PHYSIOLOGY OF RETINA

Sanket Parajuli

Retinal pigment epithelium • Outermost layer

• 4-6 million RPE cells/eye

• RPE cells have long villous processes on their apical surfaces that interdigitate with outer segments of photoreceptor cells

• Basal surface – adjacent to Bruch’s membrane

• On their apical side RPE cells are joined by tight junctions—blocks passage of water and ions—contribute to blood retinal barrier

(Biological filter for the neurosensory retina)

• RPE cells are rich in melanin granules and phagosomes

• Also are rich in microperoxisomes -active in detoxifying large number of free radicals and oxidized lipids

Physiological Functions:• Visual pigment regeneration• Phagocytosis of shed photoreceptor cells outer disks• Transport of essential nutrients and ions to photoreceptors and

removing waste products from photoreceptors• Absorption of scattered and out of focus light via pigmentation• Adhesion of retina• Synthesis and remodeling of IPM• Formation of blood retinal barrier• Elaboration of growth factors

Visual pigment regeneration

• Regeneration of visual pigment rhodopsin involves both photoreceptor and RPE

• RPE plays important role in uptake storage and mobilization of vitamin A for use in visual cycle

• Basic function of RPE cell is to regenerate 11-cis-retinaldehyde

• Photoreceptor cell synthesizes opsin-used by 11 cis retinaldehyde in generation of rhodopsin

• In photoreceptor cell rhodopsin is photolysed and undergoes cis to trans isomerization

• All trans retinaldehyde is released and converted to all trans retinol by retinol dehydrogenase

• Retinol is returned to RPE in presence if IRBP

• In RPE retinol is converted to retinyl eser in presence of enzyme lecithin retinol acyltransferase

• When needed retinyl ester is converted by isomerohydrolase (isomerase) to 11-cis retinol and converted to11-cis retinal by dehydrogenase

• 11 cis retinal is returned to photoreceptor cell along with IRBP

• Opsin combines with 11-cis retinal to form rhodopsin

• Proteins RPE65 plays a role in isomerization

RPE acquires vitamin A in 3 ways

• Through release during bleaching of rhodopsin and return via regeneration process of visual cycle

• From circulation-receptor mediated mechanism

• Via phagocytosis of shed photoreceptor outer segment disk

Phagocytosis :

• RPE cells play crucial role in turn over of photosensitive membrane of rods and cones

• One of the best studied function of RPE is their role in outer segment renewal

• Disc shedding is the process by which small packets of disk membranes are eliminated from tip of outer segment

• Ingestion and subsequent degradation of outer segment fraction which are shed daily from distal ends of photoreceptors• Overall process can be divided into 3 stepsi. Recognition and binding of outer segments by

receptors in RPE plasma membraneii. Generation of transmembrane signal across

RPE plasma membrane with production of second messengers

iii. Ingestion of outer segments via RPE cytoskeletal mobilization

• Each outer segment is completely replaced in humans in every 10 days

• Disc shedding is a daily process- occurring in a burst at the same time each day

• Each RPE cell is in contact with 200 photoreceptor outer segments

• Each photoreceptor sheds approx. 100 outer segments per day

• Each RPE cell ingest more than 4000 discs daily

In rods shedding is most vigorous within 2 hrs. of light onset and in cones occurs at onset of darkness(rods shed more vigorously in the morning whereas cones shed more in the darkness)

• With age and pathological process deficient degradation of the phagolysosomes leads to formation of lipofuscin granules

Transport, barrier and metabolism

• RPE controls the volume and composition of fluid in subretinal space through transport of ions, fluid and metabolites

• Tight junctions of RPE cells form barrier against diffusion of proteins and other metabolites, thus most transport occurs through cytoplasm-RPE controls the metabolic provisions to photoreceptor

• RPE --also a barrier to diffusion in opposite direction so helps to conserve vitamin A and lipids that might be lost from the eye

• Aqueous environment of subretinal space is maintained by ion-transport systems of RPE in turn maintains integrity of RPE photoreceptor interface

• Active transport of ions(K,Ca,Na,Cl,Hco3) occur across RPE

• Na is actively transported from choriocapillaries towards the subretinal space, K is transported in opposite direction

• Apical membrane of RPE-major locus of transport –Na/K ATPase is present at the apical portion

• Active bicarbonate transport systems(Hco3 ATPase) also present

• Net ionic influxes in RPE are responsible for transepithelial electrical potential-can be measured across the RPE apical membrane

• Trans-RPE potential basis for EOG—most common electrophysiological test for evaluation of RPE

Pigmentation:• A characteristic feature of RPE is presence of melanin pigment

• Pigment granules abundant in cytoplasm(most in apical and mid portions)

• As age increases melanin pigment fuse with lysosomes so elderly fundus is less pigmented than that of young

• Helps in minimization of light scatter-acts as a neutral density filter protective role

• Melanin is a free radical stabilizer ---can bind to toxic substances

• Role in retinal development

• Melanin levels when is below critical level –aberrant neuronal migration in visual pathway, lack of foveal development, low vision nystagmus, strabismus

Retinal adhesion :Factors keeping the retina attached

1. Mechanical forces outside the sub retinal space: a. Fluid pressure: hydrostatic & osmotic fluid

Aqueous is driven from the vitreous towards the choroid, but the posterior route is limited because the retina & RPE provide substantial resistance to water movement. Hence as an effect of this outward push of fluid the retina remains on the wall of the eye.

b. Pressure difference across the retina

c. Vitreous adhesion:

-the vitreous has a physical structure of a gel that may help to keep the retina in place

2. Forces in the sub retinal space:a. RPE pump: -the RPE can pump fluid out of the subretinal space to the choroid at the rate of

0.3microL\hr\ sqmm. -this is an active energy dependent process and keeps the subretinal space dry.

b. Mechanical interdigitation: -the RPE microvilli wrap closely around the tips of the outer segments of the

photoreceptors.

c. Inter photoreceptor matrix: -Cell-cell adhesion mediated by cell adhesion molecules, which are intrinsic

membrane glycoproteins. -Cell-matrix adhesion mediated by matrix molecules like fibronectin,

laminin, collagen and proteoglycans.

Interphotoreceptor Matrix(IPM) and inter Photoreceptor Retinoid Binding Protein (IRBP)

IPM • occupies space between Photoreceptor OS and RPE • Contains: proteins, glycoprotiens, GAGs and proteoglycans (chondroitin sulfate)

Functions:• Retinal attachment and adhesion molecular trafficking• Facilitation of phagocytosis• arrangement of photoreceptor OS

IRBP:• 70% of soluble proteins in IPM• Only binding protein found in retina• Produced mainly by cones (PR)• Binds all- trans – retinol, 14-cis-retinal, ὰ-tocopherol, retinoic acid and

cholesterol

Function:• Efficient transport of retinoids between PR and RPE• Minimize fluctuation of retinoid availability• Protect plasma membrane form damaging effect of high concentration of

retinoid

Produces growth factors

• Secretes vascular endothelial growth factor (VEGF): maintain function of choriocapillaries

• Over-production of VEGF--- neovascularization

• So, also produces an antiangiogenic factor, pigment epithelial derived factor (PEDF): maintains balance

Retinitis Pigmentosa

• Autosomal dominant retinal dystrophy• Progressive loss of RPE and photoreceptor

function• Apoptosis of PR cells• Rods (functional in periphery) • Cones (functional in fovea)

• RPE degenerates, pigment migrates into the sensory retina, and accumulates around blood vessels in a characteristic bone-spicule pattern

Stargardt’s Macular Dystrophy

• autosomal recessive• an early age vision loss• gene defect in production of protein

(transport to and from photoreceptor cells)• RPE degenerates in early stage • lipofuscin-like, yellow and fleck-shaped

deposits accumulate in macular area in later stage• atrophy RPE followed by changes PR• progressive loss of vision• by age 50, 50% of patients affected can

have reduction of visual acuity to 20/200 or worse

Photo of right fundus with Stargardt’s macular dystrophy,RPE degeneration and lipofuscindepositition in the macular area.

Best’s disease (vitelliform macular dystrophy)

• Autosomal dominant• Malfunctioning transport protein • Deposition between the RPE and

neural retina• Striking yellow or orange egg

yolk like elevated lesion in the macula

Photo : right fundus of patient with Best’s disease;tissue disruption and mottling are evident in macular area.

Rods Phototransduction

• Rods are sensitive to nocturnal light • Rods are more in number than cone • 1000 discs within a rod outers segment--- million

rhodopsin molecules in each sac

• Light falling upon retina is absorbed by photosensitive pigments in rods and cones, initiates photochemical changes which in turn initiates electrical changes and the process of vision begins

• Light is absorbed by rhosopsin concentrated in outer segment of rods

• Phosphorylation site exists on cytoplasmic side of protein where rhodopsin is inactivated and sugar is attached on intradiscal side

• 11-cis retinal is bound to a AA 296 on the 7 h membrane loop by a protonated Schiff base linkage

• Rhodopsin absorbs green light best at wavelength of about 510nm

• Absorbs blue and yellow light and is insensitive to longer wavelength red light

• Once rhodopsin absorbs quantum of light the 11 cis bond of retina is broken- the opsin molecule undergoes configurational changes leading to activated state –meta rhodopsin 11

• Activated rhodopsin starts a reaction that controls the inflow of cations into rod outer segment

• Target is a cGMP gated cationic channel located on outer membrane of outer segment

• This channel controls the flow of Na and Ca ions into rod

• In dark Na and Ca ion flow in through this channel kept open by cGMP-ionic balances is maintained by Na-K Atpase pump in inner segment and Na-K Ca exchanger in outer segement

• Depolarisation of rod causes the release of transmitter glutamate from synaptic terminal starting the neural signals for vision

• Hyperpolarisation stops glutamate release from synaptic vesicle

• The reduction in cGMP is responsible for producing electrical response which makes the beginning of nerve impulse

• When light goes off –rod turns to its dark state as the reaction cascade turns off

Cone Phototransduction:

• Cone phototransduction is comparatively insensitive but fast capable of adapting enormously to ambient levels of illumination

• Respond to specific wavelength of blue, green and red sensitive cones --about 435,535 and 580 nm

• Light activated cone opsin start an enzymatic cascade that hydrolyses cGMP and closes cone specific cGMP

• Without cones one is legally bling losing the ability to read and to see colours

• There is neutrally mediated negative feedback on cones

• Horizontal cells of inner nuclear layer synapse back on to cones releaseing GABA(Inhibitory )

• When light hyperpolarizes a cone --the cone hyperpolarizes neighboring horizontal cells

• This inhibits horizontal cells and stops the release of GABA which depolarizes the cone by recurrent synapse-this depolarization antagonizes the hyperpolarization produced by light and tries to put cone back in the dark

• Life span of activated cone opsin must be shorter—its turn off

• Turning off of cone transducing is faster than of rods

Inner nuclear layer:• Consists of bipolar cells, horizontal cells, amacrine cells

anf glial cell(Muller cell)Bipolar cells:• Separate bipolar cells for cones and rods• 2 types of cone bipolars:• On-bipolar• Off-bipolar

• When light hyperpolarizes the cones the on bipolar is excited and off bipolar is inhibited• When shadow depolarized the cones, reverse of this occurs• Some bipolars synapse only with L cones and other with

M cones which is necessary for colour vision

• In fovea some cone bipolars synapse with a single L or M cone which provide highest spatial activity

• Separate L and M cone on-bipolar and off bipolars transmit faster phasic signal to a parallel system of larger ganglion cells

• Rods and S cones have only bipolar cells

• S cones are involved in colour vision and rod in twilight vision

Horizontal cells

• Antagonistic interneurons that inhibit photoreceptors by releasing GABA when depolarized

• Dendrites of horizontal cells go to cones. One class goes to L and M cones and another class goes to S cones

• Axon from cell body of horizontal cell send dendrites to rods

• Dendrites of horizontal cells receive glutamate from rods and cones and release GABA back to cones and rods-provides negative feedback

• When light causes the cones to hyperpolarize and stop its transmitter release horizontal cell is also hyperpolarized(turned off) – stops the release of GABA from horizontal cell on to cone and thus depolarize the cone

Amacrine cells

• Cone amacrine cells mediate antagonistic interaction among on-bipolars and off-bipolars and ganglion cells

• Rods have unusual amacrine cell that receives the input of rod bipolar and delivers signal to on an off bipolar ganglion cells

• Rod signal undergoes additional synaptic delay before they reach the ganglion cell output

Ganglion cells

Types:• On (center cells excited)• Off (center cells inhibited by light in center of their receptive field)• A shadow initiates opposite reaction in these 2 cell types• Ganglion cells are excited by short waves entering and long waves leaving

their receptive field• Project to lateral geniculate nucleus and mediate both high spatial resolution

and colour vision

Muller cells:• Play a supportive role to the neural tissue extending from inner

segments of photoreceptors to inner limiting membrane

• Buffer the ionic concentration in extracellular space seal off subretinal space by forming the ELM

• Plays a role in Vit A metabolism of cones

Non neural cells of retina• Macroglia (astrocytes, oligodendroglia, schwann cells)• Microglia:

These cells provide:• Respond to retinal cell injury• Regulate the ionic and chemical composition of extracellular mileau• Participate in blood retinal barrier• Form the myelination of optic nerve• Guide neuronal migration during development and exchange metabolites with

neurons

Visual adaptation: dark and light

Dark adaptations:

• The ability of visual system both rods and cones mechanisms to recover sensitivity following exposure to light

• The recovery is faster in cone but the sensitivity is greatest in rods

• The time taken to see in dim illumination is called dark adaptation time

Mechanism of dark adaptations:• Its based on the changes in visual pigments

• The process is slower than in light adaptation

• When the person remains in darkness for a long time the retinaldehyde and opsins in rods and cones are converted back into light sensitive pigments

• Vitamin A reconverted back into retinal give additional light sensitive pigments the final limit being determined by the amount of opsins in rods and cones

Parameters affecting dark adaptations:• Preadaptation• Retinal location• Wavelength

Light adaptation • The process by which retina adapts itself to bright light• Is quicker and occurs in a period of 5 minutes

Mechanism:• When a person remains in bright light—large proportion of photochemicals in

rods and cones is reduced to retinal and opsins much of the retinal of both rods and cones is converted to vitamin A

Retinal Electrophysiology:

• Changes in light flux on the retina produce electrical changes in all of the retinal cells including RPE-Muller cells-neurons

• Electrical change result from ionic current that flow when ion specific channels are opened or closed

• These current reach the cornea and vitreous where can be detected as electroretinogram

• Human eye behaves like a dipole along AP axis—with cornea being positive than posterior pole

• Movement of eye causes changes of potential in one electrode placed near inner canthus relative to another placed near the outer canthus of eye

• Record of eye movement is obtained by this means is called electro oculo gram

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