京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18644号 / 医博第3943号 / 新制||医||1006(附属図書館) / 31558 / 京都大学大学院医学研究科医学専攻 / (主査)教授 河野 憲二, 教授 福山 秀直, 教授 渡邉 大 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23102号 / 医博第4729号 / 新制||医||1050(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 花川 隆, 教授 髙橋 良輔, 教授 伊佐 正 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Although in conventional models of visual information processing, object identity and spatial information are processed separately and independently in ventral and dorsal cortical visual pathways respectively, some recent studies have shown that information about both object’s identity (of shape) and space are present in both visual pathways. However, it is still unclear whether the presence of identity and spatial information in both pathways have functional roles or not. In a recent study (Han & Sereno, in press), we have tried to answer this question through computational modeling. Our simulation results suggested that two separate cortical visual pathways for identity and space (1) actively retain information about both identity and space; (2) retain information about identity and space differently; (3) that this differently retained information about identity and space in the two pathways may be necessary to accurately and optimally recognize and localize objects. However, in these simulations, there was only one object in each image. In reality, there may be more than one object in an image. In this master’s thesis, I have tried to run visual recognition simulations with two objects in each image. My two object simulations suggest that (1) the two separate cortical visual pathways for identity and space (orientation) still retain information about both identity and space (orientation) when there are two objects in each image; (2) the retained information about identity and space (orientation) in the two pathways may be necessary to accurately and optimally recognize objects’ identity and orientation. These results agree with our one object simulation results.

Rett syndrome (RTT), a severe neurodevelopmental disorder, affects females resulting from loss-of-function mutations in the X-linked transcription factor methyl-CpG-binding protein 2 (MECP2). RTT patients show severe verbal, motor, respiratory, and intellectual impairments. We studied two forms of activity-dependent plasticity in Mecp2 mutant mice to better understand the loss of MECP2 function in neuronal circuit and sensory processing. Sensory deprivation was applied by trimming one whisker to 3 mm to study long-term cortical plasticity in Mecp2-/y mice. Intrinsic optical signaling (IOS) imaging showed the neuronal response to wiggling a non-trimmed was consistent from day 0 to 14 but reduced for the trimmed whisker by 49.0 ± 4.3% in wild type (WT) and 22.7 ± 4.6% (p=0.0135) in RTT mice. Primary hindlimb (HL) somatosensory cortical responses to vibratory stimulation were assessed by IOS and intracortical local field potential (LFP). Responses were assessed before, during and, after 1 hour of repeated HL vibratory stimulation (100Hz,1sec, ISI 6 sec) in symptomatic male (4-6 week), female (10-12 month) and pre-symptomatic young female (4 week) RTT model mice. After 1-hour, cortical responses to test vibrations were reduced by approximately 40% in RTT and WT mice as assessed by both methods. Recovery of the IOS responses (1 sec vibration at 100Hz) and LFP (300µm below pia, 7 stimuli, 100mse ISI) were tested at 15 min intervals for 1 hour after ceasing the repeated stimulation. Reduced responses persisted for at least 60 min in WT but recovered to 90-100% of normal within 15-30 min in RTT. Analysis of the LFP responses within the test train indicated that the reduced cortical sensitivity during and after continuous stimulation resulted primarily from an increase in adaptation during the 7-stimulus test train rather than a reduction in the response to a single vibratory stimulus in all groups. Retention of this increased STA is the primary cause of the persistently reduced tactile response in young WT female mice, while in RTT mice the rapid recovery of tactile sensitivity was due to the return of STA to lower, baseline levels. Male RTT mice exhibited a marked increased excitability to the first stimulus in the test train resulting in hypersensitivity to a single vibration by 45 minutes. Old females exhibited the same pattern of adaptation and recovery but retention of adaptation was less pronounced in both WT and RTT compared to younger animals suggesting an age-dependent reduction in neural plasticity may mask deficits specific to RTT. Recording sciatic nerve sensory afferent activity did not reveal any STA, persistent adaptation or sensitization of peripheral afferent endings in any groups. I propose persistent sensory adaptation mediated by increased short-term adaptation may reflect enhanced feedback by inhibitory elements of circuits within the sensory pathway. The rapid recovery of responsiveness in young female RTT mice may therefore reflect a deficit in the capacity for activity dependent plasticity to consolidate and thus could provide a platform to understand the causes of learning and cognitive deficits in RTT patients. / Graduate

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Second language acquisition has proven to impact the brain in many ways. Studies have shown a distinct difference between the monolingual and the bilingual brain. These structural differences have included an observable increase in cortical thickness in bilingual individuals when compared to monolinguals. This is a significant observation since many neurological diseases and impairments have been connected to a decrease in cortical thickness. However, previous studies have focused solely on bilinguals who had acquired their second language early on in life. These studies have failed to focus on the potential impact that could be observed on the cortical thickness of individuals who acquire a language after the age of 40. This study focused on monolingual students 40 years and older, who participated in an entry level university level Spanish course and examined how their brains structurally changed after a three-month course. These results were compared to a group of adults not participating in a language course who acted as a control group. This was achieved through MRI imaging of all the participants' brains and measuring any changes in their cortical thickness. Upon completing the second round of MRI imaging, the comparison of the pre and post MRI scans yielded an observable difference between the experimental group who had participated in the Spanish course and the control group. However, these differences did not prove to be significant and should be viewed as exploratory. Future research opportunities should entail studies with a longer duration combined with a curriculum better suited to this age group.

Modelling and simulation of the human body during an impact situation such as a car accident, can lead to better designed safety features on vehicles. In order to achieve this, investigation into the material properties and the creation of a numerical model of cortical bone is needed. One approach to creating a material model of cortical bone suitable for these situations is to describe the material model as visco-elastic, as reported by Shim et al. [1], Bekker et al. [2] and Cloete et al. [3]. The work by Shim et al. and Bekker et al. developed three-dimensional models, but do not accurately capture the transition in behaviour in the intermediate strain rate region, while Cloete et al. developed a phenomenological model which captures the intermediate strain rate behaviour in one dimension. This work aims to verify and extend these models. The intermediate strain rate regime (1 s−1 to 100 s−1 ) is of particular interest because it is a key characteristic of the behaviour of cortical bone and several studies have been conducted to gather experimental data in this region [3, 4, 5, 6]. The behaviour can be captured using non-linear viscoelastic models. This dissertation focuses on the development and implementation of a material model of cortical bone based on non-linear visco-elastic models to capture the intermediate strain rate regime behaviour. The material model was developed using uni-axial test results from cortical bone. The model by Cloete et al. has been improved and extended, and issues of local and global strain rate with regards to the viscosity have been clarified. A hereditary integral approach was taken in the analysis and implementation of discrete models and was found to be consistent with mathematical models. The model developed was extended to three dimensions in a manner similar to that of Shim et al. and Bekker et al. for implementation in commercial finite element software (LS-Dyna and Abaqus).

When exploring movement production, motor control researchers have been interested in investigating the relative contributions to different types of movement. In a research setting, a startling acoustic stimulus (SAS) can be used as a tool to explore the neural processes that are occurring when preparing and initiating a movement. Additionally, suprathreshold TMS is another tool which can induce a suppression of the cortical region of the brain, resulting in RT delays which provides us with the ability to assess the corticospinal contributions to a particular movement. The aim of the current study was to investigate potential differences in the planning and execution of bimanual versus unimanual wrist extension movements. It was of particular interest as to whether bimanual coupling occurs at the cortical level or in lower parts of the output pathway (reticulospinal). Participants (N=6) were instructed to complete a unimanual or bimanual wrist extension following a control go-signal or a SAS. For subset of trials, in order to explore the level of corticospinal excitability of the movement, suprathreshold TMS was applied over the left M1 during the task to induce a cortical silent period (CSP). Results revealed that theimpact of TMS on response initiation was not significantly different for unimanual task versus a bimanual task. Furthermore, the SP (silent period) only had an impact on the right limb and not the left during the bilateral task. Lastly, SAS did lead to shorter RTs for both the unimanual and bimanual wrist extension task, but the RT delay induced by TMS in the right limb was not shorter in SAS trials compared to control. The findings of the present study suggest that bimanual coupling may be occurring at the cortical level and in lower parts of the output pathway as there may be correlated neural activity in the two hemispheres occurring during bimanual wrist extension movements.

Part 1: Monitoring Response to Single-Dose (1000cGy) Radiotherapy in a RIF-1 Tumor Model The current standard of measure for monitoring chemotherapeutic and radiotherapeutic treatment response is tumor volume. Unfortunately, changes in tumor volume are generally slow and tumor volume does not necessarily indicate the degree of tumor viability. The development of marker(s) with the ability to detect an early therapeutic response would greatly aid in patient management, opening the possibility for both rapid dose optimization and replacement of ineffective therapies with alternative treatment. Previous studies have shown that diffusion measurements using magnetic resonance imaging (MRI) techniques are sensitive to therapy-induced changes in cellular structure, allowing demarcation between regions of necrosis and viable tumor tissue. This sensitivity, based on the correlation between water apparent diffusion coefficient (ADC) values and tumor cellular density, may allow diffusion measurements to be employed in non-invasive monitoring of treatment response. Therapy-induced increases in tumor ADC preceding tumor regression have been reported in a variety of experimental tumor models and several human brain tumors. Despite the demonstrated diffusion sensitivity to therapeutic response in these particular studies, shortcomings still remain that hinder the efficacy of clinical application in oncology. Earlier studies have concentrated on the mean ADC present within the tumor, either within the entire tumor volume or a region of-interest (ROI) defined by the user, and their evolution pre-treatment and post-treatment. Because of inter- and intra-tumor heterogeneity, volume-averaged ADC measurements suffer from poor correlation with treatment efficacy. In addition, most studies make little or no attempt to characterize the entire tumor volume (necrotic, viable, edema). The identification of regions of differing tissue viability should aid in the staging of treatment, therefore making accurate and reproducible tissue segmentation an important goal. The results of earlier, single-parameter studies indicate that a multi-parametric approach in which several MR parameters are monitored (ADC, T2, M0) may provide greater power than that of the single parameter approach. A multi-parametric or multi-spectral (MS) analysis uses pattern-recognition techniques, such as clustering, for image segmentation. Clustering algorithms use characteristics of the multiple MR-parameter dataset to group tissue of similar type, e.g., fat, muscle, viable tumor, necrosis. Specifically, k-means (KM) is an unsupervised segmentation algorithm that groups together similar tissue based on the difference in MR parameter space between the image voxel of interest and the mean parameter values of the voxels in that cluster. In the first step of the classification algorithm, it is applied to separate the data into two clusters (k = 2), tissue and background noise voxels. All voxels classified as background noise are set to zero and removed from further processing. In the second step, KM is applied to the remaining tissue voxels to segment the data into multiple tissue types. In the case of tumors, it is not clear in advance how many different types of tissue exist. The number of clusters, k, should be varied to ensure that all relationships between tissues are found. In the final step, the resulting KM maps may be compared to histological slices taken from the same tissue as the imaging slices in order to identify the tissue type of each cluster. In line with the studies and analyses described above, quantitative MRI was performed to investigate the spatial correlation between ADC, spin-spin (T2) relaxation times, and proton density (M0) in murine radiation-induced fibrosarcoma (RIF-1) tumors following single-dose (l000cGy) radiotherapy using the KM algorithm (Chapters 3 and 4) and different combinations of features and/or clusters. For all cluster/feature combinations, an in-depth comparison between KM-derived volume estimates and conventional histology via the hematoxylin-eosin (H&E) staining procedure (for identification of viable tumor versus necrosis), as well as via hypoxic-inducible factor-lα. (HIF-1α) immunohistochemistry (for identification of regions of hypoxia versus well-oxygenated tissue) was performed (Chapter 3). The optimal cluster/feature combination was determined by minimizing the sum-of-squared-differences (SSD) between the actual datapoints and the ideal one-to-one correlation that should exist between KM-derived volume estimates and histology-derived volume estimates. The optimal cluster/feature combination was determined to be a 2-feature (ADC, T2) and 4-c1uster (2 regions each of viable tissue and necrosis) segmentation. This KM method was then applied in analysis of the radiotherapeutic response: first, to gain insight into the various processes whose combination yield the total ADC response over time; second, to identify the contribution of tissue heterogeneity to the treatment response and changes in tumor growth kinetics and cell kill (Chapter 4). Comparisons between control and various time-points out to 14 days post-radiotherapy permitted more accurate tissue characterization and prediction of therapeutic outcome over analysis using ADC alone. The results based on histological validation demonstrated: (1) MS analysis provides an improved tissue segmentation method over results obtained from conventional methods employing ADC alone; (2) MS analysis permits subdivision based on the degree of necrosis, as well as delineation between well-oxygenated and hypoxic viable tissue; and (3) Individual KM volumes corresponded well with both H&E volumes and regions with increased HIF-1α expression. The results based on the radiotherapeutic response demonstrated: (1) MS analysis provides a method for monitoring the range of tissue viability as a function of time post-treatment; (2) MS analysis permits assessment of the various contributions to the total ADC response post-treatment; (3) The relative fractions of well-oxygenated (i.e., radiosensitive) versus hypoxic (i.e., radioresistant) tissue pretreatment may be predictive of treatment response; and (4) The early ADC increase did not seem to be a result of radiation-induced vasogenic edema, but instead was most likely due to a slight reduction in cellular density following therapy. These studies provide a non-invasive method of tissue characterization that may be used in monitoring treatment response and optimizing drug dose-timing schemes, with the potential for predicting treatment efficacy. Part 2: Role of Spreading Depression in Ischemic Stroke Stroke is a prevalent disease that ranks as the 3rd leading cause of death and disability in the United States, according to NIH statistics, costing millions of dollars in medical costs and lost wages. At present, the mechanism by which focal ischemia evolves into infarction remains poorly understood. By determining the patho-physiological mechanisms involved in the evolution of focal brain ischemia, therapeutic strategies may be designed for instances of acute ischemic stroke. In the late 1980s, researchers discovered MRI techniques that allow the detection of stroke very early after onset. Such techniques as diffusion-weighted imaging and perfusion-weighted imaging (DWI and PWI) have been applied both clinically and experimentally. Previous studies employing these techniques suggest that cortical spreading depression plays a detrimental role in the evolution of focal brain ischemia. Spreading depression (SD) is characterized by a spontaneous and reversible depression of cortical electrical activity that spreads from the site of onset as a wave with a speed of 2-5 mm/min. It is accompanied by an ionic redistribution, with efflux of potassium ions (K+) and influx of sodium, chloride, and calcium (Na+, Cl-, Ca2+) ions, as well as water. This results in cellular swelling and a decreased extracellular space (ES), yielding a decline in ADC. A positive correlation between the number of both spontaneous and induced SDs and infarct volume has well been documented, supporting the idea that SD inhibition might be neuroprotective if initiated early after ischemic onset. Even though these studies show promise in their ability to track SD using diffusion mapping, changes in ADCs reflect cytotoxic edema and do not necessarily correspond to SD or SD-like depolarizations or calcium (Ca2+) influx, leading to cell death. Recent studies have reported the use of manganese ions (Mn2+) as a depolarization-dependent contrast agent in monitoring brain activation through the application of glutamate, as well as in the study focal ischemia. Since extracellular accumulation of potassium (K+) ions or glutamate in ischemic tissue is believed to play a central role in the initiation and propagation of SDs, and knowing that Mn2+, having an ionic radius similar to that of Ca2+, is handled in a manner similar to Ca2+, these studies suggest the possible use of manganese ions (Mn2+) in tracking SD or SD-like depolarizations in the evolution of focal brain ischemia. In order to determine the utility of Mn2+ as a marker for SD, two sets of T1-weighted MRI experiments were performed before applying Mn2+ in an experimental stroke model (Chapter 6). First, for verification purposes, a glutamate administration group was evaluated to validate our use of the manganese-enhanced MRI (MEMRI) method previously developed by Aoki et al, a modification of the original by Lin and Koretsky. When satisfied that the contrast enhancement was specific to glutamate only, a second set of experiments was performed. Here, experimental SD was elicited by chemical stimulation (direct application of concentrated potassium chloride [KC1] on the exposed cortical surface) and compared with control conditions (perfusion of sodium chloride [NaC1] on exposed brain cortex). This study demonstrated: (1) Mn2+, specific to Ca2+ channel activity, is a more accurate marker for SD than DWI or T2 methods; (2) Cortical restriction of MEMRI enhancement supports the contention that apical dendrites are necessary for SD propagation; (3) Subcortical enhancement is a result of corticalsubcortical neuronal connectivity; and (4) Because of the relatively slow clearance of Mn2+, MEMRI permits higher spatial resolution and signal-to-noise ratios (SNRs) via increased signal averaging. Based on these results, preliminary experiments involving the study of SD in focal ischemia using Mn2+ were performed (Chapter 7). Initial results indicate: (1) MEMRI of ischemia, when compared with standard DWI/PWI methods, may provide a method for estimating the likelihood of progression to infarct at acute time points post onset of stroke. These studies provide a foundation for further investigation into the role of SD in stroke, and the application of Mn2+ towards the design of therapeutic strategies targeting SD inhibition. Conclusions and Medical Significance The research within this dissertation employed magnetic resonance imaging techniques for monitoring the temporal evolution of pathological disease states such as focal ischemia and cancer, with and without therapeutic intervention. Optimization of these techniques in experimental models will open the possibility for future application in a clinical setting. Clinical availability of these non-invasive methods, with the ability to detect an early therapeutic response or to provide staging and prediction of tissue fate, would greatly aid in patient management of both cancer and stroke.