A method for microscopic studies of cerebral angioarchitecture and vascular-parenchymal relationships, based on the demonstration of 'paravascular' fluid pathways in the mammalian central nervous system

A new method is described for morphological studies of blood vessels and related cellular elements in the mammalian central nervous system (CNS). The tracer protein, horseradish peroxidase (HRP), in solution, is infused intraventricularly or intracisternally in anesthetized animals over 5-10 min. During this period, HRP in the subarachnoid space enters the perivascular spaces around penetrating arterioles and rapidly permeates the gliovascular basal laminae surrounding capillaries. After fixation by intravascular perfusion of aldehydes, brain sections are incubated with the highly sensitive chromogen, tetramethylbenzidine. Intraparenchymal blood vessels throughout the CNS are vividly demonstrated for light microscopy by HRP reaction product in their perivascular spaces or basal laminae. Correlative ultrastructural investigations of specific blood vessels and related parenchymal elements can be conducted using adjacent sections.     

Laminar distribution of beta-amyloid deposits in dementia with Lewy bodies and in Alzheimer's disease

This study tested whether the laminar distribution of the beta-amyloid (Abeta) deposits in dementia with Lewy bodies (DLB) cases with significant Alzheimer's disease (AD) pathology (DLB/AD) was similar to "pure" AD. In DLB/AD, the maximum density of the diffuse and primitive deposits occurred either in the upper laminae or a bimodal distribution was present with density peaks in the upper and lower laminae. A bimodal distribution of the classic Abeta deposits was also observed. Compared with AD, DLB/AD cases had fewer primitive deposits relative to the diffuse and classic deposits; the primitive deposits exhibited a bimodal distribution more frequently, and the diffuse deposits occurred more often in the upper laminae. These results suggest that Abeta pathology in DLB/AD may not simply represent the presence of associated AD.     

Pericapillary haem-rich deposits: evidence for microhaemorrhages in aging human cerebral cortex.

In this post mortem study, we examined haem-rich deposits (HRDs) in patients with and without dementia, using a histochemical label (Prussian blue) to show haem, autofluorescence to detect red blood cells (RBCs), and immunohistochemistry for clotting-related factors and collagen IV. The patients studied had no clinical or post mortem evidence of macrovascular stroke. To allow examination of the spatial relationships between HRDs and the microvasculature, we cut 45-microm sections. Haem-rich deposits were small (<200 microm diameter). They were rare in younger (<50 years) patients but were more common in older (>70 years) patients, particularly in cerebral cortex, and were most abundant in cases with senile plaques. Wherever HRDs appeared they were perivascular and appeared to form around capillaries or small arterioles. Using a software package (Proxan) developed to outline vessels and HRDs, and to analyse the distances between them, a tight spatial correlation between HRDs and capillaries was shown. In addition, HRDs were rich in von Willebrand factor (vWF), fibrinogen, collagen IV and RBCs. These observations suggest that HRDs are the residua of capillary bleeds (microhaemorrhages), and that microhaemorrhages are a common feature of the aging cerebral cortex, particularly where plaque pathology is present.     

Dispersion of prion protein deposits around blood vessels in variant Creutzfeldt-Jakob disease

In variant Creutzfeldt-Jakob disease (vCJD), a disease linked to bovine spongiform encephalopathy (BSE), florid-type prion protein (PrP(sc)) deposits are aggregated around the larger diameter (> 10 μm) cerebral microvessels. Clustering of PrP(sc) deposits around blood vessels may result from blood-borne prions or be a consequence of the cerebral vasculature influencing the development of the florid deposits. To clarify the factors involved, the dispersion of the florid PrP(sc) deposits was studied around the larger diameter microvessels in the neocortex, hippocampus, and cerebellum of ten cases of vCJD. In the majority of brain regions, florid deposits were clustered around the larger diameter vessels with a mean cluster size of between 50 μm and 628 μm. With the exception of the molecular layer of the dentate gyrus, the density of the florid deposits declined as a negative exponential function of distance from a blood vessel profile suggesting that diffusion of molecules from blood vessels is a factor in the formation of the florid deposits. Diffusion of PrP(sc) directly into the brain via the microvasculature has been demonstrated in vCJD in a small number of cases. However, the distribution of the prion deposits in vCJD is more likely to reflect molecular 'chaperones' diffusing from vessels and promoting the aggregation of pre-existing PrP(sc) in the vicinity of the vessels to form florid deposits.     

Cerebral small vessel disease-induced apolipoprotein E leakage is associated with Alzheimer disease and the accumulation of amyloid beta-protein in perivascular astrocytes

Apolipoprotein E (apoE) plays a role in the pathogenesis of Alzheimer disease (AD). It is involved in the receptor-mediated cellular clearance of the amyloid beta-protein (Abeta) and in the perivascular drainage of the extracellular fluid. Microvascular changes are also associated with AD and have been discussed as a possible reason for altered perivascular drainage. To further clarify the role of apoE in the perivascular and vascular pathology in AD patients, we studied its occurrence and distribution in the perivascular space, the perivascular neuropil, and in the vessel wall of AD and control cases with and without small vessel disease (SVD). Apolipoprotein E was found in the perivascular space and in the neuropil around arteries of the basal ganglia from control and AD cases disclosing no major differences. Western blot analysis of basal ganglia tissue also revealed no significant differences pertaining to the amount of full-length and C-terminal truncated apoE in AD cases compared with controls. In contrast, Abeta occurred in apoE-positive perivascular astrocytes in AD cases but not in controls. In blood vessels, apoE and immunoglobulin G were detected within the SVD-altered vessel wall. The severity of SVD was associated with the occurrence of apoE in the vessel wall and with that of Abeta in perivascular astrocytes. These results point to an important role of apoE in the perivascular clearance of Abeta in the human brain. The occurrence of apoE and immunoglobulin G in SVD lesions and in the perivascular space suggests that the presence of SVD results in plasma-protein leakage into the brain. It is therefore tempting to speculate that apoE represents a pathogenetic link between SVD and AD.     

Beta-amyloid deposition in the temporal lobe of patients with dementia with Lewy bodies: comparison with non-demented cases and Alzheimer's disease

beta-Amyloid (Abeta) deposition in regions of the temporal lobe in patients with dementia with Lewy bodies (DLB) was compared with elderly, non-demented (ND) cases and with Alzheimer's disease (AD). The distribution, density and clustering patterns of diffuse, primitive and classic Abeta deposits were similar in 'pure' DLB and ND cases. The distribution of Abeta deposits and the densities of the diffuse and primitive deposits were similar in 'mixed' DLB/AD cases compared with AD. However, the density of the classic deposits was significantly lower in DLB/AD compared with AD. In addition, the primitive Abeta deposits occurred more often in small, regularly spaced clusters in the tissue and less often in a single large cluster in DLB/AD compared with 'pure' AD. These results suggest that pure DLB and AD are distinct disorders which can coexist in some patients. However, the Abeta pathology of DLB/AD cases is not identical to that observed in patients with AD alone.     

Deposition of mouse amyloid beta in human APP/PS1 double and single AD model transgenic mice

The deposition of amyloid beta (Abeta) peptides and neurofibrillary tangles are the two characteristic pathological features of Alzheimer's disease (AD). To investigate the relation between amyloid precursor protein (APP) production, amyloid beta deposition and the type of Abeta in deposits, i.e., human and/or mouse, we performed a histopathological analysis, using mouse and human specific antibodies, of the neocortex and hippocampus in 6, 12 and 19 months old APP/PS1 double and APP and PS1 single transgenic mice. There was a significant correlation between the human amyloid beta deposits and the intrinsic rodent amyloid beta deposits, that is, all plaques contained both human and mouse Abeta, and the diffuse amyloid beta deposits also colocalized human and mouse Abeta. Furthermore, some blood vessels (mainly leptomeningeal vessels) show labeling with human Abeta, and most of these vessels also label with mouse Abeta. Our findings demonstrate that the human amyloid deposits in APP/PS1 transgenic mice are closely associated with mouse Abeta, however, they do not precisely overlap. For instance, the core of plaques consists of primarily human Abeta, whereas the rim of the plaque contains both human and mouse amyloid beta, similarly, human and mouse Abeta are differentially localized in the blood vessel wall. Finally, as early as amyloid beta deposits can be detected, they show the presence of both human and mouse Abeta. Together, these data indicate that mouse Abeta is formed and deposited in significant amounts in the AD mouse brain and that it is deposited together with the human Abeta.     

Experimental chronic lead poisoning.

In the peripheral nerve, the distal part of the nerve fibers was remarkably damaged--periaxial segmental demyelination--while the proximal part of nerve fibers was only slightly affected. In the CNS, perivascular edema of the small blood vessels and capillaries was observed in the cerebral cortex and cerebellar cortex. All endothelial cells of these blood vessels showed a high electron density with many pinocytotic vesicles and ribosomes. Following these changes, the nerve fibers adjacent to the edema were destroyed. The above-mentioned findings seem to indicate that lead induces a toxic effect on the blood vessel and produces perivascular edema in the CNS of adult animals. This may induce brain dysfunction.     

Occlusion of cortical ascending venules causes blood flow decreases, reversals in flow direction, and vessel dilation in upstream capillaries

The accumulation of small strokes has been linked to cognitive dysfunction. Although most animal models have focused on the impact of arteriole occlusions, clinical evidence indicates that venule occlusions may also be important. We used two-photon excited fluorescence microscopy to quantify changes in blood flow and vessel diameter in capillaries after occlusion of single ascending or surface cortical venules as a function of the connectivity between the measured capillary and the occluded venule. Clotting was induced by injuring the target vessel wall with femtosecond laser pulses. After an ascending venule (AV) occlusion, upstream capillaries showed decreases in blood flow speed, high rates of reversal in flow direction, and increases in vessel diameter. Surface venule occlusions produced similar effects, unless a collateral venule provided a new drain. Finally, we showed that AVs and penetrating arterioles have different nearest-neighbor spacing but capillaries branching from them have similar topology, which together predicted the severity and spatial extent of blood flow reduction after occlusion of either one. These results provide detailed insights into the widespread hemodynamic changes produced by cortical venule occlusions and may help elucidate the role of venule occlusions in the development of cognitive disorders and other brain diseases.     

Experimental Chronic Lead Poisoning

In the peripheral nerve, the distal part of the nerve fibers was remarkably damaged—periaxial segmental demyelination—while the proximal part of nerve fibers was only slightly affected. In the CNS, perivascular edema of the small blood vessels and capillaries was observed in the cerebral cortex and cerebellar cortex. All endothelial cells of these blood vessels showed a high electron density with many pinocytotic vesicles and ribosomes. Following these changes, the nerve fibers adjacent to the edema were destroyed. The above-mentioned findings seem to indicate that lead induces a toxic effect on the blood vessel and produces perivascular edema in the CNS of adult animals. This may induce brain dysfunction.     

Capillary and arterial cerebral amyloid angiopathy in Alzheimer's disease: defining the perivascular route for the elimination of amyloid beta from the human brain

Accumulation of amyloid beta (Abeta) in the extracellular spaces of the cerebral cortex and in blood vessel walls as cerebral amyloid angiopathy is a characteristic of Alzheimer's disease (AD) and the ageing human brain. Studies in animals suggest that Abeta is eliminated from the brain either directly into the blood or along perivascular interstitial fluid drainage channels. The aim of the present study is to define the perivascular route for the drainage of Abeta from the human brain. Smears and paraffin sections of post-mortem cortical tissue from 17 cases of AD and from two controls were stained with thioflavin and for Abeta by immunohistochemistry. Histology and confocal microscopy showed that deposits of Abeta in the cortical parenchyma were continuous with Abeta in capillary walls but Abeta in artery walls was not in continuity with Abeta in brain parenchyma. Quantitative studies supported these observations. The results of this study suggest that when Abeta is eliminated from the extracellular spaces of the human brain by the perivascular route, it enters pericapillary spaces and from there drains along the walls of cortical arteries to leptomeningeal arteries. Factors such as overproduction of Abeta, entrapment of Abeta in drainage pathways and poor drainage of Abeta due to functional changes in ageing arteries might result in the failure of elimination of Abeta from the ageing brain and play a major role in the pathogenesis of AD. Such factors might affect therapies for AD that entail administration of anti-Abeta antibodies to eliminate Abeta from the human brain.     

A morphometric study of intrafascicular vessels of mammalian sciatic nerve

Intrafascicular vessel profiles were measured with a Quantimet image analyzer in 1-micron transverse sections of sciatic nerves of rats and compared with those of ("white") skeletal muscle. Blood vessels occupy the same percentage of the total tissue area in nerve and muscle tissues. The nerve, however, has much larger capillaries (about 1.7 X) spaced farther apart (about 2.5 X). Comparison of 3 1/2 month with 1-year-old rats showed that muscle capillaries increased in diameter (slightly, but significantly) at maturity, but those of the nerve decreased (also significantly). Vascular percentages of total tissue areas did not change. Nerve and muscle samples from monkey, rabbit, mouse, and cat show that the same striking pattern of large, widely spaced vessels distinguishes the nerve from other tissues. Minor interspecies variations exist. The nerve may contain as much blood as the muscle but its angioarchitecture suggests a lower exchange capacity.     

Effects of aging on the structural and permeability characteristics of cerebrovasculature in normotensive and hypertensive strains of rats

Summary This study demonstrates that markedly different patterns of age-related changes in blood pressure and body weight occur among normotensive Wistar-Kyoto (WKY) and Sprague-Dawley (SD) rats and spontaneously hypertensive rats (SHR). In addition, a variety of age-related structural alterations occurred in the walls of arterioles, capillaries, and venules of the frontal cortex. These changes include: (1) an increase in the thickness of the vascular wall by deposits of collagen and basal lamina which, in some cases, extended into the surrounding neuropil; (2) the presence of a flocculent material in the adventitia of intracerebral arterioles; (3) vesicular inclusions in perivascular macrophages, pericytes and smooth muscle cells which were labelled with i.v. administered horseradish peroxidase (HRP); (4) fragmentation of smooth muscle cells; and (5) accumulation of lipofuscin-like pigments in perivascular glial processes. The hypertensive rats exhibited these changes, but they were more advanced and more widely distributed throughout the cerebral cortex. The aged hypertensive rats occasionally had large bundles of 10 nm diameter, intermediate filaments in the endothelial cells. Whereas no change in blood-brain barrier permeability to HRP was observed in the aged normotensive rats, all age groups of the hypertensive rats exhibited increased permeability to HRP in the initial segment of penetrating arterioles in laminae I and II of the cerebral cortex.     

The spatial patterns of prion protein deposits in cases of variant Creutzfeldt-Jakob disease

The spatial patterns of the prion protein (PrP) deposits were studied in immunostained sections of areas of the cerebral cortex, hippocampus, dentate gyrus, and the molecular layer of the cerebellum in 11 cases of variant Creutzfeldt-Jakob disease (vCJD). Clustering of PrP deposits, with a regular distribution of the clusters parallel to the tissue boundary, was the most common spatial pattern observed. Two morphological types of PrP deposit were recognised, those consisting of a condensed core (florid deposits) and those deposits lacking a condensed core (non-florid deposits). The florid and non-florid PrP deposits exhibited a different profile of spatial patterns. First, the florid deposits exhibited a regularly distributed pattern of clusters more frequently than the non-florid deposits. Second, the florid deposits formed larger clusters (greater than 1,600 micro m in diameter) less frequently than the non-florid deposits. In the areas of the cerebral cortex that exhibited a regular distribution of PrP deposit clusters, the cluster size of the deposits approximated that of the groups of cells of the cortico-cortical pathway origin in only 12% of analyses. No significant differences in the frequency of the different types of spatial pattern were observed in different brain regions, or in the cerebral cortex between the upper and lower laminae. It was concluded that the spatial patterns of the PrP deposits in the cerebral cortex in vCJD are unlikely to reflect the degeneration of the cortico-cortical pathways as has been reported in sporadic CJD (sCJD). In addition, different factors could be involved in the development of the deposits with and without a condensed core.     

Quantitation of apoE domains in Alzheimer disease brain suggests a role for apoE in Abeta aggregation

Apolipoprotein E (apoE) and apoE-derived proteolytic fragments are present in amyloid deposits in Alzheimer disease (AD) and cerebral amyloid angiopathy (CAA). In this study, we examined which apoE fragments are most strongly associated with amyloid deposits and whether apoE receptor binding domains were present. We found that both apoE2- and apoE4-specific residues were present on plaques and blood vessels in AD and CAA. We quantified Abeta plaque burden and apoE plaque burdens in 5 AD brains. ApoE N-terminal-specific and C-terminal-specific antibodies covered 50% and 74% of Abeta plaque burden, respectively (p < 0.003). Double-labeling demonstrated that the plaque cores contained the entire apoE protein, but that outer regions contained only a C-terminal fragment, suggesting a cleavage in the random coil region of apoE. Presence of N- and C-terminal apoE cleavage fragments in brain extracts was confirmed by immunoblotting. The numbers of plaques identified by the apoE N-terminal-specific antibodies and the apoE C-terminal-specific antibody were equal, but were only approximately 60% of the total Abeta plaque number (p < 0.0001). Analysis of the size distribution of Abeta and apoE deposits demonstrated that most of the Abeta-positive, apoE-negative deposits were the smallest deposits (less than 150 microm2). These data suggest that C-terminal residues of apoE bind to Abeta and that apoE may help aid in the progression of small Abeta deposits to larger deposits. Furthermore, the presence of the apoE receptor binding domain in the center of amyloid deposits could affect surrounding cells via chronic interactions with cell surface apoE receptors.     

P2X (purinergic) receptor distributions in rat blood vessels

The distribution of purinergic (P2X1 and P2X2) receptors on smooth muscle cells in relation to autonomic nerve varicosities in rat blood vessels has been determined using immunofluorescence and confocal microscopy. P2X1 and P2X2 receptors were visualised using rabbit polyclonal antibodies against the extracellular domain of the receptors and varicosities visualised using a mouse monoclonal antibody against the ubiquitous synaptic vesicle proteoglycan SV2. Two size classes of P2X1 receptor clusters were observed on the smooth muscle cells of mesenteric, renal, and pulmonary arteries as well as in the aorta and in veins: a large approximately elliptical cluster 1.32+/-0.21 microm long and 0.96+/-0.10 microm in diameter; and a smaller spherical cluster with a diameter of 0.32+/-0.05 microm. The latter occurred throughout the media of arteries of all sizes, whereas the former were restricted to the adventitial surface of the media and to endothelial cells, except for the pulmonary artery, in which large receptor clusters were found throughout the media of the vessel. At the adventitial surface, the large clusters are in general located beneath SV2 labelled varicosities. None of the small clusters was associated with varicosities. Three-dimensional reconstruction of the P2X and SV2 labelling at individual varicosities showed that the varicosities were immediately apposed to the P2X receptor clusters. P2X2 receptors were located on nerves and on endothelial cells. They were also found in low density on the smooth muscle cells in the media. These observations are discussed in relation to the mechanism of purinergic transmission to the smooth muscle cells of blood vessels.     

Spatial patterns of β-amyloid (Aβ) deposits in familial and sporadic Alzheimer's disease

The spatial patterns of the diffuse, primitive, and classic β -amyloid (Aβ) deposits were compared in cortical regions in early-onset familial Alzheimer's disease (EO-FAD) linked to mutations of the amyloid precursor protein APP) or presenilin 1 (PSEN1) genes, late-onset familial AD (LO-FAD), and sporadic AD (SAD). The objective was to determine whether genetic factors influenced the spatial patterns of the A β deposits. A β deposits were distributed either in clusters which were regularly distributed parallel to the pia mater or in larger, non-regularly distributed clusters. There were no significant differences in spatial pattern of the diffuse deposits between patient groups but mean cluster size of the diffuse deposits was larger in FAD compared with SAD. Primitive A β deposits were more frequently distributed in regular clusters and less frequently distributed in large clusters in FAD compared with SAD. Classic A β deposits were more frequently distributed in regularly spaced clusters and less frequently distributed in large clusters in LO-FAD compared with EO-FAD. There were no significant differences in the spatial patterns or cluster sizes of A β deposits in cases classified according to apolipoprotein E (APOE) genotype. These results suggest (1) greater deposition of A β in the form of clusters of diffuse deposits in FAD, (2) a greater proportion of diffuse deposits may be converted to primitive deposits in SAD, (3) classic deposits are more widely distributed in EO-FAD, and (4) the presence of APOE allele ε4 has little effect on the spatial patterns of A β deposits.     

Relationships in Alzheimer's disease between the extent of Abeta deposition in cerebral blood vessel walls, as cerebral amyloid angiopathy, and the amount of cerebrovascular smooth muscle cells and collagen

The relationship between degree of cerebral amyloid angiopathy (CAA) and the amount of smooth muscle cells (SMCs) and deposition of collagen IV fibres (COL IV) was investigated in the frontal and occipital cortex of 70 patients with autopsy confirmed Alzheimer's disease (AD). The extent of CAA was significantly greater in occipital than in frontal cortex, although SMC loss was greater in frontal than in occipital cortex. COL IV staining was significantly higher in occipital than in frontal cortex. The degree of SMC loss correlated with CAA, as Abeta40 but not as Abeta42 or total Abeta, in frontal cortex, but not in occipital cortex. Leptomeningeal arteries within occipital cortex showed significantly greater external diameter, greater wall thickness and greater luminal area than those in frontal cortex. The degree of CAA correlated with thickness of blood vessel wall and external diameter in frontal cortex, whereas extent of SMC loss correlated with thickness of blood vessel wall in occipital cortex. There were significant negative correlations between duration of disease and thickness of vessel wall, external diameter and luminal area. In patients with disease durations exceeding 10 years, external vessel diameter and thickness of the vessel wall were both halved compared with patients with durations less than 5 years; luminal area was reduced by about 75%. Blood vessels in AD undergo degenerative changes involving deposition of Abeta and COL IV with loss of SMC. SMC loss may relate to increasing Abeta deposition in early stages of disease, but this relationship may be lost with disease progression.     

Microvascular sprouting,extension, and creation of new capillary connections with adaptation of the neighboring astrocytes in adult mouse cortex under chronic hypoxia

The present study aimed to determine the spatiotemporal dynamics of microvascular and astrocytic adaptation during hypoxia-induced cerebral angiogenesis. Adult C57BL/6J and Tie2-green fluorescent protein (GFP) mice with vascular endothelial cells expressing GFP were exposed to normobaric hypoxia for 3 weeks, whereas the three-dimensional microvessels and astrocytes were imaged repeatedly using two-photon microscopy. After 7 to14 days of hypoxia, a vessel sprout appeared from the capillaries with a bump-like head shape (mean diameter 14 μm), and stagnant blood cells were seen inside the sprout. However, no detectable changes in the astrocyte morphology were observed for this early phase of the hypoxia adaptation. More than 50% of the sprouts emerged from capillaries 60 μm away from the center penetrating arteries, which indicates that the capillary distant from the penetrating arteries is a favored site for sprouting. After 14 to 21 days of hypoxia, the sprouting vessels created a new connection with an existing capillary. In this phase, the shape of the new vessel and its blood flow were normalized, and the outside of the vessels were wrapped with numerous processes from the neighboring astrocytes. The findings indicate that hypoxia-induced cerebral angiogenesis provokes the adaptation of neighboring astrocytes, which may stabilize the blood–brain barrier in immature vessels.     

Fine structure of psammoma bodies at the outer aspect of blood vessels in meningioma

Summary Psammoma bodies at the perivascular area in five cases of meningioma were examined with the electron microscope. In general, meningocytic cells invest the outer aspect of blood vessels, which are constituted by multilayered basal laminae, collagen fibers, microfibrils, and pericytes. Remnants of degenerated cells are observed in some areas of the perivascular space. Matrix vesicles and matrix giant bodies with or without mineralized deposits are also present in those areas. Energy disperisve, X-ray microanalysis evidenced the presence of both calcium and phosphorus (probably hydroxyapatite) mineralized precipitates. Production of psammoma bodies in the perivascular area may indicate that matrix vesicles and matrix giant bodies are derived from degenerated cells, which then sequestrate hydroxyapatite crystals to form psammoma bodies.