Cornelia Garz

Blood brain barrier breakdown as the starting point of cerebral small vessel disease? - New insights from a rat model.

Cerebral small vessel disease (CSVD, cerebral microangiopathy) leads to dementia and stroke-like symptoms. Lacunes, white matter lesions (WML) and microbleeds are the main pathological correlates depicted in in-vivo imaging diagnostics. Early studies described segmental arterial wall disorganizations of small penetrating cerebral arteries as the most pronounced underlying histopathology of lacunes. Luminal narrowing caused by arteriolosclerosis was supposed to result in hypoperfusion with WML and infarcts.We have used the model of spontaneously hypertensive stroke-prone rats (SHRSP) for a longitudinal study to elucidate early histological changes in small cerebral vessels. We suggest that endothelial injuries lead to multiple sites with blood brain barrier (BBB) leakage which cause an ongoing damage of the vessel wall and finally resulting in vessel ruptures and microbleeds. These microbleeds together with reactive small vessel occlusions induce overt cystic infarcts of the surrounding parenchyma. Thus, multiple endothelial leakage sites seem to be the starting point of cerebral microangiopathy. The vascular system reacts with an activated coagulatory state to these early endothelial injuries and by this induces the formation of stases, accumulations of erythrocytes, which represent the earliest detectable histological peculiarity of small vessel disease in SHRSP.In this review we focus on the meaning of the BBB breakdown in CSVD and finally discuss possible consequences for clinicians.

The Pathologic Cascade of Cerebrovascular Lesions in SHRSP: Is Erythrocyte Accumulation an Early Phase?

Cerebral small vessel disease (CSVD) is associated with vessel wall changes, microbleeds, blood—brain barrier (BBB) disturbances, and reduced cerebral blood flow (CBF). As spontaneously hypertensive stroke-prone rats (SHRSP) may be a valid model of some aspects of human CSVD, we aimed to identify whether those changes occur in definite temporal stages and whether there is an initial phenomenon beyond those common vascular alterations. Groups of 51 SHRSP were examined simultaneously by histologic (Hematoxylin—Eosin, IgG-Immunohistochemistry, vessel diameter measurement) and imaging methods (Magnetic Resonance Imaging, 201-Thallium-Diethyldithiocarbamate/99m-Technetium-HMPAO Single Photon Emission Computed Tomography conducted as pilot study) at different stages of age. Vascular pathology in SHRSP proceeds in definite stages, whereas an age-dependent accumulation of erythrocytes in capillaries and arterioles represents the homogeneous initial step of the disease. Erythrocyte accumulations are followed by BBB disturbances and microbleeds, both also increasing with age. Microthromboses, tissue infarctions with CBF reduction, and disturbed potassium uptake represent the final stage of vascular pathology in SHRSP. Erythrocyte accumulations—we parsimoniously interpreted as stases—without cerebral tissue damage represent the first step of vascular pathology in SHRSP. If that initial phenomenon could be identified in patients, these erythrocyte accumulations might be a promising target for implementing prophylactic and therapeutic strategies in human CSVD.

Interplay Between Age, Cerebral Small Vessel Disease, Parenchymal Amyloid-β, and Tau Pathology: Longitudinal Studies in Hypertensive Stroke-Prone Rats

BACKGROUND Accumulation of amyloid-β (Aβ) and hyperphosphorylated tau (ptau) accompany cerebral small vessel disease (CSVD) in the aging brain and in Alzheimers disease. CSVD is characterized by a heterogeneous spectrum of histopathological features possibly initiated by an endothelial dysfunction and blood-brain barrier (BBB) breakdown. OBJECTIVE We test the hypothesis that characteristic features of CSVD are associated with the accumulation of Aβ and ptau in non-transgenic spontaneously hypertensive stroke-prone rats (SHRSP). METHODS Amyloid-β protein precursor (AβPP) and tau were investigated by western blotting (n = 12 SHRSP, age 20 weeks). Lectin staining and plasma protein immunocytochemistry for BBB examination were performed in 38 SHRSP (age 12-44 weeks) and Aβ (n = 29) and ptau (n = 17) immunocytochemistry in 20-44 week-old SHRSP. We assessed the correlation between extracellular amyloid deposits and features of CSVD (n = 135, 12-44 weeks). RESULTS In 20 week-old SHRSP, cortical AβPP expression was significantly increased compared to Wistar controls but tau levels were unchanged. At ages of 20-44 weeks, SHRSP exhibited an age-dependent increase in extracellular Aβ. Ptau was observed in 26-44 week-old SHRSP. Distinct features of CSVD pathology developed from the age of 12 weeks on. CONCLUSION We demonstrate that in a hypertensive rat model that displays features of CSVD from 12 weeks, there is an age-dependent extracellular deposition of Aβ observed from 20 weeks onwards, increased AβPP expression at 20 weeks and ptau accumulation from 26 weeks on. This study suggests that CSVD associated with hypertension results in an age-related failure of Aβ clearance, increase in AβPP expression, and intraneuronal tau hyperphosphorylation.

Hypertension drives parenchymal β‐amyloid accumulation in the brain parenchyma

There is substantial controversy regarding the causative role of amyloid β (Aβ) deposition in Alzheimers disease (AD). The cerebrovasculature plays an important role in the elimination of Aβ from the brain and hypertension is a well‐known risk factor for AD. In spontaneously hypertensive stroke‐prone rats (SHRSP), an animal model of chronic arterial hypertension, cerebral small vessel disease (CSVD) leads to age‐dependent parenchymal Aβ accumulation similar to that observed in AD. These data approve the neuropathological link between CSVD and AD, confirm the challenge that parenchymal Aβ deposition is a specific marker for AD and disclose the meaning of SHRSP as valid experimental model to investigate the association between hypertension, CSVD, and Aβ plaques.

Stases are associated with blood–brain barrier damage and a restricted activation of coagulation in SHRSP

Cerebral small vessel disease (CSVD) is a chronically proceeding pathology of small brain vessels associated with white matter lesions, lacunar infarcts, brain atrophy and microbleeds. CSVD leads to slowly increasing cognitive and functional deficits but may also cause stroke-like symptoms, if vessels in critical brain areas are affected. Spontaneously hypertensive stroke-prone rats (SHRSP) exhibit several vascular risk factors, develop infarcts and hemorrhages and therefore represent a relevant model for the study of CSVD. Using this animal model, we recently demonstrated that intravasal accumulations of erythrocytes, we interpreted as stases, stand at the beginning of a pathological vascular cascade. After stases microbleeds occur, which are followed by reactive microthromboses. Bleeds and thromboses finally cause hemorrhagic infarcts. Immunohistochemical stainings show, that plasma proteins like IgG are deposited in the walls of vessels affected by stases. Further, we found small clots and thread-shaped aggregations of thrombocytes as well as thread-shaped structures of von Willebrand-Factor within stases. Thus, we conclude that blood-brain barrier damages occur in the neighborhood of stases and stases seem to be associated with a restricted activation of blood coagulation without formation of obstructive thromboses. Finally, we demonstrate that small vessel damage rarely appears in the cerebellum. Even animals with multiple cerebral infarcts may be free of any cerebellar vascular pathology.

Hypercholesterolemia induced cerebral small vessel disease

Background While hypercholesterolemia plays a causative role for the development of ischemic stroke in large vessels, its significance for cerebral small vessel disease (CSVD) remains unclear. We thus aimed to understand the detailed relationship between hypercholesterolemia and CSVD using the well described Ldlr-/- mouse model. Methods We used Ldlr-/- mice (n = 16) and wild-type (WT) mice (n = 15) at the age of 6 and 12 months. Ldlr-/- mice develop high plasma cholesterol levels following a high fat diet. We analyzed cerebral capillaries and arterioles for intravascular erythrocyte accumulations, thrombotic vessel occlusions, blood-brain barrier (BBB) dysfunction and microbleeds. Results We found a significant increase in the number of erythrocyte stases in 6 months old Ldlr-/- mice compared to all other groups (P < 0.05). Ldlr-/- animals aged 12 months showed the highest number of thrombotic occlusions while in WT animals hardly any occlusions could be observed (P < 0.001). Compared to WT mice, Ldlr-/- mice did not display significant gray matter BBB breakdown. Microhemorrhages were observed in one Ldlr-/- mouse that was 6 months old. Results did not differ when considering subcortical and cortical regions. Conclusions In Ldlr-/- mice, hypercholesterolemia is related to a thrombotic CSVD phenotype, which is different from hypertension-related CSVD that associates with a hemorrhagic CSVD phenotype. Our data demonstrate a relationship between hypercholesterolemia and the development of CSVD. Ldlr-/- mice appear to be an adequate animal model for research into CSVD.

Vascular basement membrane alterations and β-amyloid accumulations in an animal model of cerebral small vessel disease

Non-amyloid cerebral small vessel disease (CSVD) and cerebral amyloid angiopathy (CAA) may be interrelated through the damaged basement membranes (BMs) and extracellular matrix changes of small vessels, resulting in a failure of β-amyloid (Aβ) transport and degradation. We analyzed BM changes and the pattern of deposition of Aβ in the walls of blood vessels in spontaneously hypertensive stroke-prone rats (SHRSP), a non-transgenic CSVD model. In 45 SHRSP and 38 Wistar rats aged 18 to 32 weeks: (i) the percentage area immunostained for vascular collagen IV and laminin was quantified; (ii) the capillary BM thickness as well as endothelial and pericyte pathological changes were analysed using transmission electron microscopy (TEM); and (iii) the presence of vascular Aβ was assessed. Compared with controls, SHRSP exhibited a significantly higher percentage area immunostained with collagen IV in the striatum and thalamus. SHRSP also revealed an age-dependent increase of the capillary BM thickness and of endothelial vacuoles (caveolae) within subcortical regions. Endogenous Aβ deposits in the walls of small blood vessels were observed in the cortex (with the highest incidence found within fronto-parietal areas), striatum, thalamus and hippocampus. Vascular β-amyloid accumulations were frequently detected at sites of small vessel wall damage. Our data demonstrate changes in the expression of collagen IV and of the ultrastructure of BMs in the small vessels of SHRSP. Alterations are accompanied by vascular deposits of endogenous Aβ. Impaired β-amyloid clearance along perivascular and endothelial pathways and failure of extracellular Aβ degradation may be the key mechanisms connecting non-amyloid CSVD and CAA.

Impact of N-Acetylcysteine on cerebral amyloid-β plaques and kidney damage in spontaneously hypertensive stroke-prone rats.

BACKGROUND Cerebral small vessel disease (CSVD) in spontaneously hypertensive stroke prone rats (SHRSP) is accompanied by parenchymal amyloid-β (Aβ) deposition in the brain and by hypertensive nephropathy with tubulointerstitial damage. N-acetylcysteine (NAC) promotes blood-brain barrier (BBB) breakdown in SHRSP and may thus accelerate the failure of vascular and perivascular clearance of Aβ. OBJECTIVE In this study, we test the hypothesis that treatment with NAC increases the cerebral Aβ load and improves renal damage in the SHRSP model. METHODS A total of 46 SHRSP (ages 18-44 weeks) were treated daily with NAC (12 mg/kg body weight) and 74 no-treated age-matched SHRSP served as controls. The prevalence of parenchymal Aβ load, IgG positive small vessels, and small perivascular bleeds was assessed in different brain regions. Tubulointerstitial kidney damage was assessed through a) the presence of erythrocytes in peritubular capillaries and b) tubular protein cylinders. RESULTS SHRSP treated with NAC had an age-dependent increase of BBB breakdown (assessed by the presence of IgG positive small vessels) and small perivascular bleeds, mainly in the cortex. NAC significantly increased the Aβ plaque load in the cortex while the number of parenchymal amyloid deposits in the remaining brain areas including basal ganglia, hippocampus, thalamus, and corpus callosum were unchanged. There were no significant treatment effects on tubulointerstitial kidney damage. CONCLUSION The impact of NAC on cerebral cortical plaque load increase may result from the vascular pathology of SHRSP that accompanies BBB breakdown, leading to the failure of amyloid clearance mechanisms. It remains to be seen whether in humans chronic NAC intake may increase amyloid load in the aging human brain and dementia.

Common impact of chronic kidney disease and brain microhemorrhages on cerebral Aβ pathology in SHRSP.

While chronic kidney disease seems to be an independent risk factor for cognitive decline, its impact on cerebral amyloid‐β (Aβ) depositions, one hallmark of Alzheimers Disease (AD) pathology, has not been investigated.

Do basophile structures as age dependent phenomenon indicate small vessel wall damage

Here we demonstrate basophile structures located in the arteriolar wall and being associated with a plasma-protein-leakage. We assume, that the structures indicate blood-brain-barrier-disturbances and degenerative small vessel wall alterations.