abstract: The unique anatomical and functional properties of vasculature determine the susceptibility of the spinal cord to ischemia. The spinal cord vascular architecture is designed to withstand major ischemic events by compensating blood supply via important anastomotic channels. One of the important compensatory channels of the arterial basket of the conus medullaris (ABCM). ABCM consists of one or two arteries arising from the anterior spinal artery (ASA) and circumferentially connecting the ASA and the posterior spinal arteries. In addition to compensatory function, the arterial basket can be involved in arteriovenous fistulae and malformations of the conus. The morphometric anatomical analysis of the ABCM was performed with emphasis on vessel diameters and branching patterns. A significant ischemic event that overcomes vascular compensatory capacity causes spinal cord injury (SCI). For example, SCI complicating thoracoabdominal aortic aneurysm repair is associated with ischemic injury. The rate of this devastating complication has been decreased significantly by instituting physiological methods of protection. Traumatic spinal cord injury causes complex changes in spinal cord blood flow (SCBF), which are closely related to a severity of injury. Manipulating physiological parameters such as mean arterial pressure (MAP) and intrathecal pressure (ITP) may be beneficial for patients with a spinal cord injury. It was discovered in a pig model of SCI that the combination of MAP elevation and cerebrospinal fluid drainage (CSFD) significantly and sustainably improved SCBF and spinal cord perfusion pressure. In animal models of SCI, regeneration is usually evaluated histologically, requiring animal sacrifice. Thus, there is a need for a technique to detect changes in SCI noninvasively over time. The study was performed comparing manganese-enhanced magnetic resonance imaging (MEMRI) in hemisection and transection SCI rat models with diffusion tensor imaging (DTI) and histology. MEMERI ratio differed among transection and hemisection groups, correlating to a severity of SCI measured by fraction anisotropy and myelin load. MEMRI is a useful noninvasive tool to assess a degree of neuronal damage after SCI. / Dissertation/Thesis / Doctoral Dissertation Neuroscience 2016

<p>Transition metals, such as iron, manganese, and copper are essential in the development and function of biological systems. However, disrupted levels of transition metals are highly cytotoxic, and metal homeostasis is strictly maintained in cells under normal conditions. The neuropathology of several brain disorders, such as Alzheimer’s disease and Parkinson’s disease has been linked to altered metal levels. This work focused on the measurement of iron, manganese, and copper, with the aim of better elucidating their role in brain disease.</p> <p>Two experiments were carried out in C57Bl/6 mice looking at metal homeostasis: <em>1.</em> following manganese injections typically administered in manganese-enhanced MRI animal studies, and <em>2.</em> following copper deficiency in a cuprizone model of demyelination. Metal measurements were made in the brain and visceral organs using X-ray fluorescence to measure iron and copper concentrations, and neutron activation analysis to measure manganese concentrations.</p> <p>In the MEMRI study in this work, in addition to the expected manganese concentration increases in brain regions, a statistically significant decrease in iron concentration in the thalamus was found. This change in iron levels in the thalamus following manganese injections should serve as a caution that care should be taken when interpreting signal changes in brain regions.</p> <p>The cuprizone study in this thesis confirmed that copper levels are reduced following cuprizone administration. Surprisingly, manganese concentrations were significantly higher in several brain regions that have demyelination in this model, but not iron or copper. The mechanism of cuprizone toxicity was related to manganese neurotoxicity that may contribute to demyelination.</p> / Master of Science (MSc)

Recent progress in magnetic resonance imaging (MRI) has opened the way for micron-scale resolution, and thus for imaging biological cells. In this thesis work, we performed magnetic resonance microscopy (MRM) on the nervous system of Aplysia californica, a model particularly suited due to its simplicity and to its very large neuronal cell bodies, in the aim of studying cellular-scale processes with various MR contrasts. Experiments were performed on a 17.2 Tesla horizontal magnet, at resolutions down to 25 µm isotropic. Initial work consisted in conceiving and building radiofrequency microcoils adapted to the size of single neurons and ganglia. The first major part of the project consisted in using the manganese ion (Mn2+) as neural tract tracer in the buccal ganglia of Aplysia. Manganese is an MR contrast agent that enters neurons via voltage-gated calcium channels. We performed the mapping of axonal projections from motor neurons into the peripheral nerves of the buccal ganglia. We also confirmed the existence of active Mn2+ transport inside the neural network upon activation with the neurotransmitter dopamine. In the second major part of the project, we tested the potential of two diffusion MRI sequences for microscopy. On the one hand, we explored a very original mechanism for diffusion weighting, DESIRE (Diffusion Enhancement of SIgnal and REsolution), particularly suited for small samples. The two-dimensional DESIRE sequence was implemented and successfully tested on phantoms. The measured enhancement was consistent with theoretical predictions. Using this sequence to produce diffusion weighted images with an unprecedented contrast in biological tissue remains a challenge. On the other hand, a more "standard" sequence was implemented to measure the apparent diffusion coefficient (ADC) in nervous tissue with MRM. This sequence was a three-dimensional DP-FISP (Diffusion Prepared Fast Imaging with Steady-state free Precession), which met criteria for high resolution in a short acquisition time, with minimal artifacts. Using this sequence, we studied the changes in water ADC at different scales in the nervous system, triggered by cellular challenges. The challenges were hypotonic shock or exposure to ouabain. ADC measurements were performed on single isolated neuronal bodies and on ganglia tissue, before and after challenge. Both types of stress produced an ADC increase inside the cell and an ADC decrease at tissue level. The results favor the hypothesis that the increase in membrane surface area associated with cell swelling is responsible for the decrease of water ADC in tissue, typically measured in ischemia or other conditions associated with cell swelling.

The retina is a thin layer of tissue lining the back of the eye and is primarily responsible for sight in vertebrates. The neural retina has a distinct layered structure with three dense nuclear layers, separated by plexiform layers comprising of axons and dendrites, and a layer of photoreceptor segments. The retinal and choroidal vasculatures nourish the retina from either side, with an avascular layer comprised largely of photoreceptor cells. Diseases that directly affect the neural retina like retinal degeneration as well as those of vascular origin like diabetic retinopathy can lead to partial or total blindness. Early detection of these diseases can potentially pave the way for a timely intervention and improve patient prognosis. Current techniques of retinal imaging rely mainly on optical techniques, which have limited depth resolution and depend mainly on the clarity of visual pathway. Magnetic resonance imaging is a versatile tool that has long been used for anatomical and functional imaging in humans and animals, and can potentially be used for retinal imaging without the limitations of optical methods. The work reported in this thesis involves the development of high resolution magnetic resonance imaging techniques for anatomical and functional imaging of the retina in rats. The rats were anesthetized using isoflurane, mechanically ventilated and paralyzed using pancuronium bromide to reduce eye motion during retinal MRI. The retina was imaged using a small, single-turn surface coil placed directly over the eye. The several physiological parameters, like rectal temperature, fraction of inspired oxygen, end-tidal CO2, were continuously monitored in all rats. MRI parameters like T1, T2, and the apparent diffusion coefficient of water molecules were determined from the rat retina at high spatial resolution and found to be similar to those obtained from the brain at the same field strength. High-resolution MRI of the retina detected the three layers in wild-type rats, which were identified as the retinal vasculature, the avascular layer and the choroidal vasculature. Anatomical MRI performed 24 hours post intravitreal injection of MnCl2, an MRI contrast agent, revealed seven distinct layers within the retina. These layers were identified as the various nuclear and plexiform layers, the photoreceptor segment layer and the choroidal vasculature using Mn54Cl2 emulsion autoradiography. Blood-oxygenlevel dependent (BOLD) functional MRI (fMRI) revealed layer-specific vascular responses to hyperoxic and hypercapnic challenges. Relative blood volume of the retina calculated by using microcrystalline iron oxide nano-colloid, an intravascular contrast agent, revealed high blood-volume in the choroidal vasculature. Fractional changes to blood volume during systemic challenges revealed a higher degree of autoregulation in the retinal vasculature compared to the choroidal vasculature, corroborating the BOLD fMRI data. Finally, the retinal MRI techniques developed were applied to detect structural and vascular changes in a rat model of retinal dystrophy. We conclude that retinal MRI is a powerful investigative tool to resolve layer-specific structure and function in the retina and to probe for changes in retinal diseases. We expect the anatomical and functional retinal MRI techniques developed herein to contribute towards the early detection of diseases and longitudinal evaluation of treatment options without interference from overlying tissue or opacity of the visual pathway.

"Manganese (Mn2+) has recently gained acceptance as a magnetic resonance imaging (MRI) contrast agent useful for generating contrast in the functioning brain. The paramagnetic properties of Mn2+, combined with the cell's affinity for Mn2+ via voltage-gated calcium channels, makes Mn2+ sensitive to cellular activity in the brain. Compared with indirect measures of brain function, such as blood oxygenation level dependent (BOLD) functional MRI, manganese-enhanced MRI (MEMRI) can provide a direct means to visualize brain activity. MEMRI of the brain typically involves osmotic opening of the blood-brain barrier (BBB) to deliver Mn2+ into the interstitial space prior to initiation of a specific neuronal stimulus. This method assumes that the BBB-disruption process itself does not induce any apparent stimuli or cause tissue damage that might obscure any subsequent experimental observations. However, this assumption is often incorrect and can lead to misleading results for particular types of MRI applications. One aspect of these studies focused on characterizing the confounding effects of the BBB-opening process on MRI measurements typically employed to characterize functional activity or disease in the brain (Chapters 4 and 5). The apparent diffusion coefficient (ADC) of tissue water was found to decrease (relative to the undisrupted contralateral hemisphere) following BBB opening, obscuring similar ADC changes associated with ischemic brain tissue following stroke. Brain regions exhibiting reduced ADC values following osmotic BBB disruption also experienced permanent tissue damage, as validated by histological measures in the same vicinity of the brain. Non-specific MEMRI-signal enhancement was also observed under similar conditions and was found to be correlated to regions with BBB opening as verified by Evans Blue histological staining. In this case, MEMRI may prove to be a useful alternative for monitoring BBB-permeability changes in vivo. MEMRI was also investigated as a method for visualizing regions of BBB damage following ischemic brain injury (Chapter 6). BBB disruption following stroke has been investigated using gadolinium-based MRI contrast agents (e.g., Gd-DTPA). However, as an extracellular MRI contrast agent, Gd-DTPA is not expected to provide information regarding cell viability or function as part of MR image contrast enhancement. By comparison, brain regions with ischemia-induced BBB damage, and blood-flow levels sufficient to deliver Mn2+, show MEMRI-signal enhancement that correlates to regions with tissue damage as verified by histological staining. This approach should allow us to better understand the factors responsible for ischemia-induced BBB damage. Furthermore, MEMRI should be a useful tool for monitoring therapeutic interventions that might mitigate the damage associated with BBB disruption following stroke. "

Les progrès technologiques récents en imagerie par résonance magnétique (IRM) ont ouvert la voie à une résolution spatiale de l’ordre de quelques microns, et donc à l’imagerie de cellules biologiques. Dans le cadre de ce projet, nous avons réalisé des expériences de microscopie IRM sur le système nerveux de l’aplysie (Aplysia californica), particulièrement adapté de par sa simplicité et de par la très grande taille de ses neurones, en vue d’étudier des processus à échelle cellulaire avec divers contrastes IRM. Les expériences d’imagerie ont été effectuées sur un aimant horizontal 17.2 Tesla, à des résolutions spatiales jusqu’à 25 µm isotrope. Le travail initial a consisté en la conception et fabrication de micro-antennes radiofréquences adaptées à la taille de neurones uniques et de ganglions. La première partie du projet a porté sur l’utilisation de l’ion manganèse (Mn2+) comme traceur de réseaux neuronaux dans le ganglion buccal de l’aplysie. Le manganèse (Mn) est un agent de contraste IRM qui pénètre dans les neurones par les canaux de calcium. La cartographie des projections axonales des neurones moteurs du ganglion dans chacun des nerfs périphériques a été établie. Il a également été démontré l’existence d’un transport actif du Mn2+ au sein du réseau neuronal activé par le neurotransmetteur dopamine. Dans un second temps, on s’est intéressé à deux méthodes de mesure de diffusion par IRM, à échelle microscopique. D’une part, un mécanisme de pondération en diffusion, DESIRE (Diffusion Enhancement of SIgnal and REsolution), original et particulièrement adapté à des échantillons petits, a été exploré. La séquence DESIRE a été implémentée en deux dimensions et testée avec succès sur fantôme. Le rehaussement mesuré était en accord avec les prévisions théoriques. Le grand défi à venir sera d’utiliser cette séquence pour acquérir des images de tissu biologique pondérées en diffusion avec un contraste unique. D’autre part, une séquence plus « classique » a été implémentée pour mesurer le coefficient de diffusion apparent (ADC) dans le tissu nerveux. Il s’agit d’une DP-FISP (Diffusion Prepared Fast Imaging with Steady-state free Precession) en trois dimensions, qui répond aux critères de résolution spatiale et de rapidité, avec un minimum d’artefacts. Cette séquence a permis d’étudier l’évolution de l’ADC de l’eau à différentes échelles du tissu nerveux en réponse à un stress cellulaire. Les deux sollicitations retenues étaient un choc hypotonique ou l’ajout d’ouabaïne. Des mesures d’ADC ont été effectuées sur des corps neuronaux isolés et sur du tissu de ganglion, avant et après sollicitation. Les deux types de stress ont entraîné une augmentation de l’ADC dans la cellule et une diminution globale de l’ADC dans le tissu. Ces résultats soutiennent l’hypothèse que la diffusion ralentie de l’eau habituellement observée dans un tissu ischémié (ou dans d’autres conditions associées à un gonflement cellulaire) est due à l’augmentation de surface membranaire. / Recent progress in magnetic resonance imaging (MRI) has opened the way for micron-scale resolution, and thus for imaging biological cells. In this thesis work, we performed magnetic resonance microscopy (MRM) on the nervous system of Aplysia californica, a model particularly suited due to its simplicity and to its very large neuronal cell bodies, in the aim of studying cellular-scale processes with various MR contrasts. Experiments were performed on a 17.2 Tesla horizontal magnet, at resolutions down to 25 µm isotropic. Initial work consisted in conceiving and building radiofrequency microcoils adapted to the size of single neurons and ganglia. The first major part of the project consisted in using the manganese ion (Mn2+) as neural tract tracer in the buccal ganglia of Aplysia. Manganese is an MR contrast agent that enters neurons via voltage-gated calcium channels. We performed the mapping of axonal projections from motor neurons into the peripheral nerves of the buccal ganglia. We also confirmed the existence of active Mn2+ transport inside the neural network upon activation with the neurotransmitter dopamine. In the second major part of the project, we tested the potential of two diffusion MRI sequences for microscopy. On the one hand, we explored a very original mechanism for diffusion weighting, DESIRE (Diffusion Enhancement of SIgnal and REsolution), particularly suited for small samples. The two-dimensional DESIRE sequence was implemented and successfully tested on phantoms. The measured enhancement was consistent with theoretical predictions. Using this sequence to produce diffusion weighted images with an unprecedented contrast in biological tissue remains a challenge. On the other hand, a more “standard” sequence was implemented to measure the apparent diffusion coefficient (ADC) in nervous tissue with MRM. This sequence was a three-dimensional DP-FISP (Diffusion Prepared Fast Imaging with Steady-state free Precession), which met criteria for high resolution in a short acquisition time, with minimal artifacts. Using this sequence, we studied the changes in water ADC at different scales in the nervous system, triggered by cellular challenges. The challenges were hypotonic shock or exposure to ouabain. ADC measurements were performed on single isolated neuronal bodies and on ganglia tissue, before and after challenge. Both types of stress produced an ADC increase inside the cell and an ADC decrease at tissue level. The results favor the hypothesis that the increase in membrane surface area associated with cell swelling is responsible for the decrease of water ADC in tissue, typically measured in ischemia or other conditions associated with cell swelling.

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