Impaired Glymphatic Function and Pulsation Alterations in a Mouse Model of Vascular Cognitive Impairment

Copyright \ua9 2022 Li, Kitamura, Beverley, Koudelka, Duncombe, Lennen, Jansen, Marshall, Platt, Wiegand, Carare, Kalaria, Iliff and Horsburgh. Large vessel disease and carotid stenosis are key mechanisms contributing to vascular cognitive impairment (VCI) and dementia. Our previous work, and that of others, using rodent models, demonstrated that bilateral common carotid stenosis (BCAS) leads to cognitive impairment via gradual deterioration of the neuro-glial-vascular unit and accumulation of amyloid-β (Aβ) protein. Since brain-wide drainage pathways (glymphatic) for waste clearance, including Aβ removal, have been implicated in the pathophysiology of VCI via glial mechanisms, we hypothesized that glymphatic function would be impaired in a BCAS model and exacerbated in the presence of Aβ. Male wild-type and Tg-SwDI (model of microvascular amyloid) mice were subjected to BCAS or sham surgery which led to a reduction in cerebral perfusion and impaired spatial learning acquisition and cognitive flexibility. After 3 months survival, glymphatic function was evaluated by cerebrospinal fluid (CSF) fluorescent tracer influx. We demonstrated that BCAS caused a marked regional reduction of CSF tracer influx in the dorsolateral cortex and CA1-DG molecular layer. In parallel to these changes increased reactive astrogliosis was observed post-BCAS. To further investigate the mechanisms that may lead to these changes, we measured the pulsation of cortical vessels. BCAS impaired vascular pulsation in pial arteries in WT and Tg-SwDI mice. Our findings show that BCAS influences VCI and that this is paralleled by impaired glymphatic drainage and reduced vascular pulsation. We propose that these additional targets need to be considered when treating VCI

Imaging learned fear circuitry in awake mice using fMRI

Functional magnetic resonance imaging (fMRI) of learned behaviour in ‘awake rodents’ provides the opportunity for translational preclinical studies into the influence of pharmacological and genetic manipulations on brain function. fMRI has recently been employed to investigate learned behaviour in awake rats. Here, this methodology is translated to mice, so that future fMRI studies may exploit the vast number of genetically modified mouse lines that are available. One group of mice was conditioned to associate a flashing light (conditioned stimulus, CS) with foot shock (PG; paired group), and another group of mice received foot shock and flashing light explicitly unpaired (UG; unpaired group). The blood oxygen level-dependent signal (proxy for neuronal activation) in response to the CS was measured 24 h later in awake mice from the PG and UG using fMRI. The amygdala, implicated in fear processing, was activated to a greater degree in the PG than in the UG in response to the CS. Additionally, the nucleus accumbens was activated in the UG in response to the CS. Because the CS signalled an absence of foot shock in the UG, it is possible that this region is involved in processing the safety aspect of the CS. To conclude, the first use of fMRI to visualise brain activation in awake mice that are completing a learned emotional task is reported. This work paves the way for future preclinical fMRI studies to investigate genetic and environmental influences on brain function in transgenic mouse models of disease and aging

Multicentre evaluation of MRI variability in the quantification of infarct size in experimental focal cerebral ischaemia

Ischaemic stroke is a leading cause of death and disability in the developed world. Despite that considerable advances in experimental research enabled understanding of the pathophysiology of the disease and identified hundreds of potential neuroprotective drugs for treatment, no such drug has shown efficacy in humans. The failure in the translation from bench to bedside has been partially attributed to the poor quality and rigour of animal studies. Recently, it has been suggested that multicentre animal studies imitating the design of randomised clinical trials could improve the translation of experimental research. Magnetic resonance imaging (MRI) could be pivotal in such studies due to its non-invasive nature and its high sensitivity to ischaemic lesions, but its accuracy and concordance across centres has not yet been evaluated. This thesis focussed on the use of MRI for the assessment of late infarct size, the primary outcome used in stroke models. Initially, a systematic review revealed that a plethora of imaging protocols and data analysis methods are used for this purpose. Using meta-analysis techniques, it was determined that T2-weighted imaging (T2WI) was best correlated with gold standard histology for the measurement of infarctbased treatment effects. Then, geometric accuracy in six different preclinical MRI scanners was assessed using structural phantoms and automated data analysis tools developed in-house. It was found that geometric accuracy varies between scanners, particularly when centre-specific T2WI protocols are used instead of a standardised protocol, though longitudinal stability over six months is high. Finally, a simulation study suggested that the measured geometric errors and the different protocols are sufficient to render infarct volumes and related group comparisons across centres incomparable. The variability increases when both factors are taken into account and when infarct volume is expressed as a relative estimate. Data in this study were analysed using a custom-made semi-automated tool that was faster and more reliable in repeated analyses than manual analysis. Findings of this thesis support the implementation of standardised methods for the assessment and optimisation of geometric accuracy in MRI scanners, as well as image acquisition and analysis of in vivo data for the measurement of infarct size in multicentre animal studies. Tools and techniques developed as part of the thesis show great promise in the analysis of phantom and in vivo data and could be a step towards this endeavour

Imaging of activated complement using ultrasmall superparamagnetic iron oxide particles (USPIO) - conjugated vectors: an in vivo in utero non-invasive method to predict placental insufficiency and abnormal fetal brain development.

In the current study, we have developed a magnetic resonance imaging-based method for non-invasive detection ofcomplement activation in placenta and foetal brain in vivo in utero. Using this method, we found that anti-complementC3-targeted ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles bind within the inflamed placenta and foetal braincortical tissue, causing a shortening of the T2* relaxation time. We used two mouse models of pregnancy complications: a mousemodel of obstetrics antiphospholipid syndrome (APS) and a mouse model of preterm birth (PTB). We found that detection of C3deposition in the placenta in the APS model was associated with placental insufficiency characterised by increased oxidative stress,decreased vascular endothelial growth factor and placental growth factor levels and intrauterine growth restriction. We alsofound that foetal brain C3 deposition was associated with cortical axonal cytoarchitecture disruption and increasedneurodegeneration in the mouse model of APS and in the PTB model. In the APS model, foetuses that showed increased C3in their brains additionally expressed anxiety-related behaviour after birth. Importantly, USPIO did not affect pregnancyoutcomes and liver function in the mother and the offspring, suggesting that this method may be useful for detecting complementactivation in vivo in utero and predicting placental insufficiency and abnormal foetal neurodevelopment that leads toneuropsychiatric disorders

Serelaxin as a potential treatment for renal dysfunction in cirrhosis: Preclinical evaluation and results of a randomized phase 2 trial

<div><p>Background</p><p>Chronic liver scarring from any cause leads to cirrhosis, portal hypertension, and a progressive decline in renal blood flow and renal function. Extreme renal vasoconstriction characterizes hepatorenal syndrome, a functional and potentially reversible form of acute kidney injury in patients with advanced cirrhosis, but current therapy with systemic vasoconstrictors is ineffective in a substantial proportion of patients and is limited by ischemic adverse events. Serelaxin (recombinant human relaxin-2) is a peptide molecule with anti-fibrotic and vasoprotective properties that binds to relaxin family peptide receptor-1 (RXFP1) and has been shown to increase renal perfusion in healthy human volunteers. We hypothesized that serelaxin could ameliorate renal vasoconstriction and renal dysfunction in patients with cirrhosis and portal hypertension.</p><p>Methods and findings</p><p>To establish preclinical proof of concept, we developed two independent rat models of cirrhosis that were characterized by progressive reduction in renal blood flow and glomerular filtration rate and showed evidence of renal endothelial dysfunction. We then set out to further explore and validate our hypothesis in a phase 2 randomized open-label parallel-group study in male and female patients with alcohol-related cirrhosis and portal hypertension. Forty patients were randomized 1:1 to treatment with serelaxin intravenous (i.v.) infusion (for 60 min at 80 μg/kg/d and then 60 min at 30 μg/kg/d) or terlipressin (single 2-mg i.v. bolus), and the regional hemodynamic effects were quantified by phase contrast magnetic resonance angiography at baseline and after 120 min. The primary endpoint was the change from baseline in total renal artery blood flow.</p><p>Therapeutic targeting of renal vasoconstriction with serelaxin in the rat models increased kidney perfusion, oxygenation, and function through reduction in renal vascular resistance, reversal of endothelial dysfunction, and increased activation of the AKT/eNOS/NO signaling pathway in the kidney. In the randomized clinical study, infusion of serelaxin for 120 min increased total renal arterial blood flow by 65% (95% CI 40%, 95%; <i>p <</i> 0.001) from baseline. Administration of serelaxin was safe and well tolerated, with no detrimental effect on systemic blood pressure or hepatic perfusion. The clinical study’s main limitations were the relatively small sample size and stable, well-compensated population.</p><p>Conclusions</p><p>Our mechanistic findings in rat models and exploratory study in human cirrhosis suggest the therapeutic potential of selective renal vasodilation using serelaxin as a new treatment for renal dysfunction in cirrhosis, although further validation in patients with more advanced cirrhosis and renal dysfunction is required.</p><p>Trial registration</p><p>ClinicalTrials.gov <a href="https://clinicaltrials.gov/ct2/show/NCT01640964" target="_blank">NCT01640964</a></p></div