1st INCF Workshop on Genetic Animal Models for Brain Diseases

The INCF Secretariat organized a workshop to focus on the “role of neuroinformatics in the processes of building, evaluating, and using genetic animal models for brain diseases” in Stockholm, December 13–14, 2009. Eight scientists specialized in the fields of neuroinformatics, database, ontologies, and brain disease participated together with two representatives of the National Institutes of Health and the European Union, as well as three observers of the national INCF nodes of Norway, Poland, and the United Kingdom

Preliminary evidence of increased striatal dopamine in a nonhuman primate model of maternal immune activation.

Women exposed to a variety of viral and bacterial infections during pregnancy have an increased risk of giving birth to a child with autism, schizophrenia or other neurodevelopmental disorders. Preclinical maternal immune activation (MIA) models are powerful translational tools to investigate mechanisms underlying epidemiological links between infection during pregnancy and offspring neurodevelopmental disorders. Our previous studies documenting the emergence of aberrant behavior in rhesus monkey offspring born to MIA-treated dams extends the rodent MIA model into a species more closely related to humans. Here we present novel neuroimaging data from these animals to further explore the translational potential of the nonhuman primate MIA model. Nine male MIA-treated offspring and 4 controls from our original cohort underwent in vivo positron emission tomography (PET) scanning at approximately 3.5-years of age using [18F] fluoro-l-m-tyrosine (FMT) to measure presynaptic dopamine levels in the striatum, which are consistently elevated in individuals with schizophrenia. Analysis of [18F]FMT signal in the striatum of these nonhuman primates showed that MIA animals had significantly higher [18F]FMT index of influx compared to control animals. In spite of the modest sample size, this group difference reflects a large effect size (Cohen's d = 0.998). Nonhuman primates born to MIA-treated dams exhibited increased striatal dopamine in late adolescence-a hallmark molecular biomarker of schizophrenia. These results validate the MIA model in a species more closely related to humans and open up new avenues for understanding the neurodevelopmental biology of schizophrenia and other neurodevelopmental disorders associated with prenatal immune challenge

Adult Human Neurogenesis: From Microscopy to Magnetic Resonance Imaging

Neural stem cells reside in well-defined areas of the adult human brain and are capable of generating new neurons throughout the life span. In rodents, it is well established that the new born neurons are involved in olfaction as well as in certain forms of memory and learning. In humans, the functional relevance of adult human neurogenesis is being investigated, in particular its implication in the etiopathology of a variety of brain disorders. Adult neurogenesis in the human brain was discovered by utilizing methodologies directly imported from the rodent research, such as immunohistological detection of proliferation and cell-type specific biomarkers in postmortem or biopsy tissue. However, in the vast majority of cases, these methods do not support longitudinal studies; thus, the capacity of the putative stem cells to form new neurons under different disease conditions cannot be tested. More recently, new technologies have been specifically developed for the detection and quantification of neural stem cells in the living human brain. These technologies rely on the use of magnetic resonance imaging, available in hospitals worldwide. Although they require further validation in rodents and primates, these new methods hold the potential to test the contribution of adult human neurogenesis to brain function in both health and disease. This review reports on the current knowledge on adult human neurogenesis. We first review the different methods available to assess human neurogenesis, both ex vivo and in vivo and then appraise the changes of adult neurogenesis in human diseases

In Vivo Imaging Biomarkers in Mouse Models of Alzheimer's Disease: Are We Lost in Translation or Breaking Through?

Identification of biomarkers of Alzheimer's Disease (AD) is a critical priority to efficiently diagnose the patients, to stage the progression of neurodegeneration in living subjects, and to assess the effects of disease-modifier treatments. This paper addresses the development and usefulness of preclinical neuroimaging biomarkers of AD. It is today possible to image in vivo the brain of small rodents at high resolution and to detect the occurrence of macroscopic/microscopic lesions in these species, as well as of functional alterations reminiscent of AD pathology. We will outline three different types of imaging biomarkers that can be used in AD mouse models: biomarkers with clear translational potential, biomarkers that can serve as in vivo readouts (in particular in the context of drug discovery) exclusively for preclinical research, and finally biomarkers that constitute new tools for fundamental research on AD physiopathogeny

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

Focused ultrasound for safe and effective release of brain tumor biomarkers into the peripheral circulation

The development of noninvasive approaches for brain tumor diagnosis and monitoring continues to be a major medical challenge. Although blood-based liquid biopsy has received considerable attention in various cancers, limited progress has been made for brain tumors, at least partly due to the hindrance of tumor biomarker release into the peripheral circulation by the blood-brain barrier. Focused ultrasound (FUS) combined with microbubbles induced BBB disruption has been established as a promising technique for noninvasive and localized brain drug delivery. Building on this established technique, we propose to develop FUS-enabled liquid biopsy technique (FUS-LBx) to enhance the release of brain tumor biomarkers (e.g., DNA, RNA, and proteins) into the circulation. The objective of this study was to demonstrate that FUS-LBx could sufficiently increase plasma levels of brain tumor biomarkers without causing hemorrhage in the brain. Mice with orthotopic implantation of enhanced green fluorescent protein (eGFP)-transfected murine glioma cells were treated using magnetic resonance (MR)-guided FUS system in the presence of systemically injected microbubbles at three peak negative pressure levels (0.59, 1.29, and 1.58 MPa). Plasma eGFP mRNA levels were quantified with the quantitative polymerase chain reaction (qPCR). Contrast-enhanced MR images were acquired before and after the FUS sonication. FUS at 0.59 MPa resulted in an increased plasma eGFP mRNA level, comparable to those at higher acoustic pressures (1.29 MPa and 1.58 MPa). Microhemorrhage density associated with FUS at 0.59 MPa was significantly lower than that at higher acoustic pressures and not significantly different from the control group. MRI analysis revealed that post-sonication intratumoral and peritumoral hyperenhancement had strong correlations with the level of FUS-induced biomarker release and the extent of hemorrhage. This study suggests that FUS-LBx could be a safe and effective brain-tumor biomarker release technique, and MRI could be used to develop image-guided FUS-LBx

Accelerating drug discovery for Alzheimer's disease: best practices for preclinical animal studies

Animal models have contributed significantly to our understanding of the underlying biological mechanisms of Alzheimer's disease (AD). As a result, over 300 interventions have been investigated and reported to mitigate pathological phenotypes or improve behavior in AD animal models or both. To date, however, very few of these findings have resulted in target validation in humans or successful translation to disease-modifying therapies. Challenges in translating preclinical studies to clinical trials include the inability of animal models to recapitulate the human disease, variations in breeding and colony maintenance, lack of standards in design, conduct and analysis of animal trials, and publication bias due to under-reporting of negative results in the scientific literature. The quality of animal model research on novel therapeutics can be improved by bringing the rigor of human clinical trials to animal studies. Research communities in several disease areas have developed recommendations for the conduct and reporting of preclinical studies in order to increase their validity, reproducibility, and predictive value. To address these issues in the AD community, the Alzheimer's Drug Discovery Foundation partnered with Charles River Discovery Services (Morrisville, NC, USA) and Cerebricon Ltd. (Kuopio, Finland) to convene an expert advisory panel of academic, industry, and government scientists to make recommendations on best practices for animal studies testing investigational AD therapies. The panel produced recommendations regarding the measurement, analysis, and reporting of relevant AD targets, th choice of animal model, quality control measures for breeding and colony maintenance, and preclinical animal study design. Major considerations to incorporate into preclinical study design include a priori hypotheses, pharmacokinetics-pharmacodynamics studies prior to proof-of-concept testing, biomarker measurements, sample size determination, and power analysis. The panel also recommended distinguishing between pilot 'exploratory' animal studies and more extensive 'therapeutic' studies to guide interpretation. Finally, the panel proposed infrastructure and resource development, such as the establishment of a public data repository in which both positive animal studies and negative ones could be reported. By promoting best practices, these recommendations can improve the methodological quality and predictive value of AD animal studies and make the translation to human clinical trials more efficient and reliable