Hanneke De Waal
email: h.dewaal@vumc.nl

Cornelis J Stam
email: cj.stam@vumc.nl

Marinus A Blankenstein
email: ma.blankenstein@vumc.nl

Yolande Al Pijnenburg
email: y.pijnenburg@vumc.nl

Philip Scheltens
email: p.scheltens@vumc.nl

Wiesje M Van Der Flier

EEG abnormalities in early and late onset Alzheimer's Disease: understanding heterogeneity

Keywords: Alzheimer Disease, young onset, electroencephalography, visual analysis

Objective: To compare differences in severity and type of EEG abnormalities between early and late onset Alzheimer's disease (AD) and to assess the influence of APOE genotype on this association, in order to understand the biological differences in AD according to age at onset Method: Of 460 probable AD patients and 336 patients with subjective complaints, serving as controls, EEG and APOE genotype were obtained. Subjects were categorised by age into a younger (≤ 65 years) and an older group (> 65 years), based on age at diagnosis. Severity and type of EEG abnormalities were visually assessed. Severity of EEG abnormalities ranged from normal to slightly abnormal to moderately severe. EEG abnormalities were characterized as only focal abnormalities, only diffuse abnormalities or both focal and diffuse abnormalities.

Results: Logistic regression revealed that younger AD patients more often had EEG abnormalities, which were more severe, with a predominance of both focal and diffuse abnormalities. In controls we observed the opposite, as older controls more often had EEG abnormalities than younger controls. Furthermore, APOE ε4 negative AD patients had more severe EEG abnormalities than APOE ε4 positive AD patients, while no such effect was observed in controls. There was no interaction between age at onset and APOE ε4 genotype.

Conclusion:

Early onset and APOE ε4 negative AD patients present with more severe EEG abnormalities than late onset and APOE ε4 positive AD patients. These results suggest that in younger patients, AD manifests with more prominent functional brain changes.

Introduction

Alzheimer's disease (AD) is the most common cause of dementia. Although it is typically regarded as a disease of old age, it does occur in younger patients (i.e. before the age of 65 years), commonly referred to as early onset AD. The characteristic clinical presentation of AD in the elderly consists of progressive memory impairment, followed by global cognitive decline. Early onset AD often presents in a different way compared to AD with late onset. Early onset AD patients more often show focal impairments like aphasia and apraxia, [START_REF] Seltzer | A comparison of clinical features in early-and lateonset primary degenerative dementia. One entity or two?[END_REF][START_REF] Greicius | Presenile dementia syndromes: an update on taxonomy and diagnosis[END_REF][START_REF] Licht | Cognitive differences between early-and late-onset Alzheimer's disease[END_REF][START_REF] Koedam | Early-versus Late-Onset Alzheimer's Disease: More than Age Alone[END_REF] and a more rapid cognitive decline.(5; 6) APOE ε4 genotype is associated with age at onset. [START_REF] Strittmatter | Apolipoprotein E and Alzheimer disease[END_REF] Moreover, it has been suggested that APOE ε4 carriers present with more severe memory impairment, while conversely non-carriers present with more pronounced impairment in other cognitive domains .(8; 9) These differences in presentation suggest the existence of heterogeneity within the spectrum of AD.

EEG can be used to distinguish AD patients and healthy controls, with a positive predictive value between 75-80% in visual as well as quantitative analysis. [START_REF] Jonkman | The role of the electroencephalogram in the diagnosis of dementia of the Alzheimer type: an attempt at technology assessment[END_REF] Hallmarks of EEG abnormalities in AD are an increase in diffuse slow activity and a reduction in alpha and beta activities. [START_REF] Jeong | EEG dynamics in patients with Alzheimer's disease[END_REF] Studies on EEG in early onset AD are sparse. In a small group of early onset AD patients an increase of power in the slow frequency bands and a decrease of power in the fast frequency bands was found, compared to age-matched controls. [START_REF] Schreiter-Gasser | Quantitative EEG analysis in early onset Alzheimer's disease: a controlled study[END_REF] However, no comparison was made between early onset and late onset AD. In another study, correlations were found between several EEG parameters and age at onset of AD. A more 'abnormal' EEG occurred in patients with a younger age of onset. [START_REF] Pucci | EEG power spectrum differences in early and late onset forms of Alzheimer's disease[END_REF] In the present study we aimed to compare differences in severity and type of EEG abnormalities between early onset and late onset AD by visual EEG analysis in a large population of AD patients. A group of controls was included for comparison.

Furthermore we assessed how APOE genotype influenced this association. We hypothesized that patients with early onset AD have more often and more severe EEG abnormalities compared to patients with late onset AD. 16) Presence of diffuse abnormalities was defined as a dominant frequency of rhythmic background activity below 8 Hz, diffuse slow-wave activity or diminished reactivity of the rhythmic background activity to the opening of the eyes. These two variables were combined in a third 4-level variable: (i) no abnormalities, (ii) only focal abnormalities, (iii) only diffuse abnormalities and (iv) both focal and diffuse abnormalities. In addition, severity of EEG abnormalities was rated using a 4point scale, ranging from no abnormalities to severe abnormalities. Due to very small group size for severe abnormalities (n=4), the groups moderately severe and severe abnormality were merged, resulting in 3 levels in the severity variable: none, mild, moderate to severe abnormalities. In previous reports by our study group kappavalues for inter observer agreement between .60 and .87 have been reported. (

Methods

Subjects

Results

Baseline characteristics are presented for patients and controls according to age (table 1). The mean age in the AD group was higher than in the control group, for both the younger and the older patients. AD patients were more often APOE ε4 positive than controls. In the group of AD patients there was no difference in sex, estimated disease duration or APOE ε4 carrier ship according to age. The younger AD group had a lower MMSE-score than the older AD group. In controls there was no difference in sex, MMSE-score or APOE ε4 carrier ship according to age. 1. In controls we found the opposite pattern:

EEG abnormalities were found more often in older controls than in younger controls (p < .01), although there was no significant difference in severity of abnormalities (p = .54).

Figure 2 shows the association between EEG abnormalities and APOE ε4 genotype.

We observed that APOE ε4 negative AD patients had more severe EEG abnormalities than APOE ε4 positive patients (p <. 05); we found no effect of APOE ε4 genotype on type of EEG abnormalities. In controls there was no effect of APOE ε4 genotype on type or severity of EEG abnormalities.

Subsequently, we used logistic regression to adjust for sex, use of psychotropic medication and estimated disease duration (see table 3).

Discussion

We found that younger AD patients more often had a combination of focal and diffuse EEG abnormalities than older AD patients. They also had more severe EEG abnormalities than older AD patients. In controls, the opposite effect was found: more often and more severe EEG abnormalities occurred in older controls. In APOE ε4

negative AD patients severe EEG abnormalities occurred more often than in APOE ε4

positive patients. This effect was independent of the effect of age.

Our results are in line with the few earlier studies that reported more severe EEG abnormalities in early onset AD. In a quantitative EEG study on patients with early onset AD opposed to controls, early onset AD patients showed an increase of power in the slow frequency bands.( 12 Strengths of this study are the large cohort size including AD patients as well as controls with a wide age range, and the use of visual analysis, which together make the findings in our study quite robust. Visual analysis is a relatively easy and fast way of analysing EEG's and, for clinical purposes, the visual assessment of EEG is equal to quantitative analysis, with a high sensitivity for moderate-to-severe dementia.( 25)

A former study demonstrated that visual EEG analysis can be used to differentiate between different diagnoses in a memory clinic population. [START_REF] Liedorp | Associations between patterns of EEG abnormalities and diagnosis in a large memory clinic cohort[END_REF] Another tool for visual EEG analysis, the Grand Total of EEG (GTE) score, has proven to be useful in the diagnostic evaluation of AD [START_REF] Claus | The diagnostic value of electroencephalography in mild senile Alzheimer's disease[END_REF] and in differentiating between DLB and AD with good sensitivity and specificity. [START_REF] Roks | The use of EEG in the diagnosis of dementia with Lewy bodies[END_REF] In the present study, we show that visual analysis can also differentiate subgroups within the spectrum of AD.

A possible limitation of this study is the use of patients with subjective complaints as control group, since these patients are known to have an increased risk of progression to dementia [START_REF] Visser | Ten-year risk of dementia in subjects with mild cognitive impairment[END_REF] and could comprise of a complex group. However, we believe that by excluding patients with major psychiatric disorders, our control group is generalizable to a normal population.

The results of this study give further evidence for the existence of biological differences between early onset and late onset AD. We demonstrated that early onset AD patients show more EEG abnormalities than late onset AD patients. This implies that early onset AD patients behave differently than late onset patients not only on a structural, behavioural and cognitive level, but also on the level of brain function. An underlying structural and functional difference in different patient groups within AD is of great clinical importance, since it can help making an early diagnosis, it can influence the kind of treatment needed and it might imply a different prognosis.

:

  We included 460 probable AD patients and 336 controls. All patients had been referred to the memory clinic of the Alzheimer center of the VU university medical center, Amsterdam, the Netherlands between March 2001 and June 2009. Standardised dementia screening included a history and, when available, an informant based history, a standard neurological examination, a cognitive examination including Mini Mental State Examination (MMSE), electroencephalography (EEG), Magnetic Resonance Imaging (MRI) of the brain, neuropsychological evaluation and laboratory tests. Patients were diagnosed with probable AD according to the NINCDS-ADRDA criteria (14) during a multidisciplinary consensus meeting. The control group consisted of patients who presented at our memory clinic with subjective complaints, but who had normal clinical investigations and did not have significant cognitive deficits (i.e. MCI criteria were not fulfilled) or major psychiatric disorder. To rule out the influence of past medical history on EEG characteristics, we excluded 54 patients, with a past medical history of epilepsy, serious head trauma, ischemic stroke, haemorrhagic stroke, subarachnoid haemorrhage, multiple sclerosis, meningitis, encephalitis or intracranial space occupying lesions. Patients were categorised in younger (65 years or younger; n= 154) and older (older than 65 years; n=280) category, based on age at diagnosis at the multidisciplinary consensus meeting. Controls were also categorised in a younger (n=211) and an older (n=97) age group, based on age at time of discussion at the multidisciplinary consensus meeting. The study has been approved by the ethical review board of the VU University Medical Center. All patients gave written informed consent to use their clinical data for research purposes. EEG recording All EEGs were recorded using the Nihon Kohden digital EEG apparatus (EEG 2100), and since September 2003, OSG digital equipment (Brainlab®; OSG b.v., Rumst, Belgium) at the positions of the 10-20 system: Fp2, Fp1, F8 F7, F4, F3, A2, A1, T4, T3, C4, C3, T6, T5, P4, P3, O2, O1, Fz, Cz, Pz. Sample frequency was 200 Hz for the Nihon Kohden system and 500 Hz for the OSG Brainlab system. Electrode impedance was below 5kΩ. Initial filter settings were: time constant 1s; low pass filter, 70 Hz. Patients were seated in a slightly reclined chair in a sound attenuated room. Patients sat mainly with eyes closed, EEG technicians were alert on keeping patients awake by sound stimuli. Visual EEG assessment: Board certified clinical neurophysiologists assessed all EEG recordings, without knowledge of clinical information. Type of EEG abnormalities consisted of two dichotomous variables: focal abnormalities and diffuse abnormalities.(15) Presence of focal abnormalities was defined as (transients of) slow or sharp wave activity in 1 or more EEG leads, excluding benign temporal theta of the elderly (BTTE).(

15 )

 15 APOE genotyping APOE genotyping was performed after DNA isolation from 10 ml EDTA blood, with the Light Cycler APOE mutation detection method (Roche Diagnostics GmbH, Mannheim, Germany). APOE ε4 carrier ship was dichotomized in negative or positive, with positive containing both homozygous and heterozygous APOE ε4 carriers.Statistics: SPSS 15.0 for Windows was used for statistical analyses. Differences between groups for baseline characteristics were investigated with t-tests and χ 2 -tests where appropriate. Differences in prevalence of EEG abnormalities between groups were investigated by χ 2 -tests. Subsequently, to adjust for sex, use of psychotropic medication and estimated disease duration and to assess the combined effect of age and APOE genotype on EEG abnormalities, logistic regression analyses were performed. Presence of EEG abnormalities, dichotomized as no abnormalities versus any abnormalities, was used as dependent variable. In model 1, the effects of both age and APOE genotype were assessed unadjusted in separate models. In model 2 we adjusted for sex and use of psychotropic medication. In model 3 both age and APOE genotype, adjusted for sex, use of psychotropic medication and estimated disease duration were entered simultaneously. Interaction between age group and APOE genotype was assessed. For age, older age group was used as reference group and for APOE the positive genotype was used as reference group. Odds Ratio (OR) and 95% confidence intervals are reported.

Table 1 .

 1 Baseline characteristics of memory clinic populationValues are mean (SD) or n (%). a early onset AD group versus younger controls: p < .05. b late onset AD group compared to older controls: P<.05. c early onset AD versus late onset AD: p < .05.

	In the control group, 65% did not report any clinically significant past medical
	history, 14% reported cardiovascular disease (e.g. atrial fibrillation, myocardial
	infarction, angina, lower extremity claudication, aortic aneurysm), 8% reported
	depression in the past, 2% reported pulmonary disease (COPD, asthma), 2% suffered
	from type 2 diabetes mellitus, 2% obstructive sleep apnea syndrome, 1% reported

patients (p < .05). Examples of characteristic EEGs of both a younger AD patient and an older AD patient are shown in figure

Table 3 .

 3 Influence of age at onset of AD and APOE ε4 genotype on EEG abnormalities. effects remained of comparable strength. There was no interaction between APOE ε4 genotype and age at onset of AD (p = .70).

	Data are presented as Odds Ratio (95% Confidence Interval). For age: 'late' is
		Model 1	Model 2	Model 3
	Age (early/late)	1.8 (1.1-2.8)*	1.7 (1.1-2.8)*	1.7 (1.1-2.8)*
	APOE genotype	1.5 (0.9-2.5)	1.7 (1.0-2.7)*	1.7 (1.0-2.8)*
	(positive/negative)
	reference category. For APOE ε4 genotype: 'positive' is reference category
	Model 1: univariate; Model 2: sex, medication and disease duration as covariates;
	Model 3: both age and APOE genotype are entered in the model.
	EEG abnormalities were dichotomized as no abnormalities or any severity of
	abnormalities. Younger AD patients had an increased risk of EEG abnormalities,
	compared to older patients (OR 1.7, 95% CI 1.1-2.8). APOE ε4 negative AD patients

had an increased risk of EEG abnormalities, as opposed to APOE ε4 positive patients (OR 1.5, 95% CI 0.9-2.4). When we entered both age and APOE ε4-genotype in one

  ) In a study comparing early onset AD patients with late onset AD patients, significant correlations were found between age at onset and In addition to the effect of age, we also explored the influence of APOE ε4 genotype on EEG abnormalities. Since it has been shown before that the effect of age at onset may be modified by APOE genotype,(7; 8; 20) we examined if there was an interaction between age at onset and APOE ε4 genotype on the prevalence of EEG abnormalities. This was not the case, but we did find a main effect of APOE ε4 genotype, with APOE ε4 negative AD patients having more prominent EEG abnormalities than APOE ε4 positive patients. This is in contrast to most earlier studies, which found more EEG slowing in APOE ε4 positive patients.(8; 21; 22)

	relative power in different frequency bands.(13) We extend on these former studies by
	including a large sample of AD patients and controls, showing that the effect of age
	on EEG has opposite directions in these two groups.
	Earlier research on the difference between early onset and late onset AD showed
	evidence of heterogeneity in other modalities as well: early onset AD patients show
	more global atrophy with a faster atrophy rate,(17) disproportionate precuneus
	atrophy (18) and a more severe glucose hypometabolism in parietal, frontal and
	subcortical areas.(19) These findings suggest the involvement of different regions,
	and perhaps of different neuronal networks. Our findings show that brain function is
	differentially affected in AD patients with early and late onset. Further study is
	needed to take regional changes in brain function into account.

Studies on functional connectivity report conflicting results: one study found a higher connectivity in APOE ε4 positive patients,

[START_REF] Kramer | EEG functional connectivity and ApoE genotype in Alzheimer's disease and controls[END_REF]

whereas another study found a reduced connectivity in APOE ε4 positive patients.

[START_REF] Jelic | Apolipoprotein E epsilon4 allele decreases functional connectivity in Alzheimer's disease as measured by EEG coherence[END_REF]

In both studies no slowing in quantitative EEG analysis for APOE ε4 positive patients was found. We are not sure what the reason is of our finding more prominent EEG abnormalities in APOE ε4 negative patients, but we feel that these findings are in line with former observations of APOE ε4 negative AD patients more often showing a more aggressive clinical course, especially when they are young.

(6; 17)

Acknowledgement

The Alzheimer Center VUmc is supported by Alzheimer Nederland and Stichting VUmc fonds. The clinical database structure was developed with funding from Stichting Dioraphte.

Copyright Licence Statement

I, Hanneke de Waal, the corresponding author of this article ("the Contribution") has the right to grant on behalf of all authors and does grant on behalf of all authors, a licence to the BMJ Publishing Group Ltd and its licensees, to permit this Contribution (if accepted) to be published in Journal of Neurology, Neurosurgery and Psychiatry (JNNP) and any other BMJ Group products and to exploit all subsidiary rights, as set out in our licence set out at (http://jnnp.bmj.com/site/about/licence.pdf).

I am one author signing on behalf of all co-owners of the Contribution.

List of names and email addresses of all co-authors:

Hanneke de Waal h.dewaal@vumc.nl Patient is a 79 year old female, MMSE score 27.

APPENDICES

Figure 2 Bar graph representing the association between APOE ε4 genotype and severity of EEG abnormalities. In AD, APOE ε4 negative patients had more severe EEG abnormalities than APOE ε4 positive patients (p < .05). In controls there was no effect of APOE ε4 genotype on severity of EEG abnormalities.


MD Alida A Gouw
email: aa.gouw@vumc.nl

MD Alexandra Seewann

PhD Wiesje M Van Der Flier

PhD Frederik Barkhof

Annemieke M Rozemuller

PhD Philip Scheltens

PhD Jeroen J G Geurts

PhD J M Rozemuller

Heterogeneity of small vessel disease: a systematic review of MRI and histopathology correlations

Keywords:

come   L'archive ouverte pluridisciplinaire

Background

MRI and clinical expression of small vessel disease

White matter hyperintensities (WMH), lacunes, and microbleeds are regarded as MRI expressions of small vessel disease (SVD) and are commonly found on brain MRI of elderly subjects. WMH are visible as hyperintense areas on T2-weighted MRI scans (including FLAIR), while lacunes are identified on MRI as small cavities with a diameter of 3mm to 10-15mm, and signal intensities comparable to cerebrospinal fluid. Lacunes are located in the white matter (WM) or subcortical gray matter and often have a surrounding hyperintense halo. Microbleeds are small, round, hypointense foci on gradient-echo T2*-weighted MRI, and are mostly located in the basal ganglia or corticalsubcortical areas. 1;2 Examples of these abnormalities are given in Figure 1. Unfortunately, definition and quantification of these MRI expressions of SVD vary between studies. This warrants a standardization of SVD rating on MRI, as the extrapolation of results from different studies to more general conclusions may be severely hampered otherwise.

In non-demented elderly subjects, WMH, lacunes and microbleeds have been associated with cognitive decline, including reduced mental speed and impaired executive functions. [START_REF] Van Der Flier | Small vessel disease and general cognitive function in nondisabled elderly: the LADIS study[END_REF][START_REF] Vermeer | Silent brain infarcts and the risk of dementia and cognitive decline[END_REF][START_REF] Gold | Cognitive consequences of thalamic, basal ganglia, and deep white matter lacunes in brain aging and dementia[END_REF][START_REF] Werring | Cognitive dysfunction in patients with cerebral microbleeds on T2*-weighted gradient-echo MRI[END_REF][START_REF] Won | Clinical significance of microbleeds in subcortical vascular dementia[END_REF] WMH have also been related to other potentially disabling symptoms, such as gait disturbances, depression and urinary incontinence. [START_REF] O'brien | Cognitive associations of subcortical white matter lesions in older people[END_REF][START_REF] Guttmann | White matter abnormalities in mobility-impaired older persons[END_REF][START_REF] Baezner | Association of gait and balance disorders with age-related white matter changes: the LADIS study[END_REF][START_REF] Poggesi | Urinary Complaints in Nondisabled Elderly People with Age-Related White Matter Changes: The Leukoaraiosis And DISability (LADIS) Study[END_REF] SVD is even more common in subjects with AD, and it might interact with the neurodegenerative changes in AD and with their effect on cognitive decline. 12;13 Thus, SVD probably contributes significantly to clinical disability in the elderly. As it can potentially be treated or prevented, increased insight in underlying pathological mechanisms of SVD is of paramount importance.

The value of postmortem MRI studies

The association between SVD features on MRI and clinical symptoms is modest. An explanation for this may be heterogeneity of the neuropathological substrates underlying SVD. T2-weighted MRI dichotomizes the white matter as 'hyperintense' (WMH) or 'normal', whereas the hyperintense areas may reflect pathological tissue changes that vary in type and severity. It further reveals the presence of lacunes and microbleeds, but it has been suggested that expressions of SVD that are not readily detectable on conventional MRI, i.e., cortical microinfarcts and tissue changes in the normal appearing white matter (NAWM), may play an even more important clinical role in terms of clinical symptomatology (Figure 2). These abnormalities can now only be revealed post mortem. [START_REF] Kovari | Cortical microinfarcts and demyelination significantly affect cognition in brain aging[END_REF] To better understand the pathological changes involved in SVD, postmortem MRI scanning and direct correlation with pathology is a valuable tool, as it bridges the gap between MRI findings and clinical studies. 15;16 As it has been shown for other neurological diseases, such as multiple sclerosis, 15;17 postmortem MRI-histopathology correlation studies may help to solve the weak clinicalradiological associations in SVD.

Aim

This paper aimed to investigate the published pathological substrates of WMH and other features of SVD, by comprehensively reviewing studies that have directly compared postmortem MRI and histopathology. Another aim was to pinpoint the gaps in our knowledge and provide the readership with suggestions for further studies, which will hopefully contribute to the development of future treatment options for demented and non-demented elderly suffering from SVD.

A small proportion of patients with SVD features on their MRI suffer from genetic disorders such as cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) or hereditary cerebral amyloid angiopathy (CAA). These diseases have a distinct etiology, and we will only focus on SVD that is observed in 'normal' aging and AD here.

Method (search strategy)

We have systematically searched PubMed for scientific reports correlating postmortem MRI and histopathological assessment of WMH, lacunes and microbleeds until December 2009. The following search terms were used: postmortem, MRI, magnetic resonance, white matter (hyperintensities/lesion[s]), lacune(s), lacunar infarct(ion), microbleed(s).

White matter hyperintensities (WMH)

Studies having correlated postmortem MRI to histopathology of WMH are summarized in Table 1.

These studies confirmed in-vivo studies, stating that WMH are highly prevalent (94%) in elderly populations [START_REF] Fernando | Vascular pathologies and cognition in a population-based cohort of elderly people[END_REF] . The first studies are small and descriptive. However, subsequent studies have specified WMH by distinguishing PVL versus DWMH and the extent of DWMH.

Box: Sensitivity and specificity of postmortem MRI

All studies identified by the abovementioned search criteria have used formalin-fixed brain specimens.

Fixation duration and time to autopsy influence the reliability of postmortem MRI measurements. It has been shown that tissue fixation decreases both T1-and T2-relaxation times in both gray and white matter. 19;20 Early descriptive studies have claimed that postmortem MRI of 0.25T to 1.5T can already visualize WMH with sufficient image quality. 16;21;22 However, the sensitivity of postmortem MRI does appear to be dependent on the size of WMH. Smaller punctate WMH, thought to have little clinical impact, can be less clearly visible on postmortem MRI. [START_REF] Fazekas | The morphologic correlate of incidental punctate white matter hyperintensities on MR images[END_REF][START_REF] Fernando | Comparison of the pathology of cerebral white matter with postmortem magnetic resonance imaging (MRI) in the elderly brain[END_REF][START_REF] Inzitari | Risk of rapid global functional decline in elderly patients with severe cerebral age-related white matter changes: the LADIS study[END_REF] A sensitivity of 95% (range: 87-99%) and specificity of 71% (range: 44-90%) was found for PVL on postmortem T2-weighted post mortem MRI, which could be directly compared to myelin loss in Luxol Fast-Blue stainings. For DWMH, the sensitivity was 86% (range: 79-93%) and specificity was 80% (range: 72-88%). 24;26 Overall, postmortem MRI was considered a valuable technique for translating pathological findings to the clinical setting.

Descriptive MRI and histopathology studies

Using direct post mortem MRI and histopathology correlations, a plethora of histopathological alterations in WMH was described in several studies, including studies with AD patients, patients with cortical infarctions and with Binswanger's disease. WMH was shown to reflect partial loss of myelin, axons and oligodendroglial cells, astrogliosis, dilatation of perivascular spaces, activated macrophages and fibrohyalinotic vessel changes. 16;21;22;27 This range of tissue changes was suggested to be collectively suggestive of incomplete infarcts. Also, complete deep white matter infarcts were found, mostly in WMH with arteriolosclerotic vessel changes. 16;27;28 Distinct types of WMH In clinical studies using in vivo MRI, an attempt to improve specificity for WMH was made by distinguishing between periventricular WMH (thin hyperintense line, smooth halo or irregular bands / caps) and WMH in the deep WM (punctate, early confluent and confluent WMH). [START_REF] Fazekas | MR signal abnormalities at 1.5 T in Alzheimer's dementia and normal aging[END_REF] Postmortem MRI and histopathology correlation studies have described that each type of WMH reflects distinct pathological changes. 23;30-33 Mild periventricular WMH presents with discontinuity of the ependyma, mild-moderate gliosis in the subependymal layer, loosening of the fibre network and myelin loss around so-called 'tortuous venules' and dilated perivascular spaces. No arteriolosclerotic vessel changes were found in these regions. 23;34 Irregular PVL was shown to correspond to more severe, partly confluent, areas with varying fibre and myelin loss and reactive gliosis. Some complete infarcts were seen in irregular PVL regions, in combination with fibrohyalinotic and arteriosclerotic vessels. Punctate, early confluent and confluent WMH in the deep WM were found to be associated with increasing severity of tissue changes. In punctate DWMH, tissue changes were generally mild and confined to the area around dilated perivascular spaces with myelin loss and atrophic neuropil around fibrohyalinotic arterioles. In early confluent DWMH, perivascular rarefaction of myelin was accompanied by varying degrees of axonal loss and astrogliosis. In confluent DWMH, diffuse areas of incomplete parenchymal destruction were observed, together with loss of myelin, axons and oligodendrocytes, astrogliosis, spongiosis and focal transitions to complete infarcts. 23;26;30-32;35 Examples of pathological samples with periventricular and deep WMH, defined on postmortem MRI, are shown in Figure 3.

The above described studies imply that smooth periventricular WMH and punctate WMH are mild forms of WMH and may therefore not be clinically relevant or even detectable. [START_REF] Scarpelli | MRI and pathological examination of post-mortem brains: the problem of white matter high signal areas[END_REF] Irregular periventricular WMH and confluent DWMH, however, correspond to more severe tissue changes, probably of ischemic origin, and are more likely to produce clinical symptoms. 28;30;31;35 Of note is the dependence on subject selection: in the relatively healthy NUN-study cohort, evidence of ischemia was not found in extensive DWMH. [START_REF] Smith | Periventricular white matter hyperintensities on MRI: correlation with neuropathologic findings[END_REF] Pathogenetic mechanisms underlying WMH Recently, several studies further assessed possible pathogenetic mechanisms of WMH by quantitative assessment of immunohistochemical stainings. These studies include important work on the Medical Research Council Cognitive Function and Ageing Study (MRC CFAS) cohort that prospectively collects unselected brain specimens from a large community-based cohort. [START_REF]Pathological correlates of late-onset dementia in a multicentre, community-based population in England and Wales. Neuropathology Group of the Medical Research Council Cognitive Function and Ageing Study (MRC CFAS)[END_REF] First, the role of hypoxia in the pathogenesis of WMH was investigated using specific markers for vascular morphology and tissue hypoxia. [START_REF] Fernando | White matter lesions in an unselected cohort of the elderly: molecular pathology suggests origin from chronic hypoperfusion injury[END_REF] Thicker vessel walls and larger perivascular spaces were found in WMH. In DWMH specifically, capillary endothelial cells were found to be activated and an increased expression of several hypoxia markers was observed. Other studies on characterization of afferent vessels showed arteriolar tortuosity and decreased vessel densities in WMH. 38;39 These findings support an ischemic pathogenesis of WMH, especially in DWMH.

Second, blood-brain barrier dysfunction was demonstrated in a proportion of WMH. 28;40 This was shown by the presence of swollen, eosinophilic, GFAP-positive astrocytes in both DWMH and PVL. [START_REF] Sahlas | Clasmatodendrosis correlating with periventricular hyperintensity in mixed dementia[END_REF] These clasmatodendritic astrocytes stained positively for serum fibrinogen implying leakage of the blood-brain barrier. [START_REF] Tomimoto | Alterations of the blood-brain barrier and glial cells in white-matter lesions in cerebrovascular and Alzheimer's disease patients[END_REF] Furthermore, vascular integrity (as determined by CD31 staining) and P-glycoprotein, an important constituent of the blood-brain barrier, were decreased in WMH. [START_REF] Young | Neuropathologic correlates of white matter hyperintensities[END_REF] Concentric collagen deposition in venular walls may cause intramural thickening, stenosis and eventually venous insufficiency. Venous collagenosis then induces ischemic stress and may cause dysfunction of the blood-brain barrier. 39;41 Third, the role of microglial cells in the pathogenesis of WMH was investigated. [START_REF] Simpson | Microglial activation in white matter lesions and nonlesional white matter of ageing brains[END_REF] Microglial cells in PVL showed a greater tendency to be immunologically activated than DWMH, as shown by the expression of MHC class II. DWMH contained microglial cells with an amoeboid morphology, which were less immune-activated but were likely involved in the phagocytosis of myelin breakdown products. Alternative pathogenetic mechanisms of WMH included altered cerebral blood flow autoregulation, axonal depletion from Wallerian degeneration or toxic effects of amyloid on vascular permeability in AD patients. [START_REF] O'sullivan | Normal-appearing white matter in ischemic leukoaraiosis: a diffusion tensor MRI study[END_REF] These findings have illustrated that MRI-visible WMH are associated with various underlying pathological features and (patho)biological responses in the MRC CFAS cohort. [START_REF] Pantoni | Pathophysiology of age-related cerebral white matter changes[END_REF] This cohort, however, consists of a heterogeneous community-based group of subjects, including healthy elderly, AD patients and subjects with other neurological disorders. The large heterogeneity encountered in this group may therefore be partly artificial and WMH may be pathologically distinct for patient (and control) groups. [START_REF] Simpson | White matter lesions in an unselected cohort of the elderly: astrocytic, microglial and oligodendrocyte precursor cell responses[END_REF] WMH in AD patients MRI expressions of SVD are more prevalent in patients with Alzheimer's disease than in nondemented elderly. [START_REF] Scheltens | White matter lesions on magnetic resonance imaging in clinically diagnosed Alzheimer's disease. Evidence for heterogeneity[END_REF] In addition to the prototypical neuropathological characteristics of AD, i.e. amyloid plaques and neurofibrillary tangles, cerebrovascular pathology is also more frequently observed in AD compared to the general elderly population. 36;47 The impact of cerebrovascular pathology on cognitive decline in AD patients remains to be established. For WMH, some studies have suggested a synergistic effect with common AD pathology on cognitive decline, 13;48 whereas others have not found a distinct role for WMH in AD. [START_REF] Hirono | Impact of white matter changes on clinical manifestation of Alzheimer's disease: A quantitative study[END_REF] Although WMH was found to be more extensive in AD patients than in controls, the nature of pathological correlates, including vascular morphological changes and specific markers for hypoxia were comparable between these groups. 37;50;51 An exception is microglial activation, which was specific for WMH in AD patients. [START_REF] Gouw | Heterogeneity of white matter hyperintensities in Alzheimer's disease: post-mortem quantitative MRI and neuropathology[END_REF] The severity of tissue changes, however, differed with more severe loss of myelinated axons, ependyma denudation, gliosis and thicker adventitia of the deep white matter arteries in AD patients. 50;51 In vascular dementia (VaD) patients, the histopathological profile of WMH was comparable to that of AD patients. [START_REF] Englund | Neuropathology of white matter changes in Alzheimer's disease and vascular dementia[END_REF] This generally comparable pathology suggests that WMH associated with aging, AD and VaD does not have a distinct pathogenesis, but instead may be part of a pathological continuum. [START_REF] Fernando | White matter lesions in an unselected cohort of the elderly: molecular pathology suggests origin from chronic hypoperfusion injury[END_REF] A specific pathology possibly linking SVD and AD is CAA. 54;55 CAA is characterized by amyloid deposition in the smooth muscle cells of cortical, subcortical, and leptomeningeal small arteries and arterioles. 32;56 Patients with CAA can present with intracerebral hemorrhage, transient neurologic events and cognitive decline. [START_REF] Chao | Cerebral amyloid angiopathy: CT and MR imaging findings[END_REF] In CAA patients, the severity of CAA was found to be associated with WMH severity, [START_REF] Greenberg | Amyloid angiopathy-related vascular cognitive impairment[END_REF] possibly due to global vascular dysfunction, which includes the vasculature in the white matter. [START_REF] Greenberg | Amyloid angiopathy-related vascular cognitive impairment[END_REF] In AD, some studies have found weak correlations between WMH and CAA in AD, 37;57;58 whereas other studies failed to find any correlations. 27;28;47;50

Other expressions of SVD

Lacunes

In pathological terms a 'lacune' corresponds to small (lacunar) infarcts, dilated perivascular spaces or old, small hemorrhages. 59;60 However, the term 'lacune' in MRI studies is generally used for a lacunar infarct. These are focal cerebrospinal fluid-filled cavities, often surrounded by a hyperintense rim on FLAIR images. Lacunes are typically located in the areas supplied by the deep thalamoperforant, lenticulostriate, or pontine paramedian arterioles, i.e. basal ganglia, thalamus, internal capsule, pons and centrum semiovale. 61;62 The few postmortem MRI studies that have focused on lacunes are summarized in Table 2. On histological examination, MRI-defined lacunes were found to correspond to irregular cavitations with scattered fat-laden macrophages, which can be accompanied by surrounding reactive gliosis and myelin and axonal loss. 22;60;63;64 With increasing age of the lacune, the density of macrophages diminishes and gliosis becomes more fibrillary. [START_REF] Matsusue | White matter changes in elderly people: MR-pathologic correlations[END_REF] A subtype of lacunes may be seen that is not yet cavitated, but shows selective neuronal loss with relative preservation of glial elements. [START_REF] Lammie | Incomplete lacunar infarction (Type Ib lacunes)[END_REF] Several postmortem MRI studies have compared lacunes to dilated perivascular (Virchow-Robin) spaces, as these structures appear similar on MRI, which generally hinders a clear distinction. 21;30;32 The clinical relevance of enlarged perivascular spaces, if any, is not yet fully elucidated. In general, enlarged perivascular spaces are considered to be asymptomatic, but a relation with SVD may exist. A discriminating feature between lacunes and enlarged perivascular spaces may be that lacunes are commonly larger (>3 mm), and can be accompanied by perifocal signal changes. 66;67 Focal cavities in the anterior perforated substance and the lower part of the basal ganglia / putamen have been reported to generally refer to perivascular spaces rather than to lacunes. [START_REF] Adachi | Dilated Virchow-Robin spaces: MRI pathological study[END_REF][START_REF] Song | MR imaging and histologic features of subinsular bright spots on T2-weighted MR images: Virchow-Robin spaces of the extreme capsule and insular cortex[END_REF][START_REF] Pullicino | Infraputaminal 'lacunes'. Clinical and pathological correlations[END_REF] The most frequently reported cause of lacunes is acute arteriolar occlusion by arteriosclerosis/thrombosis, but the existence of non-cavitated lacunes and the relationship with WMH suggest that there may be other pathogenetic mechanisms with a more gradual development. 59;65;71-75 Possible alternatives include thrombo-embolism, general, ongoing hypoxia or tissue damage by extravasated toxic serum proteins due to blood-brain barrier leakage. 71;76 Future postmortem MRI studies with histopathological confirmation is warranted to further investigate these mechanisms.

Microbleeds

Clinical MRI studies have generally regarded small foci of signal loss on gradient-echo T2*-MRI sequences as microbleeds. 1;77 They are not only a predictor of future lobar intracerebral hemorrhage, but are also independently associated with cognitive decline. 6;7;78 Only few studies have used direct postmortem MRI-pathological correlations to establish the pathological changes responsible for these MRI-hypointensities (see Table 3). [START_REF] Fazekas | Histopathologic analysis of foci of signal loss on gradient-echo T2*-weighted MR images in patients with spontaneous intracerebral hemorrhage: evidence of microangiopathy-related microbleeds[END_REF][START_REF] Tanaka | Small chronic hemorrhages and ischemic lesions in association with spontaneous intracerebral hematomas[END_REF][START_REF] Tatsumi | Direct comparison of histology of microbleeds with postmortem MR images: a case report[END_REF] A recent study that systematically correlated susceptibility weighted imaging, an advanced T2*-MRI sequence, to tissue pathology of hypointensities in AD patients, [START_REF] Schrag | Correlation of hypointensities in susceptibility-weighted images to tissue histology in dementia patients with cerebral amyloid angiopathy: a postmortem MRI study[END_REF] found that most lesions indeed seem to be microscopic bleedings. A minority of these lesions, however, corresponded to small lacunes, dissections of a vessel wall or to microaneurysms. Microbleeds may also correspond to focal accumulations of hemosiderincontaining macrophages in the perivascular space, and there is evidence of heme degradation activity with a surrounding inflammatory reaction with activated microglial cells, late complement activation and apoptosis. [START_REF] Schrag | Correlation of hypointensities in susceptibility-weighted images to tissue histology in dementia patients with cerebral amyloid angiopathy: a postmortem MRI study[END_REF] Microbleeds were found to be occasionally surrounded by gliosis and incomplete ischemic changes. The walls of ruptured arterioles may show CAA related vascular damage, with thickened, acellular morphology, lack of the muscularis layer, and β amyloid deposition. CAA-related microbleeds tended to be localized at the grey-white matter junction and in superficial cortical layers of the parietal and occipital lobes. Microbleeds in hypertensive subjects, however, were more often seen in the basal ganglia, brain stem and cerebellum. [START_REF] Roob | Frequency and location of microbleeds in patients with primary intracerebral hemorrhage[END_REF] Arteriosclerosis of the vessel walls was often present in these subjects. 79;84

MRI-'invisible' expressions of SVD

As noted above, there is accumulating evidence that there are also pathological changes associated with SVD which are 'invisible' to conventional MRI, such as tissue changes in white matter areas appearing normal on postmortem MRI (NAWM) and cortical microinfarcts. Pathologically, NAWM may correspond to mild tissue changes with a slightly lower myelin density, activated endothelium, a looser but still largely intact axonal network and a normal glial density 18;26 . Furthermore, it has been shown that the density of small afferent vessels is not only decreased in WMH, but extends into NAWM and cortex. [START_REF] Moody | Quantification of afferent vessels shows reduced brain vascular density in subjects with leukoaraiosis[END_REF] Cortical microinfarcts are microscopically small lesions. They are attributed to ischemia, consisting of complete or incomplete cavitation with myelin pallor and neuronal loss, surrounded by glial cells and/or macrophages. [START_REF] Haglund | Cerebral amyloid angiopathy and cortical microinfarcts as putative substrates of vascular dementia[END_REF] Cystic microinfarcts tend to be larger (up to 5 mm) than non-cystic microinfarcts (0.05-0.4 mm). 85;86 Several population-based prospective autopsy studies suggested that cortical microinfarcts are major determinants of dementia. [START_REF] Haglund | Cerebral amyloid angiopathy and cortical microinfarcts as putative substrates of vascular dementia[END_REF][START_REF] White | Cerebrovascular pathology and dementia in autopsied Honolulu-Asia Aging Study participants[END_REF][START_REF] Sonnen | Pathological correlates of dementia in a longitudinal, population-based sample of aging[END_REF] Microinfarcts also have an independent influence on cognitive decline in the non-demented elderly, with only little or moderate AD changes on histology. 14;88 Moreover, microinfarcts were associated with CAA in patients with VaD. [START_REF] Haglund | Cerebral amyloid angiopathy and cortical microinfarcts as putative substrates of vascular dementia[END_REF] All these findings suggest that SVD is a widespread disease and has various expressions throughout the brain of which only some aspects can be visualized with conventional MRI. As illustrated by Figure 2, MRI-'invisible' pathologies, including cortical microinfacts and tissue changes in the NAWM, hence contribute to the clinical-radiological association that is found in SVD.

Postmortem quantitative MRI: more specific for SVD-related pathology than T2-weighted MRI?

To be able to draw conclusions on the clinical relevance of SVD, additional pathology-specific tools are needed in vivo. Recently, quantitative MRI techniques (QMRI) have been suggested to be more specific for underlying pathology. 24;26;53 It has been shown that Magnetization Transfer Imaging (MTI) and Diffusion Tensor Imaging (DTI) distinghuish between WMH and NAWM in elderly subjects. 45;89 When correlating postmortem QMRI and pathology, it needs to be taken into account that both the time to autopsy and fixation duration have an effect on T1-and T2-relaxation times and diffusivity measures. 90;91 Although several postmortem studies using QMRI have been performed in patients with multiple sclerosis and other neurological diseases, [START_REF] Schmierer | Quantitative magnetization transfer imaging in postmortem multiple sclerosis brain[END_REF][START_REF] Vrenken | Whole-brain T1 mapping in multiple sclerosis: global changes of normal-appearing gray and white matter[END_REF][START_REF] Simonyan | Focal white matter changes in spasmodic dysphonia: a combined diffusion tensor imaging and neuropathological study[END_REF] postmortem QMRI studies in elderly with SVD are still scarce. Two small studies showed that DTI measures in WMH seem to correspond to the degree of myelin loss. Also, the area of diffusivity and pathological changes was found to be more spatially extensive than indicated by the hyperintense areas on conventional T2-weighted MRI. 95;96 Pathological correlates of fractional anisotropy (FA) in DTI and T1-relaxation time were established in a recent study on WMH in AD patients and controls. FA reflected axonal loss, whereas T1relaxation time corresponded with axonal loss, myelin loss and microglial activation. [START_REF] Gouw | Heterogeneity of white matter hyperintensities in Alzheimer's disease: post-mortem quantitative MRI and neuropathology[END_REF] These few studies reveal that QMRI techniques may be promising in assessing tissue damage in vivo, because they sensitively and specifically reflect the severity of pathological substrates and reveal tissue changes in areas that appear normal on conventional MRI (NAWM).

Conclusions and considerations for future research

This review has considered the pathological correlates of SVD, as reflected on MRI. The available literature suggests that several explanations may exist for the weak clinical-pathological associations.

First, pathological substrates of SVD expressions on MRI, such as WMH, lacunes and microbleeds, are heterogeneous in nature and differ in severity. Relative to WMH, postmortem MRIpathology correlation studies of lacunes and microbleeds are still scarce. For lacunes, pathological correlation studies are certainly warranted to be able to further investigate their hypothesized multiple etiologies including acute thromboembolism, continuing moderate ischemia with eventual focal tissue loss and inflammation. For microbleeds, the pathogenetic mechanisms and their relationship with WMH and lacunes also needs to be further unraveled. It should be noted that in this review, we have only focused on normal aging and dementia. In specific brain diseases, such as CADASIL and hereditary CAA, SVD features are also present, but may differ with regard to their MRI and histopathology profiles.

Second, until recently, clinical MRI studies have often focused on separate aspects of SVD, such as WMH, and found only weak associations with clinical symptoms. However, not only WMH, but also lacunes and microbleeds are bound to contribute to clinical symptoms such as cognitive decline. 3;6 Furthermore, the previously discussed MRI-'invisible' lesions, i.e. microinfarcts and tissue changes in the NAWM, may be clinically relevant, independently of MRI-'visible' characteristics of SVD. [START_REF] Kovari | Cortical microinfarcts and demyelination affect cognition in cases at high risk for dementia[END_REF] The combination of SVD features is therefore a better predictor of cognitive decline than separate SVD expressions. [START_REF] Gold | Sorting out the clinical consequences of ischemic lesions in brain aging: a clinicopathological approach[END_REF] Moreover, SVD should be assessed together with frequently co-existing large vessel infarcts, which may improve insight in the mechanisms of vascular cognitive impairment.

In addition to vascular disease, other (degenerative) brain changes, e.g. AD pathology, CAA or cortical Lewy bodies, may interact and modulate their specific contributions to cognitive decline. 87;98- 101 Future studies should therefore consider the full spectrum of SVD expressions, together with vascular and degenerative pathologies co-existing in the aging brain.

Unfortunately, MRI sequences that are commonly used in radiological practice, are insufficiently sensitive and specific to detect all the tissue changes related to SVD. Novel QMRI techniques, such as DTI, MTI, T1-and T2 relaxation time measurements have two advantages over conventional MRI. First, QMRI better reflects the severity of underlying pathological substrates.

Second, it may better reveal clinically relevant tissue changes in the WM that appears normal on conventional MRI. 95;96 QMRI techniques are therefore more adequate at reflecting the full range of SVD expressions. Several QMRI abnormalities were confirmed histopathologically, and these techniques are promising, pathology-specific tools for future in vivo studies. It should be noted that, even though study logistics will become more complex, the use of fresh brain tissue in exploratory post mortem MRI studies is preferred above the use of fixed specimens, as formalin fixation has been shown to influence tissue proton relaxation characteristics. Unlike tissue changes in the NAWM, QMRI techniques have not yet been investigated to visualize cortical microinfarcts. The future use of high resolution MRI, using 7 Tesla MRI scanners, may achieve in vivo assessment of these lesions, which are probably beyond the resolution of 1.5 or 3 Tesla MRI. Vascular wall changes = arteriosclerotic changes with thickening, fibrosis and splitting of the internal elastic lamina, dilated perivascular spaces.

Van Swieten 1991 [START_REF] Van Swieten | Periventricular lesions in the white matter on magnetic resonance imaging in the elderly. A morphometric correlation with arteriolosclerosis and dilated perivascular spaces[END_REF]

White matter hyperintensities Lacunes Microbleeds

A B C

  AD = Alzheimer's disease, neur (-) controls = non-demented subjects without neurological disease, neur (+) controls = non demented patients with neurological disease. FTD = frontotemporal dementia, VaD = vascular dementia. HE = Haematoxylin-Eosin, LFB = Luxol Fast Blue, LFB-PAS = Luxol Fast Blue/ periodic acid-Schiff, KB = Klüver-Barrera, EvG = Elastic van Gieson, MBP = myelin basic protein, GFAP = glial fibrilary acidic protein, HIF = hypoxia inducible factor, IgG = immunoglobulin, PTAH = Phosphotungstic Acid Hematoxylin, MMP7 = matrix metalloproteinase 7, ICAM1 = intercellular adhesion molecule, Ngb = neuroglobin, NMBR = neuromedin B receptor, VEGFR2 = vascular endothelial growth factor receptor 2, Mcm2, PCNA and Ki67 = cell proliferation-related molecules, B7-2, CD40 and CD40-Ligand = immune costimulatory molecules, PDGFaR = platelet-derived growth factor a receptor, OPC = oligodendrocyte precursor cells, MAP-2 +13 = microtubule-associated protein-2 expressing exon 13, CD31 = vascular integrity, hGLUT-5 = human glucose transporter-5, APP = amyloid precursor protein, HLA-DR = human leukocyte antigen-DR, P-gp = P-glycoprotein, (D)WMH = (deep) white matter hyperintensities, PVL = periventricular white matter hyperintensities, DTI = diffusion tensor imaging, (Q)MRI = (quantitative) magnetic resonance imaging, CAA = cerebral amyloid angiopathy, PA = pathology.

  neur (-) controls = non-demented subjects without neurological disease, neur (+) controls = non demented patients with neurological disease, VaD = vascular dementia. HE = Haematoxylin-Eosin, LFB = Luxol Fast Blue, LFB-PAS = Luxol Fast Blue/ periodic acid-Schiff, KB = Klüver-Barrera, EvG = Elastic van Gieson, GFAP = glial fibrilary acidic protein, PTAH = Phosphotungstic Acid Hematoxylin, VRS = Virchow Robin spaces, WM = white matter.

  CAA related vascular damage: thickened, acellular, arteriolar walls with βamyloid deposition and lacking muscularis layer. Activated microglia (CD68), evidence of heme degradation (HO-1), late complement activation, apoptosis. Inflammatory reaction along local microvasculature. neur (+) controls = non demented patients with neurological disease, CADASIL = cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy. MB = microbleed HE = Haematoxylin-Eosin, KB = Klüver-Barrera, EvG = Elastic van Gieson, HO-1 = heme oxygenase 1.

Figure 1 .

 1 Figure 1. MRI expressions of small vessel disease. Axial Fluid Attenuated Inversion Recovery (FLAIR)-images with periventricular and deep white matter hyperintensities (A); and an illustration of two lacunes in the right hemisphere (arrows) and deep white matter hyperintensities (B). T2*-gradient echo image with multiple cortical-subcortical microbleeds (C).

Figure 2 .

 2 Figure 2.A schematic representation of small vessel disease (SVD) expressions is shown including illustrative postmortem MRI and histological sections. SVD expressions that are visible on MRI are illustrated as: a pre-frontal coronal FLAIR image and a matching Bodian Silver stained section of white matter hyperintensities (WMH); a parietal coronal FLAIR image and a Klüver-Barrera stained section of two lacunes (two arrows in the magnification); and a cerebellar axial T2*-image and Haematoxylin Eosinstained section of a microbleed (reproduced with permission from Fazekas, AJNR 1999[START_REF] Fazekas | Histopathologic analysis of foci of signal loss on gradient-echo T2*-weighted MR images in patients with spontaneous intracerebral hemorrhage: evidence of microangiopathy-related microbleeds[END_REF] ). SVD expressions that are not readily detected by conventional MRI include: cortical microinfarcts, illustrated by microglial/macrophage activation on a HLA-DR stained section; and changes in the normal appearing white matter, e.g. astrogliosis (GFAP stained section). Future studies should be directed to assess the whole spectrum of SVD, because all expressions may contribute to clinical symptoms in the elderly subject.

Figure 3 .

 3 Figure 3. Prefrontal coronal FLAIR image (A) of an 88-year-old female Alzheimer's disease patient. Regions of interest represent white matter hyperintensities (WMH) in the periventricular area (green; B1 to E1); WMH in the deep white matter (yellow; B2 to E2); and an area of normal appearing white matter (white, B3 to E3). Bodian Silver stained sections (B, original magnification 200x) showed lower axonal density in WMH (B1 and B2) than in NAWM (B3); more microglial activation (C) was observed in WMH (C1 and C2) than in NAWM (C3) on HLA-DR immunohistochemical sections (original magnification 200x); WMH also showed more myelin loss (D1 and D2) compared to NAWM (D3) in Luxol-Fast-Blue/Cresyl-Violet stained sections (original magnification 100x); the severity of astrogliosis (E) (GFAPimmunostained sections, original magnification 400x) was not clearly different between WMH and NAWM in this patient. Adapted with permission from Gouw, Brain 2008 52 .

Figure 1 .

 1 Figure 1.

Table 1 .

 1 Postmortem studies on characterization of WMH using postmortem MRI -pathology correlation

	Reference	Subjects	Histochemistry	Immunohisto-	MRI	Main findings
				chemistry
	Awad 1986 21	7 neur (-) controls	HE, LFB	GFAP	1.5T	WMH correspond to a spectrum of histological alterations.
						Mild tissue changes = enlarged perivascular spaces, vascular ectasia.
						Severe tissue changes =degeneration axons, gliosis.
	Englund 1987 16	21 AD	HE, LFB		0.25T	T1 and T2 relaxation times increased with severity of tissue changes in WMH.
		19 neur (+) controls
	Marshall 1988 28	16 neur (-) controls	HE, Congo Red, Alcian	GFAP, IgG,	0.35T	WMH correspond to intermediate and old infarctions:
			Blue, LFB	Albumin		-intermediate infarctions = necrosis, minimal cavitation, gliosis.
						-old infarction = cavitation, surrounded by demyelination, fibrohyalinosis and
						isomorphic gliosis (swollen reactive astrocytes, positive for IgG and albumin). Findings
						imply blood-brain barrier leakage.
						No correlation with CAA.
	Braffmann 1988 66	23 neur (+/-) controls HE, Weil, LFB, Bodian		1.5T	WMH vary from subtle gliosis and demyelination to frank infarction.
	Mascalchi 1989 102	1 VaD	HE, Woelke, Congo Red		0.5T	Case report: WMH reflect demyelination, axonal loss, moderate astrogliosis,
						oligodendroglial swelling, a few macrophages and slight edema. Arterial/ arteriolar wall
						changes: severe thickening with stenosis, hyperplasia and hyalinosis. Scarce vascular
						amyloid deposition.
	Revesz 1989 22	4 VaD	HE, KB, Nissl, Holzer		0.5T	Good correlation of extent of WMH on MRI and pathological changes.
			axons, cresyl violet, EvG,
			Heidenhain's myelin,
			Bielschowsky, PTAH

Table 2 .

 2 Postmortem studies on characterization of lacunes using postmortem MRI -pathology correlation

	Reference	Subjects	Histochemistry	Immuno-	MRI	Main findings
				histochemistry
	Braffman 1988 66 36 neur (+/-) controls	HE, Weil, LFB, Bodian		1.5T	-lacunes in 6 subjects: 4 subjects with 6 lacunes in basal ganglia, 2 subjects
						with 3 lacunes in posterior fossa and 2 subjects with 5 supratentorial white
						matter lacunes.
						Usual morphology: slitlike or ovoid, size 3 -14mm, varying degrees of
						cavitation.
						-état criblé in 4 subjects. Usual morphology was round or linear, 1mm -
						5mm.
	Revesz 1989 22	4 VaD	HE, KB, Nissl, Holzer axons,		0.5T	All subjects: lacunes in the white matter, basal ganglia and pons. 1 subject: a
			cresyl violet, EvG,			lacune in internal capsule. In 2 cases old cystic cavities were densely lined by
			Heidenhain's myelin,			haemosiderin-containing macrophages (possibly reflect microbleeds)
			Bielschowsky, PTAH
	Pullicino 1995 70	2 subjects postmortem	HE, LFB-PAS, Gomori's	actin	-	Infraputaminal cavities or 'lacunes' are single or grouped enlarged
		MRI, 1 in vivo MRI.	trichrome, Verhoeff's elastic			perivascular spaces.
		not specified				Histology: regular and clearly demarcated walls, some arteries within cavity
						were tortuous (not occluded), thickened adventitia. Surrounding white matter
						showed corpora amylacea, vacuolation, myelin loss and minimal gliosis, but
						no infarction.
	Bokura 1998 67	9 neur (+) controls	HE, KB		1.5T	114 asymptomatic lesions detected on PA. Basal ganglia n= 87 (76%): 29
		(cerebrovascular				lacunes, 58 VRS. WM: 11 lacunes, 8 VRS. Thalamus: 2 lacunes, 3 VRS.
		disease)				Brainstem: 3 lacunes, no VRS. In basal ganglia more VRS, in WM more
		3 neur (-) controls				lacunes.
						Morphology: VRS more round/ linear, lacunes more wedge shaped, but both
						could be ovoid. VRS are smaller than lacunes (72% of VRS < 2x1mm, 60%
						of lacunes > 2x2mm).
	Matsusue 2006 64 Not specified.	HE, LFB, axon staining	GFAP	1.5T	Description of old lacune: cystic cavity with ill-defined and irregular margin,
						with surrounding mild loss of myelin and axons with mild gliosis.

Table 3 .

 3 Postmortem studies on characterization of microbleeds using postmortem MRI -pathology correlation. MRI hypointensities: all could be identified as brown spots on the cut surface. 8 hemosiderin-laden macrophages, 1 vascular pseudocalcification (left pallidum). 5/8 had vascular abnormalities: degenerated endothelial lining and hyalinosis. Size of hemosiderin deposit similar with MRI hypointensity. Associated with tissue rarefaction, gliosis or arteriolar changes. Some hemosiderin deposits not observed on MRI. Ante-and postmortem MRI comparable.

	Reference	Subjects	Histochemistry	MRI	Main findings
	Fazekas 1999 79	11 subjects with	HE, Masson trichrome,	1.5T	MRI signal loss in 7/11 subjects. 2 pts: only cortical-subcortical, 1 pt only basal
		intracerebral	KB, congo red, iron		ganglia/infratentorial, 4 pts both locations. WMH in all patients, lacunes in 5/7 pts.
		hemorrhage			In 62% of MRI signal loss: focal accumulation of hemosiderin-containing macrophages adjacent to
					small blood vessels, sometimes minute areas of tissue necrosis. The remainder of MRI signal loss:
					no pathological substrate.
					Also MR-negative hemosiderin deposits: smaller, only a few perivascular, hemosiderin-laden
					macrophages. No calcification or vascular malformations.
					2 subjects had cerebral amyloid angiopathy of variable extent in multiple vessels, associated with
					foci of remote blood leakage. Brains with fibrohyalinosis showed microbleeds preferentially in the
					basal ganglia/thalami, but also cortical-subcortical.
	Tanaka 1999 80	3 neur (+) controls,	HE, Masson trichrome,	1.0T	Foci of old hemorrhages caused by rupture of arteriosclerotic microvessels <200 μm, surrounded
		with MRI signal loss	KB, Berlin Blue	In vivo MRI	by gliosis and incomplete ischemic necrosis. Identified as hemosiderin pigments within the
					perivascular space and as an organised pseudoaneurysm (1 case).
	Tatsumi 2008 81 9 Schrag 2009 82 1 neur (+) control HE, Berlin Blue 1.5T, both postmortem and in vivo MRI 8 AD (of which 6 Aβ1-42, CD68, HO-1, 3.0T 38 MBs: correspond to variety of pathological changes = 16 old hematomas, 7 small cavities, 3
		advanced CAA), 2	complement C6, CD3,		microscopic hemosiderin granules + hematoidin deposition, 1 dissection vessel wall, 1
		neur (-) controls	CD20, Prussian Blue,		microaneurysm.
			fluorescent study (HO-1 +		Location: 79% grey-white junction, 21% superficial cortex.
			MAP-2), TUNEL

Acknowledgements

We would like to acknowledge Miriam Cornella, MSc, for her help with the literature search.