Comparison of stimulus-evoked cerebral hemodynamics in the awake mouse and under a novel anesthetic regime

Neural activity is closely followed by a localised change in cerebral blood flow, a process termed neurovascular coupling. These hemodynamic changes form the basis of contrast in functional magnetic resonance imaging (fMRI) and are used as a correlate for neural activity. Anesthesia is widely employed in animal fMRI and neurovascular studies, however anesthetics are known to profoundly affect neural and vascular physiology, particularly in mice. Therefore, we investigated the efficacy of a novel ‘modular’ anesthesia that combined injectable (fentanyl-fluanisone/midazolam) and volatile (isoflurane) anesthetics in mice. To characterize sensory-evoked cortical hemodynamic responses, we used optical imaging spectroscopy to produce functional maps of changes in tissue oxygenation and blood volume in response to mechanical whisker stimulation. Following fine-tuning of the anesthetic regime, stimulation elicited large and robust hemodynamic responses in the somatosensory cortex, characterized by fast arterial activation, increases in total and oxygenated hemoglobin, and decreases in deoxygenated hemoglobin. Overall, the magnitude and speed of evoked hemodynamic responses under anesthesia resembled those in the awake state, indicating that the novel anesthetic combination significantly minimizes the impact of anesthesia. Our findings have broad implications for both neurovascular research and longitudinal fMRI studies that increasingly require the use of genetically engineered mice

Functional ultrasound reveals effects of MRI acoustic noise on brain function

Loud acoustic noise from the scanner during functional magnetic resonance imaging (fMRI) can affect functional connectivity (FC) observed in the resting state, but the exact effect of the MRI acoustic noise on resting state FC is not well understood. Functional ultrasound (fUS) is a neuroimaging method that visualizes brain activity based on relative cerebral blood volume (rCBV), a similar neurovascular coupling response to that measured by fMRI, but without the audible acoustic noise. In this study, we investigated the effects of different acoustic noise levels (silent, 80 dB, and 110 dB) on FC by measuring resting state fUS (rsfUS) in awake mice in an environment similar to fMRI measurement. Then, we compared the results to those of resting state fMRI (rsfMRI) conducted using an 11.7 Tesla scanner. RsfUS experiments revealed a significant reduction in FC between the retrosplenial dysgranular and auditory cortexes (0.56 ± 0.07 at silence vs 0.05 ± 0.05 at 110 dB, p=.01) and a significant increase in FC anticorrelation between the infralimbic and motor cortexes (−0.21 ± 0.08 at silence vs −0.47 ± 0.04 at 110 dB, p=.017) as acoustic noise increased from silence to 80 dB and 110 dB, with increased consistency of FC patterns between rsfUS and rsfMRI being found with the louder noise conditions. Event-related auditory stimulation experiments using fUS showed strong positive rCBV changes (16.5% ± 2.9% at 110 dB) in the auditory cortex, and negative rCBV changes (−6.7% ± 0.8% at 110 dB) in the motor cortex, both being constituents of the brain network that was altered by the presence of acoustic noise in the resting state experiments. Anticorrelation between constituent brain regions of the default mode network (such as the infralimbic cortex) and those of task-positive sensorimotor networks (such as the motor cortex) is known to be an important feature of brain network antagonism, and has been studied as a biological marker of brain disfunction and disease. This study suggests that attention should be paid to the acoustic noise level when using rsfMRI to evaluate the anticorrelation between the default mode network and task-positive sensorimotor network.journal articl

Functional Ultrasound Imaging of Spinal Cord Hemodynamic Responses to Epidural Electrical Stimulation: A Feasibility Study

This study presents the first implementation of functional ultrasound (fUS) imaging of the spinal cord to monitor local hemodynamic response to epidural electrical spinal cord stimulation (SCS) on two small and large animal models. SCS has been successfully applied to control chronic refractory pain and recently was evolved to alleviate motor impairment in Parkinson's disease and after spinal cord injury. At present, however, the mechanisms underlying SCS remain unclear, and current methods for monitoring SCS are limited in their capacity to provide the required sensitivity and spatiotemporal resolutions to evaluate functional changes in response to SCS. fUS is an emerging technology that has recently shown promising results in monitoring a variety of neural activities associated with the brain. Here we demonstrated the feasibility of performing fUS on two animal models during SCS. We showed in vivo spinal cord hemodynamic responses measured by fUS evoked by different SCS parameters. We also demonstrated that fUS has a higher sensitivity in monitoring spinal cord response than electromyography. The high spatial and temporal resolutions of fUS were demonstrated by localized measurements of hemodynamic responses at different spinal cord segments, and by reliable tracking of spinal cord responses to patterned electrical stimulations, respectively. Finally, we proposed optimized fUS imaging and post-processing methods for spinal cord. These results support feasibility of fUS imaging of the spinal cord and could pave the way for future systematic studies to investigate spinal cord functional organization and the mechanisms of spinal cord neuromodulation in vivo

Chronic assessment of cerebral hemodynamics during rat forepaw electrical stimulation using functional ultrasound imaging

Functional ultrasound imaging is a method recently developed to assess brain activity via hemodynamics in rodents. Doppler ultrasound signals allow the measurement of cerebral blood volume (CBV) and red blood cells' (RBCs') velocity in small vessels. However, this technique originally requires performing a large craniotomy that limits its use to acute experiments only. Moreover, a detailed description of the hemodynamic changes that underlie functional ultrasound imaging has not been described but is essential for a better interpretation of neuroimaging data. To overcome the limitation of the craniotomy, we developed a dedicated thinned skull surgery for chronic imaging. This procedure did not induce brain inflammation nor neuronal death as confirmed by immunostaining. We successfully acquired both high-resolution images of the microvasculature and functional movies of the brain hemodynamics on the same animal at 0, 2, and 7 days without loss of quality. Then, we investigated the spatiotemporal evolution of the CBV hemodynamic response function (HRF) in response to sensory-evoked electrical stimulus (1 mA) ranging from 1 (200 mu s) to 25 pulses (5 s). Our results indicate that CBV HRF parameters such as the peak amplitude, the time to peak, the full width at half-maximum and the spatial extent of the activated area increase with stimulus duration. Functional ultrasound imaging was sensitive enough to detect hemodynamic responses evoked by only a single pulse stimulus. We also observed that the RBC velocity during activation could be separated in two distinct speed ranges with the fastest velocities located in the upper part of the cortex and slower velocities in deeper layers. For the first time, functional ultrasound imaging demonstrates its potential to image brain activity chronically in small animals and offers new insights into the spatiotemporal evolution of cerebral hemodynamics. (C) 2014 The Authors. Published by Elsevier Inc

Estudios funcionales mediante resonancia magnética en pequeños animales

This thesis is framed within the field of preclinical biomedical imaging, and specifically devoted to the study of functional magnetic resonance imaging (fMRI) technique in small animals. The experimental and technological complexity of this modality has greatly limited its use, and therefore it is not a routine imaging modality. However, it provides valuable information both at the physiological level, to study the mechanisms of normal brain during neuronal activity, and at the pathological level, to study drugs intended for different brain dysfunctions. In this work we have studied techniques and methods that intend to alleviate these difficulties and facilitate their use by the scientific community. The work includes contributions at several stages: the experimental setup, the data acquisition and reconstruction, and the quantitative image analysis. The first section addresses the problem of using anesthesia during the experiment. In order to perform functional measurements, it is necessary to establish a protocol to induce anesthetic sedation of the animal rather than a deep anesthetic state. Moreover, the use of non-toxic drugs with fast induction and recovery is desirable. In this section of the thesis we conducted fMRI experiments in rats sedated with sevoflurane, and since this agent had not been previously reported for fMRI, it was necessary to conduct strategies in order to determine the optimum dose-response and stimulation frequency. Furthermore, the signal obtained in the cerebral cortex was compared with a more traditional protocol sedation, subdermal medetomidine. The signal obtained was similar to that obtained under medetomidine, but the animal preparation time increased considerably, which constitutes a serious practical drawback for the use of sevoflurane. The second section is devoted to the study of a compressed sensing framework that allows a substantial reduction on the acquisition time without degrading image quality. The acquisition of a much reduced amount of data, thus at high rates of acceleration that violate the Nyquist-Shannon criterion, is possible by means of a wise exploitation of the temporal information redundancy and by the use of nonlinear iterative reconstruction algorithms. In this study we evaluated the performance of three compressed-sensing reconstruction algorithms that exploit temporal redundancy to recover the BOLD contrast and which have proved successful in other applications or imaging modalities such as: X-ray tomography, dynamic cardiac MRI, and resting state MRI studies. The comparison was performed in two signal-to-noise ratio scenarios and the conclusion drawn is that the algorithm which uses an a priori image (PICCS) yields the best reconstruction. The third section deals with the post-processing and image analysis. There are several open-source tools available to this purpose, but they were originally designed for human studies. Their adaptation to rodent images requires the use of additional tools or some image transformation processing that involve programming skills. Moreover, to obtain quantitative values, the user would need to use additional extensions or external software. In this work we have studied the existing tools and proposed and developed a new software, fMRat, which automatically performs a full multi-subject analysis, from the initial format conversion to the extraction of numerical values from the regions interest chosen by the user. The tool was programmed in Matlab as an extension of the existing SPM package, and was validated with 460 real rat studies. The code has been published as "open-software" in Github website and is accessible to the neuroscience community.Esta tesis se enmarca dentro del ámbito de la imagen biomédica preclínica, y específicamente trata sobre la técnica de imagen de resonancia magnética funcional (fMRI) en pequeños animales. La complejidad de dicha técnica tanto a nivel experimental como tecnológico ha limitado considerablemente su ámbito de uso, y por ello no es una modalidad de imagen que se realice de manera habitual. Sin embargo ofrece información muy valiosa tanto a nivel fisiológico, para el estudio de los mecanismos del cerebro normal durante la actividad neuronal, como a nivel patológico, para la búsqueda y estudio de fármacos aplicables a diferentes disfunciones cerebrales. En esta tesis se han estudiado técnicas y métodos para intentar aliviar estas dificultades y facilitar su utilización por parte de la comunidad científica. El trabajo incluye aportaciones en los ámbitos de la configuración del experimento, de la adquisición de los datos y su reconstrucción, y por último del análisis cuantitativo final de las imágenes. En el primer capítulo se trata el problema del uso de anestesia durante el experimento. Para obtener medidas funcionales es necesario establecer un protocolo anestésico que facilite la sedación del animal pero sin llegar a un estado anestésico profundo. Por otra parte, es deseable que sea de rápida inducción y recuperación, y que no sea tóxico para que pueda usarse en estudios longitudinales. En esta parte de la tesis se realizaron experimentos de fMRI en rata sedada con sevofluorano, para lo cual fue necesario realizar un estudio dosis-respuesta y un barrido de frecuencias de estimulación. Además, la señal obtenida en la corteza cerebral se comparó con la de otro protocolo de sedación más tradicional, con medetomidina subdérmica. La señal obtenida fue de intensidad similar a la obtenida con medetomidina, pero el tiempo de preparación del animal se incrementó considerablemente, lo cual constituye un grave inconveniente práctico para el uso de este anestésico. El segundo capítulo está dedicado al estudio de un entorno de adquisición comprimida o “compressed sensing” que permita reducir sustancialmente el tiempo de adquisición sin degradar la calidad de la imagen, gracias a la adquisición de una cantidad mucho menor de datos. En este trabajo se muestra que sería posible acelerar la adquisición a altas tasas que incumplen el criterio de Nyquist-Shannon siempre y cuando se explote la redundancia de información temporal y al mismo tiempo se empleen algoritmos de reconstrucción de imagen iterativos no lineales. En concreto se compara la eficacia de tres algoritmos de reconstrucción que explotan la redundancia temporal para recuperar el contraste BOLD y que han arrojado buenos resultados en otras aplicaciones o modalidades de imagen: tomografía por rayos X, estudios dinámicos de corazón por resonancia magnética, y resonancia funcional en reposo o “resting state”. La comparativa se realizó en dos escenarios de relación señal a ruido y se concluye que el algoritmo que utiliza una imagen a priori (PICCS) es el que mejores resultados obtiene en la reconstrucción. El tercer capítulo aborda el postprocesado y análisis de las imágenes. Existen varias herramientas gratuitas y de código abierto para este fin, pero fueron diseñadas para imagen de cerebro humano, y su adaptación a imágenes de roedores requiere el uso de herramientas adicionales o la realización de transformaciones en la imagen que implican conocimientos de programación. Además, para obtener valores cuantitativos es imprescindible el uso de extensiones o herramientas adicionales. En este trabajo se han estudiado las herramientas existentes y se ha propuesto y desarrollado un nuevo software, fMRat, que realiza el análisis completo de varios sujetos de manera automática, desde el cambio de formato de las imágenes hasta la obtención de valores numéricos de las regiones de interés elegidas por el usuario. La herramienta está programada en Matlab como una extensión de un paquete SPM ya existente, y fue validada con 460 estudios reales de ratas. El código está publicado como “opensoftware” en el sitio web de Github y es accesible a cualquier neurocientífico que desee utilizarlo.Programa Oficial de Doctorado en Multimedia y ComunicacionesPresidente: Pedro Ramos Cabrer.- Secretario: Juan Miguel Parra Robles.- Vocal: María Jesús Ledesma Carbay

Structural and functional brain imaging using extended-focus optical coherence tomography and microscopy

Neuroimaging techniques aim at revealing the anatomy and functional organisation of cerebral structures. Over the past decades, functional magnetic resonance imaging (fMRI) has revolutionized our understanding of human cerebral physiology through its ability to probe neural activity throughout the entire brain in a non-invasive fashion. Nevertheless, despite recent technological improvements, the spatial resolution of fMRI remains limited to a few hundreds of microns, restricting its use to macroscopic studies. Microscopic imaging solutions have been proposed to circumvent this limitation and have enabled revealing the existence of various cerebral structures, such as neuronal and vascular networks and their contribution to information processing and blood flow regulation within the brain. Optical imaging has proven, through its increased resolution and available contrast mechanisms, to be a valuable complement to fMRI for cellular-scale imaging. In this context, we demonstrate here the capabilities of an extension of optical coherence tomography, termed extended-focus optical coherence tomography (xf-OCT), in imaging cerebral structure and function at high resolution and very high acquisitions rates. Optical coherence tomography is an interferometric imaging technique using a low-coherence illumination source to provide fast, three-dimensional imaging of the back-scattering of tissue and cells. By multiplexing the interferometric ranging over several spectral channels, Fourier-domain OCT performs depth-resolved imaging at very high acquisition rates and high sensitivity. Increasing the lateral resolution of optical systems typically reduces the available depth-of-field and thus hampers this depth multiplexing advantage of OCT. Extended-focus systems aim at alleviating this trade-off between imaging depth and lateral resolution through the use of diffraction-less beams such as Bessel beams, providing high resolution imaging over large depths. The xf-OCT system therefore combines fast acquisition rates and high resolution, both characteristics required to image and study the structure and function of microscopic constituents of cerebral tissue. In this work, we performed functional brain imaging using the ability of xf-OCT to obtain quantita- tive measurements of blood flow in the brain. Changes in blood velocity evoked by neuronal activation were monitored and maps of hemodynamic activity were generated by adapting tools routinely used in fMRI to xf-OCT imaging. Additionally, three novel xf-OCT instruments are presented, wherein the advantages of different spectral ranges are exploited to reach specific imaging parameters. The increased contrast and resolution afforded by an illumination in the visible spectral range was used in two extended-focus optical coherence microscopy (xf-OCM) implementations for subcellular imaging of ex-vivo brain slices and cellular imaging of neurons, capillaries and myelinated axons in the superficial cortex in-vivo. Subsequently, an xf-OCT system is presented, operating in the infrared spectral range, wherein the reduced scattering enabled imaging the smallest capillaries deep in the murine cortex in-vivo