Massachusetts Eye and Ear. Flaum Eye Institute
Definitions:
Retina: the layer of nerve cells lining the back wall inside the eye that senses light. The retina communicates with your brain to help you see. The retina is responsible for visual prcoessing and converts light energy from photons into electrical signals. The retina consists of five main types of nuerons: photoreceptor cells (including rod cells and cone cells), horizontal cells, bipolar cells, amacrine cells and retinal ganglion cells.
The layer closest to the external surface of the eyeball consists of two kinds of photoreceptor cells; the rods and cones. Rods, which get their name from the shape of their outer segment, are responsible for black and white vision when the illumination is dim. In contrast, cones are responsible for high visual acuity (sharpenss) and oclar vision. Cones have a cone shaped outer segment. The next layer contains bipolar cells and the layer closest to the cavity of the eye is composed of ganglion cells. Thus, light must first pass through the ganglion cells and bipolar cells in order to reach the photoreceptors. This is because photoreceptors are located at the back of the retina, and light travels through the outer layers of the retina, including the ganglion cell layer and the inner plexiform layer, before reaching them. When light enters the eye, it first encounters the ganglion cells and then the inner plexiform layer before reaching the photoreceptors at the back of the retina. Light interacts with the photoreceptors, causing them to produce electrical signals. These signals then travel through the bipolar cells and eventually to the ganglion cells, which send visual information to the brain.
The rods and cones synapse with the bipoplar cells, and the bipolar cells synapse with the ganglion cells, which transmit impulses to the brain via the optic nerve. Ganglion cells are the only neurons of the retina capable of sending action potentials to the brain. The flow of sensory information in the retina is thus opposite to the path of light through the retina.
Ganglion cells are located in the inner layer of the retina, closer to the front of the eye and the optic nerve, which connects to the brain. Light needs to travel through the retina to reach the photoreceptors at the back. When light enters the eye and strikes the retina, it must pass through all the neuronal cell layers before reaching and activating the photoreceptors. The photoreceptors then initiate the synaptic communication back toward the ganglion cells.
Due to its isolation from the other tissues and organs by the inner and outer blood-retina-barrier (BRB), the retina is considered as an immune-privileged organ that serves as an ideal target for gene therapy and gene editing. (Jin “Reitnal organoid and gene editing for basic and translational research” Vision Research 210 (2023)
Macula: An area in the center of the retina that helps you see objects in front of your clearly.
Cornea: the window in the front of your eye that focuses light. The cornea is located at the very front of the eye, while the retina is situated at the back of the eye. The cornea is the clear, outer covering that helps focus light, and the retina is the light-sensitive tissue that converts light into nerve signals
The cornea is a transparent tissue that works as a “lens” within the eye to focus light onto the retina. Consequently, it must retain its transparency if it is to serve this function. This transparency is maintained by the corneal endothelium, which regulates water flow between the aqueous humor and the corneal stroma by pump-and-leak barrier functions. However, the corneal endothelia cells (CECs) that perform this function have severely limited proliferative capacity, so any severe damage to the corneal endothelium, such as that arising from pathological conditions like Fuchs endothelia corneal dystrophy or from iatrongenic damage during cataract surgery, cuases irreversible cell loss. (Koizumi, “Feasibility of a cryopreservation of cultured human corenal endothelial cells, PLOS One, June 21, 2019).
Cornea endothelium (CE):
The CE plays a critical role in the regulation of corneal hydration, maintaining corneal thickness and keeping the cornea transparent. The human CE has very limited propensity to proliferate in vivo. Accordingly, in order to replace dead of damaged corneal endothelial cells (CECs), the existing cells spread out to maintain functional integrity and sustain corneal deturgenscence. In a situation where an individual experiences accelerated or acute corneal endothelial cell loss due to either accidental or surgical trauma, endothelial dysfunction of the CE layer may occur. This results in their inability to pump fluid out of the stroma, causing stromal and epitheilial edema, loss of corneal clarity and visual acuity and will eventually lead to the clinical condition of bullous keratopathy. (Mehta, “Cultivation of human corneal endothelial cells isolated form paired donor corneas” 2011).
Optic nerve: the nerve in the back of your eye that sends signals to your brain.
Dilation: a procedure in which a doctor uses eye drops to widen your pupil, allowing for a close look inside your eye.
Legal blindness: when vision can not be made better than 20/200, meaning that you have to be 20 feet or lower to see an object that someone with normal vision could see 200 feet away.
Sensory Transduction in Photoreceptors:
The transduction of light energy into nerve impulses follows a sequence that is the opposite of the usual way that sensory stimuli are detected. In the dark, the photoreceptor cells release an inhibitory neurotransmitter that hyperpolirizes the bipolar neurons. This prevents the bipolar neurons from releasing excitatory neurotransmitter to the ganglion cells that signal to the brain. The production of inhibitory neurotransmitter by photoreceptor cells is due to the presence of ligand gated Na+ channels. In the dark, many of these channels are open, allowing an influx of Na+ which depolarizes the membrane of photoreceptor cells. In this state, the cells produce inhibitory neurotrasmitter that hyperpolarizes the membrane of bipolar cells.
In the light, the process works in the opposite way. When a photopigment absorbs light, cis-retinal isomerizes and dissociates from the receptor protein, opsin, in which is known as the bleaching reaction. As a result of this dissociation, the opsin receptor protein changes shape, activating its associated G protein. The activated G protein then activates its effector protein, phosphodiesterase, which cleaves cGMP to GMP. The loss of cGMP causes the cGMP gated Na+ channels to close, reducing the dark current. Each opsin is associated with over 100 regulatory G proteins, which, when activated, release subunits that activate hundreds of molecules of the phosphodiesterase enzyme. Each enzyme molecule can convert thousands of cGMP to GMP, closing the Na+ channels at a rate of about 1000 per socond and inhibiting the dark current. The absorption of a single photon of light can block the entry of more than a million Na+, without changing K+ permeability. The photoreceptor becomes hyperpolarized and releases less inhibitory neurotransmitter. Freed from inhibition, the bipolar cells release excitatory neurotransmitter to the ganglion cells, which send impulses ot the brain.
Evolution of the Eye:
In the early 19902, biologists studied the development of the eye in both vertebrates and insects. In each case, a gene was discovered that codes for a transcription factor important in lens formation; the mouse gene was called Pax6 and the fly gene was termed eyeless. A mutation in the eless gene led to a lack of production of the transcription factor and compelte absence of eye development, giving the gene its name. When the genes were sequenced, it became apparent that they were highly similar. In fact, insertion of the mouse version of the Pax6 into the genome of a fuit fy created a transgene fly. In this fly, the Pax6 gene was turned on by regulatory factors in the fly’s leg and an eye formed ont he leg of the fly.
Common Vision Disorders
The incidence of vision related disorders increases significantly with advancing age. The most serious age related disorders include cataract, glaucoma, and age-related macular degeneration (AMD). Collectively, these three diseases account for about 65% of new cases of legal blindness in the U.S. 8.4 million office visits and 426k hospitalizations annually are attributable to these and related conditions (WO 95/17673).
Age-Related Macular Degeneration (AMD)
Cataract is associated with an increase in the pressure inside of the eye “intraocular pressure or IOP” which eventually results in the degeneration of a portion or the retina. Once detected, glaucoma usually can be arrested by treatment with pressure lowering drugs. Delayed detection and treatment, however, leads to impaired vision and eventual blindess. Glaucoma can be effectively treated by replacing diseased lens with a plastic intraocular lens or IOL.
The number one reason you have cataracts is age. By 50, we can usually start to see some changes in the transparency of the lens that are an indication of early cataracts.
Corneal endothelia decompensation:
In the past the only treatment for corneal endothelial decompensation was transplantation of a donor corea. However, tissue engineering technology is now receiving increased attention as a way to overcome problems of corneal transplantations, which include a shortage of donor corneas, late graft failure due to continuous cell loss, graft rejection and the learning curve involved in performing corneal transplant procedures. (Koizumi, “Feasibility of a cryopreservation of cultured human corenal endothelial cells, PLOS One, June 21, 2019).
Koizumi, (“Feasibility of a cryopreservation of cultured human corneal endothelial cells, PLOS One, June 21, 2019) disclosed in 2013 cell based therapy involving injection of a suspension of cultured human corneal endothelial cells (HCECs) in combination with a Rho kinase inhibitor, into the anterior chamber. All 11 cases recovered corneal transparency and none experience any severe adverse effects. The HCECs were obtained form donor corneas and expanded in vitro culture. The HCECs were harvested form a culture plate, placed in a tube in the form of a cell suspension and immediately transported to the operating room in the same facility. (Koizumi, “Feasibility of a cryopreservation of cultured human corenal endothelial cells, PLOS One, June 21, 2019).
–Cell Culture of human corneal endothelial cells (HCECs):
Mehta, (“Cultivation of human corneal endothelial cells isolated form paired donor corneas” 2011) discloses culturing isolated primary hCECs in 4 different medium, expanding the cells for two passages and anlyzing them for their propensity to adhere and proliferate., their expression of characteristic corneal endothelium markers; Na+K+/ATPase and ZO-1 and (3) their cellular morphology. hCECs established in the four media exhibited different proliferation profiles with striking norphological differences. Corneal endothelial cells cultured in M1 and M3 could not be propagated beyond the frist and secone passage, respectively. The hCECs cultured in M2 and M4 were significant more proliferative and epxressed markers characteristic of human coneal endothelium.
–Cryopreservation of human corneal endothelial cells (HCECs):
Koizumi, “Feasibility of a cryopreservation of cultured human corenal endothelial cells, PLOS One, June 21, 2019) further screened several cyroperservation reagents for their effectiveness in preserving HCECs. Bambanker hRM enabled the cryopreservation of HCECs by maintaing cell viability and cell density.
Glaucomas are a group of neuropathic eye diseases characterized by increased intraocular pressure (IOP) and damage to the optic nerve. Glaucoma can lead to reduction or loss of vision, and is the second leading cause of blindness. Current treatments involve reduction of intraocular pressure by chemical or mechanical means. For example, the most common treatment is trabeculectomy, a surgical procedure in which part of the trabecular meshwork of the eye is removed to allow drainage of the aqueous humor out of the eye into the conjunctiva, thereby reducing intraocular pressure. However, trabeculectomy is generally followed by inflammation and fibrosis.
A comprehensive dilated-eye exam is the best way to detect glaucoma. Every adult over 50 should get this test every year, but if you have a fmaily history of glaucoma, you may need the test more often. If glacoma is caught early, it is a very treatable disease. When diagnosied late or not at all, glaucoma is extremely aggressive because the optic nerve is already damaged, and a damanged nerve is more vulnerable. Later-stage glaucoma attacks central vision and can cause irreversible blindess.
Corneal Neovascularization (KNV)
The cornea bears an “angiogenic privilege” and is avascular allowing maximal entry of incident light. This angiogenic privilege is maintained by a fine balance between anti-angiogenic factors and angiogenic factors in the cornea. Insults of chemical, mechanical or infectious nature can trigger inflammatory and immune-mediated pathways which upregulate expression of VEGF (vascular endothelial growth factor), the key player in KNV, and its signalling cascades. Common infectious diseases associated with KNV are the following:
1. Aspergillosis, Candidiasis, Chamydia trachomatis, Fusarium, Herpes simplex keratisis:
Herpes stromal keratitis (recurrent infection on the cornea by herpes simplex virus) is the most common cause of infectious corneal blindness in the western world. In the US an estimated 400,000 persons are affected, with 20,000 new cases occurring annually. Each episode increases the risk of future episodes. Current treatment consists of topical steroids in addiiton to prophylactice oral (acyclovir or valacyclovir) or topical (trifuridine) anti-viral drug therapy. Despite this treatment, patients develop severe corneal scarring due to repeated episodes of the disease, which often require corneal transplantaiton.
2. Onchocerciasis, Pseudomonas, Syphillis:
Common inflammatory conditions assocaited with KNV are graft rejection, acne rosacea, Stevens-Johnson syndrome, GVHD, Pemphigoid and Atopic conjunctivitis.
Diabetic Retinopathy:
Uncontrolled, elevated blood sugar damages blood vessels in the retina, cuasing a leakage of fluid into these tissues, a conditions called diabetic retinopathy. The early stage of the disease can be controlled with lifestyle changes by keeping A1c (a diabetes test result) below 6.5 with diet and exercise, as well as maintaining healthy blood pressue and lipid levels.
Diabetic retinopathy is the top cause of blindness in working age adults in the U.S. The later, more symptomatic stage of DR is called profliferative, in which new, but weeak, blood vessels grow in the retina to compensate for those that were damanged by blood sugar. When caught at this later stage, when symptoms like burriness or glare arise, you may need additional eye injections or even surgery.
Ocular Fibrosis:
–Treatments for Ocular Fibrosis:
A proposed method of treatment of ocular fibrosis is inhibition of certain lysyl oxidase-type enzymes which have been found to occur in parallel with the fibrotic damage that can follow trabeculectomy. A lysyl oxidase-type enzyme refers to a member of a family of proteins that catalyzes formation of aldehydes from lysine residues in collagen and elastin precursors. The aldehyde residues of allysine are reactive and can spontaneously condense with other allysine and lysine residues, resulting in crosslinking of collagen molecules to form collagen fibrils. The first member of this family to be isolated was lysyl oxidase, also known as protein-lysine 6-oxidase, protein-L lysine or LoX. Additional lysyl oxidase type enzymes were subsequently discovered and termed LOX-like or LOXL Although all lysyl oxidase-tyep enzymes share a common catalytic domain, they differ, particularly in their amino terminal regions.
Chemical Injuries of the eye: are opthalmic emergecies that require immediate evaluation adn management. These in juries often result in significant occular morbidity. Injuries caused by alkali agents are more common and generally more serious than those casued by acids. There are three main goals in managing a chemical injury,: enhance recovery of the corneal epithelium; augment collagen synthesis while minimizing collagen breakdown and sterile ulceration and control inflammation. (US 2020/0289580).
Dry Eye Disease: (see outline)
Rare Eye Diseases
Purtscher’s retinopathy: is a rare retinal disorder characterized by acute visual loss and retinal findings such as cotton-wool spots, intraretinal hemorrhages and retinal whitening following head or chest trauma. When the etiology is not a trauma, the disease is called Purtscher-like retinopathy. Nuermous conditions such as acute pancreatitis, connetive tissue disorders, autoimmune diseases, pregnancy-related diseases, and thrombotic microangiopathic diseases can cause Purtscher-like retinopathy. It has also been associated with aHUS (Turk J. Ophthalmol 47; 6: 2017).
Retinoblastoma (Rb) is the most common intraocular malignancy, with a high incidence in children aged 2-3 years and an average survival rate of only 30% worldwide. (Jin “Reitnal organoid and gene editing for basic and translational research” Vision Research 210 (2023)
Retinitis pigmentosa (RP) (“night blindness”): is a congenital inherited eye disease with an incidence rate of about 1/4000 worldwide. The main symptoms of the patients are decreased visual acuity and reduced visual field at night. As the disease worsens, the patients’ vision gradually decreases during the day and they even become totally blind. 3D organoids carrying mutations in one of the most common RP causing genes, RPGR, has been denerated. (Jin “Reitnal organoid and gene editing for basic and translational research” Vision Research 210 (2023)
Models for Retinal Diseases:
Three-dimensional (3D) culture organoids: consist of a suspension growth of a diverse set of cells in a qliuqid medium or semi-liquid matrix, can mimic the development process of many tissues and organs. 3D retinal organoids (ROs) with human retinal charateristics can be differentiated form embryonic stem cells and induced pluripotent stem cells (iPSCs) under certain conditions. They not only contain a variety of retinal cell types such as photoreceptors, ganglion cells and RPI, but also form laminal layers very close to teh retina. They have many advantages include (1) use to study the retina development in vitro and the mechanism of retinal diseases, (2) high-throughput drug screening, (3) preclinical evaluation of the treatment for reitnal degenerative diases and the establihsment of personalized therapies such as gene editing and stem cell replacement thearapy.Composite organoids such as the eye-brin organoid, SEAM and assembloids, consist of the retina and other neural or ocular tissues. (Jin “Reitnal organoid and gene editing for basic and translational research” Vision Research 210 (2023)
Three-dimensional bioprinting pints multiple cells, tissues, and organoids in a programmed sequence, such that an assembloid in designed can be created from scratch. For example, iPSC-derived fibroblasts, endothelial cells and pericytes have been bioprinted on the basal side of a biodegradable scaffold, and iPSC derived RPE placed on top of the scaffold. This 3D model closely recapitulates the 3D outer-blood-retina-barreir of the eye under healthy and diseased conditions. (Jin “Reitnal organoid and gene editing for basic and translational research” Vision Research 210 (2023)
Treatments of Eye Diseases:
Gene editing: can be in vitro gene editing, where patients’ induced pluripotent stem cells (iPSCs) are corrected in vitro and then the validated retinal stem cells are transplanted into the eye or in vivo gene editing, which uses viruses or liposomes and other vectors to deliver gene editing substances such as DNA, protein or mRNA directly intothe patients’ eye for gene editing to acheive the treatment purpose. With the continous updating and upgrading of gene editing tools, researchers can select appropriate gene editing tools to modify disease genes according to the characteritics of disease. (Jin “Reitnal organoid and gene editing for basic and translational research” Vision Research 210 (2023)
–Prime editing (PE): uses the fusion protein of Cas9 nickase and engineered revsearch transcriptase such that the target fragment can be transferred to the target DNA site and editing by prime editing buide RNA (pegRNA). The advantage of PE is that it can precisely target insertion, deletion, and 12 types of point mutations without the need for DNA double-strand breaks and donor DNA tempaltes. Compared with the Cas9-mediated HDR, alhtough the editing efficiency is imilar or lower, PE produces fewer byproducts in the boidng process, and the off-target or indels are much lower than those of HDR.
Prox1 protein is a key factor preventing retinal regeneration in mammals, but blocking its activity can restore this ability. Researchers have discovered that Prox1, produced in surrounding neurons and transferred to Müller glia (MG) cells, inhibits MG from reprogramming into retinal progenitor cells, which are necessary for regeneration. By blocking this transfer, especially with antibodies, they have been able to induce retinal regeneration in mice, even restoring vision in models of retinitis pigmentosa