Imaging the subthalamic nucleus in Parkinson’s disease

This thesis is comprised of a set of work that aims to visualize and quantify the anatomy, structural variability, and connectivity of the subthalamic nucleus (STN) with optimized neuroimaging methods. The study populations include both healthy cohorts and individuals living with Parkinson's disease (PD). PD was chosen specifically due to the involvement of the STN in the pathophysiology of the disease. Optimized neuroimaging methods were primarily obtained using ultra-high field (UHF) magnetic resonance imaging (MRI). An additional component of this thesis was to determine to what extent UHF-MRI can be used in a clinical setting, specifically for pre-operative planning of deep brain stimulation (DBS) of the STN for patients with advanced PD. The thesis collectively demonstrates that i, MRI research, and clinical applications must account for the different anatomical and structural changes that occur in the STN with both age and PD. ii, Anatomical connections involved in preparatory motor control, response inhibition, and decision-making may be compromised in PD. iii. The accuracy of visualizing and quantifying the STN strongly depends on the type of MR contrast and voxel size. iv, MRI at a field strength of 3 Tesla (T) can under certain circumstances be optimized to produce results similar to that of 7 T at the expense of increased acquisition time

Personalizing functional Magnetic Resonance Protocols for Studying Neural Substrates of Motor Deficits in Parkinson’s Disease

Parkinson’s disease (PD) is a progressive neurodegenerative movement disorder characterized by a large number of motor and non-motor deficits, which significantly contribute to reduced quality of life. Despite the definition of the broad spectrum of clinical characteristics, mechanisms triggering illness, the nature of its progression and a character of therapeutic effects still remain unknown. The enormous advances in magnetic resonance imaging (MRI) in the last decades have significantly affected the research attempts to uncover the functional and structural abnormalities in PD and have helped to develop and monitor various treatment strategies, of which dopamine replacement strategies, mainly in form of levodopa, has been the gold standard since the late seventies and eighties. Motor, task-related functional MRI (fMRI) has been extensively used to assess the pathological state of the motor circuitry in PD. Several studies employed motor paradigms and fMRI to review the functional brain responses of participants to levodopa treatment. Interestingly, they provided conflicting results. Wide spectrum of symptoms, variability and asymmetry of the disease presentation, several treatment approaches and their divergent outcomes make PD enormously heterogeneous. In this work we hypothesized that not considering the disease heterogeneity might have been an adequate cause for the discrepant results in aforementioned studies. We show that not accounting for the disease variability might indeed compromise the results and invalidate the consequent interpretations. Accordingly, we propose and formalize a statistical approach to account for the intra and inter subject variability. This might help to minimize this bias in future motor fMRI studies revealing the functional brain dysfunction and contribute to the understanding of still unknown pathophysiological mechanisms underlying PD

MPTP 투여 마모셋 파킨슨병 모델의 확립과 도파민성 신경전구세포의 치료 효과 평가

학위논문 (박사) -- 서울대학교 대학원 : 의과대학 의학과, 2020. 8. 강병철.Parkinson's disease (PD) is one of the most important neurodegenerative diseases. Studies investigating cell transplantation as an alternative to L-3,4-dihydroxyphenylalanine administration or deep brain stimulation surgery are being actively conducted. Many PD animal models are used for PD treatment or prevention. However, most of them are rodent models, and the most representative is the model established with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Compared to other models, nonhuman primate (NHP) MPTP-treated models show clinical symptoms similar to human patients and facilitate behavioral evaluation, suggesting the use of various MPTP injection models according to experimental needs. Most NHP MPTP-treated models are optimized for short-term studies within three months and are not suitable for long-term studies such as cell transplantation. Since fetal mesenchymal cell transplantation in early studies, studies using mesenchymal stem cells or embryonic stem cells (ESCs) have been conducted. Studies have also been conducted using induced pluripotent stem cells, which can resolve ethical concerns and immune rejection. Despite advances in efficacy evaluation and safety of cell transplantation, studies on differentiation and discovery of homogeneous classification marker have yet to be investigated systematically since the degree of differentiation and homogeneity of cells after differentiation are directly related to clinical recovery and reduction of side effects. Accordingly, a Parkinson's disease model was established by subcutaneous administering "2-2-1-1-1" mg/kg of MPTP to common marmosets (Callithrix jacchus) to induce a long-term and stable clinical manifestations. Daily observation showed stable and persistent clinical symptoms. The results of tower test also reduced the motor function compared with pre-treatment with MPTP. In striatal positron emission tomography (PET) images, radioactivity was significantly reduced compared with prior MPTP administration. Immunohistrochemical analysis showed loss of tyrosine hydroxylase (TH)-positive cells and fibers in substantia nigra. In addition, 2.0 × 106 cells were implanted intracranially into the stratum of marmoset PD model to evaluate the therapeutic effect of dopaminergic (DAergic) precursor cells from human ESCs differentiating into DAergic neurons associated with PD symptoms using trophoblast glycoprotein, a new differentiation marker. The results of daily observation showed that the clinical symptoms recovered significantly from the third week after the cell transplant compared with the group exposed to MPTP. The tower test result confirmed that significant increase in the number of levels the marmosets climbed from the seventh week after the cell transplant. In the striatal PET image, the specific uptake ratio value was significantly increased from the fourteenth week after the cell transplant compared to the MPTP treatment group. The histopathological analysis revealed no excessive inflammatory reactions or tumor-like neoplasms, and TH-positive cells developed from implanted DAergic precursor cells in the cell transplant site. Based on the above results, it is purposed that the marmoset model produced by the new MPTP treatment method is suitable for long-term studies such as cell transplantation, and it is suggested that DAergic precursor cells represent potential as PD treatments for human patients.Since animal models of Parkinsons disease (PD) are useful research tools to investigate human patients, it is most appropriate and important to select the optimal research model for treatment or prevention. Because most of the characteristics of PD patients can be expressed, MPTP is mostly used to generate various animal model of PD, including NHP. In the case of a NHP model, various methods have been introduced depending on the experimental purpose. However, acute dosing is associated with a high incidence of early deaths due to the toxicity of MPTP itself. PD symptoms and lesions do not appear completely at low doses administered long term. In addition, most of the known method using MPTP are models suitable for short-term research and not for experiments that require sufficient time, such as cell or tissue transplants. Based on these findings, a new method of subcutaneous treatment using 2-2-1-1-1 mg/kg MPTP was administered to common marmosets (Callithrix jacchus) with stable PD symptoms over a long-term period without animal death. After MPTP treatment, stable clinical symptoms were observed continuously based on evaluation criteria of 10 or higher in daily observation. Based on the tower test, marmosets did not show an elevation of 5.61 ± 0.72 levels compared to levels before MPTP administration. In the striatal PET image, radioactivity after treatment decreased by 33.35 ± 1.23% compared to levels before MPTP treatment. Immunohistochemistry showed a loss of TH-positive cells and fibers in the SN after MPTP treatment. It is proposed that the marmoset model developed by the novel MPTP treatment method may be an optimal model for studies requiring long-term cell transplantation.Cell transplantation is as an alternative to existing treatments for PD such as conventional L-DOPA administration and DBS surgery. The degree of differentiation and the homogeneity of cells after differentiation are directly linked to the recovery of clinical symptoms and the reduction of side effects in cell transplantation. Therefore, efforts to discover new markers of differentiation and homogeneous classification that are most effective in PD treatment are ongoing as transplanted cells differentiate into dopamine neurons. Accordingly, a total of 2.0 × 106 cells were implanted into striatum of the marmoset MPTP model intracranially to evaluate the therapeutic effects of dopaminergic (DAergic) precursor cells obtained using trophoblast glycoprotein, a newly discovered marker that uniquely divides into ventral midbrain DAergic neurons associated with PD clinical symptoms. Observations of daily behavior showed a significant recovery compared to the MPTP treatment group at 3 weeks after cell transplantation, resulting in a difference of up to 11.17 ± 0.83 points based on evaluation criteria. In the tower test, it was significantly higher than in the MPTP treatment group at 7 weeks after cell transplantation, confirming an average difference of up to 5.67 ± 0.33 levels. In addition, the PET image analysis of the striatum showed a significant difference from 14 weeks after cell transplantation compared with the MPTP treatment group, with an increase of up to 0.26 ± 0.01 in SUR value. In addition, histopathologic assessment showed that no excessive inflammatory cell erosion or tumor-like tissue was observed. TH-positive cells observed were identified as those derived from the transplanted DAergic precursor cells in the cell transplant site. The results suggest that DAergic precursor cells represent a potential treatment modality for PD patients.파킨슨병은 가장 중요한 신경퇴행성 질환 중 하나이고, L-3,4-hydroxyphenylalanine 투여법이나 뇌심부자극 수술법을 현재 치료법으로 사용하고 있다. 하지만 기존 치료법으로는 완전 회복이 되지 않아 대안 치료법으로써 세포이식에 대한 연구가 활발히 진행되고 있다. 이러한 파킨슨병 치료나 예방을 위하여 많은 동물 모델이 사용되고 있고, 대부분 설치류 모델이 사용되고 있다. 파킨슨병 동물모델을 제작하는 방법으로는 1-metyl-4-phenyl-1,2,3,6-tetrahyrdopyridine (MPTP)를 투여한 모델 제작법이 가장 대표적이다. 다른 모델에 비해 영장류 MPTP 투여 모델은 파킨슨병 환자와 임상증상이 동일하다는 것과 행동학적 평가 적용이 용이하다는 장점을 가지고 있기 때문에 실험 목적에 따라 다양한 MPTP 투여법을 사용한 영장류 모델을 사용하고 있다. 하지만 대부분 영장류 MPTP 투여 모델은 세 달 이내의 단기간 연구에 최적으로 개발되어, 세포이식과 같은 장시간 연구에는 적합하지 않다. 초기 연구에서 태아 유래 중뇌조직을 이식하는 방법에서 중간엽줄기세포나 배아줄기세포를 이용한 연구가 진행되었고, 최근에는 윤리적 문제와 면역거부반응 문제를 해결할 수 있는 유도만능줄기세포를 이용한 연구가 진행되고 있다. 여러 연구를 통해 세포이식에 대한 효능 평가, 안전성 확보와 관련하여 많은 진보가 있었으나, 분화 정도와 분화 이후 세포 균질성이 임상증상 회복과 부작용 감소에 직접적으로 연관이 되어 있기 때문에 새로운 분화와 균질성 마커 발굴에 대한 연구가 꾸준히 진행되고 있다. 이러한 점들을 바탕으로 장시간 안정적인 임상증상이 발현되는 영쟝류 PD 모델을 제작하기 위하여 마모셋에 2-2-1-1-1 mg/kg MPTP 피하투여법을 적용하여 새로운 영장류 PD 모델을 확립하였다. 일생행동 평가 결과 마모셋 모델은 장시간 동안 안정적인 임상증상을 보였고 tower test 결과 역시 마모셋 모델은 MPTP 투여 전에 비해 운동기능이 저하된 상태로 유지됨을 관찰하였다. 또한 마모셋 모델의 선조체 양전자방출단층촬영 (PET) 영상에서 MPTP 투여 전에 비해 유의하게 방사선 발현도가 감소함을 확인하였고, 마모셋 모델의 뇌조직 면역염색 결과 흑색질에서 티로신 수산화효소 (TH)-양성 세포와 섬유체가 소실됨을 확인하였다. 또한 새로운 분화 마커인 영양막 당단백질을 사용하여 파킨슨병 증상과 관련된 배쪽중뇌 도파민성 신경세포로 분화하는 도파민성 신경전구세포에 대한 치료 효과를 평가하기 위하여 위의 마모셋 모델의 선조체에 2.0 × 106 개 세포를 뇌내에 이식하였다. 일상행동 평가 결과 세포 이식군은 MPTP 투여군에 비해 세포이식 후 3주째부터 임상증상이 유의하게 회복됨을 관찰하였고, tower test 결과 세포 이식군은 MPTP 투여군에 비해 세포이식 후 7주째부터 올라간 계단이 유의하게 증가됨을 확인하였다. 세포 이식군의 선조체 PET 영상에서 MPTP 투여군에 비해 세포이식 후 14주째부터 specific uptake ratio 값이 유의하게 증가됨을 확인하였다. 조직병리학적 평가 결과 세포이식 부위에서 과도한 염증반응이나 종양성 신생조직은 관찰하지 못했고, 관찰된 TH-양성 세포는 뇌내에 이식한 도파민성 신경전구세포에서 유래됨을 확인하였다. 위 결과들을 종합하였을 때, 새로운 MPTP 투여법으로 제작한 마모셋 모델은 세포이식과 같은 장시간 연구에 적합하고, 도파민성 신경전구세포는 파킨슨병 치료법으로써 고려될 수 있을 것으로 제안한다.LITERATURE REVIEW 1 CHAPTER Ⅰ Establishment of a novel Parkinson's disease modelin common marmoset for cell therapy evaluation 52 ABSTRACT 53 INTRODUCTION 55 MATERIALS AND METHODS 61 Animals 61 MPTP-induced PD model 61 Behavioral assessment 64 PET imaging analysis 67 Microscopic assessment 69 Statistical analysis 69 RESULTS 71 Stable parkinsonian symptoms without death for 32 weeks after MPTP treatment with novel method 71 Motor dysfunctions without recovery for 32 weeks after MPTP treatment with novel method 73 Amelioration of clinical symptoms temporarily after administration of L-DOPA following MPTP treatment 77 Lower radioactivity in the striatum based on 18F-FP-CIT PET images without recovery for 32 weeks after MPTP treatment with novel method 79 Loss of tyrosine hydroxylase-positive cells and fibers in the substantia nigra and striatum after MPTP treatment with novel method 81 DISCUSSION 83 CHAPTER Ⅱ Evaluation of therapeutic effects ofhuman embryonic stem cell-deriveddopaminergic precursor cells transplanted intoa marmoset model of Parkinson's disease 91 ABSTRACT 92 INTRODUCTION 94 MATERIALS AND METHODS 99 Animals 99 MPTP-induced PD model 100 Cell collection 102 Cell transplantation 102 Behavioral assessment 105 PET-CT imaging and analysis 107 Histopathologic examination 108 Statistical analysis 110 RESULTS 112 No significant difference in body weight between MPTP-treated and cell-transplanted marmosets due to intensive care 112 Progressive recovery of motor symptoms in MPTP pre-treated cell-transplanted marmosets compared to MPTP-treated marmosets 115 Significant, but not full recovery of motor function in cell-transplanted PD marmosets compared to MPTP-treated marmosets 117 Weak recovery pattern in striatal PET images and SUR in cell-transplanted marmosets 119 No tumor-like lesions, but increased TH-positive neurons and fibers at transplant sites at 28 weeks after cell transplantation in MPTP-treated marmosets 122 Identification of cells expressing DAergic markers in transplanted cells at 28 weeks after cell transplantation 126 DISCUSSION 129 REFERENCES 138 ABSTRACT IN KOREAN (국문초록) 203Docto

Quantitative whole body imaging at high field

In this thesis methods of accurately and reproducibly measuring intestinal content and transverse relaxation to study the fate of food in the gastrointestinal tract are reported. In addition to this a technique for measuring relaxation parameters in the brain at ultra-high field is also investigated. Several methods can be used when quantifying the fate of food through the gastrointestinal tract; there are many factors that can be measured using MRI and these are discussed along with other non-MRI techniques. In this work a method for quantifying small bowel water content (SBWC) is optimised and validated for use at 3.0 T and a technique for measuring T2 in the abdomen is developed and optimised called T2-prepared balanced turbo field echo (T2-prep bTFE). These two methods are then used, in conjunction with other established MRI techniques, to study the fate of food in the gastrointestinal tract from the stomach all the way through to the colon. A hybrid gradient echo-spin echo (GESSE) sequence is also investigated and optimised for measuring T2 and T2* simultaneously in the brain at 7.0 T. This sequence is also proved to have applications in the liver at lower field strengths. The GESSE sequence is used to measure the first T2 values in deep grey structures in the brain at 7.0 T. In this work cross-field (1.5, 3.0 and 7.0 T) variations in T2 are studied. Also differences in T2 and T2* are measured in the brain to determine variations between white matter tracts and to ascertain any effects of Parkinson’s disease on deep grey matter structures

A multi-shell multi-tissue diffusion study of brain connectivity in early multiple sclerosis.

BACKGROUND: The potential of multi-shell diffusion imaging to produce accurate brain connectivity metrics able to unravel key pathophysiological processes in multiple sclerosis (MS) has scarcely been investigated. OBJECTIVE: To test, in patients with a clinically isolated syndrome (CIS), whether multi-shell imaging-derived connectivity metrics can differentiate patients from controls, correlate with clinical measures, and perform better than metrics obtained with conventional single-shell protocols. METHODS: Nineteen patients within 3 months from the CIS and 12 healthy controls underwent anatomical and 53-direction multi-shell diffusion-weighted 3T images. Patients were cognitively assessed. Voxel-wise fibre orientation distribution functions were estimated and used to obtain network metrics. These were also calculated using a conventional single-shell diffusion protocol. Through linear regression, we obtained effect sizes and standardised regression coefficients. RESULTS: Patients had lower mean nodal strength (p = 0.003) and greater network modularity than controls (p = 0.045). Greater modularity was associated with worse cognitive performance in patients, even after accounting for lesion load (p = 0.002). Multi-shell-derived metrics outperformed single-shell-derived ones. CONCLUSION: Connectivity-based nodal strength and network modularity are abnormal in the CIS. Furthermore, the increased network modularity observed in patients, indicating microstructural damage, is clinically relevant. Connectivity analyses based on multi-shell imaging can detect potentially relevant network changes in early MS

The Phenomenology, Pathophysiology and Progression of the Core Features of Lewy Body Dementia

Lewy body dementias – Dementia with Lewy bodies (DLB) and Parkinson’s disease dementia (PDD) - are disabling neurodegenerative conditions defined pathologically by the presence of intraneuronal α-synuclein rich aggregates (‘Lewy bodies’ and ‘Lewy neurites’). These disorders are characterized by a set of ‘core’ clinical features, namely cognitive fluctuations, visual hallucinations, motor parkinsonism, and most recently added, REM sleep behaviour disorder. These features are central to the diagnosis of Lewy bodies dementias (especially DLB) and discriminate them from other neurodegenerative disorders. Despite decades of research, the etiopathogenesis underlying Lewy body disorders is poorly understood. This accounts for the relative lack of objective biomarkers and both symptomatic and disease modifying therapies. The present thesis comprises a series of investigations that seeks to understand the phenomenology, pathophysiology, and clinical progression of Lewy body dementias through focus on each of the core clinical features. Systematic review and empiric studies are organized under the respective headings of cognitive fluctuations, visual hallucinations, REM sleep behaviour disorder, motor features, interrelationships, and clinical progression of the core features. Novel clinical and pathophysiological insights are obtained which have implications for the prediction and diagnosis of core features, the development of new objective biomarkers, and clinical endpoints of disease progression. From these studies, a shared pathophysiological basis for the core features is postulated and potential avenues for future directions are highlighted, focusing on replication and validation of new biomarkers and clinical measures, discovery of new biomarkers and mechanisms, and translation to prodromal and patient cohorts