Comparative route of administration studies using therapeutic siRNAs show widespread gene modulation in Dorset sheep

siRNAs comprise a class of drugs that can be programmed to silence any target gene. Chemical engineering efforts resulted in development of divalent siRNAs (di-siRNAs), which support robust and long-term efficacy in rodent and nonhuman primate brains upon direct cerebrospinal fluid (CSF) administration. Oligonucleotide distribution in the CNS is nonuniform, limiting clinical applications. The contribution of CSF infusion placement and dosing regimen on relative accumulation, specifically in the context of large animals, is not well characterized. To our knowledge, we report the first systemic, comparative study investigating the effects of 3 routes of administration - intrastriatal (i.s.), i.c.v., and intrathecal catheter to the cisterna magna (ITC) - and 2 dosing regimens - single and repetitive via an implanted reservoir device - on di-siRNA distribution and accumulation in the CNS of Dorset sheep. CSF injections (i.c.v. and ITC) resulted in similar distribution and accumulation across brain regions. Repeated dosing increased homogeneity, with greater relative deep brain accumulation. Conversely, i.s. administration supported region-specific delivery. These results suggest that dosing regimen, not CSF infusion placement, may equalize siRNA accumulation and efficacy throughout the brain. These findings inform the planning and execution of preclinical and clinical studies using siRNA therapeutics in the CNS

Chemogenetic dissection of the primate prefronto-subcortical pathways for working memory and decision-making

「何を買うんだっけ」と「どれにしよう」を処理する2つの脳回路を明らかに --霊長類の生体脳で神経経路を可視化・操作する技術で解明、高次脳機能の理解へ大きく前進--. 京都大学プレスリリース. 2021-06-24.The primate prefrontal cortex (PFC) is situated at the core of higher brain functions via neural circuits such as those linking the caudate nucleus and mediodorsal thalamus. However, the distinctive roles of these prefronto-subcortical pathways remain elusive. Combining in vivo neuronal projection mapping with chemogenetic synaptic silencing, we reversibly dissected key pathways from dorsolateral part of the PFC (dlPFC) to the dorsal caudate (dCD) and lateral mediodorsal thalamus (MDl) individually in single monkeys. We found that silencing the bilateral dlPFC-MDl projections, but not the dlPFC-dCD projections, impaired performance in a spatial working memory task. Conversely, silencing the unilateral dlPFC-dCD projection, but not the unilateral dlPFC-MDl projection, altered preference in a decision-making task. These results revealed dissociable roles of the prefronto-subcortical pathways in working memory and decision-making, representing the technical advantage of imaging-guided pathway-selective chemogenetic manipulation for dissecting neural circuits underlying cognitive functions in primates

Closed-Loop Interruption of Hippocampal Ripples through Fornix Stimulation in the Non-Human Primate

AbstractBackgroundHippocampal sharp-wave ripples (SWRs) arising from synchronous bursting in CA3 pyramidal cells and propagating to CA1 are thought to facilitate memory consolidation. Stimulation of the CA3 axon collaterals comprising the hippocampal commissure in rats interrupts sharp-wave ripples and leads to memory impairment. In primates, however, these commissural collaterals are limited. Other hippocampal fiber pathways, like the fornix, may be potential targets for modulating ongoing hippocampal activity, with the short latencies necessary to interrupt ripples.ObjectiveThe aim of this study is to determine the efficacy of closed-loop stimulation adjacent to the fornix for interrupting hippocampal ripples.MethodStimulating electrodes were implanted bilaterally alongside the fornix in the macaque, together with microelectrodes targeting the hippocampus for recording SWRs. We first verified that fornix stimulation reliably and selectively evoked a response in the hippocampus. We then implemented online detection and stimulation as hippocampal ripples occurred.ResultsThe closed-loop interruption method was effective in interrupting ripples as well as the associated hippocampal multi-unit activity, demonstrating the feasibility of ripple interruption using fornix stimulation in primates.ConclusionAnalogous to murine research, such an approach will likely be useful in understanding the role of SWRs in memory formation in macaques and other primates sharing these pathways, such as humans. More generally, closed-loop stimulation of the fornix may prove effective in interrogating hippocampal-dependent memory processes. Finally, this rapid, contingent-DBS approach may be a means for modifying pathological high-frequency events within the hippocampus, and potentially throughout the extended hippocampal circuit

Intrathecal Adeno-Associated Virus Vector Delivery for Mucopolysaccharidosis Type I

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disease resulting from deficiency of the enzyme α-L-iduronidase (IDUA). Accumulation of the IDUA substrates heparan and dermatan sulfate causes widespread organ pathology. While many of the somatic manifestations of MPS I can be treated with intravenous enzyme replacement, the devastating CNS sequelae—cognitive impairment, spinal cord compression, and hydrocephalus—do not respond to treatment. Partial preservation of cognitive function is possible with early hematopoietic stem cell transplantation, although transplant is associated with substantial morbidity and mortality. Gene transfer using adeno-associated virus (AAV) vectors offers a potential alternative approach to deliver the IDUA enzyme to the CNS. Introducing a functional IDUA gene to a subset of quiescent cells could provide a permanent source of secreted enzyme beyond the blood-brain barrier. However, preclinical studies evaluating direct injection of AAV vectors into the brain have shown that transduction is limited to small regions surrounding the injection site, and that injection can be associated with a localized inflammatory response and immune-mediated killing of transduced cells. In order to overcome these limitations, we evaluated delivery of an AAV serotype 9 vector into the cerebrospinal fluid as a less invasive method to achieve widespread brain transduction. Studies in nonhuman primates demonstrated that intrathecal AAV9 delivery results in transduction of cells throughout the brain and spinal cord without eliciting destructive immune responses to the transgene product. Intrathecal injection of AAV9 vectors expressing IDUA in canine and feline models of MPS I replicated the widespread transduction observed in primate studies, and demonstrated resolution of storage lesions throughout the CNS. Antibodies against the enzyme were detected in the CSF of some animals, which coincided with lower CSF IDUA activity and less efficient correction of storage lesions. We found that immunological tolerance could be induced to IDUA by exposing newborn MPS I dogs to the enzyme, which enhanced the efficacy of subsequent gene transfer. These results were replicated in rhesus macaques, supporting the potential to translate neonatal tolerance induction to clinical applications. Intrathecal AAV delivery offers the potential for widespread gene transfer in the CNS with a single minimally invasive vector injection, which could prove transformative for the field of gene therapy for inherited neurological disorders

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

Injury induced neuroplasticity and cell specific targeting of the lumbar enlargement for gene therapy.

This dissertation is an examination of spinal cord injury induced neuroplasticity and tests whether noninvasive gene therapy can successfully target neurons in the lumbar spinal cord. It begins with an overview of neural control of locomotion and a brief summary of therapeutics that are used and/or in development for treating spinal anatomically characterize s subset of neurons in the spinal cord, long ascending propriospinal neurons, that are involved in interlimb coordination. Characterization of these neurons allows for subsequent evaluation of their potential involvement in injury induced neuroplasticity. This dissertation is divided into five chapters, covering spinal cord injury and therapeutics. Chapter One gives background on locomotor control, propriospinal neurons, spinal cord injury, and therapeutics. Chapter Two develops and characterizes viral tracing methods for spinal cord anatomy. Chapter Three then uses these methods to characterize long ascending propriospinal neurons and evaluate their involvement in injury induced plasticity. Chapter Four then focuses on the development of noninvasive delivery of gene transfer to the lumbar enlargement. This involves optimizing focused ultrasound and intravenous microbubble delivery to focally and transiently permeabilize the blood spinal cord barrier of the lumbar spinal cord. This optimization then allows for successful gene transfer in neurons in the lumbar spinal cord following intravenous delivery of viral vector. Lasty, Chapter Five discusses the implications for all of these findings and how these findings have contributed to our understanding spinal cord anatomy and injury, and how the proof-of-concept in Chapter 4 provides a promising new avenue for spinal cord injury therapeutics

Divergent Whole Brain Projections from the Ventral Midbrain in Macaques

To understand the connectome of the axonal arborizations of dopaminergic midbrain neurons, we investigated the anterograde spread of highly sensitive viral tracers injected into the ventral tegmental area (VTA) and adjacent areas in 3 macaques. In 2 monkeys, injections were centered on the lateral VTA with some spread into the substantia nigra, while in one animal the injection targeted the medial VTA with partial spread into the ventro-medial thalamus. Double-labeling with antibodies against transduced fluorescent proteins (FPs) and tyrosine hydroxylase indicated that substantial portions of transduced midbrain neurons were dopaminergic. Interestingly, cortical terminals were found either homogeneously in molecular layer I, or more heterogeneously, sometimes forming patches, in the deeper laminae II-VI. In the animals with injections in lateral VTA, terminals were most dense in somatomotor cortex and the striatum. In contrast, when the medial VTA was transduced, dense terminals were found in dorsal prefrontal and temporal cortices, while projections to striatum were sparse. In all monkeys, orbitofrontal and occipito-parietal cortex received strong and weak innervation, respectively. Thus, the dopaminergic ventral midbrain sends heterogeneous projections throughout the brain. Furthermore, our results suggest the existence of subgroups in meso-dopaminergic neurons depending on their location in the primate ventral midbrain

Recent advances in radiotracers targeting norepinephrine transporter: structural development and radiolabeling improvements

The norepinephrine transporter (NET) is a major target for the evaluation of the cardiac sympathetic nerve system in patients with heart failure and Parkinson's disease. It is also used in the therapeutic applications against certain types of neuroendocrine tumors, as exemplified by the clinically used 123/131I-MIBG as theranostic single-photon emission computed tomography (SPECT) agent. With the development of more advanced positron emission tomography (PET) technology, more radiotracers targeting NET have been reported, with superior temporal and spatial resolutions, along with the possibility of functional and kinetic analysis. More recently, fluorine-18-labelled NET tracers have drawn increasing attentions from researchers, due to their longer radiological half-life relative to carbon-11 (110 min vs. 20 min), reduced dependence on on-site cyclotrons, and flexibility in the design of novel tracer structures. In the heart, certain NET tracers provide integral diagnostic information on sympathetic innervation and the nerve status. In the central nervous system, such radiotracers can reveal NET distribution and density in pathological conditions. Most radiotracers targeting cardiac NET-function for the cardiac application consistent of derivatives of either norepinephrine or MIBG with its benzylguanidine core structure, e.g. 11C-HED and 18F-LMI1195. In contrast, all NET tracers used in central nervous system applications are derived from clinically used antidepressants. Lastly, possible applications of NET as selective tracers over organic cation transporters (OCTs) in the kidneys and other organs controlled by sympathetic nervous system will also be discussed

Proceedings of the Sixth Deep Brain Stimulation Think Tank Modulation of Brain Networks and Application of Advanced Neuroimaging, Neurophysiology, and Optogenetics

The annual deep brain stimulation (DBS) Think Tank aims to create an opportunity for a multidisciplinary discussion in the field of neuromodulation to examine developments, opportunities and challenges in the field. The proceedings of the Sixth Annual Think Tank recapitulate progress in applications of neurotechnology, neurophysiology, and emerging techniques for the treatment of a range of psychiatric and neurological conditions including Parkinson’s disease, essential tremor, Tourette syndrome, epilepsy, cognitive disorders, and addiction. Each section of this overview provides insight about the understanding of neuromodulation for specific disease and discusses current challenges and future directions. This year’s report addresses key issues in implementing advanced neurophysiological techniques, evolving use of novel modulation techniques to deliver DBS, ans improved neuroimaging techniques. The proceedings also offer insights into the new era of brain network neuromodulation and connectomic DBS to define and target dysfunctional brain networks. The proceedings also focused on innovations in applications and understanding of adaptive DBS (closed-loop systems), the use and applications of optogenetics in the field of neurostimulation and the need to develop databases for DBS indications. Finally, updates on neuroethical, legal, social, and policy issues relevant to DBS research are discussed

Animal Models of Thrombosis From Zebrafish to Nonhuman Primates: Use in the Elucidation of New Pathologic Pathways and the Development of Antithrombotic Drugs

Thrombosis is a leading cause of morbidity and mortality worldwide. Animal models are used to understand the pathological pathways involved in thrombosis and to test the efficacy and safety of new antithrombotic drugs. In this review, we will first describe the central role a variety of animal models of thrombosis and hemostasis has played in the development of new antiplatelet and anticoagulant drugs. These include the widely used P2Y12 antagonists and the recently developed orally available anticoagulants that directly target factor Xa or thrombin. Next, we will describe the new players, such as polyphosphate, neutrophil extracellular traps, and microparticles, which have been shown to contribute to thrombosis in mouse models, particularly venous thrombosis models. Other mouse studies have demonstrated roles for the factor XIIa and factor XIa in thrombosis. This has spurred the development of strategies to reduce their levels or activities as a new approach for preventing thrombosis. Finally, we will discuss the emergence of zebrafish as a model to study thrombosis and its potential use in the discovery of novel factors involved in thrombosis and hemostasis. Animal models of thrombosis from zebrafish to nonhuman primates are vital in identifying pathological pathways of thrombosis that can be safely targeted with a minimal effect on hemostasis. Future studies should focus on understanding the different triggers of thrombosis and the best drugs to prevent each type of thrombotic event