Marco Foddis

Metabotropic P2Y1 receptor signalling mediates astrocytic hyperactivity in vivo in an Alzheimer’s disease mouse model

Astrocytic network alterations have been reported in Alzheimers disease (AD), but the underlying pathways have remained undefined. Here we measure astrocytic calcium, cerebral blood flow and amyloid-β plaques in vivo in a mouse model of AD using multiphoton microscopy. We find that astrocytic hyperactivity, consisting of single-cell transients and calcium waves, is most pronounced in reactive astrogliosis around plaques and is sometimes associated with local blood flow changes. We show that astroglial hyperactivity is reduced after P2 purinoreceptor blockade or nucleotide release through connexin hemichannels, but is augmented by increasing cortical ADP concentration. P2X receptor blockade has no effect, but inhibition of P2Y1 receptors, which are strongly expressed by reactive astrocytes surrounding plaques, completely normalizes astrocytic hyperactivity. Our data suggest that astroglial network dysfunction is mediated by purinergic signalling in reactive astrocytes, and that intervention aimed at P2Y1 receptors or hemichannel-mediated nucleotide release may help ameliorate network dysfunction in AD.

Neurovascular coupling in rat brain operates independent of hemoglobin deoxygenation

Recently, a universal, simple, and fail-safe mechanism has been proposed by which cerebral blood flow (CBF) might be coupled to oxygen metabolism during neuronal activation without the need for any tissue-based mechanism. According to this concept, vasodilation occurs by local erythrocytic release of nitric oxide or ATP wherever and whenever hemoglobin is deoxygenated, directly matching oxygen demand and supply in every tissue. For neurovascular coupling in the brain, we present experimental evidence challenging this view by applying an experimental regime operating without deoxy-hemoglobin. Hyperbaric hyperoxygenation (HBO) allowed us to prevent hemoglobin deoxygenation, as the oxygen that was physically dissolved in the tissue was sufficient to support oxidative metabolism. Regional CBF and regional cerebral blood oxygenation were measured using a cranial window preparation in anesthetized rats. Hemodynamic and neuronal responses to electrical forepaw stimulation or cortical spreading depression (CSD) were analyzed under normobaric normoxia and during HBO up to 4 ATA (standard atmospheres absolute). Inconsistent with the proposed mechanism, during HBO, CBF responses to functional activation or CSD were unchanged. Our results show that activation-induced CBF regulation in the brain does not operate through the release of vasoactive mediators on hemoglobin deoxygenation or through a tissue-based oxygen-sensing mechanism.

Vascular change and opposing effects of the angiotensin type 2 receptor in a mouse model of vascular cognitive impairment

Our aims were to assess the spatiotemporal development of brain pathology in a mouse model of chronic hypoperfusion using magnetic resonance imaging (MRI), and to test whether the renin-angiotensin system (RAS) can offer therapeutic benefit. For the first time, different patterns of cerebral blood flow alterations were observed in hypoperfused mice that ranged from an immediate and dramatic to a delayed decrease in cerebral perfusion. Diffusion tensor imaging revealed increases in several quantitative parameters in different brain regions that are indicative of white-matter degeneration; this began around 3 weeks after induction of hypoperfusion. While this model may be more variable than previously reported, neuroimaging tools represent a promising way to identify surrogate markers of pathology. Vascular remodelling was observed in hypoperfused mice, particularly in the anterior part of the Circle of Willis. While the angiotensin II receptor type 2 agonist, Compound 21 (C21), did not influence this response, it did promote expansion of the basilar artery in microcoil animals. Furthermore, C21-treated animals exhibited increased brain lymphocyte infiltration, and importantly, C21 had opposing effects on spatial reference memory in hypoperfused and sham mice. These results suggest that the RAS may have a role in vascular cognitive impairment.

Elevating intracranial pressure reverses the decrease in deoxygenated hemoglobin and abolishes the post-stimulus overshoot upon somatosensory activation in rats

BOLD fMRI localizes activated brain areas by measuring decreases of deoxygenated hemoglobin (deoxy-Hb) caused by neurovascular coupling. To date, it is unclear whether intracranial pressure (ICP) modifies deoxy-Hb signaling for brain mapping. In addition, ICP elevation can test whether the BOLD post-stimulus undershoot, a transient hypo-oxygenation following functional activation, is due to vascular compliance rather than elevated cerebral metabolic rate of oxygen (CMRO(2)). We addressed these questions by studying the effect of ICP elevation on neurovascular coupling. In anesthetized rats, a cranial window was implanted over the somatosensory cortex. Using laser Doppler flowmetry and optical spectroscopy, changes in cerebral blood flow (CBF), cerebral blood volume (CBV) and deoxy-Hb were measured during electrical forepaw stimulation. Neuronal activity was monitored by somatosensory evoked potentials. ICP was elevated by subarachnoideal and intracisternal infusion of artificial cerebrospinal fluid. ICP elevation did not abrogate neurovascular coupling. However, the concomitant deoxy-Hb decrease was reduced (ICP=14mmHg) and reversed (ICP=28mmHg). Therefore, the validity of BOLD fMRI has to be questioned during increased ICP. Moreover, the amplitude of the deoxy-Hb post-stimulus overshoot was reduced with ICP elevation. CMRO(2) was not elevated during the post-stimulus response. Therefore, these data provide experimental evidence that the BOLD post-stimulus undershoot is a passive vascular phenomenon.

A role of the sodium pump in spreading ischemia in rats

In rats, spreading depolarization induces vasodilation/hyperemia in naïve tissue but the inverse response when artificial cerebrospinal fluid is topically applied to the brain containing (a) a nitric oxide–lowering agent and (b) elevated K+. The inverse response is characterized by severe vasoconstriction/ischemia. The perfusion deficit runs together with the depolarization in the tissue (=spreading ischemia). Here, we found in male Wistar rats that pre-treatment with artificial cerebrospinal fluid containing elevated K+ in vivo led to a selective decline in α2/α3 Na+/K+-ATPase activity, determined spectrophotometrically ex vivo. Moreover, spreading ischemia, recorded with laser-Doppler flowmetry and electrocorticography, resulted from artificial cerebrospinal fluid containing a nitric oxide–lowering agent in combination with the Na+/K+-ATPase inhibitor ouabain at a concentration selectively inhibiting α2/α3 activity. Decline in α2/α3 activity results in increased Ca2+ uptake by internal stores of astrocytes, vascular myocytes, and pericytes since Ca2+ outflux via plasmalemmal Na+/Ca2+-exchanger declines. Augmented Ca2+ mobilization from internal stores during spreading depolarization might enhance vasoconstriction, thus, contributing to spreading ischemia. Accordingly, spreading ischemia was significantly shortened when intracellular Ca2+ stores were emptied by pre-treatment with thapsigargin, an inhibitor of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA). These findings might have relevance for clinical conditions, in which spreading ischemia occurs such as delayed cerebral ischemia after subarachnoid hemorrhage.

Interaction of ARC and Daxx: A Novel Endogenous Target to Preserve Motor Function and Cell Loss after Focal Brain Ischemia in Mice

The aim of this study was to explore the signaling and neuroprotective effect of transactivator of transcription (TAT) protein transduction of the apoptosis repressor with CARD (ARC) in in vitro and in vivo models of cerebral ischemia in mice. In mice, transient focal cerebral ischemia reduced endogenous ARC protein in neurons in the ischemic striatum at early reperfusion time points, and in primary neuronal cultures, RNA interference resulted in greater neuronal susceptibility to oxygen glucose deprivation (OGD). TAT.ARC protein delivery led to a dose-dependent better survival after OGD. Infarct sizes 72 h after 60 min middle cerebral artery occlusion (MCAo) were on average 30 ± 8% (mean ± SD; p = 0.005; T2-weighted MRI) smaller in TAT.ARC-treated mice (1 μg intraventricularly during MCAo) compared with controls. TAT.ARC-treated mice showed better performance in the pole test compared with TAT.β-Gal-treated controls. Importantly, post-stroke treatment (3 h after MCAo) was still effective in affording reduced lesion volume by 20 ± 7% (mean ± SD; p < 0.05) and better functional outcome compared with controls. Delayed treatment in mice subjected to 30 min MCAo led to sustained neuroprotection and functional behavior benefits for at least 28 d. Functionally, TAT.ARC treatment inhibited DAXX–ASK1–JNK signaling in the ischemic brain. ARC interacts with DAXX in a CARD-dependent manner to block DAXX trafficking and ASK1–JNK activation. Our work identifies for the first time ARC–DAXX binding to block ASK1–JNK activation as an ARC-specific endogenous mechanism that interferes with neuronal cell death and ischemic brain injury. Delayed delivery of TAT.ARC may present a promising target for stroke therapy. SIGNIFICANCE STATEMENT Up to now, the only successful pharmacological target of human ischemic stroke is thrombolysis. Neuroprotective pharmacological strategies are needed to accompany therapies aiming to achieve reperfusion. We describe that apoptosis repressor with CARD (ARC) interacts and inhibits DAXX and proximal signals of cell death. In a murine stroke model mimicking human malignant infarction in the territory of the middle cerebral artery, TAT.ARC salvages brain tissue when given during occlusion or 3 h delayed with sustained functional benefits (28 d). This is a promising novel therapeutic approach because it appears to be effective in a model producing severe injury by interfering with an array of proximal signals and effectors of the ischemic cascade, upstream of JNK, caspases, and BIM and BAX activation.

Neuroimaging Biomarkers Predict Brain Structural Connectivity Change in a Mouse Model of Vascular Cognitive Impairment

Background and Purpose— Chronic hypoperfusion in the mouse brain has been suggested to mimic aspects of vascular cognitive impairment, such as white matter damage. Although this model has attracted attention, our group has struggled to generate a reliable cognitive and pathological phenotype. This study aimed to identify neuroimaging biomarkers of brain pathology in aged, more severely hypoperfused mice. Methods— We used magnetic resonance imaging to characterize brain degeneration in mice hypoperfused by refining the surgical procedure to use the smallest reported diameter microcoils (160 &mgr;m). Results— Acute cerebral blood flow decreases were observed in the hypoperfused group that recovered over 1 month and coincided with arterial remodeling. Increasing hypoperfusion resulted in a reduction in spatial learning abilities in the water maze that has not been previously reported. We were unable to observe severe white matter damage with histology, but a novel approach to analyze diffusion tensor imaging data, graph theory, revealed substantial reorganization of the hypoperfused brain network. A logistic regression model from the data revealed that 3 network parameters were particularly efficient at predicting group membership (global and local efficiency and degrees), and clustering coefficient was correlated with performance in the water maze. Conclusions— Overall, these findings suggest that, despite the autoregulatory abilities of the mouse brain to compensate for a sudden decrease in blood flow, there is evidence of change in the brain networks that can be used as neuroimaging biomarkers to predict outcome.

Increased homocysteine levels impair reference memory and reduce cortical levels of acetylcholine in a mouse model of vascular cognitive impairment

HighlightsMthfr+/− mice fed FADD with microcoils (VCI) had impaired reference memory.All FADD mice had elevated levels of plasma homocysteine.Arterial remodeling was present in Mthfr+/− microcoil mice.Acetylcholine was reduced in FADD microcoil mice. ABSTRACT Folates are B‐vitamins that are vital for normal brain function. Deficiencies in folates either genetic (methylenetetrahydrofolate reductase, MTHFR) or dietary intake of folic acid result in elevated levels of homocysteine. Clinical studies have shown that elevated levels of homocysteine (Hcy) may be associated with the development of dementia, however this link remains unclear. The purpose of this study was to evaluate the impact of increased Hcy levels on a mouse model of vascular cognitive impairment (VCI) produced by chronic hypoperfusion. Male and female Mthfr+/+ and Mthfr+/− mice were placed on either control (CD) or folic acid deficient (FADD) diets after which all animals underwent microcoil implantation around each common carotid artery or a sham procedure. Post‐operatively animals were tested on the Morris water maze (MWM), y‐maze, and rotarod. Animals had no motor impairments on the rotarod, y‐maze, and could learn the location of the platform on the MWM. However, on day 8 of testing of MWM testing during the probe trial, Mthfr+/− FADD microcoil mice spent significantly less time in the target quadrant when compared to Mthfr+/− CD sham mice, suggesting impaired reference memory. All FADD mice had elevated levels of plasma homocysteine. MRI analysis revealed arterial remodeling was present in Mthfr+/− microcoil mice not Mthfr+/+ mice. Acetylcholine and related metabolites were reduced in cortical tissue because of microcoil implantation and elevated levels of homocysteine. Deficiencies in folate metabolism resulting in increased Hcy levels yield a metabolic profile that increases susceptibility to neurodegeneration in a mouse model of VCI.

Atlas registration for edema-corrected MRI lesion volume in mouse stroke models:

Lesion volume measurements with magnetic resonance imaging are widely used to assess outcome in rodent models of stroke. In this study, we improved a mathematical framework to correct lesion size for edema which is based on manual delineation of the lesion and hemispheres. Furthermore, a novel MATLAB toolbox to register mouse brain MR images to the Allen brain atlas is presented. Its capability to calculate edema-corrected lesion size was compared to the manual approach. Automated image registration performed equally well in in a mouse middle cerebral artery occlusion model (Pearson r = 0.976, p = 2.265e-11). Information encapsulated in the registration was used to generate maps of edema induced tissue volume changes. These showed discrepancies to simplified tissue models underlying the manual approach. The presented techniques provide biologically more meaningful, voxel-wise biomarkers of vasogenic edema after stroke.

Correction: Individual and temporal variability of the retina after chronic bilateral common carotid artery occlusion (BCCAO)

Animal models of disease are an indispensable element in our quest to understand pathophysiology and develop novel therapies. Ex vivo studies have severe limitations, in particular their inability to study individual disease progression over time. In this respect, non-invasive in vivo technologies offer multiple advantages. We here used bilateral common carotid artery occlusion (BCCAO) in mice, an established model for ischemic retinopathy, and performed a multimodal in vivo and ex vivo follow-up. We used scanning laser ophthalmoscopy (SLO), ocular coherence tomography (OCT) and electroretinography (ERG) over 6 weeks followed by ex vivo analyses. BCCAO leads to vascular remodeling with thickening of veins starting at 4 weeks, loss of photoreceptor synapses with concomitant reduced b-waves in the ERG and thinning of the retina. Mononuclear phagocytes showed fluctuation of activity over time. There was large inter-individual variation in the severity of neuronal degeneration and cellular inflammatory responses. Ex vivo analysis confirmed these variable features of vascular remodeling, neurodegeneration and inflammation. In summary, we conclude that multimodal follow-up and subgroup analysis of retinal changes in BCCAO further calls into question the use of ex vivo studies with distinct single end-points. We propose that our approach can foster the understanding of retinal disease as well as the clinical translation of emerging therapeutic strategies.