Ординатура / Офтальмология / Английские материалы / The Retina and its Disorders_Besharse, Bok_2011

They also possess minimal to no elastic lamina, but have a relatively dense reticular lining. The adventitia is continuous with collagen fibers of the intravascular space, and the endothelium is covered by a basement membrane.

Precapillaries and capillaries have a very thin wall and their basement membranes stain positively with the periodic acid-Schiff reaction. The endothelium, basement membrane, and mural cells of the venous structures are similar to that of the capillaries. The veins of the optic nerve are composed of an inner lining of endothelial cells, elastic fibers, thin adventitia, and intermittently, irregularly spaced, smooth muscle cells. When the venules enlarge, the basement membrane thickens accordingly.

Within the anterior ONH, the histology of the vasculature resembles that of the CNS, as the vessels contain a nonfenestrated endothelium with tight junctions. Capillaries predominate within the anterior ONH, and larger vessels are seldom visualized. Posteriorly, larger arterioles may be seen entering the lamina cribrosa. These capillaries and arterioles lack the internal elastic lamina and elastic tissue in the media that is characteristic of the larger vessels of the laminar and retrolaminar regions.

Regulation of Ocular Blood Flow

Autoregulation is the intrinsic ability of a system to maintain constant blood flow despite changes in perfusion pressure and local vascular parameters. The intrinsic control of blood flow involves chemical secretion by the cells in the immediate vicinity of the blood vessels. Within the eye, autoregulation is defined as local vascular constriction or dilation to alter vascular resistance, thereby maintaining a constant nutrient supply in response to perfusion pressure changes. Perfusion pressure is equal to the difference between the mean arterial pressure (MAP) and the venous pressure. The MAP is defined as the diastolic blood pressure plus one-third of the difference between systolic and diastolic blood pressure. Since the pressure in the central vein is normally slightly higher than the intraocular pressure (IOP), the IOP is used as an estimate of ocular venous pressure:

Mean ocular perfusion pressure ¼ 2 MAP IOP 3

MAP ¼ Diastolic BP þ 1 ðSystolic BP Diastolic BPÞ 3

The maintenance of appropriate ocular blood flow is challenged by physiological changes in IOP, blood pressure, ocular perfusion pressure, and local tissue metabolic demands. As per Poiseuille’s law, vascular resistance is inversely proportional to the fourth power of the radius of a vessel, and is directly proportional to the blood viscosity

and length of the vessel. In the eye, vascular resistance is therefore dependent on the regulation of vessel diameter. In normal subjects, autoregulation is usually maintained until the IOP reaches approximately 40–45 mmHg. Failure of stable blood flow regulation may lead to ischemic damage of the optic nerve or retinal ganglion cells, which likely contributes to further impairment in vascular regulation. Several mechanisms, including neurogenic-, metabolic-, myogenic-, humoral-, and endothelial-mediated factors, have been demonstrated to play a role in the vascular regulation of ocular blood flow.

Fed primarily by the CRA, the retinal system is generally a low-flow, constant rate system that supplies a highly metabolically active tissue. Although it may only account for as little as 15% of the total ocular circulation, the retinal circulation is capable of providing relatively constant blood flow over a substantial range of IOPs. The retinal and anterior optic nerve head do not possess direct autonomic innervations. Although the retinal and optic nerve head have adrenergic and cholinergic receptors their role remains unclear. Consequently, retinal blood flow is locally autoregulated.

Several vasoactive molecules mediate retinal vascular autoregulation. Endothelial tone is determined by the balance between the vasoconstricting and vasodilating effects of secreted factors. Nitric oxide (NO) is produced by the oxidation of L-arginine by endothelial-derived nitric oxide synthase, which is present in both a constitutively active, membrane-bound form, and an inducible, cytosolic form. NO diffuses to nearby pericytes and smooth muscle, where it activates guanylyl cyclase, leading to the increase of cGMP and subsequent vasodilation. There are numerous stimuli for the production of NO, including increased shear force, bradykinins, insulin-like growth factor 1, acetycholine, and thrombin. Additionally, NO also inhibits platelet aggregation, platelet granule secretion, and leukocyte adhesion.

Vasoconstriction of the retinal microvasculature is stimulated by several vasoactive molecules. Endothelins, the most potent vasoconstricting agents known, are molecules that bind to receptors on pericytes and smooth muscle cells. A second vasoconstrictive substance is angiotensin II. Angiotensinogen is an inactive molecule that is constitutively produced by the liver. In response to physiologic stimuli, the kidneys release renin, which converts angiotensinogen into angiotensin I. Angiotensin converting enzyme (ACE), which is present on the surface of luminal endothelial cells, converts angiotensin I to angiotensin II, and also inactivates bradykinin. Once in its active form, angiotensin II moderates retinal vasoconstriction through the activation of smooth muscle cells and pericytes.

The choroidal vasculature is controlled extrinsically through hormonal influence and stimulation from the autonomic nervous system. It is characterized as a highflow, variable-rate system which is tightly regulated by

the autonomic nervous system. The outer, larger vessels are composed of nonfenestrated endothelium, while the inner, smaller vessels form a richly anastomotic network of fenestrated capillaries. The vascular tone of the choroid is dominated by the sympathetic nervous system. Neurons course from the cranial cervical ganglion to the vascular bed, where vasoconstriction is mediated by the release of neuropeptide Y. The parasympathetic nervous system plays only a moderate and poorly defined role in the regulation of vascular tone. The presence of choroidal autoregulation is controversial and classically considered absent, though some autoregulatory response was reported during perfusion pressure changes.

Technology for Measuring Ocular Blood Flow

Color Doppler Imaging

Although originally developed for monitoring blood flow in the heart, carotid arteries, and peripheral vasculature, color doppler imaging (CDI) has proven to be useful in the study of retrobulbar vessels as well. By combining B-scan ultrasound images with velocity of measurements calculated from the Doppler shift of moving erythrocytes, CDI can be used to assess the velocity of blood flow through the retrobulbar vessels. The peak systolic velocity (PSV) and end diastolic velocity (EDV) can be measured and used to

calculate the mean flow velocity (MFV). An index of resistance (RI) can be calculated as RI = (PSV – EDV)/ PSV (Figure 8). Further research is necessary to determine the usefulness and application of the RI as it applies to the retrobulbar vasculature. Until recently, one critical limitation of this imaging technique had been that no quantitative information on vessel diameter was obtained, and therefore the calculation of total blood flow or flux was not possible. A recently developed analysis technique has made it possible to determine the diameter of the OA, allowing volumetric blood flow to be assessed. Further research is necessary to apply this technique to the assessment of blood flow through the other major vessels.

Angiography

Using sodium fluoroscein, angiography allows direct visualization of retinal blood flow. Most techniques measure the amount of time that it takes for the fluoroscein to pass through the retinal circulation. Using this technique, data can then be used to assess blood velocity through the retinal and optic disc circulation. Choroidal vessels are visualized in a similar fashion using indocyanine green, which is selected due to its increased binding to plasma proteins, preventing leakage from vessels to the surrounding tissue. Using videoangiography and scanning laser ophthalmoscopy, the arterio-venous passage time and retinal circulation time can be determined. This technology

also has limitations, as it is based on the assumption that all of the blood of an area supplied by a specific artery is drained by a single corresponding vein.

Blue Field Entotpic Technique

The blue field entoptic phenomenon is produced by the different absorption of red and white blood cells when the retina is illuminated with blue light. Red blood cells absorb the short wavelength light, while passing white blood cells do not, thereby allowing the flux of the perimacular white blood cells to be estimated. This technique is limited by the assumption that leukocyte flux is proportional to retinal blood flow.

Laser Doppler Velocimetry

Laser Doppler velocimetry (LDV) is a technique that uses the optical Doppler shift of light to measure the blood flow velocities in retinal arterioles and venules. The Doppler shift of light is directly proportional to the blood velocity when the vessel is illuminated with a laser beam. The flow velocity in the vessel can be extrapolated from the range of frequency shifts of the power spectrum of the reflected laser light. The maximum frequency shift corresponds with the maximum velocity in the center of the vessel, assuming laminar flow.

Retinal Vessel Diameters

The aforementioned techniques can be utilized to provide information about ocular blood velocity, but lack the ability to calculate flux or flow rate. To determine blood flow, it is necessary to accurately measure the diameter of the vessel through which the blood is flowing. There are now commercially available systems that permit real-time assessment of retinal vessel diameter. The retinal vessel analyzer (RVA) is composed of a fundus camera and a sophisticated computer system which record vessel size in real time. One such system is the Canon Laser Doppler blood flowmeter, which combines the techniques of LDV and RVA. This approach is still limited to the study of larger vessels, and can only be performed on patients with clear ocular media.

Laser Speckle Technique

When the rough surface of the fundus is illuminated by coherent light, the backscatter of light produces a rapidly varying pattern. The rate of variation of this pattern produced by this laser speckle phenomenon can be measured to compute an estimate of the velocity of blood flowing through the retinal vessels. This laser speckle technique is limited by the fact that it provides only velocity information, as it cannot determine vessel

diameter and therefore cannot be used to measure volumetric flow.

Laser Doppler Flowmetry

The scattering theory for light in tissue, formulated by Bonner and Nossal, assumes a randomization of light directions impinging on the erythrocytes. By directing a laser light on vascularized tissue that contains no large vessels, relative mean velocity of erythrocytes and blood volume can be calculated. Through two-dimensional mapping of the optical Doppler shift, blood flow to the juxtapapillary retina and ONH can be accurately evaluated. There is, however, significant variation in scattering of light between test subjects, likely a result of varying vascular densities and orientations. Thus, this technique can be used to compare changes in a given subject, but has less use in comparison of values between subjects.

Laser Doppler flowmetry can be combined with scanning laser tomography to provide a two-dimensional map of blood flow to the optic nerve and surrounding retina. The Heidelberg retina flowmeter (HRF) is one such commercially available system (Figure 9). This technique, however, is most sensitive to blood flow changes in the superficial layers of the ONH, and therefore provides only limited information about the deeper regions. This limits the ability to account for the retinal blood flow that is supplied by the choriocapillaris from the uveal system.

Pulsatile Ocular Blood Flow

Based on the changes in ocular volume and pressure during the cardiac cycle, it is possible to estimate pulsatile ocular blood flow. The pulse amplitude, which is the maximum IOP change during a cardiac cycle, is measured using a modified pneumotonometer. Alternatively, the

Figure 9 Confocal scanning laser Doppler flowmetry (Heidelberg retinal flowmeter) of optic nerve head and peripapillary retina. The patient is seated with the chin and forehead against the bar. The picture acquisition is performed without the need to dilate the patient’s eye. The conventional 40 40 pixel measurement window collects flow values in arbitrary units from the entire retina except for large vessels.

ocular fundus pulsation amplitude can be determined by calculating the maximum change in distance between the retina and the cornea during a cardiac cycle. These two values have been shown to be useful in the calculation of pulsatile ocular blood flow, but lack information of the nonpulsatile component of ocular blood flow.

Optical Doppler Tomography

This technique combines the high-resolution cross-sec- tional imaging of optical coherence tomography with laser Doppler to measure velocity of blood flow in retinal arteries in real time.

Future Studies

Each of the previously discussed technologies quantifies some aspect of ocular blood flow. It is impossible, however, to interpret the impact of any single blood flow parameter measured within a single vascular bed on total retinal metabolism. The measurement of ocular blood flow is only a surrogate assessment of the metabolic status of the retina. Direct measurement of retinal tissue oxygenation would reveal the true impact of ischemia on retinal ganglion cell health and function.

New and emerging tools that assess metabolic parameters may help to reveal the relationship between reductions in ocular blood flow and tissue hypoxia. For example, Michelson and colleagues conducted a study in which they used imaging spectrometry to measure the oxygen saturation in retinal arterioles and venules in patients with glaucomatous optic neuropathy. In all examined eyes, the arteriolar oxygen saturation and the retinal arterio-venous differences in oxygenation were found to significantly correlate with the area of the patient’s optic rim. Eyes with normal tension glaucoma, but not those with primary open angle glaucoma, showed significantly decreased arteriolar oxygen saturation. Although further advancements are still needed, these metabolic assessment tools may be very valuable in the evaluation of retinal hypoxia and in elucidating the effects of ocular ischemia. Obtaining accurate measurements of ocular tissue metabolism will greatly improve our understanding of disease

pathophysiology, and will therefore lead to advancements in both diagnosis and treatment.

See also: Optic Nerve: Inherited Optic Neuropathies; IOP and Damage of ON Axons; Ischemic Optic Neuropathy; Optic Nerve: Optic Neuritis; Retinal Ganglion Cell Apoptosis and Neuroprotection.

Further Reading

Bron, A. J., Tripathi, R. C., and Tripathi, B. J. (1997). The choroid and uveal vessels. Wolff’s Anatomy of the Eye and Orbit, 8th edn. London: Chapman and Hall Medical.

Drexler, W. and Fujimoto, J. G. (2008). State-of-the-art retinal optical coherence tomography. Progress in Retinal and Eye Research

27: 45–88.

Hardarson, S. H., Harris, A., Karlsson, R. A., et al. (2006). Automatic retinal oximetry. Investigative Ophthalmology and Visual Science

47: 5011–5016.

Harris, A., Jonescu-Cuypers, C. P., Kagemann, L., Ciulla, T. A., and Krieglstein, G. K. (2003). Atlas of Ocular Blood Flow. Philadelphia, PA: Elsevier.

Harris, A. and Rechtman, E. (2008). Optic nerve blood flow measurement. In: Yanoff, M. and Duker, J. (eds.) Ophthalmology, 3rd edn., ch. 10.8, section 2, pp. 52–55. Edinburgh: Elsevier.

Hayreh, S. S. (2001). Blood flow in the optic nerve head and factors that may influence it. Progress in Retinal and Eye Research

20: 595–624.

Hayreh, S. S. (2008). Patholophysiology of glaucomatous optic neuropathy: Role of optic nerve head vascular insufficiency.

Journal of Current Glaucoma Practice 2: 6–17. Mackenzie, P. J. and Cioffi, G. A. (2008). Vascular anatomy of

the optic nerve head. Canadian Journal Ophthalmology

43: 308–312.

Michelson, G. and Scibor, M. (2006). Intravascular oxygen saturation in retinal vessels in normal subjects and open-angle glaucoma subjects. Acta Ophthalmologica Scandinavica 84: 289–295.

Morrison, J. C. and van Buskirk, E. M. (1984). American Journal of Ophthalmology 97: 372–383.

Orgu¨l, S. and Cioffi, G. A. (1996). Embryology, anatomy, and histology of the optic nerve vasculature. Journal of Glaucoma 5: 285–294.

Orgu¨l, S., Gugleta, K., and Flamer, J. (1999). Physiology of perfusion as it relates to the optic nerve head. Survey of Ophthalmology 43: S17–S26.

Schmetterer, L. and Garhofer, G. (2007). How can blood flow be measured? Survey of Ophthalmology 52: 134–138.

Simon, B., Moroz, I., Goldenfeld, M., and Melamed, S. (2004). Scanning laser Doppler flowmetry of nonperfused regions of the optic nerve head in patients with glaucoma. Ophthalmic Lasers, Surgery, and Imaging 34: 245–250.

Glossary

BAC – Bacterial artificial chromosome. It is a DNA construct based on a functional fertility plasmid, used for cloning in bacteria. The bacterial artificial chromosome’s usual insert size is 150–350 kbp. BAX – Proapoptotic BCL2-associated X protein. BCL2 is an integral outer mitochondrial membrane protein that blocks the apoptotic death of some cells. Retrobulbar space – The area located behind the globe of the eye.

Synechia – An eye condition where the iris adheres to either the cornea (anterior synechia) or the lens (posterior synechia).

Tonometry – The procedure to determine the intraocular pressure.

TUNEL – Terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate nick end labeling for detection of DNA fragmentation resulting from apoptotic programmed cell death.

Glaucoma is a complex disease, the initiation and progression of which involves interactions between different parts of the eye and brain. It is difficult to perform experiments directed toward elucidating pathogenic molecular mechanisms and potential treatments for glaucoma in human subjects and, as a rule, only postmortem material can be used for biochemical analysis. Experiments in cell culture or organ culture systems may only partially reproduce the complexity of the natural ocular environment. It is now well recognized that animal models may provide a very useful tool for understanding the underlying molecular mechanisms involved in glaucoma and for identifying new genetic components of the disease, including both causative and modifier genes. In addition, appropriate animal models are used to develop and test new regiments of glaucoma treatment as a prerequisite for clinical trials in humans. A number of animal models of glaucoma have been developed over the years. Since elevated intraocular pressure (IOP) is the most important risk factor in glaucoma, most of the animal models of glaucoma are based on elevation of IOP by surgical procedures or by genetic manipulations. Several models used to study death of the retinal ganglion cells (RGCs) include optic nerve crush or transaction, intravitreal injection of excitory amino acids (glutamate and N-methyl-D-aspartic acid (NMDA)), or retinal ischemia. Although these are not true glaucoma

models, they allow the comparison of processes leading to RGC death induced by different initial insults. Such comparative analysis may lead to the identification of changes that are specific to glaucoma versus changes that are involved in more general RGC dysfunction. While none of the existing animal models is perfect, some of the existing models have been successfully used to uncover important features of glaucoma pathology in humans. Several factors should be considered in selecting a particular animal model of glaucoma for experimentation: (1) the similarity of the model visual system to the human eye; (2) the similarity in the time course of pathological changes in the model and human eyes; (3) ability to apply genetic manipulations; (4) training necessary to produce affected animals; (5) the size of the eye; (6) availability and difficulties of methods of analysis; (7) availability of animals; and (8) cost. This article briefly describes available animal models of glaucoma with emphasis on the strengths and weaknesses of each model.

Mammalian Models

Primate Models of Glaucoma

Monkey and human eyes are very similar both anatomically and functionally, making monkey models very attractive to study different eye pathologies including glaucoma. IOP in monkeys is measured using the same equipment that is used to measure IOP in humans. Moreover, tonometry and visual-field analysis can be performed in conscious, trained monkeys. This is an important factor since it is well documented that general anesthesia that is necessary to measure IOP in most other animal models results in rapid ocular hypotension. The main disadvantage of monkey models is that experiments with monkeys are expensive and require a highly skilled team of investigators. Moreover, large numbers of animals are required to assess effects of elevated IOP on the optic nerve head (ONH) and retina because of genetic variations between animals.

Several approaches have been used to develop pressureinduced glaucoma models in nonhuman primates. The most common method of IOP elevation in the monkey was originally developed more than 30 years ago and involves circumferential laser photocoagulation treatment of the trabecular meshwork. Several laser sessions are normally required to produce a sustained elevation of IOP. In the treated eyes, IOP rises several days after the laser treatment, normally to between 25 and 60 mmHg, and may last for more than a year. Other methods that have

been used to produce elevated IOP elevation in monkeys are less consistent than laser coagulation. They include injection of ghost red cells, latex microspheres, cross-linked polyacrylamide gels, or enzymes into the anterior chamber or application of topical steroids. A non-IOP-related monkey model of glaucoma involves the delivery of endothelin- 1 to the retrobulbar space through osmotic pump for 6–12 months; this induces ischemia and leads to the preferential loss of large RGC axons. Ischemia-induced focal axonal loss is similar to human glaucoma and this model may reproduce some aspects of normal tension glaucoma.

A number of important observations have been made using the monkey photocoagulation model. Apoptosis as the primary mechanism of glaucomatous RGC death was first demonstrated in this model before later being confirmed in other models and in human glaucoma. Multifocal electroretinogram (ERG) techniques were used in monkeys to demonstrate that not only RGCs but also cells in the inner and outer nuclear layers are damaged in advanced glaucoma. The monkey glaucoma model has been successfully used to study changes in retinal gene expression patterns after the induction of ocular hypertension. It is also being used to efficiently test new drugs and techniques to reduce

IOP. For instance, recombinant adenoviral delivery of the human p21WAF-1/cip-1 gene to cause cell cycle arrest before

filtration surgery in ocular hypertensive monkey eyes has shown a beneficial effect in long-term control of IOP.

Rodent Models of Glaucoma

Several rodent models of glaucoma have been developed over the last 20 years and new models are at different stages of development in several laboratories. These models have proven useful because the drainage structures of the rodent eye are similar to those in humans. Their utility was enhanced further by the development of new methods to measure IOP and analyze glaucomatous changes in these small eyes. Rodent models, and especially mouse models, are relatively cheap and allow extensive genetic manipulations. Rodent models are preferred when a significant number of animals are required to conduct genetic screens or to test different drugs and agents for neuroprotective or IOPlowering effects. One of the main disadvantages of rodent models is that there are anatomical differences between rodent and human eyes, including the arterial blood supply to the ONH and the absence of a well-developed, collagenous lamina cribrosa. These variations, as well as differences in general physiology, may explain why expression of certain genes in mouse and human eyes (e.g., mutated myocilin) have differential effects.

Rat Models

Rats are easy to handle. The relatively large size of their eyes allows multiple noninvasive IOP measurements

in awake trained animals with commercially available equipment. The TonoPen was the instrument of choice for IOP measurements for many years but has recently been superseded by an induction/impact tonometer, marketed as the TonoLab rebound tonometer. This instrument is easy to operate and can be used in both rats and mice.

Several rat models of pressure-induced glaucoma have been developed over the last 15 years. IOP elevation in the rat eye may be achieved by injection of hypertonic saline solution into the episcleral vein that leads to sclerosis of the aqueous humor outflow pathway. Sustained IOP elevation occurs 7–10 days after injection in most but not all rats. The saline injection generally produces a range of IOP elevation in different animals from a very minimal rise to twofold increase over IOP in control eyes, which can remain elevated for up to several months. Cauterization of two or more of the four large episcleral veins is another method of IOP elevation. In this model, IOP elevation occurs very quickly and there are some indications that this procedure impedes blood outflow from the globe and leads to ischemia. Reports indicate that IOP elevation may last from several weeks to several months without requiring retreatment. IOP increase can be also achieved by laser photocoagulation of the trabecular meshwork with or without injection of Indian ink into anterior chamber. Intracameral injection of hyaluronic acid or latex microspheres is another method of IOP elevation in rats. However, the repeated weekly injections required by this method may produce undesirable effects and are labor consuming. Topical application of dexamethasone for 4 weeks may also be used to induce ocular hypertension. These methods of chronic IOP elevation in rats are accompanied by death of the RGCs by apoptosis, optic nerve degeneration, and ONH remodeling similar to those observed in glaucoma in humans. Acute ocular hypertension, on the other hand, may be produced in rats by cannulation of the anterior chamber with a needle attached to a saline reservoir. Although such treatment leads to retinal ischemic injury, it has been suggested that this model mimics acute angleclosure glaucoma in humans.

A mutant rat strain with a unilateral or bilateral globe enlargement and IOPs that range from 25 to 45 mmHg have been described. In this strain, cupping of the ONH as well as reduction in the number of RGCs progress with age. Unfortunately, this strain was obtained from the Royal College of Surgeons colony that has a mutation in the receptor tyrosine kinase gene, leading to degeneration of the photoreceptors. This drastically limits the utility of this strain to study phenomena that are specific to glaucoma and not confounded by other neurodegenerative processes.

Rat models of glaucoma have been used to study effects of elevated IOP on the ERG, changes in the gene expression patterns in the retina, RGCs and optic nerve, and

changes in the protein spectrum of the retina. Rat models also are often used to study neuroprotection. For instance, the hypertonic saline model was used to demonstrate for the first time that agents targeting multiple phases of the amyloid-b pathway provide a therapeutic avenue in glaucoma management.

Mouse Models

Mouse models of glaucoma recently have become very popular. Although most mouse models of glaucoma are based on the elevation of IOP, information about IOP is essential even for the models that do not include experimental IOP manipulation. The mouse eye is much smaller than the human eye, and devices designed for tonometry in humans do not produce reliable data in the mouse. Thus, new methods to measure IOP in mice have been developed and, as a result, the development and acceptance of mouse models of glaucoma have been accelerated. Currently, several invasive and noninvasive methods of IOP measurements in mice exist. The oldest method remains as one of the most reliable and accurate methods and does not depend upon the mechanical properties of the cornea. It involves the insertion of a glass microneedle connected to a pressure transducer into anterior chamber of the eye. However, this procedure cannot be performed too frequently in the same eye, as adequate time is required for corneal wound healing. In addition, cannulation tonometry is technically difficult and training is required to develop sufficient expertise to obtain reliable IOP readings. Cannulation tonometry was used to demonstrate that common mouse strains exhibit different average IOPs in the range between 10 and 20 mmHg. Other methods of IOP measurements in mice were later developed including noninvasive techniques (TonoLab tonometer). Noninvasive techniques allow multiple IOP measurements within short periods of time without extensive training.

Pressure-induced mouse models

Surgical approaches similar to those that were used to produce elevated IOP in rats have also been developed in mice. Significant elevation of IOP in the C57BL/6J mouse eye is accomplished by combined injection of indocyanine green dye into the anterior chamber and diode laser treatment of the trabecular meshwork and episcleral vein region. IOP in operated eyes is significantly elevated 10 days after the surgery but returns back to normal 60 days after the procedure. Histological analysis of the treated eyes 65 days after the surgery revealed development of anterior synechia, loss of RGCs, thinning of all retinal layers, and damage to the optic nerve structures without evidence of prominent cupping. A reduction in the function of all retinal layers, as assessed by ERG studies, indicates that this model produces more dramatic

changes in the retina compared to glaucoma in humans. Elevation of IOP may also be induced by argon laser photocoagulation of the episcleral and limbal veins in C57BL/6J mouse eyes or by cauterization of three episcleral veins in CD1 mouse eyes. In one study, mean IOP in the eyes that underwent laser treatment was about 1.5 times higher than in control eyes for 4 weeks. RGC loss was 22.4 7.5% at 4 weeks after treatment with the majority of terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL)- positive apoptotic cells detected in the peripheral areas of the retina. Episcleral vein cauterization produced a maximum IOP elevation within 2–9 days after the procedure, which decreased progressively after that to baseline values in the following 24–33 days. This was associated with a 20% decline in the number of RGCs 2 weeks after the surgery.

The DBA/2J strain has become a popular mouse model of secondary-angle-closure glaucoma and is one of the best-characterized mouse models of glaucoma in general. DBA/2J mice have mutations in two genes, Tyrp1 and Gpnmb, which lead to pigment dispersion, iris transillumination, iris atrophy, and anterior synechia. IOP is elevated in most mice by the age of 9 months. IOP elevation was accompanied by the death of the RGCs, optic nerve atrophy, and optic nerve cupping. Although no group of the RGCs appears especially vulnerable or resistant to degeneration, fan-shaped sectors of cell death and survival radiating from the ONH have been detected. It has been suggested that axon damage at the ONH might be a primary lesion in this model. Several important observations have been made using DBA/2J model. It was shown that proapoptotic protein BAX is required for RGC death but not for RGC axon degeneration in this model of glaucoma, suggesting that BAX may be a candidate human glaucoma susceptibility gene. Unexpectedly, high dose of g-irradiation accompanied with syngenic bone marrow transfer protected RGCs in DBA/2J. Similar to the results obtained with rat and monkey models, genes involved in the glial activation and immune response are activated in DBA/2J retina as shown by array hybridization. Complement component C1q is upregulated in the retina in several animal models of glaucoma and human glaucoma with timing, suggesting that complement activation plays a significant role in glaucoma pathogenesis. Recent data suggest that complement proteins opsonize central nervous system synapses during a distinct window of postnatal development and that the complement proteins C1q and C3 are required for synapse elimination in the developing retinogeniculate pathway. In DBA/2J mice, C1q relocalizes to adult retinal synapses at an early stage of glaucoma prior to obvious neurodegeneration. These data indicate that C1q in adult glaucomatous retina marks synapses for elimination at early stages of disease, suggesting that the complement cascade mediates synapse loss in glaucoma.

Another DBA/2 substrain, DBA/2NNia, also develops elevated IOP and demonstrates RGC loss and optic nerve degeneration when aged. However, depletion of cells in the inner and outer nuclear layers and significant damage of the photoreceptor cells in 15-month-old mice have also been observed.

Transgenic and knock-out approaches have been used to prospectively develop several mouse models of glaucoma. The main advantage of these approaches is that animals within a particular line produce more uniform responses in terms of IOP elevation and damage to the retina and optic nerve as compared to surgically induced models. A large number of animals may be obtained and no training is needed to produce affected mice. Several lines of transgenic mice have been developed that contain BAC DNAs with a Tyr423His point mutation in the mouse or Tyr437His point mutation in the human MYOCILIN (MYOC) genes. Tyr437His mutation in the MYOC gene leads to severe glaucoma cases in humans, and mouse Tyr423His mutation corresponds to this human mutation. However, expression of mutated mouse or human myocilin in the eye-drainage structures of mice leads to moderate (about 2 mmHg at daytime and 4 mmHg at nighttime) elevation of IOP which is much less dramatic than IOP elevation in humans carrying the same mutation in the MYOC gene. Since these mice demonstrate progressive degenerative changes in the peripheral RGC layer and optic nerve with normal organization of the drainage structures, it has been suggested that these mice represent a mouse model of primary open-angle glaucoma. Another model of primary open-angle glaucoma was developed by the expression of a mutated gene for the a1 subunit of collagen type I. This mutation blocks the cleavage of collagen by matrix metalloproteinase-1. Transgenic mice expressing mutated collagen demonstrate elevated IOP which increases to a maximum of 4.8 mmHg greater than controls at 36 weeks.

A transgenic model of acute angle-closure glaucoma was developed by expression of calcitonin-receptor-like receptor under the control of a smooth muscle a-actin promoter. Overexpression of this receptor in the papillary sphincter muscle results in enhanced adrenomedullininduced sphincter muscle relaxation that leads to abrupt transient rises in IOP in some mice up to a mean level of about 50 mmHg between 30 and 70 days of age. Although the aberrant ocular functions of adrenomedullin and cal- citonin-gene-related peptide have not been associated with the pathogenesis of human acute glaucoma, it has been suggested that adrenomedullin and its receptor in the iris sphincter may present novel targets for the treatment of angle-closure glaucoma.

Normal-tension mouse models

Mice deficient in the glutamate transporters GLAST or EAAC1 show RGC death and typical glaucomatous

damage of the optic nerve without elevation of IOP. It has been shown that the glutathione levels are decreased in Mu¨ller cells of GLAST-deficient mice, while administration of glutamate receptor blocker prevents loss of RGCs. RGCs are more sensitive to oxidative stress in EAAC1deficient mice. These mice represent a model of normal tension glaucoma and are currently being used to develop therapies directed at IOP-independent mechanisms of RGC loss.

Developmental mouse models

Defects in genes involved in the development of the anterior eye segment may lead to relatively rare developmental glaucomas, which account for less than 1% of all human glaucoma cases. Several genes have been implicated in congenital glaucoma and anterior segment dysgenesis. They include CYP1B1, FOXC1, FOXC2, PITX2, LMX1b, and PAX6. Although Cyp1b1 knock-out mice do not develop elevated IOP, they have ocular abnormalities similar to defects in humans with primary congenital glaucoma: small or absent Schlemm’s canal, defects in the trabecular meshwork, and attachment of the iris to the trabecular meshwork and peripheral cornea. Foxc1–/– mice die at birth, while Foxc1+/– animals are viable but have defects in the eye-drainage structures in the absence of IOP changes. Similar eye defects are observed in Foxc2+/– mice. It has been suggested that Foxc1+/– and Foxc2+/– mice are useful models for studying anterior segment development and its anomalies, and they may allow identification of genes that interact with Foxc1 and Foxc2 to produce a phenotype with elevated IOP and glaucoma.

Transgenic mice overexpressing the ocular develop- ment-associated gene (ODAG) in photoreceptors under the control of mouse Crx promoter exhibit gradual protrusion of the eyeballs with dramatically increased IOP that is not attributable to mechanical block of the aqueous humor outflow. These transgenic mice demonstrate optic nerve atrophy and impaired retinal development. All retinal layers of these transgenic mice are affected, thereby differentiating this model from a typical glaucomatous retina where morphological changes are detected only in the RGC layer.

Other Mammalian Models

Several other mammalian models of glaucoma have been developed. Pig eyes are relatively large and, although the drainage outflow system of the pig eye is slightly different from that of the human eye, the porcine retina is more similar to the human retina than that of other large mammals (i.e., dog, goat, and cow). Cauterization of three porcine episcleral veins leads to a 1.3-fold elevation of IOP that is apparent 3 weeks after the surgery and persists for at least 21 weeks. It has been shown that endothelium leukocyte adhesion molecule 1 (ELAM-1), a molecular marker

for human glaucoma, is also elevated in the trabecular meshwork of pigs with elevated IOP.

Rabbits are a standard ophthalmic animal model for glaucoma filtration surgery and are often used for the development of new devices (e.g., drainage implants and degradable biopolymers) and medical therapies including gene therapy. At the same time, due to the unique anatomy of the rabbit eye, laser-induced elevation of IOP, like that in the monkey eye, is difficult to achieve. Alternatively, application of glucocorticoids has been successfully used to induce ocular hypertension in rabbit model. In addition, a line of rabbits with congenital glaucoma has been developed. Thick subcanalicular tissues and the deposition of extracellular matrix in the trabecular meshwork appear to contribute to the ocular hypertension exhibited by this model.

Several purebred dogs develop glaucoma with high frequency. Among North American breeds, the highest prevalence of primary glaucoma is observed in the American cocker spaniel (5.52%), basset hound (5.44%), and chow chow (4.70%), exceeding that in humans. Lens displacement resulting in secondary glaucoma is common in terrier breeds. The high prevalence of the glaucomas in these canine breeds suggests a genetic basis of pathophysiology.

It has been reported that topical application of corticosteroid induces reproducible elevation of IOP in the cow. The large amount of tissues available from the cow eye makes this model useful for biochemical studies.

Nonmammalian Models

Zebrafish

The zebrafish is an excellent model system to study complex diseases as it allows one to combine forward and reverse genetic approaches. The general organization of the zebrafish eye is similar to the human eye, although the fine details of individual ocular structures are rather different. In particular, there are significant differences in the organization of the iridocorneal angle between zebrafish and mammals. They include the trabecular meshwork and lack of iris muscles as well as ciliary folds in zebrafish as compared to mammals. Even with these limitations in mind, zebrafish have been used as a model organism for glaucoma studies. An accurate method exists to measure IOP in zebrafish which is based on servo-null electrophysiology. Using this method, baseline IOP differences have been demonstrated in genetically distinct zebrafish strains. Among tested strains, the long fin strain (LF) had the highest IOP (20.5 1.2 mm Hg) while the Oregon AB strain (AB) has the lowest IOP (10.8 0.3 mm Hg). At the same time, these differences in IOP do not lead to detectable defects of the retina or in visual function. Zebrafish have also been used to determine the function of several genes (foxc1, lmx1b, wdr36, olfactomedin 1, and olfactomedin 2)

implicated in glaucoma. It has been shown that wdr36 functions in ribosomal RNA processing and interacts with the p53 stress-response pathway, while olfactomedin 1 is essential for optic nerve growth and targeting of the optic tectum. Thus, zebrafish system may be very useful to complement studies with other model organisms, but by itself should be used with caution to study glaucoma.

Other Nonmammalian Models

Open-angle glaucoma characterized by elevated IOP can be induced in domestic chickens or in Japanese quails when they are reared under continuous light. Besides, an unknown autosomal dominant mutation in a Slate line of domestic turkeys has been identified that leads to secondary angle-closure glaucoma. Although these models might be useful to study certain aspects of glaucoma in humans, one should remember that structural and physiological differences between human and bird eyes complicate direct comparison.

Drosophila eyes have been suggested as a useful system for the discovery of genes that are associated with glaucoma. However, the general organization of human and Drosophila eyes are very different and data obtained with Drosophila may not always be relevant to glaucoma in humans.

Conclusion

Animal models have already provided interesting new information about potential mechanisms of glaucoma in humans. However, even in monkey models which most closely mimic the human form of the disease, the time course of changes in the glaucomatous eyes may be significantly accelerated as compared with human glaucomatous eyes. Indeed, all of the previously discussed systems are, after all, just models of human glaucoma. Reactions to the same insult (IOP, expression of the same mutated protein, etc.) may be somewhat different between various animal models and humans. Results obtained with these models should not automatically be applied to human condition and should be confirmed by testing in human subjects when possible. Nevertheless, information on molecular mechanisms of glaucoma obtained using animal models might be extremely valuable to develop new therapeutic approaches for glaucoma treatment and prevention in humans.

Further Reading

Anderson, M. G., Libby, R. T., Gould, D. B., et al. (2005). High-dose radiation with bone marrow transfer prevents neurodegeneration in an inherited glaucoma. Proceedings of the National Academy of Sciences of the United States of America 102: 4566–4571.

Baulmann, D. C., Ohlmann, A., Flu¨gel-Koch, C., et al. (2002). Pax6 heterozygous eyes show defects in chamber angle differentiation that are associated with a wide spectrum of other anterior eye segment abnormalities. Mechanisms of Development 118: 3–17.

Harada, T., Harada, C., Nakamura, K., et al. (2007). The potential role of glutamate transporters in the pathogenesis of normal tension glaucoma. European Journal of Clinical Investigation 117: 1763–1770.

Iwata, T. and Tomarev, S. (2008). Animal models for eye diseases and therapeutics. In: Conn, P. M. (ed.) Sourcebook of Models for Biomedical Research, pp. 279–287. Totowa, NJ: Humana Press.

Levkovitch-Verbin, H., Quigley, H. A., Martin, K. R., et al. (2002). Translimbal laser photocoagulation to the trabecular meshwork as a model of glaucoma in rats. Investigative Ophthalmology and Visual Science 43: 402–410.

Libby, R. T., Anderson, M. G., Pang, I., et al. (2005). Inherited glaucoma in DBA/2J mice: Pertinent disease features for studying the neurodegeneration. Visual Neuroscience 22: 637–648.

McMahon, C., Semina, E. V., and Link, B. A. (2004). Using zebrafish to study the complex genetics of glaucoma. Comparative Biochemistry and Physiology – Part C: Toxicology and Pharmacology 138: 343–350.

Morrison, J. C., Johnson, E. C., Cepurna, W., and Jia, L. (2005). Understanding mechanisms of pressure-induced optic nerve damage. Retinal Eye Research 24: 217–240.

Pang, I.-H. and Clark, A. F. (2007). Rodent models for glaucoma retinopathy and optic neuropathy. Glaucoma 16: 483–505.

Rasmussen, C. A. and Kaufman, P. L. (2005). Primate glaucoma models. Journal of Glaucoma 14: 311–314.

Senatorov, V., Malyukova, I., Fariss, R., et al. (2006). Expression of mutated mouse myocilin induces open-angle glaucoma in transgenic mice. Journal of Neuroscience 26: 11903–11914.

Smith, R. S., John, S. W. M., Nishina, P. M., and Sundberg, J. P. (eds.) (2002). Systematic Evaluation of the Mouse Eye. Boca Raton, FL: CRC Press.

Weinreb, R. N. and Lindsey, J. D. (2005). The importance of models in glaucoma research Volume. Journal of Glaucoma 14: 302–304.