Two-photon microscopy : sequential imaging studies in vivo

Microscopists have always desired to look inside various organ tissues to study structure, function and dysfunction of their cellular constituents. In the past, this has frequently required tissue extraction and histological preparation to gain access. Traditional optical microscopy techniques, which use linear (one-photon) absorption processes for contrast generation, are limited to use near the tissue surface (< 80 µm) because at greater depths strong and multiple light scattering blurs the images. Scattering particularly strongly affects signal strength in confocal microscopy, which achieves three-dimensional resolution and optical sectioning with a detection pinhole that rejects all light that appears not to originate from the focus. New optical microscopy techniques have been developed that use nonlinear light-matter interactions to generate signal contrast only within a thin raster-scanned plane. Since its first demonstration over a decade ago, two-photon microscopy has been applied to a variety of imaging tasks and has now become the technique of choice for fluorescence microscopy in thick tissue preparations and in live animals. The gain in resolution over conventional in vivo imaging techniques has been several orders of magnitude. Neuroscientists have used it to measure calcium dynamics deep in brain slices and in live animals, blood flow measurement, neuronal plasticity and to monitor neurodegenerative disease models in brain slices and in live rodents. These types of applications define the most important niche for two-photon microscopy - high-resolution imaging of physiology, morphology and cell-cell interactions in intact tissue. Clearly the biggest advantage of two-photon microscopy is in longitudinal monitoring of rodent models of disease or plasticity over days to weeks. The aim of this article is to discuss some methodological principles, and show some applications of this technique obtained from our laboratory in the area of acute experimental stroke research.peer-reviewe

From mouse to man and back : closing the correlation gap between imaging and histopathology for lung diseases

Lung diseases such as fibrosis, asthma, cystic fibrosis, infection and cancer are life-threatening conditions that slowly deteriorate quality of life and for which our diagnostic power is high, but our knowledge on etiology and/or effective treatment options still contains important gaps. In the context of day-to-day practice, clinical and preclinical studies, clinicians and basic researchers team up and continuously strive to increase insights into lung disease progression, diagnostic and treatment options. To unravel disease processes and to test novel therapeutic approaches, investigators typically rely on end-stage procedures such as serum analysis, cyto-/chemokine profiles and selective tissue histology from animal models. These techniques are useful but provide only a snapshot of disease processes that are essentially dynamic in time and space. Technology allowing evaluation of live animals repeatedly is indispensable to gain a better insight into the dynamics of lung disease progression and treatment effects. Computed tomography (CT) is a clinical diagnostic imaging technique that can have enormous benefits in a research context too. Yet, the implementation of imaging techniques in laboratories lags behind. In this review we want to showcase the integrated approaches and novel developments in imaging, lung functional testing and pathological techniques that are used to assess, diagnose, quantify and treat lung disease and that may be employed in research on patients and animals. Imaging approaches result in often novel anatomical and functional biomarkers, resulting in many advantages, such as better insight in disease progression and a reduction in the numbers of animals necessary. We here showcase integrated assessment of lung disease with imaging and histopathological technologies, applied to the example of lung fibrosis. Better integration of clinical and preclinical imaging technologies with pathology will ultimately result in improved clinical translation of (therapy) study results

Replacing vascular corrosion casting by in-vivo micro-CT imaging for building 3D cardiovascular models in mice

The purpose of this study was to investigate if in vivo micro-computed tomography (CT) is a reliable alternative to micro-CT scanning of a vascular corrosion cast. This would allow one to study the early development of cardiovascular diseases. Datasets using both modalities were acquired, segmented, and used to generate a 3D geometrical model from nine mice. As blood pool contrast agent, Fenestra VC-131 was used. Batson's No. 17 was used as casting agent. Computational fluid dynamics simulations were performed on both datasets to quantify the difference in wall shear stress (WSS). Aortic arch diameters show 30% to 40% difference between the Fenestra VC-131 and the casted dataset. The aortic arch bifurcation angles show less than 20% difference between both datasets. Numerically computed WSS showed a 28% difference between both datasets. Our results indicate that in vivo micro-CT imaging can provide an excellent alternative for vascular corrosion casting. This enables follow-up studies

Understanding the Structural and Functional Correlates of Acute Lung Inflammation in Two Murine Models

The outcome of lung inflammation is important to host survival as lungs are necessary for oxygen exchange and fighting pathogens or any injurious stimuli. Thus, diagnosing and understanding the kinetics of lung inflammation is an emerging technological area in the field of imaging research and development. Dr. Aulakh’s lab has two separate established models of neutrophilic murine acute lung injury namely, acute low-dose (0.05 ppm) ozone-induced and intranasal bacterial lipopolysaccharide (LPS)-induced lung inflammation. In order to characterize the dynamics of these models, there are two research hypotheses of my project, which are a) acute low-dose ozone exposure causes lung [18F]F-FDG retention because of increased leukocyte glucose uptake due to inflammation as assessed by sequential micro-Positron Emission Tomography-Computed tomography (microPET-CT) in murine lungs, similar to the effects of intranasal bacterial lipopolysaccharide, LPS and b) acute low-dose ozone exposure induces an increase in ultra-small-angle scatter (USAXS) (due to alveolar recruitment), absorption (due to alveolar edema) and a decrease in refraction (due to peri-bronchiolar edema) comparable to intranasal LPS induced changes in these X-ray optical properties as assessed by Lung Multiple Image X-Radiography (MIR). Thus, the premise of my thesis is to test the utility of longitudinal non-invasive imaging modalities, namely sequential [18F]-fluoro-deoxy glucose ([18F]F-FDG) positron emission tomography-computed tomography (PET-CT) and synchrotron multiple image X-radiography (MIR), to assess the progression of acute murine low-dose ozone or intranasal bacterial lipopolysaccharide (LPS) induced lung inflammation over 24 and 70 h time periods, respectively. Both ozone and LPS induced an increase in murine lung [18F]F-FDG standard uptake ratio (SUR) and a heterogenous lung distribution which was unlike the craniocaudal [18F]F-FDG gradient observed in lungs before any exposure (called as baseline or control [18F]F-FDG). The whole-body distribution profiles revealed that lung [18F]F-FDG activity was higher and prolonged up to 28 h in LPS compared to ozone exposed mice. While [18F]F-FDG is a useful marker to highlight areas with high metabolic uptake of glucose in cells such as neutrophils and macrophages recruited during inflammation, the resolution of PET-CT (hundreds of μm) precludes the evaluation of microscopic histopathologic changes especially in the alveoli. Using lung hematoxylin and eosin stained cryosections, the ratios of total lung tissue to air spaces and specifically alveolar parenchyma to air spaces were assessed in mice lungs exposed to 0.05 ppm ozone for 2 h. Results from the X-ray CT lung tissue volume quantifications as well as the histologically derived percent-stained lung or alveolar area quantifications suggest significant damage that is observed as reduced percentage area as well as variability or standard deviation (S.D.) of binary lung images in mice immediately i.e., at 0 h and 6 h after exposure to 2 h of 0.05 ppm ozone. Alveolar damage was also significant at 0 h as shown by reduction in percentage area and S.D. in the binary image region restricted to alveoli. The synchrotron study aimed at following mice lungs before, immediately i.e., at 0 h, and thereafter at 24, 48 or 70 h after saline, bacterial lipopolysaccharide (LPS, 50 μg), or low dose (0.05 ppm for 2 h) ozone exposure. Our results indicate that the lung ultra-small-angle scatter (USAXS), which is a metric of air-tissue boundaries and refraction (which is due to bending of X-rays across air-tissue conducting airways) reduces, especially in the cranial part of left lung, with a corresponding increase in absorption upon exposure to LPS or ozone and is detectable up to 70 h. The changes in lung X-ray optical properties are indicative of the gross inflammatory changes, in response to LPS or ozone exposure, as indicated by increases in lung absorption but reduction in refraction and USAXS. Overall, the results from my project indicate that for a comprehensive analysis of lung inflammation, a combination of lung histological analysis along with objective lung image analysis as described in the longitudinal microPET-CT and lung MIR experiments form powerful techniques for sensitive delineation of inflammatory changes in gross lung structure and function

Magnetic Resonance Imaging Techniques for Brown Adipose Tissue Detection

Magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) methods can non-invasively assess brown adipose tissue (BAT) structure and function. Recently, MRI and MRS have been proposed as a means to differentiate BAT from white adipose tissue (WAT) and to extract morphological and functional information on BAT inaccessible by other means. Specifically, proton MR (1H) techniques, such as proton density fat fraction mapping, diffusion imaging, and intermolecular multiple quantum coherence imaging, have been employed to access BAT microstructure; MR thermometry, relaxometry, and MRI and MRS with 31P, 2H, 13C, and 129Xe have shown to provide complementary information on BAT function. The purpose of the present review is to provide a comprehensive overview of MR imaging and spectroscopy techniques used to detect BAT in rodents and in humans. The present work discusses common challenges of current methods and provides an outlook on possible future directions of using MRI and MRS in BAT studies

Cine and tagged cardiovascular magnetic resonance imaging in normal rat at 1.5 T: a rest and stress study

BACKGROUND: The purpose of this study was to measure regional contractile function in the normal rat using cardiac cine and tagged cardiovascular magnetic resonance (CMR) during incremental low doses of dobutamine and at rest. METHODS: Five rats were investigated for invasive left ventricle pressure measurements and five additional rats were imaged on a clinical 1.5 T MR system using a cine sequence (11-20 phases per cycle, 0.28/0.28/2 mm) and a C-SPAMM tag sequence (18-25 phases per cycle, 0.63/1.79/3 mm, tag spacing 1.25 mm). For each slice, wall thickening (WT) and circumferential strains (CS) were calculated at rest and at stress (2.5, 5 and 10 microg/min/kg of dobutamine). RESULTS: Good cine and tagged images were obtained in all the rats even at higher heart rate (300-440 bpm). Ejection fraction and left ventricular (LV) end-systolic volume showed significant changes after each dobutamine perfusion dose (p < 0.001). Tagged CMR had the capacity to resolve the CS transmural gradient and showed a significant increase of both WT and CS at stress compared to rest. Intra and interobserver study showed less variability for the tagged technique. In rats in which a LV catheter was placed, dobutamine produced a significant increase of heart rate, LV dP/dtmax and LV pressure significantly already at the lowest infusion dose. CONCLUSION: Robust cardiac cine and tagging CMR measurements can be obtained in the rat under incremental dobutamine stress using a clinical 1.5 T MR scanner

Development of an Awake Behaving model for Laser Doppler Flowmetry in Mice

Bien que le cerveau ne constitue que 2% de la masse du corps chez les humains, il présente l’activité métabolique la plus élevée dans le corps, et en conséquence, constitue un organe hautement vascularisé. En fait, l’approvisionnement en sang dans le cerveau est strictement modulé au niveau régional par un mécanisme fondamental nommé couplage neurovasculaire (CNV), qui associe les besoins métaboliques locaux au flux sanguin cérébral [1, 2]. Notre compréhension du CNV sous des conditions physiologiques et pathologiques a été améliorée par un large éventail d’études menées chez les rongeurs. Néanmoins, ces études ont été réalisées soit sous anesthésie, soit chez la souris éveillée et immobilisée, afin d’éviter le mouvement de la tête pendant l'acquisition de l'image. Les anesthésiques, ainsi que le stress induit par la contention, peuvent altérer l'hémodynamique cérébrale, ce qui pourrait entraver les résultats obtenus. Par conséquent, il est essentiel de contrôler ces facteurs lors de recherches futures menées au sujet de la réponse neurovasculaire. Au cours de l’étude présente, nous avons développé un nouveau dispositif pour l'imagerie optique éveillée, où la tête de la souris est immobilisée, mais son corps est libre de marcher, courir ou se reposer sur une roue inclinée. En outre, nous avons testé plusieurs protocoles d'habituation, selon lesquels la souris a été progressivement entraînée pour tolérer l’immobilisation de tête, afin de minimiser le stress ressenti lors des sessions d'imagerie. Enfin, nous avons, pour la première fois, cherché à valider l'efficacité de ces protocoles d'habituation dans la réduction du stress, en mesurant l'évolution des taux plasmatiques de corticostérone tout au long de notre étude. Nous avons noté que les souris s'étaient rapidement adaptées à la course sur la roue et que les signes visibles de stress (luttes, vocalisations et urination) étaient nettement réduits suite à deux sessions d'habituation. Néanmoins, les taux de corticostérone n'ont pas été significativement réduits chez les souris habituées, par rapport aux souris naïves qui ont été retenues sur la roue sans entraînement préalable (p> 0,05). Ce projet met en évidence la nécessité d'une mesure quantitative du stress, car une réduction des comportements observables tels que l'agitation ou la lutte peut être indicative non pas d'un niveau de stress plus faible, mais plutôt d'un désespoir comportemental. Des recherches supplémentaires sont nécessaires pour déterminer si la fixation de la tête lors de l'imagerie optique chez la souris peut être obtenue avec des niveaux de stress plus faibles, et si le stress induit par la contrainte effectuée avec notre dispositif est associé à des changements de la réponse hémodynamique.Whilst the brain only constitutes 2% of total body weight in humans, it exhibits the highest metabolic activity in the body, and as such is a highly vascularized organ. In fact, regional blood supply within the brain is strictly modulated through a fundamental process termed neurovascular coupling (NVC), which couples local metabolic needs with cerebral blood flow [1, 2]. A wide array of optical imaging studies in rodents has enhanced our understanding of NVC under physiological and pathological conditions. Nevertheless, these studies have been performed either under anesthesia, or in the awake mouse using restraint to prevent head-motion during image acquisition. Both anesthetics and restraint-induced stress have been clearly shown to alter cerebral hemodynamics, thereby potentially interfering with the obtained results [3, 4]. Hence, it is essential to control for these factors during future research which investigates the neurovascular response. In the present study, we have developed a new apparatus for awake optical imaging, where the mouse is head-restraint whilst allowed to walk, run or rest on an inclined wheel. In addition, we have tested several habituation protocols, according to which the mouse was gradually trained to tolerate head-restraint, in order to minimize the stress experienced during imaging sessions. Lastly, we have, for the first time, sought to validate the efficiency of these habituation protocols in reducing stress, by measuring the evolution of plasma corticosterone levels throughout the study. We noted that the mice had quickly adapted to running on the wheel, and that the overt signs of stress (struggling, vocalizations and urination) were clearly reduced within two habituation sessions. Nevertheless, corticosterone levels were not significantly reduced in habituated mice, relative to naïve mice that were restrained on the wheel without prior training (p > 0.05). This project highlights the necessity for a quantitative measure of stress, as a reduction in observable behaviors such as agitation or struggling may be indicative not of lower stress, but rather, of behavioral despair. Further research is needed to determine whether head-fixation during optical imaging in mice can be achieved with lower stress levels, and if restraint-induced stress using our apparatus is associated with changes in the hemodynamic response

How bold is blood oxygenation level dependent (BOLD) magnetic resonance imaging of the kidney? Opportunities, challenges and future directions

Renal tissue hypoperfusion and hypoxia are key elements in the pathophysiology of acute kidney injury and its progression to chronic kidney disease. Yet, in vivo assessment of renal haemodynamics and tissue oxygenation remains a challenge. Many of the established approaches are invasive, hence not applicable in humans. Blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) offers an alternative. BOLD-MRI is non-invasive and indicative of renal tissue oxygenation. Nonetheless recent (pre-)clinical studies revived the question as to how bold renal BOLD-MRI really is. This review aims to deliver some answers. It is designed to inspire the renal physiology, nephrology, and imaging communities to foster explorations into the assessment of renal oxygenation and haemodynamics by exploiting the powers of MRI. For this purpose the specifics of renal oxygenation and perfusion are outlined. The fundamentals of BOLD-MRI are summarized. The link between tissue oxygenation and the oxygenation sensitive MR biomarker T2 * is outlined. The merits and limitations of renal BOLD-MRI in animal and human studies are surveyed together with their clinical implications. Explorations into detailing the relation between renal T2 * and renal tissue partial pressure of oxygen (pO2 ) are discussed with a focus on factors confounding the T2 * versus tissue pO2 relation. Multi-modality in vivo approaches suitable for detailing the role of the confounding factors that govern T2 * are considered. A schematic approach describing the link between renal perfusion, oxygenation, tissue compartments and renal T2 * is proposed. Future directions of MRI assessment of renal oxygenation and perfusion are explored