Magnetic Resonance Q Mapping Reveals a Decrease in Microvessel Density in the arcAβ Mouse Model of Cerebral Amyloidosis

Alterations in density and morphology of the cerebral microvasculature have been reported to occur in Alzheimer's disease patients and animal models of the disease. In this study we compared magnetic resonance imaging (MRI) techniques for their utility to detect age-dependent changes of the cerebral vasculature in the arcAβ mouse model of cerebral amyloidosis. Dynamic susceptibility contrast (DSC)-MRI was performed by tracking the passage of a superparamagnetic iron oxide nanoparticle in the brain with dynamic gradient echo planar imaging (EPI). From this measurements relative cerebral blood volume [rCBV(DSC)] and relative cerebral blood flow (rCBF) were estimated. For the same animal maps of the relaxation shift index Q were computed from high resolution gradient echo and spin echo data that were acquired before and after superparamagnetic iron oxide (SPIO) nanoparticle injection. Q-values were used to derive estimates of microvessel density. The change in the relaxation rates ΔR∗2 obtained from pre- and post-contrast gradient echo data was used for the alternative determination of rCBV [rCBV(ΔR∗2)]. Linear mixed effects modeling found no significant association between rCBV(DSC), rCBV(ΔR∗2), rCBF, and Q with genotype in 13-month old mice [compared to age- matched non-transgenic littermates (NTLs)] for any of the evaluated brain regions. In 24-month old mice there was a significant association for rCBV(DSC) with genotype in the cerebral cortex, and for rCBV(ΔR∗2) in the cerebral cortex and cerebellum. For rCBF there was a significant association in the cerebellum but not in other brain regions. Q-values in the olfactory bulb, cerebral cortex, striatum, hippocampus, and cerebellum in 24-month old mice were significantly associated with genotype. In those regions Q-values were reduced between 11 and 26% in arcAβ mice compared to age-matched NTLs. Vessel staining with CD31 immunohistochemistry confirmed a reduction of microvessel density in the old arcAβ mice. We further demonstrated a region- specific association between parenchymal and vascular deposition of β-amyloid and decreased vascular density, without a correlation with the amount of Aβ deposition. We found that Q mapping was more suitable than the hemodynamic read-outs to detect amyloid-related degeneration of the cerebral microvasculature

Pathophysiological Interference with Neurovascular Coupling – When Imaging Based on Hemoglobin Might Go Blind

Assessing neuronal activity by non-invasive functional brain imaging techniques which are based on the hemodynamic response depends totally on the physiological cascade of metabolism and blood flow. At present, functional brain imaging with near infrared spectroscopy (NIRS) or BOLD-fMRI is widely used in cognitive neuroscience in healthy subjects where neurovascular coupling and cerebrovascular reactivity can be assumed to be intact. Local activation studies as well as studies investigating functional connectivity between brain regions of the resting brain provide a rapidly increasing body of knowledge on brain function in humans and animals. Furthermore, functional NIRS and MRI techniques are increasingly being used in patients with severe brain diseases and this use might gain more and more importance for establishing their use in the clinical routine. However, more and more experimental evidence shows that changes in baseline physiological parameters, pharmacological interventions, or disease-related vascular changes may significantly alter the normal response of blood flow and blood oxygenation and thus may lead to misinterpretation of neuronal activity. In this article we present examples of recent experimental findings on pathophysiological changes of neurovascular coupling parameters in animals and discuss their potential implications for functional imaging based on hemodynamic signals such as fNIRS or BOLD-fMRI. To enable correct interpretation of neuronal activity by vascular signals, future research needs to deepen our understanding of the basic mechanisms of neurovascular coupling and the specific characteristics of disturbed neurovascular coupling in the diseased brain

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

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

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 levelsof homocysteine. Clinical studies have shown that elevated levels of homocysteine (Hcy) may be associ-ated with the development of dementia, however this link remains unclear. The purpose of this study wasto 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 eithercontrol (CD) or folic acid deficient (FADD) diets after which all animals underwent microcoil implantationaround each common carotid artery or a sham procedure. Post-operatively animals were tested on theMorris 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 MWMtesting during the probe trial, Mthfr+/−FADD microcoil mice spent significantly less time in the targetquadrant when compared to Mthfr+/−CD sham mice, suggesting impaired reference memory. All FADDmice 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

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 endogenousARCprotein 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 (1ug 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

Elevated levels of plasma homocysteine, deficiencies in dietary folic acid and uracil–DNA glycosylase impair learning in a mouse model of vascular cognitive impairment

Dietary deficiencies in folic acid result in elevated levels of plasma homocysteine, which has been associated with the development of dementia and other neurodegenerative disorders. Previously, we have shown that elevated levels of plasma homocysteine in mice deficient for a DNA repair enzyme, uracil–DNA glycosylase (UNG), result in neurodegeneration. The goal of this study was to evaluate how deficiencies in folic acid and UNG along with elevated levels of homocysteine affect vascular cognitive impairment, via chronic hypoperfusion in an animal model. Ung+/+ and Ung−/− mice were placed on either control (CD) or folic acid deficient (FADD) diets. Six weeks later, the mice either underwent implantation of microcoils around both common carotid arteries. Post-operatively, behavioral tests began at 3-weeks, angiography was measured after 5-weeks using MRI to assess vasculature and at completion of study plasma and brain tissue was collected for analysis. Learning impairments in the Morris water maze (MWM) were observed only in hypoperfused Ung−/− FADD mice and these mice had significantly higher plasma homocysteine concentrations. Interestingly, Ung+/+ FADD produced significant remodeling of the basilar artery and arterial vasculature. Increased expression of GFAP was observed in the dentate gyrus of Ung−/− hypoperfused and FADD sham mice. Chronic hypoperfusion resulted in increased cortical MMP-9 protein levels of FADD hypoperfused mice regardless of genotypes. These results suggest that elevated levels of homocysteine only, as a result of dietary folic acid deficiency, don’t lead to memory impairments and neurobiochemical changes. Rather a combination of either chronic hypoperfusion or UNG deficiency is required

Tracking CNS and systemic sources of oxidative stress during the course of chronic neuroinflammation

The functional dynamics and cellular sources of oxidative stress are central to understanding MS pathogenesis but remain elusive, due to the lack of appropriate detection methods. Here we employ NAD(P)H fluorescence lifetime imaging to detect functional NADPH oxidases (NOX enzymes) in vivo to identify inflammatory monocytes, activated microglia, and astrocytes expressing NOX1 as major cellular sources of oxidative stress in the central nervous system of mice affected by experimental autoimmune encephalomyelitis (EAE). This directly affects neuronal function in vivo, indicated by sustained elevated neuronal calcium. The systemic involvement of oxidative stress is mirrored by overactivation of NOX enzymes in peripheral CD11b(+) cells in later phases of both MS and EAE. This effect is antagonized by systemic intake of the NOX inhibitor and anti-oxidant epigallocatechin-3-gallate. Together, this persistent hyper-activation of oxidative enzymes suggests an "oxidative stress memory" both in the periphery and CNS compartments, in chronic neuroinflammation

Implikationen für die funktionelle Bildgebung in Patienten

Functional magnetic resonance imaging (fMRI) with BOLD (blood oxygen level- dependent) has become a standard tool for psychological and neurological research. It relies on the local adaptation of blood flow to increased neuronal activity - a phenomenon known as neurovascular coupling. The effects of pathophysiological conditions on neurovascular coupling and its corresponding BOLD signal are largely unknown, restricting its implementation as a clinical tool for research and diagnosis. This PhD-thesis summarizes findings relevant to this issue from three studies each exposing neurovascular coupling in the rat somatosensory cortex to a different pathophysiological challenge. As common experimental procedure, anesthetized rats underwent surgical preparation of a closed cranial window over the somatosensory cortex. Using Laser Doppler Flowmetry and optical spectroscopy, changes in cerebral blood flow (CBF), cerebral blood volume, deoxygenated hemoglobin (deoxy-Hb) and cerebral metabolic rate of oxygen were measured during electrical forepaw stimulation. Neuronal activity was monitored by somatosensory evoked potentials. The first study evaluated the effect of hypothermia. Cooling down the brain by 10 °C preserved neurovascular coupling. Functional brain imaging (e.g. fMRI or near infrared spectroscopy) therefore offers the potential to evaluate neurovascular function in a patient undergoing hypothermia. The second study investigated pharmacological blocking of the neurovascular coupling response employing the cyclooxygenase (COX)-inhibitor indomethacin and the adenosine-receptor-inhibitor theophyllin, medications commonly used in patients. While CBF responses were reduced by 70 - 80 %, the deoxy-Hb response (correlate of the BOLD signal) was abolished. It was shown that medications potentially can interfere with neurovascular coupling, thereby rendering BOLD- fMRI impossible. The third study explored the influence of raised intracranial pressure (ICP). A stepwise ICP elevation to 28 mmHg reduced and eventually reversed the deoxy-Hb response to somatosensory activation. In addition, the post-stimulus response of deoxy-Hb regularly detectable at physiological conditions was abolished by increased ICP. Intracranial hypertension, a common condition in patients with an intracranial lesion or disease, can seriously disturb neurovascular coupling and preclude reliable brain mapping with fMRI. Future studies investigating these pathophysiological states in patients should be performed prior to an establishment of fMRI in the clinical setting.Die funktionelle Kernspintomographie (fMRT) mit BOLD (blood oxygen level dependent) ist zu einem Standardinstrument der psychologischen und neurologischen Forschung geworden. Sie beruht auf der lokalen Adaptation des Blutflusses an erhöhte neuronale Aktivität-ein Phänomen, welches als neurovaskuläre Kopplung bekannt ist. Die Effekte pathophysiologischer Bedingungen auf die neurovaskuläre Kopplung und auf das korrespondierende BOLD-Signal sind grösstenteils unbekannt und beschränken somit die Implementierung der Kernspintomographie als klinisches diagnostisches Instrument. Die vorliegende Doktorarbeit fasst relevante Ergebnisse aus drei verschiedenen Studien zusammen. In jeder Teilstudie wurde die neurovaskuläre Kopplung im somatosensorischen Cortex der Ratte verschiedenen pathophysiologischen Konditionen ausgesetzt. Hierfür wurde anästhesierten Ratten ein geschlossenes kranielles Fenster über dem somatosensorischen Cortex präpariert. Unter Verwendung von Laser Doppler Flowmetry und optischer Spektroskopie wurden Veränderungen im zerebralen Blutfluss (CBF), zerebralen Blutvolumen (CBV), deoxygeniertem Hämoglobin (deoxy-Hb) und in der zerebralen metabolischen Rate von Sauerstoff während elektrischer Vorderpfotenstimulation gemessen. Die neuronale Aktivität wurde durch die Ableitung von somatosensorisch evozierten Potentialen überprüft. Die erste Studie evaluierte den Effekt der Hypothermie. Die neurovaskuläre Kopplung blieb während der Abkühlung des Gehirns um 10°C intakt. Die funktionelle Bildgebung (z.B. fMRT oder Nahinfrarotspektroskopie) bietet demzufolge das Potenzial, neurovaskuläre Funktionen in Patienten unter hypothermen Bedingungen zu bestimmen. Die zweite Studie untersuchte den Effekt einer pharmakologischen Inhibition der neurovaskulären Kopplungsantwort unter Verwendung des Cyclooxygenase- Inhibitors Indomethacin und des Adenosin-Rezeptor-Inhibitors Theophyllin, die in der klinischen Praxis häufig Anwendung finden. Während die CBF-Antworten um 70-80% reduziert waren, wurde die Antwort des deoxy-Hb (Korrelat des Bold- Signals) aufgehoben. Dieses Ergebnis impliziert, dass Medikationen mit der neurovaskulären Kopplung interferieren können und somit BOLD-fMRI unmöglich machen. Die dritte Studie erforschte den Einfluss von gesteigerten intrakraniellen Druck (ICP). Eine stufenweise Erhöhung des ICP auf 28mmHg reduzierte und kehrte letztlich das deoxy-Hb-Signal um. Desweiteren konnte die post-Stimulus-Antwort, die regelmässig unter physiologischen Bedingungen nachweisbar ist, nicht mehr detektiert werden. Intrakranielle Hypertension könnte demzufolge die neurovaskuläre Kopplung ernsthaft stören und die verlässliche Bildgebung des Gehirns mittels fMRT in Frage stellen. Zukünftige Studien, die diese pathophysiologischen Konditionen in Patienten erforschen, sollten im Vorfeld der Etablierung des fMRT für den klinischen Einsatz durchgeführt werden

Determination of the brain–blood partition coefficient for water in mice using MRI

Cerebral blood flow (CBF) quantification is a valuable tool in stroke research. Mice are of special interest because of the potential of genetic engineering. Magnetic resonance imaging (MRI) provides repetitive, noninvasive CBF quantification. Many MRI techniques require the knowledge of the brain–blood partition coefficient (BBPC) for water. Adopting an MRI protocol described by Roberts et al (1996) in humans, we determined the BBPC for water in 129S6/SvEv mice from proton density measurements of brain and blood, calibrated with deuterium oxide/water phantoms. The average BBPC for water was 0.89±0.03 mL/g, with little regional variation within the mouse brain