Fergus N Doubal
email: fergus.doubal@ed.ac.uk

Martin S Dennis

Joanna M Wardlaw

Characteristics of patients with minor ischaemic strokes and negative MRI: a cross-sectional study

Keywords: Minor stroke, MRI, DWI-MRI

Background: Diffusion weighted (DWI) MRI is recommended in UK guidelines to evaluate minor strokes, yet can produce negative results.

Objective:

We determined the rate of negative MRI (including DWI) and associated features in patients presenting to hospital with minor strokes.

Methods:

We performed a prospective observational cross sectional study in a teaching hospital of patients with a clinical diagnosis of ischaemic lacunar or minor cortical stroke. We performed MRI (DWI, T2, FLAIR, T2* and T1) as soon as possible after presentation. We used multivariate analysis to determine predictors of negative DWI and MRI (all sequences). Gold standard for clinical diagnosis of stroke was the opinion of an expert panel.

Results:

We recruited 246 patients, mean age 68.1 years (SD 11.6 years), 162 were males (66%) and the median NIHSS was 2 (range 0-8). The median time from stroke onset to MR scan was 12 days (IQR 4-27 days).

Eighty-one patients (33%) did not show any ischaemia on DWI. Sixty patients (24%) did not show the recent infarct on MRI (DWI/T2/FLAIR). With multivariate analysis, less severe stroke, younger age, female gender and increased time from stroke onset to scan were associated with negative DWI.

With multivariate analysis, younger age and female gender were associated with negative MRI (DWI or T2 or FLAIR) scans.

Introduction

UK guidelines recommend magnetic resonance imaging (MRI) in patients presenting with minor stroke. 1;2 Diffusion weighted MR imaging (DWI) is more sensitive than Computed Tomography (CT) scanning at detecting acute cerebral ischaemia in minor stroke. 3;4 DWI can be positive for ischaemia for several weeks after minor stroke, 5;6 but there is also a negative rate for DWI in patients with clinically definite stroke. 7;8 It is unclear which features are associated with negative DWI. We investigated the rate and associations of negative DWI and MRI in patients presenting with minor ischaemic stroke.

Methods

We recruited patients prospectively between April 2005 and December 2007.

We included patients presenting to our teaching hospital (which sees 550 patients/year with stroke) with a clinical diagnosis of minor ischaemic stroke (defined as lacunar or partial anterior circulation stroke [START_REF] Bamford | Classification and natural history of clinically identifiable subtypes of cerebral infarction[END_REF] ). We defined stroke if it was diagnosed by a panel of experts (neuroradiologist, vascular neurologist and stroke physician all blinded to the study hypothesis) with full access to MRI (to exclude other structural causes of the symptoms) and other investigation results. We excluded patients with transient ischaemic attacks (symptom duration < 24 hours), contraindications to MRI and those without a clinical diagnosis of minor ischaemic stroke. In August 2009, we reviewed all case records in those with negative imaging to see if an alternative diagnosis had emerged and re-reviewed the initial diagnosis of stroke. The follow up period ranged from 21 to 52 months.

Patients had MRI after presentation on a 1.5-T MR scanner (Signa LX; General Electric) with 22 mTm -1 maximum strength gradients, including axial DWI, T2-weighted, fluid-attenuated inversion recovery (FLAIR), gradient echo and sagittal T1 weighted sequences. A consultant neuroradiologist, blinded to the study hypothesis but not clinical information, coded acute relevant infarcts (DWI increased signal, reduced signal on apparent diffusion coefficient map, or increased signal with/without swelling without evidence of cavitation on FLAIR/T2) using a validated morphological schema. [START_REF] Wardlaw | A simple practical classification of cerebral infarcts on CT and its interobserver reliability[END_REF] Patients had carotid Doppler ultrasound, electrocardiogram, routine blood tests, and further clinical investigations as indicated. We defined time to scan as the time from stroke onset to scan, the persistence of symptoms was recorded at the time of the scan and age at presentation to hospital. We recorded stroke severity (NIHSS) and gender.

We defined minor cortical and lacunar stroke syndromes as previously (but did not use a subclassification system to diagnose stroke). [START_REF] Doubal | Retinopathy in ischemic stroke subtypes[END_REF] We further classified stroke subtype using radiological criteria, that is whether any recent infarct on MRI was cortical or lacunar, and used both clinical and radiological classification to subtype stroke. Where classifications differed, the radiological classification was used.

To determine which variables were included in the multivariate model we used parametric and non-parametric tests as appropriate to assess univariate associations between variables and the presence of a negative scan. We used multivariate logistic regression to assess contributing factors with the binary outcome of positive/negative scan as the dependent variable.

Calculations were performed with Minitab (version 15; Minitab Inc., State College, PA, US) and the alpha level for significance was 0.05. We excluded 1 patient with an incomplete dataset. There were no suitable data for sample size calculations. The Lothian Regional Ethics Committee approved this study. Patients provided written informed consent.

Results

We screened 355 patients and recruited 253 patients. We excluded 7 patients upon further review of case notes who did not have a clinical diagnosis of ischaemic stroke when final follow up information was taken into account (3 patients had functional disease, one patient had a TIA and 3 patients had migraine) leaving 246 patients with definite stroke for analysis.

The mean age of those included was 68.1 years (Standard Deviation (SD) 11.6 years). There were 162 males (66%) and the median NIHSS score 2 (range 0-8). The median time from stroke onset (not presentation) to MR scan was 12 days (IQR 4-27 days).

Of 246 patients, 81 (33%) had no ischaemia on DWI and 60 (24%) did not show the recent infarct on any MRI sequence (DWI/T2/FLAIR). In 21 patients there was a recent symptomatic infarct seen on T2/FLAIR not seen on DWI.

With univariate analysis there was no association between lacunar stroke and negative DWI or MRI (T2/FLAIR/DWI) scan and this was excluded from the multivariate model.

With multivariate analysis a negative DWI was associated with younger age, lower NIHSS, female gender and increased time from stroke onset to scan after correcting for each other and the presence of persisting weakness. The odds of having a negative scan increased by 2% (95% CI 1-3%) for each additional day lapsed between stroke and scan (OR 1.02). The proportion of patients with negative DWI for those scanned between 0-6 days was 12%, 7-13 days 30%, 14-21 days 34% and >21 days 56%.

With multivariate analysis a negative MRI (DWI/FLAIR/T2) was associated with younger age and female gender after correcting for each other and time to scan, the presence of persisting symptoms and NIHSS (table).

Discussion

In patients presenting with minor ischaemic stroke in the anterior circulation, 33% had negative DWI and 24% had negative MRI (DWI/T2/FLAIR).

Negative DWI was associated with increasing time between stroke and scan, There are some weaknesses. Despite our best efforts and reference panel discussion, some of the subjects may have had a stroke mimic, especially as some of the negative scans were in younger females whose symptoms were compatible with migraine or functional disease. Previous studies have confirmed a diagnosis of stroke in patients with initially negative DWI with infarcts on follow up scans [START_REF] Oppenheim | False-negative diffusionweighted MR findings in acute ischemic stroke[END_REF] but some patients may never develop a relevant visible lesion. This was a cross sectional study so we can only report associations. The duration of DWI lesions in longitudinal studies after stroke demonstrates high inter-individual variability [START_REF] Schulz | Evolution of the diffusion-weighted signal and the apparent diffusion coefficient in the late phase after minor stroke[END_REF] and we cannot comment on how long DWI lesions remain on an individual basis. New asymptomatic DWI lesions may appear after ischaemic stroke. [START_REF] Kang | Early ischemic lesion recurrence within a week after acute ischemic stroke[END_REF] We only reported a DWI lesion as positive if in an arterial territory relevant to the most recent symptoms. Our results do not generalize to patients with posterior circulation stroke. If stroke awareness and services improve, the time from stroke symptoms to scan will decrease. The generalisability of our results may be limited by the relatively late median time to scan of 12 days in this mainly outpatient population, attributable to delays in patients presenting to hospital.

less
How do our results compare with other studies? A similar rate of negative DWI (30%) is reported in minor stroke patients scanned at a median 17 days post stroke, 8 but this study did not distinguish between stroke and TIA in the multivariate regression. Studies of DWI within 24 hours of acute stroke found negative DWI rates ranging from 6% 12;14 to 17%, 4 but were retrospective [START_REF] Oppenheim | False-negative diffusionweighted MR findings in acute ischemic stroke[END_REF] or reclassified patients presenting with TIA but who had an acute ischaemic lesion on DWI as stroke. 4 Other studies did not report negative DWI rates in TIA and stroke separately. [START_REF] Sylaja | When to expect negative diffusionweighted images in stroke and transient ischemic attack[END_REF] Lower NIHSS (lower stroke severity) was associated with negative DWI. In general, small infarcts are less likely to be identified on imaging than large infarcts and although infarct size was shown in a small longitudinal study not to predict duration of DWI-visible lesions, [START_REF] Schulz | Evolution of the diffusion-weighted signal and the apparent diffusion coefficient in the late phase after minor stroke[END_REF] it is probable that small infarcts resolve quicker than larger infarcts.

Increased time to imaging was associated with negative DWI although not negative T2/FLAIR MRI. This confirms other findings [START_REF] Schulz | Diffusion-Weighted MRI in 300 Patients Presenting Late With Subacute Transient Ischemic Attack or Minor Stroke[END_REF] and is attributable to normalisation of the DWI signal as cellular oedema resolves, but this does not mean that the tissue damage has resolved. Consistent with this, we have demonstrated that T2/FLAIR identifies more infarcts than DWI in patients presenting subacutely with minor stroke. Female gender was associated with both negative DWI and negative T2/FLAIR MRI. Gender did not previously predict negative DWI in patients with TIA/minor stroke combined. [START_REF] Schulz | Diffusion-Weighted MRI in 300 Patients Presenting Late With Subacute Transient Ischemic Attack or Minor Stroke[END_REF] The cause of this discrepancy is not clear -possibly our result was due to residual confounding from baseline imbalances.

We have shown that a third of patients presenting with minor stroke have negative DWI which is associated with decreased stroke severity, increased time to scan, younger age and female gender. Had we been less critical with our clinical diagnosis of stroke and included even some of the 355 patients with suspected stroke that were initially screened, the proportion with negative DWI would have been higher. These results should alert clinicians about when to expect a negative MRI in a patient with minor stroke and encourage them not to automatically exclude the diagnosis of stroke if DWI is negative.

Copyright

The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article (if accepted) to be published in the Journal of Neurology, Neurosurgery & Psychiatry editions and any other BMJPGL products to exploit all subsidiary rights, as set out in our licence (http://jnnp.bmjjournals.com/ifora/licence.pdf)."

Table .

 . Multivariate associations of a negative DWI and MRI (T2 or FLAIR or DWI). The OR are per unit increase in the explanatory variable and all ORare corrected for all of the explanatory variables in the table.

		Negative DWI	Negative MRI (T2/FLAIR/DWI)
	Variable
		OR (95% CI)	P value	OR (95% CI)	P value
	Age (years)	0.97	0.047	0.96	0.001
		(0.95 to 1.00)		(0.93 to 0.98)
	Male sex	0.44	0.008	0.46	0.018
		(0.24 to 0.81)		(0.24 to 0.87)
	NIHSS	0.70	0.008	0.76	0.059
	score	(0.54 to 0.91)		(0.58 to 1.01)
	Persisting	0.61	0.191	0.50	0.077
	symptoms at	(0.29 to 1.28)		(0.23 to 1.08)
	time of
	scan(yes/no)
	Time from	1.02	0.003	1.01	0.132
	stroke to	(1.01 to 1.03)		(1.00 to 1.02)
	scan (days)
	DWI -diffusion weighted imaging, OR -odds ratio, CI -confidence interval,
	NIHSS -National Institute for Health Stroke Scale, Persisting symptoms = at
	time of scan.

Conclusions:

There is a high rate of negative MRI and DWI amongst patients with minor stroke (a third) which has important management and research implications. A negative MRI or DWI does not exclude the diagnosis of stroke.

Acknowledgements and Funding

Declaration of sources of funding: FD was funded by the Wellcome Trust

Competing Interests

All authors declare that the answers to the questions on your competing interest form are all No and therefore have nothing to declare. The authors are independent from the sources of funding.

Contributors

FND conceived the study, collected and analysed data and wrote the paper.

MSD helped conceive the study, collected data and commented on drafts of the paper. JMW helped conceived the study, obtained original study funding, analysed all MRI scans and commented on drafts of the paper. All authors had full access to all of the data and take responsibility for the integrity of the data and the accuracy of the data analysis.


Samuel ; Hylander
email: samuel.hylander@limnol.lu.se

Lars Anders

Limnology Ecology

Lars-Anders Hansson

Anders

UV effects on zooplankton community composition

Keywords: zooplankton, UV, community, vertical migration

come

Vertical migration mitigates UV effects on zooplankton community composition

8 plastic cylinders (diameter 0.37 m, height 1.0 m) arranged randomly in two rows. A mixture 81 of tap water (80 L) and filtered lake water (27 L from a nearby lake (Dalby Quarry); 50-µm 82 mesh size) was added to each mesocosm, and the water was constantly and gently aerated. A 83 certain volume of a mixture of zooplankton coming from Dalby Quarry and a small nearby 84 pond was added to each mesocom giving an initial dominance of calanoid copepods 85 (Eudiaptomus gracilis, approximately 56 L -1 ) and Daphnia sp. (0.93 L -1 ). Pre-cultured algal 86 food (Scenedesmus sp. and Clamydomonas sp.) was added to each cylinder in original 87 concentrations of approximately 120 000 cells mL -1 and 3 000 cell mL -1 , respectively. This 88 food was added to the mesocosms once a week. Water from Dalby Quarry had an absorption 89 coefficient of 2.0 and an attenuation depth of 1.6 m (320 nm, 1% remaining). The water in the 90 mesocosms had an absorption coefficient of 9.4 and an attenuation depth of approximately 0.4 91 m. Two treatments (n=4) were applied, consisting of visible light (V) or the entire solar 92 spectrum (UV). In addition to the natural sunlight, extra UV radiation was supplied with one 93 fluorescent lamp placed above each cylinder. The fluorescent lamps (Philips,CLEO 94 Performance, 80 W) were turned on for 16 hours each day, approximately between sunrise 95 and sunset. The lamps have a spectrum with maximum intensity around 350 nm. For spectral 96 composition of the same kinds of lamps see Hylander et al. (Hylander et al., 2009b). The UV 97 radiation reaching the water surface of the cylinders was controlled by Plexiglas (Röhm GS 98 233) that screened out most of the UV radiation (i.e. the V-treatment; effectively cutting off 99 radiation below ~370 nm), or by UV-transparent Plexiglas (Röhm GS 2458) that let both 100 visible and UV radiation through (i.e. the UV-treatment). The lamps provided additional 101 radiation to the UV treatment, adding approximately 40% extra UVA compared with ambient 102 conditions (daily integrated values during a clear day); and the UV-blocking Plexiglas 103 lowered UVA by circa 70% in the V treatment (Fig. 1). Experimental treatments also 104 increased UVB radiation by approximately 30% in the UV treatment and reduced the 105 exposure by approximately 100% in the V treatment (Fig. 1). For transmittance of the 106 Plexiglas see [START_REF] Hansson | Escape from UV threats in 362 zooplankton: A cocktail of behavior and protective pigmentation[END_REF]. Ambient UVA and UVB radiation were 107 also monitored (Fig. 1; UV sensors SUL 033 and SUL 240, connected to a logging meter IL 108 1400A, International Light). Ambient integrated daily UVA radiation was 58 (±23) J cm -2 109 day -1 and UVB was 1.2 (±0.2) J cm -2 day -1 (mean for the entire experimental period measured 110 outside the experiment with the same sensors as above). To estimate penetration of UV into 111 the water, absorbance at 320 nm (A 320 ) was regularly measured (Beckman DU 800 112 (at 320 nm) and the attenuation depth (1%, 320 nm) were calculated using the relationship 114 between the absorption coefficient at 320 nm and the diffuse attenuation coefficient (K 320 , 115 [START_REF] Kirk | Optics of UV-B radiation in natural waters[END_REF], Morris et al., 1995). The UV threat estimate of radiation still remaining at a depth 116 of 0.1 m (I 0.1 ) was calculated [START_REF] Hansson | Plasticity in pigmentation induced by conflicting threats from predation 355 and UV radiation[END_REF]) from I 0.1 =I 0 e (-K320*0.1) , where I 0 is the daily 117 integrated UVA radiation from the sun and the fluorescent lamps. One of the main 118 zooplankton groups in this study, Daphnia, has a receptor within the compound eye in the 119 UVA spectrum [START_REF] Smith | UV photoreceptor in the compound eye of Daphnia 416 magna[END_REF] making UVA suitable for the threat estimate. In 120 order to keep periphyton production down, the walls of the cylinders were swept with a brush 121 once a week, which clouded the water for a couple of hours. Evaporation lead to reduced 122 water levels and therefore we added new water every month. 123

Throughout the experiment surface oxygen, temperature and fluorescence were 124 monitored (fluorescence was recalculated to chlorophyll with a standard curve; Turner 125 Designs, Aquafluor Handheld Fluorometer/Turbidimeter). Chlorophyll a values ranged 126 between 12 and 139 µg L -1 with a mean value of 40 µg L -1 for both treatments over the entire 127 experiment, and there were no differences between treatments (RM ANOVA; F 1,6 = 0.7; p > 128 0.05). A depth profile of chlorophyll was measured during a sunny day and there were no 129 differences between treatments or between depths and no significant interaction (Treatment: 130 Two-way ANOVA; F 1,12 = 0.8; p > 0.05; depth: Two-way ANOVA; F 1,12 = 1.1; p > 0.05; 131 treatment x depth interaction: Two-way ANOVA; F 1,12 = 1.0; p > 0.05). Temperature varied 132 throughout the experiment from 9.5 -27.1 ºC and there were no differences between 133 treatments (Fig. 2; RM ANOVA; F 1,6 = 0.1; p > 0.05). A depth profile of temperature was 134 measured during a sunny day and there were no differences between treatments but 135 approximately one degree Celsius lower temperature at the bottom compared with surface 136 water (Treatment: Two-way ANOVA; F 1,12 = 0.6; p > 0.05; depth: Two-way ANOVA; F 1,12 = 137 45.6; p < 0.05). There was no significant interaction between treatment and depth (Two-way 138 ANOVA; F 1,12 = 2.3; p > 0.05). Also oxygen did not differ between treatments and ranged 139 from 7.9 -14.4 mg L -1 (Fig. 2; RM ANOVA; F 1,6 = 0.6; p > 0.05). The phytoplankton 140 community composition was also monitored in surface waters three times throughout the 141 experimental period. there were no differences between treatments (two-way MANOVA, F 6,13 = 2.4, p > 0.05). 148

Depth profiles for phytoplankton species composition are not available but chlorophyll 149 measurements indicate that there were no differences in algal biomass between depths and 150 treatments (see above). Samples for nutrients were collected at the end of the experiment and 151 analyzed according to standard methods (Tyler and Olsson 2001; ICP MS ELAN-600). Mean 152 total phosphorous concentrations (± 1SD) were 57. 8 (±51.6) and44.3 (±30.4) in the V and153 UV treatments, respectively and there were no differences between treatments (t = 0.396, p > 154 0.05) 155

Experimental treatments started on 10-May and then zooplankton population dynamics and 156 vertical migration were monitored approximately once a month, between 18-May and 5-Sept-157 2006, by collecting samples at noon from the surface (depth 0.1 m) and the bottom (depth 0.9 158 m) of the cylinders. A four-litre sample was taken from each level through tubing in the 159 cylinder walls. The animals were retrieved on a 50 µm net and preserved with Lugol's 160 solution. All zooplankton in the entire sample, i.e. not a subsample, were counted using a 161

Petri dish with eight sub-chambers, each 26 × 33 mm (Nalge Nunc, USA), and a dissecting 162 microscope. Each sampling also included counting of egg-and ephippia-carrying Daphnia 163 (i.e. normal eggs and resting eggs). 164 Analysis of population dynamics, community composition, and reproduction was made by 165 means of repeated measures ANOVA (RM ANOVA) on log-or angular-transformed data. 166 Zooplankton densities were calculated from mean values of the 0.1 and 0.9 m samplings, and 167 the total filtered volume from each sampling represents approximately 7.5% of the total water 168 volume in the mesocosms. Vertical position, expressed as surface-to-bottom ratio, was 169 assessed by regression analysis, plotted against the different UV threats (se threat estimate 170 above, i.e. I 0.1 ). Note that each point in the regression analysis represents one mesocosm at 171 one sampling date. Statistical analysis was performed in SPSS 15.0. 172 F o r P e e r R e v i e w similar when comparing the two treatments throughout the experiment (V and UV; Fig. 3). 177

The most common taxa by numbers were Chydorus sp., Daphnia sp., calanoid copepods and 178 copepod nauplii, whereas Eurycercus sp., cyclopoid copepods and rotifers occurred in lower 179 abundances (Table 1). Community differences between treatments were small, although there 180 was a significant negative effect of UV on the proportion of Daphnia sp. and copepod nauplii 181 (RM ANOVA; p ≤ 0.05; Table 2). The proportion of Daphnia of the total zooplankton 182 population was higher in UV treatments in May but then decreased and was generally lower 183 in the UV treatment than in the V treatment (Table 2; Fig. 3). Copepod nauplii relative 184 abundance was generally lower in the UV treatment than in the V treatment throughout the 185 entire experiment (Fig. 3). None of the zooplankton taxa showed any response in absolute 186 density to the treatments (i.e. Number L -1 ; RM ANOVA; p > 0.05; Table 2). Daphnia started 187 (in May) at very low abundances (< 1 L -1 ), but already in June it had reached between 10 and 188 30 individuals L -1 (Fig. 4). There was no overall treatment effect (p>0.05; Table 2), but a 189 significant treatment by time interaction illustrated by higher numbers of Daphnia early in the 190 season in the UV than in the V treatment. This was followed by a shift to higher absolute 191 densities in the V treatment during September (p<0.05; Table 2; Fig. 4). Chydorus increased 192 in abundance over time but did not display any differences in abundance between treatments 193 over time (Fig. 4). 194

Behavioural responses to UV 195

Along a UV threat gradient some taxa, including Eurycercus sp., Chydorus and rotifers 196 showed no behavioural response to UV radiation, whereas the depth distribution (expressed as 197 surface-to-bottom ratio) of copepod nauplii, cyclopoid and calanoid copepods, and Daphnia,198 was inversely related to UV threat (Table 3; p < 0.05; Fig. 5). Chydorus sp. and Eurycercus 199 sp. rarely visited surface waters, as indicated by a surface-to-bottom ratio below zero (Fig. 5). 200

Reproductive responses 201

The proportion of egg-carrying Daphnia increased from less than 5% to between 30 and 40% 202 from June to September (Fig. 6). Overall, the proportion of egg-carrying females was lower in 203 the UV treatment compared with the V treatment during June and July but there was a shift 204 towards higher proportions in the UV treatment during August and September (Fig. 6; RM 205 ANOVA; treatment*time; F 9,36 = 3.5; p < 0.05). In contrast, the proportion of the Daphnia 206 population that had induced ephippia (resting eggs) was always higher in the presence of UV 207 In open-water habitats of lakes and oceans, there are considerable temporal and spatial 212 variations in mortality risk; organisms, such as zooplankton, use behavioural and 213 morphological traits to meet these challenging variations (Hansson et al., 2007a;Hylander et 214 al., 2009aHylander et 214 al., , 2009b)). Several factors, such as temperature (Loose and Dawidowitcz, 1994;215 Winder et al., 2003), pH, and food limitation [START_REF] Gliwicz | The role of predation in zooplankton succession[END_REF]Winder et al., 216 2003), but also ultraviolet radiation (UV), have been shown to affect zooplankton mortality 217 rate and behaviour [START_REF] Tartarotti | Survivorship of Cyclops 423 abyssorum tatricus (Cyclopoida, Copepoda) and Boeckella gracilipes (Calanoida, 424 Copepoda) under ambient levels of solar UVB radiation in two high-mountain lakes[END_REF][START_REF] Leech | In situ exposure to ultraviolet radiation alters the 381 depth distribution of Daphnia[END_REF]Williamson et al., 218 2001;Hansson et al., 2007a). UV is known to affect accumulation of UV protective 219 compounds, behaviour and potentially also zooplankton community community (Alonso et 220 al., 2004;[START_REF] Marinone | UV radiation as a potential driving force for zooplankton 386 community structure in Patagonian lakes[END_REF][START_REF] Persaud | Even if we applied elevated levels of UV compared with ambient levels, 306 attenuation in the water was relatively high. In systems where UV penetrates to deeper depths 307 it cannot be ruled[END_REF]Hylander et al., , 2009b)). Even though we applied an 221 elevated UV level (both UVA and UVB) for several months in a shallow system (~1m) we 222 only saw moderate effects on the zooplankton community composition and population 223 dynamics. This indicates that the community is well adapted to handle changes in UV 224 radiation through their natural UV defences. Zooplankton have a number of different defence 225 mechanisms in response to UV radiation, including vertical migration, photoenzymatic repair 226 and accumulation of UV protective compounds [START_REF] Hansson | Effects of ultraviolet radiation on pigmentation, 359 photoenzymatic repair, behavior, and community ecology of zooplankton[END_REF]. 227

The behavioural response (vertical migration) to increased UV radiation differed 228 considerably among taxa and Daphnia showed the strongest response; although showing a 229 less dramatic response, copepod nauplii, calanoid and cyclopoid copepods also spent more 230 time at the surface under low UV threat, compared to high UV. Chydorus and Eurycercus, 231 regardless of UV treatment, remained mainly at the bottom during daytime, as illustrated by 232 the almost constant low surface-to-bottom ratio. Species spending most of the time at the 233 bottom are decoupled from any UV threat; but they are also restricted to bottom waters during 234 daytime, which may be a competitive disadvantage (Loose and Dawidowitcz, 1994). 235

Accordingly, from our study three different strategies can be identified regarding daytime 236 vertical distribution: strong response to the instantaneous UV-threat, illustrated by Daphnia; a 237 weak, albeit significant response, such as in copepods and rotifers; and finally, a stationary 238 position at depth as a refuge from UV radiation, such as by Chydorus and Eurycercus. Such 239 taxon-specific differences have previously been identified for several other zooplankton taxa 240 [START_REF] Tartarotti | Survivorship of Cyclops 423 abyssorum tatricus (Cyclopoida, Copepoda) and Boeckella gracilipes (Calanoida, 424 Copepoda) under ambient levels of solar UVB radiation in two high-mountain lakes[END_REF][START_REF] Boeing | Damaging UV 335 radiation and invertebrate predation: conflicting selective pressures for zooplankton 336 vertical distribution in the water column of low DOC lakes[END_REF][START_REF] Marinone | UV radiation as a potential driving force for zooplankton 386 community structure in Patagonian lakes[END_REF][START_REF] Obertegger | Multifactorial nature of rotifer water 395 layer preferences in an oligotrophic lake[END_REF]. 241 The strength of the behavioural response to UV has been attributed to the amount of 242 photoprotective pigmentation, for example melanin and carotenoids, such that organisms with 243 high amounts of pigments tend to have weaker behavioural responses compared to organisms 244 with low amounts of pigments (Tollrian and Heibl, 2004;Hansson et al., 2007a;Obertegger et 245 al., 2008;Hylander et al., 2009b). Melanin pigmentation in Daphnia from temperate regions 246 is generally low (Hebert and Emery, 1990;Hansson et al., 2007a), which may explain the 247 strong migratory response to UV threat that we observed in this study. Also differences in 248 temperature and food availability may change vertical migration patterns (Orcutt and Porter, 249 1983;Johnsen and Jakobsen, 1987). In this study, there were no differences between 250 treatments in surface chlorophyll a and temperature. The temperature was slightly lower deep 251 down in the mesocoms but there were no significant interactions between depth and 252 treatment. Furthermore, vertical position of different algal species was not studied but algal 253 biomass (chlorophyll a) was evenly distributed throughout the water column and there were 254 no effects of depth or treatment x depth interaction effects. Overall, this indicates that UV was 255 the most important factor in explaining the vertical position of the animals. 256

Daphnia reproduction was altered by the treatments. Effects on the proportion of egg-257 carrying females were minor but the production of ephippia (resting eggs) increased upon UV 258 exposure. This may reflect one of the costs associated with UV exposure in natural 259 populations. Ephippia induction has previously been shown to be triggered by both abiotic 260 and biotic factors, for example temperature, photoperiod, and food level (reviewed in 261 Gyllström and [START_REF] Hansson | Plasticity in pigmentation induced by conflicting threats from predation 355 and UV radiation[END_REF]. Diapause is generally assumed to arise as a way of escaping 262 periods of harsh environmental conditions, but, to our knowledge, UV has not previously 263 been shown to be an induction agent. Since UV varies temporally over hours, days, and 264 seasons, it is likely that ephippia production would be beneficial as a way of escaping periods 265 of intense radiation. In nature, Daphnia populations in rock pools with high solar exposure 266 have been observed to have high occurrence of ephippia, but the phenomenon has not been 267 quantified systematically (D.O. Hessen, Oslo, personal communication). 268

Long-term population dynamics were relatively similar between treatments for all taxa 269 investigated; none showed any overall treatment response to increased UV radiation levels 270 compared to non-UV treatments. Also the community composition was rather stable with the 271 same number of species occurring in approximately the same proportions when comparing the 272 UV and non-UV treatments. However, after several months small differences occurred with 273 slightly lower relative abundance of copepod nauplii and Daphnia in the UV treatment, 274 indicating that these two groups were negatively affected by the radiation. Others have 275 [START_REF] Marinone | UV radiation as a potential driving force for zooplankton 386 community structure in Patagonian lakes[END_REF]. However, our study underlines the importance of allowing the 310 organisms to use their inducible defences, in this case vertical migration, in order to assess the 311 true net effect of UV on the community composition. 312

During recent years much research has focused on how predicted climate change, including 313 alterations in temperature, rainfall [START_REF] Suttle | Species interactions reverse grassland 421 responses to changing climate[END_REF], as well as UV radiation (Williamson, 314 1995), may affect natural ecosystem processes. In our investigation of UV effects on 315 freshwater zooplankton and responses in behaviour and community composition, we conclude 316 that one component of global change (UV radiation) may to a large extent be overruled and 317 buffered against by biological processes such as vertical migration. Even though we applied 318 elevated levels of UV throughout a summer we only saw moderate community changes. 319

Daphnia and copepod larvae were relatively less common in the UV treatment but differences 320 were small. Both relative and absolute abundances for all other species were stable between 321 treatments, which indicates that organisms that are able to use efficient UV defences (like 322 vertical migration) will withstand elevated UV levels. Instead, alterations in other climatic 323 variables and interspecific competition may have stronger effects on community dynamics. 324

  the surface waters of all cylinders. The diffuse attenuation coefficient 113

  experiment. Regarding the abundances of all algal species present, 147

  to absence of UV (Fig.6; means 8.7 and 5.0%, respectively; F 1,6 = 8.6

Fig. 4 .

 4 Figure legends 460Fig. 1. Daily UVA and UVB exposures measured weekly in ambient conditions and estimated 461 in UV and V treatments. Net emission from fluorescent lamps is calculated as the emission 462 coming from the lamps in a dark room minus shading from experimental setup and the 463 transmission of the Plexiglass. 464 465 Fig. 2. Temperature (ºC) and oxygen levels (mg L -1 ) throughout the experiment. Standard 466 deviations are sometimes not visible. 467 468 Fig. 3. Community response: Relative abundance (%) of the different zooplankton species 469 throughout the experiment (note that it is copepod nauplii). Treatments are visible light (V; 470 panel A) or the entire solar spectrum (UV; panel B). Density estimates are based on a mean 471 value from the 0.1 and 0.9 m samplings. 472 473 Fig. 4. Population dynamics: Mean abundances (number L -1 ; ± 1SD) of the two most common 474 species of zooplankton, Daphnia sp. and Chydorus sp. throughout the experimental period. 475 Treatments are visible light (V) or the entire solar spectrum (UV). Density estimates are based 476 on a mean value from the 0.1 and 0.9 m samplings. 477 478 Fig. 5.. Depth distribution of zooplankton (calanoid copepods, Chydorus sp., cyclopoid 479 copepods, Daphnia sp., Eurycercus sp., copepod nauplii, and rotifers) expressed as surface-480 to-bottom ratio at different UV threats. Significant relationships are indicated by p-values. 481

  figure represents data from one mesocom at one sampling. The variability in UV threat that is 483 seen within treatments is due to changes in ambient radiation and also changing absorption 484 coefficients over time. 485 486 Fig. 6.. Mean proportion (%, ±1SD) egg-(A) and ephippia-(B) carrying Daphnia during the 487 experiment. Treatments are visible light (V) or the entire solar spectrum (UV). Ephippia and 488 egg production estimates are based on a mean value from the 0.1 and 0.9 m samplings. 489 490

  Fig. 3.

  The community was initially dominated by Chlamydomonas sp., 142

	143	Scenedesmus sp., Rhodomonas sp., Fragillaria sp., Aphanothece sp., and some
	144	Ankistrodesmacae. Eventually the diversity decreased, and Scenedesmus sp. and
	145	Chlamydomonas sp. became dominant, constituting approximately 90% and 6.5% of the total
	146	algal numbers, respectively. All other algal species constituted less than 1% of total algal

Table 1 V -treatment Month Calanoids Eurycercus Chydorus Cyclopoids Rotifers Daphnia Nauplii

 1

	May	19.8 (13.7)	0 0)	0.4 (0.2)	0.7 (0.3)	0 (0)	3.2 (1.9)	0 (0)
	June	4.2 (1.6)	0 (0)	11.5 (2.1)	10.8 (5.9) 3.4 (2.5) 19.9 (4.7) 4.0 (0.9)
	July	4.5 (1.8)	0.3 (0.1	59.5 (5.8)	3.8 (0.3)	0.6 (0.2) 15.0 (5.2) 14.7 (2.7)
	Aug	0.1 (0.1)	3.0 (1.6)	34.3 (9.0)	6.6 (2.7)	0.3 (0.1) 8.5 (2.2) 19.6 (5.5)
	Sep	0 (0)	5.0 (3.1)	19.6 (5.8)	4.6 (3.2)	2.9 (2.7) 22.7 (3.9) 21.4 (8.1)
	UV -treatment
	Month	Calanoids	Eurycercus Chydorus Cyclopoids Rotifers Daphnia Nauplii
	May	5.7 (1.6)	0 (0)	0.1 (0.02)	0.7 (0.2)	0 (0)	2.3 (1.0)	0 (0)
	June	3.0 (1.0)	0 (0)	36.4 (19.1)	3.3 (0.3)	4.0 (2.5) 31.9 (6.9) 3.0 (1.1)
	July	1.9 (0.6)	0.6 (0.2)	83.4 (1.8)	4.1 (0.9)	0.8 (0.4) 21.1 (7.8) 11.6 (3.4)
						0.1
	Aug	0.1 (0.04)	3.7 (1.4)	36.7 (20.4)	7.1 (2.2)	(0.04)) 13.8 (2.7) 15.1 (3.2)
	Sep	0.02 (0.02)	3.4 (1.8)	32.6 (29.8) 10.2 (5.5) 0.2 (0.1) 8.0 (3.7) 11.3 (6.6)

http://mc.manuscriptcentral.com/jplankt Journal of Plankton Research

The number of zooplankton species and the proportions of the different species was rather

the UV environment[START_REF] Williamson | Ultraviolet radiation and zooplankton community structure following 440 deglaciation in Glacier Bay, Alaska[END_REF]. It can be difficult, however, to single out the

daphnia. Funct. Ecol., 4,[703][704][705][706][707][708][709][710]

Hylander, S., Boeing, W. J., Granéli, W., Karlsson, J., von Einem, J., Gutseit, K. and

Tyler, G. and Olsson, T. (2001) Concentrations of 60 elements in the soil as related to the soil

Acknowledgements 328

The study was funded by the Swedish Research Council (VR). Bruce Jeffs provided linguistic 329 corrections. We also thank two anonymous reviewers for valuable comments. 330


MD Suzanne Persoon
email: s.persoon@umcutrecht.nl

Md ; Merel

J A Luitse

Md ; Gert

Jan De Borst

PhD; Albert Van Der Zwan

PhD Ale Algra

PhD ; L Jaap Kappelle

Catharina J M Klijn

Symptomatic Internal Carotid Artery Occlusion: A long-term follow-up study

Keywords: Carotid Artery Diseases, Stroke, Follow-Up Studies

Background: Information on outcome of patients with occlusion of the internal carotid artery (ICA) is limited by short duration of follow-up and lack of haemodynamic studies of the brain.

Methods:

We prospectively investigated 117 consecutive patients with transient or moderately disabling cerebral or retinal ischaemia associated with ICA occlusion between September 1995 and July 1998 and followed them until June 2008. We determined the risk of recurrent ischaemic stroke and other vascular events and prognostic factors including collateral pathways and transcranial Doppler CO 2 -reactivity.

Results: Patients (mean age 61 ± 9 years; 80% male) were followed for a median time of 10.2 years; 22 patients underwent endarterectomy for contralateral ICA stenosis and 16 extracranial/intracranial bypass surgery. Recurrent ischaemic stroke occurred in 23 patients, resulting in an annual rate of 2.4% (95% confidence interval [CI], 1.5-3.6). Risk factors for recurrent ischaemic stroke were age (hazard ratio [HR] 1.07, 1.02-1.13), cerebral rather than retinal symptoms (HR 8.0, 1.1-60), recurrent symptoms after documented occlusion (HR 4.4,, limb-shaking TIAs at presentation (HR 7.5,, history of stroke (HR 2.8, 1.2-6.7) and leptomeningeal collaterals (HR 5.2, 1.5-17), but not CO 2 -reactivity (HR 1.01, 0.99-1.02). The composite event of any vascular event occurred in 57 patients, resulting in an annual rate of 6.4% (95%CI, 4.9-8.2).

Conclusion:

The prognosis of patients with TIA or minor stroke and ICA occlusion depends on age, several clinical factors and on the presence of leptomeningeal collaterals.

The long-term risk of recurrent ischaemic stroke is much lower than that of other vascular events.

INTRODUCTION

Patients with transient ischaemic attack (TIA) or ischaemic stroke associated with an occlusion of the internal carotid artery (ICA) have a risk of recurrent stroke of approximately 5-6% per year. [START_REF] Klijn | Outcome in patients with symptomatic occlusion of the internal carotid artery or intracranial arterial lesions: a meta-analysis of the role of baseline characteristics and type of antithrombotic treatment[END_REF] In the subgroup of patients with symptomatic ICA occlusion in whom a compromised haemodynamic state of the brain has been demonstrated, this risk is around 12% per year. [START_REF] Klijn | Symptomatic carotid artery occlusion. A reappraisal of hemodynamic factors[END_REF][START_REF] Grubb | Importance of hemodynamic factors in the prognosis of symptomatic carotid occlusion[END_REF] The information on long-term outcome in patients with symptomatic ICA occlusion is limited by short duration of follow-up [START_REF] Klijn | Symptomatic carotid artery occlusion. A reappraisal of hemodynamic factors[END_REF][START_REF] Grubb | Importance of hemodynamic factors in the prognosis of symptomatic carotid occlusion[END_REF][START_REF] Paciaroni | Outcome in patients with stroke associated with internal carotid artery occlusion[END_REF] or by lack of information about the flow state of the brain. [START_REF] Faught | Presentation and natural history of internal carotid artery occlusion[END_REF][START_REF] Fields | Joint study of extracranial arterial occlusion. X. Internal carotid artery occlusion[END_REF][START_REF] Furlan | Long-term prognosis after carotid artery occlusion[END_REF][START_REF] Flaherty | Population-based study of symptomatic internal carotid artery occlusion: incidence and long-term follow-up[END_REF][START_REF] Persson | The natural history of total occlusion of the internal carotid artery[END_REF][START_REF] The | Failure of extracranial-intracranial arterial bypass to reduce the risk of ischemic stroke. Results of an international randomized trial[END_REF] Since the large extracranial/intracranial (EC/IC) bypass trial did not show any benefit of bypass surgery in preventing recurrent stroke in patients with symptomatic ICA occlusion in general, [START_REF] The | Failure of extracranial-intracranial arterial bypass to reduce the risk of ischemic stroke. Results of an international randomized trial[END_REF] the current standard treatment consists of antithrombotic medication and management of vascular risk factors. Whether an EC/IC bypass operation or carotid endarterectomy (CEA) of a contralateral ICA stenosis can prevent stroke in a subgroup of patients with symptomatic ICA occlusion is uncertain. [START_REF] Grubb | Importance of hemodynamic factors in the prognosis of symptomatic carotid occlusion[END_REF][START_REF] Markus | Carotid endarterectomy improves haemodynamics on the contralateral side: implications for operating contralateral to an occluded carotid artery[END_REF][START_REF] Rutgers | Sustained bilateral hemodynamic benefit of contralateral carotid endarterectomy in patients with symptomatic internal carotid artery occlusion[END_REF][START_REF] Garrett | The efficacy of direct extracranial-intracranial bypass in the treatment of symptomatic hemodynamic failure secondary to athero-occlusive disease: a systematic review[END_REF][START_REF] Baracchini | Cerebral hemodynamics after contralateral carotid endarterectomy in patients with symptomatic and asymptomatic carotid occlusion: a 10-year follow-up[END_REF] We previously reported the short-term outcome of a prospectively collected cohort of patients with symptomatic ICA occlusion. [START_REF] Klijn | Recurrent ischemia in symptomatic carotid occlusion: prognostic value of hemodynamic factors[END_REF] The aim of the current follow-up study was to investigate the longterm outcome of this cohort and to study risk factors including haemodynamic characteristics for recurrent ischaemic stroke.

PATIENTS AND METHODS

Patients

We prospectively included 117 consecutive patients with symptoms of transient or at most moderately disabling (modified Rankin scale, mRS ≤3) [START_REF] Banks | Outcomes validity and reliability of the modified Rankin scale: implications for stroke clinical trials: a literature review and synthesis[END_REF] cerebral or retinal ischaemia associated with an ipsilateral ICA occlusion, who were referred to the Department of Neurology at the University Medical Center Utrecht, The Netherlands between September 1995 and July 1998. All patients had had symptoms within 6 months prior to inclusion in the study. The presence of the ICA occlusion was confirmed by digital subtraction angiography showing absence of filling of the extracranial ICA or common carotid artery (CCA). Patients were excluded if the ICA occlusion was caused by dissection or radiation therapy. All patients were interviewed about clinical characteristics and risk factors as listed in Table 2, with special attention for clinical characteristics suggesting a haemodynamic cause of symptoms such as limb-shaking, [START_REF] Baquis | Limb shaking--a carotid TIA[END_REF] precipitation of symptoms by rising from a sitting or lying position, exercise, transfer from a cold to a warm environment, a decrease of blood pressure, or retinal claudication.[18] Furthermore, we documented whether patients had had ongoing symptoms after occlusion of the carotid artery had been demonstrated (but before inclusion in the study). All patients underwent magnetic resonance imaging (MRI) or computed tomography (CT) scan of the brain. Cerebral infarcts were considered symptomatic if their location corresponded with the patients' symptoms and were classified as territorial, watershed, lacunar (diameter ≤15 mm) or large subcortical. [START_REF] Damasio | A computed tomographic guide to the identification of cerebral vascular territories[END_REF] The degree of stenosis of the contralateral ICA was measured on the angiograms according to the NASCET criteria. [START_REF] Fox | How to measure carotid stenosis[END_REF] The presence of collateral pathways was assessed by the combined information from the angiogram and transcranial Doppler (TCD).

Collateral pathways via either the anterior communicating artery (ACoA) or the posterior communicating artery (PCoA) were considered present if either of these showed at least filling of middle cerebral artery (MCA) branches on the angiogram or if TCD showed reverse flow in the first part of the anterior cerebral artery ipsilateral to the symptomatic ICA occlusion. Reversal of flow in the ipsilateral ophthalmic artery on TCD was considered a sign of collateral circulation via the external carotid artery. Leptomeningeal collaterals were considered present if pial branches from the posterior cerebral artery (PCA) extending as far as the vascular territory of the MCA or anterior cerebral artery (beyond the usual PCA territory) were visualised on the angiogram after selective catheterisation of one of the vertebral arteries beyond normal variability. [START_REF] Van Der Zwan | A quantitative investigation of the variability of the major cerebral arterial territories[END_REF] Patients underwent TCD with measurement of the CO 2 -reactivity to investigate cerebrovascular reserve capacity. The CO 2 -reactivity after carbogene inhalation was measured as the relative change in blood flow velocity in the MCA and expressed as a percentage as described previously. [START_REF] Klijn | Recurrent ischemia in symptomatic carotid occlusion: prognostic value of hemodynamic factors[END_REF] Treatment consisted of antithrombotic medication and management of vascular risk factors. Patients with a 70 to 99% stenosis of the contralateral ICA were offered CEA.

Patients with symptoms of cerebral ischaemia that continued after documentation of the ICA occlusion and evidence of presumably haemodynamic origin (i.e. the presence of specific symptoms associated with a haemodynamic cause, a watershed infarct or low CO 2 -reactivity) were offered treatment with the high flow EC/IC bypass. [START_REF] Klijn | Excimer laser-assisted high-flow extracranial/intracranial bypass in patients with symptomatic carotid artery occlusion at high risk of recurrent cerebral ischemia: safety and long-term outcome[END_REF] The study was approved by the institutional review board of the University Medical Center Utrecht and written informed consent was obtained from all patients.

Follow-up and outcome

Patients were followed at regular intervals in the outpatient clinic until November 1999.

Final follow-up information was obtained by structured telephone interviews of the patients in June 2008. If the patient had died we obtained information of their relatives or their general practitioner. In case of a possible outcome event, we determined the mRS by a standardized questionnaire [START_REF] Wilson | Improving the assessment of outcomes in stroke: use of a structured interview to assign grades on the modified Rankin Scale[END_REF] and retrieved medical records and CT or MRI scans of the brain in case of recurrent stroke. The primary outcome was any first recurrent ischaemic stroke defined as the acute onset of new focal neurological deficit of cerebral origin persisting for more than 24 hours without hemorrhage on CT or MRI scan of the brain. The secondary outcome event was defined as the composite event of a non-fatal ischaemic or haemorrhagic stroke, non-fatal myocardial infarction (MI), or death due to vascular causes, whichever happened first. [START_REF] Klijn | Recurrent ischemia in symptomatic carotid occlusion: prognostic value of hemodynamic factors[END_REF] Vascular death was defined as death from fatal stroke, fatal MI, sudden death, terminal heart failure, systemic bleeding, pulmonary embolism or complications after vascular surgery. Fatal stroke was defined as death within 30 days after stroke or death after major stroke (mRS≥4), in the absence of other clear causes. [START_REF] Halkes | Classification of cause of death after stroke in clinical research[END_REF] Any death that was not clearly non-vascular, or if no information was available about the cause of death, was classified as 'other vascular.' [START_REF] The | A randomised, blinded, trial of clopidogrel versus aspirin in patients at risk of ischaemic events (CAPRIE)[END_REF]

Data analysis

We determined annual rates of recurrent ischaemic stroke and of the combined outcome of vascular events with the Kaplan Meier method. The survival analysis started from the time of inclusion in the study. We used a Cox proportional hazards model for univariable analysis of pre-defined risk factors for the primary and secondary outcome events, resulting in hazard ratios (HR) with 95% confidence intervals (CI). The primary outcome recurrent ischaemic stroke was subdivided into any recurrent ischaemic stroke and only ipsilateral ischaemic stroke. Variables with a p-value < 0.15 in the univariable analysis were included in a multivariable model. To exclude the influence of surgical treatment on the effect of pre-defined risk factors on the occurrence of the primary outcome event we also performed the Cox proportional hazards analysis with censoring of patients at the time of CEA of a contralateral ICA stenosis or at the time of an EC/IC bypass.

RESULTS

In total we included 117 patients (mean age 61 years, range 35-79, 80% male). Ninetythree (80%) patients had presented with cerebral ischaemic symptoms, whereas 24 (20%) patients had had only retinal ischaemic symptoms (retinal infarction in three, transient monocular blindness in 19 and chronic ocular ischaemic syndrome in five patients; three patients had two of these symptoms). Symptoms with haemodynamic characteristics were present in 16 patients; limb-shaking in nine, retinal claudication in three, symptoms after rising in five, after exercise in three and after low blood pressure in two patients.

Seventy-eight (66%) patients had a symptomatic infarct on their CT or MRI, that we classified as territorial in 31 patients, watershed in 32, lacunar in 11 and large subcortical in four patients. Of 117 patients, 40 (34%) patients underwent a surgical revascularization procedure after a median of 46 days after inclusion (range 3-623 days). Twenty-two patients underwent CEA of a 70% to 99% contralateral ICA stenosis. One of them also had angioplasty for severe stenosis of the proximal part of the CCA on the side of the occlusion 14 days after the CEA because of recurrent TIAs. One patient underwent stenting of the contralateral ICA 20 months after inclusion in the study because of recurrent TIAs due to a contralateral ICA stenosis that had progressed from moderate to severe (>70%). Another patient underwent endarterectomy of a 90% stenosis of the ECA.

Sixteen patients underwent EC/IC bypass surgery. Six patients with a 70-99% stenosis of the contralateral ICA chose not to be operated on and in another patient CEA was not possible because the stenosis extended to the carotid siphon.

Risk of recurrent ischaemic stroke

The median follow-up time until recurrent ischaemic stroke or death was 10.2 years (range 7 days-12.8 years). None of the patients was lost to follow-up. Of the 117 patients, 52 (44%) died during follow-up. Any recurrent ischaemic stroke occurred in 23 (20%) patients, resulting in an annual rate of 2.4% (95% CI, 1.5-3.6). The risk of any recurrent ischaemic stroke was highest in the first 1.5 years after presentation (Figure 1) (annual rate 8.0%, 95% CI 4.4-13.0). As shown in Table 1, 15 of the 23 patients had an ipsilateral ischaemic stroke, whereas eight ischaemic strokes occurred in another vascular territory.

Nine of the 23 recurrent ischaemic strokes were major resulting in a mRS≥4 (of which six were fatal) and 13 were at most moderately disabling strokes (mRS≤3). In one patient we could not reliably determine the mRS because of co-morbidities. In three patients the (minor) ischaemic stroke occurred during the time they were waiting for the planned operation. Recurrent ischaemic stroke after the intervention occurred in six patients after an EC/IC bypass operation (four within 30 days, one after eight months and one almost 12 years after the operation), in two patients after CEA of the contralateral ICA (three and four years after surgery) and in one patient immediately after angioplasty of the CCA. Eleven of the 77 (14%) patients who did not undergo a revascularization procedure had a recurrent ischaemic stroke. Two of them were known to have a contralateral ICA stenosis. The annual rate of recurrent ischaemic stroke in patients who did not undergo a revascularization operation was 2.1% (95% CI 1.2-3.6).

Risk factors for recurrent ischaemic stroke

Table 2 shows the relationship between baseline characteristics and the recurrence of ischaemic stroke. Patients with cerebral symptoms had an eight times (age and sex adjusted HR 8.0, 95% CI 1.1-60) higher risk of recurrent ischaemic stroke than patients with retinal symptoms only. Continuation of symptoms after documented occlusion was associated with a four times (HR 4.5, 95% CI 1.6-12) higher risk of recurrent ischaemic stroke. Other risk factors were older age (HR 1.07, 95% CI 1.02-1.13), the presence of limb-shaking (HR 7.7, 95% CI 2.6-22), history of stroke (HR 2.9, 95% CI 1.2-6.7) and the presence of leptomeningeal collaterals on angiography (HR 5.1, 95% CI 1.5-17).

TCD CO 2 -reactivity was not predictive for the risk of recurrent ischaemic stroke (HR 1.01, 95% CI 0.99-1.02). Bilateral ICA occlusion tended to be associated with a lower risk of recurrent ischaemic stroke (HR 0.2, 95% CI 0.02-1.3). Because of the relatively small number of recurrent ischaemic strokes we refrained from further multivariable analysis. When we restricted the analysis to ipsilateral recurrent ischaemic stroke, age and sex adjusted risk factors were continuation of symptoms after documented carotid occlusion (HR 7.3, 95% CI 1.7-33), limb-shaking (HR 7.8, 95% CI 2.4-26) and leptomeningeal collaterals (HR 5.3, 95% CI 1.2-23). After censoring the patients at the time of intervention, the age and sex adjusted factors continuation of symptoms after documented carotid occlusion (HR 3.7, 95% CI 1.2-11) and older age (HR 1.07, 95% CI 1.00-1.13) remained significantly associated with recurrent ischaemic stroke.

Risk of the composite outcome of any vascular event

Vascular outcome events occurred in 57 (49%) patients. This included 28 vascular deaths (including six fatal ischaemic strokes, one fatal intracerebral haemorrhage, one fatal MI, six terminal heart failures, 11 sudden deaths, three other vascular causes), 17 non-fatal ischaemic strokes, one non-fatal intracerebral haemorrhage in the basal ganglia and 11 non-fatal MIs. Overall, of the 52 patients who died during follow-up the causes of death were vascular in 40 patients (in 28 vascular death as first event, in 12 vascular death after non-fatal stroke or MI as first event), and non-vascular in 12 patients (malignant disease in seven, infectious disease in three, renal disease in one, and an undiagnosed gradually progressive disease in one patient). The annual rate of the composite vascular endpoint was 6.4% (95% CI, 4.9-8.2; Figure 1). Multivariable analysis showed that age (HR 1.06, 95% CI 1.02-1.09; Table 3), a history of stroke (HR 1.9, 95% CI 1.1-3.5) and a history of ischaemic heart disease (HR 1.8, 95% CI 1.1-3.2) were independent risk factors for the occurrence of any vascular event.

DISCUSSION

This 10-years follow-up study shows that patients with TIA or minor ischaemic stroke and ICA occlusion, treated with EC/IC bypass in case of recurrent symptoms of presumably haemodynamic origin or treated with CEA in case of contralateral ICA stenosis, have an annual risk of recurrent ischaemic stroke of 2.4%, with the highest risk in the first 1.5 years after presentation. Elderly patients, those who present with cerebral instead of retinal ischaemic symptoms, patients with limb-shaking, recurrent symptoms after documented occlusion, a history of stroke, and those who have leptomeningeal collaterals on their angiogram have an increased risk of recurrent ischaemic stroke. CO 2reactivity as measured with TCD did not predict recurrence of ischaemic stroke.

We were able to obtain a complete and very long duration of follow-up of a large cohort of well-documented prospectively studied patients. The largest study on outcome in patients with symptomatic ICA occlusion to date is the EC/IC bypass trial, [START_REF] The | Failure of extracranial-intracranial arterial bypass to reduce the risk of ischemic stroke. Results of an international randomized trial[END_REF] in which 423 patients who received best medical treatment were followed for 4.5 years. This study reported an annual rate of ischaemic stroke of 6.3%, but measurements of the haemodynamic state of the brain were not performed. Other prospective follow-up series of patients with symptomatic ICA occlusion had a mean duration of follow-up of at most 4 years and found annual rates of ischaemic stroke between 5 and 10%, [START_REF] Grubb | Importance of hemodynamic factors in the prognosis of symptomatic carotid occlusion[END_REF][START_REF] Paciaroni | Outcome in patients with stroke associated with internal carotid artery occlusion[END_REF][START_REF] Fields | Joint study of extracranial arterial occlusion. X. Internal carotid artery occlusion[END_REF][START_REF] Vernieri | Outcome of carotid artery occlusion is predicted by cerebrovascular reactivity[END_REF][START_REF] Hankey | Prognosis of Symptomatic Carotid Artery Occlusion[END_REF] except for one study that found an annual rate of 2%. [START_REF] Persson | The natural history of total occlusion of the internal carotid artery[END_REF] Some of these studies also included patients who presented with major stroke. [START_REF] Paciaroni | Outcome in patients with stroke associated with internal carotid artery occlusion[END_REF][START_REF] Fields | Joint study of extracranial arterial occlusion. X. Internal carotid artery occlusion[END_REF] The comparatively low recurrent stroke rate that we found can be explained in three ways. First, we showed that patients who present with symptomatic ICA occlusion have the highest risk of recurrent ischaemic stroke in the first 1.5 years and the risk of stroke is relatively low thereafter.

Second, a considerable proportion of the patients in our study had presented with retinal ischaemic symptoms only. Patients with retinal symptoms only have a lower risk of ischaemic stroke than patients who present with cerebral ischaemic symptoms. [START_REF] Grubb | Importance of hemodynamic factors in the prognosis of symptomatic carotid occlusion[END_REF][START_REF] Streifler | The risk of stroke in patients with first-ever retinal vs hemispheric transient ischemic attacks and high-grade carotid stenosis. North American Symptomatic Carotid Endarterectomy Trial[END_REF] Third, over the last decades medical secondary prevention has improved with the introduction of statins, [START_REF] Amarenco | Lipid management in the prevention of stroke: review and updated meta-analysis of statins for stroke prevention[END_REF] dipyridamole in combination with aspirin [START_REF]The Esprit study group. Aspirin plus dipyridamole versus aspirin alone after cerebral ischaemia of arterial origin (ESPRIT): randomised controlled trial[END_REF] and more rigorous control of blood pressure. [START_REF] Zhang | Blood pressure lowering for primary and secondary prevention of stroke[END_REF] In contrast to other studies, [START_REF] Vernieri | Outcome of carotid artery occlusion is predicted by cerebrovascular reactivity[END_REF][START_REF] Kleiser | Course of carotid artery occlusions with impaired cerebrovascular reactivity[END_REF] TCD CO 2 -reactivity in this study was not predictive of recurrent ischaemic stroke. In the previous studies the results may have been confounded by including asymptomatic as well as symptomatic patients. Asymptomatic patients have a relatively low stroke risk [START_REF] Powers | Benign prognosis of never-symptomatic carotid occlusion[END_REF] and a relatively high cerebrovascular reactivity. [START_REF] Vernieri | Outcome of carotid artery occlusion is predicted by cerebrovascular reactivity[END_REF] Similar to what we found after 2 years follow-up, [START_REF] Klijn | Recurrent ischemia in symptomatic carotid occlusion: prognostic value of hemodynamic factors[END_REF] we found that the presence of leptomeningeal collaterals on the angiogram was predictive of recurrent ischaemic stroke.

Although the role of these collaterals in the flow state of the brain needs further investigation, [START_REF] Brozici | Anatomy and functionality of leptomeningeal anastomoses: a review[END_REF][START_REF] Liebeskind | Collateral circulation[END_REF] it is suggested that the finding of leptomeningeal collaterals is indicative of haemodynamic compromise. [START_REF] Hofmeijer | Collateral circulation via the ophthalmic artery or leptomeningeal vessels is associated with impaired cerebral vasoreactivity in patients with symptomatic carotid artery occlusion[END_REF][START_REF] Muller | Vasomotor reactivity and pattern of collateral blood flow in severe occlusive carotid artery disease[END_REF][38] The risk of the composite endpoint of any vascular event of 6.4% per year is relatively high when compared to the risk of 4% per year in patients with TIA or minor stroke not selected because of carotid disease, [START_REF]The Esprit study group. Aspirin plus dipyridamole versus aspirin alone after cerebral ischaemia of arterial origin (ESPRIT): randomised controlled trial[END_REF] probably indicating that patients with a symptomatic ICA occlusion have relatively severe generalised vascular disease.

This study was a single-center study. Patients with a symptomatic ICA occlusion were referred from all over the Netherlands to a tertiary university hospital and referral bias may have played a role. However, patients with frequent and ongoing symptoms are probably more likely to be referred than those with only one event and no further symptoms. Therefore, the risk of recurrent ischaemic stroke that we report would rather be over-than underestimated.

Another limitation of our study is that it does not reflect true natural history as we advised CEA in patients with a >70% stenosis of the contralateral ICA and EC/IC bypass operation in selected patients with recurrent symptoms of cerebral ischaemia of presumed haemodynamic origin. Recently, another observational study showed that CEA of the contralateral ICA in 39 patients with symptomatic or asymptomatic ICA occlusion resulted in improvement of the vasomotor reactivity compared with 32 control patients who did not undergo CEA. However, they did not find a beneficial effect of CEA on the rate of recurrent stroke or death on long-term. [START_REF] Baracchini | Cerebral hemodynamics after contralateral carotid endarterectomy in patients with symptomatic and asymptomatic carotid occlusion: a 10-year follow-up[END_REF] Although there is no evidence from randomized controlled trials that CEA of a contralateral ICA stenosis or EC/IC bypass operation can prevent stroke, we advised these operations because we estimated the risk of recurrent stroke to be high if patients were treated with best medical treatment only.

Whether this treatment strategy may have prevented ischaemic stroke in some patients remains unclear. In fact, 5 operated patients suffered a stroke within 30 days of the operation and 4 of the 35 patients who were operated on without any complications suffered a recurrent stroke after on average 5 years. Because of the selection bias with regard to treatment, we could not statistically compare outcome in medically and surgically treated patients.

A third limitation is that the follow-up of our patients did not include serial duplex scanning or CT-angiography to reconfirm the occlusion after several years. Although infrequently, it has been described that spontaneous recanalization of an occluded ICA can occur, and a remaining high-grade stenosis would expose a patient at increased risk of an embolic event. [START_REF] Nguyen-Huynh | Spontaneous recanalization of internal carotid artery occlusion[END_REF] Despite complete occlusion of the ICA, patients who have not suffered a major stroke at the time of occlusion have a relatively low risk of recurrent stroke, in particular once no stroke has occurred in the first 18 months. While this information is reassuring to the patients, it should be noted that their risk of other vascular events is significant and meticulous control of vascular risk factors is of key importance. Patients who continue to have symptoms after documentation of the ICA occlusion have a relatively high risk of recurrent ischaemic stroke. Although our study was not designed to evaluate the effectiveness of several treatment strategies, we suggest that particularly in patients with ongoing symptoms after documented occlusion and a contralateral ICA stenosis a CEA may be considered. EC/IC bypass surgery is associated with a considerable risk of postoperative stroke, in particular in unstable patients with repeated TIAs. [START_REF] Klijn | Excimer laser-assisted high-flow extracranial/intracranial bypass in patients with symptomatic carotid artery occlusion at high risk of recurrent cerebral ischemia: safety and long-term outcome[END_REF] Whether or not EC/IC bypass surgery can prevent stroke in patients with demonstrated haemodynamic compromise will be answered by the ongoing Carotid Occlusion Surgery Study. [START_REF] Grubb | The Carotid Occlusion Surgery Study[END_REF] Table 1. Overview of the primary and secondary outcome events * Analysis with censoring at the time of the first revascularization procedure ** HR for age is expressed as the increase in hazard for every incremental year. † Defined as patients with either a history of hyperlipidaemia, patients on drugs because of hyperlipidaemia or patients with levels of cholesterol, triglycerides, or high density lipoprotein cholesterol beyond the normal ranges.

21 ‡ In 24 patients the presence of collateral circulation via either the ACoA or PCoA could not be determined. § In 24 patients the flow direction in the ophthalmic artery could not be determined. ¶ In 41 patients the angiography was insufficient to define the presence of leptomeningeal collaterals.

|| CO 2 -reactivity ipsilateral to the symptomatic ICA occlusion could be measured in 21 of 23 patients and in 88 of 94 patients without a recurrent ischaemic stroke.

HR is expressed as the increase in hazard for every % increase in CO 2 -reactivity.

# In 32 of 78 patients with symptomatic ischaemic lesion on CT or MRI, we classified the infarct as watershed.

NE, not estimable.

*

  One patient underwent stenting of the contralateral ICA, another patient underwent CEA of the ipsilateral external carotid artery ** One patient had the recurrent ischaemic stroke after angioplasty of the ipsilateral common carotid artery 14 days after CEA of the contralateral ICA

Figure 1 .

 1 Figure 1. Time-to-event curves for recurrent ischaemic stroke (upper part) and for any

Table 2 .

 2 Analysis of risk factors for recurrent ischaemic stroke in patients with symptomatic ICA occlusion (n=117)

	Ischaemic stroke	Univariable analysis Age and sex	Censored age and sex adjusted
				adjusted analysis	analysis *
	Yes	No
	n = 23	n = 94	HR (95% CI)	HR (95% CI)	HR (95% CI)

Table 3 .

 3 Analysis of risk factors for any stroke, myocardial infarction or vascular death in patients with symptomatic ICA occlusion (n=117)

	Any stroke, myocardial	Univariable analysis	Multivariable analysis
	infarction or vascular death
	Yes	No
	N = 57	N = 60	HR (95% CI)	HR (95% CI)

ACKNOWLEDGMENTS:

S. Persoon is supported by a grant from the Netherlands Heart Association (grant number 2003B263) and the Foundation "De Drie Lichten" (grant number 41/09). C.J.M. Klijn was supported by a clinical fellowship from the Netherlands Organization for Health Research and Development (grant number 907-00-103).

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