M Caru

D Curnier

M Bousquet

L Kern

Evolution of depression during rehabilitation program in patients with cardiovascular diseases

Keywords: Depression, cardiac rehabilitation, exercise, cardiovascular diseases, depression scales

Background: Cardiovascular diseases represent the main cause of death in the world. Rehabilitation through exercise is more and more used in cardiac patients. Given that these patients suffer from depressive symptoms, the risk of having recurrent cardiovascular problems increases. Thus, the aim of this study is to identify the effects of a rehabilitation program on the physiological and psychological parameters; with a particular attention on the depression scores between the scales. Methods: Twenty-eight cardiac patients participated in this study during a cardiovascular rehabilitation program. They are tested at their entry and at their exit with an evaluation of their physical fitness on an electromagnetic cycle ergometer and by four depression scales (Beck Depression Inventory, Hospital Anxiety and Depression Scale (HADS), Center for Epidemiologic Studies -Depression and Geriatric Depression Screening). Results: We observe that 21.4-50% of these patients have depressive symptoms, according to depression scales. The women have depression scores significantly higher than the men. The rehabilitation program improves their maximal oxygen consumption and their maximal aerobic power. At the end of the rehabilitation program, our analysis identifies a significant decrease in the depression score for the HADS. Regardless of the physical deconditioning level and of the improvement of the maximal oxygen consumption, our results show an effect of the rehabilitation program on the depression scores. No correlation between the physical deconditioning and the different depression scores is observed. Conclusions: This study shows the importance of measuring depression and its severity to improve the care of patients. Our findings show that between 21.4% and 50% of patients have depressive symptoms which challenges the categorical approach of the self-report depression scales.

ä IMPLICATIONS FOR REHABILITATION Depression and cardiovascular diseases have an impact on the patients' physical fitness. The rehabilitation program, primarily based around exercise, reduces depressive symptoms. As soon as cardiovascular diseases patients enter in a rehabilitation program, the depression should be measured by a psychologist. Taking into account the depressive symptoms of the patients as soon as their cardiac event occurs makes it possible to improve the care of patients.

Introduction

Cardiovascular diseases (CVDs) represent the main cause of death in the world with 17.5 million deaths each year (31% of global mortality) [START_REF]Global status report on noncommunicable diseases[END_REF]. In Europe, recent data suggest that about half of the deaths are due to CVDs, with almost 4 million of people dying each year [START_REF] Nichols | Cardiovascular disease in Europe 2014: epidemiological update[END_REF]. The CVDs are favored by a sedentary population [START_REF] Myers | Physical activity and cardiorespiratory fitness as major markers of cardiovascular risk: their independent and interwoven importance to health status[END_REF] and by the presence of multiple cardiovascular risk factors (i.e., hypertension, obesity, overweight, dyslipidemia, diabetes, smoking, depression) [START_REF]Cardiovascular diseases (CVDs)[END_REF].

Depression is an important psychological factor to predict mortality [START_REF] Kachur | Significance of comorbid psychological stress and depression on outcomes after cardiac rehabilitation[END_REF] and is known to be associated with CVDs [START_REF] Kubzansky | Shared and unique contributions of anger, anxiety, and depression to coronary heart disease: a prospective study in the normative aging study[END_REF][START_REF] Jonas | Symptoms of depression as a prospective risk factor for stroke[END_REF]. Indeed, depression and anxiety are predictors of coronary heart disease [START_REF] Kubzansky | Shared and unique contributions of anger, anxiety, and depression to coronary heart disease: a prospective study in the normative aging study[END_REF] and of infarcts [START_REF] Jonas | Symptoms of depression as a prospective risk factor for stroke[END_REF]. Also, depression is a consequence of CVDs since the patients may suffer from a depressive disorder or a severe depression after the development of a heart disease [START_REF] Gary | Combined exercise and cognitive behavioral therapy improves outcomes in patients with heart failure[END_REF][START_REF] Bounhoure | Influence of depression on the prognosis of cardiovascular diseases[END_REF]. Seldenrijk et al. show, in currently depressive patients, a dose-response association between symptom severity and CVDs with a risk of two-fold in a follow-up period of six years [START_REF] Seldenrijk | Depression, anxiety and 6-year risk of cardiovascular disease[END_REF]. A metaanalysis shows that patients with both depressive symptoms and CVDs have a higher risk of having recurrent cardiovascular problems than patients who have no depressive symptoms [START_REF] Barth | Depression as a risk factor for mortality in patients with coronary heart disease: a meta-analysis[END_REF]. In those with depression without CVDs, physical exercise is associated with a reduction of depressive symptoms [START_REF] Craft | The benefits of exercise for the clinically depressed[END_REF], which is also observed in depressive patients with CVDs [START_REF] Blumenthal | Effects of exercise training on depressive symptoms in patients with chronic heart failure: the HF-ACTION randomized trial[END_REF][START_REF] Ernstsen | Protective effect of regular physical activity on depression after myocardial infarction: the HUNT study[END_REF]. The physical mechanisms underlying the antidepressant effects of physical activity is in the state of assumptions [START_REF] Craft | The benefits of exercise for the clinically depressed[END_REF]. Indeed, several physiological and psychological mechanisms of the body could explain the relationship between physical activity and depression (thermogenic hypothesis, endorphin hypothesis, monoamine hypothesis, distraction hypothesis).

Another significant factor in the prognostic of patients with CVDs is their physical fitness [START_REF] Myers | Physical activity and cardiorespiratory fitness as major markers of cardiovascular risk: their independent and interwoven importance to health status[END_REF]. Depression and anxiety are predictors of physical decline in patients with heart failure [START_REF] Shen | Depression and anxiety predict decline in physical health functioning in patients with heart failure[END_REF]. Thus, the care of patients has evolved in recent years by integrating physical activity in cardiovascular rehabilitation programs [START_REF] Anderson | Exercisebased cardiac rehabilitation for coronary heart disease: Cochrane systematic review and meta-analysis[END_REF][START_REF] Lavie | Impact of cardiac rehabilitation and exercise training on psychological risk factors and subsequent prognosis in patients with cardiovascular disease[END_REF]. Physical activity has been consistently associated with a reduced risk of CVDs [START_REF] Thompson | Exercise and physical activity in the prevention and treatment of atherosclerotic cardiovascular disease[END_REF], a means of prevention against depression [START_REF] Ernstsen | Protective effect of regular physical activity on depression after myocardial infarction: the HUNT study[END_REF] and a treatment of depression [START_REF] Milani | Exercise training as treatment of depression in heart failure[END_REF]. Current clinical recommendations include the screening and treating of depression as the "standard of care" for the patient to avoid re-hospitalization within the first 6 months [START_REF] Lichtman | Depression and coronary heart disease: recommendations for screening, referral, and treatment: a science advisory from the American Heart Association Prevention Committee of the Council on Cardiovascular Nursing, Council on Clinical Cardiology, Council on Epidemiology and Prevention, and Interdisciplinary Council on Quality of Care and Outcomes Research: endorsed by the American Psychiatric Association[END_REF]. This is important because the symptoms associated with depression such as fatigue, lack of energy, loss of appetite, sleep problems, psychomotor and attention problems are also associated with a poor prognosis for CVDs outcomes [START_REF] Michal | Differential associations of depressive symptom dimensions with cardio-vascular disease in the community: results from the Gutenberg health study[END_REF].

Several depression scales are used to assess depressive symptoms in patients with CVDs [START_REF] Yohannes | Depression and anxiety in chronic heart failure and chronic obstructive pulmonary disease: prevalence, relevance, clinical implications and management principles[END_REF][START_REF] Delville | A systematic review of depression in adults with heart failure: instruments and incidence[END_REF], allowing the observation of a prevalence depression in people with a heart disease of about 30% [START_REF] Yohannes | Depression and anxiety in chronic heart failure and chronic obstructive pulmonary disease: prevalence, relevance, clinical implications and management principles[END_REF]. However, the prevalence of depression is variable within the same scale [START_REF] Westlake | Depression in patients with heart failure[END_REF][START_REF] Freedland | Prevalence of depression in hospitalized patients with congestive heart failure[END_REF]. The aim of this study is to identify the effects of a rehabilitation program on the physiological and psychological parameters of CVDs patients; with a particular attention on the depression scores between the scales. Therefore, the first hypothesis is that patients with CVDs will have high depression scores that will differ according to the scales used, while the second hypothesis is that the rehabilitation program will improve the depression score.

Methods

Participants

It is a retrospective study where all data are collected in May 2008. Informed consent is obtained from participants. This investigation is conducted in accordance to the World Medical Association Declaration of Helsinki, and is depended upon country rules (law n 2004-806; CCTIR 24 October 2005). A total of 28 cardiac patients (22 men and six women) participate in this study consisting of an ambulant cardiovascular rehabilitation program of 4 weeks. The inclusion criteria are all patients with coronary, infarct, heart failure or valvulopathy, whereas the exclusion criteria are applied when the patients are under 18 years old or have a chronic lower respiratory disease. All patients are tested at their entry (time 1) and at their exit (time 2) with at each moment, an evaluation of the physical fitness by an exercise stress test on ergocycle. The patients complete four depression scales. Between time 1 and time 2, they follow a rehabilitation program.

Exercise stress test

All patients perform an exercise stress test at their entry and at their exit of the rehabilitation program; the tests are performed on an electromagnetic cycle ergometer (Ergoline 900; Bunnik, The Netherlands). A cardiopulmonary exercise test combined with a breath-by-breath combined exercise metabolic and cardiac system (CardiO2; Medical Graphic Corp, St Paul, MN) allows measuring of the maximal aerobic power (MAP) and the maximal oxygen consumption ( _ VO 2 peak). The exercise stress test is composed of a warm-up at 20% of the predetermined maximal load followed by an increment of 10 W per minute.

Depression scales

The depression scales enable measuring of the depressive symptoms in cardiac patients. This study examines the main depression scales of the scientific literature and especially those studied in the works of Yohannes and Delville [START_REF] Yohannes | Depression and anxiety in chronic heart failure and chronic obstructive pulmonary disease: prevalence, relevance, clinical implications and management principles[END_REF][START_REF] Delville | A systematic review of depression in adults with heart failure: instruments and incidence[END_REF]. The Beck Depression Inventory (BDI) is used to measure depressive symptoms [START_REF] Beck | Beck depression inventory-II[END_REF]. It has excellent psychometric properties with Cronbach's alpha coefficient of 0.93 and retests Pearson's r of 0.93. The BDI is a 21-item multiple-choice self-report inventory for measuring the severity of depression. Statements are reported on a four-points scale ranging from 0 (not at all) to 3 (very much so) in terms of severity; the total score range is of 0-63. All patients are categorized into four groups of depressive symptoms based on standard clinical categories for the BDI.

The patients with a score of 0-13 are categorized as having no depressive symptoms, 14-19 as mild, 20-28 as moderate and 29-63 as having severe depressive symptoms [START_REF] Beck | Depression: clinical, experimental, and theoretical aspects[END_REF].

The Hospital Anxiety and Depression Scale (HADS) is used to identify patients with a clinically significant anxiety and depression [START_REF] Zigmond | The hospital anxiety and depression scale[END_REF]. The HADS is widely recommended as the screening tool in the cardiac setting [START_REF] Doyle | The Hospital Anxiety and Depression Scale depression subscale, but not the Beck Depression Inventory-Fast Scale, identifies patients with acute coronary syndrome at elevated risk of 1-year mortality[END_REF]. It has excellent psychometric properties with Cronbach's alpha coefficient of 0.85 and Pearson's r of 0.73. This depression scale is composed of two subscales: an anxiety scale (HADS-A) and a depression scale (HADS-D). Each subscale includes seven-item rated on a four points scale (0-3); a high score indicates more symptoms. Scores for each subscale (anxiety and depression) range from 0 to 21 with scores categorized as follows: no anxiety and depression 0-7, anxiety and depressive disorders 8-10, severe anxiety and depression 11-21.

The Center for Epidemiologic Studies -Depression (CES-D) is designed to identify depression among the general population [START_REF] Radloff | The CES-D scale a self-report depression scale for research in the general population[END_REF]. It has excellent psychometric properties with Cronbach's alpha coefficient of 0.85 and Pearson's r of 0.70. The CES-D is composed of 20-item, which evaluate various aspects of depressive symptoms: depressed mood, feelings of guilt, hopelessness, psychomotor retardation, anorexia and sleep disorders, according to the previous week. Each answer is rated from 0 (never, rarelyless than a day) to 3 (often, all time -5-7 days) depending on the frequency of symptoms. A score higher than 17 for men and 23 for women is indicative of depressive disorders. The Geriatric Depression Screening (GDS) uses a 30-item selfreport assessment designed to identify geriatric depression [START_REF] Yesavage | Development and validation of a geriatric depression screening scale: a preliminary report[END_REF]. The patients can answer yes or no to the questions depending on the state in which they felt during the previous week. A positive response to 20 of the 30 questions and a negative response to the 10 other questions (

Rehabilitation program

The rehabilitation program is especially based around exercise where all patients, within a group, perform exercise training during 4 weeks with 5-6 training sessions per week. Each training session is composed of 30-45 min on a stationary electromagnetic bicycle with a 5-min warm-up at 20% of the exercise workload and with a 10-min recovery. The exercise workload is determined to entail a heart rate equivalent to a percentage of oxygen uptake (mostly starting at 50% to attain 80% at the end of the rehabilitation) prescribed by a cardiologist. The patients perform calisthenics exercises, relaxation, and rest in warm water five times a week to favor the social exchange between patients. Also, therapeutic education sessions are offered in groups of patients for 15-20 min per week. The proposed workshops discuss cardiovascular risk factors, signs and symptoms of the disease, lifestyle, control of stress related to daily management of the disease and exercise (i.e., intensity management, security, benefits).

Statistical analysis

Statistical analysis is performed using SPSS (SPSS Inc., Chicago, IL). We perform descriptive statistics to describe our population.

The quantitative variables are reported as mean 6 standard deviation (SD). A Student t-test is used to observe an effect of the rehabilitation program on the physiological and psychological parameters between time 1 and 2. We use an analysis of variance (ANOVA) to assess the effect of the rehabilitation program on depressive symptoms. A Pearson correlation coefficient test is realized to identify a possible relationship between physical deconditioning and the different depression scales. The differences are considered significant when p value is p < 0.05 with an alpha risk of 5%.

Results

Our study is composed of 28 patients (22 men and six women) with a mean age of 60.11 6 11.76 years, a body mass index of 26.80 6 5.54 kg m À2 , a _ VO 2 peak of 18.8 6 5.9 ml kg À1 min À1 and a physical fitness level of 63.3%. Within our population, there are 19 patients with a coronary artery disease (67.9%), seven patients with a valvulopathy (25%), one patient with an aneurysm rupture (3.6%) and one patient with hypertension (3.6%). We observe, depending on the severity of depression determined by scales (Table 1) in our population, that 50% (HADS) to 78.6% (BDI) of patients have no symptoms of depression and that 3.6% (BDI) to 14.3% (HADS) have symptoms of severe depression. Overall, we observe that 21.4-50% of patients have depressive symptoms, according to depression scales. ).

Values are expressed as mean ± standard deviation (standard error).

a Does not mean effect of gender or time, but an interaction effect between men and women (p < 0.01).

A significant difference has been shown by gender on the scores of BDI, GDS, and HADS (Table 2). The women have depression scores significantly higher than the men on these three scales at time 2; only CES-D do not show a significant difference. Moreover, depression scores in men seem to decrease at time 2, comparatively to time 1. Also, at time 1, men have a _ VO 2 peak that is significantly higher than women.

The rehabilitation program improves the _ VO 2 peak and the MAP (Table 3). HADS have a significant decrease in the depression scores at the end of the rehabilitation program, whereas CES-D and the BDI present a tendency to decrease. No significant improvement of the heart rate at the end of the rehabilitation program is shown.

The results show no correlation between physical deconditioning and the different scores of the depression scales (Table 4).

The patients are separated into two groups according to the median to observe the effect of improving the _ VO 2 peak on their depression scores. The group 1 has the lowest improvement of _ VO 2 peak. The effect of the rehabilitation program according to the improvement of _ VO 2 peak and to each depression scale is visible in Table 5. The repeated measures ANOVA's analyses show an effect of time on intra-patients for the BDI, CES-D, and HADS; no inter-group effect and no interaction effect are observed. The effect of the rehabilitation program according to physical deconditioning and to each depression scale is visible in Table 5. The repeated measures ANOVA's analyses show an effect of time on intra-patients for the BDI, CES-D, and HADS; no inter-group effect and no interaction effect are observed.

Discussion

The aim of this research is to study the physiological and psychological dimensions in patients during a cardiac rehabilitation program. Overall, we observe that patients have improved their depressive symptoms and their physical fitness at their exit of the rehabilitation program, comparatively to their entry. It is particularly important to target different depression scales in a same studied population because, even if they are standardized tools, there is not a scale that is favorized in comparison to another for older adults [START_REF] Delville | A systematic review of depression in adults with heart failure: instruments and incidence[END_REF]. Also, the patients with CVDs have relatively high scores of depression, which seems to differ from one scale to another [START_REF] Yohannes | Depression and anxiety in chronic heart failure and chronic obstructive pulmonary disease: prevalence, relevance, clinical implications and management principles[END_REF]. According to the categorical approach and the scales used, we observe that between 21.4% and 50% of the patients evaluated have depressive symptoms; this observation is in accordance with the literature [START_REF] Yohannes | Depression and anxiety in chronic heart failure and chronic obstructive pulmonary disease: prevalence, relevance, clinical implications and management principles[END_REF][START_REF] Delville | A systematic review of depression in adults with heart failure: instruments and incidence[END_REF]. For example, it is observed in chronic heart failure that the prevalence of depression is high (10-60% depression) [START_REF] Yohannes | Depression and anxiety in chronic heart failure and chronic obstructive pulmonary disease: prevalence, relevance, clinical implications and management principles[END_REF]. The sensitivity of these scales in our population may be due to the fact that some are generic while others are specific. Indeed, patients with a cardiac disease have a rate of depression twofold higher than the general population [START_REF] Bounhoure | Influence of depression on the prognosis of cardiovascular diseases[END_REF][START_REF] Katon | The association of depression and anxiety with medical symptom burden in patients with chronic medical illness[END_REF]. However, according to the scales used, we identify different depression scores from one scale to another. Our results show that between 3.6% and 14.3% of patients received a diagnosis of severe depression. These results are important because all scales which examine the prevalence of depression are self-report measures. A patient with a score inferior to 9/63 by the GDS is considered to have no depression. If a patient has a score superior to 10/63, he would then be considered to have a depression ranging from mild to severe. The categorical approach allows the classification of patients by the presence or absence of a trait. Moreover, a patient may be categorized as having symptoms of depression by the GDS, whereas the categorization by the BDI is reversed. This illustrates that the sensitivity of the depression scales is not yet perfect and deserves to be studied. We note that with the BDI, one on five patients is categorized as being depressive, whereas for the HADS, the depression concerns one of two patients. For the HADS, we observe no consensus on the values of the thresholds used to classify patients. The cutoff for establishing the sensitivity and specificity values is different, according to studies [START_REF] Stafford | Validity of the Hospital Anxiety and Depression Scale and Patient Health Questionnaire-9 to screen for depression in patients with coronary artery disease[END_REF][START_REF] Bjelland | The validity of the Hospital Anxiety and Depression Scale. An updated literature review[END_REF]. According to the optimal cutoff to screen depression in patients with CVDs, the score in the HADS !8 [START_REF] Stafford | Validity of the Hospital Anxiety and Depression Scale and Patient Health Questionnaire-9 to screen for depression in patients with coronary artery disease[END_REF], whereas, according to Bjelland et al., the cutoff range is from 4 to 11 [START_REF] Bjelland | The validity of the Hospital Anxiety and Depression Scale. An updated literature review[END_REF]. A cutoff too low has for effect an increase in the number of false positives and inversely [START_REF] Pittam | Appraisal of screening for depression: a report for the UK National Screening Committee[END_REF]. Some depression scales overestimate the depression symptoms while other underestimate these symptoms. Our results suggest that the prevalences for the HADS are different from the other three depression scales, which are similar to one another. Our findings show that men have depression scores significantly lower than women after a rehabilitation program (time 2) for the BDI and GDS. This is consistent with previous work, which shows that women have significantly higher depression scores on the BDI [START_REF] Azevedo | Depressive symptoms and heart failure stages[END_REF]. This can be explained by the fact that men give more importance to goal-oriented factors (i.e., improvement of _ VO 2 peak), whereas women give more importance to interpersonal relationships while internalizing symptoms [START_REF] Bartels | Exploring the association between well-being and psychopathology in adolescents[END_REF][START_REF] Kendler | Sex differences in the pathways to major depression: a study of opposite-sex twin pairs[END_REF].

Regardless of the gender, the rehabilitation program has an effect on the depression score of patients at their exit. Our results show that the practice of physical activity improves the physiological parameters (i.e., MAP and _ VO 2 peak) and decreasing the depression scores in patients with CVDs. The improvement in physical fitness level (i.e., _ VO 2 peak) is shown to be correlated with improvements of the depression factors [START_REF] Milani | Impact of cardiac rehabilitation on depression and its associated mortality[END_REF]. This reduces the deleterious effects associated with the disease because patients with depression and CVDs observe a quicker loss of physical function [START_REF] Gary | Combined exercise and cognitive behavioral therapy improves outcomes in patients with heart failure[END_REF] resulting in the appearance of a physical disability [START_REF] Vaccarino | Depressive symptoms and risk of functional decline and death in patients with heart failure[END_REF]. Indeed, the CVDs patients who have a low functional autonomy have a greater severity of their symptoms and a weaker health-related quality of life, comparatively with CVDs patients without depression [START_REF] Jiang | Relationship between depressive symptoms and long-term mortality in patients with heart failure[END_REF][START_REF] Juenger | Health related quality of life in patients with congestive heart failure: comparison with other chronic diseases and relation to functional variables[END_REF]. Nevertheless, contrary to what the literature observes [START_REF] Milani | Impact of cardiac rehabilitation on depression and its associated mortality[END_REF], the physical deconditioning level does not influence the depression score between time 1 and time 2. Indeed, our results show an effect of the rehabilitation program on the depression score, independently of the patient's physical deconditioning level and of the improvement of the _ VO 2 peak. Patients who have improved their _ VO 2 peak the most do not show a significant decrease in their depression scores, compared to patients who have not improved their _ VO 2 peak. The absence of a control group forces us to remain careful with our results. However, these results may be explained by the social component of our study, which is also the strength of our rehabilitation program. Where most studies put the spotlight on cardiorespiratory response at the expense of psychological responses, our study brings an innovative aspect to current cardiac rehabilitation programs. Indeed, in this study, there is social exchange since the majority of exercise sessions are conducted in a group several times a day. Patients have time to talk and exchange between the activities. The gymnastics activities are an essential component of this rehabilitation program because they consist of short exercise sessions with recovery phases enabling patients to interact with each other more easily. The inclusion of psychological components in cardiac rehabilitation programs should be standard practice by adapted physical activity professionals to improve patient care.

Compared to other studies, which usually take place between 4 and 12 months [START_REF] Taylor | Exercise-based rehabilitation for patients with coronary heart disease: systematic review and meta-analysis of randomized controlled trials[END_REF][START_REF] Lavie | Benefits of cardiac rehabilitation and exercise training programs in elderly coronary patients[END_REF][START_REF] Kachur | Impact of cardiac rehabilitation and exercise training programs in coronary heart disease[END_REF], the duration of our program is shorter. Indeed, the duration of the exercise training program is twofold less than other studies [START_REF] Taylor | Exercise-based rehabilitation for patients with coronary heart disease: systematic review and meta-analysis of randomized controlled trials[END_REF] and the impact of rehabilitation on improving _ VO 2 peak and depressive symptoms could be underestimated compared to longer rehabilitation programs. Nevertheless, this does not prevent an improvement in _ VO 2 peak of 17.5% in our study. Despite a relatively short rehabilitation program, it is extremely encouraging to observe such effects. Our results are similar to previous studies [START_REF] Lavie | Benefits of cardiac rehabilitation and exercise training programs in elderly coronary patients[END_REF][START_REF] Lavie | Disparate effects of improving aerobic exercise capacity and quality of life after cardiac rehabilitation in young and elderly coronary patients[END_REF] and are higher than others [START_REF] Jelinek | Cardiac rehabilitation outcomes following a 6-week program of PCI and CABG Patients[END_REF]. In the literature, a cardiac rehabilitation program reduces from 20% to 32% probability of all-cause mortality [START_REF] Taylor | Exercise-based rehabilitation for patients with coronary heart disease: systematic review and meta-analysis of randomized controlled trials[END_REF]. Although these effects are known, many patients often stop the program after a few months of rehabilitation due to external constraints; it is essential to help them become autonomous. The depressive symptoms worsen these aspects, which does not lead to a healthy lifestyle [START_REF] Sarris | Lifestyle medicine for depression[END_REF]. These results are encouraging in the actual context of patient care which promotes, mostly, the improvement of physiological capacities over the psychological aspects.

Limitations

Our study identifies some limitations. The first one concerns our number of patients (n = 28) which is low compared to other studies in the same field. Moreover, our population is unbalanced regarding the number of men and women, but this did not prevent us from observing interesting differences between them. The second one is that we observe in this study a high prevalence between depression scores. Thus, we invite the reader to be more careful in using these depression scales. A diagnosis of depression should not be identified only with one depression scale. In order to have a better diagnosis, it is necessary to couple the depression scales with clinical interviews. Also, it might have been interesting to study the PHQ-9 questionnaire to complete our results. This study being retrospective (data collected in May 2008), we did not know the American Heart Association recommends concerning the PHQ-9 [START_REF] Lichtman | AHA science advisory. Depression and coronary heart disease. Recommendations for screening, referral, and treatment. A science advisory from the American Heart Association Prevention Committee to the Council on Cardiovascular Nursing, Council on Clinical Cardiology, Council on Epidemiology and Prevention, and Interdisciplinary Council on Quality of Care Outcomes Research. Endorsed by the American Psychiatric Association[END_REF]. Finally, patients could have different physical fitness level related to environment (i.e., physical activity), which might vary the response of each patient in the rehabilitation program.

Conclusions

This study shows the importance of measuring depression and its severity to improve the care of patients. However, our findings show that between 21.4% and 50% of the patients have depressive symptoms and that 3.6-14.3% of patients received a diagnosis of severe depression. The categorical approach which allows classifying patients is questioned. Finally, an effect of the rehabilitation program on both the physiological and psychological parameters is observed but readers should be careful with our results, promote a multidimensional approach and confirm the diagnosis by a psychologist. Following this study, the clinic has a psychologist in their team allowing the establishment of a psychological and physiological follow-up.

Table 1 .

 1 Categorical analysis of the depression in our population by scales.

	Scales	Categories	N	%	Depressive patients (%)
	BDI	No depression	22	78.6	21.4
		Mild depression	5	17.9
		Moderate depression	0	0.0
		Severe depression	1	3.6
	GDS	No depression	21	75.0	25.0
		Mild depression	5	17.9
		Severe depression	2	7.1
	CES-D	No depression	20	71.4	28.6
		Depression	8	28.6
	HADS	No depression	14	50.0	50.0
		Depressive disorders	10	35.7
		Severe depression	4	14.3

BDI: beck depression inventory; GDS: geriatric depression screening; CES-D: center for epidemiologic studiesdepression; HADS: hospital anxiety and depression scale.

Table 2 .

 2 Comparison of scores on depression scales by gender.

		MD	-6.41	-4.70	-4.64	-5.17	-	3.61	VO 2
		p	0.01	0.02	0.18	0.02	-	0.34
		DF	26	26	26	26	-	25
		t	-2.68	-2.46	-1.38	-2.46	-	0.98
		95% CI	(1.49;11.32)	(0.77;8.62)	(-2.27;11.55)	(0.84;9.49)	-	(-11.20;3.98)
	Time 2	Men Women	5.59 ± 4.38 (0.93) 12.00 ± 7.72 (3.15)	3.64 ± 3.19 (0.68) 8.33 ± 6.83 (2.79)	7.36 ± 7.22 (1.54) 12.00 ± 7.64 (3.12)	8.50 ± 3.61 (0.77) 13.67 ± 7.34 (3.00)	--	22.69 ± 6.84 (1.46) 19.08 ± 10.01 (4.48)
		MD	0.17	-4.64	-3.98	-1.55	2.85	7.15
		p	0.96	0.16	0.29	0.57	0.80	0.01
		DF	26	26	26	26	25	25
		t	0.06	-1.46	-1.07	-0.58	0.25	2.77
		95% CI	(-6.21;5.88)	(-1.88;11.16)	(-3.64;11.61)	(-3.96;7.05)	(-12.46;-1.83)	(-26.01;20.32)
	Time 1	Variables Men Women	BDIa 9.00 ± 6.68 (1.42) 8.83 ± 4.96 (2.02)	GDS 5.36 ± 5.98 (1.28) 10.00 ± 9.82 (4.01)	CES-D 10.68 ± 7.41 (1.58) 14.67 ± 10.33 (4.22)	HADS 11.95 ± 5.88 (1.25) 13.50 ± 5.54 (2.26)	Physical deconditioning _ VO 2 peak 20.06 ± 5.21 (1.11) 35.01 ± 20.49 (4.37) 32.16 ± 31.90 (14.27) 12.91 ± 5.22 (2.33)	peak: maximal oxygen consumption (ml kg À1 min À1

DF: degree freedom; MD: mean difference; BDI: beck depression inventory; GDS: geriatric depression screening; CES-D: center for epidemiologic studies -depression; HADS: hospital anxiety and depression scale; _

Table 3 .

 3 Statistical analyses between time 1 and time 2 on different variables studied.

	Variables	Time 1	Time 2	95% CI	t	DF	p Value
	BDI	8.96 ± 6.27 (1.18)	6.96 ± 5.76 (1.09)	(-0.19;4.19)	1.87	27	0.07
	GDS	6.36 ± 7.03 (1.33)	4.64 ± 4.52 (0.85)	(-0.57;4.00)	1.53	27	0.14
	CES-D	11.54 ± 8.08 (1.53)	8.36 ± 7.42 (1.40)	(-0.15;6.50)	1.96	27	0.06
	HADS	12.29 ± 5.74 (1.08)	9.61 ± 4.98 (0.94)	(0.92;4.43)	3.13	27	0.004
	MAP	85.97 ± 45.75 (8.80)	104.89 ± 53.63 (10.32)	(-33.79;-4.05)	-2.62	26	0.02
	MHR _ VO 2 peak	109.27 ± 24.45 (4.71) 18.74 ± 5.84 (1.12)	118.56 ± 32.51 (6.26) 22.02 ± 7.43 (1.43)	(-23.38;4.80) (-5.84;-0.73)	-1.35 -2.65	26 26	0.19 0.01
	DF: degree freedom; BDI: beck depression inventory; GDS: geriatric depression screening; CES-D: center for epidemiologic studies -depression; HADS: hospital anxiety and depression scale; MAP: maximal aerobic power; MHR: maximal heart rate; _ VO 2 peak: maximal oxygen consumption (ml kg À1 min À1 ).
	Values are expressed as mean ± standard deviation (standard error).

Table 4 .

 4 Pearson's correlation coefficient test between the physical deconditioning and the different depression scales.

			BDI		GDS	CES-D	HADS
	Variable	Time 1	Time 2	Time 1	Time 2	Time 1	Time 2	Time 1	Time 2
	Physical deconditioning	-0.05	0.09	-0.08	-0.12	0.11	-0.02	-0.13	-0.24
	BDI: beck depression inventory; GDS: geriatric depression screening; CES-D: center for epidemiologic studies -depression; HADS:
	hospital anxiety and depression scale.

Table 5 .

 5 Repeated measures ANOVA's analyses according to improving maximum oxygen consumption ( _ VO 2 peak) and physical deconditioning level of patients.

	Scales	Test	Condition	DF	MS	F	pValue
	According to improving maximum oxygen consumption ( _ V O 2 peak)
	BDI	Intra-patients	Time	1	80.42	5.60
	Inter-patients	Time _ VO 2 peak improvement _ VO 2 peak improvement	1 1	11.97 66.92	0.83 1.42	0.37 0.24
	GDS	Intra-patients Inter-patients	Time Time _ VO 2 peak improvement _ VO 2 peak improvement	1 1 1	45.81 8.03 34.58	2.49 0.44 0.77	0.13 0.51 0.39
	CES-D	Intra-patients Inter-patients	Time Time _ VO 2 peak improvement _ VO 2 peak improvement	1 1 1	184.62 11.28 19.08	5.16 0.32 0.24	0.03 0.58 0.63
	HADS	Intra-patients Inter-patients	Time Time _ VO 2 peak improvement _ VO 2 peak improvement	1 1 1	112.18 0.18 99.73	10.78 0.02 2.34	<0.001 0.90 0.14
	According to physical deconditioning level
	BDI	Intra-patients	Time	1	76.48	5.24	0.03
			Time  physical deconditioning	1	6.41	0.44	0.51
		Inter-patients	Physical deconditioning	1	2.64	0.05	0.82
	GDS	Intra-patients	Time	1	45.00	2.41	0.13
			Time  physical deconditioning	1	1.44	0.08	0.78
		Inter-patients	Physical deconditioning	1	30.89	0.68	0.42
	CES-D	Intra-patients	Time	1	182.43	5.04	0.03
			Time  physical deconditioning	1	1.39	0.04	0.85
		Inter-patients	Physical deconditioning	1	29.78	0.38	0.54
	HADS	Intra-patients	Time	1	115.19	11.57	<0.005
			Time  physical deconditioning	1	11.49	1.15	0.29
		Inter-patients	Physical deconditioning	1	17.44	0.38	0.54

DF: degree freedom; MS: mean square; BDI: beck depression inventory; GDS: geriatric depression screening; CES-D: center for epidemiologic studiesdepression; HADS: hospital anxiety and depression scale.

Acknowledgements

We would like to thank the cardiac rehabilitation center, all the staff of service of rehabilitation and all participants of this study. We appreciate the invaluable assistance of Ariane Levesque (McGill University) for her expert review of the article in the English language. The contents of this article are solely the responsibility of the authors and none has received funding to carry out this work.

Disclosure statement

The authors take responsibility for all aspects of the reliability and freedom from bias of the data presented and their interpretation discussed. The authors report no declarations of interest.


Damien Letellier

Olivier Sandre

Christine Ménager

Valérie Cabuil

Michel Lavergne

Magnetic tubules

Keywords: Magnetic fluids, Self-assemblies, Tubules, Phospholipids, Electrostatic interaction

la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The present work describes the use of molecular selfassemblies of amphiphilic molecules to synthesize new microstructures which exhibit original properties suitable for technical applications. Many recent works report the use of molecular self assemblies of lipids molecules as templates for the crystallization of inorganic oxides in order to obtain new morphologies [1,2] or to synthesize porous materials [3,4]. Due to their morphology, size, hollowness and rigidity, lipidic tubules composed of multibilayers wrapped in a cylinder [5] are well suited for technical applications [6]. In the particular case of magnetic applications, anisotropy and rigidity are both required and an electroless deposition technique has already been described in order to obtain permalloy-coated tubules [7].

Here we describe the synthesis of tubules which get a magnetic behavior when they are well combined with magnetic colloids made of nanoscopic magnetic particles suspended in a liquid solvent [START_REF] Rosensweig | Ferrohydrodynamics[END_REF].

The compatibility between self-assemblies of tensioactive molecules with magnetic colloidal dispersions has already been considered. The first hybrid system combining a lyotropic lamellar phase and magnetic particles was described by Fabre et al. [START_REF] Fabre | [END_REF]. In this system the tensioactive membrane was fluid. as the one of the liposomes into which we had then introduced magnetic particles [10]. The main problem for the synthesis of the so-called magnetic liposomes was the impossibility to encapsulate high volume fractions of particles because liposomes can not afford the heavy osmotic pressure due to the high ionic strength of the encapsulated magnetic dispersions. In the case of magnetic tubules the rigidity of the membrane avoids this problem. Using magnetic fluids with a high volume fraction of particles allows to observe a response of tubules to low intensity magnetic fields. We interpret the magnetic behavior ofthe magnetized tubules as monitored by the nature of the interaction between the phospholipidic membrane and the magnetic nanoparticles coated either by positive or negative surface charges.

Materials and methods

The tubules

Observation of tubules has been described in systems containing synthetic chiral amphiphiles [11], amino-acid-based surfactants [12], sugar-based surfactants [13] and biological galactocerebrosides [14]. Tubules formation is driven by a reversible first order phase transition from an interlamellar chain-melted L α phase to a chain-frozen L β' phase, the tubule phase being the one in which the hydrocarbon chains are highly ordered. Here we use a symmetric phosphatidylcholine lipid: 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine or DC 8,9 PC obtained from Avanti Polar Lipids and used as purchased. Polymerizable lipids with diacetylenic fatty acyl chains as DC 8,9 PC are known to form tubular microstructures when Iiposornes of these lipids are cooled through their chain melting transition [11]. DC 8,9 PC chain melting transition temperature is approximately 43°C [5]. Above this temperature DC 8,9 PC forms liposomes in aqueous dispersion. To obtain tubules we employ the precipitation method firstly described by Schoen and co-workers [5] which consists in the solubilisation of lipids in an appropriate solvent and a further precipitation by addition of water.

The method is as follows: a solution of lipids DC 8,9 PC (1 mg) is dissolved in ethanol (1 mL) at room temperature. Then 1 mL of water is added drop by drop, a flocculent precipitate of tubules is observed. This precipitate is centrifuged at 6000 rpm for 15 min and the resulting pellet is dispersed in distilled water.

The magnetic fluids

An aqueous magnetic fluid is a colloidal dispersion of magnetic ferric oxide particles in water [15]. Magnetite particles are firstly synthesized according to Massart' s procedure [16], then oxidized to rnaghernite by ferric nitrate in nitric acid medium. Particles mean diameter is about 7 nm. These particles have surface charges due to the acid-base behavior of their surface. According to the pH, the sign and the density of surface charges can be monitored.

We used here two kinds of particles: i) cationic particles, which have a positive surface charge density around 0.2 C m -2 in acidic media (pH~2); ii) anionic particles, which are coated by citrate species and have negative charges (around 0.2 C m -2 ) for pH∼7.

These nanometric particles dispersed in water lead to aqueous colloidal dispersions (called ionic ferrofluid) stabilized by the electrostatic repulsions between the magnetic grains. No additional surfactant is needed. Particles never separate from the solvent, even when submitted to a high magnetic field gradient.

Mixtures of tubules with magnetic fluid

Aqueous magnetic fluids can replace the normal aqueous phase in organized systems of surfactant molecules provided that their ionic strength does not destabilize the lyotropic system [17]. That is the reason why we have dialyzed all our magnetic dispersions through a membrane of Visking type until their conductivity is inferior to 2 mS cm -1 . Then DCPC tubules have been precipitated in water as described above and dispersed, after centrifugation, in the dialyzed magnetic dispersions.

Observation of tubules behavior

All the methods we shall describe further are grounded on observation of tubules under an optical microscope. The sample is kept in a sealed glass capillary with a rectangular crosssection (50 µm × 1 mm or 200 µm × 4 mm). According to the geometry of the experiment, a normal (Zeiss, Ortholuxe) or an inverted microscope (Leica, LEITZ DMIL) is used.

The difference between refraction indices of DCPC tubules and solvent (pure water or magnetic fluid) aIlows image formation without the use of phase contrast. When it is possible to approach the objective near the sample, a magnification ×40 is used, with a numerical aperture (NA) of 0.75 or 0.65. Otherwise a magnification ×32 with NA equal to 0.40 or 0.30 is sufficient. The tubules are filmed with a charge coupled device (CCD) color camera (Vista, VPC 4130, U.K.) connected to a video tape recorder. Selected images are digitized on a computer (768 × 512 pixels, 256 gray levels) and processed with the NIH Image (National Institute of Health, USA) software. Noise reduction of the pictures is obtained with a standard algorithm (median filter).

Electrophoresis

The simple setup we used is schematized on Fig. I. Two copper wires are immersed in a glass capillary, the distance between the electrodes being approximately 4mm. The applied potential is a few Volts. In spite of its simplicity, this basic method gives qualitative informations on the electrokinetic behavior of the tubules. Nevertheless the drawbacks are the limitation of the applied voltage by water electrolysis and the lack of control of some parameters: geometry of the current lines, electroosmosis caused by the negative charge of the glass walls. That is why the results are controIled with a more sophisticated experiment built by L. Mitnik et al. [18,19]. In their setup two solvent reservoirs at different electric potentials are tightly isolated, except by a bridge in which the current lines are confined. This one is made of two glass slides separated by a spacer, containing the solution to be studied . Solvent electrolysis is no longer a problem because it appears next to the electrodes which are far from the region of observation. Tensions as high as 100 V can be applied, provided that the conductivity is low enough (18 MΩ em resistivity water). Electroosmosis (spontaneous motion of the solvent in a capillary under an electric field just because the waIls bear a surface charge) is a phenomenon which is more difficult to be independent of. Two strategies have been tested: coating the glass surfaces with bovine serum albumin (BSA), or closing the cell with an agarose plug at each end [19].

Magnetic alignment: fixed or rotating magnetic field

The glass capillary containing tubules in a magnetic fluid is set between two identical coils in serial (125 turns each, selfinductance equals to 2.5 mH). With polar pieces (soft iron) inside the coils, the magnetic field can be varied from 2 × 10 3 to 6.4 × 10 4 A m -1 (25 to 800 Gauss), and for each intensity the mean time of alignment is measured.

Another experiment to test the orienting of tubules consists in applying a rotating magnetic field with pulsation Ω. In that experiment the sample is in a square cell made of two glass coverslides (20mm side) spaced by a polymer film based on paraffin (Parafilm) after melting on a heating plate. The field is created by two pairs of coils oriented at 90° (Fig. 2). Circular polarisation is obtained when the currents I x and I y through the coils are in phase quadrature. The phase of the rotating magnetic field is known thanks to a periodic light pulse (time width: 6ms) superposed to the image in the microscope and triggered with I x .

Magnetophoresis

The aim is to observe a migration under a magnetic field gradient. The setup is described on Fig. 3. A magnetic field is applied along Ox-direction and aligns tubules parallel to Ox. A gradient is created perpendicular to the field direction (Oy) by deviating the field lines of two coils (125 turns each, selfinductance equal to 2.5mH) in serial with polar pieces edged at 45°. Measurements with Hall effect probes give a gradient of 9.6 × 10 5 A m -2 (120 G/cm) with a mean field equal to 2.4 × 10 3 A m -1 (300G) under a 3A direct current.

Thermolysis of tubules

Thermoregulation of sealed cappilaries is obtained using a usual Peltier cell with a platinum probe and a water cooling. Less common, a heating by induction was build. A radiofrequency magnetic field is produced with coils in an electric resonant circuit. The resonance occurs at 850 kHz. A cooling circuit with nonane is used instead of water because its high dielectric constant hugely decreases the quality factor.

Transmission electron microscopy (TEM)

TEM is performed with a JEOL 100CXII top entry UHR microscope, on samples deposited on carbon films and air dried. Visualization of the helical structure of the tubules is enhanced when samples are negatively stained with ammonium molybdate according to the procedure described in ref [START_REF]Liposomes: a Practical Approach[END_REF]. Nevertheless, tubules combined with magnetic particles have also been directly observed without staining, for magnetic particles provide enough contrast to make the tubules observable.

Results

Characteristic of DCPC tubules in pure water

We have firstly characterized the tubules obtained as described in section A in terms of length. thickness and surface charge. Optical microscopy (Fig. 4) reveals that tubules are long with a relatively broad distribution of lengths (few microns to few tenths of microns). It can be noticed that the synthesis we used does not allow to control precisely the length of tubules, but this parameter could be controlled according to Ref. [START_REF] Thomas | [END_REF] in which the autors describe the synthesis of tubules formed under controlled cooling rates.

On the other hand, TEM shows that tubules' diameter is constant throughout all the samples, with an average value of 0.5 µm. Tubules appear like hollow cylinders which can be made of one or much bilayers. We observed tubules and helices which appear to have only one bilayer per wall (Fig. 5). Nevertheless such tubules consisting of a single cylindrical bilayer are as straight and rigid as those with multiple bilayer walls. When an electric field is applied on DCPC tubules in pure water, they move towards the positive electrode. For a field intensity of the order 10 3 V m -1 (4 V between the eletrodes separated by 4 mm), their velocity is approximately 30 µm s -1 . Fig. 6a gives a schematic velocity profile: almost all the tubules move in the opposite direction of the applied field, except the ones which adhere to the glass walls (they are still) and those which are in a thin region next to the walls (these latter move towards the negative electrode). This backflow is much smaller than the principal flow, so that the net current is towards the anode. The dependency of this electocinetik behavior with the Debye lenght κ -1 is tested by using tubules prepared in ultrapure water (18 MΩ cm resistivity obtained thanks to Millipore filters: κ -1 up to 1 mm) instead of simply distillated water. While the measured electric current is unchanged (I=2 mA for a 4 V voltage), the tubules migration is affected: the direction of the net motion is unclear, and some trajectories of tubules make loops. The experiment on Mitnik's setup gives the same kind of velocity profile in the case when the capillary is end up by agarose plugs. When the glass slides are covered by a layer of BSA proteins, the only difference is that the backflow near the glass walls is not observed anymore.

Tubules dispersed with anionic magnetic particles in water

Tubules are mixed with a dialyzed magnetic fluid at pH ∼ 7 as described in the experimental section. The addition of the magnetic fluid does not cause any damage to the tubules according both to optical and electronic microscopy. TEM observations are performed without the use of the coloring agent; as a matter of fact ammonium, molybdate induces precipitation of the particles on the tubules surface (Fig. 7). Without coloration, the nanoparticles appear homogeneously dispersed in the solution: they form a uniform film that covers more or less the membrane of the tubule (Fig. 8). When a magnetic field is applied to the mixture, tubules align themselves along the field lines (Fig. 9). Orientation times have been measured and depend on the volume fraction of magnetic particles and on the field intensity. Below a critical value ofthe magnetic field intensity orofthe particle's concentration , orientation does not occur because the magnetic torque is too weak compared to thermal agitation. Typical times for alignment are given in Fig. 10a.

In the magnetophoresis experiment, the DCPC tubules are oriented in the field direction (Ox) and migrate along the direction of the field gradient (Oy). When dispersed in an anionic magnetic fluid (constituted by negative particles such as the volume fraction of particles is around 1.8 %), the motion of tubules is always in the direction of decreasing field intensity (towards negative y values, Fig. 11). A typical value of velocity is 1.5 µm s -1 and it increases with the field gradient. Submitted to a magnetic field with circular polarization, the tubules rotate synchronously with the field up to a critical angular velocity Ω c (Fig. 12). For a volume fraction of particles equal to 3.5 % and a field intensity of 4.0 × 10 3 A m -1 (51 G) Ω c /2π is 0.5Hz (Ω c =3.14 rad s -1 ). Fig. 12 shows a tubule rotating at 0.1Hz: the angle increment every quarter of field period (T/4= 2.5 s) being always 90°, the rotation is perfectly synchronous. However, at field frequencies higher than Ω c /2π, the rotation of the tubules is not synchronous anymore, the coupling to the field rotation being only intermittent (motion described as jingle movement by Skjeltorp et al. [22] ). It can be noticed that the critical rotation period is 2s, a value which is not so far from the time of alignment (3 s) of a tubule in the same magnetic fluid under 100G. Finally, it appeared that the thermal behavior ofthe DCPC bilayers is strongly modified by the presence of anionic particles. The tubule-vesicle transition temperature is lowered in a wide range: Tm ∼ 10°C lower in a magnetic fluid (volume fraction in particles 3.5%) than in pure water (Fig. 13).

Tubules dispersed with cationic magnetic particles in water

On the microscopic scale, the tubules dispersed in a cationic magnetic fluid have the same appearance and the same behavior as tubules dispersed in an anionic one. However electron microscopy (without coloration) shows that in this case, the tubules appear like black cylinders made of concentrated magnetic particles (Fig . 14a) in a diluted dispersion of particles. It can be noticed that the concentrations of the preparation for picture 8 and 14a are of the same order. At a higher magnification (Fig. 14b), one can see the lipid membrane which appears like two white bands delimit ing the hollow tube full of particles. At the extremity (Fig. 14c), the tubules seem to bring out the particles.

In the magnetophoresis experiment, tubules with cationic particles exhibit an opposite behavior compared to the tubules with anionic particles: almost all of them migrate in the direction of increasing field intensity (towards positive y values, Fig. 11). Nevertheless a small amount of the tubules still goes in the opposite direction. The velocities under a 9.6 × 10 5 A m -2 gradient range from 0.3 to 0.9 µm s -1 .

As in the former case, tubules in the cationic magnetic fluid (φ = 2 %, pH=3.55) can rotate synchronously with the magnetic field (Fig. 15), but the critical frequency is ten times smaller: 0.05Hz (Ω c =0.314 rad s -1 ) under a field of 6.3 × 10 3 A m -1 (80 G). The phase lag between the tubule and the field is 5° at 0.01Hz and 38° at 0.05Hz, the maximum phase lag been 45° (see discussion below). Once again the minimal rotation period (20s) is approximately the time of alignment of a tubule (25 s) measured at 8 × 10 3 A m -1 (100G) (Fig. 10b). The transition temperature of the tubules in an acidic magnetic fluid is also affected by the presence of the cationic particles. This transition leads to globular aggregates badly defined under microscope observation (Fig. 13b). In the case of cationic particles as with anionic particles, TEM observations of heated samples exhibit some vesicles.

Discussion

The experimental results described above give information concerning DCPC tubules and their behavior when they are associated to magnetic nanoparticles. The combining of the different experiments allows to get some conclusions.

Charges on DCPC tubules in water

The question of tubules' charge is very important for they will be mixed with other charged species. DCPC is a zwitterionic molecule thus the structural charge of tubules should be zero. In the electrophoresis experiment it may be discussed whether the tubules have an own electrokinetic behavior or if the motion observed is an artifact due to electroosmosis. In the electroosmosis profile between two planar walls with closed capillary condition s (Fig. 6b), the surface velocity (towards cathode) is two times higher than the backflow velocity (towards anode) in the middle of the cell [18,19]. In our experiment with tubules, the surface velocity towards cathode being much smaller than the main flow towards anode, we conclude that the profile is not a pure electroosmotic one. Thus the tubules have an electrophoretic mobility indicating that they behave as negatively charged objects.

The values for tubules electrophoretic mobilities appeared as high as 3 × 10 -8 m 2 s -1 V -1 . Such values are too high to be only due to charged impurities or to the hydrolysis of a few phosphate polar heads. However, this negative mobility can be due to the conformation of the lipid molecule in the chain frozen L β ' phase. In this solid gel state with tilted hydrocarbon chains, the polar head is also tilted and the P-N axis is in the conformation shown on Fig. 16 [START_REF]Handbook of Lipid Research: The Physical Chemistry of Lipids[END_REF]. In this case, the negative charge borne by the phosphate group can be more exposed to the solvent than the positive one on the choline group.

A negative apparent charge is also in good accordance with the behavior of the tubules with anionic or cationic particles. When tubules are dispersed with cationic magnetic particles, electron microscopy pictures shows that most tubules are full of particles, and magnetophoresis experiments confirm this result, as they show that tubules behave as objects more magnetic than the external medium (the magnetic fluid). On the contrary, when tubules are dispersed with anionic magnetic particles, these latter do not seem to enter inside the lumen of the tubules, and in the magnetophoretic experiments, tubules behave as objects less magnetic than the external medium.

These results point out the electrostatic interactions between tubules and particles, according to the sign of surface charges of the latter. When particles are positively charged, they are strongly attracted by the tubules, as if the phospholipids self-assemblies were negatively charged objects.

Behavior of tubules in a magnetic fluid: "susceptibility" of tubules

The relevant parameter to describe the orientation of a tubule in a magnetic field is the relative difference between the magnetic permeability of the tubule (µ tub ) and the one of the magnetic fluid (µ FF ). We shall define the ratio χ (which is not a real susceptibility) as:

χ = µ tub -µ FF µ FF (1)
χ is only a convenient ratio which sign indicates if the tubule is more or less magnetic than the surrounding medium.

Magnetophoresis: effect of a magnetic field gradient

Magnetophoresis is sensitive to the sign of the χ parameter. The field x H orients the tubule along the Ox axis while the field gradient causes its migration along Oy with a constant velocity v  . The tubule is described as a cylinder of radius r and length l. Its motion is driven by the magnetic force m F 

:  F m = χµ FF VH x  ∇ y H x ( ) (2)
In fact we use dilute magnetic fluids with a magnetic susceptibility smaller than 10 -3 , so that µ FF can be approximated by µ 0 . Viscous friction on the tubule surface results in a drag force v F  given by (from [START_REF] Landau | Mécanique des fluides[END_REF]):

v 4 F 4 v l ln rv   η = πη   r     (3)
where η and r are respectively the viscosity and the volumic mass of the surrounding liquid. Because the volume fraction of the magnetic colloidal particles in the magnetic fluid is of a few percents, those values may be taken the same as for pure water (η= 10 -3 Pa s and r= 10 3 kg m -3 ). Both forces being proportional to l, their balance leads to a velocity v  which does not depend on the tubule length:

( )

2 0 x y x 4 v r ln H H 4 rv   η = χµ ∇ η   r     (4)
The geometry of a field gradient (along Oy) perpendicular to the field direction (Ox) enables to sort the tubules by their χ value but not by their size, as it is observed experimentally.

Under a field H x = 2.4 × 10 4 A m -1 (300 G) and a field gradient 9.6 × 10 5 A m -2 (120 G cm -1 ), the mean velocity is -15 µm s -1 in the anionic magnetic fluid (φ = 1.8%) and +0.9 µm s -1 in the cationic magnetic fluid (φ = 2.0%). A typical value for r measured on the electronic micrographs is 0.25 µm. Thus equation 4 gives χ= -0.20 for the anionic magnetic fluid and χ= +0.12 for the cationic one.

Effect of a rotating magnetic field

When a rotating magnetic field H  is applied to tubules, their long axes (Fig. 17) are not parallel to H  , and they bear a magnetic torque Γ m . It is exactly balanced by a viscous torque Γ v in the stationary rotation regime. In this part we have to consider the tubules as elongated ellipsoids for the viscous torque on an ellipsoid rotating with angular velocity Ω in a liquid of viscosity η has a known expression proposed by Perrin for a>5b [START_REF] Perrin | [END_REF]

: v Γ = 4ηV a b     2 2 ln 2a b     -1 Ω (5)
The torque acting on an axisymetric ellipsoid of long semiaxis a, short semi-axis b, and volume V is [START_REF] Landau | Electrodynamique des milieux continus[END_REF]:

Γ m = χ 2 µ FF 2 2 + χ ( ) H 2 V sin 2α ( ) (6)
Stationary rotation is possible as long as there exists an angle α wich satisfies Γ m =Γ v . But when the angular velocity exceeds a critical value Ω c , the magnetic torque can no longer balance the visous one (the balance gives sin(2α)>1). At the critical point sin(2α)=1 and hence α=45°:

Ω c = χ 2 2 + χ µ 0 H 2 2 ln 2a b     -1 8η a b     2 (7)
In the case of a tubules in an anionic ferrofluid (volume fraction of particles 3.5%) we measured Ω c /2π=0.5Hz for H=4.0×10 3 A m -1 (51 Gauss). Assuming an aspect ratio a/b=10 (Fig. 12) this second order equation gives a negative root, χ= -0.21. In the other case, i.e. tubules in a cationic magnetic fluid (φ=1.9%), Ω c /2π=0.05Hz for H=6.3×10 3 A m -1 (80 Gauss) and a/b=20 (Fig. 15), we find a positive root χ=+0.08. The choice between the roots of opposite signs in the precedent estimations is driven by the magnetophoresis experiment, which is sensible to the sign of the χ parameter.

Even if the so calculated values of the χ parameters are only approximative, the three experiments on tubules in a magnetic fluid (alignment by a static filed, stationary rotation and magnetophoresis) lead to the same result: the tubules are less magnetic than the carrier liquid (χ<0) when this one is an anionic magnetic fluid, although they are more magnetic than the surrounding medium (χ>0) when the magnetic particles are cationic. Besides, the lack of magnetic permeability in the fisrt case is higher than the excess in the second case. This result accounts for the following conclusion: in an anionic magnetic fluid, tubules and negatively charged particles do not interact, and tubules behave as magnetic holes [START_REF] Skjeltorp | [END_REF] in a magnetic medium; in a cationic magnetic fluid, particles stick on the tubules even fill them, and tubules become more magnetic than the medium. Nevertheless, in this last case, the phospholipidic membrane which is not magnetic lowers the χ value.

Conclusion and prospects

The use of molecular self-assembly of lipids for the fabrication of micrometric magnetic tubules is reported here. Two kinds of aqueous magnetic fluid (with cationic or anionic particles) lead to different systems and well-separated behaviors. The characterization of the magnetic tubules reveals an electrostatic interaction between the tubules behaving as negative objects and the particles according to the sign of their surface charges. Electron microscopy allows to interpret these experiments: anionic particles do not interact with tubules surface, whereas cationic particles stick to the membrane and seem to fill the tubules. It appeared that the tubules dispersed in a magnetic fluid in every case orientate along the direction of an applied magnetic field of low intensity and follow a rotating magnetic field. In a magnetic field gradient, the way the tubules moves is function of the sign of particles surface charges. Tubules full of cationic particles are more magnetic than the surrounding medium and tubules mixed with anionic particles are less magnetic.

These results illustrate again the possibility to use magnetic fluids either as an "inert" medium for the orientation of biological species or as agents able to modify self-assemblies of phospholipids in order to obtain magnetic hybrid organicinorganic objects.

Fig. 1 .

 1 Fig. 1. Electrophoresis basic experiment. The electric field is about 10 3 V m -1.

Fig. 2 .

 2 Fig. 2. Magnetic alignment by a rotating magnetic field. The two pairs of coils and the appropriate electronic setup are sketched.

Fig. 3 .

 3 Fig. 3. Magnetophoresis experiment. The field lines are parallel one to each other in the region of the capillary and the field gradient is perpendicular to them.

Fig. 4 .

 4 Fig. 4. Optical micrograph picture of the tubules which appear as black cylinders with a relatively broad distribution of lengths. The bar represents 20 µm.

Fig. 5 .

 5 Fig. 5. Electron micrograph image (negative stain microscopy) of a DC8,9PC tubule. This one seems to have one bilayer wall only. This bilayer is rolled so as to delimit a hollow cylinder. The bar represents 1 µm.

Fig. 6 .

 6 Fig. 6. (a) Schematic experimental velocity profile of tubules in pure water under an applied electric field. The surface backflow is much smaller than the principal flow. (b) Electroosmosis velocity profile with closed capillary conditions as described in reference [19]. The profile is parabolic and the total flow is zero.

Fig. 7 .

 7 Fig. 7. Negative stain electron micrograph of a helix (unrolled tubule) dispersed in an anionic magnetic fluid. The coloring agent induces the precipitat ion of the particles on the membrane. The bar represents 0.5 µm.

Fig. 8 .

 8 Fig. 8. Electron micrograph image, without coloration, of a tubule combined with the same anionic magnetic fluid as in Fig. 7. The particles form a uniform film which covers partially the membrane. Bar length is 1 µm.

Fig. 9 .

 9 Fig. 9. Optical micrograph of tubules alignment under a field of 2.4 × 10 3 A m -1 (300G). Bar length is 5 µm.

Fig. 10 .

 10 Fig. 10. ( a) Orientation time of tubules in an anionic magnet ic fluid as a function of the applied magnetic field H. The symbols correspond to various volume fractions φ in magnetic particles. (b) Orientation time of tubules in a cationic magnetic fluid as a function of the applied magnetic field H. The symbols correspond to various volume fractions φ in magnetic particles. The fields allowing to align the tubules are higher than for Fig. l0a.

Fig. 11 .

 11 Fig. 11. Time-space diagram of tubules magnetophoretic motion s: ( a) ( b) (c) tubules in an anionic magnetic fluid (φ = 1.8 %) at several field gradient values (a = 9.6, b = 6.6, and c= 3.1 × 10 5 A m -2 ) ; (d ) ( e) (f) tubule, in a cationic magnetic fluid (φ = 2 %) at a given field gradient (9.6 × 10 5 A m -2 ) for several tubule lenghts (d = 9, e = 21 and f = 27 µm).

Fig. 12 .

 12 Fig. 12. Synchronous rotation of a tubule in an anionic magnetic fluid (φ = 3.5%) under a 6.3 × 10 3 A m -1 (80 G) field rotating at 0.1 Hz. Theperiod T is l0 s and the time increment between two consecutive pictures of the same column is T/4. The bar length is 5 µm.

  . (a) Tubule-vesicle transition temperature versus volume fraction φ of magnetic nanoparticles in the case of an anionic magnetic fluid (squares) and in the case of a cationic magnetic fluid (triangles). (b) Images of a tubule undergoing transition under optical microscopy.

Fig. 14 .

 14 Fig. 14. Electron micrograph pictures of tubules with cationic magnetic particles for different magnifications: a) Tubules appear like black cylinders full of particles in a diluted dispersion of the same particles. Bar length is 2 µm. b) This magnification permits to observe the membrane (two white bands) delimiting the walls of the cylinder containing the particles. Bar length is 0.5 µm. c) At the extremity the tubule seems to bring out the panicles. Bar length is 0.5 µm.

Fig. 15 .

 15 Fig. 15. Synchronous rotation of a tubule in a cationic magnetic fluid (φ = 2%) submitted to a rotating magnetic field (intensity 6.3 × 10 3 A m -1 (80 G), frequency 0.05 Hz). The period T is 20 s and the time increment between two consecutive pictures of the samc column is T/4=5s. The bar length is 5 µm.

Fig. 16 .

 16 Fig. 16. Phospholipidic bilayer in the gel state (Lβ'). The polar head is tilted.

Acknowledgements

The authors thank D. Riveline for his help with the electrophoresis experiments, J. Servais for the engineering of the electronic setups and J-C. Bacri for his comments on the manuscript.