This volume provides experimental approaches aimed to characterize the Cerebral Cavernous Malformations (CCM) disease and to define the cellular and molecular mechanisms underlying this pathology. Chapters are divided into four sections providing a general overview of the natural history, epidemiology, and pathogenetic mechanisms of CCM disease, describing methods currently used for diagnosis and treatment, production and analysis of distinct cellular and animal models in which CCM can be studied, and different methodological approaches to study the mechanisms underlying the CCM onset and…mehr
This volume provides experimental approaches aimed to characterize the Cerebral Cavernous Malformations (CCM) disease and to define the cellular and molecular mechanisms underlying this pathology. Chapters are divided into four sections providing a general overview of the natural history, epidemiology, and pathogenetic mechanisms of CCM disease, describing methods currently used for diagnosis and treatment, production and analysis of distinct cellular and animal models in which CCM can be studied, and different methodological approaches to study the mechanisms underlying the CCM onset and progression. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and cutting-edge, Cerebral Cavernous Malformations (CCM): Methods and Protocols aims to ensure successful results in the further study of this complex disease and its pathophysiologic correlates.
From Genes and Mechanisms to Molecular-targeted Therapies: the Long Climb to the Cure of Cerebral Cavernous Malformation (CCM) Disease.- Incidence, Prevalence, and Clinical Presentation of Cerebral Cavernous Malformations.- Natural History, Clinical, and Surgical Management of Cavernous Malformations.- Molecular Genetics Screening of CCM patients, An Overview.- Next Generation Sequecing (NGS) Strategies for Genetic Testing and Diagnosis of Cerebral Cavernous Malformation (CCM) Disease.- Genome-wide Genotyping of Cerebral Cavernous Malformation Type 1 Individuals to Identify Genetic Modifiers of Disease Severity.- Clinical Imaging of Cerebral Cavernous Malformations: Computed Tomography and Magnetic Resonance Imaging.- Neuroradiology: Differential Diagnosis, Follow-Up, and Reporting.- Surgical Management of Brain Cavernous Malformations.- Generation of CCM Phenotype by a Human Microvascular Endothelial Model.- Isolation and Purification of Mouse Brain Endothelial Cells to Study Cerebral Cavernous Malformation Disease.- Production of KRIT1-knockout and KRIT1-knockin Mouse Embryonic Fibroblasts as Cellular Models of CCM Disease.- CRISPR/Cas9-mediated Generation of Human Endothelial Cell Knockout Models of CCM Disease.- Dissection of the Role of CCM Genes in Tubulogenesis using the Drosophila Tracheal System as a Model.- Generation and Analysis of CCM Phenotypes in C. elegans.- Generation of Transgenic Lines of Zebrafish Expressing Fluorescently-tagged CCM Proteins to Study Their Function and Subcellular Localization within the Vasculature.- Vertebrate Models to Investigate CCM Pathogenesis: The Zebrafish and Mouse Model.- Generation of Cerebral Cavernous Malformation in Neonatal Mouse Models using Inducible Cre-LoxP Strategy.- Isolation of Cerebral Endothelial Cells from CCM1/KRIT1 Null Mouse Brain.- Identification of the KRIT1 Protein by LexA-based Yeast Two-Hybrid System.- Crystallographic Studies of the Cerebral Cavernous Malformations Proteins.- Microscopy Techniques to Investigate CCM Pathogenesis.- Preparation and Analysis of Protein Extracts to Investigate CCM Pathogenesis.- Systems Wide Analysis of CCM Signaling Complex (CSC) Alterations in CCM Deficient Models using Omics Approaches.- Study of Molecular Interactions of CCM Proteins by using a GAL4-based Yeast Two-hybrid Screening.- Study of CCM Microvascular Endothelial Phenotype by an in vitro Tubule Differentiation Model.- Bidimentional in vitro Angiogenic Assays to Study CCM Pathogenesis: Endothelial Cell Proliferation and Migration.- Measurement of Endothelial Barrier Function in Mouse Models of Cerebral Cavernous Malformations using Intravital Microscopy.- Immunofluorescence of Cell-Cell and Cell-Extracellular Matrix Adhesive Defectsin in vitro Endothelial CCM Model. Juxtacrine Role of Mutant Extracellular Matrix on Wild-Type Endothelial Cells.- Detection of p62/SQSTM1Aggregates in Cellular Models of CCM Disease by Immunofluorescence.- Notch Signaling in Familial Cerebral Cavernous Malformations and Immunohistochemical Detection of cleaved Notch1 Intracellular Domain.- Measuring the Kinase Activity of GCKIII Proteins in vitro.- Spectrophotometric Method for Determining Glyoxalase 1 Activity in Cerebral Cavernous Malformation (CCM) Disease.- Fluorescence Analysis of Reactive Oxygen Species (ROS) in Cellular Models of Cerebral Cavernous Malformation Disease.- Small RNA Library Preparation for Profiling Small Non-coding RNAs in Patients Affected by Cavernous Malformations.- Affinity Purification and Preparation of Peptides for Mass Spectrometry from C. elegans.
From Genes and Mechanisms to Molecular-targeted Therapies: the Long Climb to the Cure of Cerebral Cavernous Malformation (CCM) Disease.- Incidence, Prevalence, and Clinical Presentation of Cerebral Cavernous Malformations.- Natural History, Clinical, and Surgical Management of Cavernous Malformations.- Molecular Genetics Screening of CCM patients, An Overview.- Next Generation Sequecing (NGS) Strategies for Genetic Testing and Diagnosis of Cerebral Cavernous Malformation (CCM) Disease.- Genome-wide Genotyping of Cerebral Cavernous Malformation Type 1 Individuals to Identify Genetic Modifiers of Disease Severity.- Clinical Imaging of Cerebral Cavernous Malformations: Computed Tomography and Magnetic Resonance Imaging.- Neuroradiology: Differential Diagnosis, Follow-Up, and Reporting.- Surgical Management of Brain Cavernous Malformations.- Generation of CCM Phenotype by a Human Microvascular Endothelial Model.- Isolation and Purification of Mouse Brain Endothelial Cells to Study Cerebral Cavernous Malformation Disease.- Production of KRIT1-knockout and KRIT1-knockin Mouse Embryonic Fibroblasts as Cellular Models of CCM Disease.- CRISPR/Cas9-mediated Generation of Human Endothelial Cell Knockout Models of CCM Disease.- Dissection of the Role of CCM Genes in Tubulogenesis using the Drosophila Tracheal System as a Model.- Generation and Analysis of CCM Phenotypes in C. elegans.- Generation of Transgenic Lines of Zebrafish Expressing Fluorescently-tagged CCM Proteins to Study Their Function and Subcellular Localization within the Vasculature.- Vertebrate Models to Investigate CCM Pathogenesis: The Zebrafish and Mouse Model.- Generation of Cerebral Cavernous Malformation in Neonatal Mouse Models using Inducible Cre-LoxP Strategy.- Isolation of Cerebral Endothelial Cells from CCM1/KRIT1 Null Mouse Brain.- Identification of the KRIT1 Protein by LexA-based Yeast Two-Hybrid System.- Crystallographic Studies of the Cerebral Cavernous Malformations Proteins.- Microscopy Techniques to Investigate CCM Pathogenesis.- Preparation and Analysis of Protein Extracts to Investigate CCM Pathogenesis.- Systems Wide Analysis of CCM Signaling Complex (CSC) Alterations in CCM Deficient Models using Omics Approaches.- Study of Molecular Interactions of CCM Proteins by using a GAL4-based Yeast Two-hybrid Screening.- Study of CCM Microvascular Endothelial Phenotype by an in vitro Tubule Differentiation Model.- Bidimentional in vitro Angiogenic Assays to Study CCM Pathogenesis: Endothelial Cell Proliferation and Migration.- Measurement of Endothelial Barrier Function in Mouse Models of Cerebral Cavernous Malformations using Intravital Microscopy.- Immunofluorescence of Cell-Cell and Cell-Extracellular Matrix Adhesive Defectsin in vitro Endothelial CCM Model. Juxtacrine Role of Mutant Extracellular Matrix on Wild-Type Endothelial Cells.- Detection of p62/SQSTM1Aggregates in Cellular Models of CCM Disease by Immunofluorescence.- Notch Signaling in Familial Cerebral Cavernous Malformations and Immunohistochemical Detection of cleaved Notch1 Intracellular Domain.- Measuring the Kinase Activity of GCKIII Proteins in vitro.- Spectrophotometric Method for Determining Glyoxalase 1 Activity in Cerebral Cavernous Malformation (CCM) Disease.- Fluorescence Analysis of Reactive Oxygen Species (ROS) in Cellular Models of Cerebral Cavernous Malformation Disease.- Small RNA Library Preparation for Profiling Small Non-coding RNAs in Patients Affected by Cavernous Malformations.- Affinity Purification and Preparation of Peptides for Mass Spectrometry from C. elegans.
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