Cerebral Microbleeds in the Elderly: A Pathological Analysis

Abstract

Background and Purpose

Cerebral microbleeds in the elderly are routinely identified by brain MRI. The purpose of this study was to better characterize the pathological basis of microbleeds.

Methods

We studied post-mortem brain specimens of 33 individuals with no clinical history of stroke, age range 71–105 years. Cerebral microbleeds were identified by presence of hemosiderin (iron), identified by routine histochemistry and Prussian blue stain. Cellular localization of iron (in macrophages and pericytes) was studied by immunohistochemistry for smooth muscle actin, CD68, and, in selected cases, electron microscopy. Presence of beta-amyloid was analyzed using immunohistochemistry for epitope 6E10.

Results

Cerebral microbleeds were present in 22 cases, and occurred at capillary, small artery, and arteriolar levels. Presence of microbleeds occurred independent of amyloid deposition at site of microbleeds. While most subjects had hypertension, microbleeds were present with and without hypertension. Putamen was site of microbleeds in all but one case; one microbleed was in subcortical white matter of occipital lobe. Most capillary microbleeds involved macrophages, but the two microbleeds studied by electron microscopy demonstrated pericyte involvement.

Conclusions

These findings indicate that cerebral microbleeds are common in elderly brain and can occur at the capillary level.

Introduction

Increasing reliance on MRI of stroke patients has emphasized substantial prevalence and significance of cerebral microbleeds in the aging population. MRI using state-of-the-art gradient echo sequences has demonstrated cerebral microbleeds in 18% of individuals between 60–69 years old and in 38% of individuals over the age of 80 (1). Moreover, presence of cerebral microbleeds appears to increase risk of warfarin-associated intracerebral hemorrhage more than 80-fold (2), and use of platelet aggregation inhibitors is associated with presence of microbleeds (3). These findings underscore the importance of cerebral microbleeds.

Despite their importance, cerebral microbleeds have received only limited attention in pathological analyses. Recent work has examined in detail cerebral microbleeds in cerebral amyloid angiopathy (4), demonstrating strong correlation between microbleeds in post-mortem brain tissue and MRI lesions. The consensus view is that microbleeds of lobar location reflect underlying cerebral amyloid angiopathy, while deep subcortical microbleeds indicate hypertensive vasculopathy (1, 5–8). The purpose of the present study was to expand our knowledge of the pathology of cerebral microbleeds in the aging brain. Our focus was the vascular abnormalities underlying cerebral microbleeds, including vessel type, presence of associated amyloid angiopathy, and range of cellular involvement in the microbleeds themselves.

Materials and Methods

The investigation consisted of two studies of brain tissue from elderly subjects

Initial survey study consisted of twelve subjects, autopsied between January 2006 and December 2008 with no clinical diagnosis of cerebral vascular disease, selected consecutively by the Department of Pathology, Harbor-UCLA Medical Center. Portions of subcortical white matter from frontal, parietal, occipital, and temporal lobes were sampled. Selected portions of brains were first fixed in 10 % buffered formalin solution for two weeks then cut serially in 1.0 cm slices. To detect hemosiderin/iron, slices were immersion-stained using Prussian blue method. Slices (4 μm (microns) thick) were selected for paraffin embedding, sectioning (20–40/brain) and hematoxylin & eosin staining.

Immunohistochemistry

To detect pericytes, macrophages, and amyloid deposition, detection kits based on immunohistochemistry were used according to the manufacturers’ protocols. Specifically, to detect pericytes of cerebral capillaries a mouse monoclonal antibody against human alpha smooth muscle actin (Sigma-Aldrich Corp. St Louis, MO) was used. To detect macrophages, mouse monoclonal antibody against human CD68 (Dako North America, Inc. Carpinteria, CA) was used, and mouse monoclonal antibody against human beta-amyloid epitope 6E10 (Covance Inc.) was used for amyloid detection. Antigen retrieval was done by using 10 mmol/L sodium citrate (pH 6.0) at 95°C for 15 minutes. Slides were first incubated with primary antibodies at 1:2000 dilution for 30 minutes at room temperature. Slides were then incubated with a biotinylated secondary antibody. Staining was carried by using Cell and Tissue Staining kit (R&D System) according to the manufacturer’s instruction. The slides were counter-stained with hematoxylin and photographed by using a light microscope. Negative control samples were exposed to a secondary antibody with a similar IgG isotype (Cell Signaling) to the primary antibody.

Electron microscopy

For electron microscopy, tissues positive for Prussian blue staining of capillaries were selected and first fixed in 2.5 % glutaraldehyde and emersion-fixed in osmium tetroxide and then embedded in plastic. These sections were examined by using an electron microscopy (Hitachi 600) according to the manufacturer’s instruction.

Follow-up study focused on basal ganglia as well as cortical tissue, material obtained from the University of California Irvine Alzheimer Disease Research Center and from The 90+ Study. Blocks of lenticular nucleus from 21 subjects (who had died during eighth, ninth, tenth, and eleventh decades of life), taken at the level of the mammillary bodies from brains that had been fixed for two weeks in 4% paraformaldehyde, were embedded in paraffin and sectioned at 8 μm. In addition to hematoxylin and eosin and Prussian blue stains, immunostains using mouse anti-human beta-amyloid protein diluted 1:10,000 (6E10, Covance, Emeryville, CA), rabbit anti-human alpha-synuclein diluted 1:3000 (Chemicon, Temecula, CA), mouse anti-human CD68 (clone KP1) diluted 1:400 (Dako, Carpinteria, CA), mouse anti-human smooth muscle actin (clone 1A4) diluted 1:100 (Dako), and rabbit anti-human tau diluted 1:3000 (Dako) were performed using AEC substrate chromogen (Dako). Braak staging was performed as previously described (9). Briefly, neurofibrillary tangles were assessed semiquantitatively (0 to +++) within six cerebral neocortical regions along with CA1 of the hippocampus, subiculum, entorhinal-transentorhinal region, and amygdala; tangle severity was scored on a scale of I to VI. Microbleeds were quantified (for follow-up study only) in the following manner: Within the microscope field encompassed by a 2X objective, the mid-putamen at the level of the mammillary bodies was examined, and the number of capillary and non-capillary channels bounded by one or more hemosiderin deposits were counted. From these counts, the number of vessels per square cm (capillary density) was calculated. Unpaired student t-tests and Pearson’s correlation were used to further evaluate microbleed scores; p<.05 was considered significant.

Results

In the initial study, the age of subjects ranged from 71 to 92 years old, mean age 79.3 years; three were male, nine female. Microbleeds were found in two subjects, and were located in subcortical white matter of occipital lobe and in putamen. In these subjects, iron was present in capillary wall at the ablumenal endothelial surface at the site of location of capillary pericytes (Figure 1). Electron microscopy of these microbleeds demonstrated iron in pericytes, immediately adjacent to endothelial tight junction (Figures 1, 2). Vascular beta-amyloid deposition was not encountered at the sites of iron deposition. Nine of the twelve subjects had history of hypertension, including the two with microbleeds (Table).

Figure 1.

Occipital lobe, subcortical white matter.

A: Capillary with hemosiderin; H&E, Prussian blue. (Original magnification X520)

B, C: Capillary with hemosiderin; H&E, Prussian blue. (Original magnification X520)

D: Alpha smooth muscle actin immunostain of capillary. Immunoreactivity is adjacent to ablumenal surface of endothelium, where pericytes are located. (Original magnification X520)

Figure 2.

Electron microscopy of capillary in subcortical white matter, stained for iron (Prussian blue). Pericyte, attached to capillary wall and surrounded by basement membrane, contains dense deposits of iron. Tight junction of endothelial cell (red arrows) is immediately adjacent to pericyte. Also shown are basal lamina (green arrow), endothelium (blue arrow), hemosiderin in pericyte (white asterisk), red blood cell (yellow #), and blood vessel lumen (red arrowhead). (Original magnification X15,000)

Table.

Cerebral Microbleeds: Clinical and Pathological Characteristics

Patient	Age	Gender	HTN	BP	MB	Braak	MB Score
1	86	F	Y	130/70	N	-	-
2	79	F	Y	124/70	N	-	-
3	71	F	Y	153/70	N	-	-
4	73	F	Y	134/82	N	-	-
5	72	M	Y	131/86	N	-	-
6	80	F	Y	134/63	Y	-	-
7	78	M	Y	132/82	N	-	-
8	71	F	Y	127/82	N	-	-
9	77	F	Y	166/87	Y	-	-
10	87	F	N	111/72	N	-	-
11	80	F	N	107/43	N	-	-
12	84	M	N	140/70	N	-	-
13	84	F	N	129/86	Y	V	159
14	77	F	N	UNK	Y	VI	44
15	77	F	UNK	132/80	Y	VI	26
16	100	F	Y	105/60	Y	II	152
17	104	F	N	124/65	Y	V	159
18	83	F	N	120/70	Y	V	111
19	102	F	Y	138/64	Y	III	89
20	94	F	N	130/55	Y	VI	244
21	88	M	Y	118/70	Y	VI	85
22	96	F	Y	135/70	Y	VI	130
23	91	F	Y	160/76	Y	IV	81
24	79	M	UNK	UNK	Y	VI	30
25	91	F	Y	UNK	Y	II	111
26	97	M	Y	145/80	Y	II	448
27	102	F	Y	129/74	Y	II	56
28	94	M	Y	150/90	N	I	0
29	105	F	N	120/60	Y	I	152
30	103	F	Y	80/50	Y	II	67
31	99	F	Y	158/64	Y	II	322
32	96	F	Y	110/70	Y	II	89
33	100	F	Y	198/78	Y	II	193

Initial Study: Patients 1–12; Follow-Up Study: Patients 13–33

HTN: Hypertension

MB: Microbleeds

Braak: Braak Score

MB Score: Microbleeds Score (see Materials and Methods)

UNK: Unknown

In the follow-up study, age of subjects ranged from 77 to 105 years old, mean age 93.4 years; four were male, seventeen female. Post-mortem interval ranged from 3–21 hours, mean 6.3 hours. Thirteen subjects had history of hypertension and six were without hypertension history. In two subjects, hypertension history was uncertain; one of these subjects was not hypertensive at final reading. Microbleeds were seen within putamen in 20 of 21 subjects, including all subjects without history of hypertension. Iron was observed predominantly within macrophages adjacent to small arteries, arterioles, and particularly capillaries (Figure 3); it was also seen to be deposited free in the tissue. In most instances it was distributed widely within the putamen. One subject (#31) had evidence of cerebral amyloid angiopathy involving vessels remote from the microbleeds. For the remainder, beta-amyloid protein deposition was observed within diffuse and neuritic plaques but not within the walls of involved capillaries or arterioles. The vast majority of vessels involved in these microbleeds were capillaries, and microbleed score (capillary density) is listed in Table. There was no significant difference in microbleed score for males vs females and for subjects with and without hypertension, and there was no significant association between age and microbleed score and between microbleed score and post-mortem interval (data not shown).

Figure 3.

Pericapillary deposition of hemosiderin (brown) within macrophage (red) in putamen, using CD68 immunostain. (Original magnification X600)

Discussion

We found frequent evidence of cerebral microbleeds in the aging brain, often with capillary involvement. The vast majority of the microbleeds occurred in putamen. The microbleeds occurred in vessels without amyloid deposition and microbleeds occurred both in presence and in absence of hypertension. Moreover, blood-brain barrier pericytes in addition to brain macrophages appeared to have a role in microbleeds.

Iron uptake into brain is complex, and may occur as a consequence of hemorrhage and by receptor-mediated endocytosis of transferrin-bound iron (10, 11). The latter occurs at endothelial cells of the blood-brain barrier and results in tissue iron distribution principally in oligodendrocytes (10, 11). This is a well-described and age-related process that does not, however, include phagocytosis or inflammation (10–13).

The neurovascular unit of the blood-brain barrier consists of endothelial cells in close approximation to pericytes, separated only by basement membrane (14–16). In addition, macrophages are known to be found adjacent to neurovascular unit, either in residence or as cells migrating to that site (17). It is therefore not surprising that both cell types appear to have a role in microbleeds. Erythrophagocytosis is a well-described feature of macrophages (18), and Prussian blue stains of hemosiderin iron show punctuate staining similar to what is observed in current study (19, Fig 1). Prior work has shown macrophage involvement in cerebral microbleeds (4,6). Pericytes are also known to have phagocytic function (14, 20) and erythrophagocytosis has been observed in systemic pericytes (21, 22).

Red blood cells may pass through endothelial junctions in systemic capillaries, resulting in petechial hemorrhages (23). This is common in thrombocytopenia, along with other hemorrhagic diatheses. It is noteworthy that brain hemorrhage is rare in thrombocytopenia (24), and this is likely due to structural and functional properties of the blood-brain barrier that can prevent local hemorrhage. Presence of tight endothelial junctions in the neurovascular unit is one likely component of the brain’s armamentarium against hemorrhage. Opening of the endothelial junctions to allow passage of red blood cells might represent age-related changes in barrier function; the latter has been described in cerebral white matter disease of aging, with down-regulation of blood-brain barrier efflux transporter p-glycoprotein (25).

Pericytes are known to be preferentially located adjacent to tight junctions of the blood-brain barrier and have been localized adjacent to histamine-induced gaps between endothelial cells (14). This location is ideally suited for a “gatekeeper” function of pericytes, in which these cells scavenge and phagocytose red cells that are able to pass the barrier between adjacent endothelial cells. In this scenario, macrophages adjacent to the neurovascular unit would be able to act as secondary scavengers for those red blood cells that are able to bypass pericytes and enter brain parenchyma (Figure 4).

Figure 4.

Proposed mechanism for extravasation of red blood cells from brain capillaries. Opening of tight junction allows for pericyte erythrophagocytosis, with adjacent macrophage available as alternative or secondary site for phagocytosis.

Our study is not designed to provide pathological correlations to MRI findings of cerebral microbleeds. This represents a limitation of the present study, as our findings indicate microbleeds primarily in basal ganglia location while MRI studies show microbleeds more frequently in lobar site compared to deep brain regions (1). MRI is known to overestimate the size of microbleeds (the “blooming effect”), with MRI diameter on average more than 150% of pathological lesions (4); nevertheless, MRI may not be sensitive to the relatively small microbleed findings of the current study. Other limitations of our study include absence of younger brains, predominance of female subjects, and absence of data of cognitive function to which pathological features may be correlated. These represent potential future directions for research. Moreover, it should be noted that the two studies described herein were not conducted identically; the initial study focused primarily on subcortical white matter, while the second study focused on cortex and basal ganglia.

In conclusion, cerebral microbleeds appear to be common in aging brain, occur at the capillary level, and are particularly prevalent in the putamen. Both pericytes and macrophages at the neurovascular unit seem to play a role modulating microhemorrhage. Microbleeds occurred both in the presence and absence of hypertension, and amyloid deposition was not found at site of microbleeds; this suggests that amyloid and hypertension may not necessarily be required for cerebral microbleeds in the aging brain. Further pathological investigations may determine the relationship between cerebral microbleeds as described herein and vasculopathic amyloid deposition and hypertension.