Indiana University-Purdue University Indianapolis (IUPUI) / Although paclitaxel is a commonly used anti-neoplastic agent for the treatment of solid tumors, therapy often results in a number of side effects, the most debilitating of which is peripheral neuropathy. Peripheral neuropathy is defined as a pathology of peripheral nerves, and, depending on the type of nerves damaged, the neuropathy can be classified as sensory, motor, or autonomic neuropathy. In the case of peripheral neuropathy induced by paclitaxel, the symptoms are experienced in the extremities and are sensory in nature. Patients undergoing chemotherapy with paclitaxel often report sensory disturbances such as burning, tingling, numbness, a diminished sensation to pain and temperature, loss of vibration sense, loss of proprioception, and loss of deep tendon reflexes. Electrophysiological abnormalities including decreased sensory nerve action potential amplitude and conduction confirm damage to large myelinated fibers. However, the involvement of damage to small diameter sensory neurons in the etiology of paclitaxel – induced peripheral neuropathy is still controversial. Therefore, experiments were performed to determine if paclitaxel alters the function of small diameter sensory neurons and to examine the mechanisms responsible for the change in function. vi Sensory neuron mediated vasodilatation in paclitaxel – injected animals was examined as an indirect measure of calcitonin gene related peptide (CGRP) release and therefore of sensory neuron function. CGRP release was also directly measured from central terminals in the spinal cord. To examine mechanisms of paclitaxel – induced sensory neuron damage, CGRP release and neurite length was examined in paclitaxel – treated sensory neurons in culture. The results demonstrate that (1) paclitaxel decreases the ability of small diameter sensory neurons to produce an increase in blood flow in the skin; (2) paclitaxel alters the release of CGRP from the small diameter sensory neurons; (3) paclitaxel causes the neuronal processes of isolated sensory neurons to degenerate. This dissertation provides novel information showing that paclitaxel alters the function of small diameter sensory neurons and thus provides a better understanding of the mechanisms mediating the sensory disturbances characteristic of peripheral neuropathy resulting from chemotherapy with paclitaxel.

The retina is one of the most metabolically active tissues in the body and the high energetic demand is met by a well-organized vascular network. Aberrant vasculature is a prominent feature of many vision-threatening diseases, and although angiogenic pathways have been extensively studied the limited efficacy of therapies currently available for the treatment of these diseases suggests that there is more to be elucidated. The caspase family of proteases is best known for their roles in programmed cell death and inflammation, however members of this family have been found to have essential functions independent of cell death. Caspase-8, in particular, has been previously shown to be essential for embryonic vascular development, however, a requirement for caspase-8 in postnatal vascular development has not been established and it is unclear how caspase-8 exerts its function. In this study, we investigate the cell specific roles of caspase-8 in the development of the retinal vasculature using the postnatal mouse retina as our model and identified endothelial caspase-8 as a mediator of canonical Wnt signaling. Inducible endothelial cell-specific caspase-8 knockout (Casp8 iECKO) resulted in a delay in early angiogenesis and barrier establishment, and an increase in inflammation and premature vascular remodeling compared to littermate controls. Assessment of Lef1, a downstream effector of the Wnt pathway, confirmed that this phenotype was a result of inhibited Wnt signaling. We additionally show that caspase-8 mediates this pathway through degradation of its substrate HDAC7. HDAC7 has been shown previously to bind to β-catenin blocking its nuclear translocation. Caspase-8 mediated HDAC7 degradation restores β-catenin translocation and downstream Wnt signaling. We also explore the function of caspase-8 in myeloid cells – microglia and macrophages – during angiogenesis. We used an inducible myeloid-specific caspase-8 knockout (Casp8 imGKO) mouse and found that loss of caspase-8 in these cells did not affect angiogenesis. However, Casp8 imGKO resulted in a reduction in microglia number and a change in their morphology specifying a role for caspase-8 in mediating cell survival and activation in microglia. Altogether we show that caspase-8 exerts cell specific functions during retinal angiogenesis that are independent of cell death. We elucidate a novel role of caspase-8 in mediating Wnt/β-catenin signaling, and implicate caspase-8 as a potential therapeutic target in pathological angiogenesis.

Tissue Engineering A Blood Vessel Mimic While Monitoring Contamination Through Sterility Assurance Testing Navid Djassemi Tissue engineering blood vessel mimics has been proposed as a method to analyze the endothelial cell response to intravascular devices that are used in today’s clinical settings for the treatment of cardiovascular disease. Thus, the development of in vitro blood vessel mimics (BVMs) in Cal Poly’s Tissue Engineering Lab has introduced the possibility of assessing the characteristics of cellular response to past, present, and future intravascular devices that aim at treating coronary artery disease. This thesis aimed at improving the methods and procedures utilized in the BVM model. Initial aspects of this project focused on using an expanded polytetrafluoroethylene (ePTFE) scaffold in conjunction with human endothelial cells to replicate the innermost intimal layer of a blood vessel. Human umbilical vein endothelial cells (HUVECs) were pressure sodded onto ePTFE scaffolds through cell sodding techniques in an attempt to effectively and consistently replicate and assess the intimal layer. Through each study ePTFE grafts were subjected to different culture times and steady flow rates to observe and compare the differences in the endothelial cell deposition. Results were inconsistent, although moderate cell adhesion was noted throughout each of the BVM setups. Each study exhibited a range of cell sodding density rates. In the second phase of the thesis, contamination assessment protocols were implemented in the BVM lab. The intent of this part of the project was to assess the relative sterility in the cell culture lab, a critical component involved with the success or hindrance of cell and tissue cultures. Using microbiological validated methods, microbiological tests were conducted to examine the levels of microbial growth in and around the tissue engineering lab. Results were tracked over a two month period in the lab with several observations of aerobic microorganism growth on various counter and lab surfaces. Higher growth trends were found on surfaces outside the cell culture lab, in the general TE lab area. These findings were used to provide overall suggestions on tracking microbes for long-term durations in ongoing BVM setups to directly improve the overall sterility assurance of the studies. As the project reached its conclusion a look back at all the BVM setups and contamination assessments lead to a few suggestions for improving aseptic techniques within the TE lab, such as monitoring microbial growth in the culture processes, creating limit specifications, and creating a standardized way to regulate quality control within the lab environment. Furthermore, as the development BVM evolves, the findings from this report can be used with related research for improving the culture conditions of various BVM studies.

Colonoscopy is essential for colorectal cancer screening and disease surveillance. It can remove pre-cancerous colon polyps to reduce a patient’s cancer risk. This thesis aims to improve colonoscopy’s localization using motion tracking and colonoscopy’s coverage using surface mapping. Chapter 4 describes an endoscope motion tracker that records the scope’s insertion length, rotation, and speed during a colonoscopy. The endoscope tracker’s motion record can be combined with the endoscope’s video to localize colon polyps or cancers. In the future, the device could record highly skilled manoeuvres performed by endoscopists to help train medical residents. It is difficult to image the colon’s mucosa because the colonoscope’s camera has a limited field of view. Chapter 3 uses a 180° fisheye camera to unwrap high resolution panoramas of a colon phantom. The panoramas are then combined into a mosaic map of the colon phantom’s surface. The colon’s surface is approximated as a cylinder. Follow up experiments could test our mapping algorithm using imagery from a wide-angle, high-definition colonoscope. Chapter 2 describes another technique to localize locations where polyps have been removed—blood vessel landmarks. Colonic blood vessels from a pig were imaged to determine if they could be used to fingerprint locations on the colon’s wall. Blood vessels are also useful image features for surface mapping. The proof-of-concept experiments successfully imaged large arteries but further work is needed to image the small capillaries in the colonic mucosa and to image the veins. In summary, we have visualized colonic blood vessels to test if they could be useful landmarks, tested using an extended field of view camera to create an unwrapped map of the colon wall, and designed an endoscope tracker to help localize abnormal tissue. Combining the endoscope tracker with the other two techniques should make is possible to accurately map the colon. / Thesis / Doctor of Philosophy (PhD) / Colonoscopy is a powerful tool for colon cancer screening. A colonoscopy can decrease the chance of developing advanced cancers by removing pre-cancerous polyps before they grow. This research works to improve colonoscopy’s localization using motion tracking and its coverage using surface mapping. We have developed an endoscope motion tracker that records the scope’s insertion length, rotation, and speed during a colonoscopy. It is In described in Chapter 4. The recorded motion can be combined with the endoscope’s video to improve colon cancer localization. Next, it is difficult to image the colon’s mucosa because the colonoscope’s camera has a limited field of view. Chapter 3 uses a 180° fisheye camera to unwrap high resolution panoramas of a colon phantom. The panoramas are then combined into a cylindrical surface map. Finally, Chapter 2 images the colon’s blood vessels to determine if they can fingerprint locations on the colon’s wall.

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No description available.

Submitted in partial compliance with the requirements for the Master’s Degree in Technology: Chiropractic, Durban University of Technology, Durban, South Africa, 2015. / Background: When a soft tissue injury occurs the blood vessels and surrounding tissue are damaged leading to haemorrhaging and inflammation. Cryotherapy (cold therapy) is generally acknowledged as the preferable treatment by manual therapists during this immediate post-traumatic period of an injury. Cryotherapy has been shown to result in vasoconstriction decreasing the rate of blood flow which has a favourable effect on inflammation and pain. The commercially available cooling cuff is a relatively new cryotherapy modality offering a mechanism of cooling that does not require freezing and is easy to use. The polymer granules within the cooling cuff are activated by emersion in water therefore freezing is not required making the cooling cuff readily available compared to more traditional forms of cryotherapy. Aim: The aim of this study was to determine the effect of a moist ice pack and a commercially available cooling cuff radial artery blood flow (cm.s-1) and radial artery lumen diameter (mm) after 15 minutes of application. Method: This study was a pre-test post-test design utilising 43 asymptomatic participants that were randomly allocated to one of two groups. Each group either received a standard moist ice pack or a commercially available cooling cuff, placed on the ventral surface of the participants forearm, over the radial artery, for a duration of 15 minutes. Measurements were taken with a Doppler ultrasound to determine radial artery blood flow and lumen diameter, prior to the intervention and 15 minutes after the cryotherapy application. Data analysis was performed using IBM SPSS VERSION 20 (IBM Corp. Released 2010.IBM SPSS Statistics for Windows, Version 19.0. Armonk, New York: IBM Corp.). Statistical significance was set at a p< 0.05 level. Intra-group and inter-group comparisons were measured using repeated measures ANOVA testing. Results: Both the moist ice pack and commercially available cooling cuff resulted in a significant decrease in radial artery blood flow (p< 0.001) after 15 minutes of application with no significant changes being observed in radial artery diameter Conclusions: The commercially available cooling cuff resulted in a similar effect on radial artery blood flow and lumen diameter as moist ice, indicating that the commercially available cooling cuff may be utilised in the acute phase of an injury to alter blood flow. / M

Neste estudo avaliou-se a ação da Angiotensina II (Ang II) e do bloqueio dos seus receptores AT1 e AT2 no remodelamento da matriz extracelular (MEC) e traçamos o perfil da sinalização intracelular envolvida no processo. O estudo foi feito in vivo em camundongos isogênicos C57Bl/6J submetidos a tratamento durante 7 e 14 dias com doses subpressoras de Ang II, bloqueador do receptor AT1 (Losartan) e uma combinação destes. Os animais foram sacrificados e procedeu-se a coleta de tecidos de artérias (aorta, carótida e femoral), coração, rins e pulmão para análise da síntese e degradação de componentes da MEC. Foram feitas avaliações hemodinâmicas, morfológicas em microscopia de luz, morfométricas, imunohistoquímicas para os componentes da matriz extracelular: colágeno (tipos I, III, IV e VI), fibronectina, tenascina-C, elastina, metaloproteinases (tipos 2 e 9), e quantificação de algumas proteínas ligadas à sinalização intracelular da via das Proteínas quinases ativadas por mitógenos (MAPK - Mitogen-activated protein kinases) usando-se Western Blotting. / In this study we evaluated the action of Angiotensin II (Ang II) and the blockade of AT1 and AT2 receptors in the remodeling of extracellular matrix (ECM) and outlines the process involved in intracellular signaling. The study was done in vivo in C57Bl/6J inbred mice undergoing treatment for 7 and 14 days subpressor doses of Ang II, AT1 receptor blocker (Losartan) and a combination thereof. The animals were sacrificed and proceeded to collect tissues of arteries (aorta, carotid and femoral arteries), heart, kidneys and lungs for analysis of the synthesis and degradation of ECM components. We assessed hemodynamic, morphological light microscopy, morphometry, immunohistochemistry for extracellular matrix components: collagen (types I, III, IV and VI), fibronectin, tenascin-C, elastin, metalloproteinases (types 2 and 9) and quantification of some proteins related to intracellular signaling pathways of the mitogen-activated protein kinase (MAPK) using Western Blotting.

As imagens de angiografia por ressonância magnética (angio-RM) ou por tomografia computadorizada (angio-TC) permitem uma análise minuciosa das redes vasculares. A segmentação de redes vasculares a partir de tais imagens é uma das tarefas iniciais no diagnóstico de doenças vasculares como estenoses ou aneurismas. Porém, a grande diversidade de arquiteturas dos vasos dificulta a validação dos algoritmos de segmentação. Assim, a construção de redes vasculares sintéticas realistas permitem validar novas metodologias de segmentação de vasos. Este trabalho descreve uma metodologia de geração de redes vasculares sintéticas em três dimensões utilizando sistemas de Lindenmayer (L-systems) estocásticos. Para atingir esse objetivo, foram implementados um analisador léxico, um analisador sintático e um gerador de L-systems para a criação de vasos sintéticos baseado em gramáticas. A parametrização destas gramáticas possibilita a simulação de características naturais de vasos reais como o ângulo de bifurcação, comprimento, diâmetro médio e possibilita a simulação de anomalias vasculares. As expressões resultantes são utilizadas para criar imagens angiográficas sintéticas que simulam a distribuição de intensidades dos vasos em imagens angio-RM e angio-TC reais. As redes vasculares sintéticas podem também ser delimitadas por superfícies 3D arbitrárias de forma similar à geometria de órgãos. A flexibilidade de parametrização e natureza estocástica desta metodologia faz com que ela se torne uma ferramenta ideal para a validação de algoritmos de segmentação de vasos em imagens angiográficas. / Magnetic resonance angiography (MRA) or computed tomography angiography (CTA) images allow for a thorough analysis of the blood vessels. Vessel segmentation from MRA or CTA is thus the primary task in the diagnosis of vascular diseases such as stenosis and aneurysms. The wide architectural variability of the blood vessels, however, hinders the validation of vascular segmentation methods. The construction of synthetic realistic vascular architecture trees will aid in the validation of new vessel segmentation methodologies. This thesis describes a three-dimensional synthetic blood vessel generation methodology that employs stochastic Lindenmayer systems (L-systems). For this purpose, we implemented a parser and a generator of L-systems to create grammars that represent blood vessel architectures. The parameterization of the grammar allows one to simulate natural features of real vessels such as bifurcation angle, average length and diameter, and also accounts for vascular anomalies. The resulting expressions are used to create synthetic angiographic images that mimic real vessel intensity distributions in MRA and CTA. Blood vessel growth can also be delimited by arbitrary 3D surfaces that may represent organ geometries. The flexibility in the parameterization and stochastic nature of this methodology makes it an ideal tool for the validation of blood vessel segmentation algorithms from angiographic images.

本研究採用結紮眼血管的方法誘發短暫性視網膜缺血，針對同缺血時間同存活時間化成年金黄地鼠中視網膜節細胞的存活和小型膠質細胞的激活。首先，我們的據顯示，和假缺血手術組相對應的存活時間比較，暫時性視網膜缺血10分鐘或30分鐘沒有導致視網膜節細胞的存活明顯下。暫時性視網膜缺血60分鐘再灌注後7天，視網膜節細胞的存活下至58%，14後為51%，28後為44%。暫時性視網膜缺血120分鐘之後再灌注7天，視網膜節細胞的存活急劇下，僅保22%，至14天，僅剩17%, 之後節細胞的死亡速減緩，至28天時，仍由18%存活。視網膜缺血10分鐘、30分鐘、60分鐘和120分鐘均引起大小型膠質細胞激活，激活在第七天達到頂峰，之後在14天和28天顯著並逐步下。相關性分析發現損傷後7天，視網膜節細胞的死亡和視網膜節細胞層中的小型膠質細胞存在緊密的相關性。 / 其次，我們首次證實缺血性預處僅有於提高視網膜節細胞對抗視網膜缺血/再灌注損傷，還對視神經斷後的視網膜節細胞同樣具有保護作用。結果顯示無是5分鐘還是10分鐘的缺血性預處，無是軸突橫斷術前1天還是前3天實施預處，都對視網膜節細胞有明顯的保護作用。在缺血性預處對抗神經橫斷損傷的實驗組, 的表達只表現在陽性細胞上的明顯優勢，但占全部存活細胞的百分比存在差；而缺血性預處對抗視網膜缺血再灌注損傷的實驗組，的陽性節細胞的無論還是存活百分比都存在差。在預處組和假處組的比較中， 的表達也只是陽性細胞上較多，占全部存活細胞的百分比存在差。在缺血性預處加視網膜缺血分鐘的實驗組中，我們測視網膜矢片中各層的厚。結果顯示，缺血性預處組中，視網膜的整體厚和節細胞層的厚都與正常組相當，而假處組中，這層的厚明顯減少。 / 進一步地，我們研究遠端缺血性後處對視網膜節細胞對抗視神經軸突橫斷術的保護作用。我們選用鉗夾右股動脈作為遠端缺血性後處的方法，鉗夾股動脈分鐘，之後放開，再鉗夾再放開，共個循環。結果顯示，軸突橫斷術后分鐘實施缺血性后處組，視網膜節細胞的存活較假處組明顯增加，包括術後天和天；軸突橫斷術后小時實施缺血性後處組，視網膜節細胞的存活只在術後天較假處組明顯較多，但在天的實驗組，者的差消失；軸突橫斷術小時實施缺血性後處組，視網膜節細胞的存活比假處組多。在缺血性後處的實驗中，視神經橫斷術后分鐘實施遠端缺血性後處的實驗組與假處組比較，的表達僅表現在陽性細胞上的明顯增加，而且占全部存活細胞的百分比也明顯增加。的表達與預處實驗組的結果相似，只存在上的優勢。 / 我们的實驗證明，缺血性預處在對抗視神經橫斷和視網膜缺血的損傷中，可以為節細胞提供有效的保護作用，遠端缺血性後處可以對抗視神經橫斷損傷提高節細胞的存活。陽性節細胞在三個同條件的實驗中，表現出同的結果，这可能暗示遠端缺血性後處對抗視神經橫斷術的損傷，節細胞的再生能較優，與遠端缺血性後處對抗視神經橫斷術的神經保護作用有一定關。的表達在三個實驗組中，處組與假處組比較，均只表現出陽性細胞上的優勢，占存活細胞的百分比就存在差，可能意味著與缺血性預處和後處的保護作用關係不大。 / Ligature of the ophthalmic vessels (LOV) was used as an animal model to study transient retinal ischemia/reperfusion in adult hamsters. Firstly, we quantified the loss of retinal ganglion cells (RGCs) and activation of microglia after10 min, 30 min, 60 min or 120 min retinal ischemia at 7, 14 and 28 days post-ischemia. The results showed that after 10-min or 30-min retinal ischemia, the number of RGCs had no significant decrease compared to sham LOV group at 7 days. In the retinal ischemia 60 min group, there were 58% of the RGCs population remained alive at 7 days, 51% at 14 days and 44% at 28 days post-ischemia, respectively. In the retinal ischemia 120 min group, the number of RGCs was reduced to 22% at 7 days and 17% at 14 days, but cell death slowed down from 14 to 28 days. Meanwhile, the number of microglia was increased sharply at 7 days and decreased gradually from 7 to 28 days. At the same time, it was found that the loss of RGCs and activation of microglia in the ganglion cell layer at 7 days post-insult existed strong positive correlation. / Secondly, the effects of ischemic preconditioning (IPC) were proved to promote RGCs survival after axotomy or retinal ischemia 120 min. It was presented firstly that a 5 or 10 min brief IPC which performed 1 or 3 days prior to axotomy enhanced the RGCs survival at 7 days and 14 days post-axotomy. The number of HSP27-positive RGCs was significantly higher in the IPC plus axotomy subgroup compared with the sham-operated subgroup, while the percentage of HSP27-positive RGCs did not show significant difference between subgroups. For the IPC plus retinal ischemia 60 min group, both the number and the percentage of HSP27-positive RGCs had no significant difference between IPC and sham-operated subgroups. The number of HSP70-positive RGCs exhibited significant difference but not the percentage in IPC plus axotomy or retinal ischemia 60 min experimental groups. The thicknesses of the whole retina and GCL were similar to the normal value in the IPC plus ischemia 60 min subgroup, while in the sham-operated subgroup, these two values decreased significantly. / Consequently, the effect of remote ischemic postconditioning (RIPostC) was also explored to promote RGCs survival after axotomy. Four cycles of 10 min occlusion and 10 min release of the right femoral artery were initiated on animals at 10 min, 6 h or 24 h after axotomy. In the10 min group, the effect of RIPostC on promoting RGCs survival was significant at both 7 and 14 days post-injury. In the 6 h group, the survival of RGCs was more in the RIPostC treatment subgroup at 7 days, while there was no significant difference at 14 days post-axotomy. In the 24 h group, RGC survival was not significantly different at 7 days post-axotomy. Both the number and the percentage of HSP27-positive RGCs were significantly higher in the RIPostC treatment subgroup. The results of the induction of HSP70 only showed a priority in absolute number of the HSP70-positive RGCs in the RIPostC treatment subgroup. / In summary, the effect of IPC has been proved that it could protect RGCs against axotomy and retinal ischemia/reperfusion injury, in addition, the application of RIPostC also protected RGCs from axotomy. The proportion of HSP27-positive RGCs increased significantly in the process of RIPostC against axotomy, which may clue that the ability of axonal regeneration is stronger which induced by the RIPostC intervention. The upregulation of HSP27 might play a role in the neuroprotection of the RIPostC against axotomy. The expression of HSP70 maybe plays a little role in the neuroprotection of the IPC and RIPostC. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Liu, Xia. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 182-196). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / Abstract in Chinese --- p.iv / Acknowledgements --- p.vii / Table of Abbreviations --- p.viii / Table of Contents --- p.ix / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter Chapter 2 --- Changes of retinal ganglion cells and microglia after different types of injuries / Introduction --- p.38 / Materials and Methods --- p.43 / Results --- p.49 / Discussion --- p.57 / Figures and tables --- p.71 / Chapter Chapter 3 --- Ischemic preconditioning protect retinal ganglion cells against axotomy and retinal ischemia/reperfusion injury and expression of heat shock protein 27 and 70 / Introduction --- p.93 / Materials and Methods --- p.98 / Results --- p.103 / Discussion --- p.109 / Figures and tables --- p.116 / Chapter Chapter 4 --- Remote ischemic postconditioning protect retinal ganglion cells against axotomy and expression of heat shock protein 27 and 70 / Introduction --- p.143 / Materials and Methods --- p.147 / Results --- p.150 / Discussion --- p.154 / Figures and tables --- p.161 / Chapter Chapter 5 --- General Discussion --- p.175 / References --- p.182