Clinical Anatomy and Physiology of Vitreous and Retina

Rohan  Chawla; Atul  Kumar; Raghav  Ravani; Divya  Agarwal; Aman  Kumar; Yogita  Gupta

SECTION 1

1Basic Sciences and Diagnostics

Clinical Anatomy and Physiology of Vitreous and Retina

Raghav Ravani, Divya Agarwal, Aman Kumar, Yogita Gupta, Rohan Chawla, Atul Kumar
Retinal Imaging

Atul Kumar, Devesh Kumawat, Divya Agarwal, Raghav Ravani, Kavitha Duraipandi, Anu Sharma
Electrophysiology

Raghav Ravani, Devesh Kumawat, Priyanka Ramesh, Divya Agarwal, Atul Kumar
Diagnostic Ophthalmic Ultrasonography

Raghav Ravani, Divya Agarwal, Yogita Gupta, Meghal Gagrani, Atul Kumar2

Clinical Anatomy and Physiology of Vitreous and RetinaCHAPTER 1

Raghav Ravani,

Divya Agarwal,

Aman Kumar,

Yogita Gupta,

Rohan Chawla,

Atul Kumar

THE VITREOUS

The word vitreous literally means like a glass (in appearance or properties). The vitreous is a transparent, hydrophilic, optically clear media that constitutes about 80% of the eye volume. Anteriorly, it is limited by the ciliary body, the zonules, and the lens while posteriorly, it is limited by the retina and forms the vitreoretinal interface. Topographically, vitreous may be classified into: The central or core vitreous and the peripheral or cortical vitreous.

The anterior cortex, consists of condensation of collagenous fibers that attach to the posterior surface of capsule of lens forming the Wieger's ligament or hyaloideocapsular ligament (Fig. 1.1). The presence of crystalline lens leads to a concave, retrolental indentation of the anterior cortex called as the patellar fossa. The potential space between lens and anterior vitreous (anterior hyaloid) which is bordered by Wieger's ligament is called the Berger's space (Fig. 1.1). The pre-equatorial and postequatorial lens zonules enclose a space called the Canal of Hannover. The space between postequatorial zonules and hyaloid zonules is called Canal of Petit. Vitreous base is an area that extends about 2 mm anterior and 3 mm posterior to the ora serrata where the collagen fibers are especially dense and insert firmly (Fig. 1.2). The vitreous cortex is firmly adherent to the internal limiting membrane (ILM) in certain areas: at the region of the vitreous base,¹ around the optic disc, at the retinal vessels and around foveola.^2,3

Embryology and Development of Vitreous

Embryologically, the vitreous can be divided into primary (primitive), secondary (definitive) and tertiary vitreous, which represent different phases of development of vitreous from various layers of the developing embryo (Table 1.1).

Structural development: In the first month of gestation, at 5–13 mm fetus stage, a fibrillar vascular structure, the primary vitreous, forms from the mesenchymal layer and fills the space formed as the optic cup grows with its lens vesicle. Later the cells of the hyaloid arterial wall presumably secrete the fibrillar material forming the primary vitreous.^4–8 It has a dense vascular plexus (anterior and posterior tunica vasculosa lentis) that mainly provides nourishment to the developing lens.

Fig. 1.1: Gross anatomy of vitreous.

4In the second month of gestation at 14–70 mm fetus stage, primary vitreous with its vasculature is seen to regress and secondary vitreous forms which is an avascular and compact network of type II collagen fibrils secreted from the neuroectoderm of the optic cup by the 6th week. The primary vitreous is thus eventually replaced by secondary vitreous and the main hyaloid artery disappears and leaves a residual tube of primary vitreous, called Cloquet's canal (Fig. 1.1), surrounded by the secondary vitreous, which extends from the retrolental space to the optic nerve (area of Martegiani). If the regression of primary vitreous fails to occur, it leads to a condition known as persistent fetal vasculature (PFV) (formerly known as persistent hyperplastic primary vitreous or PHPV).

Fig. 1.2: Anatomy of vitreous base.

TABLE 1.1 Embryological phases of vitreous development.

Developmental vitreous	Origin
Primary (primitive) vitreous	Mesenchymal (secreted by embryonic retinal cells and later from cells of penetrating hyaloid artery)^4–8
Secondary (definitive) vitreous	Neuroectodermal (neuroectoderm of optic cup)
Tertiary vitreous	Neuroectodermal (neuroectoderm of the ciliary region)

The tertiary vitreous develops from neuroectoderm in the third month of gestation at stage of 71–110 mm fetal length, which develops as the suspensory fibrils, contains no vessels or nerves and has two main parts: collagen fibers and hyaluronic acid (HA) with high (~98%) water content.

Molecular and cellular development: The two main components of vitreous, i.e., the collagen and HA, are produced in the primary and secondary vitreous. The cells in the primary vitreous differentiate as hyalocytes and fibroblasts in secondary vitreous. The hyalocytes supposedly produces glycosaminoglycans (GAGs), especially HA.⁹ The collagen may be synthesized from fibroblast or from retina.^10,11 The hyalocytes are mononuclear cells found in the posterior vitreous cortex, approximately 30 microns (20–50 microns) from the ILM, with highest density near the vitreous base and posterior pole, and lowest near the equator.^12,13

Biochemical Properties of the Vitreous

Vitreous consists of three major structural components: water, collagen fibers, and GAGs. The vitreous contains more than 99% of water. The vitreous exists as a gel-like structure due to the arrangement of long, nonbranching, collagen fibrils which are suspended in a network of HA.^15–17 The most common type of collagen fibrils are collagen type II, which are composed of helices made of three α-chains, stabilized by hydrogen bonds between opposing residues in different chains.⁸ Collagen type IX functions as a link between type II collagen fibrils.^16,18,19

Posterior Vitreous Detachment and Anomalous Posterior Vitreous Detachment

The vitreoretinal interface is a complex formed by the posterior vitreous cortex, the ILM and an intervening extracellular matrix, consisting of fibronectin, laminin, etc. The vitreous at this interface is believed to be adhered to ILM by an extracellular matrix, causing the adhesion to be fascial.^20,21 Chondroitin sulfate exists at sites of strong vitreoretinal adhesions like vitreous base and optic disc, and hence the rationale for pharmacologic vitreolysis using chondroitinase derivatives for disorders of vitreoretinal interface. Posterior vitreous detachment (PVD) means separation of posterior vitreous cortex from ILM. PVD begins in the perifoveal region. This is due to vitreous degeneration which may be age-related or due to other secondary causes. The mechanism of 5age-related vitreous degeneration and hence PVD is briefly discussed in following sections.

Synchysis (Liquefaction)^22,23

There is age-related increase in central liquid volume of vitreous and decrease in the gel volume leading to formation of vitreous pockets (lacunae) which coalesce to form larger posterior lacuna or bursa or precortical pocket.²⁴ A study observed 20% of the total volume as liquid vitreous as the human eye reaches adult size.²² Changes in collagen or conformational change in HA with subsequent cross-linking of fibrils is postulated mechanism for liquefaction of vitreous.^19,25 Changes in minor GAGs and chondroitin sulfate may also play a role in vitreous liquefaction.²⁶ Biochemically, vitreous HA concentration is steady after the age of 20 years²² and liquid vitreous increases with age. With age, an increase in HA content of the liquid vitreous and a concomitant decrease in HA content of the gel vitreous is seen. Thus, a reduction of vitreous HA concentration results in decreased viscosity of vitreous gel, which may be accelerated by cataract surgery or a breach in the posterior capsule of the lens.

Syneresis

Along with age-related liquefaction, there occurs thickening and tortuosity of vitreous fibers and resulting collapse of the vitreous. This collapse of vitreous body is called as syneresis. This is an age-related process, but may occur earlier in some cases like high myopia,²⁷ post-trauma to ocular structures, ocular inflammation and congenital vitreoretinopathies (arthro-ophthalmopathies).^28,29

A clean separation between the cortical vitreous and retinal internal limiting lamina (ILL) is called an innocuous PVD.³⁰ In most cases, this may be asymptomatic leading to total separation of cortical vitreous from ILL, resulting in a ring of tissue composed of fibrous astrocytes and collagen at its attachment to the optic disc, called as Weiss ring. This may lead to symptom of a floater. Incidence of PVD is 66% between the ages of 66 and 86 years,³¹ and 53% after 50 years.³²

Anomalous Posterior Vitreous Detachment

This results from liquefaction of vitreous without concurrent weakening of vitreoretinal adherence, leading to various manifestation depending on the level of separation, site of firm adherence and liquefaction. This may lead to either partial thickness detachment called as vitreoschisis,^14,33 wherein there is splitting of the posterior vitreous cortex with anterior displacement of a part of cortex, with posterior layer still attached to the retina. Note, this is not to be confused with partial PVD, which means full thickness but incomplete separation of cortical vitreous.

RETINA

The retina (Latin: rete = net) is the innermost layer and the most comprehensive sensory structure of the eye. It is derived from the optic vesicle and grossly is a thin, transparent membrane with two main components: (1) a pigmented layer (called as retinal pigment epithelium or RPE), and (2) a sensory layer (the neurosensory retina). Embryologically, both are derived from outer and inner layers of the optic vesicle, respectively (Fig. 1.3).

The two layers are attached loosely to each other by various mechanisms, failure of which leads to separation of neurosensory retina from the RPE with accumulation of subretinal fluid, a clinical entity called as retinal detachment.

Fig. 1.3: Formation of lens vesicle and optic cup. The thin outer wall of optic cup forms the pigmented layer of retina. The thick inner wall forms the neurosensory retina.

6Forces that keep the retina attached can be divided into:

Mechanical forces
Subretinal fluid transport
Metabolic factors

Mechanical Forces

Fluid pressure and resistance to flow:
- Hydrostatic pressure: Contributed by intraocular pressure (IOP)
- Osmotic pressure: Contributed by extracellular fluid in the choroid
  - Both of the above mentioned forces enhance absorption of fluid out of subretinal space which in turn enhance the binding properties of interphotoreceptor matrix (IPM).
- Retinal flow resistance because of retina and RPE^34,35: Acts to push neurosensory retina against RPE.
Vitreous support: Does not directly contribute to attachment of retina, but intact gel form vitreous has a role in preventing pathologic fluid access to subretinal space.^36–41
Mechanical interdigitation: RPE microvilli wrap closely around the tips of outer segments, thus acting as mechanical interdigitation between the RPE and neurosensory retina. Exact mechanism is unknown, but close ensheathment might provide frictional resistance to separation of neurosensory retina and RPE. Electrostatic forces that oppose separation of the membranes may also play a role.⁴²
Interphotoreceptor matrix properties: Various properties of IPM like the presence of the viscous material,⁴³ largely due to proteins and glycoproteins and the presence of GAGs⁴⁴ helps in attachment of the two layers. The presence of cone matrix sheath that remains attached to both RPE and photoreceptors cells may also play a role in attachment of retina.^45–47

Subretinal Fluid Transport

Retinal pigment epithelium actively transports water from the subretinal space to the choroid.^37,48,49 Conditions that keep the subretinal space dehydrated and thereby keeping the matrix viscous, contribute to keep the retina closely apposed, e.g., raising systemic osmolality with mannitol passively increases subretinal fluid absorption⁵⁰ and thus increases adhesiveness of retina.^51–53

Metabolic Factors

Metabolic factors like oxygenation, pH, calcium concentration which affect RPE activity and bonding of IPM influence attachment of RPE and neurosensory retina.

Topography of the Retina

The retina proper has a surface area of about 266 mm². The major landmarks of the retina are: the optic disc, area centralis (macula lutea), and the peripheral retina.

The retina is thickest near the optic disc, where it measures approximately 0.56 mm. The retina becomes thinner in the periphery (approximately 0.18 mm at equator to 0.3 mm at the ora serrata).^54–57

The Optic Disc

It is a circular to oval well-defined pale pink structure of about 1.5 mm in diameter. All the retinal layers terminate at the optic disc, except the nerve fiber layer (NFL), which pass through the lamina cribrosa and form the optic nerve. The depression within the optic disc is called as the physiological cup. Increase in the size of the cup and/or difference in the size of cup of two eyes should arouse suspicion of glaucomatous damage to nerve fibers and should be evaluated.

Area Centralis⁵⁵

This region of the retina, located in the posterior fundus temporal to the optic disc, is divided into the fovea and foveola with parafoveal and a perifoveal ring around the fovea. The area is a horizontally elliptical area demarcated approximately by the upper and lower arcuate and temporal retinal vessels with an average diameter of about 5.5 mm. This area corresponds to approximately 15° of the visual field. Histologically, it is the area that contains two or more ganglion cell layers.

Fovea Centralis (Figs. 1.4 and 1.5)

It is the central depressed part of the macula around 1.85 mm in diameter and 0.25 mm in thickness and is approximately 3–4 mm temporal (approximately 2 disc diameter) and 0.8 mm below the center of the optic disc. It corresponds to visual field of 5°.⁵⁵

Foveola

It measures 0.35 mm in diameter, 0.13 mm in thickness, and corresponds to the 1° of visual field. It represents area of the highest visual acuity in the retina, consists solely of cone photoreceptors and is avascular.

Parafoveal and Perifoveal Zone

These are areas around the fovea about 0.5 mm and 1.5 mm in diameter, respectively.

Peripheral Retina

It can be divided into following regions:^54,55

Near periphery—is a circumscribed region of 1.5 mm around the area centralis.
Mid periphery—is a 3 mm wide zone around the near periphery.
Far periphery is a region that extends from the optic disc, 9–10 mm on the temporal side and 16 mm on the nasal side in the horizontal meridian.

7Ora Serrata

It is the anterior most region of the retina, where the retina ends and ciliary body starts.

Dentate processes: These are jetties of retinal tissue extending anteriorly into the pars plana. These are more prominent nasally.
Ora bays: These are posterior extension of the pars plana towards the retinal side.
Enclosed ora bay: Dentate processes may wrap around a portion of ora bay to form an enclosed ora bay.
Meridional fold: It is prominent thickening of retinal tissue extending into the pars plana.
Meridional complex: Meridional fold when aligned with a ciliary process is known as meridional complex.

Fig. 1.4: Schematic diagram of microscopic structure of fovea centralis.

Fig. 1.5: Optical coherence tomography picture of the macula depicting all the layers.

Microscopic Architecture of the Retina

As seen by light microscopy, the cross-section of the retina consists of different cell types and their synapses, arranged in 10 layers from without inwards as follows (Figs. 1.6 to 1.8):

Retinal pigment epithelium (RPE)
Photoreceptor layer of rods and cones
External limiting membrane (ELM)
Outer nuclear layer
Outer plexiform layer (OPL)
Inner nuclear layer
Inner plexiform layer
Ganglion cell layer
Nerve fiber layer
Internal limiting membrane

Retinal Pigment Epithelium

It is the outermost layer of retina consisting of a monolayer of hexagonal pigmented cells derived from the outer layer of the optic cup. The RPE cells in the macula are taller and denser than in the periphery. It maintains apex-to-apex arrangement with Müllerian glia. RPE layer is continuous with the pigment epithelium of the ciliary body and iris.

Electron microscopy shows that each RPE cell has an apical portion with microvilli which envelope the photoreceptor outer segments. Its basal portion shows plasma membrane infolding and is firmly adherent to the underlying basal lamina of the choroid (Bruch's membrane). These cells are connected with each other by tight junctions (zonula occludens and zonula adherens) near the apices, and thus forming the outer blood-retinal barrier.

Functions of RPE with clinical implications:

Fundus on examination has a granular appearance due to unequal pigmentation of the RPE cells giving mottled appearance.
The pigment granules of RPE has melanin pigment that absorbs photons of light and minimizes light scatter within the retina.⁵⁸
As explained above, the potential space between RPE and sensory retina is called as subretinal space, and fluid collection in this layer is called as subretinal fluid which leads to retinal detachment. RPE pumps this fluid from the subretinal space at a rate of about 0.3 µL/h/mm² of RPE (measured in microliters per hour per millimeter square area).^59–63 Resorption rate of detachment in human eyes has been measured as 0.11 µL/h/mm² of RPE⁶⁴ which is about 3.5 mL of fluid per day.
The tight junctions between the RPE cells forms the outer blood-retinal barrier. Thus, the selective transport properties of the RPE regulating transepithelial diffusion through paracellular spaces helps maintain environment of the photoreceptors.⁶⁵
RPE is responsible for transport of metabolites through the blood-retinal barrier and for elaboration of the extracellular matrix.⁶⁶
It phagocytoses outer segments of photoreceptors being shed according to their circadian rhythm.^67,68 (Rods shed discs at dawn and cones shed at dusk).
It helps in polyunsaturated fatty acid metabolism.
8Majority of the regeneration process of 11-cis retinaldehyde (the chromophore found in rhodopsin) occurs in the RPE. This helps in perpetuation of the Wald's visual cycle. Defect in RPE65 (retinal pigment epithelium-specific 65-kDa) gene on chromosome 17 leads to a condition called as Leber's congenital amaurosis (LCA type 2) and retinitis pigmentosa.

Fig. 1.6: Optical coherence tomography image depicting various layers of the retina.

Fig. 1.7: Schematic diagram of microscopic structure of the retina.Source: Modified with permission from textbook on ‘Anatomy and Physiology of Eye, 2nd edition, CBS, 2011 by Dr AK Khurana

9Recently, gene therapy for LCA with subretinal injection of recombinant adeno-associated virus gene vector carrying altered human RPE65 is being tried and trials are on.⁶⁹

Fig. 1.8: Photograph of histological specimen of human retina.Courtesy: Dr Seema Kashyap, Department of Ocular Pharmacology, Dr RP Centre for Ophthalmic Sciences, AIIMS, New Delhi.

Photoreceptor Layer of Rods and Cones

The photoreceptor layer of the retina consists of the rods and cones. These are the end organs of the visual pathway that transform light energy into visual impulse. On an average, there are about 120 million rods and 6.5 million cones in the human eye. As seen on electron microscopy, these photoreceptors are arranged as mosaic, composition of which varies in different regions of the retina depending on the density variation of both rods and cones in different regions of the retina (Figs. 1.9 and 1.10).

Rod photoreceptor: These are about 40–60 microns long. The highest density of rods are below the optic disc and their number gradually reduces towards the periphery. Rods are absent at the fovea in an area of 0.35 mm, which corresponds to 1.25° of the visual field. It consists of the following:

Outer segment: It is cylindrical, refractile, transversely serrated and contains a photosensitive substance—rhodopsin. The rod photoreceptor outer segment is composed of numerous lamellar lipoprotein discs (around 6,000–10,000/rod, each about 22.5–24.5 nm thick) stacked together and surrounded by a cell membrane.
Inner segment: It is thicker than the outer segment consisting of two regions, ellipsoid and myoid.
- Ellipsoid (the outer portion) contains abundant mitochondria.
- Myoid (the inner portion) contains glycogen and other organelles.
Cell body and nucleus: Lies in the outer nuclear layer. It arises from the inner end of the rod, passes through the ELM and swells into a densely staining nucleus called the rod granule. This terminates into a bulbous structure called the spherule.

Fig. 1.9: Microscopic (schematic) structure of rod cell.

Fig. 1.10: Microscopic (schematic) structure of cone cell.

Cone photoreceptor: It has following parts—

Outer segment: Cone outer segment is conical in shape, shorter than that of the rod. It contains pigment–iodopsin. The outer segment is composed of lamellar discs (around 1,000–1,200 discs/cone) which are in continuity with the surface plasma membrane.
10Inner segment: It is similar in structure to rod inner segment, consisting of ellipsoid and myoid. Ellipsoid is larger than that of rod and has more numerous mitochondria.
Cell body and nucleus: Cone inner segment becomes directly continuous with its nucleus and lies in the outer nuclear layer. A stout cone inner fiber runs from the nucleus which ends in lateral processes called cone pedicle.

On OCT, interphase between inner and outer segment of photoreceptors is seen as a hyper-reflective band, previously known as IS-OS junction. The term is now disputed. The correct terminology is ellipsoid zone which is hyper-reflective due to increased density of mitochondria. Myoid zone is a hyporeflective region situated between ellipsoid zone and external limiting membrane. It contains inner segment of photoreceptors and is hyporeflective due to paucity of mitochondrias.

Interphotoreceptor matrix: The IPM occupies the space between the photoreceptor outer segments and the RPE. It is a viscous structure consisting of proteins, glycoproteins, GAGs and proteoglycans. Functionally, IPM plays a role in physiological attachment of retina, facilitation of phagocytosis and probably in alignment of photoreceptor outer segment. The interphotoreceptor retinoid binding protein (IRBP) accounts for 70% of the soluble proteins in the IPM. Its primary function is transport of retinoids between the photoreceptors and the RPE. Thus, minimizes the fluctuations in retinoid availability and protects the plasma membranes from the toxic effects of high retinoid concentration.

External Limiting Membrane

It is a fenestrated membrane through which the processes of the photoreceptors pass. It is not a true basement membrane and electron microscopy studies show that the ELM is formed by the zonula adherens between the Müller cells and the plasma membrane of photoreceptors.

Outer Nuclear Layer

This multilayered outer nuclear layer is formed by the nuclei of rods and cones. Rod nuclei forms the major bulk of this layer except in the foveal region which is dominated by cone nuclei.

Outer Plexiform Layer

This layer consists of synapses between photoreceptors with bipolar cells and processes of the horizontal cells. Thus, it represents junction of the end organs of visual pathway and its first-order neurons in the retina.⁵⁵ This layer is thickest at the macula and consists predominantly of oblique fibers from the fovea known as Henle's layer.

Fig. 1.11: Fundus fluorescein angiography showing typical petalloid leak in cystoid macular edema (CME).

Consists of intercellular junctions and synapses, which may act as a functional barrier to diffusion of fluids and metabolites. This functional barrier may retard or prevent the spread of exudates, hemorrhages, and cysts from spreading through the entire retinal thickness.
It is in a watershed zone of vascular supply of the retina and is sensitive to variations in the circulatory supply from either of the vascular sources, i.e., choroidal or retinal. Thus, it is vulnerable to metabolic insult due to senile choriocapillary atrophy or age-related thickening of Bruch's membrane.
The OPL is established site of formation of retinal cysts during aging, advanced stage of which is represented by senile retinoschisis.
OPL is the most frequent site of exudate and hemorrhage accumulation in patients with retinal vasculopathy.

Inner Nuclear Layer

The inner nuclear layer consists of 8–12 rows of closely packed nuclei of the bipolar cells, horizontal cells, amacrine cells, interplexiform cells and supportive Müller cells.⁵⁵

Horizontal cells: The flat horizontal cells serve to modulate and transform visual information received from the photoreceptors. The concentration of horizontal cells is highest at the fovea and decreases towards the periphery. Horizontal cells are seen by light microscopy to have short processes and long processes that stem from the base of a short process or directly from the perikaryon. A characteristic feature of horizontal cells 11is the presence of an intracytoplasmic inclusion, the Kolmer crystalloid or body.

Bipolar cells: The bipolar cells are the second-order neurons in the visual pathway and are radially oriented in the retina. Nine main type of bipolar cells have been distinguished⁷¹ based on morphology and synaptic relationships.

Rod or mop
Invaginating midget
Flat midget
Flat diffuse or brush
Invaginating diffuse
On-center blue cone
Off-center blue cone
Giant bistratified
Giant diffuse invaginating.

Rod bipolar cells constitute 20% of the total population and are present 1 mm from the fovea. The invaginating midget cells are the smallest of the bipolar cells. There is a one-to-one ratio (1:1) of midget bipolar cells and the cones at the fovea.⁷¹ All types of bipolar cells have a similar ultrastructure. Most of the midget and diffuse bipolar cells are glutamatergic. A subpopulation of bipolar exhibits strong immunolabeling for glycine. The bipolar cells transmit signals from the photoreceptors and pass it on to the ganglion cells either directly or indirectly via amacrine cells.

Müller cells: Müller cells are the largest of all cells in the retina, and extend from the external to the ILMs. The cell bodies of Müller cells occupy most of the inner intermediate layer of the inner nuclear layer. Embryonically, Müller cells are derived from the inner layer of the optic vesicle. During development of the retina, these cells have an important role in the orientation, displacement and positioning of the developing neurons. They are the principal glial cells of the retina and conserve the structural alignment of its neuronal elements.

Amacrine cells: The cell body of the amacrine cells lie internal to the nuclei of the Müller cells. Each amacrine cell has a single process with extensive branching. The amacrine processes thus extends over a wide area in the inner plexiform layer. The neurotransmitter substances associated with amacrine cell function include both neuroactive substances [acetylcholine, gamma aminobutyric acid (GABA), glycine, dopamine, serotonin] and neuropeptides (cholecystokinin, enkephalin, glucagon, neurotensin, somatostatin, substance P, neuropeptide Y and vasoactive intestinal peptide). Two or more of these neuromodulating chemicals may be present in one cell. Most amacrine cells contain γ-aminobutyric acid and glycine, which have an inhibitory action on the ganglion cells.^72,73

Inner Plexiform Layer

It is the junction of the bipolar cells (the second-order neurons) with ganglion cells (the third-order neurons). The bipolar cells act as afferent and the ganglion cell acts as efferent to this layer. The amacrine cells mediate interactions within the layer which in turn provides input to the interplexiform cells. Thus, bipolar cells synapse with process of amacrine cells and the dendrites of ganglion cells. In addition to synapses between various cell types, this layer contains processes of the Müller cells and an abundant microvasculature. This layer is absent from the foveola. The dendrites of all bipolar cells have receptors for GABA-A suggesting that inhibition feedback from amacrine cells is mediated by GABA-A.

Ganglion Cell Layer

This layer is composed of the cell bodies of ganglion cells (the third-order neurons). Processes of Muller cells and branches of retinal vessels are also present. It forms two layers at the temporal side of the optic disc, about 6–8 layers at the edge of the foveola and is single layered in the peripheral retina. Ganglion cell layer is absent at foveola and at optic nerve head. Ganglion cells are packed closely together except at the periphery. There are about 1.2 million ganglion cells in the retina with overall cone: ganglion cell ratio of 2.9:1–7.5:1. The axons of these cells form the optic nerve. The ganglion cells can be divided into two major types—M cells and P cells. The M cells project to the magnocellular layer of the lateral geniculate body and exhibit nonopponent responses. The P cells project to the parvocellular layers of the lateral geniculate body. The P cells are further divided into P1 cells or midget and P2 or small bistratified.

Nerve Fiber Layer

The nerve fiber layer contains the axons of the ganglion cells (also called as the centripetal fibers), a rich capillary bed and centrifugal (efferent) fibers along with glial cells. The axons remain unmyelinated until they reach the lamina cribrosa of the optic nerve. The nerve fiber layer is thickest at the nasal edge of the disc (about 20–30 µm). The thickness decreases from the optic disc to the ora serrata. The papillomacular bundle represents the thinnest portion of the nerve fiber layer around the optic disc.

Arrangement of nerve fibers in the retina (Fig. 1.12):

The fibers within the nerve fiber layer course parallel to the surface of the retina, compared to the rest of the fibers of the sensory retina which are perpendicular to it.
Nasal retina: Fibers of the nasal half of the retina reach to the optic nerve as superior and inferior radiating fibers.
Temporal retina: Fibers from the macular region pass straight in the temporal part of the optic disc as papillomacular bundle.
Fibers from rest of the temporal retina arch above and below the macular and papillomacular bundle as superior and inferior arcuate fibers.

Nerve fiber layer thickness at the disc (From thinnest to thickest): In eyes with normal optic nerves, the RNFL at the 12optic disc border shows a double hump configuration with the mean highest mean thickness in the inferior quadrant (mean ± S.D: 266 ± 64 micron), followed by the superior quadrant (240 ± 57 micron), the nasal quadrant (220 ± 70 micron), and finally the temporal quadrant (170 ± 58 micron).

Clinical implications

Glaucoma: One of the earliest indications for glaucoma (preperimetric glaucoma) is retinal nerve fiber layer (RNFL) thinning. Arcuate nerve fibers (temporal) are most sensitive to glaucomatous damage and lead to corresponding visual field loss documented on perimetry. Macular fibers occupying the lateral quadrant are most resistant to glaucomatous damage and thus cause a preservation of the central vision even in most advanced stages of glaucoma.
Papilledema: Papillomacular fibers being the thinnest fibers form the last affected portion of the optic disc in papilledema. Papilledema appears first of all in the thickest quadrant of the optic disc, i.e., upper nasal and lower nasal quadrants.

Fig. 1.12: Arrangement of nerve fibers in the retina.

Internal limiting membrane: It forms the innermost layer of the retina. The ILM along with posterior vitreous cortex form the vitreoretinal interface. The fibrils of the vitreous merge with the internal lamellae of the ILM. This layer mainly consists of a periodic acid-Schiff (PAS) positive true basement membrane, unlike ELM. The ILM consists mainly of four elements:

Collagen fibrils
Proteoglycans of the vitreous (especially HA)
The basement membrane
The plasma membrane of the Müller cells and other glial cells of the retina.

In posterior retina, the ILM attains a thickness of 0.5–2.0 µm. It is thickest at the fovea, but is absent at the edge of the optic disc. The ILM generally thickens with aging and is interrupted at the ora serrata.

Fig. 1.13: Schematic diagram showing arrangement of retinal capillaries.

Blood Supply of the Retina (Fig. 1.13)

Outer four layers of retina: Receive nutrition from the choriocapillaris.
Inner six layers of the retina: Receive blood supply from the central retinal artery.
Capillary network exists in most of the extramacular fundus, which can be divided into superficial and deep.
- Superficial capillary network lies at the level of the nerve fiber layer.
- 13Deep capillary network lies between inner nuclear layer and outer plexiform layer.
Fovea is an avascular zone and receives its nutrition from choriocapillaris.
Macula receives its blood supply by branches from superior and inferior temporal branches of the central retinal artery. In approximately 10% cases, cilioretinal artery (from the ciliary system) is seen to supply the macula.
Foveal avascular zone is a capillary-free zone in the fovea of about 500 µm in diameter.
Parafoveal zone has a three-layered capillary network, which becomes four-layered in the peripapillary region to support the extremely thick nerve fiber layer.

Choroidal circulation: The nutrition to retina is from two different circulatory systems—the retinal circulation and the choroidal or uveal circulation. Both the circulatory systems are derived from the ophthalmic artery, the first branch of the internal carotid artery.

The major branches of the ophthalmic artery are the central retinal artery, the posterior ciliary arteries, and the muscular branches. Typically, two posterior ciliary arteries exist—a medial and a lateral—but occasionally a third superior posterior ciliary artery is seen.

The posterior ciliary arteries further divide into two long posterior ciliary arteries and numerous short posterior ciliary arteries. The outer layer of choroidal vessels, known as the Haller's layer merges with smaller vessels in middle Sattler's layer. The posterior choriocapillaris is supplied by these short posterior ciliary arteries, which enter the choroid in the peripapillary and submacular region. The anterior choriocapillaris is supplied by recurrent branches from the long ciliary arteries and anterior ciliary arteries. The watershed zone of the anterior and posterior choroidal circulatory systems is at the equator.

The choroid is by far the most vascular portion of the eye with following functions:

It is responsible for the nourishment of the photoreceptor–RPE complex.
Acts as a heat sink and removes the large amount of heat that develops as a result of the metabolic processes initiated when photons strike the photopigments and the melanin of the RPE and choroid.
It probably also serves as a mechanical cushion for the internal structures of the eye.

PHYSIOLOGY OF THE VISION

The term visual cycle was given by George Wald (1906–1997; who received the Nobel Prize in 1967 for this cycle named after him). It is a chain of biochemical reactions following exposure to light so that a steady state equilibrium is maintained between the rate of photo decompensation and photo regeneration. The processing of visual information begins with the detection of light by photoreceptor cells. In the photoreceptors, there occurs a cycle of rhodopsin bleaching and regeneration.

Rhodopsin is a photosensitive visual pigment in the rod outer segments. It has protein opsin and a carotenoid called retinal. The first step in the visual cycle is the absorption of photon's energy by 11-cis retinal inducing it to convert into a more stable all-trans-retinal. This process is called bleaching. Then a series of conformational changes in rhodopsin leads to the formation of photoexcited metarhodopsin II (Fig. 1.14). Rhodopsin (500 nm)→Bathorhodopsin (543 nm) →Lumirhodopsin (497 nm)→Metarhodopsin-I (480 nm) →Metarhodopsin-II (380 nm).

After release from opsin, the fate of all-trans-retinal differs in rods and cones. In cones, re-isomerization can occur in neural retina regenerating 11-cis retinal that recombines with the bleached rhodopsin. In rods all-trans-retinal is converted to all-trans-retinol by retinol dehydrogenase and transported to interphotoreceptor retinoid binding protein to the RPE. In the RPE, all-trans-retinol is esterified to all-trans-retinyl ester which is re-isomerized to 11-cis-retinol. It is then stored in RPE as 11-cis-retinyl palmitate or converted back to 11-cis-retinal and transported to rod outer segments.

Fig. 1.14: Wald's visual cycle.(NAD: nicotinamide adenine dinucleotide; NADH: nicotinamide adenine dinucleotide reduced)

14Transduction

The process of translation of the information in light stimulus into electrical signals is known as visual transduction. Metarhodopsin-II activates the heterotrimeric G-protein—transducins. Transducin is a G protein that consists of alpha, beta and gamma subunits. It binds to opsin after a conformational change at metarhodopsin II. Guanosine diphosphate (GDP) is bound to the T alpha subunit and during transduction, it is displaced by guanosine-5’-triphosphate (GTP)-causing transducing molecule to separate into a T-alpha GTP and a beta-gamma complex. Activated phosphodiesterase (PDE) catalyzes conversion of cyclic adenosine monophosphate (cAMP) to 5’ guanosine monophosphate (GMP) (Fig. 1.15).

Fig. 1.15: Schematic diagram depicting the process of transduction(GDP: guanosine diphosphate; GTP: guanosine-5’-triphosphate; GMP: guanosine monophosphate; cGMP: cyclic guanosine monophosphate; PDE: phosphodiesterase)

This leads to the hyperpolarization of these cells, which changes the transmission of glutamate-mediated neuronal signals. This initiates a downstream action potential by ganglion cells that convey signals to the brain. The cells return to its resting state (depolarized) by production of cyclic GMP by guanylate cyclase (GC) activated by Ca⁺² entering the cells.

Recoverin Cycle

Inhibition of the photocascade can occur at several stages. Activated rhodopsin may be switched off by phosphorylation or by binding of arrestin to its phosphorylation sites. Activated PDE will continue to hydrolyze cyclic GMP until it recombines with the PDE gamma subunits. Tα-GTP is then inactivated by hydrolysis to Tα-GDP which recombines with Tβγ complex. During light stimulation calcium influx through cGMP controlled channels is inhibited. This stimulates the activity of recoverin which in turn activates GC. This enzyme increases cyclic GMP production leading to reopening of ion channels and membrane depolarization.

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Chapter Notes

Clinical Anatomy and Physiology of Vitreous and RetinaCHAPTER 1

Clinical implication

Clinical implication

Clinical implication

Clinical implication

Clinical implications

Clinical implication