Ординатура / Офтальмология / Английские материалы / Mechanisms of the Glaucomas_Shields, Tombran-Tink, Barnstable_2008

a contributing factor toward glaucomatous optic neuropathy (118–120). Non-human primate models of experimental glaucoma have been useful in this area by allowing the use of techniques that cannot be tested in humans. However, early studies that examined the effects of elevated IOP on optic nerve blood flow provided conflicting results. Alm and Bill (121) employed radiolabeled microspheres to determine the effect of shortterm elevation of IOP on blood flow rates in different ocular tissues of non-human primates. They reported reduced blood flow in the choroid and prelaminar optic nerve for moderate increments of IOP, and explained their results by reduced autoregulation of blood flow through the optic disc. Using similar techniques, Geiger and Bill (122) found no decrease in optic nerve blood flow in monkeys following IOP elevations lasting several hours. Quigley et al. (123) measured blood flow at the LC in monkeys with short-term and long-term experimental glaucoma and found that ONH blood flow was unaffected by IOP elevations up to 75 mmHg. More recent evidence for a vascular involvement in glaucoma comes from the finding that endothelin-1 (ET-1), a potent vasoconstrictor produced primarily by the vascular endothelium, is increased in many patients with normal tension as well as POAG (124–126). This finding has prompted investigators to explore the effects of optic nerve ischemia induced by chronic administration of ET-1. Cioffi and colleagues (127) found that ET-1 delivered continuously through osmotic mini-pumps to the perineural region of the anterior optic nerve for 6–12 months decreased optic nerve blood flow, as determined by the microspheres technique, and caused a significant loss of optic nerve axons (128). On the contrary, Brooks at al. (129) did not observe any significant changes in ONH morphology or blood flow in monkeys receiving chronic delivery of ET-1 to the optic nerve for 18 months, but did notice subtle pathological changes in visual cortex. Although these studies provide supportive evidence for ocular blood flow involvement in the pathogenesis of glaucomatous optic neuropathy, they also indicate that there are many areas in need of further investigation, a role in which non-human primate models clearly have a potential. The biochemical theory of optic nerve injury is based primarily on data from glaucomatous human eyes where it has been shown that astrocytes and microglia within the ONH, when activated by elevated IOP, are capable of releasing a number of different factors, including nitric oxide, tumor necrosis factor alpha, and MMPs, which can be neurotoxic and disruptive to the ECM of the ONH (82,99–102,130–132). Of particular interest here are reports of elevated levels of TNF-alpha receptors in the ganglion cell and optic nerve regions of the glaucomatous eye, suggesting that activated ONH glia might play a primary, and not merely secondary, role in nerve and ganglion cell degeneration (131,132).

A common feature of glaucoma is the progressive loss of retinal ganglion cells, and thus progressive loss of vision, associated with the disease. Although studies in human and monkey experimental glaucoma eyes also have identified changes with respect to the photoreceptors (133), the degree of involvement at this level of the retina is less clear (134) and appears to be much less severe than that seen within the ganglion cell layer. At present, however, the mechanism(s) by which injured retinal ganglion cells die remains unclear. Two theories concerning ganglion cell death that have received considerable attention are the neurotrophic and excitotoxic theories. The neurotrophic theory is based on the premise that pressure-induced injury to the optic

nerve results in a decrease in the level of trophic materials ganglion cells receive from their primary target neurons in the dorsal lateral geniculate of the thalamus. Indirect support for this theory comes from the work noted previously showing blockade of axonal transport in the ONH of monkeys with experimental glaucoma. More direct evidence is provided by Pease et al. (135), who used immunohistochemistry to demonstrate that at least two of those blocked components are brain-derived neurotrophic factor, a potent retinal neuroprotectant (10,136,137), and its associated tyrosine receptor kinase, TrkB. The excitotoxic theory derives from the fact that glutamate is a major neurotransmitter within the retina, and ganglion cell layer, and that elevated levels of glutamate can lead to overactivation of neurons, which results in excessive levels of intracellular calcium and cell death (138). In the case of glaucoma, this theory gained early support from the work of Dreyer et al. (139) in which they reported elevated levels of glutamate in the vitreous of glaucomatous eyes from humans and monkeys. Additional indirect support has come from non-human primate studies describing elevated levels of glutamine and glutathionine in Müller cells, glia involved closely with the regulation of extracellular retinal glutamate (140,141). However, further direct measurement studies by Carter-Dawson et al. (142) and Wamsley et al. (143) have failed to find any significant differences with respect to the vitreal glutamate concentrations of normal versus glaucomatous monkey eyes. Although this does not rule out a possible role for glutamate-induced excitotoxicity in glaucoma-related ganglion cell death in primates, it does indicate that the mechanisms by which this might occur are much more complex and perhaps involve interactions among multiple neurotransmitter and receptor systems (137), as well as modifications to the normal level and distribution of glutamate transporters within the retina (144,145).

At a more cellular level, Quigley et al. (146) have demonstrated using the TUNEL method for identifying DNA fragmentation that some ganglion cells in the glaucomatous primate retina die through programmed cell death, or apoptosis. Although this does not rule out involvement of the other major form of cell death, necrosis, at present there is no direct evidence for ganglion cell death by necrosis during glaucoma, although the form of cell death might depend on the degree of insult to the individual neurons affected (10,137). Our intracellular analyses suggest a gradual, pre-apoptotic degradation of ganglion cell health in the glaucomatous eye. By combining the monkey model of glaucoma with intracellular staining and recording techniques, we were able to identify the pattern of degenerative changes single ganglion cells go through as a result of chronic elevation of IOP (28,30). The morphological work indicates that both major classes of ganglion cells in the primate retina (midget and parasol) undergo similar patterns of degeneration. In brief, the earliest signs of degeneration appear at the level of the dendritic arbor, with shrinkage of the cell body, and presumably removal of the cell by apoptosis, occurring later in the process (see Fig. 3A–D). Because ganglion cells receive all of their synaptic input from more distal retinal elements through their dendrites, it was not surprising to find in the physiology study (30) that the parasol cells from the glaucomatous eyes showed a decrease in their overall visual responsiveness, and in particular with respect to their ability to respond to patterned stimuli and stimuli of increasing temporal frequency (see Fig. 3E–G). Interestingly, these cells

Fig. 3. Intracellularly-stained parasol (A, B) and midget (C, D) ganglion cells from normal (A, C) and glaucomatous (B, D) primate retinae. Comparison of the visual responsiveness, grating responsiveness, and temporal frequency response for parasol cells in normal and glaucomatous eyes [Republished with permission of the Association For Research in Vision and Ophthalmology from Weber et al. (1998) Morphology of single ganglion cells in the glaucomatous primate retina. Investig. Ophthalmol. Vis. Sci. 39(12), 2304–2320, and Weber and Harman (2005) Structure-function relations of parasol cells in the normal and glaucomatous primate retina. Investig. Ophthalmol. Vis. Sci. 46(9), 3197–3207].

retained most of their normal intrinsic electrical properties. Additional work is needed to determine whether similar changes also are seen with respect to the midget cells.

The relatively distinct anatomical and physiological characteristics of the different types of ganglion cells in the primate retina has long begged the question of whether there are differences in their susceptibility to injury as a result of chronic elevation of IOP, and if so, could this information be used toward the development of more specific non-invasive tests aimed at earlier detection and monitoring of the disease process. Human and monkey studies that have compared the axon diameters (147,148), soma size (149,150), neurofilament content (151), and dendritic field sizes (28) of ganglion cells from normal and glaucomatous eyes, suggest that large ganglion cells, and thus

parasol cells and the M-pathway, might be affected preferentially. Although a similar bias has been reported with respect to changes at the level of the LGN for monkeys with experimental glaucoma (152,153), the issue of selective versus non-selective deficits in glaucoma remains controversial; not all ganglion cell loss can be accounted for by a selective loss of parasol cells (137,149,150,154,155); immunohistochemical analyses of LGNs of glaucomatous monkeys suggest either no difference (156) or even a bias toward the parvocellular pathway (157), and numerous psychophysical studies have demonstrated early functional changes considered to be associated with both the M- and P-pathways (155).

NON-INVASIVE ASSESSMENT OF GLAUCOMA

ONSET AND PROGRESSION

Glaucoma leads to irreversible structural and functional changes to the retina and optic nerve. Consequently, early detection and treatment are critically important for preventing or minimizing permanent vision loss. New clinical tests are being developed for better characterization and quantification of structural and functional damage in glaucomatous eyes. However, longitudinal clinical studies to validate these new tests will take years to complete. Primate models of glaucoma have proven to be very useful in this regard because of the many similarities of the disease process to the human condition that can be observed on an accelerated time scale and the possibility to obtain histological verification of procedures. Furthermore, techniques that have been established as useful and reliable have been employed to monitor disease progression and gain insight into the disease mechanism itself.

In Vivo Measurement of Neural Structure

It is important to identify glaucomatous eyes with early structural changes, as these eyes are at risk of developing progressive damage. Primate models of glaucoma have been used to validate in vivo imaging techniques such as confocal scanning laser ophthalmoscopy (CSLO), scanning laser polarimetry (SLP), and optical coherence tomography (OCT) by determining whether the structural changes assessed by these instruments correlate with histomorphometric measurements. In one of the earliest studies of primate glaucoma that employed the CSLO technique to image the ONH, Burgoyne et al. (158) compared the optic disc surface changes in eyes with experimental glaucoma and optic nerve transection. They concluded that early changes in the optic disc surface under conditions of elevated IOP are unlikely to be due to axon loss alone, but also include damage to the load-bearing connective tissues of the ONH. Yucel et al. (159) reported that the optic disc topographic parameters from eyes with experimental glaucoma correlated significantly with optic nerve fiber numbers, and concluded that CSLO technology can potentially evaluate optic nerve damage in glaucomatous eyes. Weinreb and colleagues (160) showed that the SLP technique, using eye-specific corneal polarization compensation, provides meaningful retinal nerve fiber layer (RNFL) thickness measurements in primate eyes with experimental glaucoma (see Fig. 4). Although the previous studies were cross-sectional, Shimazawa et al. (161) have reported longitudinal changes in optic disc topography and RNFL thickness in eyes with experimental glaucoma using CSLO and SLP techniques, respectively.

Fig. 4. Optic disc photographs (A, C, F, H) and scanning laser polarimetry (SLP) retardation maps with eye-specific corneal birefringence compensation (B, D, G, I) from two monkeys with experimental glaucoma of the right eye (C, H) and normal left eyes (A, F) (photographs from the two animals shown in separate rows). Panels E and J show parapapillary retinal nerve fiber layer (RNFL) thickness as a function of polar position around the optic disc. Panels B, G, D, and I include polarization artifacts from the area of parapapillary atrophy and lamina cribrosa (Republished from Weinreb et al. (2002) Scanning laser polarimetry in monkey eyes using variable corneal polarization compensation. J. Glaucoma 11, 378–384, with permission from Lippincott Williams and Wilkins).

They found that in vivo imaging parameters show good correlations with RNFL thickness evaluated from histology, thereby demonstrating the potential for longitudinal assessment of structural changes in glaucomatous eyes using these technologies. More recently, using OCT measurements, Vilupuru et al. (162) showed that structural changes from experimental glaucoma can be detected earlier at the ONH than in the RNFL. This same group (163) also demonstrated that RNFL thickness may be a more sensitive measurement for early stages of glaucoma and perimetry a better measure for moderate to advanced stages of glaucoma.

In Vivo Measures of Neural Function (Perimetry)

Standard threshold automated perimetry is the gold standard for testing and following functional vision loss secondary to glaucoma in humans. Early studies have demonstrated excellent agreement between monkeys and humans for psychophysical functions of foveal vision (164–166). Harwerth and co-workers (167) pioneered peripheral visual sensitivity testing in monkeys, demonstrated that normal visual field characteristics of monkeys and humans are quite similar, and showed that visual fields of eyes with experimental glaucoma undergo progressive changes in sensitivity similar to the visual field losses observed in human glaucoma (see Fig. 5). In later studies, comparing retinal ganglion cell densities and visual thresholds at various locations in the visual field, Harwerth and co-workers (168) demonstrated that neural losses from glaucoma are predictable from visual sensitivity measurements by clinical perimetry; however, a relatively large proportion of ganglion cells (40–50%) must be lost before the threshold measurements exceeds the normal variability and reaches statistical significance. These findings, obtained under very tightly controlled experimental conditions from primates

In Vivo Measures of Neural Function (Electrophysiology)

Although behavioral perimetric tests can be very useful for assessing the impact of neural damage on vision, they do not provide direct information on the level or the visual pathway affected by the disease. Furthermore, training non-human primates to perform behavioral tasks is very time consuming and quite demanding. Non-invasive electrophysiological recordings such as the electroretinogram (ERG) and visually evoked potentials (VEP) can bypass these disadvantages, and have been more widely used for estimating neural function in animal models of glaucoma. In certain cases, primate glaucoma models have been used to validate electrophysiological tests of retinal ganglion cell origin. Pattern ERGs (PERG) elicited to contrast modulations of patterned stimuli depend on the integrity of RGCs (172) and have been widely used in estimating retinal ganglion cell activity in experimental glaucoma (173–176) (see Fig. 6A). More recently components of the full-field flash ERG have been used to assess functional damage to ganglion cells in experimental glaucoma. In particular, these components include the scotopic threshold response (STR), a very sensitive negativegoing potential of the dark-adapted ERG recorded to stimuli close to the absolute visual threshold, and the photopic threshold response (PhNR), another negative-going potential of the cone ERG recorded under photopic conditions. Both have been used to assess the function of retinal ganglion cells (177,178) (see Fig. 6B and C). In fact, the dependence of the STR and the PhNR on retinal ganglion cell activity was first demonstrated in a primate model of experimental glaucoma. Introduction of the multifocal ERG-recording technique has made it possible to record ERG responses from multiple retinal areas simultaneously over a short duration (179–181) and has demonstrated the potential for monitoring neuronal dysfunction from focal areas over the course of the disease (see Fig. 6D). Large-field pattern and flash VEPs have been employed in most of these studies in conjunction with ERG recordings to estimate the retinocortical transmission (173–175,179). On the whole, the investigation of electrophysiological techniques in primate models of glaucoma has provided a reasonable measure of the degree and level of functional damage, and they lend support to the use of these techniques as objective measures for assessing neuronal dysfunction in human glaucoma.

SUMMARY

The development of a clear understanding of the complex mechanisms underlying human disease requires the availability of animal models from which different levels of knowledge can be gained, and hypotheses tested, with a high degree of confidence. In addition, these models should allow the greatest degree of generalization to the human condition. With respect to human glaucoma, the monkey model of experimental glaucoma has provided, and will continue to provide, important basic, clinical, and translational data. To date, this model has facilitated major advances in our understanding of the pathobiology and physiology of glaucoma. In addition, it also has allowed the development of new surgical (182,183) and pharmacological approaches aimed at pressure reduction (70,71,184–189), as well as exploration of neuroprotective

Fig. 6. Electroretinogram (ERG) changes in experimental glaucoma. (A) Transient pattern ERG (PERG) responses to three different stimulus spatial frequencies; (B) Scotopic threshold responses; (C) Photopic Negative responses; (D) Progressive changes in the first order response of the multifocal ERG (mfERG) responses from upper nasal (UN), upper temporal (UT), lower temporal (LT), and lower nasal (LN) retinal quadrants with progression of mean deviation (MD) in behavioral perimetry [Panels B, C, and D republished from Harwerth et al. (2002) Visual field defects and neural losses from experimental glaucoma. Prog. Retin. Eye Res. 21, 91–125, with permission from Elsevier].

(190,191) and gene therapy-related strategies (192) for mitigating or reversing neuronal degeneration, and thus preserving sight.

REFERENCES

1.Hart W.M., Jr. (1989) The epidemiology of primary open-angle glaucoma and ocular hypertension, in The Glaucomas (Ritch R., Shields M.B., Krupin T., eds), St. Louis, C.V. Mosby Co., pp 789–795.

2.Sommer A, Tielsch J.M., Katz J, Quigley H.A., Gottsch J.D., Javitt J, and Singh K. (1991) Relationship between intraocular pressure and primary open angle glaucoma among White and Black Americans. Arch. Ophthalmol. 109, 1090–1095.

3.Teilsch J.M., Sommer A., Katz J., Royall R.M., Quigley H.A., Javitt J. (1991) Racial variations in the prevalence of primary open-angle glaucoma. The Baltimore eye survey. JAMA 266, 369–374.

4.Quigley H.A (1993) Open-angle glaucoma. N. Engl. J. Med. 328, 1097–1106.

5.Gelatt K.N. (1977) Animal models for glaucoma. Invest. Ophthalmol. Vis. Sci. 16, 592–596.

6.Levkovitch-Verbin H. (2004) Animal models of optic nerve diseases. Eye 18, 1066–1074.

7.Weinreb R.N. and Lindsey J.D. (2005) The importance of models in glaucoma research.

J. Glaucoma 14, 302–304.

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

9.Quigley H.A., Nickells R.W., Kerrigan L.A., Pease M.E., Thibault D.J., and Zack D.J. (1995) Retinal ganglion cell death in experimental glaucoma and after axotomy occurs by apoptosis. Invest. Ophthalmol. Vis. Sci. 36, 774–786.

10.Nickells R.W. (1996) Retinal ganglion cell death in glaucoma: the how, they why, and the maybe. J. Glaucoma 5, 345–356.

11.Harwerth R.S., Crawford M.L., Frishman L.J., Viswanathan S., Smith E.L. III, and CarterDawson L. (2002) Visual field defects and neural losses from experimental glaucoma.

Prog. Retin. Eye Res. 21, 91–125.

12.Rodieck, R.W. (1998) The First Steps in Seeing. Sinauer, Sunderland, MA.

13.Kalvin N.H., Hamasaki D.I., and Gass J.D.M. (1966) Experimental glaucoma in monkeys.

Arch. Ophthalmol. 76, 82–93.

14.Zimmerman L.E., de Venecia G., and Hamasaki D.I. (1967) Pathology of the optic nerve in experimental acute glaucoma. Invest. Ophthalmol. Vis. Sci. 6, 109–125.

15.Lessell S. and Kuwabara T. (1969) Experimental -chymotrypsin glaucoma. Arch. Ophthalmol. 81, 853–864.

16.Levy N.S. (1974) The effects of elevated intraocular pressure on slow axonal protein flow. Invest. Ophthalmol. 13, 691–695.

17.Gaasterland D. and Kupfer C. (1974) Experimental glaucoma in the rhesus monkey.

Invest. Ophthalmol. Vis. Sci. 13, 455–457.

18.Quigley H.A. and Hohman R.M. (1983) Laser energy levels for trabecular meshwork damage in the primate eye. Invest. Ophthalmol. Vis. Sci. 24, 1305–1307.

19.Wang R.F., Schumer R.A., Serle J.B., and Podos S.M. (1998) A comparison of Argon laser and diode laser photocoagulation of the trabecular meshwork to produce the glaucoma Monkey model. J. Glaucoma 7, 45–49.

20.Pederson J.E. and Gaasterland D.E. (1984) Laser-induced primate glaucoma. Arch. Ophthalmol. 102, 1689–1692.

21.Konstas A.G., Mantziris D.A., and Stewart W.C. (1997) Diurnal intraocular pressure in untreated exfoliation and primary open-angle glaucoma. Arch. Ophthalmol. 115, 182–185.

22.Sacca S.C., Rolando M., Marletta A., Macri A., Cerqueti P., and Ciurlo G. (1998) Fluctuations of intraocular pressure during the day in open-angle glaucoma, normal-tension glaucoma and normal subjects. Ophthalmologica 212, 115–119.

23.Komaromy A.M., Brooks D.E., Kubilis P.S., Dawson W.W., Sapp H.L., Jr., Nelson G., Collins B.R., and Sherwood M.B. (1998) Diurnal intraocular pressure curves in healthy rhesus macaques (Macaca mulatta) and rhesus macaques with normotensive and hypertensive primary open-angle glaucoma. J. Glaucoma 7, 128–131.

24.Liu J.H., Kripke D.F., Hoffman R.E., Twa M.D., Loving R.T., Rex K.M., Gupta N., and Weinreb R.N. (1998) Nocturnal elevation of intraocular pressure in young adults. Invest. Ophthalmol. Vis. Sci. 39, 2707–2712.

25.Asrani S., Zeimer R., Wilensky J., Giesser D., Vitale S., and Lindenmuth K. (2000) Large diurnal fluctuations in intraocular pressure are an independent risk factor in patients with glaucoma. J. Glaucoma 9, 134–142.

26.Ollivier F.J., Brooks D.E., Kallberg M.E., Sapp H.L., Komaromy A.M., Stevens G.R., Dawson W.W., Sherwood M.B., and Lambrou G.N. (2004) Time-specific intraocular

pressure curves in Rhesus macaques (Macaca mulatta) with laser-induced ocular hypertension. Vet. Ophthalmol. 7, 23–27.

27.Quigley H.A. and Addicks E.M. (1980) Chronic experimental glaucoma in primates. II. Effect of extended intraocular pressure elevation on optic nerve head and axonal transport.

Invest. Ophthalmol. Vis. Sci. 19, 137–152.

28.Weber A.J., Kaufman P.L., and Hubbard W.C. (1998) Morphology of single ganglion cells in the glaucomatous primate retina. Invest. Ophthalmol. Vis. Sci. 39, 2304–2320.

29.Weber A.J. and Zelenak D. (2001) Experimental glaucoma in the primate induced by latex microspheres. Invest Ophthalmol. Vis Sci. 111, 39–48.

30.Weber A.J. and Harman C.D. (2005) Structure-function relations of parasol cells in the normal and glaucomatous primate retina. Invest. Ophthalmol. Vis. Sci. 46, 3197–3207.

31.Dawson W.W., Brooks D.E., Hope G.M., Samuelson D.A., Sherwood M.B., Engel H.M., and Kessler M.J. (1993) Primary open angle glaucomas in the rhesus monkey. Br. J. Ophthalmol. 77, 302–310.

32.Dawson W.W., Brooks D.E., Dawson J.C., Sherwood M.B., Kessler M.J., and Garcia A. (1998) Signs of glaucoma in rhesus monkeys from a restricted gene pool. J. Glaucoma 7, 343–348.

33.Rohen J.W., Futa R., and Lütjen-Drecoll E. (1981) The fine structure of the cribiform meshwork in normal and glaucomatous eyes as seen in tangential sections. Invest. Ophthalmol. Vis. Sci. 21, 574–585.

34.Samuelson D.A. (1996) A reevaluation of the comparative anatomy of the eutherian iridocorneal angle and associated ciliary body musculature. Vet. Comp. Ophthalmol. 6, 153–172.

35.Ethier C.R., Kamm R.D., Palaszewski B.A., Johnson M.C., and Richardson T.M. (1986) Calculation of flow resistance in the juxtacanalicular meshwork. Invest. Ophthalmol. Vis. Sci. 27, 1741–1750.

36.Mäepea O. and Bill A. (1989) The pressures in the episcleral veins, Schlemm’s canal and the trabecular meshwork in monkeys: effects of changes in intraocular pressure. Exp. Eye Res. 49, 645–663.

37.Fine B.S. (1964) Observations on the drainage angle in man and rhesus monkey: a concept of the pathogenesis of chronic simple glaucoma. Invest. Ophthalmol. 3, 609–646.

38.Inomata H., Bill A., and Smelser G.K. (1972) Aqueous humour pathways through the trabecular meshwork and into Schlemm’s canal in the cynomolgus monkey (Macaca irus). And electron microscopic study. Am. J. Ophthalmol. 73, 760–789.

39.Epstein D.L. and Rohen J.W. (1991) Morphology of the trabecular meshwork and innerwall endothelium after cationized ferritin perfusion in the monkey eye. Invest. Ophthalmol. Vis. Sci. 32, 160–171.

40.Parc C.E., Johnson D.H., and Brilakis H.S. (2000) Giant vacuoles are found preferentially near collector channels. Invest. Ophthalmol. Vis. Sci. 41, 2984–2990.

41.Johnstone M.A. and Grant W.M. (1973) Pressure-dependent changes in the structures of the aqueous outflow system of human and monkey eyes. Am. J. Ophthalmol. 75, 365.

42.Grierson I .and Lee W.R. (1974) Changes in the monkey outflow apparatus at graded levels of intraocular pressure: a qualitative analysis by light microscopy and scanning electron microscopy. Exp. Eye Res. 19, 21–33.

43.Johnstone M.A. (1979) Pressure-dependent changes in nuclei and the process origins of the endothelial cells lining Schlemm’s canal. Invest. Ophthalmol. Vis. Sci. 18, 44–51.

44.Ye W., Gong H., Sit A., Johnson M., and Freddo T.F.(1997) Interendothelial junctions in normal human Schlemm’s canal respond to changes in pressure. Invest. Ophthalmol. Vis. Sci. 38, 2460–2468.

45.Brilakis H.S. and Johnson D.H. (2001) Giant vacuole survival time and implications for aqueous humour outflow. J. Glaucoma 10, 277–283.