Kristof Govaerts

Deletion or Inhibition of the Oxygen Sensor PHD1 Protects against Ischemic Stroke via Reprogramming of Neuronal Metabolism

The oxygen-sensing prolyl hydroxylase domain proteins (PHDs) regulate cellular metabolism, but their role in neuronal metabolism during stroke is unknown. Here we report that PHD1 deficiency provides neuroprotection in a murine model of permanent brain ischemia. This was not due to an increased collateral vessel network. Instead, PHD1(-/-) neurons were protected against oxygen-nutrient deprivation by reprogramming glucose metabolism. Indeed, PHD1(-/-) neurons enhanced glucose flux through the oxidative pentose phosphate pathway by diverting glucose away from glycolysis. As a result, PHD1(-/-) neurons increased their redox buffering capacity to scavenge oxygen radicals in ischemia. Intracerebroventricular injection of PHD1-antisense oligonucleotides reduced the cerebral infarct size and neurological deficits following stroke. These data identify PHD1 as a regulator of neuronal metabolism and a potential therapeutic target in ischemic stroke.

Persistent Impact of In utero Irradiation on Mouse Brain Structure and Function Characterized by MR Imaging and Behavioral Analysis.

Prenatal irradiation is known to perturb brain development. Epidemiological studies revealed that radiation exposure during weeks 8–15 of pregnancy was associated with an increased occurrence of mental disability and microcephaly. Such neurological deficits were reproduced in animal models, in which rodent behavioral testing is an often used tool to evaluate radiation-induced defective brain functionality. However, up to now, animal studies suggested a threshold dose of around 0.30 Gray (Gy) below which no behavioral alterations can be observed, while human studies hinted at late defects after exposure to doses as low as 0.10 Gy. Here, we acutely irradiated pregnant mice at embryonic day 11 with doses ranging from 0.10 to 1.00 Gy. A thorough investigation of the dose-response relationship of altered brain function and architecture following in utero irradiation was achieved using a behavioral test battery and volumetric 3D T2-weighted magnetic resonance imaging (MRI). We found dose-dependent changes in cage activity, social behavior, anxiety-related exploration, and spatio-cognitive performance. Although behavioral alterations in low-dose exposed animals were mild, we did unveil that both emotionality and higher cognitive abilities were affected in mice exposed to ≥0.10 Gy. Microcephaly was apparent from 0.33 Gy onwards and accompanied by deviations in regional brain volumes as compared to controls. Of note, total brain volume and the relative volume of the ventricles, frontal and posterior cerebral cortex, cerebellum, and striatum were most strongly correlated to altered behavioral parameters. Taken together, we present conclusive evidence for persistent low-dose effects after prenatal irradiation in mice and provide a better understanding of the correlation between their brain size and performance in behavioral tests.

Longitudinal, in vivo assessment of invasive pulmonary aspergillosis in mice by computed tomography and magnetic resonance imaging

Invasive aspergillosis is an emerging threat to public health due to the increasing use of immune suppressive drugs and the emergence of resistance against antifungal drugs. To deal with this threat, research on experimental disease models provides insight into the pathogenesis of infections caused by susceptible and resistant Aspergillus strains and by assessing their response to antifungal drugs. However, standard techniques used to evaluate infection in a preclinical setting are severely limited by their invasive character, thereby precluding evaluation of disease extent and therapy effects in the same animal. To enable non-invasive, longitudinal monitoring of invasive pulmonary aspergillosis in mice, we optimized computed tomography (CT) and magnetic resonance imaging (MRI) techniques for daily follow-up of neutropenic BALB/c mice intranasally infected with A. fumigatus spores. Based on the images, lung parameters (signal intensity, lung tissue volume and total lung volume) were quantified to obtain objective information on disease onset, progression and extent for each animal individually. Fungal lung lesions present in infected animals were successfully visualized and quantified by both CT and MRI. By using an advanced MR pulse sequence with ultrashort echo times, pathological changes within the infected lung became visually and quantitatively detectable at earlier disease stages, thereby providing valuable information on disease onset and progression with high sensitivity. In conclusion, these non-invasive imaging techniques prove to be valuable tools for the longitudinal evaluation of dynamic disease-related changes and differences in disease severity in individual animals that might be readily applied for rapid and cost-efficient drug screening in preclinical models in vivo.

In vivo evidence for long-term vascular remodeling resulting from chronic cerebral hypoperfusion in mice.

We have characterized both acute and long-term vascular and metabolic effects of unilateral common carotid artery occlusion in mice by in vivo magnetic resonance imaging and positron emission tomography. This common carotid artery occlusion model induces chronic cerebral hypoperfusion and is therefore relevant to both preclinical stroke studies, where it serves as a control condition for a commonly used mouse model of ischemic stroke, and neurodegeneration, as chronic hypoperfusion is causative to cognitive decline. By using perfusion magnetic resonance imaging, we demonstrate that under isoflurane anesthesia, cerebral perfusion levels recover gradually over one month. This recovery is paralleled by an increase in lumen diameter and altered tortuosity of the contralateral internal carotid artery at one year post-ligation as derived from magnetic resonance angiography data. Under urethane/α-chloralose anesthesia, no acute perfusion differences are observed, but the vascular response capacity to hypercapnia is found to be compromised. These hemispheric perfusion alterations are confirmed by water [15O]-H2O positron emission tomography. Glucose metabolism ([18F]-FDG positron emission tomography) or white matter organization (diffusion-weighted magnetic resonance imaging) did not show any significant alterations. In conclusion, permanent unilateral common carotid artery occlusion results in acute and long-term vascular remodeling, which may have immediate consequences for animal models of stroke but also vascular dementia.

Bronchoscopic fibered confocal fluorescence microscopy for longitudinal in vivo assessment of pulmonary fungal infections in free-breathing mice

Respiratory diseases, such as pulmonary infections, are an important cause of morbidity and mortality worldwide. Preclinical studies often require invasive techniques to evaluate the extent of infection. Fibered confocal fluorescence microscopy (FCFM) is an emerging optical imaging technique that allows for real-time detection of fluorescently labeled cells within live animals, thereby bridging the gap between in vivo whole-body imaging methods and traditional histological examinations. Previously, the use of FCFM in preclinical lung research was limited to endpoint observations due to the invasive procedures required to access lungs. Here, we introduce a bronchoscopic FCFM approach that enabled in vivo visualization and morphological characterisation of fungal cells within lungs of mice suffering from pulmonary Aspergillus or Cryptococcus infections. The minimally invasive character of this approach allowed longitudinal monitoring of infection in free-breathing animals, thereby providing both visual and quantitative information on infection progression. Both the sensitivity and specificity of this technique were high during advanced stages of infection, allowing clear distinction between infected and non-infected animals. In conclusion, our study demonstrates the potential of this novel bronchoscopic FCFM approach to study pulmonary diseases, which can lead to novel insights in disease pathogenesis by allowing longitudinal in vivo microscopic examinations of the lungs.

Transcriptional upregulation of myelin components in spontaneous myelin basic protein-deficient mice

Myelin is essential for efficient signal transduction in the nervous system comprising of multiple proteins. The intricacies of the regulation of the formation of myelin, and its components, are not fully understood. Here, we describe the characterization of a novel myelin basic protein (Mbp) mutant mouse, mbp(jive), which spontaneously occurred in our mouse colony. These mice displayed the onset of a shaking gait before 3 weeks of age and seizure onset before 2 months of age. Due to a progressive increase of seizure intensity, mbp(jive) mice experienced premature lethality at around 3 months of age. Mbp mRNA transcript or protein was undetectable and, accordingly, genetic analysis demonstrated a homozygous loss of exons 3 to 6 of Mbp. Peripheral nerve conductance was mostly unimpaired. Additionally, we observed grave structural changes in white matter predominant structures were detected by T1, T2 and diffusion weighted magnetic resonance imaging. We additionally observed that Mbp-deficiency results in an upregulation of Qkl, Mag and Cnp, suggestive of a regulatory feedback mechanism whereby compensatory increases in Qkl have downstream effects on Mag and Cnp. Further research will clarify the role and specifications of this myelin feedback loop, as well as determine its potential role in therapeutic strategies for demyelinating disorders.