In vivo real-time data of ultrasound and microbubble assisted blood-brain barrier disruption is centrally based on low-resolution magnetic resonance images. Additional information can be gained using online microscopic monitoring. This study presents the first ever in vivo two-photon microscopy, four-dimensional data sets of ultrasound and microbubble assisted blood-brain barrier disruption. It characterized the threshold pressures and mechanical index needed to disrupt the vasculature with 800 kHz ultrasound, and found three different leakage constants from the compromised vasculature. Furthermore, using numerical models, an ultrasound array was designed and optimized to perform specifically with our two-photon microscope. It was fabricated, fully characterized, and its performance met both the required pressure field profile and the pressure values needed for our in vivo two-photon microscopy experiments. This array is an important step in microscopically characterizing ultrasound and microbubble assisted blood-brain barrier disruption.

In vivo real-time data of ultrasound and microbubble assisted blood-brain barrier disruption is centrally based on low-resolution magnetic resonance images. Additional information can be gained using online microscopic monitoring. This study presents the first ever in vivo two-photon microscopy, four-dimensional data sets of ultrasound and microbubble assisted blood-brain barrier disruption. It characterized the threshold pressures and mechanical index needed to disrupt the vasculature with 800 kHz ultrasound, and found three different leakage constants from the compromised vasculature. Furthermore, using numerical models, an ultrasound array was designed and optimized to perform specifically with our two-photon microscope. It was fabricated, fully characterized, and its performance met both the required pressure field profile and the pressure values needed for our in vivo two-photon microscopy experiments. This array is an important step in microscopically characterizing ultrasound and microbubble assisted blood-brain barrier disruption.

Cerebral ischaemia by mechanism of thrombosis is one of the leading causes of disability and/or death worldwide, the outcome thereof increasing in severity with advancing age. Cerebral ischaemia triggers a cascade of events including inflammation, blood-brain barrier disruption and apoptosis. It is well known that oestrogen is neuroprotective through various mechanisms including the interruption of inflammation, regulation of thrombosis and delay of apoptosis. This creates a strong factorial interconnection in predicting the consequences of cerebral ischaemia. Since platelets have a central role in thrombosis and inflammation, their ultrastructure being altered in conditions of inflammatory and thrombotic derivation, the question arises whether chemical analysis of coagulation factors and ultrastructural analyses of platelet morphology may provide further insight into the role of oestrogen during ischaemic insult associated with stroke. Accordingly, an exclusively hyperglycaemic modification of the two-vessel occlusion model for inducing experimental cerebral ischaemia was established, since pre-ischaemic hyperglycaemia is known to intensify the outcome of cerebral ischaemic injury. Consequent neural tissue injury levels were correlated for three experimental groups (males, cyclic and acyclic females) of Sprague Dawley rats at vital times, to the presence of oestrogen as well as changes in coagulation factors and ultrastructure. This design allowed for an association to be formed between cerebral ischaemia, inflammation and thrombotic potential. Collectively the results strongly suggest that oestrogen is indeed neuroprotective through various actions including roles in the regulation of thrombosis and inflammation, targeting neural cells through the inhibition of apoptosis and exerting anti-inflammatory and antioxidant effects. It is evident that under the influence of oestrogen in cyclic females, there is reduced neural tissue injury as well as a lesser degree of inflammation evident in coagulation factor analysis and platelet activation morphology when compared to males and acyclic females. Oestrogen therefore exerts positive effects on the outcome of cerebral ischaemia through mechanisms which regulate inflammation, thrombosis and apoptosis. Furthermore it is unmistakeable that neural injury is closely shadowed, if not preceded, by inflammatory changes in the coagulation system, particularly manifested in platelet ultrastructure. It is therefore suggested that platelets may be used successfully to follow the progression of events of cerebral ischaemia and possibly assist in the assessment of treatment strategies and their effects on haemostasis. This research advances the understanding that inflammation is evident soon after ischaemic insult and if such inflammation is not curbed, necrosis of platelets and more severe injury to neural tissue may follow. Therefore, the development of agents which not only target thrombosis, but also which control inflammation must be explored to advance treatment strategies. It is proposed that even before it is determined whether a stroke has been caused by thromboembolism or haemorrhage; it will be beneficial to immediately target inflammation in order to prevent most severe consequences in human patients. / Thesis (PhD)--University of Pretoria, 2013. / gm2013 / Anatomy / unrestricted

The most common and aggressive malignant brain tumor, glioblastoma (GBM), demonstrates on average a 5-year survival rate of only 6.8%. Difficulties arising in the treatment of GBM include the inability of large molecular agents to permeate through the blood-brain barrier (BBB); migration of highly invasive GBM cells beyond the solid tumor margin; and gross, macroscopic intratumor heterogeneity. These characteristics complicate treatment of GBM with standard of care, resulting in abysmal prognosis. Electroporation-based therapies have emerged as attractive alternates to standard of care, demonstrating favorable outcomes in a variety of tumors. Notably, irreversible electroporation (IRE) has been used for BBB disruption and nonthermal ablation of intracranial tumor tissues. Despite promising results, IRE can cause unintended muscle contractions and is susceptible to electrical heterogeneities. Second generation High-frequency IRE (H-FIRE) utilizes bursts of bipolar pulsed electric fields on the order of the cell charging time constant (~1 μs) to ablate tissue while reducing nerve excitation, muscle contraction, and is far less prone to differences in electrical heterogeneities. Throughout my dissertation, I discuss investigations of H-FIRE for the treatment of malignant gliomas and other intracranial disorders. To advance the versatility, usability, and understanding of H-FIRE for intracranial applications, my PhD thesis focuses on: (1) characterizing H-FIRE-mediated BBB disruption effects in an in vivo healthy rodent model; (2) the creation of a novel, real-time impedance spectroscopy technique (Fourier Analysis SpecTroscopy, FAST) using waveforms compatible with existing H-FIRE pulse generators; (3) development of FAST as an in situ technique to monitor ablation growth and to determine patient-specific ablation endpoints; (4) conducting a preliminary efficacy study of H-FIRE ablation in an orthotopic F98 rodent glioma model; and (5) establishing the feasibility of MRI-guided H-FIRE for the ablation malignant gliomas in a spontaneous canine glioma model. The culmination of this thesis advances our understanding of H-FIRE in intracranial tissues, as well as develops a novel, intraoperative impedance spectroscopy technique towards determining patient-specific ablation endpoints for intracranial H-FIRE procedures. / Doctor of Philosophy / The most aggressive malignant brain tumor, glioblastoma (GBM), demonstrates on average a 5-year survival rate of only 6.8%. Difficulties arising in the treatment of GBM include the inability of chemotherapy agents to diffuse into brain tumor tissue as these molecular are unable to pass the so-called blood-brain barrier (BBB). This tumor tissue also presents with cells with the propensity to invade healthy tissue, to the point where diagnostic scans are unable to capture this migration. These characteristics complicate treatment of GBM with standard of care, resulting in abysmal prognosis. Electroporation-based therapies have emerged as attractive alternates to standard of care, demonstrating favorable outcomes in a variety of tumors. For instance, irreversible electroporation (IRE) has been used to successfully treat tumors in the prostate, liver, kidney, and pancreas. Second generation High-frequency IRE (H-FIRE) may possess even greater antitumor qualities and this is the focus of my dissertation. Throughout my dissertation, I discuss investigations of H-FIRE with applications to treat malignant gliomas and other intracranial disorders. My PhD thesis focuses on: (1) characterizing H-FIRE effects for enhanced drug delivery to the brain; (2) the creation of a new, real-time electrical impedance spectroscopy technique (Fourier Analysis SpecTroscopy, FAST) using waveforms compatible with existing H-FIRE pulse generators; (3) development of FAST as a technique to determine H-FIRE treatment endpoints; (4) conducting a preliminary efficacy study of H-FIRE to ablate rodent glioma tumors; and (5) establishing the feasibility of MRI-guided H-FIRE for the ablation malignant gliomas in a spontaneous canine glioma model. The culmination of this thesis advances our understanding of H-FIRE in intracranial tissues, as well as develops a new impedance spectroscopy technique to be used in determining patient-specific ablation endpoints for intracranial H-FIRE procedures.

Glioblastoma Multiforme (GBM) is a highly aggressive and prevalent brain tumor with an average 5-year survival rate of approximately 6.9%. Its complex pathophysiology, characterized by the capacity to invade surrounding tissues beyond the visible tumor margin, intratumor heterogeneity, hypoxic core, and the presence of the blood-brain barrier (BBB) that restricts the penetration of large therapeutic agents, all pose formidable challenges for effective therapeutic intervention. The standard of care for GBM has thus far exhibited limited success, and patients often face a poor prognosis. Electroporation-based therapies, such as irreversible electroporation (IRE), have emerged as promising alternatives to conventional treatments. By utilizing high amplitude pulsed electric fields, IRE is able to permeabilize cells, disrupt the BBB, and induce non thermal ablation of soft tissues. However, IRE is oftentimes accompanied by undesirable secondary effects such as muscle contractions, complex anesthetic protocols, and susceptibility to electrical heterogeneities, which have impeded its clinical translation. To address these limitations, high-frequency IRE (H-FIRE) was developed. H-FIRE employs short bursts of bipolar pulses, similar in duration to the cell charging time constant, enabling the desired tissue ablation while minimizing nerve excitation and muscle contractions. Additionally, H-FIRE reduces susceptibility to electrical heterogeneities, allowing for more predictable treatment volumes, thus enhancing the feasibility of clinical translation. This dissertation investigates H-FIRE for targeting malignant gliomas while looking into improved efficacy when administering the therapy in conjunction with other treatment forms and technologies. Specifically, the presented work focuses on several key areas: (1) determining the effect of pulsing protocol and geometric configuration selection on the biological outcomes from electroporation; (2) using a tumor bearing rodent glioma model to evaluate the effects of H-FIRE as a standalone therapy and as a combinatorial therapy with liposomal doxorubicin; (3) investigating the effects of waveform shape on biological outcomes; (4) utilizing real-time Fourier Analysis SpecTroscopy (FAST) to accurately model rises in temperature during treatment; and (5) modifying real-time FAST methods to determine treatment endpoints for safe and effective ablation volumes. / Doctor of Philosophy / Glioblastoma Multiforme (GBM) is one of the deadliest tumors, with an overall five-year survival rate of approximately 6.9%. Unfortunately, it also holds the position of being the most prevalent malignant brain tumor, constituting nearly 50.1% of all primary malignant brain tumor diagnoses. Despite its widespread occurrence, there has been limited success in improving survival rates. The tumor's infiltrative nature and its location behind the blood-brain barrier (BBB), which often screens out large drug molecules like chemotherapeutics, contribute significantly to these unfavorable treatment outcomes. This dissertation explores the potential of high-frequency irreversible electroporation (H-FIRE) as a solution to these challenges. H-FIRE employs bursts of pulsed electric fields to induce nanoscale defects in the cell membrane. The response to these defects may involve temporary pores that facilitate the uptake of therapeutic molecules into the cell, or larger and longer lasting pores that disrupt cell homeostasis, ultimately leading to cell death. Furthermore, this pulsed field therapy has shown success in enabling molecules to bypass the BBB. Thus, this dissertation aims to elucidate the various biophysical phenomena associated with H-FIRE, shedding light on how to manipulate treatment protocols to maximize BBB disruption and enhance therapy when used in conjunction with combinatorial agents. Additionally, this work aims to further develop technologies to provide real-time feedback, ensuring the safe and effective delivery of the treatment. Through these efforts, this dissertation aspires to offer valuable insights into optimizing H-FIRE for the treatment of malignant gliomas and advancing the understanding of combinatorial therapies in this specific context.

This thesis project serves to fill experimental gaps needed to advance the goal of performing pre-clinical trials using an orthotopic rat glioblastoma model to evaluate the efficacy of high-frequency electroporation (H-FIRE) and QUAD-CTX tumor receptor-targeted cytotoxic conjugate therapies, individually and in combination, in selectively and thoroughly treating glioblastoma multiforme. In order to achieve this, an appropriate model must be developed and characterized. I have transduced F98 rat glioma cells to express red-shifted firefly luciferase, which will facilitate longitudinal tumor monitoring in vivo through bioluminescent imaging. I have characterized their response to H-FIRE relative to DI TNC1 rat astrocytes. I have demonstrated the presence of the molecular targets of QUAD in F98 cells. The in vitro characterization of this model has enabled preclinical studies of this promising glioblastoma therapy in an immunocompetent rat model, an important step before advancing ultimately to clinical human trials. / Master of Science / Treating glioblastoma multiforme (GBM), a form of cancer found in the brain, has not been very successful; patients rarely live two years following diagnosis, and there have been no major breakthrough advances in treatment to improve this outlook for decades. We have been working on two treatments which we hope to combine. The first is high-frequency electroporation (H-FIRE), which uses electrical pulses to kill GBM cells while leaving healthy cells alive and blood vessels intact. The second is QUAD-CTX, which combines a toxin with two types of protein that attach to other proteins that are more common on the surface of GBM cells than healthy cells. We have shown these to be effective at disproportionately killing human GBM cells growing in a lab setting. Before H-FIRE and QUAD-CTX may be tested on humans, we need to show them to be effective in an animal model, specifically rats. I have chosen rat glioma cells that will behave similarly to human GBM and a rat species that will not have an immune response to them. I have made these cells bioluminescent so that we may monitor the tumors as they grow and respond to our treatments. I have also shown that QUAD-CTX kills these rat glioma cells, as does H-FIRE. Because of this work, we are ready to begin testing these two treatments in rats.

No description available.

Glioblastoma (GBM) is the most common and deadliest of the malignant primary brain tumors in humans, with a reported 5-year survival rate of only 6.8% despite years of extensive research. Failure to improve local tumor control rates and overall patient outcome is attributed to GBM's inherent therapeutic resistance. Marked heterogeneity, extensive local invasion within the brain parenchyma, and profound immunosuppression within the tumor microenvironment (TME) are some of the unique features that drive GBM therapeutic resistance. Furthermore, tumor cells are sequestered behind the blood-brain barrier (BBB), limiting delivery of effective therapeutics and immune cell infiltration into the local tumor. Electroporation-based therapies, such as irreversible electroporation (IRE) and second generation, high-frequency IRE (H-FIRE) represent attractive alternative approaches to standard GBM therapy given their ability to induce transient BBB disruption (BBBD), achieve non-thermal tumor cell ablation and stimulate local and systemic anti-tumor immune responses without significant morbidity. The following work explores the use of H-FIRE to overcome GBM-induced therapeutic resistance and improve treatment success. Chapter 1 opens with an overview of GBM and known barriers to treatment success. Here, we emphasize the utility of spontaneous canine gliomas as an ideal translational model for investigations into novel treatment approaches. Chapter 2 introduces novel ablation methods (i.e. IRE/H-FIRE) capable of targeting treatment-resistant cancer stem cells. The focus of Chapter 3 is to highlight IRE applications in a variety of spontaneous tumor types. In Chapter 4, we investigate the feasibility and local immunologic response of percutaneous H-FIRE for treatment of primary liver tumors using a spontaneous canine hepatocellular carcinoma (HCC) model. In chapter 5, we characterize the mechanisms of H-FIRE-mediated BBBD in an in vivo healthy rodent model. In Chapter 6, we characterize the local and systemic immune responses to intracranial H-FIRE in rodent and canine glioma models to enhance the translational value of our work. Collectively, our work demonstrates the potential for H-FIRE to overcome therapeutic resistance in GBM, thereby supporting its use as a novel, alternative treatment approach to standard therapy. / Doctor of Philosophy / Glioblastoma (GBM) is the most common and deadliest form of primary brain cancer in humans, with only 6.8% of people surviving 5-years after their diagnosis. GBM is characterized by a number of unique features that make it resistant to standard treatments, such as surgery, radiation and chemotherapy. Examples include: (1) extensive invasion of tumor cells into the brain, making complete removal via surgery very difficult; (2) tumor cells are protected by a structure called the blood-brain barrier (BBB), which restricts the entry of most drugs (i.e. chemotherapy) and many immune cells, into the brain, thereby preventing them from reaching tumor cells; (3) tumor cells produce substances that block the immune system from being able to detect the tumor itself, which allows it to continue to grow undetected. High-frequency irreversible electroporation (H-FIRE) represents a new approach for the treatment of GBM. H-FIRE uses electric pulses to temporarily or permanently injure cell membranes without the use of heat, which allows for very precise treatment. The following work explores the ways in which H-FIRE can interfere with specific GBM features that drive its resistance to treatment. Here, we demonstrate that H-FIRE is capable of temporarily disrupting the BBB and characterize the mechanisms by which this occurs. This allows for drugs and immune cells within the blood to enter the brain and access the tumor cells, particularly those extending beyond the visible tumor mass and invading the brain. We also illustrate the potential for H-FIRE treatment within the brain to stimulate local and systemic immune responses by causing the release of proteins from injured cells. Similar to a vaccine, these proteins are recognized by the immune system, which becomes primed to help fight off cancer cells within the body. The end result is an anti-tumor immune response. Collectively, this work supports the use of H-FIRE as an alternative treatment approach to standard therapy for GBM given its potential to overcome certain causes of treatment resistance.

Le glioblastome (GBM) est la tumeur cérébrale primaire la plus fréquente et agressive chez l’adulte. Son traitement, une exérèse chirurgicale maximale suivi d’un traitement adjuvant (radiothérapie et témozolomide [TMZ]), n’offre qu’un bénéfice modeste de survie médiane (14.6 mois vs. 12.1 mois pour radiothérapie post-chirurgie seule) (STUPP et al., 2005). Le TMZ demeure l’agent de choix pour le traitement du GBM. Malgré sa biodisponibilité approchant 100% suivant son administration per os (PO) (Diez et al., 2009), sa pénétration dans le liquide céphalorachidien n’est que de 20% (Ostermann et al., 2004). Ainsi, il se peut que les limites thérapeutiques du TMZ soient reliées aux barrières hémato-encéphalique (BHE) et hémato-tumorale (BHT). Plusieurs stratégies alternatives tentent de contourner ces barrières comme l’administration intra-artérielle (IA) avec une ouverture osmotique de la BHE (OBHE). Cette technique permet une plus grande distribution d’agent thérapeutique au système nerveux central (SNC). L’utilisation de cette stratégie avec le témozolomide n’a jamais été étudiée à ce jour. Nous avons émis l’hypothèse que son utilisation permettra d’augmenter la concentration de TMZ dans le SNC et que, lorsque combiné avec la radiothérapie, permettra de rehausser son activité anti-tumorale. Les objectifs du projet sont : (1) l’évaluation de la sensibilité des cellules F98 au TMZ in vitro; (2) la caractérisation de la neuropharmacocinétique du TMZ in vivo, selon différents modes d’administration; et (3) l’évaluation de l’effet anti-tumoral du TMZ in vivo, selon différents modes d’administration. Les expérimentations in vivo ont été exécutées dans le modèle syngénique Fischer-F98, porteur de tumeur gliale. L’expérimentation in vitro a démontré une résistance importante des cellules F98 au TMZ. La méthodologie développée a permis de démontrer que l’infusion IA avec et sans OBHE augmente la concentration maximale et l’aire sous la courbe du TMZ dans la tumeur cérébrale et dans le parenchyme cérébral ipsilatéral du rat Fischer-F98. Par contre, aucun bénéfice de survie n’a été observé en utilisant ces stratégies alternatives. Au contraire, l’acheminement augmenté du TMZ au SNC semble toxique. Un bénéfice de survie a été mesuré suite à l’ajout d’un traitement de radiothérapie, mais de façon indépendante au mode de livraison de TMZ ou de solution saline normale (groupe contrôle). Enfin, nos résultats témoignent de l’impact du mode d’acheminement sur la distribution d’un agent thérapeutique au SNC. En détournant la BHE, l’utilisation judicieuse d’approches alternatives combinée à un agent thérapeutique approprié a un grand potentiel clinique dans le traitement des GBM. / Abstract : Glioblastoma (GBM) is the most frequent and aggressive primary brain tumor in adults. Its’ standard treatment, maximal surgical resection followed by an adjuvant treatment (radiotherapy and temozolomide [TMZ]) offers only a modest median survival benefit of 14.6 months (vs. 12.1 months with post-surgery radiotherapy alone) (Stupp et al., 2005). TMZ remains the therapeutic agent of choice for the treatment of GBM. Despite its bioavailability approaching 100% after a per os administration (Diez et al., 2009), its cerebrospinal fluid penetration is only of 20% (Ostermann et al., 2004). Thus, TMZ’s therapeutic limitations could be due to the blood-brain barrier (BBB) and blood-tumor barrier (BTB). Alternative routes of drug delivery attempt to bypass these barriers. For example, intra-arterial (IA) administration with an osmotic blood-brain barrier disruption (OBBBD) allows greater drug distribution to the central nervous system (CNS). Its use with TMZ, with or without radiotherapy, has never been studied. We hypothesized that it will increase TMZ concentration in the CNS and that, when combined to radiotherapy, it will intensify its anti-neoplastic activity. The project was divided in three parts: (1) the evaluation of F98 cells’ in vitro sensitivity to TMZ; (2) the in vivo caracterization of TMZ’s neuropharmacokinetics, following different routes of administration; and (3) the in vivo evaluation of TMZ’s anti-tumoral effect, following different routes of administration. The syngenic glioma Fischer-F98 model was used in all in vivo experiments. Our results showed the F98 cells to be resistant to TMZ in vitro. The methodology developed showed that an IA infusion with and without OBBBD increased TMZ’s peak concentration and area under the curve in the brain tumor and ipsilateral brain parenchyma in the Fischer-F98 rat. All the while limiting systemic exposure. However, no survival benefit was observed with the use of these alternative strategies. More so, TMZ’s enhanced delivery to the CNS seemed toxic. A survival benefit was measured following the addition of radiotherapy. This was independent of the route of delivery of TMZ or normal saline. In summary, our results provide evidence that the method of TMZ administration does impact its CNS delivery. By bypassing the BBB, the judicious use of local delivery approaches combined with the appropriate therapeutic agent can have a great clinical potential in the treatment of glioblastomas.

La barrière hémato-encéphalique (BHE) est une protection naturelle du système nerveux central. Son étanchéité constitue néanmoins un frein à de nombreuses thérapies médicamenteuses. Elle peut être temporairement perméabilisée grâce à une exposition à des ultrasons, couplée à une injection de microbulles dans la circulation sanguine. Dans ce manuscrit, l'ouverture de la BHE avec un dispositif ultrasonore non focalisé et implantable est étudiée. Une méthode automatique de quantification du volume d'ouverture grâce aux images IRM issues d'une étude clinique de phase 1/2a chez des patients atteint d'un glioblastome multiforme récurrent a été développée et validée. Une corrélation entre la probabilité d'ouverture et la pression acoustique locale a été trouvée. L'activité de cavitation des microbulles a été étudiée in vitro pour affiner la compréhension de son lien avec l'ouverture de la BHE. L'incertitude de quantification de cette activité à l'aide d'un capteur mono-élément utilisé passivement (PCD) à travers le crâne a été évaluée. Une correction se basant sur la position du PCD par rapport à la source de cavitation a été proposée et validée. L'influence du volume couvert par un nuage de cavitation dans le champ ultrasonore non focalisé sur les amplitudes des signaux enregistrés par le PCD pendant le traitement clinique a été discutée. Deux méthodes de localisation et de différenciation de sources de cavitation multiples dans un contexte transcrânien ont été évaluées par simulations et in vitro / The blood-brain barrier (BBB) is a natural protection of the central nervous system. However, it limits the delivery of many drugs to the brain tissues. It can be temporarily disrupted by ultrasound exposure combined with intravenous injection of microbubbles. In this manuscript, BBB disruption with an implantable unfocused ultrasound device is studied. An automatic method for quantifying the volume of BBB disruption using MR images from a phase 1/2a clinical study in patients with reccurent glioblastoma was assessed and validated. A correlation between the probability of disruption and the local acoustic pressure was found. Microbubbles cavitation activity was studied in vitro to better understand its effect on BBB disruption. The uncertainty on the amplitudes of cavitation signals recorded with a passive single-element detector (PCD) through the skull was quantified. A position-based correction of the PCD signal was assessed and validated. The effect of the volume of a cavitation cloud in the unfocused ultrasound field on the signal amplitude recorded by the PCD during the clinical treatment was discussed. Two methods for localizing and discriminating cavitation sources in a transcranial context were evaluated by simulations and in vitro