Perivascular space dilation is associated with vascular amyloid-ß accumulation in the overlying cortex.

Perivascular spaces (PVS) are compartments surrounding cerebral blood vessels that become visible on MRI when enlarged. Enlarged PVS (EPVS) are commonly seen in patients with cerebral small vessel disease (CSVD) and have been suggested to reflect dysfunctional perivascular clearance of soluble waste products from the brain. In this study, we investigated histopathological correlates of EPVS and how they relate to vascular amyloid-ß (Aß) in cerebral amyloid angiopathy (CAA), a form of CSVD that commonly co-exists with Alzheimer's disease (AD) pathology. We used ex vivo MRI, semi-automatic segmentation and validated deep-learning-based models to quantify EPVS and associated histopathological abnormalities. Severity of MRI-visible PVS during life was significantly associated with severity of MRI-visible PVS on ex vivo MRI in formalin fixed intact hemispheres and corresponded with PVS enlargement on histopathology in the same areas. EPVS were located mainly around the white matter portion of perforating cortical arterioles and their burden was associated with CAA severity in the overlying cortex. Furthermore, we observed markedly reduced smooth muscle cells and increased vascular Aß accumulation, extending into the WM, in individually affected vessels with an EPVS. Overall, these findings are consistent with the notion that EPVS reflect impaired outward flow along arterioles and have implications for our understanding of perivascular clearance mechanisms, which play an important role in the pathophysiology of CAA and AD.

Influence of different isoflurane anesthesia protocols on murine cerebral hemodynamics measured with pseudo-continuous arterial spin labeling.

Arterial spin labeling (ASL)-MRI can noninvasively map cerebral blood flow (CBF) and cerebrovascular reactivity (CVR), potential biomarkers of cognitive impairment and dementia. Mouse models of disease are frequently used in translational MRI studies, which are commonly performed under anesthesia. Understanding the influence of the specific anesthesia protocol used on the measured parameters is important for accurate interpretation of hemodynamic studies with mice. Isoflurane is a frequently used anesthetic with vasodilative properties. Here, the influence of three distinct isoflurane protocols was studied with pseudo-continuous ASL in two different mouse strains. The first protocol was a free-breathing set-up with medium concentrations, the second a free-breathing set-up with low induction and maintenance concentrations, and the third a set-up with medium concentrations and mechanical ventilation. A protocol with the vasoconstrictive anesthetic medetomidine was used as a comparison. As expected, medium isoflurane anesthesia resulted in significantly higher CBF and lower CVR values than medetomidine (median whole-brain CBF of 157.7 vs 84.4 mL/100 g/min and CVR of 0.54 vs 51.7% in C57BL/6 J mice). The other two isoflurane protocols lowered the CBF and increased the CVR values compared with medium isoflurane anesthesia, without obvious differences between them (median whole-brain CBF of 138.9 vs 131.7 mL/100 g/min and CVR of 10.0 vs 9.6%, in C57BL/6 J mice). Furthermore, CVR was shown to be dependent on baseline CBF, regardless of the anesthesia protocol used.

Transit time mapping in the mouse brain using time-encoded pCASL.

The cerebral blood flow (CBF) is a potential biomarker for neurological disease. However, the arterial transit time (ATT) of the labeled blood is known to potentially affect CBF quantification. Furthermore, ATT could be an interesting biomarker in itself, as it may reflect underlying macro- and microvascular pathologies. Currently, no optimized magnetic resonance imaging (MRI) sequence exists to measure ATT in mice. Recently, time-encoded labeling schemes have been implemented in rats and humans, enabling ATT mapping with higher signal-to-noise ratio (SNR) and shorter scan time than multi-delay arterial spin labeling (ASL). In this study, we show that time-encoded pseudo-continuous arterial spin labeling (te-pCASL) also enables transit time measurements in mice. As an optimal design that takes the fast blood flow in mice into account, time encoding with 11 sub-boli of 50 ms is proposed to accurately probe the inflow of labeled blood. For perfusion imaging, a separate, traditional pCASL scan was employed. From the six studied brain regions, the hippocampus showed the shortest ATT (169 ± 11 ms) and the auditory/visual cortex showed the longest (284 ± 16 ms). Furthermore, ATT was found to be preserved in old wild-type mice. In a mouse with an induced carotid artery occlusion, prolongation of ATT was shown. In conclusion, this study shows the successful implementation of te-pCASL in mice, making it possible, for the first time, to measure ATT in mice in a time-efficient manner.

Loss of spontaneous vasomotion precedes impaired cerebrovascular reactivity and microbleeds in a mouse model of cerebral amyloid angiopathy.

Background: Cerebral amyloid angiopathy (CAA) is a cerebral small vessel disease in which amyloid-ß accumulates in vessel walls. CAA is a leading cause of symptomatic lobar intracerebral hemorrhage and an important contributor to age-related cognitive decline. Recent work has suggested that vascular dysfunction may precede symptomatic stages of CAA, and that spontaneous slow oscillations in arteriolar diameter (termed vasomotion), important for amyloid-ß clearance, may be impaired in CAA. Methods: To systematically study the progression of vascular dysfunction in CAA, we used the APP23 mouse model of amyloidosis, which is known to develop spontaneous cerebral microbleeds mimicking human CAA. Using in vivo 2-photon microscopy, we longitudinally imaged unanesthetized APP23 transgenic mice and wildtype littermates from 7 to 14 months of age, tracking amyloid-ß accumulation and vasomotion in individual pial arterioles over time. MRI was used in separate groups of 12-, 18-, and 24-month-old APP23 transgenic mice and wildtype littermates to detect microbleeds and to assess cerebral blood flow and cerebrovascular reactivity with pseudo-continuous arterial spin labeling. Results: We observed a significant decline in vasomotion with age in APP23 mice, while vasomotion remained unchanged in wildtype mice with age. This decline corresponded in timing to initial vascular amyloid-ß deposition (∼8-10 months of age), although was more strongly correlated with age than with vascular amyloid-ß burden in individual arterioles. Declines in vasomotion preceded the development of MRI-visible microbleeds and the loss of smooth muscle actin in arterioles, both of which were observed in APP23 mice by 18 months of age. Additionally, evoked cerebrovascular reactivity was intact in APP23 mice at 12 months of age, but significantly lower in APP23 mice by 24 months of age. Conclusions: Our findings suggest that a decline in spontaneous vasomotion is an early, potentially pre-symptomatic, manifestation of CAA and vascular dysfunction, and a possible future treatment target.

Spontaneous vasomotion propagates along pial arterioles in the awake mouse brain like stimulus-evoked vascular reactivity.

Sensory stimulation evokes a local, vasodilation-mediated blood flow increase to the activated brain region, which is referred to as functional hyperemia. Spontaneous vasomotion is a change in arteriolar diameter that occurs without sensory stimulation, at low frequency (∼0.1 Hz). These vessel diameter changes are a driving force for perivascular soluble waste clearance, the failure of which has been implicated in neurodegenerative disease. Stimulus-evoked vascular reactivity is known to propagate along penetrating arterioles to pial arterioles, but it is unclear whether spontaneous vasomotion propagates similarly. We therefore imaged both stimulus-evoked and spontaneous changes in pial arteriole diameter in awake, head-fixed mice with 2-photon microscopy. By cross-correlating different regions of interest (ROIs) along the length of imaged arterioles, we assessed vasomotion propagation. We found that both during rest and during visual stimulation, one-third of the arterioles showed significant propagation (i.e., a wave), with a median (interquartile range) wave speed of 405 (323) µm/s at rest and 345 (177) µm/s during stimulation. In a second group of mice, with GCaMP expression in their vascular smooth muscle cells, we also found spontaneous propagation of calcium signaling along pial arterioles. In summary, we demonstrate that spontaneous vasomotion propagates along pial arterioles like stimulus-evoked vascular reactivity.

Reduced excitatory neuron activity and interneuron-type-specific deficits in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is characterized by progressive memory loss and cognitive decline. These impairments correlate with early alterations in neuronal network activity in AD patients. Disruptions in the activity of individual neurons have been reported in mouse models of amyloidosis. However, the impact of amyloid pathology on the spontaneous activity of distinct neuronal types remains unexplored in vivo. Here we use in vivo calcium imaging with multiphoton microscopy to monitor and compare the activity of excitatory and two types of inhibitory interneurons in the cortices of APP/PS1 and control mice under isoflurane anesthesia. We also determine the relationship between amyloid accumulation and the deficits in spontaneous activity in APP/PS1 mice. We show that somatostatin-expressing (SOM) interneurons are hyperactive, while parvalbumin-expressing interneurons are hypoactive in APP/PS1 mice. Only SOM interneuron hyperactivity correlated with proximity to amyloid plaque. These inhibitory deficits were accompanied by decreased excitatory neuron activity in APP/PS1 mice. Our study identifies cell-specific neuronal firing deficits in APP/PS1 mice driven by amyloid pathology. These findings highlight the importance of addressing the complexity of neuron-specific deficits to ameliorate circuit dysfunction in Alzheimer's disease.

Cerebral blood flow and cerebrovascular reactivity are preserved in a mouse model of cerebral microvascular amyloidosis.

Impaired cerebrovascular function is an early biomarker for cerebral amyloid angiopathy (CAA), a neurovascular disease characterized by amyloid-ß accumulation in the cerebral vasculature, leading to stroke and dementia. The transgenic Swedish Dutch Iowa (Tg-SwDI) mouse model develops cerebral microvascular amyloid-ß deposits, but whether this leads to similar functional impairments is incompletely understood. We assessed cerebrovascular function longitudinally in Tg-SwDI mice with arterial spin labeling (ASL)-magnetic resonance imaging (MRI) and laser Doppler flowmetry (LDF) over the course of amyloid-ß deposition. Unexpectedly, Tg-SwDI mice showed similar baseline perfusion and cerebrovascular reactivity estimates as age-matched wild-type control mice, irrespective of modality (ASL or LDF) or anesthesia (isoflurane or urethane and α-chloralose). Hemodynamic changes were, however, observed as an effect of age and anesthesia. Our findings contradict earlier results obtained in the same model and question to what extent microvascular amyloidosis as seen in Tg-SwDI mice is representative of cerebrovascular dysfunction observed in CAA patients.

Progression and Classification of Granular Osmiophilic Material (GOM) Deposits in Functionally Characterized Human NOTCH3 Transgenic Mice.

CADASIL is a NOTCH3-associated cerebral small vessel disease. A pathological ultrastructural disease hallmark is the presence of NOTCH3-protein containing deposits called granular osmiophilic material (GOM), in small arteries. How these GOM deposits develop over time and what their role is in disease progression is largely unknown. Here, we studied the progression of GOM deposits in humanized transgenic NOTCH3Arg182Cys mice, compared them to GOM deposits in patient material, and determined whether GOM deposits in mice are associated with a functional CADASIL phenotype. We found that GOM deposits are not static, but rather progress in ageing mice, both in terms of size and aspect. We devised a GOM classification system, reflecting size, morphology and electron density. Six-month-old mice showed mostly early stage GOM, whereas older mice and patient vessels showed predominantly advanced stage GOM, but also early stage GOM. Mutant mice did not develop the most severe GOM stage seen in patient material. This absence of end-stage GOM in mice was associated with an overall lack of histological vascular pathology, which may explain why the mice did not reveal functional deficits in cerebral blood flow, cognition and motor function. Taken together, our data indicate that GOM progress over time, and that new GOM deposits are continuously being formed. The GOM staging system we introduce here allows for uniform GOM deposit classification in future mouse and human studies, which may lead to more insight into a potential association between GOM stage and CADASIL disease severity, and the role of GOM in disease progression.

TGFß pathway deregulation and abnormal phospho-SMAD2/3 staining in hereditary cerebral hemorrhage with amyloidosis-Dutch type.

Hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D) is an early onset hereditary form of cerebral amyloid angiopathy (CAA) pathology, caused by the E22Q mutation in the amyloid ß (Aß) peptide. Transforming growth factor ß1 (TGFß1) is a key player in vascular fibrosis and in the formation of angiopathic vessels in transgenic mice. Therefore, we investigated whether the TGFß pathway is involved in HCHWA-D pathogenesis in human postmortem brain tissue from frontal and occipital lobes. Components of the TGFß pathway were analyzed with quantitative RT-PCR. TGFß1 and TGFß Receptor 2 (TGFBR2) gene expression levels were significantly increased in HCHWA-D in comparison to the controls, in both frontal and occipital lobes. TGFß-induced pro-fibrotic target genes were also upregulated. We further assessed pathway activation by detecting phospho-SMAD2/3 (pSMAD2/3), a direct TGFß down-stream signaling mediator, using immunohistochemistry. We found abnormal pSMAD2/3 granular deposits specifically on HCHWA-D angiopathic frontal and occipital vessels. We graded pSMAD2/3 accumulation in angiopathic vessels and found a positive correlation with the CAA load independent of the brain area. We also observed pSMAD2/3 granules in a halo surrounding occipital vessels, which was specific for HCHWA-D. The result of this study indicates an upregulation of TGFß1 in HCHWA-D, as was found previously in AD with CAA pathology. We discuss the possible origins and implications of the TGFß pathway deregulation in the microvasculature in HCHWA-D. These findings identify the TGFß pathway as a potential biomarker of disease progression and a possible target of therapeutic intervention in HCHWA-D.

No evidence for the presence of neuronal surface autoantibodies in plasma of patients with schizophrenia.

The immune system has been implicated in the etiology of schizophrenia. Autoimmunity by antibodies against neuronal cell surface antigens has been proposed as one of the pathological mechanisms. We examined plasma samples of 104 patients diagnosed with schizophrenia for the presence of autoantibodies against neuronal cell surface antigens using cultured hippocampal neurons and transfected HeLa cells. None of the samples tested positive for the presence of these autoantibodies. Based on our results it seems unlikely that autoantibodies against neuronal cell surface antigens play a role in the pathogenesis of schizophrenia, although further studies using cerebrospinal fluid are needed.