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(Stroke. 1996;27:2069-2074.)
© 1996 American Heart Association, Inc.

Articles

Alterations of the Blood-Brain Barrier and Glial Cells in White-Matter Lesions in Cerebrovascular and Alzheimer's Disease Patients

Hidekazu Tomimoto, MD; Ichiro Akiguchi, MD; Toshihiko Suenaga, MD; Masaki Nishimura, MD; Hideaki Wakita, MD; Shinichi Nakamura, MD Jun Kimura, MD

the Department of Neurology, Kyoto University, Faculty of Medicine, Japan.

Correspondence to Hidekazu Tomimoto, Department of Neurology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan.

	Abstract

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Background and Purpose The underlying cause of white-matter lesions, which are frequent findings in cerebrovascular disease (CVD) and Alzheimer's disease (AD), remains uncertain. We performed immunohistochemical analysis of serum protein extravasation to investigate the function of the blood-brain barrier in white-matter lesions.

Methods White-matter lesions were estimated by use of Kluver-Barrera staining in patients diagnosed clinicopathologically as having ischemic CVD (n=14) and AD (n=12) and from nonneurological control subjects (n=6). Axonal damages were investigated by use of immunohistochemistry for amyloid protein precursor. Alteration of the blood-brain barrier was examined with fibrinogen and immunoglobulins used as markers. The numbers of HLA-DR–positive microglia and glial fibrillary acidic protein–positive astroglia were examined comparatively.

Results White-matter lesions were graded as normal (grade 0) in 14 of the 32 cases (44%), slight (grade I) in 10 cases (31%), moderate (grade II) in 6 cases (19%), and severe (grade III) in 2 cases (6%). Amyloid precursor protein was accumulated most frequently in grade II white-matter lesions. Immunohistochemistry for serum proteins labeled astroglial cell bodies and their processes, which seemed to have sequestered extravasated proteins. The groups with detectable white-matter lesions had significantly higher grading scores for fibrinogen and immunoglobulins than the control group (P<.05). Although the higher scores for serum protein extravasation were statistically significant in ischemic CVD cases (P<.05), there was no significant increase in AD cases. Activated microglia and astroglia were more numerous in the groups with white-matter lesions in both ischemic CVD and AD cases, although this increase in the number of astroglia was not evident in regions with clasmatodendrosis.

Conclusions Dysfunction of the blood-brain barrier is more prominent in white-matter lesions seen in ischemic CVD than in AD and may have a role in the pathogenesis of cerebrovascular white-matter lesions.

Key Words: Alzheimer's disease • astrocytes • Binswanger's disease • blood-brain barrier • white matter

	Introduction

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Diffuse areas of hypodensity on CT or hyperintensity on T2-weighted MRI are often encountered in the periventricular or subcortical WM of the elderly. This finding, often referred to as "leu-koaraiosis," may correspond to rarefaction of myelin and axons in the deep WM of autopsied brains,^{1 2 3} although some of these may not be related to pathological lesions.⁴ They are frequent in patients with ischemic CVD and AD and may be correlated with the deterioration of cognitive function.⁵ Multi-infarct dementia is not the sole category of vascular dementia, and the significance of these WM lesions in dementia has been reviewed recently.^{6 7}

The mechanisms whereby the WM is rarefied remain unknown. In Binswanger's disease, a form of vascular dementia characterized by WM lesions, the cause may be chronic cerebral ischemia subsequent to lipohyalinosis of the long penetrating arteries of the deep WM.⁸ Alternatively, others have hypothesized that WM lesions may have been caused by hypertension and the subsequent exudation of fluid.^{9 10} From this viewpoint, the lipohyalinosis of the WM blood vessels may have been a result of chronic cerebral edema.¹⁰ However, no direct information is available on BBB function in WM lesions. In the present report, we demonstrate by immunohistochemically examining extravasated serum proteins in autopsied brains that BBB abnormalities are present in WM lesions of patients diagnosed clinicopathologically as having ischemic CVD.

Under various pathological conditions, a large number of studies have indicated the presence of BBB abnormalities by the immunohistochemical detection of extravasated serum proteins.^{11 12 13 14} However, there are controversies as to the specificity of serum protein extravasation in autopsy materials, because improper fixation¹⁵ or the postmortem migration of serum proteins into the perivascular neural parenchyma may cause the same finding.¹⁶ In the present investigation, we sought to minimize any artifactual findings by evaluating BBB function solely on the basis of immunopositive cellular constituents, because extravasated proteins are most likely to be taken up by neurons^{17 18} or glial cells¹⁹ in vivo.

	Materials and Methods

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Tissue Procurement
Brains were obtained from the brain bank in the Neuropathology Laboratory at Kyoto University Hospital. As part of the brain bank protocol, the brains were weighed fresh at autopsy and then perfusion fixed in either 4% paraformaldehyde and 0.35% glutaraldehyde or 4% paraformaldehyde in 0.1 mol/L PB, pH 7.4, for 20 minutes. Half the brain was divided in the midsagittal plane and postfixed in 4% paraformaldehyde in 0.1 mol/L PB for 24 to 48 hours. It was sliced in the coronal plane at 1.0- to 1.5-cm intervals and stored in 20% sucrose in 0.1 mol/L PB (pH 7.4) at 4°C. The other half of the brain was fixed in 10% neutral buffered formalin for 3 to 7 days and sliced in the coronal plane. Paraffin sections were prepared and examined with hematoxylin and eosin, Holzer, Kluver-Barrera, Bielschowsky, and Masson's trichrome staining for general histological examination.

We selected and examined 14 brains with ischemic CVD, 12 brains with AD, and 6 control brains. The diagnosis of AD was made on the basis of the Consortium to Establish a Registry for Alzheimer's Disease diagnostic neuropathologic criteria.²⁰ The cases with ischemic CVD were selected clinicopathologically but irrespective of the presence of dementia or leukoaraiosis on CT and MRI. They had multiple foci of cerebral infarction with varying sizes but had no histological evidence of AD. Cases with recent massive infarction were excluded from the present investigation. Control brains were from 3 patients with heart disease, 1 with liver cancer, 1 with lung cancer, and 1 with nephropathy, all of whom had no underlying neurological disease.

Immunohistochemical Staining
We examined tissue blocks at the anterior tip of the caudate nucleus, which included the cingulate and superior, middle, and inferior frontal gyri. In some cases, two brain blocks were used. In cases with ischemic CVD, the sections having a frank cerebral infarction were excluded to focus on nonlocalizing diffuse WM lesions. The tissue blocks were snap-frozen, cut with a freezing microtome into 20-µm sections, and treated as free-floating sections for immunohistochemical staining. The rest of these blocks were then embedded in paraffin. In the paraffin sections stained with Kluver-Barrera, the WM lesions were graded as normal (grade 0), low (grade I; reduced meshwork density with scattered, irregularly widened axons), moderate (grade II; further reduction in meshwork density compared with grade I, mainly composed of relatively short axons), and high (grade III; depletion of axon meshwork with a few remaining long axons) according to modified criteria by Englund and Brun.²¹

The frozen sections were incubated with a series of primary antibodies diluted in 0.1 mol/L PBS containing 0.3% Triton X-100 for 2 days at 4°C. The following antibodies were used in the present study: anti-fibrinogen (Dakoppats, rabbit IgG, diluted 1:5000), anti–HLA-DR (Cosmo Bio, mouse IgG, diluted 1:200) as a marker for activated microglia, and anti-GFAP (Dakoppats, rabbit IgG, diluted 1:20 000) as a marker for astroglia. APP was used as a marker of damaged axons and labeled by anti-APP592 (rabbit antiserum raised against recombinant N-terminal 592 residues of APP fusion protein; a generous gift from Dr Y. Tokushima, Asahi Chemical Industry Co Ltd; 0.05 µg/mL). After incubation with the primary antibodies, the sections were treated with the appropriate biotinylated secondary antibodies (Vector Laboratories; diluted 1:200).

To detect the immunoglobulins, untreated sections were incubated overnight with biotinylated anti-human IgG or IgM because the direct method of immunohistochemistry for immunoglobulins gave less background staining than an indirect immunohistochemistry. These sections were subsequently treated with avidin biotin complex (Vector Laboratories; diluted 1:200) and visualized with 0.01% diaminobenzidine tetrahydrochloride and 0.005% H₂O₂ in 50 mmol/L Tris HCl (pH 7.6). To test for the specificity of the immunohistochemical reaction, control sections were treated with normal mouse or rabbit IgG and normal rabbit serum instead of the primary antibody.

Morphometry
Parts of the sections were counterstained with hematoxylin, and the numerical density of immunopositive cells was counted in 10 representative square fields of 0.25 mm². The APP immunoreactivity was graded as 0 (none), 1 (slight), 2 (moderate), and 3 (severe). Grading for the immunoglobulins and fibrinogen was as follows: grade 0 (none), grade 1 (a small number of axons and glial processes and a few glial cell bodies), grade 2 (many axons and glial processes and a few glial cell bodies), and grade 3 (many axons, glial processes, and cell bodies), at a magnification of x200. Representative photomicrographs of each grade are depicted in Fig 3A through 3C. The periventricular regions were excluded from the above estimation because they are in contact with the CSF directly, and rarefaction of these areas can occur in the normal aging process. Statistical analysis was done by use of nonparametric group tests (Spearman's correlation coefficient, Mann-Whitney U test) with the use of StatView II software (Abacus Concepts, Inc) for a Macintosh computer.

View larger version (590K): [in this window] [in a new window]   Figure 3. Photomicrographs of IgM (A through C) and fibrinogen (D, E) immunoreactivity in the WM. The intensity of the immunohistochemical staining for IgM was graded as 1 (A), 2 (B), and 3 (C). The perivascular neuropil was diffusely stained for fibrinogen in the absence of any WM lesions (D). There was immunolabeling of glial cells and their processes and axons in the rarefied WM (E). Bars indicate 100 µm.

	Results

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In control sections, no specific immunohistochemical staining was observed. The Table summarizes the characteristics of the patients and the histological gradings of their WM lesions. WM lesions were observed in 1 (16.7%) of 6 control subjects, 9 (64.3%) of 14 ischemic CVD patients, and 8 (66.7%) of 12 AD patients. The mean age of the patients was not significantly different between the groups.

View this table: [in this window] [in a new window]   Table 1. Characteristics of Control Subjects and Patients With Ischemic CVD and AD

Histological alterations in the axons and glial cells were examined with immunohistochemistry for APP, HLA-DR, and GFAP and then estimated semiquantitatively. The variability of grading for IgM between two observers was checked by Spearman's correlation coefficient (r=.93, P=.0013). In the regions with WM lesions, there were APP-immunoreactive fiber bundles (Fig 1), which have been considered to be indicative of axonal damages.^{22 23 24 25} These APP-immunoreactive fiber bundles were almost absent in grade 0 specimens, most frequent in grade II, and less frequent in grades I and III (Fig 2). There were diffuse perivascular immunodeposits for fibrinogen and immunoglobulins in the neuropil in both gray-matter and WM regions. These deposits were more numerous in the former, not only in cases with WM lesions, but also in normal WM (Fig 3D). Cellular constituents such as glial cell bodies, glial processes, and axons were frequently labeled for fibrinogen and immunoglobulins in the rarefied WM (Fig 3A, 3B, 3C, and 3E). Most of these glial cells showed astroglial morphology, but there were some oligodendroglia and microglia, and they were predominantly distributed in the perivascular regions. The grading scores for fibrinogen, IgM, and IgG were significantly higher (P<.05) in cases with WM lesions than in cases with normal WM (Fig 2).

View larger version (117K): [in this window] [in a new window]   Figure 1. Photomicrograph of APP immunoreactivity in brain regions with WM lesions. Note the patchy distribution of the immunoreactive fiber bundles. Bar indicates 50 µm.

View larger version (50K): [in this window] [in a new window]   Figure 2. Histochemical grading of APP, fibrinogen, IgM, and IgG immunoreactivity in regions with WM lesions due to heterogeneous causes. *P<.05, **P<.01, significantly different by Mann-Whitney U test compared with grade 0.

In cases with WM lesions, microglial cells immunostained for HLA-DR were found frequently, and most of their processes were poorly ramified and truncated, indicating activation of these cells (Fig 4A). There was mild astrogliosis with an increased number of cell bodies and processes (Fig 4B). However, in some cases with more severe rarefaction, the astroglia were swollen and had decreased GFAP immunoreactivity with intracytoplasmic vacuoles and a few beaded processes. They were also immunoreactive for fibrinogen and immunoglobulins (not shown). In the neuropil, there were scattered GFAP-positive granular structures that varied in diameter from 1 to 3 µm and could occasionally be recognized as astroglial processes (Fig 4C). The numerical densities of HLA-DR immunopositive microglia were 210.6±137.0 (per 2.5 mm²; mean±SD) in grade 0, 384.6±234.7 in grade I, 352.3±221.3 in grade II, and 499.3±89.8 in grade III, which were significantly higher in the cases with rarefied WM (P<.05) than in cases with normal WM (Fig 5). The numerical densities of astroglia were 336.0±82.2 (per 2.5 mm²; mean±SD) in grade 0, 473.3±79.7 in grade I, 398.9±96.6 in grade II, and 382.3±18.4 in grade III, which were also more numerous in grade I than in grade 0 specimens but which did not increase significantly in grades II and III (Fig 5).

View larger version (401K): [in this window] [in a new window]   Figure 4. Photomicrographs of HLA-DR (A) and GFAP (B, C) immunoreactivity in regions with WM lesions. Note the swollen cell bodies with clasmatodendrosis and the cytoplasmic vacuoles (C; arrowheads). Bars indicate 50 µm.

View larger version (54K): [in this window] [in a new window]   Figure 5. Numerical densities of glial cells immunoreactive for HLA-DR and GFAP. *P<.05, **P<.01, significantly different by Mann-Whitney U test compared with grade 0.

Fig 6 shows the different features between CVD and AD, indicating a significant extravasation of fibrinogen and immunoglobulins in the WM of CVD but not AD patients (Fig 6).

View larger version (45K): [in this window] [in a new window]   Figure 6. Comparison of the histochemical gradings for fibrinogen, IgM, and IgG immunoreactivity in regions with WM lesions among control subjects (cont), ischemic CVD patients, and AD patients. *Significantly different (P<.05) by Mann-Whitney U test compared with grade 0.

	Discussion

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Extravasated serum components may be transported via the extracellular space into the ventricular CSF and the vascular or lymphatic systems. Alternatively, these serum proteins may become sequestered in cellular compartments,²⁶ most of which are astroglia, the main buffering cell of extravasated fluids in the brain.²⁷ Thus, it is highly likely that immunopositive astroglial cell bodies and their processes are indicators of BBB function.

The exudation of serum proteins in rarefied WM has been documented previously in a case report of Binswanger's disease.²⁸ To our knowledge, the present investigation is the first to demonstrate that BBB is invariably compromised in the rarefied WM in ischemic CVD patients but barely in AD patients. WM lesions in AD brains have been attributed to either wallerian degeneration after the loss of cortical neurons²⁹ or to chronic cerebral ischemia, which is secondary to either cortical amyloid angiopathy¹ or nonamyloid lipohyalinosis of the long penetrating arteries of the WM.⁸ The lack of significant BBB dysfunction in AD patients in the present investigation may indicate a pathological process different from ischemic CVD and suggests a more important role of other etiologies, such as wallerian degeneration, in the WM lesions seen in AD.

The present investigation is in agreement with previous studies that showed a blood-CSF barrier dysfunction more prominently in CVD than in AD^{30 31 32 33} and in AD with vascular risk factors³⁴ and Binswanger's disease.³⁵ On the other hand, there are conflicting results regarding BBB abnormalities in AD, although such results have been described in autopsied brains.¹⁴ Some authors suggest the absence or, at most, the minor extravasation of serum proteins in AD, which is undetectable by usual immunohistochemical methods.^{36 37} More recently, morphometric investigations have shown subtle but significant abnormalities, which suggest a compromised BBB in AD.^{38 39}

WM lesions resembling those typically found in Binswanger's disease are frequent findings in CVD patients. These lesions are thought to arise primarily from chronic cerebral ischemia involving the WM. Irrespective of the initial triggering mechanism, the BBB dysfunction demonstrated in the present study may aggravate WM lesions, since a loss of lipid components and demyelination have been demonstrated in chronic cerebral edema.⁴⁰ A compromised BBB may allow the entry of macromolecules and other blood constituents such as proteases, immunoglobulins, complement, and cytokines into the vascular wall and perivascular neural parenchyma. These serum components may have deleterious effects on the WM myelin directly⁴¹ or by enhancing the phagocytotic activity of microglia as opsonin.⁴²

Microglia are activated in the rarefied WM of autopsied brains⁴³ and after experimental chronic cerebral hypoperfusion.⁴⁴ They may exert toxic effects on oligodendroglia and myelin, since activated microglia phagocytose myelin⁴⁵ and suppress neural growth in coculture, possibly by releasing toxic substances such as tumor necrosis factor- or nitric oxide.⁴⁶

Inconsistent results have been obtained for the astroglia in WM lesions, such as a decrease in the number of nuclei with astroglial morphology⁴⁷ or an increase of GFAP immunoreactive astroglia.^{43 48} Although the present investigation revealed a significant increase in the number of astroglia in regions with WM lesions, this did not apply to the more severe cases. The regions with marked WM lesions showed no numerical increase of astroglia, and the remaining astroglia had swollen cell bodies and disintegrated processes with a beaded appearance. These astroglia have been described previously as undergoing clasmatodendrosis (breaking up of branches).⁴⁹ This may be a terminal feature of the pathological response to the long-standing extravasation of fluids, which is supported by the observation of immunoreactivity for serum proteins, marked vacuoles within the cytoplasm, and their exclusive association with relatively severe WM lesions.

The reason for BBB breakdown remained unclear in the present investigation. In cases with lacunar infarcts in the WM, extravasated serum protein may spread from these foci. Alternatively, chronic hypertension may result in BBB dysfunction and pathological alterations in small arteries, as demonstrated in spontaneously hypertensive rats⁵⁰ and rats with induced hypertension.⁵¹ In the WM of human brains, morphological abnormalities have been revealed in arterioles and venules with advancing age and hypertension.^{52 53} In future investigations, it will be necessary to investigate vascular changes and their relationship to BBB breakdown in the rarefied WM.

	Selected Abbreviations and Acronyms

 

AD = Alzheimer's disease APP = amyloid protein precursor BBB = blood-brain barrier CSF = cerebrospinal fluid CT = computed tomography CVD = cerebrovascular disease GFAP = glial fibrillary acidic protein MRI = magnetic resonance imaging PB = phosphate buffer WM = white matter

	Acknowledgments

 
This work was supported by a grant-in-aid for scientific research on priority areas from the Japanese Ministry of Education, Science and Culture and a grant-in-aid for amyotrophic lateral sclerosis and peripheral neuropathy from the Japanese Ministry of Health and Welfare. The authors thank Dr T. Yanagihara (Osaka University) for reading the manuscript.

Received April 4, 1996; revision received June 27, 1996; accepted July 26, 1996.

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