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

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 μm). 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

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

Dopamine in the orbitofrontal cortex regulates operant responding under a progressive ratio of reinforcement in rats

Prefrontocortical dopamine (DA) plays an essential role in the regulation of cognitive functions and behavior. The orbitofrontal cortex (OFC) receives a dopaminergic projection from the ventral tegmental area and is particularly important for goal-directed appetitive behaviors and for the neural representation of reward value. We here examined the effects of DA receptor blockers locally infused into the OFC, on instrumental behavior under a progressive schedule of reinforcement. After continuous reinforcement training (lever pressing for casein pellets) rats received bilateral intra-OFC-infusions of the DA D1-receptor antagonist SCH23390 (3 microg/0.5 microl), the DA D2-receptor antagonist sulpiride (3 microg/0.5 microl), or phosphate buffered saline through chronically indwelling cannulae. Immediately after infusion they were tested under a time-constrained progressive ratio schedule of reinforcement (3, 6, 9, 12, ... lever presses for 1 casein pellet within 180 s). Both SCH23390 and sulpiride led to a significant reduction of the break point (cessation to respond to the increasing criterion of instrumental effort) compared to vehicle infusions. A food preference test revealed no drug effects on the amount of consumed pellets and on the preference of casein pellets over laboratory chow. Leftward shifts of the break point in progressive ratio tasks indicate a disturbance of the mechanisms that translate motivation into appetitive behavior under conditions of increasing instrumental effort. Therefore, our data indicate that orbitofrontal dopamine is necessary for reward-related instrumental behavior

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

Analysis of temporal changes in visual function using Ganzfeld ERG.

<p><b>A-C.</b> Graphs show representative curves of Ganzfeld ERG during the experimental time-course of a (A) sham, (B) late affected BCCAO and (C) early affected BCCAO animals. <b>D.</b> Scotopic a-wave. <b>E.</b> Scotopic b-wave. <b>F.</b> b/a-wave ratio. <b>G.</b> Oscillatory potentials.</p

Delocalization of synaptic markers in the retina after BCCAO.

<p><b>A.</b> Representative sagittal sections of the retina stained against VGLUT1 (green) showing loss and delocalization of pre-synaptic proteins in animals with BCCAO. <b>B.</b> Representative sagittal sections of the retina stained against CTBP2 (red) showing loss and delocalization of pre-synaptic proteins. <b>C.</b> Representative sagittal sections of the retina stained against PKCα (green) showing loss and delocalization of post-synaptic proteins after prolonged BCCAO. <b>D.</b> Representative sagittal sections of the retina stained against CALB (red) showing loss horizontal cells in coiled animals. All nuclei were counterstained with DAPI (blue). For all cases, scale bar = 50.</p