Retinal Degenerative Diseases - Laboratory and Therapeutic Investigations

Dedication

Preface

Contents

Contributors

Travel Awards

About the Editors

Part I Basic Science Underlying Retinal Degeneration

1 Analysis of Genes Differentially Expressed During Retinal Degeneration in Three Mouse Models

1.1 Introduction

1.2 Methods

1.2.1 RNA Preparation and cDNA Labeling

1.2.2 Hybridization of Slides, Image Acquisition and Bioinformatics

1.2.3 Real-Time PCR

1.3 Results

1.3.1 Microarray Analysis of Opsin 02550256 0/0 Model

1.3.2 Microarray Analysis of Bouse C Model

1.3.3 Microarray Analysis of MOT1 Mouse

1.4 Discussion

References

2 Regulation of Angiogenesis by Macrophages

2.1 Macrophage Polarization and Its Role in Angiogenesis

References

3 Protein Kinase C Regulates Rod Photoreceptor Differentiation Through Modulation of STAT3 Signaling

3.1 Introduction

3.2 Materials and Methods

3.2.1 Reagents

3.2.2 Animals and Retina Explant Culture

3.2.3 Cell Culture

3.2.4 Western Blot Assay

3.3 Results

3.3.1 Phorbol Esters Increase Rod Generation

3.3.2 Expression of PKC Isoforms in Developing Retina

3.3.3 Activation of PKC Decreases Phosphorylation of STAT3

3.4 Discussion

References

4 Pigment Epithelium-derived Factor Receptor (PEDF-R): A Plasma Membrane-linked Phospholipase with PEDF Binding Affinity

4.1 Introduction

4.2 Identification of a PEDF Receptor

4.3 In Silico Information

4.4 Expression and Distribution in the Retina

4.5 Transmembrane Topology

4.6 Binding to PEDF Ligands

4.7 Phospholipase Activity

4.8 PEDF-R Activity in Retinal Cells

4.9 Conclusions

References

5 The Function of Oligomerization-Incompetent RDS in Rods

References

6 The Association Between Telomere Length and Sensitivity to Apoptosis of HUVEC

6.1 Introduction

6.2 Methods

6.2.1 The Culture of HUVEC and the Construction of Cell Division Model

6.2.2 Construction of an Apoptosis Model of HUVEC with Free Hydroxyl Radicals

6.2.3 Measurement of Apoptosis Rates and Telomere Lengths

6.2.4 Statistics Analysis

6.3 Results

6.3.1 Relationship Between the Time of Culture and the Telomere Length

6.3.2 Relationship Among Apoptosis Rates, Culture Times and Oxidation

6.3.3 Oxidation Enhances the Telomere Shortening

6.4 Discussion

References

7 Photoreceptor Guanylate Cyclases and cGMP Phosphodiesterases in Zebrafish

7.1 Regulation of cGMP Levels in Photoreceptor Outer Segments

7.2 Retinal Disorders Associated with Mutations in RetGCs and PDE6

7.3 Analysis of Teleost RetGC and PDEs in Retinal Function and Disorders

References

8 RDS in Cones Does Not Interact with the Beta Subunit of the Cyclic Nucleotide Gated Channel

References

9 Increased Expression of TGF-1 and Smad 4 on Oxygen-Induced Retinopathy in Neonatal Mice

9.1 Introduction

9.2 Material and Methods

9.2.1 Animals

9.2.2 Methods

9.2.2.1 TGF- Immunohistochemistry (IH) and Smad-4 In Situ Hybridization (ISH)

9.2.3 Statistical Analysis

9.3 Results

9.4 Discussion

References

10 ZBED4, A Novel Retinal Protein Expressed in Cones and Mller Cells

10.1 Introduction

10.2 Methods and Results

10.2.1 ZBED4 mRNA is Expressed in Human Retina

10.2.2 ZBED4 mRNA is Expressed in Mouse and Human Cones

10.2.3 ZBED4 is Expressed Both in Nuclei and Cytoplasm of Human Cones

10.2.3.1 Human ZBED4 is Also Expressed in Müller Cells Endfeet

10.2.4 Human ZBED4 is Distributed Between Nuclear and Cytoplasmic Retinal Fractions

10.2.5 Subcellular Localization of ZBED4 in Stably Transfected Cells

10.2.6 Purification of His-Tagged ZBED4 and Its Dimerization In Vivo

10.2.7 Mass Spectrometry Identifies Putative Proteins Interacting with ZBED4

10.3 Discussion

References

11 Tubby-Like Protein 1 (Tulp1) Is Required for Normal Photoreceptor Synaptic Development

11.1 Introduction

11.2 Methods

11.2.1 Animals

11.2.2 Immunofluorescent Staining of Retinal Sections

11.3 Results

11.4 Discussion

References

12 Growth-Associated Protein43 (GAP43) Is a Biochemical Marker for the Whole Period of Fish Optic Nerve Regeneration

12.1 Introduction

12.2 Experimental Procedures

12.2.1 Animal

12.2.2 Immunohistochemistry

12.2.3 RT-PCR Analysis

12.2.4 Behavioral Analysis

12.3 Results

12.3.1 Immunohistochemical Studies of GAP43 Protein in the Goldfish Retina After Optic Nerve Transection

12.3.2 Time Course of GAP43 mRNA Expression in the Goldfish Retina During Optic Nerve Regeneration

12.3.3 Chasing Behavior of Two Goldfish with Treatment of Optic Nerve Transection During Optic Nerve Regeneration

12.4 Discussion

12.4.1 Termination of Optic Nerve Regeneration in Goldfish

12.4.2 GAP43 Is a Good Marker for Monitoring the Long Process of Optic Nerve Regeneration in Fish

References

13 Multiprotein Complexes of Retinitis Pigmentosa GTPase Regulator (RPGR), a Ciliary Protein Mutated in X-Linked Retinitis Pigmentosa (XLRP)

13.1 X-Linked RP (XLRP)

13.2 Retinitis Pigmentosa GTPase Regulator (RPGR)

13.3 RPGR Isoforms in the Retina

13.4 Animal Models of RPGR

13.5 Sensory Cilia

13.6 Retinal Degeneration Caused by Mutations in Ciliary Proteins

13.7 Macromolecular Complexes of RPGR ORF15

13.8 Dissection of RPGR ORF15 Complexes

13.9 Conclusion

References

14 Misfolded Proteins and Retinal Dystrophies

14.1 Endoplasmic Reticulum Stress and Retinal Degeneration

14.2 Misfolded Proteins in Photoreceptors

14.3 Misfolded Proteins in Retinal Pigment Epithelial Cells

14.4 Pharmacologic Targeting of Protein Misfolding to Prevent Retinal Degeneration

References

15 Neural Retina and MerTK-Independent Apical Polarity of v5 Integrin Receptors in the Retinal Pigment Epithelium

15.1 Introduction

15.2 Functions of Apical v5 Integrin Receptors in Retinal Phagocytosis and Adhesion

15.3 Apical Polarity of v5 Integrin Receptors is Independent of the Neural Retina

15.4 Apical Polarity of v5 Receptors is Independent of the Essential Engulfment Receptor MerTK

15.5 Motifs of the 5 Integrin Subunit Cytoplasmic Domain that May Promote Apical Polarity of v5 Integrin Receptors

15.6 Perspective

References

16 Mertk in Daily Retinal Phagocytosis: A History in the Making

16.1 Introduction

16.2 RCS Rat and MerTK Receptor: An Intimate Story

16.3 Changes Associated with Absence of MerTK in the Rat Retina

16.4 Daily Rhythmic Activation of Mertk: The Intracellular Way

16.5 The Debate About MerTK Ligands In Vivo

16.6 Perspectives

References

17 The Interphotoreceptor Retinoid Binding (IRBP)Is Essential for Normal Retinoid Processing in ConePhotoreceptors

17.1 Introduction

17.2 The Cone Population in Irbp/Mice

17.3 Implications for IRBP and Cone Function

17.4 The Cone Visual Cycle

References

18 Aseptic Injury to Epithelial Cells Alters Cell Surface Complement Regulation in a Tissue Specific Fashion

18.1 Introduction

18.2 Material and Methods

18.2.1 Reagents

18.2.2 Cell Culture

18.2.3 Flow Cytometry

18.3 Results

18.3.1 Oxidative Stress, but Not Chemical Hypoxia, Alters the Expression of Complement Regulatory Proteins on ARPE-19 Cells

18.3.2 Oxidative Stress of Renal Tubular Epithelial Cells Does Not Alter Surface Expression of Crry by the Cells

18.3.3 Expression of MCP, CD55 and CD59 on the Surface of Lung Epithelial Cells Increases After Oxidative Stress, but This Does Not Prevent Complement-Activation on the Cell Surface

18.4 Discussion

References

19 Role of Metalloproteases in Retinal Degeneration Induced by Violet and Blue Light

19.1 Introduction

19.2 Objective

19.3 Materials and Methods

19.4 Results

19.5 Conclusion

References

20 Mitochondrial Decay and Impairment of Antioxidant Defenses in Aging RPE Cells

20.1 Summary

20.2 Introduction

20.3 Materials and Methods

20.3.1 Primary Human RPE Cell Culture

20.3.2 Hydrogen Peroxide Toxicity -- PI Assays

20.3.3 Mitochondrial Morphometrics

20.3.4 Protein and Weight Estimation of RPE Cells and Mitochondria

20.3.5 Measurement of ROS, ATP and Mitochondrial Membrane Potential (00 m )

20.3.6 Measurement of ([Ca2+]c) and ([Ca2+ ]m)

20.3.7 Expression of Mitochondrial Associated Genes

20.4 Results

20.4.1 Age Related Sensitivity of RPE Cells to Oxidative Stress

20.4.2 Variation in Mitochondrial Number, Structure, and Size

20.4.3 ROS and ATP Production, and 00 m Decrease in RPE Cells with Aging

20.4.4 Age-Related Variations in ([Ca2+]c) and ([Ca 2+] m ) in RPE Cells

20.4.5 Expression of Genes Associated with Mitochondrial Function

20.5 Discussion

References

21 Ciliary Transport of Opsin

21.1 Introduction

21.2 Methods

21.3 Results

21.4 Discussion

References

22 Effect of Hesperidin on Expression of Inducible Nitric Oxide Synthase in Cultured Rabbit Retinal Pigment Epithelial Cells

22.1 Introduction

22.2 Materials and Methods

22.2.1 Preparing Hesperidin Extract of Pericarpium Citri Reticulatae

22.2.2 Identification of Hesperidin by High Performance Liquid Chromatogram (HPLC)

22.2.3 Cell Culture

22.2.4 MTT Cell Viability Assay

22.2.5 Assay of NO Production

22.2.6 Cellular Immunohistochemistry of iNOS

22.2.7 Statistical Analysis

22.3 Results

22.3.1 Identification of Hesperidin by HPLC

22.3.2 RPE Cells Morphology

22.3.3 Influence of Hesperin on RPE Cell Proliferation Under the Condition of High Glucose

22.3.4 Assay of NO and iNOS

22.4 Discussion

References

23 Profiling MicroRNAs Differentially Expressed in Rabbit Retina

23.1 Introduction

23.2 Materials and Methods

23.2.1 Rabbit Retina Tissues

23.2.2 RNA Extraction

23.2.3 miRNA Microarray Analysis

23.2.4 Data Analysis

23.2.5 Bioinformatics Analysis of the Selected Mirnas

23.3 Results and Discussion

23.3.1 miRNA Microarray Analysis

23.3.2 Putative miRNA Target Gene Prediction

References

24 Unexpected Transcriptional Activity of the Human VMD2 Promoter in Retinal Development

24.1 Introduction

24.2 Materials and Methods

24.2.1 Experiment with Animals

24.2.2 -Galactosidase Assay

24.3 Results

24.3.1 Generation of Transgenic Mice

24.3.2 Localization of Cre Function in Transgenic Mice

24.4 Discussion

References

25 Microarray Analysis of Hyperoxia Stressed Mouse Retina: Differential Gene Expression in the Inferior and SuperiorRegion

25.1 Introduction

25.2 Methods

25.3 Result

25.4 Conclusions

References

26 Photoreceptor Sensory Cilia and Inherited Retinal Degeneration

26.1 PSC Proteins Involved in Inherited Retinal Degenerations

26.2 Structure of Photoreceptor Sensory Cilium Complex

26.3 Protein Components of Photoreceptor Sensory Cilium: PSC Proteome

26.4 Novel Photoreceptor Cilia Proteins in PSC Proteome

26.4.1 Subcellular Locations of Candidate Novel PSC Proteins

26.4.2 Functional Analysis of Novel PSC Proteins in Photoreceptor and Renal Cilia

26.4.2.1 shRNAs Against Novel PSC Genes

26.4.2.2 Evaluation of Phenotypes of shRNA Knockdown in mIMCD3 Cells and PSCs

26.5 TTC21B Protein in Photoreceptor Sensory Cilia and Renal Primary Cilia

26.5.1 TTC21B Localizes to the Basal Bodies and Transition Zone of Primary and Photoreceptor Sensory Cilia

26.5.2 TTC21B is Required for Primary Cilia and Photoreceptor Sensory Cilia Formation

26.6 Future Direction: Screening Novel PSC Genes for Mutations that Cause IRDs

References

27 Role of Elovl4 Protein in the Biosynthesisof Docosahexaenoic Acid

27.1 Introduction

27.2 Materials and Methods

27.2.1 RNA Interference

27.2.2 Construction of Mouse Anti Elovl4 Gene shRNA

27.2.3 Tissue Culture

27.2.4 Fatty Acid Analysis

27.3 Results

27.3.1 661W Cells Express Elovl4 and Can Elongate 18:3n3 and 22:5n3 to Longer Chain Fatty Acids

27.3.2 Knock-Down of Endogenous Elovl4 Does Not Affect C18--C24 PUFA Synthesis

27.4 Discussion

References

Part II Molecular Genetics and Candidate Genes

28 Molecular Pathogenesis of Achromatopsia Associated with Mutations in the Cone Cyclic Nucleotide-Gated Channel CNGA3 Subunit

28.1 Introduction

28.2 Materials and Methods

28.2.1 Constructs, Cell Culture and Transfection

28.2.2 Ratiometric Measurement of Intracellular Ca2+ Concentration

28.2.3 Electrophysiological Recordings

28.2.4 SDS-PAGE and Western Blot Analysis

28.2.5 Immunofluorescence Labeling and Confocal Microscopy

28.3 Results

28.3.1 The R218C and R224W Mutations Cause Loss of Channel Function

28.3.2 The R218C and R224W Mutations Cause Channel Mis-Localization

28.3.3 Co-Expression of The R218C and R224W Mutants with the Wild Type Channel Does Not Affect the Channel Activity

28.4 Discussion

References

29 Mutation Spectra in Autosomal Dominant and Recessive Retinitis Pigmentosa in Northern Sweden

29.1 Introduction

29.2 Materials and Methods

29.2.1 Patients and Ophthalmologic Examinations

29.2.2 Molecular Genetic Analysis

29.3 Results and Discussion

29.3.1 adRP

29.3.2 Bothnia Dystrophy

29.4 Conclusions

References

30 1 Rhodopsin Mutations in Congenital Night Blindness

30.1 Introduction

30.2 Properties of Rhodopsin CSNB Mutants

30.2.1 Spectral and Photochemical Properties

30.2.2 Retinal Binding Kinetics of Rhodopsin CSNB Mutants

30.2.3 Activity of CSNB Mutants

30.2.3.1 In Vitro Assays of CSNB Mutants

30.2.3.2 Electrophysiological Studies on Transgenic Animal Models

30.3 Proposed Mechanisms of CSNB Mutations

30.3.1 Desensitization Due to Mutant Opsin Activity in Xenopus

30.3.2 Proposed Dark-Active Rhodopsin in Mouse

30.4 Future Studies

References

31 GCAP1 Mutations Associated with Autosomal Dominant Cone Dystrophy

31.1 Heterogeneity of Autosomal Dominant Cone and Cone-Rod Dystrophies

31.2 Guanylate Cyclase 1 (GC1) and GCAP1

31.3 The EF Hand Motifs of GCAP1

31.4 GUCA1A Mutations Associated with adCD and adCRD

31.5 EF3: The GCAP1(Y99C) and GCAP1(N104K) Mutations

31.6 EF4: The GCAP1(I143NT), GCAP1(L151F) and GCAP1(E155G) Mutations

31.7 Conclusion

References

32 Genotypic Analysis of X-linked Retinoschisis in Western Australia

32.1 Introduction

32.2 Methodology

32.2.1 Molecular Genetic Studies

32.2.2 Electrophysiological Studies

32.3 Results

32.3.1 RS1 Mutations in Western Australian Families

32.3.2 Compromised Full-Field and mfERG in an Obligate Carrier with 52+1G 0 T Mutation

32.3.3 Likely Pathogenicity of the Novel 289TG Genetic Variant

32.3.3.1 Family Information

32.3.3.2 Patient Information

32.3.3.3 Genetic Information

32.4 Discussion

References

33 Mutation Frequency of IMPDH1 Gene of Han Population in Ganzhou City

33.1 Introduction

33.2 Materials and Methods

33.2.1 Subjects

33.2.2 DNA Extraction

33.2.3 Amplification of IMPDH1 Genes

33.2.4 RFLP Analysis

33.2.5 Statistical Analysis

33.3 Results

33.4 Discussion

References

Part III Diagnostic, Clinical, Cytopathological and Physiologic Aspects of Retinal Degeneration

34 Reversible and Size-Selective Opening of the Inner Blood-Retina Barrier: A Novel Therapeutic Strategy

34.1 Introduction

34.2 Materials and Methods

34.2.1 Animal Experiments and Experimental Groups

34.2.2 Web-Based siRNA Design Protocols Targeting Claudin-5

34.2.3 In Vivo Delivery of siRNA to Murine Retinal Capillary Endothelial Cells by Large Volume Hydrodynamic Injection

34.2.4 Indirect Immunostaining of Retinal Flatmounts

34.2.5 Assessment of BRB Integrity by Perfusion of Hoechst (H33342)

34.2.6 Magnetic Resonance Imaging (MRI)

34.3 Results

34.3.1 Claudin-5 Levels in Retinal Flatmounts

34.3.2 Perfusion of Hoechst 33342 (562 Da) in Mice Post-Delivery of Claudin-5 Sirna

34.3.3 MRI Analysis of Ibrb Integrity Following Rnai of Claudin-5

34.4 Discussion

References

35 Spectral Domain Optical Coherence Tomography and Adaptive Optics: Imaging Photoreceptor Layer Morphology to Interpret Preclinical Phenotypes

35.1 Introduction

35.2 Materials and Methods

35.2.1 Subjects

35.2.2 Adaptive Optics Retinal Imaging

35.2.3 Spectral Domain Optical Coherence Tomography

35.3 Results

35.3.1 Cone Photoreceptor Mosaic Topography

35.3.2 Outer Nuclear Layer Thickness

35.4 Discussion

References

36 Pharmacological Manipulation of Rhodopsin RetinitisPigmentosa

36.1 Introduction

36.2 Pharmacological Strategies for Misfolding Mutant Rod Opsin

36.2.1 Pharmacological Chaperones

36.2.2 Kosmotropes

36.2.3 Molecular Chaperone Inducers

36.2.4 Autophagy Inducers

36.3 Conclusion

References

37 Targeted High-Throughput DNA Sequencing for Gene Discovery in Retinitis Pigmentosa

37.1 Introduction

37.2 Methods

37.2.1 Selection of Families

37.2.2 VisionCHIP Gene Selection

37.2.3 VisionCHIP Validation

37.2.4 Evaluating Potentially Pathogenic Variants

37.3 Conclusion

References

38 Advances in Imaging of Stargardt Disease

38.1 Introduction

38.2 Fundus Autofluorescence

38.3 OCT

38.4 Adaptive Optics Scanning Laser Ophthalmoscope

38.5 Conclusion

References

39 Protamine Sulfate Downregulates Vascular Endothelial Growth Factor (VEGF) Expression and Inhibits VEGF and Its Receptor Binding in Vitro

39.1 Materials and Methods

39.1.1 Cell Culture

39.1.2 Semi-Quantitative Assay of VEGF Expression in the Culture Cells by ICC

39.1.3 VEGF Expression was Determined by ELISA

39.1.4 Statistical Analysis

39.2 Results

39.2.1 The Maximum Inhibition of VEGF Expression by Protamine Sulfate

39.2.2 Protamine Sulfate Inhibits the RF/6A Cell VEGF Expression at the Hypoxic Condition

39.2.3 Protamine Sulfate Inhibits the Binding of VEGF to Its Receptor

39.3 Discussions

39.3.1 The Inhibition Effect of Protamine Sulfate on VEGF

39.3.2 Inhibition of the Binding Between VEGF and Its Receptor

39.3.3 The Potential Use of Protamine Sulfate Inhibition of Angiogenic Eye Diseases

References

40 Computer-Assisted Semi-Quantitative Analysis of Mouse Choroidal Density

40.1 Introduction

40.2 Methods

40.2.1 Immunohistochemial Staining of Choroidal Endothelia

40.2.2 Analysis of Choriodal Density with Photoshop 8.0

40.3 Results and Discussion

40.3.1 Analysis Of Choroidal Density

40.3.2 Usefulness of the Methodology

40.3.3 Summary

References

41 Thioredoxins 1 and 2 Protect Retinal Ganglion Cells from Pharmacologically Induced Oxidative Stress, Optic Nerve Transection and Ocular Hypertension

41.1 Introduction

41.2 Methods

41.2.1 Animals

41.2.2 RGC Counting

41.2.3 RGC Isolation

41.2.4 Western Blot Analysis

41.2.5 RGC-5 Culture and Transfection

41.2.6 Cell Viability Assay

41.2.7 In Vivo Electroporation (ELP)

41.2.8 Statistical Analysis

41.3 Results

41.3.1 Expression of TRX1, TRX2 and TXNIP in the Retina After ONT and IOP Elevation and in RGC-5 Cells with Induced Oxidative Stress

41.3.1.1 TRX Expression in RGC-5 Cells in Response to Oxidative Stress

41.3.1.2 The Levels of TRX Proteins After ONT

41.3.1.3 The Levels of TRX Proteins After IOP Elevation

41.3.2 The Effect of TRX1 and TRX2 Overexpression on RGC Survival

41.3.2.1 TRX1 and TRX2 Overexpression Protects RGC-5 cells Against Oxidative Stress

41.3.2.2 TRX1 and TRX2 Overexpression Increases RGC Survival After ONT

41.3.2.3 TRX1 and TRX2 Overexpression Increases RGC Survival After IOP Elevation

41.4 Discussion

References

42 Near-Infrared Light Protect the Photoreceptor from Light-Induced Damage in Rats

42.1 Introduction

42.2 Material and Methods

42.2.1 Animal

42.2.2 Light Damage

42.2.3 670 nm LED Treatment

42.2.4 Evaluation of Photoreceptor Cell Function by Electroretinography

42.2.5 Morphological Evaluation of Photoreceptor Rescue by Quantitative Histology

42.2.6 Statistical Analysis

42.3 Results

42.3.1 LED Attenuated the Light Damage Area in Retinas

42.3.2 LED Protected the Morphology of Light Damage Retina

42.3.3 LED Protected the Function of Light Damage Retina

42.4 Discussions

References

43 BDNF Improves the Efficacy ERG Amplitude Maintenance by Transplantation of Retinal Stem Cells in RCS Rats

43.1 Introduction

43.2 Methods

43.2.1 Animals

43.2.2 Cell Preparation and Subretinal Transplantation

43.2.3 Flash-Electroretinogram (F-ERG) Recordings

43.2.4 Histology and Quantification

43.2.5 Data Analysis

43.3 Results

43.3.1 ERG Amplitudes and Latencies

43.3.2 ONL Thickness

43.3.3 Graft Cells Survival After Subretinal Transplantation

43.4 Discussion

References

44 The Role of Purinergic Receptors in Retinal Functionand Disease

44.1 Introduction

44.2 Mechanisms of ATP Release and Degradation

44.2.1 ATP Release

44.2.2 Degradation of ATP

44.3 Purinergic Signaling in the Retina

44.3.1 Purinergic Modulation of Neuronal Signaling

44.3.2 ATP and Glial Transmission

44.4 The Role of Purinergic Receptors in Retinal Disease

44.5 Concluding Remarks

References

Part IV Macular Degeneration

45 Fundus Autofluorescence Imaging in Age-Related Macular Degeneration and Geographic Atrophy

45.1 Background

45.2 Fundus Autofluorescence Overview

45.3 FAF Findings in Early AMD with Drusen Only

45.4 FAF Findings in Late AMD with Geographic Atrophy

45.5 Progression of Geographic Atrophy

45.6 Mechanisms of Progression

45.7 Research to Prevent Progression

45.8 Discussion

References

46 Endoplasmic Reticulum Stress as a Primary Pathogenic Mechanism Leading to Age-Related Macular Degeneration

46.1 Age Related Macular Degeneration Is a Leading Cause of Vision Loss

46.2 Oxidative Stress and Complement Activation are Common Pathways in End-Stage Disease

46.3 ER Stress and Oxidative Stress Interact

46.4 ER and Oxidative Stress as Triggers for Inflammation and Disease

46.5 Future Experimental Approaches

References

47 Proteomic and Genomic Biomarkers for Age-Related Macular Degeneration

47.1 Introduction

47.2 Methods

47.3 Results

47.3.1 CEP Adducts and Autoantibodies Are Elevated in AMD Plasma

47.3.2 AMD Risk Based on CEP Biomarkers and Genotype

47.3.3 The Association Between CEP Biomarkers and AMD Risk Genotypes

47.4 Discussion

References

48 Impaired Intracellular Signaling May AllowUp-Regulation of CTGF-Synthesis and Secondary Peri-Retinal Fibrosis in Human Retinal Pigment Epithelial Cells from Patients with Age-Related Macular Degeneration

48.1 Introduction

48.2 Methods

48.2.1 Chemicals

48.2.2 Establishment and Maintenance of hRPE Cell Cultures

48.2.3 Cellular Proliferation

48.2.4 Immunoprecipitation Assay

48.2.5 Statistical Analysis

48.3 Results

48.3.1 Effect of Glucose on 14C-CTGF Synthesis in hRPE Cells

48.3.2 Effect of IGF-1 on 14C-CTGF Synthesis in hRPE cells

48.3.3 Effect of PD98059 on Glucose Stimulated 14C-CTGF Synthesis in hRPE Cells

48.3.4 Effect of PD98059 on IGF-1 Stimulated 14C-CTGF Synthesis in hRPE Cells

48.4 Discussion

References

49 PPAR Nuclear Receptors and Altered RPE Lipid Metabolism in Age-Related Macular Degeneration

49.1 Introduction

49.1.1 Current Hypotheses Surrounding Sub-RPE Deposit Formation

49.1.2 Long Chain Poly-Unsaturated Fatty Acids (LCPUFA) are Associated with ARMD Risk

49.1.3 Peroxisome Proliferator Activated Receptors (PPARs) are Expressed in ARPE19 Cells

49.2 LcPUFA Regulates Gene Expression in ARPE19 Cells

49.2.1 Purpose and Methods

49.2.2 Results

49.2.3 Discussion

References

50 The Pathophysiology of Cigarette Smoking and Age-Related Macular Degeneration

50.1 Introduction

50.2 Cigarette Smoking as a Risk Factor for AMD

50.2.1 AMD and Cigarette Smoke

50.2.2 Cigarette Smoke Constituents

50.3 Oxidative Stress

50.3.1 Oxidative Damage in AMD

50.3.2 Reactive Oxygen Species in Cigarette Smoke

50.3.3 Acrolein-Induced Oxidative Stress

50.3.4 Cadmium-Induced Oxidative Stress

50.4 Cigarette Smoke Depletion of Antioxidant Protection

50.4.1 Systemic Antioxidant Mechanisms

50.4.2 Local Ocular Antioxidants

50.5 Non-oxidative Chemical Damage by Cigarette Smoke

50.5.1 Nicotine

50.5.2 Polycyclic Aromatic Hydrocarbons

50.6 Inflammation

50.6.1 Inflammation and AMD

50.6.2 Cigarette Smoke and Complement Pathway

50.6.3 Cigarette Smoke and Other Inflammatory Mediators

50.7 Vascular Changes

50.8 Conclusions

References

51 Oxidative Stress and the Ubiquitin Proteolytic System in Age-Related Macular Degeneration

51.1 Oxidative Stress and Age-Related Macular Degeneration

51.2 The Ubiquitin Proteolytic System (UPS) and Oxidative Stress in the Retina

51.3 The UPS and the Cytoprotective Transcription Factor, Nrf2

References

52 Slit-Robo Signaling in Ocular Angiogenesis

52.1 Ocular Angiogenesis

52.2 Slit-Robo Signaling in Axon Guidance

52.3 Slit-Robo Signaling in Angiogenesis

52.4 Slit-Robo Signaling in Ocular Angiogenesis

52.5 Signaling Pathway of Slit-Robo System in Angiogenesis

52.6 Perspective

References

Part V Animal Models of Retinal Degeneration

53 Evaluation of Retinal Degeneration in P27KIP1 Null Mouse

53.1 Introduction

53.2 Materials and Methods

53.2.1 Animals and Biosafety

53.2.2 MNU-Induced Retinal Degeneration

53.2.3 Electroretinography

53.2.4 Histological Examination and Immunohistochemistry

53.3 Results

53.3.1 Fundus Examination and Histology of the Retina

53.3.2 ERG

53.3.3 BrdU Incorporation

53.3.4 Immunohistology of Nestin

53.4 Discussion

References

54 Differences in Photoreceptor Sensitivity to Oxygen Stress Between Long Evans and Sprague-Dawley Rats

54.1 Introduction

54.2 Methods

54.2.1 Animal Strains and Oxygen Exposure

54.2.2 Electroretinography

54.2.3 Immunohistochemistry and TUNEL Labeling

54.3 Results

54.3.1 Rod and Cone Components of the ERG after Hyperoxia

54.3.2 Impact of Hyperoxia on the Rate of Photo receptor Death

54.3.3 Impact of Hyperoxia on GFAP Expression

54.4 Discussion

References

55 Retinal Degeneration in a Rat Model of Smith-Lemli-Opitz Syndrome: Thinking Beyond Cholesterol Deficiency

55.1 Introduction

55.2 The AY9944 Rat Model of SLOS: Biochemical Findings

55.3 Retinal Degeneration in the SLOS Rat Model: Histology and Ultrastructure

55.4 Retinal Degeneration in the SLOS Rat Model: Electrophysiological Deficits

55.5 Effects of Feeding a High-Cholesterol Diet

55.6 Perspective: Thinking Beyond the Cholesterol Deficiency in SLOS

References

56 Do Calcium Channel Blockers Rescue Dying Photoreceptors in the Pde6brd1 Mouse?

56.1 Introduction

56.1.1 The Pde6brd1 Mouse and Increased [cGMP]

56.1.2 Calcium Regulation and Overload in the Photoreceptor Inner Segment

56.2 D-cis-diltiazem and Neuroprotection in the Retina

56.2.1 Criticism of the Frasson Study

56.2.2 Subsequent Evidence Shows L-Type Channels Are Involved in Degeneration

56.3 Other Players May Be Involved

References

57 Effect of PBNA on the NO Content and NOS Activityin Ischemia/Reperfusion Injury in the Rat Retina

57.1 Introduction

57.2 Materials and Methods

57.2.1 Animals and Reagents

57.2.2 Induction of Retinal I/R

57.2.3 Detection of MDA and NO Concentration, SOD and GSH-PX Activity

57.2.4 Statistical Analysis

57.3 Results

57.3.1 Effect of PBNA on Serum NO Content in Retinal I/R Injury

57.3.2 Effect of PBNA on T-NOS Activity in Retinal I/R Injury

57.3.3 Effect of PBNA on iNOS Activity in Retinal I/R Injury

57.3.4 Effect of PBNA on Serum eNOS Activity in Retinal I/R Injury

57.4 Discussion

References

58 Recent Insights into the Mechanisms Underlying Light-Dependent Retinal Degeneration from X. Laevis Models of Retinitis Pigmentosa

References

59 A Hypoplastic Retinal Lamination in the Purpurin Knock Down Embryo in Zebrafish

59.1 Introduction

59.2 Materials and Methods

59.2.1 Experimental Animals

59.2.2 Screening of Genomic DNA for Zebrafish Purpurin

59.2.3 Construction of the pur-GFP Reporter Vector

59.2.4 Morpholino and Microinjections

59.2.5 In Situ Hybridization

59.2.6 RNA Isolation, RT-PCR and mRNA Synthesis

59.3 Results

59.3.1 Isolation and Characterization of Zebrafish Purpurin Gene

59.3.2 Similar Phenotypes of Purpurin and Crx Morphant

59.4 Rescuing Effect of Purpurin mRNA to the Crx Morphant

59.5 Discussion

References

60 Functional Changes in Inner Retinal Neurons in Animal Models of Photoreceptor Degeneration

60.1 Introduction

60.2 Bipolar Cell Function in Retinal Degeneration

60.2.1 Glutamate Receptors of Bipolar Cells in the Normal and Degenerating Retina

60.2.2 Evidence for Bipolar Cell Dysfunction

60.2.2.1 Rod Bipolar Cells

60.2.2.2 Cone Bipolar Cells

60.3 Ganglion Cell Function in Retinal Degeneration

References

61 Photoreceptor Cell Degeneration in Abcr--/-- Mice

61.1 Introduction

61.2 Methods

61.2.1 Animals and Rearing

61.2.2 Measurement of Outer Nuclear Layer Thickness

61.2.3 Counting Photoreceptor Nuclei

61.3 Results

61.4 Discussion

References

62 Investigating the Mechanism of Disease in the RP10 Form of Retinitis Pigmentosa

62.1 Introduction

62.2 Retinitis Pigmentosa

62.3 RP10 Disease Caused by Mutations in IMPDH1

62.4 IMPDH Structure and Function

62.5 IMPDH Binds Single Stranded Nucleic Acids

62.6 Retinal Isoforms of IMPDH1

62.7 Kinetic and Nucleic Acid Binding Properties of Retinal IMPDH1

62.8 Conclusion

References

63 Congenital Stationary Night Blindness in Mice A Tale of Two Cacna1f Mutants

63.1 Introduction

63.2 Methods

63.3 Results

63.4 Discussion

63.5 Conclusion

References

64 Protection of Photoreceptors in a Mouse Model of RP10

64.1 Introduction

64.2 Results

64.2.1 Evaluation of Optimal IMPDH1 Suppressors

64.2.2 RP10 Mouse Model

64.2.3 Rescue of Photoreceptor Cells by rAAV-Mediated Downregulation of Mutant IMPDH1

64.3 Discussion

References

65 Correlation Between Tissue Docosahexaenoic Acid Levels and Susceptibility to Light-Induced Retinal Degeneration

65.1 Introduction

65.2 Methods

65.3 Results

65.4 Discussion

References

66 Activation of Mller Cells Occurs During Retinal Degeneration in RCS Rats

66.1 Introduction

66.2 Materials and Methods

66.2.1 Animal

66.2.2 Immunohistochemical Staining

66.2.3 Western Blot Test

66.2.4 Müller Cell Cultures

66.2.5 Data Analysis

66.3 Results

66.3.1 Morphology and Quantity Changes of Müller Cells

66.3.2 Expression of GFAP and ERK in RCS Rat Müller Cells

66.3.3 Effect of Mixed Retinal Cells of RCS Rats on Normal Müller Cells

66.4 Discussion

References

67 Effect of 3-Daidzein Sulfonic Sodium on theAnti-oxidation of Retinal Ischemia/Reperfusion Injury in Rats

67.1 Introduction

67.2 Materials and Methods

67.2.1 Animals and Reagents

67.2.2 Induction of RI/R

67.2.3 Detection of MDA and NO Concentration, SOD and GSH-PX Activity

67.2.4 Statistical Analysis

67.3 Results

67.3.1 The Effect of DSS on the Concentration of MDA in Serum After RI/R Injury

67.3.2 The Effect of DSS on the Activity of SOD in Serum After RI/R Injury

67.3.3 The Effect of DSS on the Activity of Serum GSH-PX After RI/R Injury

67.3.4 The Effect of DSS on the Concentration of Serum NO After RI/R Injury

67.4 Discussion

References

68 Structural and Functional Phenotyping in theCone-Specific Photoreceptor Function Loss 1 (cpfl1) Mouse Mutant -- A Model of Cone Dystrophies

68.1 Introduction

68.2 Materials and Methods

68.2.1 Animals

68.2.2 Functional Testing

68.2.3 In Vivo Imaging

68.3 Results

68.3.1 Function

68.3.2 Morphology

68.4 Discussion

References

69 The Differential Role of Jak/Stat Signaling in Retinal Degeneration

69.1 Introduction

69.2 Materials and Methods

69.2.1 Mice and Light Exposure

69.2.2 Semi-Quantitative Real Time Polymerase Chain Reaction (PCR)

69.3 Results

69.3.1 STATs Are Induced Differently in Retinas of Light-Exposed and rd1 Mice

69.3.2 Shp-1 Is Induced After Light Exposure But Not in the rd1 Mouse

69.3.3 Jak3 mRNA Is Induced Similarly in the Model of Light Induced Photoreceptor Cell Death and the rd1 Mouse Model

69.4 Discussion

References

Part VI Neuroprotection and Gene Therapy

70 Gene Therapy in the Retinal Degeneration Slow Model of Retinitis Pigmentosa

70.1 Introduction

70.2 Diseases Associated with RDS Mutations

70.3 Current Animal Models

70.4 Gene Therapy in rds Models

70.5 Viral Gene Therapy Approaches

70.6 Non-viral Approaches

References

71 PEDF Promotes Retinal Neurosphere Formation and Expansion In Vitro

71.1 Introduction

71.2 Materials and Methods

71.2.1 Retinal Stem Cell Isolation and Culture

71.2.2 Single Sphere Passaging

71.2.3 Bromodeoxyuridine Labeling

71.2.4 Retinal Stem Cell Differentiation

71.2.5 Immunofluorescence

71.3 Results

71.3.1 PEDF Promotes Retinal Neurospheres Growth and Self-Renewal

71.3.2 Retinal Neurosphere Proliferation

71.3.3 Differentiation of Retinal Cells Precursors from RSCs

71.4 Discussion

References

72 A Multi-Stage Color Model Revisited: Implications for a Gene Therapy Cure for Red-Green Colorblindness

72.1 Introduction

72.2 A Brief History of Color Vision Theory

72.3 Color Vision from an Evolutionary Perspective

72.4 Evolutionary Constraints Lead to an Extension of Devalois Model

72.5 The Possibility of Gene Therapy to Cure Red-Green Colorblindness

References

73 Achromatopsia as a Potential Candidate for Gene Therapy

73.1 Human Achromatopsia

73.1.1 Clinical Manifestations

73.1.2 Current Achromatopsia Treatments

73.2 Genetics of Human Achromatopsia

73.2.1 GNAT2 Achromatopsia

73.2.2 CNG Achromatopsia

73.2.3 Achromatopsia Gene Therapy

73.3 The Mutant Gnat2 Mouse and Gene Therapy

73.3.1 The Cnga3 Mutant Mouse and Gene Therapy

73.3.2 The Cngb3 Mutant Dog and Gene Therapy

73.4 Prospects for Achromatopsia Gene Therapy

References

74 Function and Mechanism of CNTF/LIF Signalingin Retinogenesis

74.1 Introduction

74.2 Effects of CNTF/LIF on Photoreceptor and Bipolar Neuron Differentiation

74.3 Effects of CNTF/LIF on Muller Glia Genesis and Late Progenitor Proliferation

74.4 Effects of LIF Misexpression on Retinal Vasculature Development

74.5 Expression of CNTF/LIF Signaling Components in the Developing Retina

74.6 Signaling Events Triggered by CNTF/LIF During Retinogenesis

74.7 CNTF/LIF Regulate Numerous Genes Involved in Retinogenesis

74.8 Perspective

References

75 gp130 Activation in Muller Cells is Not Essentialfor Photoreceptor Protection from Light Damage

75.1 Introduction

75.2 Conditional gp130 Knockout in the Retinal Mller Cells

75.3 Effect of Impaired gp130 Activation in Mller Cells on LIF-Induced Photoreceptor Protection

75.4 Discussion

References

76 Neuroprotectin D1 Modulates the Induction of Pro-Inflammatory Signaling and Promotes Retinal Pigment Epithelial Cell Survival During Oxidative Stress

76.1 The Importance of RPE Cell Function and Integrity for Photoreceptor Survival

76.2 The Loss of RPE Cells in Retinal Degeneration

76.3 DHA and NPD1 Properties and Neuroprotection

76.4 NPD1 Modulates the Expression of Survival and Apoptotic-Related Proteins

References

77 Adeno-Associated Virus Serotype-9 Mediated Retinal Outer Plexiform Layer Transduction is Mainly Through the Photoreceptors

77.1 Introduction

77.2 AAV9-Mediated Gene Transfer in the Retina

77.3 The Sub-Cellular Location of AAV9 Transduction in the OPL

77.4 AAV9-Mediated Retinal Gene Transfer in mdx 3cv Mice

77.5 Subretinal Injection of AAV9 Vector Did Not Cause Acute Retinal Damage

77.6 Conclusions

References

Index