Modeling Oxygen Transport in Three-Dimensional Capillary Networks

The purpose of this thesis was to examine how the use of real 3-dimensional (3D) capillary network geometries affect models of oxygen transport to tissue. Software was developed to reconstruct microvascular geometry in 3D from intravital video. Characterization of 3D reconstructions demonstrated that capillary density, length and capillary diameter were consistent with previous findings. Using reconstructed capillary networks a strategy was devised that utilized red blood cell (RBC) supply rate (SR) as a metric for flow modeling. Applying the RBC SR based flow model on baseline and perturbed flow conditions demonstrated that RBC SR is a major determinant of oxygen delivery that is insensitive to changes in flow distribution. The resulting flow solutions were used for comparing oxygen transport in 3D networks and synthetic parallel arrays. A variety of physiological conditions were simulated and it was determined that parallel arrays resulted in oxygen transport solutions with higher mean PO2 due to homogeneous distribution of vessels in the volume. Lastly, to investigate oxygen transport in a complex pathology a model of sepsis was used to investigate how incremental perfusion loss, consumption increase and change in RBC SR affect oxygen delivery. It was shown that perfusion loss did not markedly impair oxygen delivery provided that RBC SR was maintained. These results have improved our understanding of oxygen transport to tissue in normal and diseased conditions; the use of reconstructed networks and measurements of blood flow & oxygen saturation in computer models provides different solutions than those using statistical averages and synthetic networks

Microcirculation and inflammation in a numerical simulation approach

Inflammation is the response of the organism to eradicate the agent of lesion or infection in order to achieve hemostasis. This response requires the migration of specific leukocyte populations from the blood circulation towards the inflamed area. Leukocyte recruitment constitutes a complex cellular process by which leukocytes are first recruited to the endothelial vascular wall of post-capillary venules across which they further extravasate into the interstitial tissue. Recruitment is mediated via cell-cell interactions between the leukocyte and the endothelium and occurs through a multi-step cascade: tethering, rolling, slow rolling, arrest, crawling, adhesion and transmigration. However, whether or not the leukocytes adhere to the endothelium depends not only on the chemical forces generated by adhesion molecules on leukocytes and endothelial cells, but also on the physical forces that act on those cells. It has been suggested that fluid shear stress resulting from blood flow also regulates leukocyte activity which makes the fluid dynamic environment of the circulation to be considered an important aspect for leukocyte recruitment and migration during the inflammatory response. Most of the studies on the inflammatory response and in particular on leukocyte recruitment are based on animal models and involve, among others, the quantification of inflammatory mediators and cellular players, and/or the analysis of the leukocyte-endothelial cell interactions by intravital microscopy. However, the contribution of hemodynamics for leukocyte recruitment has been seldom addressed in those studies. This is mostly due to the fact that the study of hemodynamics in in vivo animal models is not straightforward and moreover, that several hemodynamic parameters cannot be experimentally determined due to technical constraints. In this work, we reasoned that these limitations could be circumvented by the development and use of numerical simulations to describe leukocyte recruitment. Many of the processes, which take place in living organisms, can be expressed as mathematical equations. This applies to leukocyte recruitment, for which scarce numerical models existed before the beginning of this work. Importantly, these mathematical simulations were performed without considering simultaneously all the players in the process, namely the vessel, the blood flow and the leukocytes. Moreover, most of these studies were two dimensional, assumed blood as a Newtonian fluid with constant viscosity and did not take into account in vivo experimental data. Taken this, our major goal with this work was to understand the contribution of hemodynamics to leukocyte recruitment in inflammation. For such purpose, we aimed here at developing numerical simulations that more adequately reproduced this process. For such, we set up animal models of inflammation to obtain the experimental data required for the development of those numerical simulations. Finally, we used these models to investigate the role of hemodynamics in leukocyte recruitment in inflammation. First, we considered the simpler case of a numerical simulation that assumed leukocytes to be rigid spheres and blood, a non-Newtonian fluid. For such, we initially developed an animal model of inflammation in Wistar rats using a lipopolysaccharide (LPS) as an inflammatory agent. Blood samples were collected for determination of TNF-α levels to ensure the triggering of the inflammatory process. Importantly, the number of rolling and adherent leukocytes in post-capillary venules was monitored using an intravital microscopy approach. As expected, our results showed that there is an increase in TNF-α concentrations after 15 minutes of LPS administration and a significant increase in the number of rolling and adherent leukocytes. The recorded intravital microcopy images, along with other recorded parameters, were then used, in collaboration with a group of mathematicians, to develop a numerical model capable of describing leukocyte recruitment in the microcirculation. To evaluate the contribution of hemodynamics, the localized velocity fields and shear stresses on the surface of leukocytes and near the vessel wall contact points have been computed in two discrete situations, namely as a single leukocyte or when a cluster of them are recruited towards the vessel wall. In the first situation, our numerical results showed the presence of one region of maximum shear stress on the surface of the leuko- cyte close to the endothelial wall and of two regions of minimum shear stress on the op- posite side of the cell. The different areas of shear stress observed in the surface of the leukocyte may be important in directing it towards the endothelial wall during an inflammatory response. The identification of a region of maximum shear stress is consistent with the molecular mechanisms that govern leukocyte rolling because it may actually cor- respond to the area that supports the interaction with the endothelium. On the other hand, the relatively lower shear stress regions may correlate with leukocyte surface areas where binding to the endothelium is not occurring at the moment, thus enabling the roll- ing of the cell along the endothelium. It was also observed that the shear stress at the endothelium gets higher as a cluster of leukocytes moves in the main stream. This sug- gests that the presence of a cluster of leukocytes may potentiate leukocyte rolling, as the increase in the shear stress promoted by the recruited leukocytes may support the migra- tion and recruitment of additional cells. Despite closely simulating leukocyte recruitment, our initial numerical simulation consid- ered the simple case of leukocytes as rigid spheres. However, while circulating leukocytes maintain an approximately spherical shape, rolling leukocytes are known to deform. In order to account for the leukocyte deformability changes that occur during its recruit- ment in inflammation, we needed to assess the deformability profile of the leukocytes under flow and therefore, to “directly” observe them regardless of the other blood cells. For such, intravital microscopy was performed in the mouse cremaster of a transgenic mice strain (Lys-EGFP-ki) in which fluorescent neutrophils can be individually tracked. By using PAF as an inflammatory agent, the analysis of the leukocyte-endothelial cell interac- tions showed a continuous increase in the number of rolling and adherent neutrophils up to 4 hours after the introduction of the inflammatory stimuli, thus confirming the devel- opment of an inflammatory response. As the properties of the red blood cells modulate blood flow properties, erythrocyte deformability was also addressed in this model. A con- tinuous decrease of this parameter was observed throughout time. The decrease in the erythrocyte deformability will most probably lead to an increase in the blood viscosity and to the decrease of the blood flow velocity. These conditions should facilitate the mi- gration of leukocytes from the mainstream to the endothelial wall and promote leukocyte slow rolling and adhesion during the inflammatory response. Importantly, in the intravital microcopy images obtained with this latter model, we clearly observed the deformation of neutrophils along the endothelial wall during rolling, as well as the formation of tethers. As such, in these images, leukocyte trajectories were tracked and their velocities and diameters were measured and further applied to the numerical simulations. Using a recent validated mathematical model describing the coupled defor- mation-flow of an individual leukocyte and the respective experimental results, numerical simulations of the recruitment of an individual leukocyte and of two leukocytes under different velocities were performed, considering a constant blood viscosity. The mathe- matical models obtained showed that under conditions of increased velocity the cell movement is accelerated along the endothelial layer, favouring the dissociation of leuko- cyte-endothelium interactions at designated attraction points. These observations lead us to propose that, in order to attain an efficient inflammatory response, the blood flow ve- locity needs so as to decrease to facilitate slow rolling and subsequent adhesion. These results are corroborated by the decrease in the erythrocyte deformability observed in our animal model, which will ultimately have an impact on the blood flow velocity. Our results further showed that in the vicinity of an adherent leukocyte there is an early slight decel- eration of the rolling leukocyte when compared with the case of an individual leukocyte. As such, these observations strongly suggest that the presence of an adherent cell in the vicinity should decrease the velocity of another leukocyte that is being recruited, thus promoting its slow rolling, and contributing to its capture by the endothelial cells. Altogether, our experimental data and numerical simulations support our working hy- pothesis that the hemodynamic properties of the flow and of the cells in circulation should play an essential role in the margination and rolling of the leukocytes to the endo- thelial wall, which in turn will impact the success of the inflammatory response. In partic- ular, our results strongly suggest that changes in hemodynamic conditions, such as de- creased flow velocities and the increase of the shear stress, will contribute to target leu- kocytes to the endothelial wall. Given our results, we propose that any change in the he- modynamic properties will certainly influence the outcome of the inflammatory response. As such, the adherence of the leukocytes to the endothelium should depend not only on the relative magnitude of the chemical forces generated by the interaction of adhesion molecules between leukocytes and endothelial cells, but also on the physical forces that act on the leukocytes. In this respect, our results suggest that alterations in the blood flow, for example in the flow velocity, will occur during an inflammatory process, thus potentiating the recruitment of more leukocytes towards the inflamed area and contrib- uting to a successful inflammatory response. Overall, the numerical simulations allowed us to better understand the contribution of the hemodynamic properties of the flow to the progression of an inflammatory response and to deepen our knowledge on leukocyte recruitment in inflammation. Importantly, our work provided numerical tools that can be used for the subsequent study and modulation of the hemodynamic parameters involved in an inflammatory response. In particular, these numerical simulations will surely enable us, in the near future, to determine or es- timate a large set of parameters which are unlikely to be recoverable by in vivo experi- ments. Moreover, our methods will allow us to analyze how the parameters evolve over time. Altogether our results further reinforce the notion that the improvement and de- velopment of animal models and numerical tools will certainly provide the medical and biological community with useful tools to study leukocyte recruitment in inflammation. By closely reproducing the microcirculation and the inflammatory process, these tools will be critical for a better comprehension of the inflammatory process and of the mecha- nisms underlying a multitude of inflammatory pathological conditions

High Resolution Intravital Imaging of Subcellular Structures of Mouse Abdominal Organs Using a Microstage Device

Intravital imaging of brain and bone marrow cells in the skull with subcellular resolution has revolutionized neurobiology, immunology and hematology. However, the application of this powerful technology in studies of abdominal organs has long been impeded by organ motion caused by breathing and heartbeat. Here we describe for the first time a simple device designated ‘microstage’ that effectively reduces organ motions without causing tissue lesions. Combining this microstage device with an upright intravital laser scanning microscope equipped with a unique stick-type objective lens, the system enables subcellular-level imaging of abdominal organs in live mice. We demonstrate that this technique allows for the quantitative analysis of subcellular structures and gene expressions in cells, the tracking of intracellular processes in real-time as well as three-dimensional image construction in the pancreas and liver of the live mouse. As the aforementioned analyses based on subcellular imaging could be extended to other intraperitoneal organs, the technique should offer great potential for investigation of physiological and disease-specific events of abdominal organs. The microstage approach adds an exciting new technique to the in vivo imaging toolbox

Spectroscopy detects skeletal muscle microvascular dysfunction during onset of sepsis in a rat fecal peritonitis model

Sepsis is a dysregulated host inflammatory response to infection potentially leading to life-threatening organ dysfunction. The objectives of this study were to determine whether early microvascular dysfunction (MVD) in skeletal muscle can be detected as dynamic changes in microvascular hemoglobin (MVHb) levels using spectroscopy and whether MVD precedes organ histopathology in septic peritonitis. Skeletal muscle of male Sprague–Dawley rats was prepared for intravital microscopy. After intraperitoneal injection of fecal slurry or saline, microscopy and spectroscopy recordings were taken for 6 h. Capillary red blood cell (RBC) dynamics and SO2 were quantified from digitized microscopy frames and MVHb levels were derived from spectroscopy data. Capillary RBC dynamics were significantly decreased by 4 h after peritoneal infection and preceded macrohemodynamic changes. At the same time, low-frequency oscillations in MVHb levels exhibited a significant increase in Power in parts of the muscle and resembled oscillations in RBC dynamics and SO2. After completion of microscopy, tissues were collected. Histopathological alterations were not observed in livers, kidneys, brains, or muscles 6 h after induction of peritonitis. The findings of this study show that, in our rat model of sepsis, MVD occurs before detectable organ histopathology and includes ~ 30-s oscillations in MVHb. Our work highlights MVHb oscillations as one of the indicators of MVD onset and provides a foundation for the use of non-invasive spectroscopy to continuously monitor MVD in septic patients

Magnetic resonance imaging and the development of vascular targeted treatments for cancer.

The main subject of the work presented in this thesis is the further development of magnetic resonance imaging (MRI) as a non-invasive method of investigating tumour microcirculation. Two different MR techniques were used: dynamic contrast enhanced (DCE)-MRI and Blood Oxygen Level Dependent (BOLD)-MRI. Intravital microscopy was used to help interpret BOLD-MRI results. The ultimate aims were to determine whether MRI methods could be relied upon to define a drug as having vascular disrupting activity and to develop techniques to predict the effectiveness of vascular disruptive agents (VDA). In DCE-MRI, tissue enhancement is continuously monitored over several minutes after intravenous injection of contrast medium. Modelling of contrast agent kinetics generates quantitative parameters related to tissue blood flow rate and permeability, e.g. Ktrans (transfer constant). In a clinical study, patients had DCE-MRI examinations before and 24 hours after cytotoxic chemotherapy to establish whether any acute ami-vascular effects could be detected. No acute reductions in Ktrans were seen. In this project, the acute effects of the VDA, combretastatin A-4-phosphate, were investigated using DCE-MRI in SW1222 tumours in mice. Responses were seen both at a clinically relevant dose and at higher doses, and a dose-response relationship established. BOLD-MRI can detect changes in oxygenation and blood flow within tumours using deoxygenated haemoglobin as an intrinsic contrast agent. Tumours contain a variable proportion of immature vessels, which may explain differential sensitivity to VDAs. In this project, BOLD-MRI was used to assess tumour vessel maturity using consequent vasoreactivity to angiotensin II and carbon dioxide (as air-5%C02 or as carbogen) in an animal model. Intravital microscopy was used to directly observe response to these agents in mouse window chambers. Results suggest that response to vasoactive agents is useful for assessing vascular maturity in tumours but that more sensitive non-invasive imaging methods than BOLD-MRI are required for clinical use

Intravital Microscopy Visualizing Immunity in Context

AbstractRecent advances in photonics, particularly multi-photon microscopy (MPM) and new molecular and genetic tools are empowering immunologists to answer longstanding unresolved questions in living animals. Using intravital microscopy (IVM) investigators are dissecting the cellular and molecular underpinnings controlling immune cell motility and interactions in tissues. Recent IVM work showed that T cell responses to antigen in lymph nodes are different from those observed in vitro and appear dictated by factors uniquely relevant to intact organs. Other IVM models, particularly in the bone marrow, reveal how different anatomic contexts regulate leukocyte development, immunity, and inflammation. This article will discuss the current state of the field and outline how IVM can generate new discoveries and serve as a “reality check” for areas of research that were formerly the exclusive domain of in vitro experimentation

Study of Liver Surface Imaging Marker to Monitor Chronic Liver Disease Progression

Ph.DDOCTOR OF PHILOSOPH

Modulating CD4+ T cell migration in the postischemic liver

Background. CD4+ T-cells play a critical role during hepatic ischemia-reperfusion (I/R) injury although the mechanisms of their migration in the post-ischemic liver remain unclear. Recent studies suggest that emigrated T-cells are colocalized with hepatic stellate cells (HSCs) during viral hepatitis. We answered the questions of whether hepatic stellate cells (HSCs) interact with CD4+ T-cells during I/R of the liver and whether modulation of HSC activity affects T-cell-dependent I/R injury. Methods. In mice, migration of CD4+ T-cells was analyzed in vivo using conventional intravital microscopy and two-photon microscopy in sham-operated mice and in mice after I/R (90 min/120min). CD4+ T-cell-HSC interactions were visualized after infusion of fluorescence-labeled CD4+ T-cells into Cx3CR1 mice (mice exhibiting GFP-labeled HSCs) after I/R. Because the activation of HSC is controlled by endocannabinoid receptors, CB-1 and CB-2, the mice received treatment before I/R with the CB-2 agonist JWH-133 to reach HSC depletion or the CB-1 agonist arachidonylcyclopropylamide to activate HSCs. Sinusoidal perfusion and liver transaminases were used as markers of I/R injury. Results. Hepatic I/R induced CD4+ T-cell recruitment in sinusoids. More than 25% of adherent CD4+ T-cells were colocalized with HSCs during reperfusion after ischemia, but not in the sham-operated mice. This is suggesting a direct cell-cell interaction. The HSC deactivation with JWH- 133 significantly attenuated the CD4+ T-cell recruitment in the post-ischemic liver and reduced I/R injury as compared to the vehicle-treated group. The HSC hyperactivation by CB-1, however, did not affect T-cell migration and even increased perfusion failure. Conclusion. Our in vivo data suggest i) that CD4+ cells interact with HSC upon their migration thought the endothelial layer; ii) a selective depletion/deactivation of HSC reduces T-cell-dependent I/R injury, whereas a HSC hyperactivation even accelerates the injury. Taken together, HSC might represent a potential target for future therapeutic strategies against T-cell-mediated I/R injury.CD4+T Zellen spielen eine zentrale Rolle während des Ischämie-Reperfusions Schadens. Dennoch sind die genauen Mechanismen der Zell Migration in der post-ischämischen Leber noch nicht verstanden sind. In unserer Studie versuchten wir zu beantworten ob a) die CD4 T-Zellen während Ischämie-Reperfusion mit hepatischen Sternzellen interagieren und ob b) die pharmakologische Beeinflussung der Sternzellaktivität einen protektiven Effekt auf den T-Zell vermittelten I/R Schaden hat. Die Migration von frisch isolierten und mit Immunfluoreszenzfarbstoff markierten CD4 positiven T-Zellen wurde mittels Intravitalmikroskopie in einer Sham- und der I/R Gruppen von Mäusen untersucht. Sinusoidale Leberperfusion und Leberenzyme wurden als Marker zur Abschätzung des Leberzellschadens verwendet. Die in Punk a) postulierte Interaktion zwischen T-Zellen und hepatischen Sternzellen wurde durch Injektion von fluoreszenzmarkierten T-Zellen in post-I/R-Mäuse mit GFP-exprimierenden Sternzellen (Cx3CR1) mittels Two-photon Mikroskopie untersucht. Nachdem in Studien gezeigt wurde, dass hepatische Sternzellen in ihrer Aktivität durch Stimulierung von Endocannabinoidrezeptoren (CB1 /CB2) beeinflusst werden können, erhielten die Tiere in unseren Versuchen entweder den CB-2 Agonist JWH-133 zur Depletion von Sternzellen oder den CB-1 Agonist ACPA zur Aktivierung dieser. Hepatische I/R führte in allen Gruppen zu T-Zell Rekrutierung in den Sinsusoiden. In der JWH-133 Gruppe führte die Deaktivierung der Sternzellen zu einer deutlich verringerten Ansammlung von T-Lymphozyten in die Sinusoide sowie signifikant geringeren hepatischen I/R Schaden verglichen mit den anderen Gruppen. HSC über-Aktivierung durch CB-1 Stimulation hatte keinen Einfluss auf T-Zell Rekrutierung, ging jedoch mit höherem hepatischem Perfusionsversagen einher. Demnach suggerieren unserer Daten, dass a) in der Tat eine Interaction zwischen HSC und T-Zellen während der Postischämie stattfindet und b) selektive Depletion/Deaktivierung von HSC durch CB-2 Agonisten zu geringerem I/R Schaden führt. Somit könnten HSC in Zukunft als ein potentielles Ziel für therapeutische medikamentöse Intervention im Rahmen der T-Zell vermittelten I/R Schaden gesehen werden

Experimental, theoretical, and translational studies of RBC distribution in capillary networks

The purpose of this thesis was to evaluate the physiology of red blood cell (RBC) distribution in skeletal muscle capillary networks. Because this is the terminal site of oxygen exchange in the microcirculation, characterization of this fundamental process informs an understanding of how microvascular blood flow regulation matches oxygen supply with local tissue demand. Prior studies in this field have focused on small groups of capillaries, and have not linked capillary network structure with functional measurements, nor evaluated the temporal complexity of RBC distribution over physiologically-relevant scales. It is also unclear how the functional units called capillary modules – comprised of parallel capillaries from arteriole to venule – relate together within large capillary networks. In this thesis, we employed multiple methodologies to achieve this goal with preclinical animal models, theoretical simulations, and translational studies in human patients. First, we used intravital videomicroscopy of resting extensor digitorum longus muscle in rats and discovered that skeletal muscle capillary networks are organized into columns of interconnected capillary modules spanning thousands of microns – a structure we called the Capillary Fascicle. We showed that capillary-RBC hemodynamics are heterogeneous within a module and between modules. Next, we evaluated capillary module hemodynamics and demonstrated that RBC flow is independent of module resistance, providing evidence for regulation of driving pressure at the level of the capillary module, that requires pre- and post-capillary mechanisms of control. Using a dual-phase mathematical model of blood flow within artificial capillary module geometries, we showed that RBC flow heterogeneity is an intrinsic property of capillary module structure, and that variations to inflow hematocrit and pressure impact RBC distribution as a consequence of the rheological properties of microvascular blood flow. Finally, we used high-resolution near-infrared spectroscopy to monitor the temporal variability of hemoglobin content in skeletal muscle of patients in the intensive care unit (ICU). We showed that RBC perfusion characteristics are not consistent between patients, and that ICU interventions directly impact microvascular RBC distribution. Together, these studies support a theory of capillary networks as active participants in microvascular blood flow regulation, with structural features of capillary networks contributing to functional characteristics of RBC distribution

Development of three-dimensional, ex vivo optical imaging

The ability to analyse tissue in 3-D at the mesoscopic scale (resolution: 2-50 µm) has proven essential in the study of whole specimens and individual organs. Techniques such as ex vivo magnetic resonance imaging (MRI) and X-ray computed tomography (CT) have been successful in a number of applications. Although MRI has been used to image embryo development and gene expression in 3-D, its resolution is not sufficient to discriminate between the small structures in embryos and individual organs. Furthermore, since neither MRI nor X-ray CT are optical imaging techniques, none of them is able to make use of common staining techniques. 3-D images can be generated with confocal microscopy by focusing a laser beam to a point within the sample and collecting the fluorescent light coming from that specific plane, eliminating therefore out-of-focus light. However, the main drawback of this microscopy technique is the limited depth penetration of light (~1 mm). Tomographic techniques such as optical projection tomography (OPT) and light sheet fluorescence microscopy (also known as single plane illumination microscopy, SPIM) are novel methods that fulfil a requirement for imaging of specimens which are too large for confocal imaging and too small for conventional MRI. To allow sufficient depth penetration, these approaches require specimens to be rendered transparent via a process known as optical clearing, which can be achieved using a number of techniques. The aim of the work presented in this thesis was to develop methods for threedimensional, ex vivo optical imaging. This required, in first instance, sample preparation to clear (render transparent) biological tissue. In this project several optical clearing techniques have been tested in order to find the optimal method per each kind of tissue, focusing on tumour tissue. Indeed, depending on its structure and composition (e.g. amount of lipids or pigments within the tissue) every tissue clears at a different degree. Though there is currently no literature reporting quantification of the degree of optical clearing. Hence a novel, spectroscopic technique for measuring the light attenuation in optically cleared samples is described in this thesis and evaluated on mouse brain. 5 Optical clearing was applied to the study of cancer. The main cancer model investigated in this report is colorectal carcinoma. Fluorescently labelled proteins were used to analyse the vascular network of colorectal xenograft tumours and to prove the effect of vascular disrupting agents on the vascular tumour network. Furthermore, optical clearing and fluorescent compounds were used for ex vivo analysis of perfusion of a human colorectal liver metastasis model