Physiology of the Retina
PHYSIOLOGY OF THE RETINA
András M. Komáromy Michigan State University [email protected]
12th Biannual William Magrane Basic Science Course in Veterinary and Comparative Ophthalmology PHYSIOLOGY OF THE RETINA
• INTRODUCTION
• PHOTORECEPTORS • OTHER RETINAL NEURONS • NON-NEURONAL RETINAL CELLS • RETINAL BLOOD FLOW Retina
©Webvision Retina
Retinal pigment epithelium (RPE)
Photoreceptor segments
Outer limiting membrane (OLM)
Outer nuclear layer (ONL) Outer plexiform layer (OPL)
Inner nuclear layer (INL)
Inner plexiform layer (IPL)
Ganglion cell layer
Nerve fiber layer
Inner limiting membrane (ILM)
©Webvision Inherited Retinal Degenerations
• Retinitis pigmentosa (RP) – Approx. 1 in 3,500 people affected
• Age-related macular degeneration (AMD) – 15 Mio people affected in U.S.
www.nei.nih.gov Mutations Causing Retinal Disease
http://www.sph.uth.tmc.edu/Retnet/
Retina Optical Coherence Tomography (OCT)
Histology
Monkey (Macaca fascicularis) fovea
Ultrahigh-resolution OCT Drexler & Fujimoto 2008 9 Adaptive Optics
Roorda & Williams 1999 6 Types of Retinal Neurons
• Photoreceptor cells (rods, cones) • Horizontal cells • Bipolar cells • Amacrine cells • Interplexiform cells • Ganglion cells Signal Transmission
1st order SPECIES DIFFERENCES!! Photoreceptors
Horizontal cells 2nd order Bipolar cells
Amacrine cells 3rd order Retinal ganglion cells Visual Pathway
lgn, lateral geniculate nucleus Changes in Membrane Potential
Net positive charge out Net positive charge in PHYSIOLOGY OF THE RETINA
• INTRODUCTION
• PHOTORECEPTORS • OTHER RETINAL NEURONS • NON-NEURONAL RETINAL CELLS • RETINAL BLOOD FLOW Photoreceptors
Retinal pigment epithelium (RPE)
Photoreceptor segments
Outer limiting membrane (OLM)
Outer nuclear layer (ONL) Outer plexiform layer (OPL)
Inner nuclear layer (INL)
Inner plexiform layer (IPL)
Ganglion cell layer
Nerve fiber layer
Inner limiting membrane (ILM)
©Webvision Photoreceptor Cells
• Rods – Vision in dim light
• Cones – Vision in bright light – Color vision – Central visual acuity ©webvision Photoreceptor Cells
Dowling 1998
cones discs are open to extracellular space (rapid flux of substances) cell body OS = outer segment IS = inner segment Photoreceptors
• Topographic variations in numbers and rod/cone ratios in different species • Spatial distribution of cones correlated to visual acuity • Domestic animals do not have a fovea, but an area centralis, area of high cone density • Visual streak is region with greatest concentration of RGC Densities of rods and cones in the human retina
Sternberg 1935
Gradient central peripheral
Peak cone densities: 64,000-212,000 cells/m2
•22 Rod density: 200,000-540,000/mm2 Peak density matched spatially with thickest part of tapetum Dorsal to visual streak.
23 PHYSIOLOGY OF THE RETINA
• PHOTORECEPTOR CELLS – PHOTOTRANSDUCTION Phototransduction
Yau & Hardie 2009 Rod Phototransduction
• Light: 11-cis-retinal all-trans-retinal • Dissociation of rhodopsin opsin and all-trans-retinal
• Activation of alpha transducin (Gtα)
• Gtα activates cGMP-PDE • cGMP-PDE hydrolyzes cGMP, which closes the OS plasma membrane channel Rod Phototransduction
• Rod stimuli by light leads to hyperpolarization, which means an increase in intrinsic negative charge
• Darkness is the actual stimulus that gives rise to a depolarization and transmitter release. Upon stimuli this continuous signal is interrupted. Phototransduction
Darkness: channel open Light: channel closed
Arshavsky et al. 2002 PHYSIOLOGY OF THE RETINA
• PHOTORECEPTOR CELLS – PHOTOTRANSDUCTION • VISUAL PIGMENT Phototransduction
Yau & Hardie 2009 Visual Pigments
• Holopigment – Opsin = protein moiety – 11-cis retinal = chromophore • Covalently linked by Schiff base
©Webvision Retinoid Structures
Picaud 2003
©Webvision
Light Rhodopsin Mutations
• Over 100 different mutations identified P = proline • Most common mutation: P23H H = histidine • Majority are gain-of-function mutations • Autosomal dominant
Arshavsky et al. 2002 Autosomal Dominant PRA
Kijas, James W. et al. (2002) Proc. Natl. Acad. Sci. USA 99, 6328-6333 Fig. 1. Consequences of clinical human retinal photography in Modest light levels, as used the RHO mutant dog in routine clinical practice, dramatically accelerate the neurodegeneration!!!
Cideciyan, Artur V. et al. (2005) Proc. Natl. Acad. Sci. USA 102, 5233-5238 A-D: standard microscope E-F: night-vision system • Severe light-induced retinal damage • Visual performance preserved • Islands of surviving photoreceptors 37 PHYSIOLOGY OF THE RETINA
• PHOTORECEPTOR CELLS – PHOTOTRANSDUCTION • VISUAL PIGMENT • COLOR VISION Different types of cone photoreceptor
©Webvision Some Specificities of Color Vision
• Human color vision depends on 3 types of cones which differ from each other in their absorption spectra • In highly diurnal animals (turtles and goldfish) there are 4 classes of cones • In less diurnal animals (dogs, cats, squirrels) there are only 2 classes of cones Canine Retina Different types of cone photoreceptor
Goldsmith 2013
©Webvision Photopigments in the Retina
Rod photoreceptors: Rhodopsin ~506-510nm (dog) Rhodopsin ©webvision Photopigments in the Retina
Rod photoreceptors: Rhodopsin ~506-510nm (dog) Red/Green cone photoreceptors: L/M-opsin ~555nm (dog) -opsin L/M ©webvision Photopigments in the Retina
Rod photoreceptors: Rhodopsin ~506-510nm (dog) Red/Green cone photoreceptors: L/M-opsin ~555nm (dog) Blue cone photoreceptors: S-opsin ~429-435nm (dog) S-opsin ©webvision Cone Photopigments – Spectral Tuning
Jacobs 2008 Cone Photopigments – Spectral Tuning
X chromosome
Neitz & Neitz 2011 Dichromatic vs. Trichromatic
Jacobs & Nathans 2009 How Primate Trichromacy Evolved
Jacobs & Nathans 2009 2 Designs for Primate Color Vision Nathans 2009 & Jacobs Old World Primates
Jacobs & Nathans 2009 New World Primates
Jacobs & Nathans 2009 https://www.youtube.com/watch?v=Ya8c6VdPwng
http://www.neitzvision.com/ PHYSIOLOGY OF THE RETINA
• PHOTORECEPTOR CELLS – PHOTOTRANSDUCTION • VISUAL PIGMENT • COLOR VISION • RETINOIDS AND THE VISUAL CYCLE Phototransduction
Yau & Hardie 2009 Retinoids and the Visual Cycle
Light 11-cis-retinal all-trans-retinal Dissociation of all-trans-retinal and opsin All-trans-retinal all-trans-retinol exits cell Binding to interphotoreceptor retinoid- binding protein (IRBP) Transport to RPE Yau & Hardie 2009
R-ester, retinyl ester RDH, retinol dehydrogenase REH, retinyl ester hydrolase LRAT, lecithin:retinol acyl tranferase
CRBP, cellular retinol-binding protein CRALBP, cellular retinaldehyde-binding protein
IRBP, interphotoreceptor retinoid binding protein
hv, photon RAL, retinal ROL, retinol R-ester, retinyl ester Photopigment Regeneration
• All-trans-retinal is reduced to all-trans-retinol
• All-trans-retinol leaves photoreceptor, traverses IPM, finds IRBP, and enters RPE
• Esterified by LRAT (lecithin:retinol acyl tranferase)
• Ester is converted to 11-cis-retinol by retinyl isomerohydrolase = RPE65
• 11-cis-retinol ester is stored or oxidized by 11-RDH, and 11-cis-retinal passes into the receptor and binds with opsin to regenerate rhodopsin
IPM, interphotoreceptor matrix Visual Pigments
Because chromophore is highly hydrophobic
Intracellular carrier proteins: CRBP and CRALBP involved in shuttling
Extracellular carrier protein: IRBP involved in shuttling
CRBP, cellular retinol-binding protein CRALBP, cellular retinaldehyde-binding protein IRBP, interphotoreceptor retinoid binding protein Substrate for Retinoids
• Bleached pigment is one of the substrates for 11-cis-retinaldehyde synthesis in RPE • Dietary uptake is main source – Vitamin A (retinol) is formed in intestine from carotenoid compounds in food – Esterified and transported to liver, bound to retinol binding protein (RBP), further transported to the eye – Specific receptors cause retinol to be taken up by the RPE cell, where it is bound to CRBP R-ester, retinyl ester RDH, retinol dehydrogenase REH, retinyl ester hydrolase LRAT, lecithin:retinol acyl tranferase
CRBP, cellular retinol-binding protein CRALBP, cellular retinaldehyde-binding protein
IRBP, interphotoreceptor retinoid binding protein
hv, photon RAL, retinal ROL, retinol R-ester, retinyl ester
Wang & Kefalov 2011 61 Pigment Cycle
• Pigment regeneration in RPE – Both rods and cones • Dedicated regeneration pathway for cones – Necessary because rod opsin outcompetes cone opsin in acquiring chromophore; acts as huge sink for for 11-cis-retinal – Müller cells • All-trans-retinol 11-cis-retinol returned to cones – Cones • 11-cis-retinol 11-cis-retinal Yau & Hardie 2009
R-ester, retinyl ester RDH, retinol dehydrogenase REH, retinyl ester hydrolase ARAT, acyl-CoA:retinol acyl tranferase
CRBP, cellular retinol-binding protein CRALBP, cellular retinaldehyde-binding protein
IRBP, interphotoreceptor retinoid binding protein
hv, photon RAL, retinal ROL, retinol R-ester, retinyl ester R-ester, retinyl ester RDH, retinol dehydrogenase REH, retinyl ester hydrolase LRAT, lecithin:retinol acyl tranferase
CRBP, cellular retinol-binding protein CRALBP, cellular retinaldehyde-binding protein
IRBP, interphotoreceptor retinoid binding protein
hv, photon RAL, retinal ROL, retinol R-ester, retinyl ester
Wang & Kefalov 2011 64 Palczewski 2012 65 Leber Congenital Amaurosis (LCA)
• Theodor Leber (1869) • Syndrome in children characterized by severe visual deficiency, with total or nearly total blindness, present at birth or shortly thereafter; pendular or searching nystagmus, with slowly progressive retinal atrophy. Inheritance is autosomal recessive and disease is heterogeneous • Mutations in at least 20 different genes cause LCA • Mutations in the RPE65 gene cause disease in 10-15% of LCA patients • Mutations in RPE65, expressed in pigment epithelium, impair retinoid cycle Leber Congenital Amaurosis (LCA) Genes den Hollander et al. 2008 RPE65-LCA
• Briard Canine Leber Congenital Amaurosis • Autosomal recessive • Mutation: 4 nucleotide deletion in exon 5 of RPE65 • Complete nightblindness, and very severe defects in day vision. Total blindness common. • Fundus normal until 5-6 years or longer • ERG - low amplitude to absent rod and cone responses; waveform altered • Visual cells initially normal or minimally diseased; very slow cell loss • Lipoidal inclusions in the RPE
Aguirre et al. 1998 Gene Therapy Strategies for Inherited Retinal Diseases
May 2016 Subretinal Injection
Cell Transplantation 2006 Subretinal Injection
Courtesy Dr. Simon Petersen-Jones Leber Congenital Amaurosis (LCA)
Nature Genetics 2001 May;28(1):92-5.
73 Dark adapted ERG
ERG photoresponse
29 Hz cone flicker
Pupillometry
Acland et al. 2001 RPE65-LCA
Untreated Treated
75
Nature Genetics 2001
Molecular Therapy 2005
Lancelot !! Lancelot
81 Ophthalmology 2016
82 Leber Congenital Amaurosis (LCA) Genes den Hollander et al. 2008 autosomal dominant rod–cone dysplasia (Rdy) Abyssinian and Somali cats Crx = cone–rod homeobox protein
single base pair deletion in exon 4 (n.546delC)
truncated the putative CRX peptide •84 Rod-cone dysplasia 2 (rcd2) in Collies akc.org RD3 is implicated in the stable expression and trafficking of guanylate cyclase (GC) in photoreceptors
85 Yau & Hardie 2009 PHYSIOLOGY OF THE RETINA
• PHOTORECEPTOR CELLS – PHOTOTRANSDUCTION • VISUAL PIGMENT • COLOR VISION • RETINOIDS AND THE VISUAL CYCLE • TRANSDUCIN Phototransduction
Yau & Hardie 2009 Transducin (Gt)
• Rh* activates Gt
• GDP-GTP exchange on α-subunit (Gtα)
• Gtα*-GTP
– dissociates from βγ subunits (Gtβγ)
– Binds to PDE γ subunit (PDEγ)
– Removes inhibition of PDEγ on catalytic PDEαβ subunits cone transducin β subunit guanine nucleotide-binding protein (GNB3) GNB3 expressed in - Cones - ON bipolar cells
primate retina (Macaca fascicularis) PHYSIOLOGY OF THE RETINA
• PHOTORECEPTOR CELLS – PHOTOTRANSDUCTION • VISUAL PIGMENT • COLOR VISION • RETINOIDS AND THE VISUAL CYCLE • TRANSDUCIN • PHOSPHODIESTERASE Phototransduction
Yau & Hardie 2009 Phosphodiesterase (PDE)
• Each PDE complex
– 1 PDEαβ
– 2 PDEγ
• PDEγ removed from catalytic PDEαβ • Hydrolyzes cGMP Rod-cone dystrophy 1 (rcd1)
Gene: PDE6B nonsense mutation in codon 807
© akc Cone-rod dystrophy 1 (crd1) American Staffordshire Terrier 3 bases deletion in exon 21 of PDE6B
© akc
Slower progression than rcd1
11 weeks 20 months 95 Rod-cone dystrophy 3 (rcd3)
© akc Phototransduction
X
X ↑↑↑cGMP
Arshavsky et al. 2002 98 PHYSIOLOGY OF THE RETINA
• ENCAPSULATED CELL-BASED DELIVERY OF CILIARY NEUROTROPHIC FACTOR (CNTF) Gene Therapy Strategies for Inherited Retinal Diseases
Neurotrophic factor
May 2016 Trophic Factors
• Where nature of retinal degeneration has not been discerned, it might be possible to delay photoreceptor cell death by delivering anti-apoptotic regulators or growth/neurotrophic factors • Generalized treatment for retinal disease Trophic Factors
• The delivery of trophic factors might be the most promising type of therapy for patients with inherited retinal degeneration, because the same treatment principle can be applied for several diseases! Tao et al. 2002
Encapsulated cell implants consist of living cells encapsulated within semipermeable polymer membranes and supportive matrices. Release CNTF.
Neurotech USA, Inc., Lincoln, RI © Neurotech Treatment of Dogs with PDE6B Mutation (rcd1)
Tao et al. IOVS. 2002
Treatment from 7 to 14 weeks of age. ENCAPSULATED CELL-BASED DELIVERY OF CNTF
CNTF capsule in CNGB3-mutant dog Tao et al. 2002 106 Sieving, Paul A. et al. (2006) Proc. Natl. Acad. Sci. USA 103, 3896-3901 No significant decrease in cone density in the CNTF-treated eyes• 108!!! 109 Gene Therapy Strategies for Inherited Retinal Diseases
Optogenetics
May 2016 Virus-mediated expression of light sensor (light-gated ionotropic glutamate receptor) in surviving retinal cells (retinal ganglion cells) following intravitreal injection of AAV2/2(4YF)-CAG-LiGluR
Rod-cone dystrophy 1 (rcd1)
111 © akc PHYSIOLOGY OF THE RETINA
• PHOTORECEPTOR CELLS – PHOTOTRANSDUCTION • VISUAL PIGMENT • COLOR VISION • RETINOIDS AND THE VISUAL CYCLE • TRANSDUCIN • PHOSPHODIESTERASE • CYCLIC NUCLEOTIDE-GATED CHANNELS Phototransduction
Yau & Hardie 2009 Cyclic Nucleotide-Gated Channels CNGA1 CNGA3 • Heterotetramers CNGB1 CNGB3 – Alpha subunit • Can form functional channels on their own • “principal” subunit – Beta subunit • Unable to form functional channels • “modulatory” subunits
• cGMP-binding site • Nonselective to cations
Kaupp, U. B. et al. Physiol. Rev. 82: 769-824 2002 Alpha Subunit: CNGA3
Wissinger et al. 2001 Image: dogtime.com Image:
116 akc.org
dog.com
117 akc.org
ARVO Conference 2016 Seattle, WA Labrador Retriever dogtime.com
dogtime.com
German Shepherd
119 Achromatopsia
• No cone photoreceptor function • Rod monochromacy
• Clinical signs: – Total color blindness ©webvision – Day-blindness – Photophobia – Low visual acuity – Nystagmus Achromatopsia in Man
• Autosomal recessive • ACHM1: HSA14 – ACHM2: HSA2q11 (CNGA3; ~27%) – ACHM3: HSA8q21-q22 (CNGB3; ~50%) – ACHM4: HSA1p13.3 (GNAT2; <2%) – PDE6C (alpha) (HSA10q24; <2%) – PDE6H (gamma) (HSA12p13) Phototransduction
transducin
cyclic nucleotide- gated channels
Arshavsky et al. 2002 Labrador Retriever dogtime.com
dogtime.com
German Shepherd
123 Awassi sheep Molecular Therapy 2015 Canine Models of CNGB3-Achromatopsia
• Autosomal recessive mutations of CNGB3: – Genomic deletion (CNGB3-/-) – D262N missense mutation exon 6 (CNGB3m/m) • Alaskan Malamute • Miniature Australian Shepherd • Siberian Husky • Alaskan sled dog ©akc •127 PHYSIOLOGY OF THE RETINA
• CONE-DIRECTED GENE THERAPY FOR ACHROMATOPSIA (cone degeneration, cd) Obstacle Course
Garcia et al., 2010 Electroretinogram (ERG) The Fate of Cones in CNGB3-Achromatopsia
Despite early and complete loss of function…
…morphologic development of cones is normal…
…and cone loss is slow!
• Rowlan, et al. 2009: – 22 – 24% of L/M- and S-cones are lost by 1 year of age
Human cone arrestin (hCAR) antibody kindly provided by Dr. Cheryl Craft (University of Southern California) Gene Therapy 2008
-4564 to -3009 -496 to 0 -496 to 0 -496 to 0 3 x 37 bp LCR at -496 end
GFP GFP L/M-Opsin L/M-Opsin wildtype CNGB3 null mutant 2.1 kb human red-cone opsin promoter provided by Dr. J. Nathans (Johns Hopkins University) Electroretinography – Short Term
Komáromy et al., 2010 Electroretinography – Long Term
Komáromy et al., 2010 Obstacle Course 136 PHYSIOLOGY OF THE RETINA
• PHOTORECEPTOR CELLS – PHOTOTRANSDUCTION • VISUAL PIGMENT • COLOR VISION • RETINOIDS AND THE VISUAL CYCLE • TRANSDUCIN • PHOSPHODIESTERASE • CYCLIC NUCLEOTIDE-GATED CHANNELS • DEACTIVATION Deactivation
Yau & Hardie 2009 G Protein-Coupled-Receptor Kinase (GRK)
• Rh* phosphorylated by GRK1 Rh*~P – Rods: GRK1 – Cones: GRK 1 and GRK7
GRK, G protein-coupled-receptor-kinase Arrestin (Arr)
Recognizes and binds Rh*~P Rh*~P-Arr All activity lost
Eventually Rh looses arrestin and is dephosphorylated
Rod and cone version of arrestin Deactivation
Yau & Hardie 2009 Deactivation
• Active Gtα*.GTP inactive Gtα.GDP
• Gtα.GDP dissociates from PDEγ and reassociates with Gtβγ
• PDEγ resumes inhibition of PDEαβ • Guanylate cyclase (GC) maintains cGMP concentration – Rods: retGC-1 and retGC-2 • Guanylate cyclase-activating proteins GCAP1 and GCAP2 – Cones: retGC-1 • GCAP1 Deactivation
Yau & Hardie 2009 Ca2+ Feedback
Decrease in Ca2+ concentration leads to…
…increased cGMP synthesis (GCAP GC)
…decreased hydrolysis of cGMP (PDE)
…increased Rh phosphorylation (GRK) via recoverin or S-modulin
…increased cGMP affinity of channel via Ca2+-calmodulin
LIGHT ADAPTATION Light Adaptation
• Is a time-dependent process in which light leads to a desensitization
• The system recovers responsiveness but requires light of higher intensity to function
• Is accomplished in the brain, inner retina, and in photoreceptors Dark Current
• Darkness: – Channels in the OS are open, and allow the passage of mainly Na ions – Inward current keeps the cell depolarized – Membrane of IS has channels that selectively allows for passage of K ions – This results in a circulating dark current • Light – Channels closed in OS, thereby reducing current and causing hyperpolarization of the photoreceptor Cones (compared to rods)
• Lower sensitivity ≈25 – 100x
– lower rate of Gt activation – GRK more abundant and with higher activity • More effective light adaptation – faster Ca2+-feedback – larger surface:volume ratio • Faster response kinetics higher temporal resolution • Different protein isoforms • Chromophore regeneration through RPE and Müller cells PHYSIOLOGY OF THE RETINA
• PHOTORECEPTOR CELLS – PHOTOTRANSDUCTION • VISUAL PIGMENT • COLOR VISION • RETINOIDS AND THE VISUAL CYCLE • TRANSDUCIN • PHOSPHODIESTERASE • CYCLIC NUCLEOTIDE-GATED CHANNELS • DEACTIVATION • PHOTORECEPTOR OUTER SEGMENT IS A CILIUM Photoreceptor Outer Segment Renewal
• Adult photoreceptors continuously replace the photosensitive disc structure of the outer segment – disc addition – disc shedding
©Webvision Membrane Renewal: Disc Addition
• New disc membranes are synthesized and assembled at the base of the OS by an evagination of newly formed plasma membrane Photoreceptor Outer Segment Renewal Segment Outer Photoreceptor Rodieck 1998; Young 1970 disc shedding disc Photoreceptor Outer Segment Renewal
disc shedding
©Webvision Membrane Renewal: Disc Shedding
• 0.5 – 2.0 µm long packets of disc membranes are eliminated daily from the tip of the OS • Rod segments are shed within 1 hour after light onset, those of cones vary among species • Shed membranes are enclosed in RPE apical processes and become primary phagosomes • Phagosomes fuse with lysosomes and become phagolysosomes which are degraded Royal College of Surgeons (RCS) rat
• Classical model of retinal degeneration – Form of Leber congenital amaurosis • RPE fails to phagocytose shed outer segments • Mutation of MERTK (c-mer proto-oncogene tyrosine kinase) – Loss of functional MERTK protein leads to ineffective phagocytosis and clearance of apoptotic cells, with resultant buildup of the retinal pigmented epithelium leading to visual loss 155 Leber Congenital Amaurosis (LCA) Genes den Hollander et al. 2008 The Retinal Ciliopathies
• Cilia and Flagella – Central bundle of microtubules, called the axoneme, in which nine outer doublet microtubules surround a central pair of singlet microtubules The Retinal Ciliopathies
• The photoreceptor outer segment is a specialized primary, non-motile cilium
Pearring et al. 2013 The Retinal Ciliopathies
• Phototransduction proteins and disc membrane lipids must be synthesized in the inner segment and then transported to the outer segment
Goldstein L S B PNAS 2001;98:6999-7003 The Retinal Ciliopathies
• With the constant turnover of rod outer segments, delivering cargo to the outer segments is essential for maintenance of the outer segments. – For example, ~2000 opsin molecules per minute
Goldstein L S B PNAS 2001;98:6999-7003 The Retinal Ciliopathies
• Selected examples of mutated genes: – RPGR (Retinitis Pigmentosa GTPase Regulator) – RPGRIP1 (RP GTPase Regulator interacting protein 1) – CEP290 (centrosomal protein with a molecular weight of 290 kDa) – Nephronophthisis genes The Retinal Ciliopathies RPGR • Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene are associated with RP that is often transmitted in X-linked recessive manner
• X-linked RP is believed to be among the most severe forms of RP with early onset of night blindness and severe decreased vision to near total blindness by the third or fourth decade of life. X-linked progressive retinal atrophy
Beltran et al. PNAS 2012
XLPRA1 - Siberian Husky - Samoyed XLPRA2 - Mongrel dog
© akc Younger = 7-28 wks; older = 36-76 wks The Retinal Ciliopathies RPGR • The exact function of RPGR is not fully understood – Interaction with RPGRIP – Disc morphogenesis – Intracellular trafficking, transport of opsins IS OS – Nucleocytoplasmic transport – Other nuclear activities • RPGR interacting protein (RPGRIP) is required for disc morphogenesis – Anchor the RPGR protein within the photoreceptor connecting cilium
PNAS 2015
Early = 5 wks Mid stage = 12 wks Late stage = 26 wks
166 The Retinal Ciliopathies RPGRIP • Mutations in the retinitis pigmentosa GTPase regulator interacting protein (RPGRIP) are associated with Leber congenital amaurosis (LCA) and possibly with cone-rod dystrophy
• RPGRIP is believed to anchor RPGR to the photoreceptor ciliary axoneme and to the centrioles
• Loss of RPGRIP leads to disorganization of the outer segments with mislocalization of opsins in the cell bodies Cone-rod dystrophy 1 (cord1/crd4) • Miniature Long-haired Dachshunds • English Springer Spaniels
© akc Considerable variation in severity of disease! Cone-rod dystrophy 1 (cord1/crd4) • In Miniature Long-haired Dachshunds the “typical” age of disease-onset has been reported to be around 2 years
• However, some affected dogs do not develop the clinical signs until after 10 years of age, or they may not develop the disease during their lifetimes. 171 Only 4/6 homozygous animals showed signs of disease! 172 Cone-rod dystrophy 1 (cord1/crd4) • Affected dogs are homozygous for mutations in the gene RPGRIP1
• However, there are other, still unknown factors or modifiers that seem to determine the age of onset and the rate of progression of the clinical symptoms 174 MAP9EORD: fusion of 2 genes due to deletion mutation microtubule-associated protein, early onset retinal degeneration MAP9 partial deficit RPGRIP1 partial deficit More serious disease 175 •176 The Retinal Ciliopathies Nephronophthisis • Nephronophthisis (NPHP) – Group of autosomal recessive cystic kidney diseases – Most frequent genetic cause for end-stage renal failure in children and young adults – With or without retinopathy and other associated conditions – Human NPHP • NPHP1 • INVS (NPHP2) • NPHP3 • NPHP4 • IQCB1 (NPHP5) • CEP290 (NPHP6) The Retinal Ciliopathies Nephronophthisis • NPHP4 (nephronophthisis 4, nephroretinin) – Mutation in standard wire-haired dachshund (180-bp deletion) lacks the domain interacting with RPGRIP1 – Cone -rod dystrophy in standard wire-haired dachshund – In humans; Mutations in NPHP4 have been associated with simultaneous eye and kidney disease – No kidney disease in dachshund http://www.allsmalldogbreeds.com/ Cone-rod dystrophy 2 (crd2) American Pit Bull Terrier cytosine insertion in exon 10 of IQCB1
(NPHP5) © akc
3 weeks 12 weeks 20 months 179 Cone-rod dystrophy 2 (crd2) American Pit Bull Terrier cytosine insertion in exon 10 of IQCB1
(NPHP5) © akc
ARVO Conference, 2016 Seattle, WA
180 Narfström et al. 2011 In addition to Abyssinian: Somali, Ocicat, American Curl, American Wirehair, Bengal, Balinese/Javanese, Colorpoint Shorthair, Cornish Rex, Munchkin, Oriental Shorthair, Peterbald, Siamese (about 33%), Singapura and Tonkinese.
Narfström et al. 2011 LCA Genes
den Hollander et al. 2008 PRA3: late onset PRA
dogtime.com dogtime.com
FAM161A (family with sequence similarity 161, member A) - Ciliary gene: Basal body, connecting cilium and centriole, and associated with microtubule network during mitosis
184 • Not PRCD or Golden Retriever PRA1 • TTC8 (tetratricopeptide repeat domain 8): • Connecting cilium, centrosomes, basal bodies
dogtime.com
185 Light-Dependent Translocations of Phototransduction Proteins
• Vertebrate rods:
– Massive translocation of Gt from OS to elsewhere in the cell under light-adapted conditions, returning in darkness. – Arrestin moves in the opposite direction • Suggestion: Translocations provide long- term light adaptation Light-Dependent Translocations of Phototransduction Proteins Pearring et al. 2013
Trojan et al. 2008 PHYSIOLOGY OF THE RETINA
• PHOTORECEPTOR CELLS • SIGNAL TRANSMISSION Signal Transmission
Photoreceptors
Horizontal cells
Bipolar cells
Amacrine cells
Retinal ganglion cells Outer Plexiform Layer
Retinal pigment epithelium (RPE)
Photoreceptor segments
Outer limiting membrane (OLM)
Outer nuclear layer (ONL) Outer plexiform layer (OPL)
Inner nuclear layer (INL)
Inner plexiform layer (IPL)
Ganglion cell layer
Nerve fiber layer
Inner limiting membrane (ILM)
©Webvision Photoreceptors synapses
• PR synapse with bipolar and horizontal cells in the OPL • Synaptic endings of rods = spherules – Pear-shaped with numerous synaptic invaginations and enclose the tips of 3 processes • Synaptic endings of cones = pedicles – Larger than those of rods, pyramidal shape with short peripheral digitations = basal processes • Synaptic ridge on spherule and pedicle, with ribbon, surrounded by synaptic vesicles Ultrastructure of rod and cone synaptic endings
Rod spherule Cone pedicle Rod triad Cone triad
©Webvision Electrophysiological Features of Rods and Cones • Signals induced in rods spread to other rods
• There is electrical coupling of rods by low resistance contacts, i.e. gap junctions
• Coupling of rods result in spatial averaging
• Many rods converge at the synapses of bipolar and horizontal cells Electrophysiological Features of Rods and Cones
• Cones are also electrically coupled: only those that bear the same photopigment are coupled • There are also gap junctions between cones and between rods and cones • The graded hyperpolarization of the rod and cone OS spreads to the synaptic terminal. The release of transmitter, stored in vesicles, is controlled by the membrane potential Electrophysiological Features of Rods and Cones
• Rods and cones are depolarized in the dark and hyperpolarized in the light • This causes a release of transmitter in the dark and light causes a fall in this release • The sharing of potentials of rods that have not themselves absorbed quanta will lead to a change also in regional rods and a large postsynaptic response. This makes the system optimized for transmission of dim light signals. PHYSIOLOGY OF THE RETINA
• PHOTORECEPTOR CELLS • SIGNAL TRANSMISSION • BIPOLAR CELLS Inner Nuclear Layer
Retinal pigment epithelium (RPE)
Photoreceptor segments
Outer limiting membrane (OLM)
Outer nuclear layer (ONL) Outer plexiform layer (OPL)
Inner nuclear layer (INL)
Inner plexiform layer (IPL)
Ganglion cell layer
Nerve fiber layer
Inner limiting membrane (ILM)
©Webvision Bipolar Cells
• Cell nuclei in INL with dendrites projecting into the OPL with synaptic input from photoreceptors • Axons synapse in IPL with dendrites from amacrine cells and RGC • Major radially conducting element of retina, transmit signals directly or indirectly from photoreceptors to RGC • 8 types of cone bipolars, 1 type of rod bipolar Bipolar Cell Types
©Webvision ON- AND OFF-CHANNELS
• 2 separate neural mechanisms in retina mediate the sensations of brightness and darkness, the ON- and OFF-channels
• These channels begin at the bipolars and have corresponding system of RGC
• They remain independent all the way to the cortex
• Separate but complementary channels ON- AND OFF-CHANNELS
Depolarizing bipolar cells = ON bipolars Rods and cones Hyperpolarizing bipolar cells = OFF bipolars Cones only
Daw, Jensen, Brunken 1990 PHYSIOLOGY OF THE RETINA
• PHOTORECEPTOR CELLS • SIGNAL TRANSMISSION • BIPOLAR CELLS – GNB3 MUTATION IN CHICKEN – CONGENITAL STATIONARY NIGHT BLINDNESS (CSNB) IN HORSES – CANCER/MELANOMA ASSOCIATED RETINOPATHY cone transducin β subunit guanine nucleotide-binding protein (GNB3) GNB3 expressed in - Cones - ON bipolar cells
primate retina (Macaca fascicularis) PHYSIOLOGY OF THE RETINA
• PHOTORECEPTOR CELLS • SIGNAL TRANSMISSION • BIPOLAR CELLS – GNB3 MUTATION IN CHICKEN – CONGENITAL STATIONARY NIGHT BLINDNESS (CSNB) IN HORSES – CANCER/MELANOMA ASSOCIATED RETINOPATHY Congenital Stationary Night Blindness (CSNB)
• Group of genetically and clinically heterogeneous retinal disorders • Disruption of the ON bipolar cell response • Severely reduced rod b-wave amplitude and slightly altered cone responses – Electronegative waveform of the dark-adapted, bright-flash ERG, in which the amplitude of the b- wave is smaller than that of the a-wave (Schubert- Bornschein type) CSNB affected normal Transient Receptor Potential Cation Channel, Subfamily M, Member 1
208 • spherical melanosomes in wild type lp/lp melanocytes, but irregularly shaped melanosomes in LP/LP melanocytes
• Differential gene expression between LP/LP and lp/lp cells • lower peak [Ca2+] uptake in LP/LP melanocytes compared to lp/lp cells, consistent with lack of TRPM1 functioning in these cells 209 ON-bipolar cells cannot depolarize and no b-wave appears 210 dogtime.com
211 PHYSIOLOGY OF THE RETINA
• PHOTORECEPTOR CELLS • SIGNAL TRANSMISSION • BIPOLAR CELLS – GNB3 MUTATION IN CHICKEN – CONGENITAL STATIONARY NIGHT BLINDNESS (CSNB) IN HORSES – CANCER/MELANOMA ASSOCIATED RETINOPATHY •213 PHYSIOLOGY OF THE RETINA
• PHOTORECEPTOR CELLS • SIGNAL TRANSMISSION • BIPOLAR CELLS • HORIZONTAL CELLS Signal Transmission
Photoreceptors
Horizontal cells
Bipolar cells
Amacrine cells
Retinal ganglion cells Horizontal Cells
• Cell bodies are found in the outer part of the INL and have long processes that run laterally in the OPL
• 2 cell types – A- type: contacts only cones – B-type: contacts both rods and cones
• Dendrites terminate at pre-synaptic ribbons in lateral terminals of photoreceptors
• Main function: spatial summation over broad retina areas / center-surround receptive fields Horizontal Cells
©Webvision Horizontal Cells
©Webvision Postreceptorial Responses
• The light response of horizontal cells is a graded hyperpolarization dependent on light intensity • They have a large receptive field because they are extensively electrically coupled • They provide the antagonistic surround that opposes the center response of bipolar cells • Center-surround antagonism is important for edge detection and in enhancing contrast Horizontal Cells
©Webvision
Lateral inhibition on cones Concentrically organized receptive fields of bipolar cells (A) Opponent ON- or OFF-surround (luminosity) (B) Color opponent responses Lee et al. 2010
221 PHYSIOLOGY OF THE RETINA
• PHOTORECEPTOR CELLS • SIGNAL TRANSMISSION • BIPOLAR CELLS • HORIZONTAL CELLS • AMACRINE CELLS Signal Transmission
Photoreceptors
Horizontal cells
Bipolar cells
Amacrine cells
Retinal ganglion cells Amacrine Cells
• Interneurons that modulate transfer of light signals from bipolars to RGC • Dendrites in IPL and cell bodies in vitreal region of INL or, less commonly, in the RGC (displaced amacrines) or within the IPL • 26 types described in primate and 22 types in cat Amacrine Cells
©Webvision Amacrine Cells
©Webvision Some Amacrine Cell Types
• AII is a narrow field, depolarizing amacrine that is postsynaptic to rod bipolars • A17 is a wide-field amacrine that establishes a feed-back synapse onto the rod bipolar endings and modifies transmission of signals from rod bipolars to AII amacrines • Starburst amacrines are postsynaptic to cone bipolars and presynaptic to bistratified RGC. May generate receptive field properties of motion-selective RGCs SPATIAL AND SPECTRAL ANTAGONISM • There is a spatial and spectral antagonism between areas of the retina mediated by inhibitory neurons: horizontal and amacrine cells • Horizontal cells are excited by cones and send inhibitory signals back to cones • Amacrine cells are excited by bipolars and send inhibitory signals back to bipolars and RGC • Lateral anatgonistic interactions enhance border contrast PHYSIOLOGY OF THE RETINA
• PHOTORECEPTOR CELLS • SIGNAL TRANSMISSION • BIPOLAR CELLS • HORIZONTAL CELLS • AMACRINE CELLS • INTERPLEXIFORM CELLS Interplexiform Cells
©Webvision
FEEDBACK PHYSIOLOGY OF THE RETINA
• PHOTORECEPTOR CELLS • SIGNAL TRANSMISSION • BIPOLAR CELLS • HORIZONTAL CELLS • AMACRINE CELLS • INTERPLEXIFORM CELLS • RETINAL GANGLION CELLS (RGC) Signal Transmission
Photoreceptors
Horizontal cells
Bipolar cells
Amacrine cells
Retinal ganglion cells Retinal Ganglion Cells (RGCs)
©Webvision Retinal Ganglion Cells (RGCs)
• 25 different cell types in mammalian retina • Classification according to cell body size, dendritic tree spread, branching pattern and branching level in 5 strata of the IPL • RGC increase in dendritic tree span with eccentricity from the central retina • Smallest fields are possessed by cells of the fovea or area centralis Signals in the Innermost Retina
• Action potentials are generated only in the innermost retina • The RGC firing rate increases or decreases depending in stimuli of spot or annulus and type of cell • Basic organization: ON-, OFF-, pathways each with a center-surround antagonism with further processing at level of the innermost retina Convergence of photoreceptors and second order neurons to cat retinal ganglion cells
©Webvision RGC Types of Primates P = parvocellular Small cells
©Webvision
M = magnocellular Large cells α, large cells Functional = Y cells
β, small cells Functional = X cells RGC Types of Cat
β, small cells α, large cells ©Webvision Functional = X cells Functional = Y cells RGC Types of Cat & Dog
• X- cells – Small receptive fields – Respond to fine detail of pattern – high resolution – beta RGC • Y-cells – Respond to coarse pattern and abrupt changes in illumination – fast – alpha RGC • W-cells – Respond to onset or termination of stimulus – Slowest type of RGC • All 3 types display some form of color coding ON- and OFF-Channels
• ON- and OFF-RGC respond to light with action potentials • When stimuli is centered within the ON-cell’s receptive field, the firing rate increases, whereas an annulus of light suppresses the spontaneous discharge • The effect of stimuli on OFF-cells is the reverse • Uniform illumination of both types has less effect on firing rate • Other RGC respond only to light moving across their receptive fields, not stationary light ON- AND OFF-CHANNELS
Dowling 1998 ON- AND OFF-CHANNELS
Dowling 1987 Directionally Selective Ganglion Cells
©Webvision PHYSIOLOGY OF THE RETINA
• PHOTORECEPTOR CELLS • SIGNAL TRANSMISSION • BIPOLAR CELLS • HORIZONTAL CELLS • AMACRINE CELLS • INTERPLEXIFORM CELLS • RETINAL GANGLION CELLS (RGC) – INTRINSICALLY PHOTOSENSITIVE RGCs Photopigments in the Retina
Rod photoreceptors: Rhodopsin ~506-510nm (dog) Red/Green cone photoreceptors: L/M-opsin ~555nm (dog) Blue cone photoreceptors: S-opsin ~429-435nm (dog) webvision Melanopsin ©
Intrinsic photosensitive RGCs: Melanopsin ~480nm 1 – 3% of RGCs Melanopsin Intrinsic photosensitive RGCs
• REGULATION OF NON-VISUAL PHOTOPHYSIOLOGY:
– Input to mammalian endogenous clock – modulation of melatonin
– Mediation of pupillary light response
– Mediation of dazzle reflex ? Melanopsin Intrinsic photosensitive RGCs
• REGULATION OF NON-VISUAL PHOTOPHYSIOLOGY:
– Input to mammalian endogenous clock – modulation of melatonin
– Mediation of pupillary light response
– Mediation of dazzle reflex ? Melanopsin Intrinsic photosensitive RGCs
• REGULATION OF NON-VISUAL PHOTOPHYSIOLOGY:
– Input to mammalian endogenous clock – modulation of melatonin
– Mediation of pupillary light reflex
– Mediation of dazzle reflex ? Melanopsin Intrinsic photosensitive RGCs
• REGULATION OF NON-VISUAL PHOTOPHYSIOLOGY:
– Input to mammalian endogenous clock – modulation of melatonin
– Mediation of pupillary light reflex
– Mediation of dazzle reflex ? Phylogenetic Tree of Opsin Family
Yau & Hardie 2009 2 Basic Types of Photoreceptors
Ciliary Rhabdomeric
– Photosensitive membranes – Photosensitive membranes derived from modified derived from microvillar cilium projections of apical cell – Vitamin A-based surface forming a rhabdom chromophore – Vitamin A-based – 7-transmembrane-helix chromophore apoprotein, opsin – Opsin = c-opsin – 7-transmembrane-helix apoprotein, opsin – Cyclic-nucleotide motif for phototransduction – Opsin = r-opsin – Light hyperpolarization – Phospholipase C (PLC) motif – Light depolarization Intrinsically Photosensitive RGCs
Yau & Hardie 2009
“A bit of fly in the mammalian eye!” -opsin S-opsin Rhodopsin L/M Melanopsin
480 nm 630 nm ©Webvision Melan-100; Biomed Vision Technologies Example 1: Normal
256 X S-opsin + Example Example Melanopsin X Rhodopsin Advanced PRA Advanced X L/M-opsin CourtesyW. Beltran 2: - Endstage PRA PRA Normal retinaNormal
©webvision Example 2: Endstage PRA
258 Example 3: OpticNerve Disease S-opsin - X Melanopsin Rhodopsin
L/M-opsin Optic nerve coloboma Optic nerve -
©webvision Example 3: Optic Nerve Disease
260 PHYSIOLOGY OF THE RETINA
• NON-NEURONAL CELLS OF THE RETINA – MÜLLER CELLS Retina
Retinal pigment epithelium (RPE)
Photoreceptor segments
Outer limiting membrane (OLM)
Outer nuclear layer (ONL) Outer plexiform layer (OPL)
Inner nuclear layer (INL)
Inner plexiform layer (IPL)
Ganglion cell layer
Nerve fiber layer
Inner limiting membrane (ILM)
©Webvision Müller Cells
• Radially oriented within neural retina • Distal ends extend past the OLM at level of photoreceptor IS • Proximal ends form the ILM at the vitreal- retinal border • Microvillar processes at distal end increase surface area and are implicated in exchange of ions and metabolites • Fine Müller cell processes extend in spaces between neurons and cover most neurons, axons as well as blood vessels Müller Cells
©Webvision Müller Cells
• Principal retinal glial cells • Participate in regulating the extracellular environment of the retina
– Buffering light-evoked variations in extracellular potassium
– Removing neurotransmitters from synapses by active uptake Müller Cells
• Structural and metabolic support to retinal neurons by
– Regulating CO2
– Participating in the retinoid metabolism
– Store glucogen and contain molecules that bind retinoids PHYSIOLOGY OF THE RETINA
• NON-NEURONAL CELLS OF THE RETINA – MÜLLER CELLS – RETINAL PIGMENT EPITHELIUM (RPE) Retina
Retinal pigment epithelium (RPE)
Photoreceptor segments
Outer limiting membrane (OLM)
Outer nuclear layer (ONL) Outer plexiform layer (OPL)
Inner nuclear layer (INL)
Inner plexiform layer (IPL)
Ganglion cell layer
Nerve fiber layer
Inner limiting membrane (ILM)
©Webvision Retinal Pigment Epithelium (RPE)
• Single layered cuboidal epithelium
• Basal membrane bounded by basal lamina that forms the most proximal layer of Bruch’s membrane
• This membrane is highly convoluted and resulting large area subserves transport
• Apical membrane, with apical microvilli, interdigitates with photoreceptor OS Retinal Pigment Epithelium (RPE)
• Melanin pigment granules in apical processes and apical part of RPE cell – Protect receptors from scattered light and light passing through the sclera
• Lipofuscin granule; represent digested remains of outer segment discs
• Liposomes and dense bodies; function in the degradative system of the RPE Main Functions of RPE
• Like an epithelium: Barrier between choroidal circulation and the neuro-retina – Transports ions, metabolites, and water
• Like a glial cell: Apical processes cover part of the OS. Apical membrane also permeable to ions and has a large potassium conductance, thus generating voltage responses and regulating retinal ion levels Functions of RPE
• Like a macrophage: Functions to phagocytize and digest discarded portions of OS. – Following retinal detachments or inflammatory disease the RPE can differentiate into macrophages and phagocytize debris within retina
• RPE has vital function in the visual cycle of retinoid metabolism and isomerization
• Significant factor for production and degradation of interphotoreceptor matrix constituents Bestrophin-1 (BEST1)
• Family of Ca2+ activated Cl-channels • Regulators of ion transport rather than ion channels? • Localized at the basolateral plasma membrane of RPE cells • Humans: vitelliform macular dystrophy (VMD) or Best macular dystrophy
Marmorstein et al. 2009 cmr1 Great Pyrenees, Mastiffs
cmr2 Coton de Tulear
cmr1 © akc
cmr1 © akc
276 basolateral plasma membrane of RPE cells •277 PHYSIOLOGY OF THE RETINA
• INTERPHOTORECEPTOR MATRIX (IPM) Retina
Retinal pigment epithelium (RPE)
Photoreceptor segments
Outer limiting membrane (OLM)
Outer nuclear layer (ONL) Outer plexiform layer (OPL)
Inner nuclear layer (INL)
Inner plexiform layer (IPL)
Ganglion cell layer
Nerve fiber layer
Inner limiting membrane (ILM)
©Webvision Interphotoreceptor Matrix
• Photoreceptors are surrounded by a unique and complex extracellular matrix called interphotoreceptor matrix (IPM)
• RPE and Müller cells contact and physically restrict the IPM
• Contains glucosaminoglycans (GAGs), glycoprotein, fatty acids, vitamin A, and other proteins such as albumin and interphotoreceptor-retinoid-binding- protein, IRBP. IRBP binds retinoids and fatty acids. PHYSIOLOGY OF THE RETINA
• INTERPHOTORECEPTOR MATRIX (IPM) • RETINAL NEUROTRANSMITTERS Retinal Neurotransmitters
• Glutamate – Transmitter released by rods, cones, some bipolars and by some amacrines
• Acetylcholine – Released by ON- and OFF-amacrine cells – Enhances firing of ON- and OFF-RGC Retinal Neurotransmitters
• GABA and glycine – Inhibitory transmitters – Open Cl channels and reduce the effect of excitatory transmitters that act on cationic channels – GABA is accumulated in horizontal cells and mediate surround inhibition of bipolars.
• Dopamine – Some amacrine and interplexiform cells – Reduces the light response of rod bipolar cells – Many effects of dopamine are exerted at distant sites PHYSIOLOGY OF THE RETINA
• INTERPHOTORECEPTOR MATRIX (IPM) • RETINAL NEUROTRANSMITTERS • RETINAL BLOOD FLOW Retinal Blood Flow
• 2 separate vascular systems – retinal system – choroidal system – Species-differences
Wild 1994 Retinal Blood Flow
• Necessary requirements for the maintenance of retinal structure and function: – Correctly regulated hemodynamics
– Delivery of oxygen
– Delivery of metabolic substrates
– Intact blood-retinal barriers Retinal Oxygenation
INL
Cioffi, Granstam, Alm 2003 (Alder, Cringle, Yu) Retinal Metabolism and Visual Activities Are Linked • Retinal oxygen consumption is lower under constant light than in darkness
• Oxygen consumption is reduced by light at the level of the inner segments
• This effect of light on the dark current is driven by the ATPase-dependent sodium- potassium pumps on the membrane of the inner segment Hemodynamics
• η, viscosity • L, length of cylindrical tube • r, radius of cylindrical tube • ΔP, pressure difference between the two ends of the tube (perfusion pressure) • Q, flow ΔP • Hagen–Poiseuille law:
4 Q Q = π R ΔP 8 η L Autoregulation
Intrinsic ability of an organ to maintain blood flow independent of changes in perfusion pressure
Perfusion pressure Autoregulation
Cioffi, Granstam, Alm 2003 Autoregulation
Retina
Choroid
Cioffi, Granstam, Alm 2003 Retinal Blood Flow
• Retinal blood flow is autoregulated – Adaptation of the vascular tone of the resistance vessels (arterioles, capillaries) to changes in the perfusion pressure or metabolic needs of the tissue
– Interaction of multiple mechanisms affecting the arteriolar smooth muscle cells and capillary pericytes • Interaction of myogenic and metabolic mechanisms through the release of vasoactive substances by the vascular endothelium and retinal tissue surrounding the arteriolar wall. Choroidal Blood Flow
• Choroidal capillaries are fenestrated and highly permeable for glucose, proteins, and other substrates
• The rate of uveal blood flow is rapid, mean retinal and choroidal circulation time is 3-4 sec. Oxygen content of choroidal blood is 95% of that of arterial blood
• Rich supply of autonomic vasoactive nerves to choroidal but not to retinal vessels Methods: Angiography
Fluorescein Indocyanine green (ICG)
Normal / 6.5 yrs / OD / 9.3 mmHg •295 Methods: Angiography
Normal / 6.5 yrs / OD / 9.3 mmHg Retinal Blood Flow
• Blood-retinal barriers – Inner blood-retinal barrier (iBRB) • Endothelium • Pericytes • Glial cells (astrocytes and Müller cells) – Outer blood-retinal barrier (oBRB) • Retinal pigment epithelium (RPE) • Bruch’s membrane Questions?