Soili M Lehto
email: soili.lehto@kuh.fi

Anu Ruusunen

Leo Niskanen

Tommi Tolmunen

Sari Voutilainen

Heimo Viinama

George A Kaplan

Jussi Kauhanen

Á H Viinama

Á S Voutilainen

Á J Kauhanen

Elevated depressive symptoms and compositional changes in LDL particles in middle-aged men

Keywords: Apolipoprotein B, Cholesterol, Depression, Low-density lipoprotein cholesterol

Depression and cardiovascular disease (CVD) are closely associated, but the mechanisms underlying this connection are unclear. Regardless of the low cholesterol levels observed in depression, a small particle size of lowdensity lipoproteins (LDL), as well as elevated apolipoprotein B (ApoB) levels, are related to increased CVD risk, even when levels of LDL cholesterol are low. We examined the association between elevated depressive symptoms and compositional changes in serum LDL particles in a sample of 2,456 middle-aged Finnish men. Depressive symptoms were assessed with the 18-item Human Population Laboratory Depression Scale, and the study population was divided into two groups (elevated depressive symptoms, n = 269; non-depressed, n = 2,187). The levels of serum total cholesterol (TC), low-and high-density lipoprotein cholesterol (LDL-C, HDL-C), triglycerides (TG), and ApoB were determined. The LDL-C/ApoB ratio, a marker of compositional changes in LDL particle size, was calculated. The group with elevated depressive symptoms had lowered levels of serum TC (P = 0.028) and LDL-C (P = 0.008). No differences were observed in the LDL-C/ApoB ratio. The likelihood for belonging to the group with elevated depressive symptoms increased 10% for each 0.5 mmol/l decrease in the levels of TC (P = 0.002) or LDL-C (P = 0.001) in regression models adjusted for age, examination years, marital and socioeconomic status, energy expenditure, body mass index, CVD history, alcohol consumption, smoking, and the use of lipid-lowering, antidepressant and antipsychotic medications. Our findings suggest that greater small-particle LDL levels are not associated with depression, and are thus unlikely to underlie the association between cardiovascular risk and depression.

Introduction

The two major conditions affecting public health, depression and cardiovascular disease (CVD), are closely associated, although the mechanisms underlying this connection are unclear [START_REF] Joynt | Depression and cardiovascular disease: mechanisms of interaction[END_REF]. Altered lipid levels have been observed in both conditions. However, while high levels of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) are associated with cardiac morbidity, low TC and LDL-C levels have been observed in depression [START_REF] Partonen | Association of low serum total cholesterol with major depression and suicide[END_REF][START_REF] ¨s | Serum cholesterol and depressive symptoms in elderly Finnish men[END_REF]. Nevertheless, these opposite depression-related observations could be explained by lipid-related factors other than the conventional cholesterol parameters. For instance, a small LDL-C particle size and elevated apolipoprotein B (ApoB) levels, both related to increased CVD risk even when levels of LDL-C are low [START_REF] Lamarche | Small, dense low-density lipoprotein particles as a predictor of the risk of ischemic heart disease in men. Prospective results from the Que ´bec Cardiovascular Study[END_REF], could underlie the connection between depression and CVD. To the best of our knowledge, however, no population-based studies have explored the role of LDL-C particle size and ApoB in depression.

Previous investigations into the associations between lipid levels and depression have provided highly discrepant findings, and the differences in study populations as well as the lack of adjustment for several possible confounders may have further increased this discrepancy. Regardless of the observed inconsistency, however, Shin and co-workers [START_REF] Shin | Are cholesterol and depression inversely related? A meta-analysis of the association between two cardiac risk factors[END_REF] concluded in their recent meta-analysis that the findings regarding an inverse association between TC levels and depressive symptoms appear to be convincing. No other lipid fractions presented such a reliable association. Nevertheless, the total number of investigations was fairly small for LDL-C and high-density lipoprotein cholesterol (HDL-C), leading to limitations in the analyses. Future investigations were recommended to focus on the roles of these less well-examined lipids in depression. Furthermore, the association between triglycerides (TG) and depressive symptomatology, previously observed to be discrepant, was not evaluated in the meta-analysis. Some studies have detected no association between TG and depression in men [START_REF] ¨s | Serum cholesterol and depressive symptoms in elderly Finnish men[END_REF][START_REF] Lindberg | Serum lipids and mood in working men and women in Sweden[END_REF][START_REF] Nakao | Relationship between major depression and high serum cholesterol in Japanese men[END_REF], while some mixed-gender samples have observed higher TG levels in depressed subjects [START_REF] Huang | Serum lipid profiles in major depression with clinical subtypes, suicide attempts and episodes[END_REF].

In order to examine the role of LDL-C particle size and ApoB in depression, and to clarify the roles of LDL-C, HDL-C and TG, we investigated the relationship between depressive symptomatology and the levels of serum lipids and lipoproteins in a sample of 2,456 Finnish middle-aged men.

Methods

The Kuopio Ischemic Heart Disease Risk Factor Study was a population-based study of risk factors for ischemic heart disease and other outcomes among middle-aged men in the Kuopio region of eastern Finland [START_REF] Salonen | Is there a continuing need for longitudinal epidemiological research? The Kuopio ischaemic heart disease risk factor study[END_REF]. A total of 2,682 participants (participation rate 82.9%) aged 42-60 years were recruited for the baseline examination, which occurred between March 1984 and December 1989. Data were incomplete for 226 participants; thus, complete data were available for 2,456 men. All participants provided written informed consent after full explanation of the study, and the study protocol was approved by the Research Ethics Committee of the University of Kuopio.

Assessment of depressive symptoms

Depressive symptoms were assessed with the 18-item Human Population Laboratory Depression Scale (HPL Depression Scale) [START_REF] Kaplan | Psychosocial predictors of depression. Prospective evidence from the human population laboratory studies[END_REF]. The scale consists of items dealing with mood disturbance, a negative self-concept, loss of energy, problems with eating and sleeping, trouble with concentration, and psychomotor agitation. It was developed especially for screening general population samples, and it also conceptually resembles other brief symptom checklists such as the Center for Epidemiological Studies Depression Scale (CES-D) [START_REF] Roberts | Prevalence of depressive symptoms among Mexican Americans[END_REF][START_REF] Roberts | Sex differences in depression reexamined[END_REF]. The HPL Depression score is generated by assigning one point for each true or false answer that is indicative of depression (range 0-18). In some items, ''often'' or ''never'' responses, whichever appropriate, were calculated as one point. A cut-off score of five or more has earlier been used to define elevated depressive symptoms [START_REF] Kaplan | Psychosocial predictors of depression. Prospective evidence from the human population laboratory studies[END_REF][START_REF] Roberts | Sex differences in depression reexamined[END_REF][START_REF] Tolmunen | Association between depressive symptoms and serum concentrations of homocysteine in men: a population study[END_REF], and thus we applied the same cut-off point. Those who scored five or more were considered to have elevated depressive symptoms (n = 269, 10.9% of participants). Poor appetite as a covariate was derived from the HPL Depression Scale.

Socioeconomic factors, illness history and medications

Participants also completed questionnaires relating to their sociodemographic background, somatic and psychiatric medications and history of physical illnesses, including CVD. Marital status was reported as a two-class variable (married or living with a partner vs. living alone). A positive CVD history was coded based on the following criteria: first, all subjects had at least one of the following physiciandiagnosed conditions: myocardial infarction, angina pectoris, other coronary conditions, cardiomyopathy, cardiac insufficiency or stroke. Second, all also used nitrates at least once per week, and had angina pectoris according to the World Health Organization angina pectoris questionnaire (the Rose Angina Questionnaire, RQ), a validated instrument to assess symptoms of typical angina pectoris in the general population [START_REF] Rose | The diagnosis of ischaemic heart pain and intermittent claudication in field surveys[END_REF][START_REF] Sorlie | Repeatability and validity of the Rose questionnaire for angina pectoris in the atherosclerosis risk in communities study[END_REF]. A variety of indicators of adulthood socioeconomic status were available, including current income, current and previous occupations, the highest level of education, the perception of financial security and housing tenure. In addition, an index of material living conditions was created by summing the number of material possessions (e.g., dishwasher, car, summer cottage) from a list of 12. The variable ''adulthood socioeconomic status'' was formed from these indicators [START_REF] Wilson | Association between plasma fibrinogen concentration and five socioeconomic indices in the Kuopio ischemic heart disease risk factor study[END_REF][START_REF] Lynch | Childhood and adult socioeconomic status as predictors of mortality in Finland[END_REF][START_REF] Lynch | Socioeconomic status and carotid atherosclerosis[END_REF].

Smoking and alcohol consumption

The current number of cigarettes, cigars and pipefuls of tobacco smoked daily and the duration of regular smoking in years were recorded using a self-administered questionnaire. The number of years smoked was defined as the sum of the years of smoking, whether it had occurred continuously or during several periods. The lifelong exposure to smoking (pack years) was estimated as the product of years smoked and the number of tobacco products smoked daily at the time of the examination, or for ex-smokers at the time when they had last smoked. Alcohol consumption (g/wk) was assessed with a structured quantity-frequency method using the Nordic Alcohol Consumption Inventory for drinking behaviour over the previous 12 months [START_REF] Kauhanen | Coping with inner feelings and stress: heavy alcohol use in the context of alexithymia[END_REF].

Physical activity

Physical activity was assessed using the 12-month Physical Activity Questionnaire [START_REF] Salonen | Assessment of physical activity in population studies-validity and consistency of the methods in the Kuopio Ischemic Heart Study[END_REF][START_REF] Ainsworth | Compendium of physical activities: classification of energy costs of human physical activities[END_REF]. The checklist included the most common physical activities of middle-aged men, for example walking, jogging, bicycling, swimming, and ball games. The subjects were asked to record the frequency, average duration, and intensity of each activity performed. A trained nurse checked and completed the questionnaire during an interview. The energy expenditure from physical activity was expressed as kcal per day.

Laboratory measurements

The weight and height of the participants were measured and the body mass index (BMI, kg/m 2 ) was calculated. The subjects came to the Research Institute of Public Health for venous blood sampling between 8 and 10 am. They had been instructed to abstain from alcohol for the previous 3 days, and from smoking and eating for 12 h before the laboratory testing. After a 30-min rest in a supine position, blood samples were drawn by venipuncture and collected into vacuum tubes (Venoject; Terumo, Leuven, Belgium) that contained ethylenediamine tetraacetic acid (EDTA) and placed on an ice-bath. No tourniquet was used. Plasma was separated by centrifugation for 15 min at ?4°C (3,500 g) and was kept in an ice-bath before further handling. Lipoprotein fractionation was started on the day of blood collection.

Measurements of serum lipid and lipoprotein concentrations have been thoroughly described elsewhere [START_REF] Salonen | Prevalence of carotid atherosclerosis and serum cholesterol levels in eastern Finland[END_REF]. In brief, lipoproteins were separated from fresh serum samples by combined ultracentrifugation and precipitation. The serum TC (reference value \5.0 mmol/l), HDL-C (reference value [1.0 mmol/l), LDL-C (reference value \3.0 mmol/l), and triglyceride (TG) (reference value \2.0 mmol/l) concentrations were measured enzymatically with the use of an autoanalyzer (Kone Specific, Kone Instruments, Espoo, Finland). Further, ApoB levels were determined by an immunoturbidimetric method (Kone Oy, Espoo, Finland). We also calculated the LDL-C/ApoB ratio, a marker of compositional LDL changes and an estimate of altered LDL particle size [START_REF] Mcnamara | Effect of gender, age, and lipid status on low density lipoprotein subfraction distribution. Results from the Framingham Offspring Study[END_REF].

Statistics

The group differences between the participants with elevated depressive symptoms (n = 269) and the rest of the cohort (n = 2,187) were examined using the Mann-Whitney U-test for continuous variables, and the v 2 test and Fisher's exact test for categorical variables. The nonparametric Mann-Whitney U-test was used due to the skewed distribution of the variables.

The lipid variables exhibiting significant group differences (P \ 0.05) were further tested with logistic regression modelling. The likelihood (odds ratio, OR; 95% confidence interval, CI) for belonging to the group with elevated depressive symptoms was examined in a model adjusted for age, examination years, marital status, adulthood socioeconomic status, energy expenditure, BMI, CVD history, alcohol consumption, smoking, and the use of lipid-lowering, antidepressant and antipsychotic medications. Examination years (all except the first year in order to form a dummy variable) were included in the regression model as technical covariates in order to control for the possible bias created by the long 6-year period of baseline data collection. The continuous measures of the examined lipid variables were inserted separately into the regression model due to their high intercorrelations (Model 1: TC; Model 2: LDL-C).

Results

The characteristics of the study population are presented in Table 1. The group with elevated depressive symptoms reported more living alone and smoking, a higher alcohol consumption, an appetite decrease and CVD history, and had a lower level of physical activity. Furthermore, they used more antidepressant and antipsychotic medications. No differences were observed in the LDL-C/ApoB ratio between the depressed and other participants. None of the participants in the group with elevated depressive symptomatology used lipid-lowering medication. Nevertheless, they had lower levels of TC and LDL-C.

In logistic regression modelling, the likelihood of belonging to the elevated depressive symptoms group increased by 10% for each 0.5 mmol/l decrease in the levels of TC (OR 1.11, 95% CI 1.04-1.18, P = 0.002) or LDL-C (OR 1.12, 95% CI 1.04-1.19, P = 0.001). The history of CVD was independently associated with depression, regardless of the tested lipid variable (Table 2).

Discussion

Principal findings

We observed lower levels of TC and LDL-C, but no alterations in the LDL-C/ApoB ratio or ApoB levels in the group with elevated depressive symptomatology. The risk of belonging to this group increased by 10% for each 0.5 mmol/l decrease in the levels of either TC or LDL-C.

Comparison with previous studies

Our data are consistent with previous investigations in showing pronounced CVD morbidity in depression [START_REF] Joynt | Depression and cardiovascular disease: mechanisms of interaction[END_REF]. Increased levels of CVD, however, have also been associated with a small LDL-C particle size and high ApoB levels [START_REF] Lamarche | Small, dense low-density lipoprotein particles as a predictor of the risk of ischemic heart disease in men. Prospective results from the Que ´bec Cardiovascular Study[END_REF][START_REF] Avogaro | Plasma level of lipoprotein A-1 and apolipoprotein B in human atherosclerosis[END_REF]. Nevertheless, the LDL-C/ApoB ratio, an estimate of LDL particle size, and ApoB levels presented no associations with depression in our data. Thus, the present findings suggest that a small LDL-C particle size and high ApoB levels do not modify the increased depression-related CVD risk in middle-aged men.

In their recently published meta-analysis, Shin et al. [START_REF] Shin | Are cholesterol and depression inversely related? A meta-analysis of the association between two cardiac risk factors[END_REF] reported that TC levels were inversely associated with depression. Our findings are in line with these observations. The levels of LDL-C, however, presented no association with depression in the same meta-analysis [START_REF] Shin | Are cholesterol and depression inversely related? A meta-analysis of the association between two cardiac risk factors[END_REF]. The total number of subjects in all studies investigating the LDL-C levels was only 6176, whereas in the TC studies the respective figure was as high as 45,098. The subject number of the present study essentially adds to the total number of subjects in LDL-C investigations, and also supports the previous findings of an inverse association between depression and LDL-C [START_REF] ¨s | Serum cholesterol and depressive symptoms in elderly Finnish men[END_REF][START_REF] Lindberg | Serum lipids and mood in working men and women in Sweden[END_REF][START_REF] Olusi | Serum lipid concentrations in patients with major depressive disorder[END_REF].

A large Finnish prospective study on 29,133 male smokers showed an inverse association between TC and depressive symptoms [START_REF] Partonen | Association of low serum total cholesterol with major depression and suicide[END_REF]. Our findings demonstrate that, regardless of smoking habits, the same association remains observable in men. Moreover, our group has previously observed low HDL-C levels, but no alterations in TC or LDL-C, in a mixed-gender sample of same-area general population subjects with a diagnosis of major depressive disorder, and a recorded 7-year history of depressive symptoms [START_REF] Lehto | Low HDL cholesterol associates with major depression in a sample with a 7-year history of depressive symptoms[END_REF]. Since the inverse association between TC levels and depression has been observable regardless of gender [START_REF] Shin | Are cholesterol and depression inversely related? A meta-analysis of the association between two cardiac risk factors[END_REF], the different gender distribution of these two investigations is unlikely to affect the observed discrepancy. Nevertheless, the baseline data collection for the present study took place more than 10 years earlier than that of the other study from the same area, and thus time-related dietary or life-style alterations could explain these discrepant observations. We observed a prevalence of 12% for elevated depressive symptoms, which is slightly higher than the previously observed 7.4% prevalence of major depressive disorder in men in Finland [START_REF] Lindeman | The 12-month prevalence and risk factors for major depressive episode in Finland: representative sample of 5993 adults[END_REF].

Possible mechanisms

The most well-known biological explanation for the effect of low cholesterol levels on depression is the theory of Engelberg [START_REF] Engelberg | Low serum cholesterol and suicide[END_REF], who suggested that low cell-membrane cholesterol levels lead to decreased serotonergic transmission. This hypothesis was further supported by neuroendocrine challenge testing that showed a positive association between serotonergic functions and cholesterol levels in men, but not in women [START_REF] Terao | Relationship between serum cholesterol levels and meta-chlorophenylpiperazine-induced cortisol responses in healthy men and women[END_REF]. Both of the previous hypotheses suggest that low cholesterol levels lead to depression. However, inverse causal relationships also need to be considered. A depression-related poor appetite may lead to lowered cholesterol levels. In our sample, however, adjustment for poor appetite did not alter the findings, although the elevated depressive symptomatology group reported a poor appetite more often than the nondepressed group.

Strengths and limitations

To the best of our knowledge, no previous investigations have examined the alterations in LDL particle size estimates in depression. The main strength of this study was the large, representative sample of middle-aged Finnish men. In addition, we were able to adjust for several major confounding factors such as appetite, BMI, CVD history, physical activity, socioeconomic factors, and relevant psychiatric and somatic medications. Due to the early period of data collection, i.e., 1984-1989, very few individuals in the study sample used lipid-lowering medications. This can be considered as a strength, because it minimises the potential confounding effect of these medications on our findings. However, there are some limitations in this study. First, the sample comprised exclusively men, and thus these findings cannot be generalized to women. Second, due to the cross-sectional nature of the present study, we are unable to draw causal conclusions on the connections between depressive symptomatology and serum lipids. Third, due to the large sample size, we were unable to perform diagnostic psychiatric interviews for the study subjects. The assessment of mood was, however, performed with a scale specifically designed for epidemiological studies [START_REF] Roberts | Prevalence of depressive symptoms among Mexican Americans[END_REF][START_REF] Roberts | Sex differences in depression reexamined[END_REF]. In addition, the HPL depression scale is highly correlated with the well-established Beck Depression Inventory [START_REF] Kaplan | Psychosocial predictors of depression. Prospective evidence from the human population laboratory studies[END_REF][START_REF] Beck | An inventory for measuring depression[END_REF].

Conclusions

Our findings suggest that greater levels of small-particle LDL, a factor predisposing to coronary heart disease, are not associated with depression. Thus, LDL particle size alterations may be unlikely to explain the association between cardiovascular risk and depression. Since the observed depression-related low levels of TC and LDL-C may be indicators of serotonergic disturbances, monitoring both low and high cholesterol levels may be warranted in all health care settings.

Table 1

 1 Characteristics of the study sample

		Elevated depressive	Non-depressed	Test statistics*	P value
		symptoms (n = 269)	(n = 2,187)
	Age, years	53.49 (4.59)	53.01 (5.10)	Z = -1.52	0.128 a
	HPL depression scale scores	6.48 (1.86)	1.32 (1.28)	Z = -27.49	\0.001 a
	Married or living with a partner, n (%)	219 (81.4)	1,912 (87.4)	v 2 = 7.54	0.006 b
	Baseline examination performed, n (%)
	1985	52 (10.7)	434 (89.3)	v 2 = 0.04	0.842 b
	1986	51 (12.0)	375 (88.0)	v 2 = 0.55	0.459 b
	1987	44 (10.3)	382 (89.7)	v 2 = 0.21	0.650 b
	1988	31 (9.3)	302 (90.7)	v 2 = 1.07	0.302 b
	1989	52 (13.0)	348 (87.0)	v 2 = 2.05	0.152 b
	Length of education, years	8.62 (3.52)	8.63 (3.40)	Z = -0.12	0.901 a
	CVD history, n (%)	159 (59.1)	771 (35.3)	v 2 = 57.9	\0.001 b
	Lipid-lowering medication, n (%)	0 (0)	12 (0.5)	-	0.384 c
	Antidepressant medication, n (%)	21 (7.8)	10 (0.5)	-	\0.001 c
	Antipsychotic medication, n (%)	18 (6.7)	26 (1.2)	-	\0.001 c
	Smoking, pack years	11.22 (19.69)	7.99 (16.14)	Z = -2.46	0.014 a
	Alcohol consumption (g/week)	95.80 (138.58)	73.55 (137.06)	Z = -2.05	0.040 a
	Poor appetite, n (%)	53 (19.7)	78 (3.6)	v 2 = 123.60	\0.001 b
	Energy expenditure, kcal/d	137.33 (196.68)	141.23 (172.75)	Z = -2.36	0.018 a
	Body mass index, kg/m 2	26.93 (3.84)	26.88 (3.49)	Z = -0.74	0.461 a
	Total cholesterol (mmol/l)	5.77 (1.03)	5.93 (1.09)	Z = -2.19	0.028 a
	LDL-C (mmol/l)	3.88 (0.94)	4.06 (1.03)	Z = -2.67	0.008 a
	HDL-C (mmol/l)	1.30 (0.31)	1.29 (0.30)	Z = -0.08	0.934 a
	Triglycerides (mmol/l)	1.35 (0.84)	1.30 (0.83)	Z = -0.49	0.622 a
	ApoB (g/l)	1.01 (0.23)	1.04 (0.25)	Z = -1.75	0.079 a
	LDL-C/ApoB	3.90 (0.60)	3.96 (0.73)	Z = -1.10	0.272

a Values are means (±SD) unless otherwise stated ApoB Apolipoprotein B, CVD cardiovascular disease, HDL-C high-density lipoprotein cholesterol, HPL human population laboratory, LDL-C low-density lipoprotein cholesterol, SD standard deviation * Presented where applicable a Mann-Whitney U-test b v 2 test c Fisher's exact test

Table 2

 2 Odds ratios (95% confidence intervals) for the likelihood of belonging to the group with elevated depressive symptoms in logistic regression analysis HDL-C high-density lipoprotein cholesterol, LDL-C low-density lipoprotein cholesterol, TC total cholesterol, TG triglycerides a The likelihood for each 0.5 mmol/l decrease in the level of the examined lipid covariate

		OR	95% CI	P
	Model 1
	Age	0.99 0.96-1.02	0.493
	Married or living with a partner	0.64 0.45-0.92	0.014
	Examination year
	1985	1.22 0.77-1.93	0.410
	1986	1.49 0.93-2.38	0.094
	1987	1.17 0.72-1.92	0.525
	1988	1.16 0.68-1.98	0.577
	1989	1.61 1.00-2.58	0.049
	High socioeconomic status	1.06 1.02-1.10	0.001
	High energy expenditure	1.00 1.00-1.00	0.610
	High body mass index	0.98 0.95-1.02	0.391
	History of cardiovascular disease (yes)	2.64 2.00-3.48 \0.001
	Smoking (high number of pack years)	1.00 0.99-1.01	0.812
	Alcohol use (high)	1.00 1.00-1.00	0.106
	Lipid-lowering medication (yes)	0.00 0.00-	1.00
	Antidepressant medication (yes)	14.9	6.45-34.5 \0.001
	Antipsychotic medication (yes)	2.92 1.39-6.13	0.005
	High TC a	1.11 1.04-1.18	0.002
	Model 2
	High LDL-C a	1.12 1.04-1.19	0.001

S. M. Lehto et al.

Acknowledgments SML was supported by Kuopio University Hospital EVO funding and research grants from the Foundation for Psychiatric Research and Finnish Medical Foundation. The authors would like to thank the personnel of the Research Institute of Public Health for their valuable contribution to this study.


Jenny L Macpherson
email: jenny.macpherson@rhul.ac.uk

Paul W Bright
email: p.bright@rhul.ac.uk

Movements of radio-tracked American mink (Neovison vison) in extensive wetland in the UK, and the implications for threatened prey species such as the water vole (Arvicola amphibius)

Keywords: Neovison vison, Reed bed, Refuge, Conservation, Arvicola amphibius, Habitat use

In a number of countries around the world, introduced American mink are acknowledged to have had a negative effect on a number of native species, many of which are of particular conservation concern. In the UK, there has been an observed correlation between the spread of mink and the decline of the, once common and widespread, water vole. Large wetlands, such as extensive reed bed, appear to mitigate the impact of mink predation on water voles and some bird species. The present study was carried out to test the hypothesis that the observed refuge effect of reed beds arises from the way mink forage in this type of wetland. The results suggest that the interior of reed bed offers a spatial refuge for water voles, and other species, from predation by mink, because more than 60% of mink foraging activity occurred within 10 m of a main (>10 m wide) channel. Where mink ventured within the reed bed itself, they associated closely with scrub. The implication is that easily navigable channels and areas of scrub probably compromise the refuge effect of reed beds. This can be used to inform management recommendations at these sites.

Introduction

In a number of countries around the world, introduced American mink are acknowledged to have had a negative effect on a number of native species, some of which are of particular conservation concern (see [START_REF] Macdonald | The American mink: the triumph and tragedy of adaptation out of context[END_REF] and references therein). In the UK, although numbers and distribution of the water vole began to decline before American mink became established [START_REF] Jefferies | The changing otter population of Britain 1700-1989[END_REF], there has been an observed correlation between the spread of mink and an acceleration in the rate of the water vole decline [START_REF] Jefferies | The water vole and mink survey of Britain 1996-1998 with a history of the long term changes in the status of both species and their causes[END_REF][START_REF] Lawton | Habitat and the distribution of water voles-why are there gaps in a species range?[END_REF][START_REF] Strachan | The Water vole Arvicola terrestris in Britain 1989-1990: its distribution and changing status[END_REF]. This has been particularly pronounced where habitat loss has resulted in water voles being constrained to narrow linear strips of suitable habitat along a water course, in what has been termed 'the tightrope hypothesis' (Barreto and Macdonald 1998). However, in some areas, this has apparently been buffered by the effect of certain habitat types-in particular non-linear wetlands such as grazing marsh (MacPherson and Bright 2010) and extensive reed beds (Barreto et al. 1998;[START_REF] Carter | Reedbeds as refuges for water voles (Arvicola terrestris) from predation by introduced mink (Mustela vison)[END_REF]. [START_REF] Carter | Reedbeds as refuges for water voles (Arvicola terrestris) from predation by introduced mink (Mustela vison)[END_REF] found that predation rate declined sharply with distance from a main water channel, suggesting that this type of habitat provides water voles with a refuge from predation. It has been demonstrated with real and artificial nests that reed beds also act as a refuge from predation for a number of bird species [START_REF] Baldi | Nest predation in European reedbeds: different losses in edges but similar losses in interiors[END_REF][START_REF] Kristiansen | Egg predation in reedbed nesting Greylag geese Anser anser in Vejlerne, Denmark[END_REF]).

Spatial refugia occur as a result of the predator's patch use behaviour and foraging aptitude [START_REF] Schmidt | Incidental predation, enemy-free space and the coexistence of incidental prey[END_REF]) and have huge potential benefit as a practical method for the conservation of endangered prey as they can be enhanced by management. However, in order to mitigate predation by manipulating the physical environment, it is essential that managers first have an understanding of the way in which predators use that environment [START_REF] Lariviere | Habitat selection and use of edges by striped skunks in the Canadian prairies[END_REF]. So, where there is an apparent refuge effect, it is important to understand the mechanism underlying this in order that its benefits can be maximised. Reed bed, wetland dominated by common reed (Phragmites australis), is an important habitat for species such as bittern (Botaurus stellaris), marsh harrier (Circus aeruginosus), Cetti's warbler (Cettia cetti) and bearded tit (Panurus biarmicus). In addition, it offers a spatial refuge from predation for the water vole [START_REF] Carter | Reedbeds as refuges for water voles (Arvicola terrestris) from predation by introduced mink (Mustela vison)[END_REF], a species of high conservation concern in the UK. In order to maximise the conservation benefits of reed bed, it was important to understand the underlying mechanism, which was likely to relate to the way in which mink utilise this type of wetland.

There have been a number of studies of mink habitat use within their native range [START_REF] Arnold | Habitat use by male mink in relation to wetland characteristics and avian prey abundances[END_REF][START_REF] Stevens | Fall early winter home ranges, movements, and den use of male mink, Mustela vison, in eastern Tennessee[END_REF] as well as along linear waterways and in coastal areas in countries where they have been accidentally or deliberately introduced [START_REF] Bonesi | Winter selection of habitats within intertidal foraging areas by mink (Mustela vison)[END_REF][START_REF] Lode | Movements of the American mink Mustela vision Schreber radiotracked on a river in Britain[END_REF]Niemimaa 1995;[START_REF] Previtali | Habitat use and diet of the American mink (Mustela vison) in Argentinian Patagonia[END_REF][START_REF] Yamaguchi | Habitat preferences of feral American mink in the Upper Thames[END_REF]Zabala et al. 2007b). However, there have been no published studies of mink habitat use in extensive wetlands in the UK.

The aim of the present study was to test the hypothesis that the observed refuge effect of reed beds for water voles from predation by mink [START_REF] Carter | Reedbeds as refuges for water voles (Arvicola terrestris) from predation by introduced mink (Mustela vison)[END_REF] arises from the way mink forage in this type of landscape.

Methods

Mink were radio-tracked at Shapwick Heath and Ham Wall (ST443, 401) in south west England. These two adjacent nature reserves are owned by Natural England and the Royal Society for the Protection of Birds, respectively. Together, the reserves form a contiguous wetland of approximately 500 ha comprising a mosaic of reed fen, marsh, wet heath and grassland.

From the end of March to the end of April, 2002, mink were trapped in single-entry commercial cage traps (Tomahawk Live Trap Co., WI, USA). Each animal was fitted with a waterproof radio transmitter attached to a collar (Biotrack Ltd, Wareham, Dorset, UK). Trapping was carried out in the main areas of reed bed at both sites under licence from DEFRA. Captured animals were weighed, sexed and approximately aged as either adult or juvenile. Males were aged by palpation of the baculum (Elder 1951) and females by the presence or absence of healed or recent mating bite wounds on the back of the neck [START_REF] Ireland | The behaviour and ecology of the American mink (Mustela vison Schreber) in a coastal habitat[END_REF][START_REF] Yamaguchi | Multiple paternity and reproductive tactics of free-ranging American minks, Mustela vison[END_REF]).

After each animal was collared, it was then located every 2 h around the clock with a Telonics TR-2 VHF receiver (Telonics Inc., Mesa, AZ, USA) and three-element Yagi flexible antenna (Biotrack Ltd.). A minimum of 2-h spacing between fixes was to ensure independence of radiolocations. Each animal was followed intensively for a minimum of 5 days [START_REF] Yamaguchi | Multiple paternity and reproductive tactics of free-ranging American minks, Mustela vison[END_REF]) or until at least 30 active fixes were recorded [START_REF] Conner | A comparison of distancebased and classification-based analyses of habitat use[END_REF]. Animals were located by homing in (White and Garrott 1990) and then taking bearings at close range from at least two locations. The position of fixes was determined to the nearest 10 m using a Garmin e-trex handheld GPS (Garmin Ltd., Romsey, Hampshire, UK). Mink were defined as active or inactive according to changes in the strength of the radio signal [START_REF] Kenward | A manual for wildlife radio tagging[END_REF]White and Garrott 1990). When an animal was stationary for more than one fix at a location, it was defined as a lie-up or, if the animal returned to that location more than once, a den.

Radio-tracking data were plotted on to high resolution recent aerial photographs of the site and entered into a Geographic Information System (MapInfo v8) so that the distances to significant features could be measured.

Generalised linear models [START_REF] Mccullagh | Activity patterns and home ranges of the American mink Mustela vison in the Finnish outer archipelago[END_REF]) were used to examine potential correlates of mink activity. The most parsimonious model was selected by first including all explanatory variables and then testing the significance of each (χ 2 statistic) by successive deletion. Explanatory variables considered were: distance from nearest tree or scrub, distance from track and distance from a main channel. A main channel was defined as being more than, or equal to, 10 m wide, as per [START_REF] Carter | Reedbeds as refuges for water voles (Arvicola terrestris) from predation by introduced mink (Mustela vison)[END_REF].

A Poisson error structure with a logarithm link function was used in the model.

Results

Five males and one female were captured and radio collared. One animal, a mature adult male, disappeared after 2 days either as a result of the transmitter failing or because the animal dispersed out of receiver range, so this animal was excluded from the analysis. The remaining five animals were each tracked for a mean of 16.8 (±3.8) days and nights (Table 1).

A generalised linear model was used to test for significance the relationship between the number of active fixes and distance from track, main channels and scrub (Table 2). The number of fixes was negatively correlated with distances above 10 m from both main channels and scrub, suggesting that for mink in this type of habitat at this time of year (March-April), easily navigable channels and scrub are highly significant features. This is shown graphically in Fig. 1.

The mean distance of den sites from a main channel was 15±5.49 m, while the mean distance of lie-up sites to a main channel was much closer at 1.75±0.19 m. These were all in willow (Salix spp.), dense stands of rush (Juncus spp.) and bramble (Rubus fruticosa) scrub.

Discussion

The results of the present study support the hypothesis that the interior of reed bed offers a spatial refuge for water voles from predation by mink, because more than 60% of mink foraging activity occurred within 10 m of a main channel.

It is widely reported that there is a strong association between intensity of habitat use by mink and other mustelids and local prey availability [START_REF] Arnold | Habitat use by male mink in relation to wetland characteristics and avian prey abundances[END_REF][START_REF] Erlinge | Spacing strategy in stoat Mustela erminea[END_REF][START_REF] Gerell | Home ranges and movements of mink Mustela vison Schreber in Southern Sweden[END_REF]. [START_REF] Carter | Reedbeds as refuges for water voles (Arvicola terrestris) from predation by introduced mink (Mustela vison)[END_REF] suggested that the refuge effect of reed bed for water voles with respect to mink predation could be a result of differences in the abundance of alternative prey along main channels or edges and within the reed bed itself. An alternative hypothesis is that predation rates are lower on water voles in reed because of the high availability of alternative prey and consequent refuge effect of relative low water vole density resulting in prey switching by mink. This is not supported by our data; if it were the case, then mink would still be active within the reed bed, hunting for alternative prey.

American and European mink are strongly associated with scrub [START_REF] Yamaguchi | Habitat preferences of feral American mink in the Upper Thames[END_REF][START_REF] Zabala | Landscape features in the habitat selection of European mink (Mustela lutreola) in south-western Europe[END_REF] ). This was supported by the present study. Where mink ventured within the reed bed itself, they associated closely with scrub. The implication is that easily navigable channels and areas of scrub probably compromise the refuge effect of reed beds. This can be used to inform management recommendations at these sites, although there will inevitably have to be a trade-off at many sites between what is optimal for water voles and the habitat requirements of other species, particularly birds.

Mink were radio-tracked during the rut in March because this is when trapping success, particularly of males, is likely to be highest [START_REF] Ireland | The behaviour and ecology of the American mink (Mustela vison Schreber) in a coastal habitat[END_REF]); however, range use is not necessarily typical at this time of year as a result of mating behaviour [START_REF] Yamaguchi | Multiple paternity and reproductive tactics of free-ranging American minks, Mustela vison[END_REF]). In a Scottish study of coastal mink, [START_REF] Ireland | The behaviour and ecology of the American mink (Mustela vison Schreber) in a coastal habitat[END_REF] showed that there was seasonal variation in the length of linear home ranges, especially among males, and that they were highest in March at the peak of the rut. Therefore, it is probable that the males radio-tracked in the present study were extending their ranges in search of females with which to mate, but the type of habitat and features used for hunting and along which to travel are likely to be similar all year. The male bias of the sample must be considered when interpreting the results of the present study. It has been shown that female American mink radio-tracked on a river system utilised narrower streams than males (Zabala et al. 2007a); however, the females' home ranges still included sections of the main (>10 m wide) river. [START_REF] Sidorovich | Individual feeding specialization in the European mink, Mustela lutreola and the American mink, M. vison in north-eastern Belarus[END_REF] also found that, in warm seasons, mink sometimes occupy marshy areas away from stream banks. Further study is required to determine if the apparent preference for wider channels in reed bed, observed in March, is affected by sex or season.

Because of their long, tubular body shape and high surface area-to-volume ratio, mink, like other mustelids, are vulnerable to extreme temperatures [START_REF] Brown | Metabolism of weasels: the cost of being long and thin[END_REF][START_REF] Dunstone | The baculum as an age criterion in mink[END_REF]. They have relatively large home range size and are not central place foragers. As a result of this, they require physical protection from the elements during periods of inactivity. It has been suggested that the availability of suitable den sites is a limiting factor in their abundance and distribution [START_REF] Halliwell | American mink Mustela vison in the upper Thames catchment: relationship with selected prey species and den availability[END_REF]. Availability of suitable den sites is of major importance to female mink when breeding, as protection for kits from predators and for shelter prior to their achievement of thermoregulatory competence. Mink are reported as using between two and ten den sites [START_REF] Birks | Studies on the home range of feral mink (Mustela vison)[END_REF] with most dens being located within 2 m of water [START_REF] Schladweiler | Den use by mink[END_REF]. Rabbit burrows are particularly important as den sites [START_REF] Yamaguchi | Habitat preferences of feral American mink in the Upper Thames[END_REF], but mink will also use tree roots, rock piles, brush piles, culverts or bridge foundations [START_REF] Birks | Studies on the home range of feral mink (Mustela vison)[END_REF][START_REF] Dunstone | The baculum as an age criterion in mink[END_REF]. Dense stands of emergent vegetation may be used by mink as temporary lie-up sites [START_REF] Arnold | Habitat use by male mink in relation to wetland characteristics and avian prey abundances[END_REF][START_REF] Birks | Studies on the home range of feral mink (Mustela vison)[END_REF][START_REF] Sargeant | Selective predation by mink, Mustela vison on waterfowl[END_REF]. In extensive wetland with a high water table, such as the sites used for the present study, rabbit warrens are scarce and in this case mink dens were all in cavities under waterside trees, scrub and other structural vegetation. Reed beds offer few suitable den sites.

Autocorrelation is a potential problem with radiotracking data, resulting in an artificial inflation of sample size. However, locations obtained at t 1 and t 2 can be considered independent provided that sufficient time has elapsed between the two fixes for the animal to have moved from one end of its home range to the other [START_REF] Salvatori | Estimating temporal independence of radio-telemetry data on animal activity[END_REF]White and Garrott 1990). Mink are recorded as achieving mean speeds on land equating to 1.73 km/ h walking and 9.4 km/h when 'bounding' [START_REF] Dunstone | The baculum as an age criterion in mink[END_REF]. The maximum distance between two points of any one of the radio-tracked mink in the present study was 2.755 km (mean 1.621). Therefore, the 2-h spacing of fixes that was used should be sufficient to ensure independence of locations.

The sample size used in the present study is equivalent to that of other published studies of habitat use by mink [START_REF] Arnold | Habitat use by male mink in relation to wetland characteristics and avian prey abundances[END_REF]Niemimaa 1995;[START_REF] Stevens | Fall early winter home ranges, movements, and den use of male mink, Mustela vison, in eastern Tennessee[END_REF][START_REF] Zabala | Landscape features in the habitat selection of European mink (Mustela lutreola) in south-western Europe[END_REF]. However, this is still relatively low. Nevertheless, the present study provides some valuable insights into the way in which mink forage in reed bed habitat and the mechanism by which this type of habitat provides a refuge for water voles and other prey species from mink predation.

Fig. 1

 1 Fig. 1 Percentage of 'active' American mink radio fixes (mean ±SE) at different distances from a main (≥10 m wide) channel and scrub

  ,

	Table 2 Results of the general-ised linear model using count of	Explanatory variable	Parameter estimate	SE	Deviance	χ 2 probability
	active fixes in each class as the
	response variable (Poisson	Constant	2.944	0.150		<0.001
	errors, log link)	10-20 m from channel	-1.609	0.367		<0.001
		20-30 m from channel	-1.721	0.385		<0.001
		30-40 m from channel	-2.156	0.465		<0.001
		40-50 m from channel	-3.86	1.04		<0.001
		>50 m from channel	-2.156	0.465		<0.001
	Estimates and standard errors	<10 m from scrub	0.110	0.206		0.598
	for the significant variables are	10-20 m from scrub	-1.258	0.319		<0.001
	shown. Parameters for factors	20-30 m from scrub	-2.944	0.670		<0.001
	are differences compared with the reference level: <10 m from channel. Regression χ 2	30-40 m from scrub 40-50 m from scrub	-3.86 -3.86	1.04 1.04		<0.001 <0.001
	probability <0.001, deviance	>50 m from scrub	-1.989	0.430	420.654	<0.001
	420.7, residual deviance=102.		Total deviance explained			80.4%

Acknowledgements This study was funded by the People's Trust for Endangered Species, English Nature, the Environment Agency and the Countryside Council for Wales to whom we are very grateful. We would also like to express our thanks to Don MacPherson for help with fieldwork and to Melvyn Yeandle and Sally Mills.


Thekla Jaeckel

Lilla Knels

Monika Valtink
email: monika.valtink@tu-dresden.de

Richard Hw Funk

Katrin Engelmann

Thekla Jäckel

Richard H W Funk

Serum-free SFM corneal organ

Keywords: human corneal endothelium, serum-free medium, protection, apoptosis, corneal organ cultivation medium

à 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

Corneal grafts can easily be preserved in organ culture, but corneal endothelial cell count declines notably about 20 -30% during cultivation. [START_REF] Armitage | Factors Influencing the Suitability of Organ-Cultured Corneas for Transplantation[END_REF] This decline continues post-surgically depending upon pre-and post-surgical risk factors. [START_REF] Abott | Long-term changes in corneal endothelium following penetrating keratoplasty[END_REF][START_REF] Lee | Influential factors on the survival of endothelial cells after penetrating keratoplasty[END_REF][START_REF] Borderie | Predicted long-term outcome of corneal transplantation[END_REF][START_REF] Bertelmann | Risk factors for endothelial cell loss post-keratoplasty[END_REF] Consequently, maintenance of graft function depends on a relatively low number of viable endothelial cells compared to healthy corneas of the same age. Hence reducing endothelial cell loss during in vitro cultivation, e.g. by ameliorating cultivation conditions, seems crucial. Most european eye banks use MEM (Minimal Essential Medium) supplemented with 2-8% fetal calf serum (FCS) as culture medium. Optimising corneal organ cultivation conditions does also include elimination of serum supplementation, because serum is a complex and varying mixture of different substances, some of them not yet determined, and underlies batch-dependent quality differences. [START_REF] Schneider | The effect of serum batch on the in vitro lifespans of cell cultures derived from old and young human donors[END_REF][START_REF] Engelmann | Quality control in the corneal bank-a necessary measure?[END_REF] Physical contact between the corneal graft and undefined, possibly pathogenic serum constituents is posing a risk to the graft recipient. Cultivation of corneal grafts in a defined serum-free medium can help avoid this risk and meets European laws and standards for handling tissue transplants. [8,9] In previous studies we investigated the effects of nine different culture media on human corneal endothelial cell (HCEC) morphology and metabolism in HCEC cultures and whole corneas. We observed that the low nutritive content of MEM is inadequate to sustain sufficient corneal endothelial metabolic activity. We could further demonstrate that corneal grafts cultured in Human Endothelial-SFM (serum-free medium, hereafter abbreviated SFM), a humanised and fully defined culture medium, showed lowest endothelial cell loss, highest RNA synthesis rate, lowest keratan sulphate leakage, highest utilisation of glucose and lactate production. [START_REF] Møller-Pedersen | Evaluation of potential organ culture media for eye banking using human donor corneas[END_REF][START_REF] Møller-Pedersen | Evaluation of potential organ culture media for eye banking using a human corneal endothelial cell growth assay[END_REF][START_REF] Hempel | Use of a serum-free medium for long-term storage of human corneas. Influence on endothelial cell density and corneal metabolism[END_REF][START_REF] Bednarz | Effect of three different media on serum free culture of donor corneas and isolated human corneal endothelial cells[END_REF] Additional experiments demonstrated that even after prolonged organ cultivation of up to 9 weeks serum-free SFM markedly supported endothelial cell survival and morphology, as well as keratocyte viability. [START_REF] Hempel | Use of a serum-free medium for long-term storage of human corneas. Influence on endothelial cell density and corneal metabolism[END_REF][START_REF] Bednarz | Effect of three different media on serum free culture of donor corneas and isolated human corneal endothelial cells[END_REF] Therefore we considered Human Endothelial-SFM superior to other conventional culture media, which needed serum supplementation. Here, we compared the effects of organ culture medium on HCEC survival and protection of apoptosis and necrosis at a subcellular level.

MATERIALS AND METHODS

Cell culture and test media

The immortalized HCEC population HCEC-12, which was established by Bednarz et al. [START_REF] Bednarz | Immortalization of human corneal endothelial cells using electroporation protocol optimized for human corneal endothelial and human retinal pigment epithelial cells[END_REF] and further characterized by Valtink et al. [START_REF] Valtink | Two Clonal Cell Lines of Immortalized Human Corneal Endothelial Cells Show either Differentiated or Precursor Cell Characteristics[END_REF], was routinely grown in F99 HCEC (Ham´s F12/Medium 199 [Biochrom AG, Berlin, Germany], 5% FCS [Invitrogen, Karlsruhe, Germany], 20µg/ml ascorbic acid [Sigma-Aldrich, Munich, Germany], 10 ng/ml human recombinant FGF-2 [Invitrogen], 20µg/ml human recombinant insulin [Sigma-Aldrich], and antibiotics [2.5µg/ml amphotericin B and 50µg/ml gentamycin, Biochrom]). For subcultivation, cells were detached with trypsin/EDTA (0.05%/0.02%, Biochrom), collected in growth medium, centrifuged at 100xg for 5min and plated at a density of 2000 cells/cm 2 . Cultures were grown in T75 tissue culture flasks coated with 10µg/ml laminin (Invitrogen) and 10mg/ml chondroitin sulphate (Sigma) solubilised in phosphate-buffered saline (PBS) for 1h at RT. Cells were maintained at 37ºC in a humidified atmosphere containing 5% CO 2 . Growth medium was changed 3x per week. For experiments, medium was changed to test media 24h after plating and cells were cultured for 5days (flow cytometry) or until confluence (immunocytochemistry). Test media were MEM (Biochrom) supplemented with 2mM Lglutamine (Biochrom), antibiotics and either 2% FCS or 5% FCS, and serum-free Human Endothelial-SFM (Invitrogen) supplemented with 10ng/ml FGF-2 and either with or without antibiotics. Medium SFM was used with and without antibiotics, because we observed a higher sensitivity of HCEC towards antibiotics in serum-free culture in preliminary experiments. HCEC cultured in growth medium F99 HCEC served as controls.

Induction of apoptosis and ROS production

Apoptosis via the mitochondrial pathway (intrinsic) was induced by incubating the cells with 0.5µmol/l staurosporine for 4h at 37°C. It was shown that staurosporine induces caspasedependent apoptosis in HCEC and that markers of apoptosis such as cleaved caspase-3 and cleaved PARP are observable within less than 4h after induction. [START_REF] Thuret | Mechanisms of staurosporine induced apoptosis in a human corneal endothelial cell line[END_REF][START_REF] Waibel | Mitochondria are not required for death receptormediated cytosolic acidification during apoptosis[END_REF] A concentration of 0.5µmol/l sufficiently induces apoptosis in the majority of susceptible cells. Generation of ROS was induced by incubating the cells with 200µmol/l H 2 O 2 for 2.5h at 37°C. Cells were photodocumented using differential interference contrast (DIC) under an IX81 fluorescence microscope (Olympus Biosystems, Hamburg, Germany) equipped with an OBS CCD FV2T camera (Olympus) driven by cell^R software (Olympus). .

Flow cytometry

Flow cytometric analyses were performed using a FACS-Calibur (Becton Dickinson GmbH, Heidelberg, Germany). 10,000 cells were recorded for each analysis and calculations were performed with BD CellQuest™software. For detection of apoptotic cells (subG1-DNA content), cells were trypsinised, pelleted at 195xg for 6min, and fixed in cold 70% ethanol for 5min on ice. 50µl of PBS containing 1µl propidium iodide solution (1mg/ml PI in H 2 O; Sigma-Aldrich) and 1µl RNase (>7 U/µl, Carl Roth GmbH, Karlsruhe, Germany) were added, followed by incubation for 30min at RT in the dark. Apoptotic cells were identified by a subdiploid DNA peak (fragmented chromatin) using a 585±21nm filter (red fluorescence).

Apoptotic cells were further identified by labelling chromatin after nuclear membrane destabilisation with YO-PRO-1. Cells were collected as above, resuspended in 50µl PBS with 1% horse serum (Sigma-Aldrich) containing 1µl YO-PRO-1 (100µmol/l in DMSO, Invitrogen) and 1µl PI solution, and incubated for 25min at RT in the dark. Cells were analysed using a 530±15nm filter for YO-PRO-1 (green fluorescence) and a 585±21nm filter for PI. The percentages of necrotic (PI high positive) and apoptotic (PI low positive, YO-PRO-1 positive) cells were calculated. Formation of intracellular reactive oxygen species (ROS) was measured using CM-H 2 DCFDA (Invitrogen), a cell-permeant indicator which becomes fluorescent in the presence of ROS. Cells were collected as above, resuspended in 50µl CM-H 2 DCFDA (10µmol/l in PBS) and incubated at 37°C for 30min in the dark. Fluorescence was recorded using a 530±15nm filter. All assays were performed at least six times, values are given as mean±SD. Statistical analysis was performed by non-parametric Mann-Whitney-Utest using SPSS (Chicago, USA) between different media (untreated cells) and between treated and untreated pairs for each medium. Alpha error adjustment by Mann-Whitney-Utest was performed with respect to the necessity of alpha error adjustment in multiple testing and with a total alpha error of 5% (0.05) for a total of15 p-values (5 comparisons of pairs treated/untreated in the same medium and 10 comparisons of untreated cells in all five media among each other, resulting in 15 p-values). An adjusted p-value <0.05/15 (0.0033) was accepted as significant.

Intravital microscopy and immunocytochemistry

Cells were seeded into 8-chamber slides (Labtek coverglass system, Nalge Nunc International, New York, USA) and grown to confluence. Intravital staining was performed with JC-1 (20µg/ml medium; Invitrogen) at 37°C for 20-30min. JC-1 is a cationic dye that selectively accumulates within the mitochondrial matrix. JC-1 forms red fluorescent Jaggregates (emission 590 nm) in the presence of a highly negative transmembrane potential, but exists as green monomers (emission 530 nm) under depolarised conditions. The red phase of JC-1 (J-aggregate) was recorded using an excitation wavelength of 535nm and an emission filter of 610/75nm. The green phase (monomer) was recorded using an excitation wavelength of 485nm and an emission filter of 540/50nm. Immunocytochemical staining was performed using rabbit anti-human cleaved caspase-3 (Asp175, 1:50, Cell Signaling, Danvers, USA), rabbit anti-human Bax (1:20, clone P-19, Santa Cruz Biotechnology, Santa Cruz, USA), mouse anti-human Bcl2 (1:20, clone 124, Dako Denmark A/S; Glostrup, Denmark), and rat anti-human caspase-8 (55kD full length and cleaved, active 18kD forms of caspase-8; 1:10, clone 1H10E4H10, Zymed Laboratories, San Francisco, USA) antibodies. Secondary antibodies were FITC-conjugated goat anti-rabbit or goat anti-mouse IgG (both 1:80, Jackson ImmunoResearch Europe Ltd., Suffolk, UK), or Alexa Fluor® 488-conjugated goat anti-rat IgG (1:200, Invitrogen). Cells were washed with PBS for 5min at RT, fixed in cold acetone/methanol at 4°C for 8min, and washed again in PBS. Cells were incubated with primary antibodies at 4°C over night, washed 2x with PBS for 5min and incubated with respective secondary antibodies at 37°C for 30min (all antibodies diluted in PBS). Cells were washed 2x with PBS for 10min and nuclei were counterstained with DAPI (0.1µg/ml, Sigma-Aldrich) at RT for 5min. Samples were covered with glycerol and immediately photodocumented using an IX81 fluorescence microscope equipped with an OBS CCD FV2T camera driven by cell^R. Primary antibodies were omitted in controls. All experiments were performed at least six times.

Western blot

Cells were collected as above and lysed in 100µl ice-cold lysis buffer (60mM TRIS-HCl pH6.8, 1% SDS, 1mM NaVO 4 [Merck KG, Darmstadt, Germany]). Samples were incubated at 99°C for 5min, homogenised by sonication for 30s, and centrifuged at 14,000rpm for 3min.

Protein concentration was determined using the BCA protein assay (Perbio Science, Bonn, Germany). 5x loading buffer (10% SDS, 15% glycerol, 0.01% bromophenol blue, 20% 2mercaptoethanol, 0.3125M TRIS-HCl pH6.8 [Merck]) was added and equal amounts of protein (40µg/lane) were loaded on 10% PAA gels and electrophoresed (running buffer 0.025mol/l Tris, 0.2mol/l glycine, 0.1% SDS pH8.8 [Merck]). Protein bands were blotted onto PVDF membranes (Roth) at 4°C over night by wet western blotting (transfer buffer 0.025mol/l Tris, 0.2mol/l glycine, 20% methanol). Blots were blocked in 5% non-fat dry milk in PBS/0.1% tween-20 at RT for 1h. Membranes were then incubated with rabbit anti-human cleaved caspase-3 (1:200) or rabbit anti-human PARP p85 (caspase-cleaved 85kD fragment; 1:200, Promega Co., Madison, USA) at 4°C over night, or were incubated with mouse anti-human gamma-tubulin (1:7500, clone GTU-88, Sigma-Aldrich) at RT for 1h (all antibodies diluted in blocking solution). Detection was performed using HRP-conjugated goat anti-rabbit IgG (1:1000 for cleaved caspase-3, 1:5000 for cleaved PARP, Cell Signaling) or HRP-conjugated horse anti-mouse IgG (1:75000, Cell Signaling) diluted in blocking solution at RT for 1h and subsequent chemiluminescence using the Amersham ECL western blotting detection system (GE Healthcare, Munich, Germany) on Hyperfilm ECL (GE Healthcare).

RESULTS

Morphology and mitochondrial membrane potential

Flow cytometric analysis of apoptosis, necrosis and oxidative stress

FACS analysis revealed that HCEC could be divided into four subpopulations after staurosporine treatment, namely healthy cells (PI and YO-PRO-1 negative), YO-PRO-1 positive (apoptotic) cells, highly PI-positive (necrotic) and PI low-positive (apoptotic, see [START_REF] Zamai | Supravital exposure to propidium iodide identifies apoptotic cells in the absence of nucleosomal DNA fragmentation[END_REF]2001) cells. [START_REF] Zamai | Supravital exposure to propidium iodide identifies apoptotic cells in the absence of nucleosomal DNA fragmentation[END_REF][START_REF] Zamai | Supravital exposure to propidium iodide identifies apoptosis on adherent cells[END_REF] The number of healthy cells in untreated cultures was significantly higher in both SFM media (95.6±1.6%, 95.6±1.9%) than in F99 HCEC (87.5±2.0%) and MEM media (79.1±2.8% and 81.9±4.4%), and was also significantly higher in F99 HCEC than in MEM+2% FCS. Adding staurosporine significantly reduced the number of healthy cells in F99 HCEC (66.2±3.5%) and MEM media (28.6±2.4% and 22.4±0.8%), but not in SFM media (93.0±3.3% and 94.4±2.3%) (fig 2A). The number of necrotic cells in untreated controls was significantly lower in both SFM media (2.1±1.3% and 1.7±1.1%) than in the other media (F99 HCEC 7.2±1.2%; MEM 13.0±2.5% and 10.8±3.6%). After staurosporine treatment, the number of highly PI-positive cells increased significantly in F99 HCEC (14.3±2.3%) and in MEM (33.2±3.6% and 33.9±3.1%) compared to their untreated counterparts (SFM 2.7±2.5% and 1.8±1.0%, not significant), (fig 2B). The number of YO-PRO-1 positive cells increased significantly after staurosporine treatment in F99 HCEC from 1.8±0.5% to 11.7±3.2% and in both MEM media from 1.0±0.2% to 21.1±6.4% and from 1.3±0.3% to 31.8±4.3%, but not in SFM media (from 0.8±0.2% to 2.1±1.1% and from 0.9±0.5% to 1.9±1.0%) compared to untreated controls (fig 2C). The number of PI low positive cells in untreated controls was significantly fewer in SFM media (1.5±0.4% and 1.8±0.9%) than in the other media (F99 HCEC 3.5±0.8%; MEM 6.8±0.3% and 6.0±0.7%) and was also significantly lower in F99 HCEC compared to MEM+2% FCS. Staurosporine-induced cell stress was significantly higher in both MEM media (17.1±4.1% and 11.9±0.8%) and in F99 HCEC (7.8±1.8%), but not in SFM media (2.2±0.6% and 1.9±0.5%) (fig 2D). The number of cells with fragmented chromatin (subG1-DNA-content) was significantly lower in untreated SFM cultures (4.2±1.0% and 4.4±1.8%) than in untreated MEM media cultures (14.2±3.7% and 8.6±1.8%). Staurosporine-induced DNA fragmentation was significantly higher in MEM media (41.9±2.2% and 48.0±2.9%) and in F99 HCEC [START_REF] Zamai | Supravital exposure to propidium iodide identifies apoptotic cells in the absence of nucleosomal DNA fragmentation[END_REF] revealed more cleaved PARP and cleaved caspase-3 after staurosporine treatment in all cultures, with cells cultured in MEM media and F99 HCEC producing markedly higher amounts of cleaved PARP and cleaved caspase-3 compared to cells cultured in SFM media. Besides, cleaved caspase-3 could also be detected in untreated cells cultured in medium F99 HCEC , and cleaved PARP could also be detected in untreated cells cultured in MEM+5% FCS.

Immunocytochemical analysis of apoptotic parameters

DISCUSSION

Endothelial cell density and morphology are the two main criteria for evaluating the quality of corneal grafts before keratoplasty, both being influenced by storage conditions. European eye banks favour long-term storage at 30-37°C, and most commonly a MEM-based medium is used. MEM has a reduced nutrient content and needs to be supplemented with fetal calf serum to maintain cell survival. Improvements in cell and organ culture techniques and updated legal guidelines for handling tissues for transplantation require serum replacement by defined, ideally humanised, factors. Previous investigations showed that metabolic requirements of corneal endothelial cells are quite particular and are only inadequately met by MEM in corneal endothelial cell and corneal organ culture. [START_REF] Møller-Pedersen | Evaluation of potential organ culture media for eye banking using human donor corneas[END_REF][START_REF] Møller-Pedersen | Evaluation of potential organ culture media for eye banking using a human corneal endothelial cell growth assay[END_REF][START_REF] Hempel | Use of a serum-free medium for long-term storage of human corneas. Influence on endothelial cell density and corneal metabolism[END_REF][START_REF] Bednarz | Effect of three different media on serum free culture of donor corneas and isolated human corneal endothelial cells[END_REF][START_REF] Rieck | Increased endothelial survival of organ-cultured corneas stored in FGF-2-supplemented serum-free medium[END_REF] The results obtained here support these former reports and clearly show that nutrient deficiency in MEM does not only lead to a reduced metabolic and proliferative activity, but results in an elevated susceptibility to apoptotic cell death in HCEC. Dramatic morphological changes of HCEC cultured in MEM, like pronounced vacuolisation, are key features of a reduced viability and are already visible in untreated control cultures. Signs of cell death, such as chromatin fragmentation, mitochondrial membrane depolarisation, and labelling with PI and YO-PRO-1, can be detected in MEM-cultured HCEC even without exogenously inducing apoptosis. Adding the apoptosis-inducer staurosporine to MEM-cultured cells resulted in a marked increase of these indicators and also in an up-regulation of the apoptotic markers caspase-3 and PARP and hence also their cleaved forms, as detected by western blotting. Furthermore, a higher staining intensity for Bax and cleaved caspase-8 was noted irrespective of the amount of serum added. This increase in staining intensity was accompanied by cytoplasmic spreading of the formerly single punctate pattern in untreated and healthy cells, which strongly resembled the staining pattern of p53, a transcription factor that regulates transcription of molecules associated to the apoptotic pathways and localises in close proximity with caspase-8 and Bax, amongst others, in healthy cells. Upon induction of apoptosis caspase-8 is up-regulated, cleaved and distributed into the cytoplasm. Likewise, Bax is up-regulated, oligomerised and also distributed to the cytoplasm. [START_REF] Han | Regulation of mitochondrial apoptotic events by p53mediated disruption of complexes between anti-apoptotic Bcl-2 members and Bim[END_REF][START_REF] Yao | Death effector domain DEDa, a self-cleaved product of caspase-8/Mch5, translocates to the nucleus by binding to ERK1/2 and upregulates procaspase-8 expression via a p53-dependent mechanism[END_REF][START_REF] Haluska | Interaction between human topoisomerase I and a novel RING finger/arginine-serine protein[END_REF] Up-regulation of Bcl-2, an anti-apoptotic protein which is expressed in organ-cultured human cornea [START_REF] Gain | In situ immunohistochemical study of Bcl-2 and heat shock proteins in human corneal endothelial cells during corneal storage[END_REF][START_REF] Yamamoto | Bcl-2 expression in the human cornea[END_REF], may be regarded as a protective mechanism sustained by the cells. For example, the higher amount of Bcl-2 in untreated cultures kept in MEM+2% FCS compared to the other untreated cultures may indicate that nutrient supply in this medium is insufficient and the cells up-regulate Bcl-2 to counteract death by starvation. Contrasting, HCEC in SFM appeared to be protected from cell death despite the lack of serum, as the examined apoptotic factors were significantly lower under control conditions and did not increase significantly after staurosporine administration. It also appeared that addition of antibiotics, which can be noxious under serum-free conditions, was well tolerated in SFM.

ROS are formed as a physiological product of respiration, hence higher ROS production in cells in SFM and F99 HCEC under control conditions reflects a higher metabolic activity. ROS production does not increase significantly under administration of H 2 O 2 in SFM, indicating that HCEC in SFM are protected against oxidative stress by utilising anti-oxidative or compensatory mechanisms. Contrasting, HCEC in MEM form only little ROS under control conditions, but increase ROS production more than double under H 2 O 2 , pointing to a reduced respiratory and metabolic activity and insufficient anti-oxidative capacity of the cells. This is further substantiated by the observed depolarised mitochondrial membranes in MEM-fed control cultures.

In summary, the results show that cells cultured in MEM media seem susceptible to cell death already in the absence of exogenous noxious stimuli, while HCEC cultured in SFM seem to be protected from cell death even when the apoptosis-inducer staurosporine is added. The nutritive conditions in MEM therefore seem to be insufficient to sustain adequate compensatory mechanisms against oxidative cell stress. Thus Human Endothelial-SFM is superior to conventionally used MEM in promoting HCEC survival in vitro cell cultures. It is known that cultured cells react more sensitive to stimuli than cells in situ, and it can be assumed that the microenvironment of corneal endothelial cells in situ may contain protective factors that prevent endothelial cell death in MEM-based organ cultured donor corneas in such an extensive way as was observed in our experiments. However, based on our results and with respect to european laws and guidelines on tissue handling we suggest that the methods of organ cultivation of donor corneas should be improved. A multi-centric clinical trial to transplant SFM-stored corneas vs. MEM-stored corneas was initiated in Germany (registered at clinicaltrials.gov, identifier NCT00623584).

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  Morphology of untreated and staurosporine-treated cells was analysed by DIC microscopy after 5days cultivation in the test media (fig1A). Untreated cells showed polygonal morphology with few vacuoles and formed regular monolayers in SFM and F99 HCEC , while cells in MEM were enlarged and had increased numbers and sizes of vacuoles. Furthermore, a markedly lesser cell density was observed in MEM cultures. Proliferation seemed also slightly reduced in SFM with antibiotics and cells contained more vacuoles than in SFM without antibiotics. Staurosporine induced cell shrinkage and loss of intercellular contacts in all cultures, and in MEM also caused fragmentation of the cells. Intravital staining (fig1B) revealed numerous mitochondria with depolarised membranes (green JC-1 fluorescence) in MEM-cultured cells irrespective of staurosporine treatment. In the other media, mitochondria of untreated cells had predominantly polarised membranes (red JC-1 fluorescence), and became depolarised only after staurosporine treatment.

  Upon staurosporine treatment, cells in MEM and F99 HCEC cultures showed stronger staining for cleaved caspase-3 (fig 3A), cleaved caspase-8 (fig 3B), Bax (fig 4A), and Bcl-2 (fig 4B), and to some extent stronger staining for cleaved caspase-8 in cells cultured in SFM with antibiotics. The staining pattern for caspase-8 and Bax changed from a single perinuclear, punctate localisation in untreated controls to a diffuse cytoplasmic appearance. Especially cells cultured in MEM media were highly susceptible to apoptosis, indicated by pronounced Bcl-2 and Bax positivity already in untreated cultures. Interestingly, cells cultured in SFM seemed less affected by staurosporine, since staurosporine failed to induce up-regulation of Bcl-2 and the other tested apoptosis markers. Omission of the primary antibodies in negative control stainings confirmed specificity of the staining (fig 5). Western blotting (fig 3C)

Figure 1

 1 Figure 1 HCEC morphology and polarization state of mitochondrial membranes. Cells were cultured in the respective test media for 5d, and then part of each culture was treated with 0.5µmol/l staurosporine for 4h at 37°C to induce apoptosis. (A) Morphology of the cells viewed by DIC microscopy. (B) Intravital staining with JC-1 indicates mitochondria with predominantly polarised (red) membranes in untreated controls in SFM and F99 HCEC ,and depolarised (green) mitochondrial membranes in untreeated MEM controls and in all media after addition of staurosporine. Scale bar 100µm.

Figure 2

 2 Figure 2 Flow cytometric analysis of HCEC. Cells were cultured in the respective test media for 5d, then part of each culture was treated with 0.5µmol/l staurosporine for 4h at 37°C to induce apoptosis (A-E) or was treated with 200µmol/l H 2 O 2 for 2.5h at 37°C to induce oxidative stress (F). (A) Number of healthy (PI-/YO-PRO-1 negative) cells, (B) number of necrotic (PI-positive) cells, (C) number of apoptotic (YO-PRO-1 positive) cells, (D) number of apoptotic (low PI-positive) cells, (E) number of cells with fragmented nuclei (sub G1 DNAcontent), (F) ROS production was visualised by means of CM-H 2 DCFDA staining, fluorescence intensity is given in arbitrary units. Data are given as mean±SD (* p<0.05). White bars: untreated control cultures, grey bars: treated with staurosporine, black bars: treated with H 2 O 2 .

Figure 3

 3 Figure 3 Determination of cleaved caspase-3, full length and cleaved caspase-8 and cleaved PARP. Cells were cultured in 5 test media for 5d and part of each culture was treated with 0.5µmol/l staurosporine for 4h at 37°C to induce apoptosis. Cells were then fixed and stained with antibodies against cleaved caspase-3 (A) and full length and cleaved caspase-8 (B). Scale bar 100µm. (C) Western blot analysis of cleaved caspase-3 and cleaved PARP (loading control: γ-tubulin).

Figure 4

 4 Figure 4 Determination of Bax and Bcl-2. Cells were cultured in 5 test media for 5d and part of each culture was treated with 0.5µmol/l staurosporine for 4h at 37°C to induce apoptosis. Cells were then fixed and stained with antibodies against Bax (A) and Bcl-2 (B). Scale bar 100µm.

Figure 5

 5 Figure 5 Negative control stainings of HCEC cultured in MEM+2% FCS and treated with staurosporine for 4h at 37°C to induce apoptosis. The primary antibodies were omitted. (A) cleaved caspase-3, (B) full length and cleaved caspase-8, (C) Bax, (D) Bcl-2; scale bar 100µm.

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  significantly lower in MEM+2% FCS than in SFM. Cells cultured in MEM or F99 HCEC showed a significantly higher ROS production after treatment with H 2 O 2 (MEM 192.1±51.3 and 97.4±7.3, F99 HCEC 238.2±50.6) compared to their untreated counterparts, but not in SFM media (116.7±14.8 and 96.9±9.7) (fig 2F).

	.2±4.5%) compared to their untreated
	counterparts, but not in SFM media (8.7±3.5% and 7.7±3.1%) (fig 2E). ROS production in
	untreated control cultures was significantly lower in MEM+5% FCS (32.1±2.7) than in the
	other media (F99 HCEC 98.2±45.7; MEM+2% FCS 44.0±5.4; SFM 86.2±19.7 and 55.9±20.2)
	and was also

Acknowledgements

We wish to thank Ms B. Rost, Ms S. Bramke and Ms A. Neisser for technical assistance.