Pressure-induced Optic Nerve Damage Research Articles

Novel glaucoma model in rats using photo-crosslinked azidobenzoic acid-modified chitosan

An experimental model of pressure-induced optic nerve damage promises to greatly expand understanding of the cellular events leading to retinal ganglion cell (RGC) death and of how they are influenced by intraocular pressure (IOP) and other risk factors associated with glaucoma. In this work, we propose a novel strategy employing photo-crosslinkable azidobenzoic acid-modified chitosan (Az-CH) for long-term, persistent elevation of IOP. For this purpose, a solution of Az-CH was injected into the anterior chamber of experimental rat eyes, which were subsequently irradiated with ultraviolet (UV) light to form an Az-CH gel that hindered aqueous outflow and effected prolonged IOP elevation thereby. The control eyes were treated as follows: (1) intracameral injection of Az-CH without UV irradiation, (2) intracameral injection of saline solution without UV irradiation or (3) no injection with UV irradiation. A significant IOP increase was observed in the experimental eyes, which was continuously higher for the whole testing period of 12 weeks after one-time treatment with Az-CH injection and UV irradiation. Also, a more significant loss of RGCs, one of the major features of glaucoma, was observed in experimental eyes than in the control eyes. Therefore, the strategy presented herein can be a novel experimental model to study the mechanism of RGC damage by elevated IOP over the course of a prolonged period.

Expression and Regulation of LOXL1 and Elastin-related Genes in Eyes With Exfoliation Syndrome

Exfoliation syndrome (XFS) is a complex, late-onset disorder of the elastic fiber system and the most common identifiable cause of open-angle glaucoma. Strong genetic risk is conferred by the lysyl oxidase-like 1 (LOXL1) gene, but additional comodulating factors are necessary for the manifestation of the disease. The aim of this study was to establish a comprehensive expression profile of LOXL1 and elastic proteins in XFS eyes and to analyze their regulation in an in vitro cell culture system. Eyes with XFS with and without glaucoma, and normal control eyes were analyzed for major elastic fiber components (elastin, fibrillin-1, fibulin-4), and lysyl oxidase (LOX) enzymes by real-time PCR, immunohistochemistry, and electron microscopy. Cultured human Tenon's capsule fibroblasts were exposed to transforming growth factor-β1, IL-6, homocysteine, oxidative stress, hypoxia, or ultraviolet radiation, and changes in the expression of LOXL1 and elastic components of XFS material were assessed by real-time PCR, immunohistochemistry, and Western blotting. LOXL1 expression in anterior eye tissues was significantly increased in early XFS stages but was decreased in advanced stages as compared with controls. LOXL1 was also found to be a major component of XFS material and to colocalize with elastin, fibrillin-1, and fibulin-4, which were upregulated in parallel to LOXL1. In contrast, in most posterior segment tissues, LOXL1 and elastic fiber proteins displayed no differential expression. Interestingly, lamina cribrosa specimens of early and late XFS stages without and with glaucoma revealed a selective downregulation of LOXL1 and elastic fiber components on the mRNA and protein level, which was associated with pronounced ultrastructural alterations of the laminar elastic fiber network in XFS eyes. Treatment of cultured cells with XFS-associated pathogenetic stimuli induced a significant increase in the expression of LOXL1 and elastic proteins and resulted in their assembly into XFS-like fibrils in vitro. The findings support the notion that both genetic and nongenetic factors may cooperate in the stable accumulation of XFS aggregates and provide evidence for a XFS-specific elastinopathy of the lamina cribrosa, possibly rendering XFS eyes more vulnerable to pressure-induced optic nerve damage and glaucoma development.

LOXL1 Deficiency in the Lamina Cribrosa as Candidate Susceptibility Factor for a Pseudoexfoliation-Specific Risk of Glaucoma

To test the hypothesis that a primary disturbance in lysyl oxidase-like 1 (LOXL1) and elastin metabolism in the lamina cribrosa of eyes with pseudoexfoliation syndrome constitutes an independent risk factor for glaucoma development and progression. Observational, consecutive case series. Posterior segment tissues obtained from 37 donors with early and late stages of pseudoexfoliation syndrome without glaucoma, 37 normal age-matched control subjects, 5 eyes with pseudoexfoliation-associated open-angle glaucoma, and 5 eyes with primary open-angle glaucoma (POAG). Protein and mRNA expression of major elastic fiber components (elastin, fibrillin-1, fibulin-4), collagens (types I, III, and IV), and lysyl oxidase crosslinking enzymes (LOX, LOXL1, LOXL2) were assessed in situ by quantitative real-time polymerase chain reaction, (immuno)histochemistry, and light and electron microscopy. Lysyl oxidase-dependent elastin fiber assembly was assessed by primary optic nerve head astrocytes in vitro. Expression levels of elastic proteins, collagens, and lysyl oxidases in the lamina cribrosa. Lysyl oxidase-like 1 proved to be the major lysyl oxidase isoform in the normal lamina cribrosa in association with a complex elastic fiber network. Compared with normal and POAG specimens, lamina cribrosa tissues obtained from early and late stages of pseudoexfoliation syndrome without and with glaucoma consistently revealed a significant coordinated downregulation of LOXL1 and elastic fiber constituents on mRNA and protein level. In contrast, expression levels of collagens and other lysyl oxidase isoforms were not affected. Dysregulated expression of LOXL1 and elastic proteins was associated with pronounced (ultra)structural alterations of the elastic fiber network in the laminar beams of pseudoexfoliation syndrome eyes. Inhibition of LOXL1 interfered with elastic fiber assembly by optic nerve head astrocytes in vitro. The findings provide evidence for a pseudoexfoliation-specific elastinopathy of the lamina cribrosa resulting from a primary disturbance in LOXL1 regulation and elastic fiber homeostasis, possibly rendering pseudoexfoliation syndrome eyes more vulnerable to pressure-induced optic nerve damage and glaucoma development and progression.

Evaluation of Lamina Cribrosa and Peripapillary Sclera Stiffness in Pseudoexfoliation and Normal Eyes by Atomic Force Microscopy

Pseudoexfoliation (PEX) syndrome is a systemic disorder of the elastic fiber system that can lead to PEX glaucoma. Elastotic alterations in the lamina cribrosa (LC) of PEX eyes suggested biomechanical implications predisposing to pressure-induced optic nerve damage. In this pilot study, the stiffness of LC and peripapillary sclera (ppSC) in eyes with and without PEX syndrome were analyzed by atomic force microscopy (AFM) nanoindentation. Unfixed cryosections (5-μm thick) were prepared from the optic nerve heads (ONH) of three donor eyes with PEX syndrome and three age-matched control eyes. AFM force mapping was performed in selected regions of the central, midperipheral, and peripheral LC and the ppSC using a spherical cantilever tip. To determine the local Young's modulus of elasticity (YME) as a measure of tissue stiffness, force curves were acquired and analyzed using the spherical Hertz model. For the LC, the median YME values calculated from single stiffness maps averaged 17.2 (±2.7) kPa in normal eyes and 10.1 (±1.4) kPa in PEX eyes, indicating a significant PEX-related decrease in stiffness by over 40% (P < 0.01). The corresponding YME values for the ppSC, which revealed a 9-fold higher tissue stiffness than in the LC, averaged 158.3 (±59.8) kPa for control and 85.8 (±16.9) kPa for PEX samples. AFM was proven suitable for determining the stiffness of ONH tissues, encouraging further large-scale analyses. The marked decrease in stiffness, implying an increased deformability of the ONH in PEX eyes, may reflect an inherent tissue weakness rendering these eyes more vulnerable to glaucomatous damage.

Mutant human myocilin induces strain specific differences in ocular hypertension and optic nerve damage in mice

Elevated intraocular pressure (IOP) is a causative risk factor for the development and progression of glaucoma. Glaucomatous mutations in myocilin (MYOC) damage the trabecular meshwork and elevate IOP in humans and in mice. Animal models of glaucoma are important to discover and better understand molecular pathogenic pathways and to test new glaucoma therapeutics. Although a number of different animal models of glaucoma have been developed and characterized, there are no true models of human primary open angle glaucoma (POAG). The overall goal of this work is to develop the first inducible mouse model of POAG using a human POAG relevant transgene (i.e. mutant MYOC) expression in mouse eyes to elevate IOP and cause pressure-induced damage to the optic nerve. Four mouse strains (A/J, BALB/cJ, C57BL/6J, and C3H/HeJ) were used in this study. Ad5.MYOC.Y437H (5 × 107 pfu) was injected intravitreally into one eye, with the uninjected contralateral eye serving as the control eye. Conscious IOP measurements were taken using a TonoLab rebound tonometer. Optic nerve damage was determined by scoring PPD stained optic nerve cross sections. Retinal ganglion cell and superior colliculus damage was assessed by Nissl stain cell counts. Intravitreal administration of viral vector Ad5.MYOC.Y437H caused a prolonged, reproducible, and statistically significant IOP elevation in BALB/cJ, A/J, and C57BL/6J mice. IOPs increased to approximately 25 mm Hg for 8 weeks (p < 0.0001). In contrast, the C3H/HeJ mouse strain was resistant to Ad5.MYOC.Y437H induced IOP elevation for the 8-week time period. IOPs were stable (12–15 mm Hg) in the uninjected control eyes. We also determined whether there were any strain differences in pressure-induced optic nerve damage. Even though IOP was similarly elevated in three of the strains tested (BALB/cJ, C57BL/6J, and A/J) only the A/J strain had considerable and significant optic nerve damage at the end of 8 weeks with optic nerve damage score of 2.64 ± 0.19 (n = 18, p < 0.001) in the injected eye. There was no statistical difference in retinal ganglion cell death or superior colliculus damage at the 8-week time point in any of the strains tested. These results demonstrate strain dependent responses to Ad5.MYOC.Y437H-induced ocular hypertension and pressure-induced optic nerve damage.

Elevated Intraocular Pressure and Optic Nerve Injury Models in the Rat

Models of experimentally elevated intraocular pressure in rats provide valuable opportunities to discover and study mechanisms of pressure-induced optic nerve damage. The structure and vasculature of the rat optic nerve head have several anatomic similarities and differences from the primate that allow useful comparisons and insights into human glaucoma. Specifically, the ultrastructural relationship between astrocytes, retinal ganglion cell axons and the connective tissues of the optic nerve head appear quite similar to the primate, and have a high potential for revealing cellular mechanisms of axonal injury. Three widely used models of creating elevated IOP in rats exist. However, they are not all equivalent and appear to differ in the relationship they exhibit between the level of pressure and extent of optic nerve damage. This indicates that these models may differ in the mechanisms by which they produce elevated eye pressure. All of these models are amenable to a variety of methods for evaluating damage. These include objective and subjective histologic assessment of the optic nerve, counting cells in the retinal ganglion cell layer of the retina and the use of retinal whole mounts to count retinal ganglion cells that have been back-labeled with dye applied to the superior colliculus. In the decade since their introduction, these versatile models have provided important insights into mechanisms of pressure-induced optic nerve damage using sensitive molecular biology techniques. They have also allowed the evaluation of several potential strategies for neuroprotection in glaucoma, ranging from currently available drugs to gene transfer studies.

Evaluation of Inducible Nitric Oxide Synthase in Glaucomatous Optic Neuropathy and Pressure-Induced Optic Nerve Damage

To determine whether inducible nitric oxide synthase (NOS-2) is involved in glaucomatous optic neuropathy. Chronic elevation of rat intraocular pressure (IOP) leading to optic nerve damage was induced by episcleral injection of hypertonic saline, which caused sclerosis and blockade of aqueous humor outflow pathways. Expression of NOS-2 in the retina and optic nerve head (ONH) was evaluated by immunohistochemistry, gene array analysis, and quantitative PCR (Q-PCR). Immunohistochemistry was also used to assess the NOS-2 level in the ONH from primary open-angle glaucoma (POAG) and nonglaucomatous human eyes. Finally, an NOS-2 inhibitor, aminoguanidine, administered orally in the drinking water, was tested for its effect on optic nerve injury in rats with ocular hypertension. Chronically elevated IOP in the rat produced optic nerve damage that correlated with pressure change (r(2) = 0.77), but did not increase NOS-2 immunoreactivity in the optic nerve, ONH, or ganglion cell layer. Retinal and ONH NOS-2 mRNA levels did not correlate with either IOP level or severity of optic nerve injury. Similarly, there was no difference in NOS-2 immunoreactivity in the optic nerve or ONH between POAG and nonglaucomatous eyes. Furthermore, aminoguanidine treatment did not affect the development of pressure-induced optic neuropathy in the rat. As demonstrated by several independent methods, glaucomatous optic neuropathy was not associated with a significant change in the expression of NOS-2 in the retina, ONH, or optic nerve.

A new experimental model of glaucoma in rats through intracameral injections of hyaluronic acid

An experimental model of pressure-induced optic nerve damage would greatly facilitate the understanding of the cellular events leading to ganglion cell death, and how they are influenced by intraocular pressure and other risk factors associated to glaucoma. The aim of the present report was to study the effect of a long-term increase of intraocular pressure in rats induced by intracameral injections of hyaluronic acid with respect to electroretinographic activity and retinal and optic nerve histology. For this purpose, hyaluronic acid was injected weekly in the rat anterior chamber of one eye, whereas the contralateral eye was injected with saline solution. The results showed a significant decrease of oscillatory potentials and a- and b-wave amplitude of the scotopic electroretinogram after 3 or 6 weeks of hyaluronic acid administration, respectively. These parameters were further reduced after 10 weeks of treatment with hyaluronic acid. No significant changes in anterior chamber angle structures from hyaluronic acid- and vehicle-injected eyes were observed, whereas a significant loss of ganglion cell layer cells and of optic nerve axons were detected in animals that received hyaluronic acid for 10 weeks, as compared to eyes injected with saline solution. In summary, present results indicate that the chronic administration of hyaluronic acid induced a significant decrease in the electroretinographic activity and histological changes in the retina and optic nerve that seem consistent with some features of chronic open-angle glaucoma. Therefore, this could be an experimental model to study the cellular mechanisms by which elevated intraocular pressure damages the optic nerve and the retina.

Understanding mechanisms of pressure-induced optic nerve damage

Patients with glaucoma can suffer progressive vision loss, even in the face of what appears to be excellent intraocular pressure (IOP) control. Some of this may be secondary to non-pressure-related (pressure-independent) factors. However, it is likely that chronically elevated IOP produces progressive changes in the optic nerve head, the retina, or both that alter susceptibility of remaining optic nerve fibers to IOP. In order to understand the nature of these progressive changes, relevant, cost-effective animal models are necessary. Several rat models are now used to produce chronic, elevated IOP, and methods exist for measuring the resulting IOP and determining the extent of the damage this causes to the retina and optic nerve. A comparison of damage, pressure and duration shows that these models are not necessarily equivalent. These tools are beginning to uncover clear evidence that elevated IOP produces progressive changes in the optic nerve head and retina. In the optic nerve head, these include axonal and non-axonal effects, the latter pointing to involvement of extracellular matrix and astrocyte responses. In the retina, retinal ganglion cells appear to undergo changes in neurotrophin response as well as morphologic changes prior to actual cell death. These, and other, as yet uncovered, abnormalities in the optic nerve head and retina may influence relative susceptibility to IOP and explain progressive optic nerve damage and visual field loss, in spite of apparent, clinically adequate IOP control. Ultimately, this knowledge may lead to the development of new treatments designed to preserve vision in these difficult patients.

A Rat Model of Chronic Pressure-induced Optic Nerve Damage

To develop unilateral, chronically elevated intraocular pressure in rats, episcleral veins were injected with hypertonic saline and the intraocular pressure was monitored with a Tono-Pen XL tonometer. Histologic analyses of eyes with differing degrees and durations of intraocular pressure elevation were performed to ascertain the effects of these pressures on the optic nerve. Out of 20 consecutive animals, nine had elevations of intraocular pressure following a single injection, while subsequent injections raised intraocular pressure in seven others. One eye became hypotonous. In the remaining animals, subsequent injections sufficient to raise intraocular pressure were deliberately withheld, to determine the possible direct effects of injections on the optic nerve. Mean sustained pressure elevations ranged from 7 to 28 mm Hg and the retinal vasculature remained perfused in all eyes. Optic nerve cross sections from eyes without intraocular pressure elevation appeared identical to those from uninjected eyes, while nerves from eyes with the greatest intraocular pressure rise demonstrated axonal damage that involved 100% of the neural area. Eyes with either less severe pressure elevations or shorter durations showed partial damage, ranging from 0.5% to 10.4% of the neural area. In 70% of these nerves, damage was concentrated in the superior temporal region. Within the optic nerve head, often associated with astrocytes, axons contained abnormal accumulations of membrane-bound vesicles and mitochondria. The anterior chamber angles showed sclerosis of the trabecular meshwork with anterior synechiae, but Schlemm's canal, collector channels and aqueous veins appeared patent. Unilateral sclerosis of the trabecular meshwork produces sustained elevation of intraocular pressure in rats with optic nerve damage that in many ways resembles that seen in human glaucoma. Understanding the mechanism of nerve damage in this model may provide new insights into the pathogenesis of human glaucoma.