Rik Paesen

Complex invasion pattern of the cerebral cortex bymicroglial cells during development of the mouse embryo.

Microglia are the immune cells of the central nervous system. They are suspected to play important roles in adult synaptogenesis and in the development of the neuronal network. Microglial cells originate from progenitors in the yolk sac. Although it was suggested that they invade the cortex at early developmental stages in the embryo, their invasion pattern remains largely unknown. To address this issue we analyzed the pattern of cortical invasion by microglial cells in mouse embryos at the onset of neuronal cell migration using in vivo immunohistochemistry and ex vivo time‐lapse analysis of microglial cells. Microglial cells begin to invade the cortex at 11.5 days of embryonic age (E11.5). They first accumulate at the pial surface and within the lateral ventricles, after which they spread throughout the cortical wall, avoiding the cortical plate region in later embryonic ages. The invasion of the cortical parenchyma occurs in different phases. First, there is a gradual increase of microglial cells between E10.5 and E14.5. From E14.5 to E15.5 there is a rapid phase with a massive increase in microglia, followed by a slow phase again from E15.5 until E17.5. At early stages, many peripheral microglia are actively proliferating before entering the parenchyma. Remarkably, activated microglia accumulate in the choroid plexus primordium, where they are in the proximity of dying cells. Time‐lapse analysis shows that embryonic microglia are highly dynamic cells.

Molecular engineering of chromophores for combined second-harmonic and two-photon fluorescence in cellular imaging

A series of chromophores with enhanced second- and third-order nonlinear optical properties were engineered for use in combined second-harmonic and two-photon fluorescence microscopy. Electron-accepting moieties imparted nonlinear optical properties to the chromophores. The electron-rich carbazole core served as a template towards one- or two-dimensional chromophores. More efficient acceptor groups (pyridinium, benzazolium, benzothiazolium) on the carbazole donor core resulted in improved second- and third-order nonlinear optical properties. A selection of these chromophores was tested in a cellular environment with a multimodal multiphoton microscope. The structural differences of the chromophores resulted in high selectivity for mitochondria or the nucleus in two-photon fluorescence and ranging from no signal to high selectivity for mitochondria in the SHG channel.

Measuring the intravitreal mobility of nanomedicines with single-particle tracking microscopy

AIM To develop a robust assay to evaluate and compare the intravitreal mobility of nanoparticles in the intact vitreous body. MATERIALS & METHODS Excised bovine eyes were prepared to preserve the fragile structure of the vitreous humor, while permitting high-resolution fluorescence microscopy and single-particle tracking analysis of intravitreally injected nanoparticles. This assay was validated by analyzing polystyrene beads and further employed to evaluate gene nanomedicines composed of poly(amido amine)s and plasmid DNA. RESULTS The assay was able to distinguish immobilized cationic nanoparticles from mobile PEGylated nanoparticles. PEGylation of the polyplexes resulted in a drastic improvement of their mobility. CONCLUSION An ex vivo eye model is presented for studying nanoparticle mobility in intact vitreous humor by single-particle tracking microscopy. These results give important guidelines for developing gene- and drug-delivery nanomedicines that are compatible with intravitreal administration.

Reactive Oxygen Species in Planarian Regeneration: An Upstream Necessity for Correct Patterning and Brain Formation

Recent research highlighted the impact of ROS as upstream regulators of tissue regeneration. We investigated their role and targeted processes during the regeneration of different body structures using the planarian Schmidtea mediterranea, an organism capable of regenerating its entire body, including its brain. The amputation of head and tail compartments induces a ROS burst at the wound site independently of the orientation. Inhibition of ROS production by diphenyleneiodonium (DPI) or apocynin (APO) causes regeneration defaults at both the anterior and posterior wound sites, resulting in reduced regeneration sites (blastemas) and improper tissue homeostasis. ROS signaling is necessary for early differentiation and inhibition of the ROS burst results in defects on the regeneration of the nervous system and on the patterning process. Stem cell proliferation was not affected, as indicated by histone H3-P immunostaining, fluorescence-activated cell sorting (FACS), in situ hybridization of smedwi-1, and transcript levels of proliferation-related genes. We showed for the first time that ROS modulate both anterior and posterior regeneration in a context where regeneration is not limited to certain body structures. Our results indicate that ROS are key players in neuroregeneration through interference with the differentiation and patterning processes.

Cardiac atrial appendage stem cells engraft and differentiate into cardiomyocytes in vivo: A new tool for cardiac repair after MI.

BACKGROUND This study assessed whether autologous transplantation of cardiac atrial appendage stem cells (CASCs) preserves cardiac function after myocardial infarction (MI) in a minipig model. METHODS AND RESULTS CASCs were isolated from right atrial appendages of Göttingen minipigs based on high aldehyde dehydrogenase activity and expanded. MI was induced by a 2h snare ligation of the left anterior descending coronary artery. Upon reperfusion, CASCs were intramyocardially injected under NOGA guidance (MI-CASC, n=10). Non-transplanted pigs (MI, n=8) received sham treatment. 3D electromechanical mapping (EMM) and cardiac MRI were performed to assess left ventricular (LV) function. MI pigs developed LV dilatation at 2 months (2M), while in the MI-CASC group volumes remained stable. Global LV ejection fraction decreased by 16 ± 8% in MI animals vs 3 ± 10% in MI-CASC animals and regional wall thickening in border areas was better preserved in the MI-CASC group. EMM showed decreased viability and wall motion in the LV for both groups POST-MI, whereas at 2M these parameters only improved in the MI-CASC. Substantial cell retention was accompanied by cardiomyogenic differentiation in 98±1% of the transplanted CASCs, which functionally integrated. Second harmonic generation microscopy confirmed the formation of mature sarcomeres in transplanted CASCs. Absence of cardiac arrhythmias indicated the safety of CASC transplantation. CONCLUSION CASCs preserve cardiac function by extensive engraftment and cardiomyogenic differentiation. Our data indicate the enormous potential of CASCs in myocardial repair.

Cellulose Amorphization by Swelling in Ionic Liquid/Water Mixtures: A Combined Macroscopic and Second‐Harmonic Microscopy Study

Amorphization of cellulose by swelling in ionic liquid (IL)/water mixtures at room temperature is a suitable alternative to the dissolution-precipitation pretreatment known to facilitate enzymatic digestion. When soaking microcrystalline cellulose in the IL 1-ethyl-3-methylimidazolium acetate containing 20 wt % water, the crystallinity of the cellulose sample is strongly reduced. As less than 4 % of the cellulose dissolves in this mixture, this swelling method makes a precipitation step and subsequent energy-intensive IL purification redundant. Second-harmonic generation (SHG) microscopy is used as a structure-sensitive technique for in situ monitoring of the changes in cellulose crystallinity. Combined optical and SHG observations confirm that in the pure IL complete dissolution takes place, while swelling without dissolution in the optimal IL/water mixture yields a solid cellulose with a significantly reduced crystallinity in a single step.

Polarization second harmonic generation by image correlation spectroscopy on collagen type I hydrogels.

Successful engineering of biomimetic tissue relies on an accurate quantification of the mechanical properties of the selected scaffold. To improve this quantification, typical bulk rheological measurements are often complemented with microscopic techniques, including label-free second harmonic generation (SHG) imaging. Image correlation spectroscopy (ICS) has been applied to obtain quantitative information from SHG images of fibrous scaffolds. However, the typical polarization SHG (P-SHG) effect, which partly defines the shape of the autocorrelation function (ACF), has never been taken into account. Here we propose a new and flexible model to reliably apply ICS to P-SHG images of fibrous structures. By starting from a limited number of straightforward assumptions and by taking into account the P-SHG effect, we were able to cope with the typically observed ACF particularities. Using simulated datasets, the resulting model was thoroughly evaluated and compared with models previously described in the literature. We showed that our new model has no restrictions concerning the fibre length for the density retrieval. For certain length ranges, the model can additionally be used to obtain the average fibre length and the P-SHG related non-zero susceptibility tensor element ratios. From experimental data on collagen type I hydrogels, values of SHG tensor element ratios and fibre thickness were determined which match values reported in the literature, thereby underpinning the validity and applicability of our new model.

Label-free mapping of microstructural organisation in self-aligning cellular collagen hydrogels using image correlation spectroscopy

UNLABELLED Hydrogels have emerged as promising biomaterials for regenerative medicine. Despite major advances, tissue engineers have faced challenges in studying the complex dynamics of cell-mediated hydrogel remodelling. Second harmonic generation (SHG) microscopy has been a pivotal tool for non-invasive visualization of collagen type I hydrogels. By taking into account the typical polarization SHG effect, we recently proposed an alternative image correlation spectroscopy (ICS) model to quantify characteristics of randomly oriented collagen fibrils. However, fibril alignment is an important feature in many tissues that needs to be monitored for effective assembly of anisotropic tissue constructs. Here we extended our previous approach to include the orientation distribution of fibrils in cellular hydrogels and show the power of this model in two biologically relevant applications. Using a collagen hydrogel contraction assay, we were able to capture cell-induced hydrogel modifications at the microscopic scale and link these to changes in overall gel dimensions over time. After 24h, the collagen density was about 3 times higher than the initial density, which was of the same order as the decrease in hydrogel area. We also showed that the orientation parameters recovered from our automated ICS model match values obtained from manual measurements. Furthermore, regions axial to cellular processes aligned at least 1.5 times faster compared with adjacent zones. Being able to capture minor temporal and spatial changes in hydrogel density and collagen fibril orientation, we demonstrated the sensitivity of this extended ICS model to deconstruct a complex environment and support its potential for tissue engineering research. STATEMENT OF SIGNIFICANCE It is generally accepted that looking beyond bulk hydrogel composition is key in understanding the mechanisms that influence the mechanical and biological properties of artificial tissues. In this manuscript, we performed label-free non-invasive imaging and extended a robust automated analysis method to characterize the microstructural organisation of cellular hydrogel systems. We underpin the sensitivity of this technique by capturing minor changes in collagen density and fibril orientation in biologically relevant systems over time. Therefore, we believe that this method is applicable in fundamental cell-matrix research and has high-throughput potential in screening arrays of hydrogel scaffolds, making it an interesting tool for future tissue engineering research.

Imaging the zebrafish dentition: from traditional approaches to emerging technologies.

The zebrafish, a model organism for which a plethora of molecular and genetic techniques exists, has a lifelong replacing dentition of 22 pharyngeal teeth. This is in contrast to the mouse, which is the key organism in dental research but whose teeth are never replaced. Employing the zebrafish as the main organism to elucidate the mechanisms of continuous tooth replacement, however, poses at least one major problem, related to the fact that all teeth are located deep inside the body. Investigating tooth replacement thus relies on conventional histological methods, which are often laborious, time-consuming and can cause tissue deformations. In this review, we investigate the advantages and limitations of adapting current visualization techniques to dental research in zebrafish. We discuss techniques for fast sectioning, such as vibratome sectioning and high-resolution episcopic microscopy, and methods for in toto visualization, such as Alizarin red staining, micro-computed tomography, and optical projection tomography. Techniques for in vivo imaging, such as two-photon excitation fluorescence and second harmonic generation microscopy, are also covered. Finally, the possibilities of light sheet microscopy are addressed.

Calcaneal Tendon Collagen Fiber Morphometry and Aging

Fibrillar collagen in tendons and its natural development in rabbits are discussed in this paper. Achilles tendons from newborn (~7 days) to elderly (~38 months) rabbits were monitored in intact (n tendons=24) and microtome sectioned (n tendons=11) states with label-free second harmonic generation microscopy. After sectioning, the collagen fiber pattern was irregular for the younger animals and remained oriented parallel to the load axis of the tendon for the older animals. In contrast, the collagen fiber pattern in the intact samples followed the load axis for all the age groups. However, there was a significant difference in the tendon crimp pattern appearance between the age groups. The crimp amplitude (A) and wavelength (Λ) started at very low values (A=2.0±0.6 µm, Λ=19±4 µm) for the newborn animals. Both parameters increased for the sexually mature animals (>5 months old). When the animals were fully mature the amplitude decreased but the wavelength kept increasing. The results revealed that the microtome sectioning artifacts depend on the age of animals and that the collagen crimp pattern reflects the physical growth and development.