = 6) or verapamil (10 mg/kg/15 days, n = 6). Each point represents the mean±S.E.M. Groups were compared with the use of nonparametric test Kruskal–Wallis for multiple comparisons. *p<0.05; ***p<0.001 compared to respective control.

 
Overexpression of NAD(P)H-oxidase components has been associated with enhanced superoxide generation in certain pathophysiological conditions [22–25]. However, sexual dimorphism on NAD(P)H-oxidase subunit expression has not been previously explored in the vasculature. To address this issue, the protein expression of NAD(P)H-oxidase subunits were studied by using a high-sensitivity Western blot analyses in extracts of isolated mesenteric arterioles. As shown in Fig. 4, arteriolar protein expression of the p22phox and gp91phox subunits of NAD(P)H-oxidase is markedly greater in male microvessels in comparison to female ones. The levels of the subunits p47phox and p67phox are also increased in mesenteric arterioles from male, although the fold increase of p47phox and p67phox expression is lower than that observed for the p22phox and gp91phox subunits. In order to correlate AT-1-mediated superoxide generation with overexpression of the NAD(P)H-oxidase components, we performed immunoblot analyses in arterioles from SHR (both sex) treated with losartan. As can be seen in Fig. 5, the overexpression of the NAD(P)H oxidase subunits observed in male was prevented by treatment with losartan, demonstrating that AT-1 receptor mediates expression of those components of NAD(P)H oxidase in male SHR.


Figure 4
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  		Fig. 4 Gender differences in the protein expression of NAD(P)H-oxidase subunits. The upper panel (A) shows the results of immunoblots analyzed in mesenteric arterioles from male and female SHR and probed with antibodies against p22phox, gp91phox, p47phox and p67phox, as indicated. The bar graphs (B) show the results of densitometric analyses from pooled data (n = 4). Values were normalized by the corresponding optical density for β-actin, used as the internal control. Each point represents the mean±S.E.M. Groups were compared with the use of nonparametric test Kruskal–Wallis for multiple comparisons. *p<0.05; **p<0.01 compared to male.

 

Figure 5
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  		Fig. 5 Effects of losartan treatment in the protein expression of NAD(P)H-oxidase subunits. The upper panel (A) shows the results of immunoblots analyzed in mesenteric arterioles from male and female SHR and probed with antibodies against p22phox, gp91phox, p47phox and p67phox, as indicated. Experiments were performed in arterioles isolated from untreated rats (control) or SHR chronically treated with losartan (15 mg/kg/15 days, n = 4). The bar graphs (B) show the results of densitometric analyses from pooled data (n = 4). Values were normalized by the corresponding optical density for β-actin, used as the internal control. Each point represents the mean±S.E.M. Groups were compared with the use of nonparametric test Kruskal–Wallis for multiple comparisons. *p<0.05; **p<0.01 compared to male {dagger}p<0.05;{dagger}{dagger}p<0.01 compared to respective control.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Gender has an important influence on blood pressure, which has been partially explained by a differential regulation of nitric oxide (NO) bioavailability. A number of data from in vitro studies have demonstrated reduced basal NO availability in males compared with females from different strains. By examining the responses of the NO synthase (NOS) inhibitor L-NAME on endothelium-dependent relaxation, some studies have described a more pronounced inhibitory effect in arteries from female than those from male [8,9]. Moreover, recent evidences have shown a greater expression/activity of NOS in normotensive female rats in comparison to males [10,11], suggesting that NO release from the endothelium is elevated in arteries of females. However, in the present study we have found that microvessels from male SHR show no apparent difference in NOS expression and activity compared to female. Although these data are contrary to reports in normotensive animals, they are in a good agreement with a report that showed no sex-associated effect on NOS activity in spontaneously hypertensive rats (SHR) [12]. Therefore, male SHR may have decreased NO availability not because of diminished NO generation, but primarily as a result of an increase in its degradation by superoxide anion (O2–). In this study, we investigated the hypothesis that male SHR may have an excessive O2– generation and explored the source of this O2– as well as the molecular mechanisms involved, such as the expression of the NAD(P)H-oxidase subunits (the main source of O2– in the vasculature).

By utilizing a methodology for in vivo measurement of O2– in the microcirculation, we have been able for the first time to detect spontaneous gender-associated oxidative changes in the resistance vasculature. In agreement with previous data obtained in aorta from normotensive animals, our study shows that hypertensive males display greater O2– generation than do females [16], an effect that may be consistent with the higher blood pressure levels described in males.

In our animals, one of the major sources of O2– is NAD(P)H-oxidase as illustrated by the attenuation of O2– generation in the presence of DPI. Besides, the finding that DPI treatment substantially attenuated the oxidation of hydroethidine in male microvessels (66%) and caused a minor decrease in vessels from females (32%), suggests a gender difference in NAD(P)H-oxidase activation in SHR. Several observations have established that NAD(P)H-oxidase accounts for the majority of superoxide generation in the vessel wall in pathological conditions, such as hypertension [26]. However, to our knowledge, this is the first report that describes sex-associated differences in the modulation of NAD(P)H-oxidase system.

Although not well known, the vascular isoform of NAD(P)H-oxidase has been described as a multi-subunit enzyme complex composed by two membrane-spanning polypeptide subunits p22phox and gp91phox (or its homologues Nox1 and Nox4), and at least three cytosolic subunits p47phox, p67phox, and the small G-protein Rac2 (or Rac1) (for review see Ref. [27]). Exposure of the cell to a variety of pathophysiological stimuli induces the association of the cytosolic with the membrane-associated components, causing activation of the basically dormant oxidase to generate superoxide [27]. Recent growing evidences have established a role for augmented expression of NAD(P)H-oxidase subunits in the increased superoxide generation in cardiovascular disease. For instance, expression of p22phox has been reported to be increased in aorta from hypertensive rats [22], to increase with age in both normotensive and hypertensive rats [23], and to be increased in atherosclerotic coronary arteries from humans [24]. In addition, a recent study has shown an overexpression of the p22phox and gp91phox components in infarcted hearts [25]. In view of these observations, we hypothesized that microvessels from male SHR may display an increased expression of the NAD(P)H-oxidase subunit(s), which may account for the overproduction of O2– observed. As shown in Fig. 4, this study provides the first evidence of overexpression of the NAD(P)H-oxidase components p22phox, gp91phox, p47phox and p67phox in arterioles from hypertensive male in comparison to female.

Growing evidences from recent studies support a role of angiotensin-II (Ang-II) in NAD(P)H-oxidase activation in the vasculature. It has been found that Ang-II increases O2– production in different arteries, via an angiotensin-II type 1 (AT-1) receptor-dependent and NAD(P)H-oxidase-mediated signaling pathway [28–31]. In this study, we found that the magnitude of inhibitory responses to DPI and the AT-1 antagonist were similar in both male and female SHR. Comparable to DPI, losartan produced about 65% of decrease in superoxide generation in male and a slightly attenuation in females (31%). Considering the similarities in the pattern of response to DPI and losartan, we are tempted to speculate that males have an increased NAD(P)H-oxidase activity/expression by a AT-1 receptor-dependent system. In fact, acute Ang-II treatment has been described to elicit activation of NAD(P)H-oxidase, by triggering different downstream signaling molecules, such as phospholipase A2, protein kinase C, extracellular signal-regulated kinase 1/2 (ERK 1/2), phosphoinositide 3-kinase (PI3-K) and c-Src [28–30]. Touyz et al. [31] have recently described that Ang-II regulates the enzyme by inducing phosphorylation of p47phox and translocation of cytosolic subunits to the membrane. Another possible mechanism whereby Ang-II regulates NAD(P)H-oxidase could be via its effects on the abundance of the oxidase components [31,32]. Recent studies have found that the expression of certain components of the NAD(P)H oxidase seems to be upregulated after a long-term (hours) Ang-II infusion [31,32].

We have recently found in SHR animals a significant gender effect on vascular AT-1 receptor gene expression, being greater in males than females (Silva-Antonialli, unpublished data, 2003). Therefore, in an effort to further address the precise role of AT-1 receptor to the gender-associated differences in NAD(P)H-oxidase activation, we analyzed the effects of losartan treatment in the protein expression of NAD(P)H-oxidase components. As can be seen in Fig. 5, the overexpression of the NAD(P)H oxidase subunits observed in male was prevented by treatment with losartan, demonstrating that AT-1 receptor mediates the upregulation of those components of NAD(P)H oxidase in male SHR. Interestingly, losartan treatment produced no effect (or just a trend of decrease) on NAD(P)H-oxidase expression in microvessels from female hypertensive rats, despite its significant inhibitory effect on superoxide generation. Reasons for this discrepancy are unclear, but may indicate that AT-1 mediates O2– production in female SHR by increasing NAD(P)H-oxidase activity rather than the abundance of the oxidase subunits. It is also possible, that a partial inhibition of NAD(P)H-oxidase expression could not be detected by the methodology used herein, since we obtained relatively weak signals in immunoblot analyses from female. Therefore, a role for AT-1 in mediating the abundance of the oxidase components in female SHR should not be ruled out.

Taken together, our data have established that males SHR present higher superoxide concentration under basal condition than do females. An AT1-dependent overexpression of the NAD(P)H-oxidase components p22phox, gp91phox, p47phox and p67phox may account for the gender difference in oxidative stress, and may play an important role in the noted gender difference on incidence of hypertension and other cardiovascular diseases.


   Acknowledgements
 
This work was supported in part by grant from the Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) and PRONEX (to the Laboratory of Hypertension). APD was supported by a fellowship award from the Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP).


   Notes
 
Time for primary review 30 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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