Mitral regurgitation (MR) is associated with increased neuronal release of norepinephrine (NE) and epinephrine (EP) into myocardial interstitial fluid (ISF) that may be necessary in sustaining left ventricular (LV) function via activation of cardiomyocyte β-adrenergic receptors (ARs). However, activation of neuronal β-ARs on cardiac neurons may lead to further catecholamine release, with an attendant risk of functional deterioration. We hypothesize that a beneficial effect of β-AR blockade may therefore mitigate excessive catecholamine release from cardiac adrenergic neurons in dogs with MR. We measured the effects of chronic β-receptor blockade (β-RB) on ISF NE and EP release using in vivo microdialysis in open-chest anesthetized dogs after 4 wk of MR with or without extended release of metoprolol succinate (100 mg/day) as well as in control dogs. Fractional shortening increased by 30% in both MR and MR + β-RB dogs after 4 wk of MR. In MR + β-RB dogs, stellate-stimulated heart rate change was attenuated compared with control and MR dogs, whereas peak change of LV pressure over time (+dP/dt) increased equally in all groups. Stellate-stimulated ISF NE increased fivefold over baseline in MR versus twofold in control dogs (<0.05), but the NE release was significantly attenuated in MR + β-RB dogs. In contrast, stellate-stimulated increases in ISF EP did not differ in control, MR, and MR + β-RB dogs. This study demonstrates that β-RB attenuates ISF NE release from cardiac neurons and that the LV functional response to MR is not dependent on an excess increase in ISF NE. Thus β1-RB may exert a beneficial effect by attenuating untoward effects of excessive sympathetic efferent neural NE release while sustaining early LV functional adaptation to MR.

Amikacin-, vancomycin- or amikacin/clindamycin-impregnated gel was placed subcutaneously on either side of horses' necks a total of 6 times each. Interstitial fluid was collected at 0, 4, 8, 12 and 24 hours, and days 2 through 10, via capillary ultrafiltration probes placed within the incision (0cm) and 1.5cm laterally. Plasma or serum was collected at days 0, 1 and 7. Biopsy samples were obtained at the completion of the study. A histomorphologic score was assigned to each sample, and the differences in mean scores between treatment (gel) and control incisions were assessed using Wilcoxon signed rank test. Amikacin and vancomycin samples were analyzed via fluorescence polarization immunoassay; clindamycin samples were analyzed via high performance liquid chromatography. Concentrations greater than 2000 times the MIC of vancomycin and clindamycin, greater than 1000 times the MIC of amikacin, and greater than 800 times the MIC of amikacin (amikacin/clindamycin gel) were obtained at 0cm. Mean concentrations remained above MIC for vancomycin and clindamycin for 10 days (0cm) and 8 days (1.5cm); for 9 days (0cm) and 7 days (1.5cm) for amikacin gel; and for 9 days (0cm) and 5 days (1.5cm) for amikacin (amikacin/clindamycin gel). Mean plasma amikacin and vancomycin concentrations were negligible; serum clindamycin concentrations were greater than MIC (0.52µg/ml and 0.63µg/ml) at 24 hours and 7 days respectively. There were no significant differences in histomorphologic scores between treatment and control incisions. Cross-linked dextran gel is a safe, effective alternative for local antibiotic delivery in horses, with substantially high local concentrations and minimal systemic absorption for amikacin- and vancomycin-impregnated gels. / Master of Science

No description available.

Gliomas are the most common and aggressive primary brain tumors. The most common treatment protocols for these brain tumors are combinations of surgery, chemotherapy and radiotherapy. However, even with the most aggressive combination of surgery and radiotherapy and/or chemotherapy schedules, gliomas almost always recur resulting in a median survival time for patients of not more than 12 months. This highly diffusive and invasive nature of brain tumors makes it very important to study the effects of these combined therapeutic strategies in an effort to improve the survival time of patients. It is also important to study the tumor microenvironment, since the complex nature of the cerebral vasculature, including the blood brain barrier and several other tumor-induced conditions such as hypoxia, high interstitial pressure, and cerebral edema affect drug delivery as well as the effectiveness of radiotherapy. Recently, a novel strategy using antiangiogenic therapy has been studied for the treatment of brain tumors. Antiangiogenic therapy interferes with the development of tumor vasculature and indirectly helps in the control of tumor growth. Recent clinical trials suggest that anti-angiogenic therapy is usually more effective when given in combination with other therapeutic strategies. In an effort to study the effects of the aforementioned therapeutic strategies, a spatio-temporal model is considered here that incorporates the tumor cell growth and the effects of radiotherapy and chemotherapy. The effects of different schedules of radiation therapy is then studied using a generalized linear quadratic model and compared against the published clinical data. The model is then extended to include the interactions of tumor vasculature and oxygen concentration, to explain tumor hypoxia and to study various methods of hypoxia characterizations including biomarker estimates and needle electrode measurements. The model predicted hypoxia is also used to analyze the effects of tumor oxygenation status on radiation response as it is known that tumor hypoxia negatively influences the radiotherapy outcome. This thesis also presents a detailed analysis of the effects of heterogenous tumor vasculature on tumor interstitial fluid pressure and interstitial fluid velocity. A mathematical modeling approach is then used to analyze the changes in interstitial fluid pressure with or without antiangiogenic therapy.

Gliomas are the most common and aggressive primary brain tumors. The most common treatment protocols for these brain tumors are combinations of surgery, chemotherapy and radiotherapy. However, even with the most aggressive combination of surgery and radiotherapy and/or chemotherapy schedules, gliomas almost always recur resulting in a median survival time for patients of not more than 12 months. This highly diffusive and invasive nature of brain tumors makes it very important to study the effects of these combined therapeutic strategies in an effort to improve the survival time of patients. It is also important to study the tumor microenvironment, since the complex nature of the cerebral vasculature, including the blood brain barrier and several other tumor-induced conditions such as hypoxia, high interstitial pressure, and cerebral edema affect drug delivery as well as the effectiveness of radiotherapy. Recently, a novel strategy using antiangiogenic therapy has been studied for the treatment of brain tumors. Antiangiogenic therapy interferes with the development of tumor vasculature and indirectly helps in the control of tumor growth. Recent clinical trials suggest that anti-angiogenic therapy is usually more effective when given in combination with other therapeutic strategies. In an effort to study the effects of the aforementioned therapeutic strategies, a spatio-temporal model is considered here that incorporates the tumor cell growth and the effects of radiotherapy and chemotherapy. The effects of different schedules of radiation therapy is then studied using a generalized linear quadratic model and compared against the published clinical data. The model is then extended to include the interactions of tumor vasculature and oxygen concentration, to explain tumor hypoxia and to study various methods of hypoxia characterizations including biomarker estimates and needle electrode measurements. The model predicted hypoxia is also used to analyze the effects of tumor oxygenation status on radiation response as it is known that tumor hypoxia negatively influences the radiotherapy outcome. This thesis also presents a detailed analysis of the effects of heterogenous tumor vasculature on tumor interstitial fluid pressure and interstitial fluid velocity. A mathematical modeling approach is then used to analyze the changes in interstitial fluid pressure with or without antiangiogenic therapy.

Glioblastoma is the most common and deadly primary brain tumor with an average survival of 15 months following diagnosis. Characterized as highly infiltrative with diffuse tumor margins, complete resection and annihilation of tumor cells is impossible following current standard of care therapies. Thus, tumor recurrence is inevitable. Interstitial fluid surrounds all of the cells in the body and has been linked to elevated invasion in glioma, which highlights the importance of this understudied fluid compartment in the brain. The primary objective of this dissertation was to identify specific interstitial fluid transport behaviors associated with elevated invasion surrounding glioma tumors. We first describe our methods to measure interstitial fluid flow in the brain using dynamic contrast enhanced magnetic resonance imaging (DCE-MRI), a clinically used, non-invasive imaging modality. We highlight the versatility of the technique and the possibilities that could arise from widespread adoption into existing perfusion-based imaging protocols. Using this method, we examined transport associated with invasion in a murine GL261 cell line. We found that elevated interstitial fluid velocity magnitudes, decreased diffusion coefficients and regions with accumulating flow were significantly associated with invasion. We tested the validity of our invasive trends by extending our analysis to multiple, clinically-relevant tumor locations in the brain. Interestingly, we found invasion did not follow the same trends across brain regions indicating location-specific structures may drive both interstitial flow and corresponding invasion heterogeneities. Lastly, we aimed to manipulate flow by engaging with the meningeal lymphatics, an established pathway for interstitial fluid drainage. Over-expression of VEGF-C in the tumor microenvironment neither enhanced drainage nor altered invasion in comparison to our control, indicating other tumor-secreted growth factors, such as VEGF-A, may play a larger role in mediating flow and invasion. Taken together, by identifying specific transport factors associated with invasion, we may be better equipped to target and treat infiltrative tumor margins, ultimately extending survival in patients diagnosed with this devastating disease. / Doctor of Philosophy / Glioblastoma is the most common and deadly primary brain tumor with an average survival of 15 months following diagnosis. Characterized as highly infiltrative with diffuse tumor margins, complete resection and annihilation of tumor cells is impossible following current standard of care therapies. Thus, tumor recurrence is inevitable. Interstitial fluid surrounds all of the cells in the body and has been linked to elevated invasion in glioma, which highlights the importance of this understudied fluid compartment in the brain. The primary objective of this dissertation was to identify specific interstitial fluid transport behaviors associated with elevated invasion surrounding glioma tumors. We first describe our methods to measure interstitial fluid flow in the brain using dynamic contrast enhanced magnetic resonance imaging (DCE-MRI), a clinically used, non-invasive imaging modality. We highlight the versatility of the technique and the possibilities that could arise from widespread adoption into existing imaging projects. Using this method, we examined transport associated with cancer cell invasion in a mouse tumor cell line. We found that interstitial fluid speeds were elevated while diffusion was decreased in regions of invasion. Further, regions that had interstitial fluid flow congregation were significantly associated with invasion. We tested the validity of these invasive trends by extending our analysis to multiple, clinically-relevant tumor locations in the brain. Interestingly, we found invasion did not follow the same trends across brain regions, indicating location-specific structures may drive both interstitial flow and invasion differences. Lastly, we aimed to manipulate flow by engaging with the meningeal lymphatics, an established pathway for interstitial fluid drainage. Following administration of a meningeal lymphatic-relevant protein, we saw no changes in flow or invasion in comparison to our untreated control, indicating other tumor-secreted proteins may play a larger role in these responses. Taken together, by identifying specific transport factors associated with invasion, we may be better equipped to target and treat infiltrative tumor margins, ultimately extending survival in patients diagnosed with this devastating disease.

Fluid flow is a complex and dynamic process in the brain, taking place at the macro- and microscopic level. Interstitial fluid in particular flows throughout the interstitial spaces within the tissue, interacting with cells and the extracellular matrix. We are coming to find that this interstitial fluid flow plays an important role in both homeostatic and pathologic conditions. It helps to transport chemokines and other molecules such as extracellular vesicles within the environment, clear waste from the brain, and provide biophysical cues to cells. When this flow is disrupted however, such as in glioblastoma or Alzheimer's disease, profound events can occur, for example the build-up of plaques or an increase in tumor cell invasion. While there has recently been an up-tick in interstitial fluid flow research, there is surprisingly little known about its exact nature within the interstitial space and its effects on brain pathology such as glioblastoma. In particular, ways to manipulate and measure brain IFF magnitude at the cellular level are lacking. In this dissertation, a set of tools is created and used to explore the role that interstitial fluid flow magnitude plays in the brain through the lens of glioma invasion. We developed and implemented a flow device that is used in conjunction with an established in vitro tissue culture insert assay to manipulate fluid flow rates through a 3D matrix of tumor cells. We showed that this flow device is biocompatible and accurately recreates flow rates that have been measured previously through the use of MRI. We quantified tumor cell invasion from several glioma cell lines using this device to show a nonlinear trend of invasion in response to increasing fluid flow magnitudes. In addition, we developed a computational model to explore one potential mechanism that fluid flow magnitude might be modulating: autologous chemotaxis. Through this model we showed that increased flow magnitudes such as those seen in gliomas cause an increase in the distribution of the chemokine gradient around a cell of interest, that the morphology of the cell is important to this gradient formation, that temporal effects should not be overlooked, and that within the tumor environment, a minimum distance is required for the invading cell to develop this gradient. Finally, we developed a novel in vivo surgical technique that allows for the manipulation and measurement of interstitial fluid flow within the brain through simultaneous multiphoton imaging. We showed that this technique can be used to modulate interstitial fluid flow, as a mechanism by which to label cells of interest, and as a means to implant and monitor glioma progression. Through these means we further characterize interstitial fluid flow in the brain, allowing for its manipulation and measurement, and examine the ability of increased interstitial fluid flow magnitudes to impact glioma invasion. / Doctor of Philosophy / Fluid flows throughout brain tissue and plays an important role in creating normal conditions for proper brain function. This fluid can also play a role in brain cancer, such as glioblastoma, by causing cancer cells to travel further into the brain which is not desirable. This dissertation seeks to understand fluid flow better by studying how its speed contributes to cancer cell movement which is accomplished through the development of several tools. One tool is a new surgical technique that allows for the measurement and manipulation of fluid flow speed within the mouse brain and visualization of cells of interest, one tool is a flow device that changes fluid flow speed through cells in a gel, and the last is a computational model that predicts how a cell might move under different flow and environmental conditions. The tools were created and utilized, showing several interesting results. Using the flow device, different cancer cell lines were seen to react differently to increased fluid flow speed with two main trends: 1) increased cancer cell movement with increased fluid flow speed and 2) a peak effect where the cell movement started to increase with increasing fluid flow speed and then decreased after a certain fluid flow speed was surpassed. The surgical technique was successful at introducing fluid flow and allowed for reproducible measurements of fluid flow speed. It also was used to introduce stains that show specific cells of interest. The computational model showed that there are specific time and spatial contributions that effect cancer cell movement and that with increased fluid flow speed, cells might be able to more easily utilize a specific mechanism to move. Altogether, this work presents novel insight into fluid flow speed that can be used to further inform the field. It is our hope that the findings from this dissertation can go towards a more comprehensive treatment of a specific type of brain cancer, glioblastoma.

The lymphatic system plays essential roles in regulating fluid balance and immunosurveillance. Across the body, local lymphatic vessels collect waste in the form of lymph and deliver it to nearby lymph nodes (LNs) to be filtered and screened for pathogens. With broad implications in adaptive immunity, cancer metastasis, and cancer treatment, developing novel in vitro models will provide new platforms to explore lymphatic function in health and disease. This dissertation sought to develop tissue-specific engineered models of the LN stroma and the meningeal lymphatics to examine the transport of cells and fluid. Within the LN, fibroblastic reticular cells (FRCs) maintain a network of extracellular matrix conduits that guide varying rates of interstitial fluid flow (IFF) based on inflammatory state. Eventually, that flow exits the LN through the afferent lymphatics, consisting of lymphatic endothelial cells (LECs). We first developed a spatially organized model of the LN stroma consisting of a monolayer of LECs on the underside of a tissue culture insert and an FRC-laden hydrogel within. We demonstrate that high magnitude IFF (3.0 µm/s) had positive impacts on FRCs but disrupted the integrity of the LEC barrier, and these effects were accompanied by increased secretion of a variety of inflammatory chemokines. We also show that IFF of any magnitude decreased T cell egress from the model. Next, we sought to apply the LN stroma model toward understanding metastasis. LN metastasis is the most important prognostic factor in breast cancer, with size of metastasis informing treatment plan. Metastasis greatly alters the structure of the LN, which in turn alters transport. However, the impact of altered transport on cancer progression is not well understood. We added different numbers of breast cancer cells to our LN stroma model to examine tumor burden. We found that tumor cells invaded the LEC barrier at similar numbers regardless of initial burden. Additionally, at the highest tumor burden, diffusivity in the stroma was significantly decreased. Most excitingly, flow velocity was positively correlated with FRC spread in the hydrogel, demonstrating the contributions of FRCs to transport. Finally, we looked to the central nervous system (CNS). The meningeal lymphatics are responsible for draining cerebrospinal fluid to the cervical lymph nodes for CNS immunosurveillance. We developed a simple model of a meningeal lymphatic vessel lumen consisting of a monolayer of LECs on the underside of a tissue culture insert and a monolayer of meningeal fibroblasts within. This is, to our knowledge, the very first in vitro model of the meningeal lymphatics. We demonstrate that our model has barrier function and is capable of immune cell transmigration and egress. We examined how systemic chemotherapy for breast cancer could cause off-target disruption of the meningeal lymphatics and found that docetaxel was significantly deleterious. We further began to explore leukemia cell behavior in our LN stroma and meningeal lymphatics model. Throughout this dissertation, we emphasize the importance of incorporating fluid and cell transport into engineered models of immunity. These models represent a step toward building up the complexity of in vitro lymphatic models to improve pre-clinical screening and understand pathophysiology. / Doctor of Philosophy / The lymphatic system plays essential roles in regulating fluid balance and immune system surveillance. Across the body, local lymphatic vessels collect waste in the form of lymph and deliver it to nearby lymph nodes (LNs) to be filtered and screened for pathogens like viruses or bacteria. With broad implications in immunity, cancer metastasis, and cancer treatment, developing novel models in the lab using human cells and 3-dimensional biomaterials will provide new platforms to explore lymphatic function in health and disease. This dissertation sought to develop engineered models that were specific to the lymph node stroma and the meningeal lymphatics to examine the transport of cells and fluid. Within the LN, fibroblastic reticular cells (FRCs) maintain a network of channels that guide varying rates of interstitial fluid flow (IFF) based on how inflamed the LN is. Eventually, that flow exits the LN through the afferent lymphatics, consisting of lymphatic endothelial cells (LECs). We first developed a spatially organized model of the LN stroma consisting of LECs on the underside of a porous membrane and an FRC-laden hydrogel above the membrane and demonstrated that high magnitude IFF altered morphology, immune cell behavior, and inflammatory protein secretion in the model. Next, we sought to apply the LN stroma model toward understanding cancer metastasis. LN metastasis is the most important prognostic factor in breast cancer, with size of metastasis informing treatment plan. Metastasis greatly alters the structure of the LN, which in turn alters the transport of lymph and immune cells. However, the impact of altered transport on cancer progression is not well understood. We added different numbers of breast cancer cells to our LN stroma model to examine tumor burden and found that tumor cells invaded the LECs at similar rates regardless of initial density, but that diffusion, a transport parameter, was significantly changed by high tumor cell density. Finally, we looked to the central nervous system (CNS). The meningeal lymphatics are responsible for draining cerebrospinal fluid to the cervical lymph nodes to screen for pathogens in the CNS. We developed a simple model of a meningeal lymphatic vessel lumen consisting of LECs and meningeal fibroblasts on either side of a porous membrane. This is, to our knowledge, the very first in vitro model of the meningeal lymphatics. We examined how systemic chemotherapy for breast cancer could cause off-target disruption of the meningeal lymphatics and found that docetaxel was significantly damaging to the model. Throughout this dissertation, we emphasize the importance of incorporating fluid and cell transport into engineered models of lymphatics. These models represent a step toward building up complexity to improve the toolset for pre-clinical screening and studying disease progression.

<p>This thesis aimed at studying mechanisms involved in control of tissue fluid homeostasis during inflammation.</p><p>The interstitial fluid pressure (P_IF) is of importance for control of tissue fluid balance. A lowering of P_IF <i>in vivo</i> will result in a transport of fluid from the circulation into the tissue, leading to edema. Loose connective tissues that surround blood vessels have an intrinsic ability to take up fluid and swell. The connective tissue cells exert a tension on the fibrous network of the tissues, thereby preventing the tissues from swelling. Under normal homeostasis, the interactions between the cells and the fibrous network are mediated by β1 integrins. Connective tissue cells are in this way actively controlling P_IF.</p><p>Here we show a previously unrecognized function for the integrin αVβ3, namely in the control of P_IF. During inflammation the β1 integrin function is disturbed and the connective tissue cells release their tension on the fibrous network resulting in a lowering of P_IF. Such a lowering can be restored by platelet-derived growth factor (PDGF) -BB. We demonstrated that PDGF-BB restored P_IF through a mechanism that was dependent on integrin αVβ3. This was shown by the inability of PDGF-BB to restore a lowered P_IF in the presence of anti-integrin β3 IgG or a peptide inhibitor of integrin αVβ3. PDGF-BB was in addition unable to normalize a lowered P_IF in β3 null mice. Furthermore, we demonstrated that extracellular proteins from <i>Streptococcus equi</i> modulated αVβ3-mediated collagen gel contraction. Because of the established concordance between collagen gel contraction <i>in vitro</i> and control of P_IF <i>in vivo</i>, a potential role for these proteins in control of tissue fluid homeostasis during inflammation could be assumed. Sepsis and septic shock are severe, and sometimes lethal, conditions. Knowledge of how bacterial components influence P_IF and the mechanisms for tissue fluid control during inflammatory reactions is likely to be of clinical importance in treating sepsis and septic shock.</p>

This thesis aimed at studying mechanisms involved in control of tissue fluid homeostasis during inflammation. The interstitial fluid pressure (PIF) is of importance for control of tissue fluid balance. A lowering of PIF in vivo will result in a transport of fluid from the circulation into the tissue, leading to edema. Loose connective tissues that surround blood vessels have an intrinsic ability to take up fluid and swell. The connective tissue cells exert a tension on the fibrous network of the tissues, thereby preventing the tissues from swelling. Under normal homeostasis, the interactions between the cells and the fibrous network are mediated by β1 integrins. Connective tissue cells are in this way actively controlling PIF. Here we show a previously unrecognized function for the integrin αVβ3, namely in the control of PIF. During inflammation the β1 integrin function is disturbed and the connective tissue cells release their tension on the fibrous network resulting in a lowering of PIF. Such a lowering can be restored by platelet-derived growth factor (PDGF) -BB. We demonstrated that PDGF-BB restored PIF through a mechanism that was dependent on integrin αVβ3. This was shown by the inability of PDGF-BB to restore a lowered PIF in the presence of anti-integrin β3 IgG or a peptide inhibitor of integrin αVβ3. PDGF-BB was in addition unable to normalize a lowered PIF in β3 null mice. Furthermore, we demonstrated that extracellular proteins from Streptococcus equi modulated αVβ3-mediated collagen gel contraction. Because of the established concordance between collagen gel contraction in vitro and control of PIF in vivo, a potential role for these proteins in control of tissue fluid homeostasis during inflammation could be assumed. Sepsis and septic shock are severe, and sometimes lethal, conditions. Knowledge of how bacterial components influence PIF and the mechanisms for tissue fluid control during inflammatory reactions is likely to be of clinical importance in treating sepsis and septic shock.