Master of Science / Department of Kinesiology / Brad J. Behnke / Introduction: Prostate tumor arterioles lack functional smooth muscle and have a diminished myogenic response. Previous research has demonstrated an enhanced prostate tumor blood flow and oxygenation associated with the augmented mean arterial pressure during exercise. Thus, we tested the hypothesis that elevations in the heart-to-prostate tumor hydrostatic gradient via adoption of the 70˚ head-up tilt (HUT) body position would enhance perfusion of the prostate tumor, which may improve tumor oxygenation and radiation therapy outcomes (Study I). Based upon those findings, we performed a secondary analysis (Study II) on previously published prostate hemodynamic responses to an identical tilt-test between young and aged animals. Methods: Study I: Dunning Cell AT-1 tumor cells (100,000) were injected into the ventral lobe of the prostate in male Copenhagen rats (4 mo.; n = 7). Four to six weeks after injection blood flow to the prostate tumor, kidneys, and soleus muscle was measured via the fluorescent microsphere technique in the supine and HUT position. Study II: A secondary analysis was performed on blood flow to the prostate (host tissue of the tumor) in young (6 mo.; n =9) and aged (24 mo.; n=7) male Fisher 344 rats from Ramsey et al., 2007 (39) to determine potential age-associated differences in conductance to this tissue. Results: Study I: No significant difference was observed in blood pressure between the two body positions. Compared to the supine posture, there was a significant reduction in blood flow to the soleus muscle. There was no difference in prostate tumor blood flow or vascular conductance between the supine and HUT position. Study II: In response to tilt, there was a significant reduction in prostate vascular conductance in young rats versus that in the supine posture (P<0.05). In the aged animals, there was no difference in prostate vascular conductance with tilt. Discussion: Contrary to our hypothesis, we did not see any significant differences in either blood flow or vascular conductance to the prostate tumor with manipulations in body position. Importantly, we believe this may be an age-associated effect. Given tumors both co-opt existing arterioles from the host tissue that retain vasomotor control and develop new vessels that lack functional smooth muscle, the enhanced vascular resistance in the prostate with young animals during tilt likely contributed to the lack of change in tumor perfusion with body position given the rats from study I were also young. Given the lack of change in vascular conductance in the prostate with tilt in aged animals, future studies should be performed in aged models of prostate cancer, of which currently there are no immunocompetent aged rodent models of prostate cancer.

A Research Report submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfillment of the requirements for the degree of Master of Medicine In the branch of Nuclear Medicine Johannesburg September 2013 / The use of Single Photon Emission Computed Tomography (SPECT) myocardial perfusion imaging (MPI), with Technetium – 99m (Tc-99m) Sestamibi in conjunction with either exercise, pharmacologic stress or both is an established tool for both the diagnosis and prognostication of patients with ischemic heart disease. For perfusion imaging with SPECT, Tc-99m labeled radiopharmaceuticals (Sestamibi or Tetrofosmin) are commonly used. The major metabolic pathway for clearance of Sestamibi is the hepatobiliary system which creates difficulty in both visual and quantitative interpretation of myocardial perfusion, particularly of the inferior and infero-septal walls after reconstruction. Diluted lemon juice, an acid-rich drink is an alimentary cholekinetic that facilitates Sestamibi transit through the liver. Whole milk stimulates liver clearance as well as increases peristaltic movement. The aim of the study was to determine which protocol would be the best to reduce interfering infra-cardiac activity and therefore result in an improvement in image quality. We had three groups, comparing the use of full fat milk, diluted lemon juice and a control group that had no intervention. All patients referred to our institution for MPI from November 2009 to May 2012 were enrolled in the study. A total of six hundred and thirty (630) patients who fulfilled the inclusion criteria were randomized without stratification into three groups. Group 0 (G0) were given diluted lemon juice, 246 patients; full fat milk to group 1 (G1), 313 patients and group 2 (G2); 71 patients, had no intervention. The latter was the control group. Raw data of both the stress and rest images were visually and quantitatively assessed by two Nuclear Medicine physicians for the presence of infra-cardiac activity. The physicians were blinded to the intervention received and the data were reviewed simultaneously. The administration of milk or lemon juice resulted in a significant decrease in the intensity of infra-cardiac activity compared to the control group. This improvement was even more significant in the milk group for patients done during rest myocardial perfusion imaging.

Developing an optimized growth environment for adipose-derived stems cells (ASCs) to obtain clinically useable cell quantities from relatively small tissue biopsies would significantly impact the field of tissue engineering. To date, ASCs have been differentiated into adipose, bone, cartilage, smooth muscle, endothelial, skeletal muscle, nervous, and cardiac tissue. Therefore, ASCs have potential for use in the treatment of a wide variety of clinical conditions ranging from myocardial infarction, to musculoskeletal disorders, and the repair of soft tissue defects. In this work, a custom-designed, 3-dimensional (3-D) scaffold-based perfusion bioreactor system was investigated in the culture of ASCs. Decellularized adipose tissue (DAT) was used to provide a 3-dimensional scaffold, as it possesses the native extracellular matrix (ECM) architecture and composition of human adipose tissue. The DAT had a permeability of 149 m2, based on a perfusion rate of 1.5 mL/min over a 200 mg DAT sample, and the culturing medium was evenly perfused throughout the DAT, thereby permitting possible cell growth within the central regions. Initial culturing studies of human ASCs on tissue culture polystyrene (TCPS) demonstrated that hypoxic (5% O2) conditions decreased the doubling time, and resulted in enhanced cell proliferation, as compared to normoxic (21% O2) conditions. The cell imaging and DNA quantification results showed that suspension seeding of the ASCs permitted cell attachment to the DAT scaffold, but did not support long-term ASC growth. In contrast, when the ASCs were seeded as multicellular aggregates, the cells attached and underwent measurable proliferation. The optimal seeding density observed was 1 x 106 ASCs/scaffold; or 50 aggregates (20,000 ASCs/aggregate) per scaffold. Based on the confocal imaging, the ASCs remained spherical in morphology during the entire culturing period. Moreover, results illustrated that the perfusion bioreactor provided an improved culturing environment for ASCs over traditional static culturing. Hypoxic (5% O2) conditions showed improved proliferation over normoxic (21% O2) conditions, within the bioreactor system. After a 14-day hypoxic culturing period in the perfusion bioreactor, the seeded ASCs retained the ability to undergo adipogenesis, as indicated by Glycerol-3-Phsophate Dehydrogenase (GPDH) enzymatic activity measurements, demonstrating the promise of this approach for soft tissue engineering applications / Thesis (Master, Chemical Engineering) -- Queen's University, 2011-11-09 20:28:34.252

Reports in the literature have shown that in man propranolol has a larger first-pass effect (70%) than pindolol (13%). The aim of this research was to make a direct comparison of first-pass extraction and other pharmacokinetic parameters of these drugs under identical experimental conditions by means of an isolated rat liver perfusion model.

Note:

Injuries to the equine distal limb are common and often involve synovial, tendinous and/or ligamentous structures. Historically, lacerations involving the equine digital flexor tendons carried a poor prognosis for return to athletic function due to contamination of the site at presentation, involvement of multiple anatomic structures and the need for immediate weight bearing after surgery. The need for weight bearing after surgery places strain on the tenorrhaphy site that exceeds the strength of the repair itself. Extrapolation of complex, stronger tenorrhaphy patterns from human literature and applying them to equine patients has been challenging. Human tenorrhaphy techniques initially focused on strong repairs, which are able to match or exceed the strength of tendon itself. Adhesion formation is problematic in human flexor tenorrhaphies, as most injuries occur to tendons surrounded by synovial structures. Human literature now focuses on using repairs that provide initial strength, minimal damage to intrinsic tendon architecture, and allow for early mobilization. This treatment protocol has greatly improved the functional outcome of human tenorrhaphies. Recent studies have evaluated the strength of complex tenorrhaphy patterns in equine superficial digital flexor tendons, using modifications of the Savage technique. The newly evaluated patterns are stronger than previously tested and commonly used techniques, such as the three-loop pulley (3LP). A review of tendon vasculature across species and healing characteristics of tendons highlights the importance of intrinsic tendon vasculature in the healing process. Using tenorrhaphy techniques that preserve this vasculature may improve the clinical outcome in these cases. Only one study has previously evaluated the effect of tenorrhaphy patterns on intrinsic tendon vasculature in equine superficial digital flexor tendon. This study compared perfusion of intrinsic tendon vasculature of equine superficial digital flexor tendon (SDFT) after 3LP and six-strand Savage (SSS) tenorrhaphies. We hypothesized that the SSS technique would significantly decrease vascular perfusion compared to the 3LP technique. Under general anesthesia, eight pairs of forelimb SDFTs were transected and either SSS or 3LP tenorrhaphy was performed on each forelimb. The horses were heparinized, euthanatized, and forelimbs perfused with barium sulfate solution then fixed with formalin under tension. The tendons were transected every 5mm and microangiographic images were obtained using a Faxitron X-ray cabinet with computed radiography imaging. Microvascular analysis of sections proximal to the tenorrhaphy, throughout the tenorrhaphy and distal to the tenorrhaphy was completed using Image J software and a custom macro. A significant reduction in the number of perfused vessels was seen in the SSS compared to the 3LP at two locations within the tenorrhaphy (p=0.004 and 0.039). The SSS technique took on average 4.7 ± 0.9 times longer to place. The SSS technique causes a reduction in tendon perfusion compared to the 3LP, which may limit its clinical use. Further research is required to elucidate the clinical significance of this difference. / Master of Science

MR allows non-invasive means of evaluating the non-mineralized components of bone, particularly the bone marrow. This thesis focuses on potential changes occurring in bone marrow perfusion and marrow fat in osteoporosis, - changes which may improve our understanding of osteoporosis pathophysiology. We know from histology studies that as osteoporosis develops and bone tissue is lost, it is replaced by fat filling the vacated marrow space. MR allows non-invasive quantification of this fat component. Although fat content may be increasing, it is not known whether any change in fat composition occurs with osteoporosis i.e. does the type of fat within bone change. Epidemiological studies have indicated a link between arterial disease and osteoporosis. It is not known, however, whether any changes occur in bone perfusion in osteoporosis and how these may be related to increasing fat within the marrow. / The hypothesis to be tested is that: Advanced magnetic resonance (MR) techniques can be applied to investigate the non-mineralised components of bone tissue in osteoporosis thereby providing more information on bone physiology both in health and disease / This thesis is based on a series of eight studies designed to study the relationship between bone marrow perfusion, bone marrow fat content, bone marrow fat composition and bone mineral density. These studies showed that as bone mineral density decreased, bone marrow perfusion decreased. A strong reciprocal relationship was found between decreasing bone marrow perfusion and increasing marrow fat. The reduction in perfusion occurred only with bone and did not affect the extra-osseous tissues alongside bone with the same arterial supply. This indicates that the reduction in bone perfusion is not simply a reflection of a more generalized circulatory impairment in subjects with osteoporosis. This same effect is seen in both males and females and in the proximal femora as well as the spine. / In animal-based studies, we found that reduction in bone perfusion was apparent as little as two weeks after orchidectomy or oorphorectomy and closely paralleled features of impaired endothelial function as well as decreased bone mineral density and a hitherto unrecognized reduction in red marrow fraction within the medullary canal. Nitric oxide synthase, produced by the endothelium, is a potent stimulator of angiogenesis and osteoblastic activity. Mesenchymal stem cell differentiation may switch from osteoblastogenesis to adipocytogenesis under hypoxic conditions, while haematopoetic stem cells also supply endothelial stem cells. Potentially endothelial dysfunction, mesenchymal stem cell differentiation and haematopoetic stem cell maturation may be implemented in the development of osteoporosis. / In normal subjects, blood perfusion was markedly reduced in the femoral head compared to the femoral neck. In osteoporotic subjects, a further reduction in blood perfusion occurred in both areas. Overall perfusion indices reduced relatively more in the femoral neck than the femoral head region. These changes in bone perfusion help explain why subjects with osteoporosis have impaired healing of femoral neck fractures though do not seem to be at increased risk of avascular necrosis. At a micro-architectural level, reduced bone perfusion may also help explain the impaired healing of microfractures seen in subjects with osteoporosis, a feature likely to contribute to reduce bone strength, microfracture accumulation and eventually clinical insufficiency fracture. / Marrow fat was increased in subjects with osteoporosis. Our studies showed that percentage marrow fat content increased even allowing for the quantitative effect increased marrow fat has on the bone densitometry measurements. This effect was shown to be of negligible clinical significance. We found a strong reciprocal relationship between increasing fat and decreasing bone perfusion in both the proximal femur and vertebral body. Although fat content increased, very little difference in marrow fat composition was apparent between normal subjects and those with osteoporosis. We found no difference was apparent in either the N3/N6 marrow fat ratio or the spectrum of individual fatty acids in the marrow of subjects with either normal bone mineral density or osteoporosis. This suggests that alternation of marrow fat composition is not likely to be a direct contributory factor in osteoporosis. Also marrow fat increase was shown to be due to an increase in number rather than size of marrow fat cells. This suggests that marrow fat increases as a result of a switch in mesenchymal cell maturation to adipocytes rather than osteoblasts. / Below average perfusion indices in the acetabulum and adductor muscle is predictive of more pronounced bone loss at the femoral neck over the ensuing four years. Perfusion indices can also predict between fast and slow losers with a high sensitivity / To summarise, in the eight studies presented, it was shown that osteoporosis is associated with a significant reduction in bone perfusion and a reciprocal increase in marrow fat content though no change in marrow fat composition. Reduction in bone perfusion is most likely due to an accompanying reduction in functioning marrow fraction. Marrow fat increase is most likely the result of a switch in mesenchymal cell maturation to adipocytes rather than osteoblasts. The studies present in this thesis confirmed the initial hypothesis that “Advanced magnetic resonance techniques can be applied to investigate the non-mineralised components of bone tissue in osteoporosis thereby providing more information on bone physiology both in health and disease. / Griffith, James Frances. / "June 2009." / Thesis (M.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 227-250). / Appendix includes Chinese. / PREFACE AND DECLARATION --- p.1 / DEDICATION --- p.2 / ACKNOWLEDGEMENT --- p.3 / PRECIS --- p.4 / PUBLICATIONS AND PRESENTATIONS OF STUDIES RELATED TO THIS THESIS --- p.8 / INTRODUCTION --- p.16 / Chapter STUDY 1 --- What is the relationship between bone perfusion, marrow fat and bone mineral density? --- p.76 / Chapter STUDY 2 --- Vertebral marrow fat content, molecular diffusion, and perfusion indices in women with varying bone density, including osteoporosis: MR evaluation --- p.94 / Chapter STUDY 3 --- Could the results of Study 1 and Study 2 be spurious due to the effect of increasing marrow fat lowering BMD estimation by DEXA? --- p.111 / Chapter STUDY 4 --- Are the same changes in perfusion and marrow fat seen in the proximal femur as were seen in the lumbar spine (Study 1 & Study 2)? --- p.128 / Chapter STUDY 5 --- What is the reproducibility of MR perfusion studies and 1H spectroscopy of bone marrow? --- p.150 / Chapter STUDY 6 --- Marrow fat content increases but does the composition of marrow fat change in osteoporosis? --- p.159 / Chapter STUDY 7 --- Likely causes of reduced bone perfusion in osteoporosis: novel findings in an ovariectomy rat model --- p.180 / Chapter STUDY 8 --- Do perfusion indices or marrow fat content predict rate of bone loss? --- p.204 / SUMMARY --- p.222 / REFERENCES --- p.227 / ABBREVIATION LIST --- p.251 / APPENDIX --- p.253

Heart transplantation is, for certain types of cardiovascular disease, the only form of treatment resulting in patient survival. Its clinical application is, however, limited by the shortage of donor organs. This shortage is largely due to the inability to consistently preserve adequate myocardial function over prolonged ischemic periods. It is the goal of this research to provide information which may contribute to techniques for heart preservation, thus improving graft survival following preservation and transplantation. Current methods for myocardial preservation generally involve the arrest and immersion of the heart in cold cardioplegic solution, the composition of which is designed to provide for the reduced metabolic demands of the cold, arrested muscle. These methods have extended the preservation period to approximately 6 hours; however, hearts cannot be held longer than this period because, although metabolism has been slowed by hypothermia, alterations take place which compromise functional recovery upon reperfusion. A variety of perfusates and perfusion techniques have been developed to protect the myocardium from the damage thought to occur as a consequence of ischemic storage of the isolated heart. However, a consistently successful technique for long-term preservation of the heart remains undefined. A growing body of knowledge has led to the hypothesis that injury to the microcirculation may result in myocardial ischemia during preservation and decreased contractile function following preservation. To test this hypothesis, standard Langendorff techniques for the measurement of left ventricular function were combined with biochemical, histological and morphological techniques to determine: (1) whether loss of microvascular function occurs in isolated hearts hypothermically perfused with an oxygenated solution; (2) the impact of microvascular dysfunction during the preservation period on the functional recovery of hearts; and (3) which mechanisms contribute to decreased microvascular function during preservation. This experimental approach will allow for characterization of the role of the microvasculature in decreased contractility of preserved hearts and will provide information regarding the contribution of specific mechanisms to the compromised contractility of preserved hearts. Systematic evaluation of mechanisms thought to be responsible for decreased contractility of isolated hearts could contribute to improved myocardial preservation techniques that can be applied to clinical transplantation.

Ce travail de thèse, principalement méthodologique, s'est intéressé aux techniques d'imagerie par résonance magnétique (IRM) permettant de mesurer le flux sanguin cérébral (CBF) et plus particulièrement aux techniques de marquage de spins artériels (ASL), qui utilisent les protons de l'eau du sang comme marqueur. Nous avons mis en place la séquence ASL de marquage pseudo-continu (pCASL) et évalué sa réponse à un stimulus hypercapnique. Nous avons évalué différentes stratégies pour optimiser l'efficacité d'inversion. Pour cela, nous avons également mis en place des outils de simulations numériques des approches ASL. Nos résultats démontrent que l'efficacité d'inversion est influencée par l'homogénéité du champ magnétique dans la région de marquage, ce qui pose un problème à haut champ magnétique. Le protocole pCASL optimisé a ensuite été évalué chez le rat à trois champ magnétiques (4.7, 7, et 11.7T) et comparé avec une approche en ASL continu classique (CASL). Cette comparaison a montré une excellente reproductibilité inter-animal et inter-champ de la méthode développée. Dans une seconde partie, nous nous sommes également intéressés à l'influence du temps de relaxation longitudinal (T1) du tissu cérébral sur les valeurs du CBF calculées. Pour cela, nous avons modifié le T1 du tissu par une injection intra-cérébrale de manganèse. Cette étude a montré la difficulté de prendre en compte le changement de T1 du tissu. Dans une troisième partie, nous avons évalué l'apport d'une antenne de marquage spécifique pour l'approche CASL en comparant les mesures de CBF obtenues avec celles de la pCASL. Nous avons observé une bonne concordance entre ces deux méthodes à travers les coupes. Nos résultats illustrent également l'importante contribution du transfert d'aimantation dans les séquences de CASL. Les outils développés au cours de cette thèse sont en cours d'application dans des protocoles d'étude de modèles de tumeurs cérébrales, d'accident vasculaire cérébral et de traumatisme crânien. / This PhD work, mainly methodological, focused on the techniques of magnetic resonance imaging (MRI) to measure cerebral blood flow (CBF) and more particularly on arterial spin labeling (ASL), which uses water protons from the blood as markers. We implemented the pseudo-continuous ASL labeling sequence (pCASL) and evaluated its response to a hypercapnic stimulus. We evaluated different strategies to maximize the labeling inversion effeciency. For this, we implemented numerical simulation tools of ASL approaches. Our results demonstrated that the inversion efficiency is influenced by the homogeneity of the magnetic field in the labeling region, which becomes a problem at high magnetic field. The optimized pCASL protocol was then evaluated in rats at three magnetic fields (4.7, 7, and 11.7T) and compared with a conventional continuous ASL approach (CASL). This comparison showed excellent inter-animal and inter-field reproducibility of the developed method. In a second part, we evaluated the influence of the longitudinal relaxation time (T1) of brain tissue on the final CBF values. For this, we modified the tissue T1 by an intracerebral injection of manganese. This study demonstrated the difficulty of taking into account the change in T1 of the tissue. In a third part, we evaluated the contribution of a specific labeling coil for the CASL approach by comparing measurements of CBF obtained with that of pCASL. We found a good agreement between these two methods for all slices. Our results also illustrate the significant contribution of magnetization transfer effects in CASL sequence. The tools developed during this thesis were also used in several preclinical studies including brain tumors, stroke and head trauma models.

L'objectif général du travail de cette thèse est de proposer des méthodes robustes pour permettre d’obtenir des critères sur l’évolution de la pathologie tumorale à partir d’études dynamiques. Actuellement, l’appréciation de l’efficacité d’un traitement antiangiogénique (destruction des vaisseaux alimentant la tumeur) repose principalement sur l’imagerie fonctionnelle dont l’objectif est de quantifier la microcirculation tumorale à partir d’acquisitions dynamiques de perfusion. Cependant, différentes limites concernant le suivi de la réponse précoce des lésions par imagerie existent (mauvaise maîtrise des mouvements respiratoires, pas de consensus sur les paramètres permettant de quantifier la microcirculation tumorale, estimation paramétrique faite à partir de données extrêmement bruitées et pour un grand nombre de zones - une estimation par voxel de la séquence dynamique d’images). Dans un contexte clinique extrêmement contraignant, nous avons mis en place un cadre rigoureux comprenant l’ensemble des étapes nécessaires pour une caractérisation plus fiable de la microcirculation tumorale à partir de séquences d’images acquises sous perfusion de produit de contraste : les contributions principales de cette thèse couvrent ainsi l’optimisation des paramètres de reconstruction, le développement d’une méthode de recalage adaptée à nos données, la sélection argumentée d’un modèle de perfusion et enfin le développement d’une méthode robuste d’estimation des paramètres. Ces travaux permettent d’envisager l’utilisation des modèles de perfusion pour la caractérisation et la prédiction de la réponse d’un patient à différents traitements antitumoraux. / The main objective is to propose robust methods to allow estimation of functional markers reflecting the tumor evolution from dynamic studies. Currently, in this domain, assessing of the efficiency evaluation of an anti-angiogenic therapy (destruction of vessels which feed the tumor) is mainly based on the functional imaging of the microcirculation, which the objective is to quantify the tumor microcirculation by dynamic acquisitions with injection of contrast product. However, several limitations are present (lack of control of the breathing movement, no consensus on the parameters permitting the quantification of tumor microcirculation, parameter estimation computed from noisy data and a large number of regions - one estimation by voxel or group of voxel of the dynamic image sequence). In a restrictive clinical context (noisy data, few number), we have developed a complete pipeline with a set of necessary steps to a reliable characterization of the tumor microcirculation from dynamic perfusion image sequence: the main contributions of this thesis cover the reconstruction parameters optimization, the development of a registration method, the argued selection of a perfusion model and the development of a robust method of parameter estimation. With these works, we can envision the utilization of these perfusion models to the characterization and the prediction of the therapy response of a patient