Research Protocols for Ophthalmic Disease Mechanisms and Therapeutics: Glaucoma - Ocular Hypertension offers a detailed, step-by-step protocols for isolating, characterizing, and analyzing cells and tissues of the eye’s anterior segment - where OHT originates - using techniques from molecular biology, pharmacology, and histology.
Across eight thematic sections, the book covers ocular anatomy, drug screening, receptor localization, cell-based assays, in vitro and in vivo models, drug formulation, and cutting-edge technologies such as gene editing, single-cell RNA sequencing, and stem cell therapies. Each protocol is designed to support reproducibility and translational relevance.
Across eight thematic sections, the book covers ocular anatomy, drug screening, receptor localization, cell-based assays, in vitro and in vivo models, drug formulation, and cutting-edge technologies such as gene editing, single-cell RNA sequencing, and stem cell therapies. Each protocol is designed to support reproducibility and translational relevance.
Key Features:
- Details validated protocols for drug discovery and disease modeling
- Demonstrates methods for studying receptor function and pharmacokinetics
- Provides animal models and ex vivo systems to evaluate therapeutic strategies
- Incorporates emerging tools such as CRISPR, metabolomics, and exosomes
- Bridges experimental design with clinical relevance for glaucoma research
Table of Contents
Chapter 1 Basic Anatomy and Physiology of the Human Eye- Fatima Muili, Anthonia Okolie, Catherine A. Opere, Ya Fatou Njie-Mbye and Sunny E.
- Introduction
- Extraocular Structures
- Eye Lids
- Lacrimal System
- Orbit
- Ocular Structures (Anterior Segment)
- Cornea
- Limbus
- Anterior Chamber
- Posterior Chambers
- Trabecular Meshwork
- Canal of Schlemm
- Iris
- Lens
- Zonule
- Ciliary Body
- Ocular Structures (Posterior Segment)
- Vitreous Humor
- Retina
- Choroid
- Sclera
- Optic Nerve
- Conclusion
- References
- Introduction to Ocular Hypertension and Glaucoma and An Overview of Drug Screening Funnels for Discovering and Characterizing New Intraocular Pressure-Reducing Compounds
- Introduction
- Screening Funnels- An Overview
- Conclusion
- References
- Introduction
- Section 1: Access to Health Authority-Approved Iop-Lowering Agents
- Section 2. Access to Investigational Iop-Lowering Agents
- Conclusion
- References
- Part 2 Target Localization and Verification Studies
- Introduction
- Rt-Pcr of Receptors in Eye Tissues of the Anterior Chamber
- Isolation of Total Rna
- Mrna Detection by Rt-Pcr
- In Situ Hybridization to Localize and Visualize Mrnas for Pg Receptors
- Tissue Preparation
- Probe Preparation
- In Situ Hybridization Procedure
- In Vitro Autoradiography to Visualize and Quantify Receptor/Enzyme Proteins
- Tissue Preparation
- Conclusion
- References
- Introduction
- Experimental Protocols
- Tissue Membrane Preparation
- Radioligand Binding Assay
- Conclusion
- References
- Introduction
- Protocols for Performing Prostaglandin Receptor Autoradiography in Vitro
- Data Acquisition and Analysis
- Conclusion
- References
- Introduction
- Experimental Protocols
- Tissue Collection and Preparation
- Immunofluorescence Labeling of Frozen Sections
- Preparation of Cryosections
- Immunofluorescence Labeling and Imaging
- Immunohistochemistry of Paraffin-Embedded Sections
- Preparation of Paraffin Sections
- Immunoenzyme Labeling and Imaging
- Immunogold Labeling of Resin-Embedded Sections
- Preparation of Ultrathin Resin Sections
- Immunogold Labeling and Imaging
- Conclusion
- References
- Part 3 Ocular Cell Types and Sources for Isolation and Characterization
- Introduction
- Procedures for Tm Isolation
- Tm Cell Isolation Procedures
- Conclusion
- References
- Introduction
- Experimental Protocols
- Preparation of Ipsc Embryoid Bodies (Ebs)
- Generation of Embryoid Bodies:
- Differentiation of Ebs
- Validation of Differentiation:
- Preparation for the Development of the Saponin Glaucomatous Cell Loss Anterior Segment Perfusion Model:
- Preparation for Transplantation of Differentiated Tm-Like Ipsc into Saponin Model and Labeling of Cells:
- Testing for Loss of Iop Homeostatic Response to 2X Sustained Pressure Challenge
- 93
- Assessing the Integration of Transplanted Tm-Like Ipscs
- Conclusion
- Acknowledgements
- References
- Introduction
- Isolation and Characterization of Ciliary Smooth Muscle Cells
- Characterization of Ciliary Smooth Muscle Cells by Immunocytochemistry
- Isolation and Characterization of Schlemm’S Canal Cells
- Characterization of Schlemm’S Canal Cells by Immunocytochemistry
- Matrix Metalloproteinases in Ocular Cells/ Tissues
- Matrix Metalloproteinases (Mmps) and Aqueous Humor Dynamics in Glaucoma
- Mmp Activity Measurement by Zymography
- Measurement of Matrix Metalloproteinases (Mmps) Expression by Western Blotting
- Conclusion
- Acknowledgements
- References
- Introduction
- Experimental Protocols
- Preparation of the Porcine Eye for Dissection
- Dissection of the Eye
- Isolation of the Npe
- Dispersion and Initial Culture of the Npe
- Trypsinization and Propagation of the Cultured Npe
- Characterization of the Cultured Npe Cells
- Cultured Npe Cells Grown on Permeable Support Form a Diffusion Barrier
- Cultured Porcine Npe Cells Express Xenobiotic Transporters
- Cultured Porcine Npe Expresses Connexin 43 and Connexin 50
- Cultured Porcine Npe Expresses Nhe1, Nhe4, Ae2, and Carbonic Anhydrases Ii and Iv 121
- Discussion and Conclusion
- Acknowledgements
- References
- Introduction
- Experimental Protocols
- Isolation of Scleral Fibroblasts
- Materials
- Cell Culture Vessels
- Culture Medium
- Fetal Bovine Serum (Fbs)
- Antibiotics
- Incubation Conditions
- Temperature
- Humidity
- Carbon Dioxide (Co2) Concentration
- Preparation
- Eye Dissection
- Tissue Digestion
- Cell Isolation
- Cell Culture
- Characterization of Scleral Fibroblasts
- Morphological Analysis
- Cell Proliferation Assay
- Brdu Incorporation Assay
- Immunocytochemistry
- Materials
- Protocol
- Protocols for Scleral Fibroblasts and Extracellular Matrix (Ecm) Production
- Isolation and Culture of Scleral Fibroblasts
- Promoting Ecm Production
- Ecm Analysis
- Sample Collection and Protein Extraction
Author
- Najam A. Sharif
- Sunny E. Ohia
