Microvascular transport is strongly influenced by the nonuniform partitioning of red blood cells at diverging microvessel bifurcations, where blood flows from one mother vessel into two daughter vessels. In such bifurcations, the volume fractions of red blood cells in the blood entering each daughter vessel typically differ significantly from the volume fraction in the mother vessel. This phenomenon is caused, to a first approximation, by nonuniform distribution of red blood cells in the cross-section of the mother vessel and the tendency of red blood cells to follow background fluid streamlines. In smaller vessels, however, red blood cell trajectories can deviate significantly from fluid streamlines. In this dissertation, the mechanical reasons for these deviations and their contributions to nonuniform partitioning are analyzed.A two-dimensional model is used to simulate the motion and deformation of flexible particles as they travel alone through a diverging microvessel bifurcation. Deviations of particle trajectories from background fluid streamlines result from migration towards the mother vessel centerline upstream of the bifurcation and flow perturbations caused by cell obstruction in the bifurcation region. Cell migration, which arises because of flexibility, causes more nonuniform partitioning while cell obstruction causes more uniform partitioning. Bifurcations with differently sized daughter vessels experience, on average, a higher red blood cell flux into the smaller branch. Partitioning is unaffected by daughter branching angles.The motion of two interacting cells is also considered. In diverging bifurcations several types of interactions were found, in which the presence of a nearby cell causes a cell to enter a different branch than it would have otherwise. The net effect of these interactions is to cause more uniform partitioning. In wall-bounded linear shear flow, a two-dimensional swapping interaction was identified, in which two cells on different background fluid streamlines approach each other, slowly move onto their partner's streamline, and then move away from each other.The simulations produced by this two-dimensional model provide insight into the effects of red blood cell deformability, bifurcation geometry and volume fraction of red blood cells on red blood cell partitioning and on the resultant distribution and transport of materials in the microvasculature.

Microvasculatures may become damaged by a variety of acute and chronic diseases. In many cases, microvessel function is irreversibly compromised, leading to the dysfunction and even death of surrounding tissues. Currently, there are few therapies that directly address the treatment of microvascular insufficiency. Responding to this need, researchers are developing methods to fabricate in vitro blood vessels. Typical strategies include; cellular sodding within polymers and/or biopolymers, the formation of cylindrical cellular monolayers around polymer mandrels, and the modification of biocompatible surfaces for cellular adhesion. Using currently available techniques, simple, individual vessel conduits have been engineered with internal diameters down to 150μm. However, no evidence has been provided illustrating the formation of patent, interconnected microvessel networks without the aid of a host circulatory system. In response to this challenge, it is hypothesized that a novel flow-based experimental system will support the in vitro development of three-dimensional microvascular tissues. Addressing this hypothesis, the presented work focused on three specific aims: Specific Aim 1. Pattern planar in vitro three-dimensional microvasculatures. Specific Aim 2. Engineer a Dynamic In vitro Perfusion Chamber (DIP Chamber) for microvascular investigation. Specific Aim 3. In vitro perfusion of microvessel fragments within the DIP Chamber. Through the supporting experiments, directed endothelial sprouting from parent isolated microvessel fragments was achieved. In addition, patent in vitro microvessel networks were successfully developed. The presented experiments are the first to achieve these experimental results. In addition, the described experimental model will provide a unique method for future investigations of microcirculatory phenomena. Since no exogenous growth factors or cell signals were introduced into the constructs, it is believed that this system presents a physiological platform for future investigations into angiogenesis, angioadaptation, and network remodeling. Moreover, this model may offer a useful platform for vascular therapeutic testing and a foundation for future tissue engineering applications.

Made available in DSpace on 2014-06-11T19:23:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-03-02Bitstream added on 2014-06-13T20:29:59Z : No. of bitstreams: 1 cavalcante_sp_me_botfm.pdf: 252896 bytes, checksum: 04649d19f170ae42a833e1b00765902d (MD5) / Fundação Pio Xii - Barretos / Avaliar a densidade microvascular (DMV) em astrocitomas de baixo grau (ABG), astrocitomas anaplásicos (AA) e glioblastoma multiforme (GBM) por imuno-histoquímica, correlacionando com índices de captação pela SPECT com SESTAMIBI (MIBI). Estudo transversal com coleta retrospectiva que avaliou 48 pacientes, com faixa etária de 20 a 73 anos, com o diagnóstico de tumores cerebrais ditos ABG (somente os difusos), AA e GBM admitidos no Hospital de Câncer da Fundação Pio XII de Barretos. As SPECT-MIBI foram classificadas como alteradas ou normais inicialmente pela análise visual. Também foram analisadas de forma semiquantitativa através do desenho de regiões de interesse (RI) com a obtenção de um índice para correlacionar com os parâmetros da DMV. Esta foi determinada com o emprego de anticorpo anti-CD34. Os GBM, AA e ABG representaram 50%, 16,7% e 33,3% da amostra, respectivamente. Treze exames foram visualmente normais, e 35 considerados alterados. A DMV média teve diferença significativa entre os AA e ABG (p=0,040), mas não entre as SPECT-MIBI normais e alteradas. Os índices de contagem média obtidos através da análise semiquantitativa das SPECT-MIBI não apresentaram correlação com a DMV. Entre os GBM não foi encontrada nenhuma significância, exceto pela maior probabilidade de encontrar-se exames alterados neste tipo histológico. A DMV demonstrou relação com o grau histológico entre os AA e ABG, mas os índices de captação das SPECT-MIBI não apresentaram correlação com a DMV. / To evaluate the microvascular density (MVD) in low-grade astrocytomas (LGA), anaplastic astrocytomas (AA) and glioblastoma multiforme (GBM) by immunohistochemistry technique using anti-CD34, correlated with SPECT-MIBI uptake parameters. This is a cross-sectional study with retrospective assessment data which evaluated 48 subjects, ages ranging from 20 to 73 years, all with diagnosis of brain tumors known as LGA (only diffuse type), AA and GBM who were admitted to the Hospital de Cancer da Fundação Pio XII de Barretos. The SPECT-MIBI images were initially classified as normal or altered by visual analysis. Then they were also considered for semiquantitative analysis through drawing of anatomical regions of interest (ROI) resulting in an index to correlate with the MVD parameters. DMV was determined with the use of monoclonal antibody anti-CD34. GBM, AA and LGA represented 50%, 16.7% and 33.3% of the sample, respectively. Thirteen images were visually normal, while 35 were considered abnormal. There were significant differences in MVD between AA and LGA (p = 0.04), but not between the normal and abnormal SPECT-MIBI. The mean counts obtained by semiquantitative analysis from SPECT-MIBI showed no correlation with MVD. Among GBM subjects it was not seen any significance, except for being most likely to find this histological test as abnormal. MVD had relationship with the histological grade between AA and LGA, but there was no correlation with SPECT-MIBI.

Objective - To assess donor site morbidity and survival of the rectus abdominis muscle with an overlying skin graft after free tissue transfer to a medial femorotibial defect in dogs. Study Design - Experimental study Sample Population: Phase one - six canine cadavers / Phase two - seven adult mixed breed dogs Methods: Phase one - The rectus abdominis muscle was removed from cadavers, muscular and vascular dimensions were recorded and angiography was performed. Phase two - Muscular transfer was performed through anastomosis of the caudal epigastric vasculature to the saphenous vasculature. Transferred tissues were evaluated on postoperative days three, six, 10, and 13. Animals were examined daily until euthanasia between postoperative days 31 and 42. Postmortem angiograms were performed and tissues collected for histopathologic evaluation. Results: Phase one - Appropriate vascular dimensions for microvascular anastomosis were confirmed and surgical technique perfected. Phase two – Muscular excision produced minimal donor site morbidity. All muscles survived after microvascular transfer and angiography confirmed vascular patency. All skin grafts survived with one graft undergoing partial necrosis. Conclusions: The rectus abdominis muscle can be successfully transferred to a medial femorotibial defect and serve as a bed for acute skin grafting. No significant donor site morbidity is associated with its removal. Clinical Relevance: Microvascular free tissue transfer of the canine rectus abdominis muscle has not been previously described. This technique provides an alternative for repair of appropriate wounds. Additional studies are needed to define its utility in clinical patients. / Master of Science

Microvascular dysfunction is believed to precede the development and contribute to the progression of obesity related diseases such as insulin resistance, hypertension, and coronary artery disease. Multiple studies have found impaired microvascular endothelium-dependent vasodilation occurs prior to the onset of disease in middle aged adults. In order to test the hypothesis that the cutaneous microvasculature of young obese (BMI>30kg/m²), but otherwise healthy, adults would exhibit impaired microvascular response, we recruited 12 obese and 12 lean (BMI<25 kg/m²) individuals. Each group was age-matched and consisted of 5 females and 7 males. Each participant was instrumented with two microdialysis probes inserted in the dermis of the non-dominant forearm for a wide dose range of infusions of either the endothelium-dependent vasodilator methacholine (MCh) or the endothelium-independent vasodilator sodium nitroprusside (SNP). Each microdialysis site was clamped at 33°C with a local heater and affixed with a laser Doppler flux (LDF) probe for determination of local red blood cell flux, an index of blood flow. LDF was recorded continuously while 7 doses of each drug (MCh: 10⁻³-10³mM; SNP: 5x10⁻⁵-50mM) were infused at a rate of 2 [mu]l/min for 8 minutes per dose. Both sites finished with heating to 43°C and infusion of 50mM SNP to confirm site specific maximal vasodilation. Blood pressure was recorded in the last minute of each stage and the corresponding LDF was used to calculate cutaneous vascular conductance (CVC). Dose response curves for CVC at each dose, as well as maximal CVC were analyzed. MCh dose response showed a trend toward endothelium–dependent impairment in obese (p=0.06) and maximal absolute CVC at the MCh site was attenuated in obese versus lean (2.70 ± 0.73 vs 3.30 ± 0.81 LDF/mmHg, p=0.027). Endothelium-independent vasodilation with SNP was impaired at the 4 highest doses of SNP (all P<0.006) and maximal CVC was attenuated in obese compared to lean (2.44 ± 0.74 vs 3.31 ± 0.65 LDF/mmHg, p=0.004). These results support the hypothesis that microvascular function is impaired in young, healthy obese, individuals; however they suggest the impairment is partially endothelium-independent. / text

The deleterious effects of Type 2 diabetes on the brain have been shown to result in a greater prevalence of age-associated cognitive impairment and an enhanced risk of age-related cognitive decline in older diabetic populations. Type 2 diabetes is a complex metabolic disorder. Apart from the negative impact of abnormalities intrinsic to diabetes, diabetes-associated cerebral microvascular disease may contribute to this accelerated cognitive ageing. Direct in vivo evaluation of the cerebral microcirculation is difficult in humans and the vessels themselves are too small to permit detailed visualisation with current neuroimaging methods. The microvasculature of the retina may offer a window into such vascular status of the brain as there is considerable homology between the retina and cerebral microcirculations. Moreover, the retinal vasculature is known to be affected by a wide range of systemic pathologies and is unique in that it is the only vasculature that can be directly visualised and photographed. Retinal microvascular abnormalities (RMAs) have been understudied risk factors in cognitive ageing epidemiological research. Few reports have comprehensively examined cognitive function in relation to diabetic retinopathy. Also the relationship between cognitive function and quantitative aspects of retinal vascular network geometry has not been investigated in people with Type 2 diabetes. The results of a systematic review reported in this thesis showed inconsistent findings on the importance of the association between retinal microvascular abnormalities and cognitive dysfunction in predominantly non-diabetic populations. This may have reflected substantial differences between studies regarding the choice of population under study, the methods applied for measuring and defining RMAs, the types of neuropsychological tests administered for assessing cognitive function, and the approach taken in data analysis. The principal aim of the original research described in this thesis was to examine the associations of cognitive test performance with severity of diabetic retinopathy and quantitative parameters of retinal vascular network in a population-based sample of older people with Type 2 diabetes. Objective, reproducible and computerized retinal image analysis was used to quantify retinal vessel calibres and arteriolar bifurcation geometry in order to detect subtle changes in retinal vascular network. A valid estimation of peak prior cognitive ability allowed the further exploration of the impact of retinal microvascular abnormalities on imputed cognitive decline from best-ever levels of cognitive function to that measured in old age. The analysis was based on a cohort of 547 men and 519 women aged 60-75 years with Type 2 diabetes, randomly sampled from the Lothian Diabetes Register, Scotland, in 2006/2007 (the Edinburgh Type 2 Diabetes Study). A battery of seven cognitive tests was administered and standard 7-field binocular digital retinal photography undertaken. The Mill Hill Vocabulary Scale was used to estimate pre-morbid cognitive ability. Diabetic retinopathy was evaluated independently by two optometrists using a standardised grading protocol (a modification of the Early Treatment of Diabetic Retinopathy Scale). Quantitative retinal vascular parameters were measured by myself from a digital image of field 1 using semi-automated, computer-based methods. Retinal vessel calibres were summarised as the central retinal arteriolar and venular equivalents (CRAE and CRVE, respectively) and arterio-venous ratio (AVR). Retinal arteriolar bifurcation geometry was expressed as arteriolar bifurcation angles (BA), arterial branching coefficient (BC), and sub-optimality (degree of deviation from optimality) of the retinal arteriolar angles. The statistical analyses were based on the 1,044 study participants who had both gradable retinal images and cognitive testing. Both general cognition, as indexed by a general cognitive factor reflecting the variance common to all the cognitive tests used, and most of the individual cognitive tests were negatively affected in participants with diabetic retinopathy relative to those without. These cognitive measures also showed a significant relationship with increasing severity of diabetic retinopathy (none, mild, and moderate-severe). Those with moderate-severe diabetic retinopathy had worst performances on general cognitive function, executive function, information processing speed, non-verbal memory and mental flexibility. When lifetime decline was estimated from peak, prior cognitive level, severity of diabetic retinopathy was significantly associated with a greater decline in information processing speed, non-verbal memory and mental flexibility and, in men for general cognition and executive function. The associations of severity of diabetic retinopathy with general cognition, executive function and information processing speed were independent of socio-demographic characteristics, cardiovascular risk factors, macrovascular disease, mood and hyperglycaemia. The associations with estimated decline in specific cognitive measures resulted principally from the impact of diabetic retinopathy on general cognitive ability. The study also showed that larger retinal arteriolar and venular calibres were both significantly associated with lower test scores on verbal memory in men. Multiple linear regression analyses demonstrated larger retinal arteriolar calibre was associated with a significantly greater decline in verbal memory after possible confounding by retinal venular calibre and vascular risk factors and disease was taken into account. In contrast, the study did not support an independent association between retinal venular calibre and cognitive decline in men or in women with Type 2 diabetes. Parameters of retinal arteriolar bifurcation geometry were not associated with cognitive outcome. Overall, these findings support the hypothesis that cerebral microvascular disease associated with Type 2 diabetes, reflected by the presence and severity of diabetic retinopathy, may exacerbate the effects of ageing on cognitive function. In particular, alterations in the blood-brain barrier may be an important pathophysiological mechanism in the occurrence of cognitive dysfunction in diabetic patients. They further may be added to the knowledge that gained from previous pathologic and brain imaging investigations demonstrating a relationship between markers of cerebral microvascular disease and cognitive dysfunction in diabetes. The role of quantitative parameters of retinal vascular network geometry in diabetes-related cognitive impairment is less clear. Prospective studies are required to clarify the temporal sequence of these associations and the eventual clinical significance of these small, early cognitive function changes. Such a follow-up project involving the present study population is underway. From a clinical perspective, if the above findings are substantiated, diabetes-associated cognitive dysfunction may be amenable to treatment and preventive strategies specifically targeted at protecting the cerebral microvasculature and reducing the risk of developing even mild microvascular disease in an ageing diabetic population.

Junctional adhesion molecule C (JAM-C) is a component of endothelial cell (EC) tight junctions that has been implicated in a number of endothelial functions, such as angiogenesis and trafficking of leukocytes through the endothelium during inflammation. Work within our lab has identified that loss of JAM-C at EC junctions results in increased reverse transendothelial migration (rTEM) of neutrophils back into the circulation, a response that has been associated with the dissemination of inflammation to distant organs. Whilst the mechanism by which JAM-C is lost or redistributed away from EC junctions has begun to be elucidated, little is known about how loss of endothelial JAM-C impacts the functions of ECs. As such, this thesis aimed to investigate the effect of JAM-C deficiency on EC functions to unravel possible molecular and cellular mechanisms of mediating neutrophil rTEM. To address the effect of JAM-C deficiency on EC functions, an in vitro RNA interference (RNAi) approach was used to efficiently knock-down (KD) JAM-C in human umbilical vein ECs (HUVECs). Importantly, KD of JAM-C did not affect expression of other key EC junctional markers such as JAM-A and VE-Cadherin and cell proliferation and apoptosis were similarly unaffected. Gene expression profiling using microarrays revealed that JAM-C depleted HUVECs exhibited a pro-inflammatory phenotype under basal conditions that was characterised by increased expression of pro-inflammatory genes such as ICAM1 and IL8. Following IL-1β-induced inflammation, no difference in expression of pro-inflammatory genes was detected between control and JAM-C KD HUVECs. However, protein levels of secreted chemokines such as IL-8 were reduced in JAM-C KD HUVECs following stimulation with IL-1β. This was corroborated by in vivo studies demonstrating reduced levels of secreted chemokines in the plasma of mice where JAM-C was conditionally deleted from ECs. A novel finding of this work is the demonstration that JAM-C KD HUVECs exhibit increased autophagy under basal conditions. This might provide a potential mechanism for the reduced chemokine secretion that is observed in this system, whereby chemokines are preferentially trafficked for autophagosome-mediated degradation. Taken together, these findings indicate a multi-functional role for JAM-C in regulating EC homeostasis under basal conditions. JAM-C KD ECs respond aberrantly to inflammatory stimuli by secreting reduced chemokine levels, a consequence that could provide novel insights into the mechanisms of neutrophil rTEM under conditions of endothelial JAM-C loss.

archives@tulane.edu / Experimental stroke in endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) knockout mice showed diverse effects on brain injury. nNOS and eNOS have been shown to uncouple in pathological conditions to produce superoxide. Oxidative stress is believed to be the underlying cause of several cardiovascular diseases including ischemic stroke. However, the role of eNOS and nNOS uncoupling in ischemic stroke is not well studied. Our objective of the study was to determine the effect of eNOS and nNOS inhibition on reactive oxygen species (ROS), NO, viability and mitochondrial bioenergetics in rat brain microvascular endothelial cells (BMECs) and rat cortical neurons following oxygen-glucose deprivation-reoxygenation (OGD/R). We found that non-specific inhibition of NOS in endothelial cells reduced ROS levels in BMECs but increased ROS levels in neurons under normoxia. This suggests that a pool of uncoupled NOS exists in the BMECs whereas the dominant functional NOS in neurons produces NO. We observed increased levels of ROS following OGD/R that is sensitive to NOS inhibition in both BMECs and neurons indicating eNOS and nNOS uncoupling during OGD/R. Furthermore, NOS inhibition reduced mitochondrial respiration while it improved cell survival rate in both BMECs and neurons following OGD/R. Thus, it is possible that decreased mitochondrial respiration in the immediate aftermath (4 hours) of OGD/R could be protective against reoxygenation injury. Moreover, we identified the expression of nNOS in BMECs from rat, human, and mouse. We observed that the nNOS in the BMECs constitutively produces superoxide under physiological conditions instead of NO. In contrast, nNOS in the neurons produces NO and doesn’t contribute to ROS. We also confirmed the nNOS expression and its function in freshly isolated rat brain microvessels. In addition, we developed a novel method to measure mitochondrial respiration in freshly isolated mouse brain microvessels using Seahorse XFe24 Analyzer. We validated the method by demonstrating impaired mitochondrial respiration in cerebral microvessels isolated from old mice compared to young mice. In summary, the present doctoral research investigated the distinct role of NOS isoforms in BMECs and Neurons leading to the identification of novel functional variant of nNOS in BMECs and brain microvessels. / 1 / RAMARAO SVNL

Transcapillary exchange is the movement of fluid and molecules through the porous capillary wall, and is important in maintaining homeostasis of bodily tissues. The classical view of this process is that of Starling's hypothesis, in which the forces driving filtration or absorption are the hydrostatic and osmotic pressure differences across the capillary wall. However, experimental evidence has emerged suggesting the importance of the capillary wall ultrastructure, and thus rather than the global differences between capillary and tissue, it is the local difference across a structure lining the capillary wall known as the endothelial glycocalyx that determines filtration. Hu and Weinbaum presented a detailed cellular level microstructural model of this phenomenon which was able to explain some experimental discrepancies. In this Thesis, rather than describing the microstructural details, the capillary wall is treated as a poroelastic material. The assumptions of poroelasticity theory are such that the detailed pore structure is smeared out and replaced by an idealized homogeneous system in which the fluid and solid phases coexist at each point. The advantage of this approach is that the mathematical problem is greatly simplified such that analytical solutions of the governing equations may be obtained. This approach also allows calculation of the stress and strain distribution in the tissue. We depart from classical poroelasticity, however, due to the fact that since there are concentration gradients within the capillary wall, the filtration is driven by both hydrostatic and osmotic pressure gradients. The model predictions for the filtration flux as a function of capillary pressure compares favourably with both experimental observations and the predictions of the microstructural models. An important factor implicated in transcapillary exchange is the endothelial glycocalyx, which was shown experimentally to protect against edema formation. Using our theory in combination with the experimental measurements of glycocalyx thickness and pericapillary space dimension (PSD), we make a quantitative comparison for the excess flow as a result of a deteriorated glycocalyx, which shows reasonably good agreement with the data. Since many of the parameters in the model are difficult to measure, a sensitivity analysis was performed on the most important of these. Finally, since there was variation in the measurements of glycocalyx thickness and PSD, we used probability distributions to represent the data, and performed further calculations to obtain ranges of likely values for the various parameters. This work could find applications in cardiovascular disease, where the glycocalyx is degraded or absent, and in cancer research, where the abnormal vasculature is an impediment to the efficient delivery of anti-cancer drugs.

The heart and microvasculature have characteristics of a complex adaptive system. Extreme challenges faced by these organ systems cause structural changes which lead to global adaptation. To assess the impact of myocardial interstitial edema on the mechanical properties of the left ventricle and the myocardial interstitium, we induced acute and chronic interstitial edema in dogs. With chronic edema, the primary form of collagen changed from type I to III and left ventricular chamber compliance significantly increased. The resulting functional adaptation allows the chronically edematous heart to maintain left ventricular chamber compliance when challenged with acute edema, thus, preserving cardiac function over a wide range of interstitial fluid pressures. To asses the effect of microvascular occlusions, we reintroduced the Pallid bat wing model and developed a novel mathematical model. We hypothesized that microvessels can switch from predominantly pressure-mediated to shear-mediated responses to ensure dilation during occlusions. Arterioles of unanesthetized Pallid bats were temporarily occluded upstream (n=8) and parallel (n=4) to vessels of interest (20-65 mm). In both cases, the vessels of interest rapidly dilated (36+24 %, 37+33 %), illustrating that they responded appropriately to either decreased pressure or increased shear stress. The model not only reproduced this switching behavior, but reveals its origin as the nonlinear shear-pressure-radius relationship. The properties of the heart and microvasculature were extended to characterize a “Research-Intensive Community” (RIC) model, to provide a feasible solution consistent with the Boyer Commission, to create a sustainable physiology research program. We developed and implemented the model with the aim of aligning diverse goals of participants while simultaneously optimizing research productivity. While the model radically increases the number of undergraduate students supported by a single faculty member, the inherent resilience and scalability of this complex adaptive system enables it to expand without formal institutionalization.