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Purpose We previously reported increased expression of cell proliferation and Jak-Stat pathway-related genes in chronic experimental glaucoma model optic nerve heads (ONH) with early, mild injury. Here, we confirm these observations by localizing, identifying, and quantifying ONH cellular proliferation and Jak-Stat pathway activation in this model. Methods Chronic intraocular pressure (IOP) elevation was achieved via outflow pathway sclerosis. After 5 weeks, ONH longitudinal sections were immunolabeled with proliferation and cell-type markers to determine nuclear densities in the anterior (unmyelinated) and transition (partially myelinated) ONH. Nuclear pStat3 labeling was used to detect Jak-Stat pathway activation. Nuclear density differences between control ONH (uninjected) and ONH with either early or advanced injury (determined by optic nerve injury grading) were identified by ANOVA. Results Advanced injury ONH had twice the nuclear density (P < 0.0001) of controls and significantly greater astrocyte density in anterior (P = 0.0001) and transition (P = 0.006) ONH regions. An increased optic nerve injury grade positively correlated with increased microglia/macrophage density in anterior and transition ONH (P < 0.0001, both). Oligodendroglial density was unaffected. In glaucoma model ONH, 80% of anterior and 66% of transition region proliferating cells were astrocytes. Nuclear pStat3 labeling significantly increased in early injury anterior ONH, and 95% colocalized with astrocytes. Conclusions Astrocytes account for the majority of proliferating cells, contributing to a doubled nuclear density in advanced injury ONH. Jak-Stat pathway activation is apparent in the early injury glaucoma model ONH. These data confirm dramatic astrocyte cell proliferation and early Jak-Stat pathway activation in ONH injured by elevated IOP.

Purpose MicroRNAs (miRNAs) are small, endogenous noncoding RNAs that have been detected in human aqueous humor (AH). Prior studies have pooled samples to obtain sufficient quantities for analysis or used next-generation sequencing. Here, we used PCR arrays with preamplification to identify and compare miRNAs from individual AH samples between patients with primary open-angle glaucoma (POAG) and normal controls. Methods AH was collected before cataract surgery from six stable, medically treated POAG patients and eight age-matched controls. Following reverse transcription and preamplification, individual patient samples were profiled on Taqman Low Density MicroRNA Array Cards. Differentially expressed miRNAs were stratified for fold changes larger than ±2 and for significance of P < 0.05. Significant Kyoto Encyclopedia of Genes and Genomes pathways influenced by the differentially expressed miRNAs were identified using the predicted target module of the miRWalk 2.0 database. Results This approach detected 181 discrete miRNAs, which were consistently expressed across all samples of both experimental groups. Significant up-regulation of miR-518d and miR-143, and significant down-regulation of miR-660, was observed in the AH of POAG patients compared with controls. These miRNAs were predicted to reduce cell proliferation and extracellular matrix remodeling, endocytosis, Wnt signaling, ubiquitin-mediated proteolysis, and adherens junction function. Conclusions This pilot study demonstrates that miRNA expression within the AH of POAG patients differs from age-matched controls. AH miRNAs exhibit potential as biomarkers of POAG, which merits further investigation in a larger case-controlled study. This technique provides a cost-effective and sensitive approach to assay miRNAs in individual patient samples without the need for pooling.

PURPOSE: To characterize early optic nerve head (ONH) structural change in rat experimental glaucoma (EG). METHODS: Unilateral intraocular pressure (IOP) elevation was induced in Brown Norway rats by hypertonic saline injection into the episcleral veins and animals were sacrificed 4 weeks later by perfusion fixation. Optic nerve cross-sections were graded from 1 (normal) to 5 (extensive injury) by 5 masked observers. ONHs with peripapillary retina and sclera were embedded, serial sectioned, 3-D reconstructed, delineated, and quantified. Overall and animal-specific EG versus Control eye ONH parameter differences were assessed globally and regionally by linear mixed effect models with significance criteria adjusted for multiple comparisons. RESULTS: Expansions of the optic nerve and surrounding anterior scleral canal opening achieved statistical significance overall (p < 0.0022), and in 7 of 8 EG eyes (p < 0.005). In at least 5 EG eyes, significant expansions (p < 0.005) in Bruch's membrane opening (BMO) (range 3-10%), the anterior and posterior scleral canal openings (8-21% and 5-21%, respectively), and the optic nerve at the anterior and posterior scleral canal openings (11-30% and 8-41%, respectively) were detected. Optic nerve expansion was greatest within the superior and inferior quadrants. Optic nerve expansion at the posterior scleral canal opening was significantly correlated to optic nerve damage (R = 0.768, p = 0.042). CONCLUSION: In the rat ONH, the optic nerve and surrounding BMO and neurovascular scleral canal expand early in their response to chronic experimental IOP elevation. These findings provide phenotypic landmarks and imaging targets for detecting the development of experimental glaucomatous optic neuropathy in the rat eye. Supported in part by NIH grants R01EY011610 (CFB), R01EY10145 (JCM) and R01EY16866 (ECJ)) from the National Eye Institute, National Institutes of Health, Bethesda, Maryland; The Legacy Good Samaritan Foundation, Portland, Oregon; the Sears Trust for Biomedical Research, Mexico, Missouri; the Alcon Research Institute, Fort Worth, Texas; and an unrestricted grant from Research to Prevent Blindness.

Small molecule delivery to the optic nerve would allow for exploration of molecular and cellular pathways involved in normal physiology and optic neuropathies such as glaucoma, and provide a tool for screening therapeutics in animal models. We report a novel surgical method for small molecule drug delivery to the optic nerve head (ONH) in a rodent model. In proof-of-principle experiments, we delivered cytochalasin D (Cyt D; a filamentous actin inhibitor) to the junction of the superior optic nerve and globe in rats to target the actin-rich astrocytic cytoskeleton of the ONH. Cyt D delivery was quantified by liquid chromatography and mass spectrometry of isolated optic nerve tissue. One day after Cyt D delivery, anterior ONH filamentous actin bundle content was significantly reduced as assessed by fluorescent-tagged phalloidin labeling, relative to sham delivery. Anterior ONH nuclear counts and axon-specific beta-3 tubulin levels, as well as peripapillary retinal ganglion cell layer nuclear counts were not significantly altered after Cyt D delivery relative to sham delivery. Lastly, the surgical delivery technique caused minimal observable axon degeneration up to 10 days post-surgery. This small molecule delivery technique provides a new approach to studying optic neuropathies in in vivo rodent models.

Injection of hypertonic saline via episcleral veins toward the limbus in laboratory rats can produce elevated intraocular pressure (IOP) by sclerosis of aqueous humor outflow pathways. This article describes important anatomic characteristics of the rat optic nerve head (ONH) that make it an attractive animal model for human glaucoma, along with the anatomy of rat aqueous humor outflow on which this technique is based. The injection technique itself is also described, with the aid of a supplemental movie, including necessary equipment and specific tips to acquire this skill. Outcomes of a successful injection are presented, including IOP elevation and patterns of optic nerve injury. These concepts are then specifically considered in light of the use of this model to assess potential neuroprotective therapies. Advantages of the hypertonic saline model include a delayed and relatively gradual IOP elevation, likely reproduction of scleral and ONH stresses and strains that may be important in producing axonal injury, and its ability to be applied to any rat (and potentially mouse) strain, leaving the unmanipulated fellow eye as an internal control. Challenges include the demanding surgical skill required by the technique itself, a wide range of IOP response, and mild corneal clouding in some animals. However, meticulous application of the principles detailed in this article and practice will allow most researchers to attain this useful skill for studying cellular events of glaucomatous optic nerve damage.

The purpose of this study is to three-dimensionally (3D) characterize the principal macroscopic and microscopic relationships within the rat optic nerve head (ONH) and quantify them in normal control eyes. Perfusion-fixed, trephinated ONH from 8 normal control eyes of 8 Brown Norway Rats were 3D histomorphometrically reconstructed, visualized, delineated and parameterized. The rat ONH consists of 2 scleral openings, (a superior neurovascular and inferior arterial) separated by a thin connective tissue strip we have termed the "scleral sling". Within the superior opening, the nerve abuts a prominent extension of Bruch's Membrane (BM) superiorly and is surrounded by a vascular plexus, as it passes through the sclera, that is a continuous from the choroid into and through the dural sheath and contains the central retinal vein (CRV), (inferiorly). The inferior scleral opening contains the central retinal artery and three long posterior ciliary arteries which obliquely pass through the sclera to obtain the choroid. Bruch's Membrane Opening (BMO) is irregular and vertically elongated, enclosing the nerve (superiorly) and CRV and CRA (inferiorly). Overall mean BMO Depth, BMO Area, Choroidal Thickness and peripapillary Scleral Thickness were 29 μm, 56.5 × 10(3) μm(2), 57 μm and 104 μm respectively. Mean anterior scleral canal opening (ASCO) and posterior scleral canal opening (PSCO) radii were 201 ± 15 μm and 204 ± 16 μm, respectively. Mean optic nerve area at the ASCO and PSCO were 46.3 × 10(3)±4.4 × 10(3) μm(2) and 44.1 × 10(3)±4.5 × 10(3) μm(2) respectively. In conclusion, the 3D complexity of the rat ONH and the extent to which it differs from the primate have been under-appreciated within previous 2D studies. Properly understood, these anatomic differences may provide new insights into the relative susceptibilities of the rat and primate ONH to elevated intraocular pressure.

Glaucoma is the leading cause of irreversible blindness and involves the death of retinal ganglion cells (RGCs). Although biomechanics likely contributes to axonal injury within the optic nerve head (ONH), leading to RGC death, the pathways by which this occurs are not well understood. While rat models of glaucoma are well-suited for mechanistic studies, the anatomy of the rat ONH is different from the human, and the resulting differences in biomechanics have not been characterized. The aim of this study is to describe a methodology for building individual-specific finite element (FE) models of rat ONHs. This method was used to build three rat ONH FE models and compute the biomechanical environment within these ONHs. Initial results show that rat ONH strains are larger and more asymmetric than those seen in human ONH modeling studies. This method provides a framework for building additional models of normotensive and glaucomatous rat ONHs. Comparing model strain patterns with patterns of cellular response seen in studies using rat glaucoma models will help us to learn more about the link between biomechanics and glaucomatous cell death, which in turn may drive the development of novel therapies for glaucoma.

A reliable method of creating chronic elevation of intraocular pressure (IOP) in rodents is an important tool in reproducing and studying the mechanisms of optic nerve injury that occur in glaucoma. In addition, such a model could provide a valuable method for testing potential neuroprotective treatments. This paper outlines the basic methods for producing obstruction of aqueous humor outflow and IOP elevation by injecting hypertonic saline (a sclerosant) into the aqueous outflow pathway. This is one of several rodent glaucoma models in use today. In this method, a plastic ring is placed around the equator of the eye to restrict injected saline to the limbus. By inserting a small glass microneedle in an aqueous outflow vein in the episclera and injecting hypertonic saline toward the limbus, the saline is forced into Schlemm's canal and across the trabecular meshwork. The resultant inflammation and scarring of the anterior chamber angle occurs gradually, resulting in a rise in IOP after approximately 1 week. This article will describe the equipment necessary for producing this model and the steps of the technique itself.

MicroRNAs are small, endogenous noncoding RNAs that modulate post-transcriptional gene expression. Recent evidence suggests that they may have a potential role in the regulation of the complex biological responses that develop in response to elevated intraocular pressure. However, contemporary microRNA assay techniques (e.g., microarrays and next-generation sequencing) typically require large amounts of RNA template that are often times difficult to obtain from glaucomatous tissue. We describe in detail an experimental protocol utilizing targeted pre-amplification and low-density polymerase chain reaction arrays to circumvent this hurdle. This approach optimizes the simultaneous high-throughput screening of small tissue samples, such as the rodent optic nerve head, for up to 754 microRNA probes while also providing an opportunity for subsequent confirmatory reactions of technical or biological replicates.

Understanding the cellular pathways activated by elevated intraocular pressure (IOP) is crucial for the development of more effective glaucoma treatments. Microarray studies have previously been used to identify several key gene expression changes in early and extensively injured ONH, as well as in the retina. Limitations of microarrays include that they can only be used to detect transcripts that correspond to existing genomic sequencing information and their narrower dynamic range. However, RNA sequencing (RNA-seq) is a powerful tool for investigating known transcripts, as well as for exploring new ones (including noncoding RNAs and small RNAs), is more quantitative, and has the added benefit that the data can be re-analyzed as new sequencing information becomes available. Here, we describe an RNA-seq method specifically developed for identifying differentially expressed genes in optic nerve heads of eyes exposed to elevated intraocular pressure. The methods described here could also be applied to small tissue samples (less than 100 ng in total RNA yield) from retina, optic nerve, or other regions of the central nervous system.

A reliable method of creating chronic elevation of intraocular pressure (IOP) in rodents is an important tool in reproducing and studying the mechanisms of optic nerve injury that occur in glaucoma. In addition, such a model could provide a valuable method for testing potential neuroprotective treatments. This paper outlines the basic methods for producing obstruction of aqueous humor outflow and IOP elevation by injecting hypertonic saline (a sclerosant) into the aqueous outflow pathway. This is one of several rodent glaucoma models in use today. In this method, a plastic ring is placed around the equator of the eye to restrict injected saline to the limbus. By inserting a small glass microneedle in an aqueous outflow vein in the episclera and injecting hypertonic saline toward the limbus, the saline is forced into Schlemm's canal and across the trabecular meshwork. The resultant inflammation and scarring of the anterior chamber angle occurs gradually, resulting in a rise in IOP after approximately 1 week. This article will describe the equipment necessary for producing this model and the steps of the technique itself.

Purpose To compare the effect of elevated intraocular pressure (IOP) on retinal capillary filling in elderly vs adult rats using optical coherence tomography angiography (OCTA). Methods The IOP of elderly (24-month-old, N = 12) and adult (6–8 month-old, N = 10) Brown Norway rats was elevated in 10 mmHg increments from 10 to 100 mmHg. At each IOP level, 3D OCT data were captured using an optical microangiography (OMAG) scanning protocol and then post-processed to obtain both structural and vascular images. Mean arterial blood pressure (MAP), respiratory rate, pulse and blood oxygen saturation were monitored non-invasively throughout each experiment. Ocular perfusion pressure (OPP) was calculated as the difference between MAP for each animal and IOP at each level. The capillary filling index (CFI), defined as the ratio of area occupied by functional capillary vessels to the total scan area but excluding relatively large vessels of > 30 μm, was calculated at each IOP level and analyzed using the OCTA angiograms. Relative CFI vs IOP was plotted for the group means. CFI vs OPP was plotted for every animal in each group and data from all animals were combined in a CFI vs OPP scatter plot comparing the two groups. Results The MAP in adult animals was 108 ± 5 mmHg (mean ± SD), whereas this value in the elderly was 99 ± 5 mmHg. All other physiologic parameters for both age groups were uniform and stable. In elderly animals, significant reduction of the CFI was first noted at IOP 40 mmHg, as opposed to 60 mmHg in adult animals. Individual assessment of CFI as a function of OPP for adult animals revealed a consistent plateau until OPP reached between 40 and 60 mmHg. Elderly individuals demonstrated greater variability, with many showing a beginning of gradual deterioration of CFI at an OPP as high as 80 mmHg. Overall comparison of CFI vs OPP between the two groups was not statistically significant. Conclusions Compared to adults, some, but not all, elderly animals demonstrate a more rapid deterioration of CFI vs OPP. This suggests a reduced autoregulatory capacity that may contribute to increased glaucoma susceptibility in some older individuals. This variability must be considered when studying the relationship between IOP, ocular perfusion and glaucoma in elderly animal models.

In this paper, we demonstrate the use of optical coherence tomography/optical microangiography (OCT/OMAG) to image and measure the effects of acute intraocular pressure (IOP) elevation on retinal, choroidal and optic nerve head (ONH) perfusion in the rat eye. In the experiments, IOP was elevated from 10 to 100 mmHg in 10 mmHg increments. At each IOP level, three-dimensional data volumes were captured using an ultrahigh sensitive (UHS) OMAG scanning protocol for 3D volumetric perfusion imaging, followed by repeated B-scans for Doppler OMAG analysis to determine blood flow velocity. Velocity and vessel diameter measurements were used to calculate blood flow in selected retinal blood vessels. Choroidal perfusion was calculated by determining the peripapillary choroidal filling at each pressure level and calculating this as a percentage of area filling at baseline (10 mmHg). ONH blood perfusion was calculated as the percentage of blood flow area over a segmented ONH area to a depth 150 microns posterior to the choroidal opening. We show that volumetric blood flow reconstructions revealed detailed 3D maps, to the capillary level, of the retinal, choroidal and ONH microvasculature, revealing retinal arterioles, capillaries and veins, the choroidal opening and a consistent presence of the central retinal artery inferior to the ONH. While OCT structural images revealed a reversible compression of the ONH and vasculature with elevated IOP, OMAG successfully documented changes in retinal, choroidal and ONH blood perfusion and allowed quantitative measurements of these changes. Starting from 30 mm Hg, retinal blood flow (RBF) diminished linearly with increasing IOP and was nearly extinguished at 100 mm Hg, with full recovery after return of IOP to baseline. Choroidal filling was unaffected until IOP reached 60 mmHg, then decreased to 20% of baseline at IOP 100 mmHg, and normalized when IOP returned to baseline. A reduction in ONH blood perfusion at higher IOP’s was also observed, but shadow from overlying retinal vessels at lower IOP’s limited precise measurements of changes in ONH capillary perfusion compared to baseline. Therefore, OCT/OMAG can be a useful tool to image and measure blood flow in the retina, choroidal and ONH of the rat eye as well as document the effects of elevated IOP on blood flow in these vascular beds.

Understanding mechanisms of glaucomatous optic nerve damage is essential for developing effective therapies to augment conventional pressure-lowering treatments. This requires that we understand not only the physical forces in play, but the cellular responses that translate these forces into axonal injury. The former are best understood by using primate models, in which a well-developed lamina cribrosa, peripapillary sclera and blood supply are most like that of the human optic nerve head. However, determining cellular responses to elevated intraocular pressure (IOP) and relating their contribution to axonal injury requires cell biology techniques, using animals in numbers sufficient to perform reliable statistical analyses and draw meaningful conclusions. Over the years, models of chronically elevated IOP in laboratory rats and mice have proven increasingly useful for these purposes. While lacking a distinct collagenous lamina cribrosa, the rodent optic nerve head (ONH) possesses a cellular arrangement of astrocytes, or glial lamina, that ultrastructurally closely resembles that of the primate. Using these tools, major insights have been gained into ONH and the retinal cellular responses to elevated IOP that, in time, can be applied to the primate model and, ultimately, human glaucoma.

In this paper, we present methods for 3D visualization and quantitative measurements of retinal blood flow in rats by the use of optical microangiography imaging technique (OMAG). We use ultrahigh sensitive OMAG to provide high-quality 3D RBF perfusion maps in the rat eye, from which the Doppler angle, as well as the diameters of blood vessels, are evaluated. Estimation of flow velocity (i.e. axial flow velocity) is achieved by the use of Doppler OMAG, which has its origins in phase-resolved Doppler optical coherence tomography. The measurements of the Doppler angle, vessel size, and the axial velocity lead to the quantitative assessment of the absolute flow velocity and the blood flow rate in selected retinal vessels. We demonstrate the feasibility of OMAG to provide 3D microangiograms and quantitative assessment of retinal blood flow in a rat model subjected to raised intra-ocular pressure (IOP). We show that OMAG is capable of monitoring the longitudinal response of absolute blood velocity and flow rate of retinal blood vessels to increased IOP in the rat, demonstrating its usefulness for ophthalmological research.

Purpose Optic nerve head (ONH) astrocytes provide support for axons, but exhibit structural and functional changes (termed reactivity) in a number of glaucoma models. The purpose of this study was to determine if ONH astrocyte structural reactivity is axon-dependent. Methods Using rats, we combine retrobulbar optic nerve transection (ONT) with acute controlled elevation of intraocular pressure (CEI), to induce total optic nerve axon loss and ONH astrocyte reactivity, respectively. Animals were euthanized immediately or 1 day post CEI, in the presence or absence of ONT. ONH sections were labeled with fluorescent-tagged phalloidin and antibodies against β3 tubulin, phosphorylated cortactin, phosphorylated paxillin, or complement C3. ONH label intensities were quantified after confocal microscopy. Retrobulbar nerves were assessed for axon injury by light microscopy. Results While ONT alone had no effect on ONH astrocyte structural orientation, astrocytes demonstrated significant reorganization of cellular extensions within hours after CEI, even when combined with ONT. However, ONH astrocytes displayed differential intensities of actin (phosphorylated cortactin) and focal adhesion (phosphorylated paxillin) mediators in response to CEI alone, ONT alone, or the combination of CEI and ONT. Lastly, label intensities of complement C3 within the ONH were unchanged in eyes subjected to CEI alone, ONT alone, or the combination of CEI and ONT, relative to controls. Conclusions Early ONH astrocyte structural reactivity to elevated IOP is multifaceted, displaying both axon dependent and independent responses. These findings have important implications for pursuing astrocytes as diagnostic and therapeutic targets in neurodegenerative disorders with fluctuating levels of axon injury.

Reduced retrograde transport of neurotrophins (NT) and their receptors has been hypothesized to contribute directly to retinal ganglion cell (RGC) loss in glaucoma. However, strategies of supplementing NT and NT receptors have failed to avert ultimate RGC death in experimental glaucoma. This study examines the response of major components of the NT system and their interacting proteins in a rat glaucoma model. Unilateral chronic intraocular pressure (IOP) elevation was produced by episcleral vein injection of hypertonic saline (N = 99). Retinas were collected and grouped by extent of optic nerve injury. Quantitative reverse transcription PCR, western blot analysis and immunohistochemistry were used to determine mRNA and protein levels and protein localization. Out of three RGC-specific Brn3 proteins (Brn3a, b, and c), only Brn3a was significantly downregulated at the message level to 35 +/- 4% of fellow values with the severest nerve injury. With IOP elevation, no significant alterations were found in retinal mRNA levels for BDNF, NGF, NT-4/5 or NT-3. The abundance of mature retinal BDNF protein was not significantly affected by elevated IOP, while proBDNF protein decreased linearly with increasing injury grade (r(2) = 0.50). In retinas with the severest nerve injury, TrkB and TrkC receptor mRNA levels significantly declined to 67 +/- 9% and 44 +/- 5% of fellow values, respectively. However, the levels of TRKB protein and its phosphorylated form were unchanged. Message level for p75(NTR) was linearly upregulated up to 219 +/- 26% with increasing injury (r(2) = 0.46), but no alteration was detected at protein level. The mRNA expression of p75(NTR) apoptosis adaptor proteins NADE, NRIF, and Lingo1 were significantly downregulated in retinas with the greatest nerve injury. A positive correlation was found between injury extent and message levels for Jun (r(2) = 0.23) as well as Junb (r(2) = 0.27), and RGC labeling of activated JUN protein increased. Atf3 mRNA levels demonstrated a positive linear correlation to the extent of injury (r(2) = 0.53), resulting in a nearly five-fold increase (482 +/- 76%) in eyes with the greatest nerve damage. Among downstream pro-survival signaling components, Erk5 mRNA expression was linearly upregulated (r(2) = 0.32) up to 157 +/- 15% of fellow values in retinas with the severest nerve injury (p < 0.01). A slight positive correlation was found between NF-kappaB message levels and injury extent (r(2) = 0.12). Bcl-xl mRNA levels in the most severely injured retinas were significantly reduced to 83 +/- 7% by elevated IOP exposure. Message levels for Erk1/2, Akt1-3 or Bcl2 appeared unaffected. Elevated IOP did not alter mRNA levels of pro-apoptotic Bim, Bax, or p53. This study demonstrates that elevated IOP exposure does not result in a dramatic decrease in retinal levels of either BDNF or its receptor, TrkB. It shows that the responses of NT pathways to elevated IOP are complex, particularly with regard to the role of p75(NTR) and Atf3. A better understanding of the roles of these proteins in IOP-induced injury is likely to suggest informed strategies for neuroprotection in glaucoma.

Glaucomatous axon injury occurs at the level of the optic nerve head (ONH) in response to uncontrolled intraocular pressure (IOP). The temporal response of ONH astrocytes (glial cells responsible for axonal support) to elevated IOP remains unknown. Here, we evaluate the response of actin-based astrocyte extensions and integrin-based signaling within the ONH to 8 hours of IOP elevation in a rat model. IOP elevation of 60 mm Hg was achieved under isoflurane anesthesia using anterior chamber cannulation connected to a saline reservoir. ONH astrocytic extension orientation was significantly and regionally rearranged immediately after IOP elevation (inferior ONH, 43.2° ± 13.3° with respect to the anterior-posterior axis versus 84.1° ± 1.3° in controls, p

Purpose To determine if retinal capillary filling is preserved in the face of acutely elevated intraocular pressure (IOP) in anesthetized rats, despite a reduction in total retinal blood flow (RBF), using optical microangiography/optical coherence tomography (OMAG/OCT). Methods OMAG provided the capability of depth-resolved imaging of the retinal microvasculature down to the capillary level. Doppler OCT was applied to measure the total RBF using an enface integration approach. The microvascular pattern, capillary density, and total RBF were monitored in vivo as the IOP was increased from 10 to 100 mm Hg in 10 mm Hg intervals and returned back to 10 mm Hg. Results In animals with mean arterial pressure (MAP) of 102 ± 4 mm Hg (n = 10), when IOP was increased from 0 to 100 mm Hg, the capillary density remained at or above 80% of baseline for the IOP up to 60 mm Hg [or ocular perfusion pressure (OPP) at 40 mm Hg]. This was then decreased, achieving 60% of baseline at IOP 70 mm Hg and OPP of 30 mm Hg. Total RBF was unaffected by moderate increases in IOP up to 30 mm Hg, beyond which total RBF decreased linearly, reaching 50% of baseline at IOP 60 mm Hg and OPP 40 mm Hg. Both capillary density and total RBF were totally extinguished at 100 mm Hg, but fully recovered when IOP returned to baseline. By comparison, a separate group of animals with lower MAP (mean = 75 ± 6 mm Hg, n = 7) demonstrated comparable decreases in both capillary filling and total RBF at IOPs that were 20 mm Hg lower than in the initial group. Both were totally extinguished at 80 mm Hg, but fully recovered when IOP returned to baseline. Relationships of both parameters to OPP were unchanged. Conclusion Retinal capillary filling and total RBF responses to IOP elevation can be monitored non-invasively by OMAG/OCT and both are influenced by OPP. Retinal capillary filling was relatively preserved down to a perfusion pressure of 40 mm Hg, despite a linear reduction in total RBF.

Glaucoma is one of the leading causes of blindness in humans and is characterized by damage to retinal ganglion cells (RGCs) and the optic nerve.1,2 While abnormally high IOP is a widely recognized risk factor,3,4 the molecular pathways linking elevated IOP and RGC loss are complex. Although several inroads toward understanding the mechanisms have recently been made,5–13 much remains to be learned, particularly regarding the responses in chronic injury. Extracellular ATP is a likely candidate to link the elevated IOP in glaucoma to perturbed signaling in the retina and optic nerve. Throughout the body, released ATP conveys information about mechanical strain and accompanies shear stress, swelling, and stretch.14–18 This mechanosensitive release of ATP can initiate a series of physiological and pathological responses including cell death, volume regulation, pain, inflammatory responses, and neuroprotection by activating ionotropic P2X and metabotropic P2Y receptors.19 Evidence is accumulating for an increased release of ATP following IOP elevation in glaucoma. For instance, humans with primary acute angle closure glaucoma (PAACG) displayed a 9-fold rise in the ATP levels in their anterior chamber.20 Bovine eyes released ATP into the vitreal chamber in levels proportional to the magnitude of applied pressure,21 while the damage caused to rat RGCs by acute rises in IOP was related to levels of extracellular ATP.22 Both RGCs and astrocytes release ATP upon mechanical strain, with pannexin hemichannels a likely pathway for release in both cell types.23–25 While these reports indicate that acute increases in IOP and/or mechanical strain lead to release of ATP in the retina, most patients with glaucoma suffer from an elevation in IOP for extended periods. Evidence for a sustained release of ATP associated with a prolonged elevation of IOP would thus have relevance for the most common forms of the disease. We have previously demonstrated that humans with primary chronic angle closure glaucoma (PCACG) have a 14-fold increase in ATP levels in the anterior chamber.26 While this finding supports the theory that a chronic elevation in IOP leads to a sustained elevation in extracellular ATP release, it was not possible to confirm this increase in the human retina as sampling was only justified from the anterior segment in these patients and not in the vitreal chamber. As the pathological changes to the retina and optic nerve head are most directly associated with vision loss, it is important to determine whether elevated extracellular levels of ATP also occur in the posterior eye during a chronic rise in IOP. Given that excessive stimulation of the P2X7 receptor for ATP can both kill RGCs and activate the inflammasome,27,28 the relationship between elevated IOP and extracellular ATP in chronic glaucoma has particular importance. The present study asked whether extracellular ATP was elevated in animal models of chronic glaucoma. As all current animal models of chronic glaucoma are imperfect, we used three different models to ensure the broadest relevance of the findings to the human condition. Specifically, chronic elevation of IOP was monitored in the Tg-MyocY437H mouse, in rat eyes following injection of hypertonic saline into episcleral veins, and in primates after laser photocoagulation of the trabecular meshwork. As ATP measurement from the small extracellular space in the retina is problematic, ATP levels in the vitreous were measured. Levels of the enzyme NTPDase1 were also compared in retinal tissue from rats, mice, and primates with sustained elevation in IOP and control IOP levels as sustained exposure to extracellular ATP increased expression of the ectoATPase NTPDase1 in optic nerve head astrocytes in vitro.

To determine if astrocyte processes label for actin and to quantify the orientation of astrocytic processes within the optic nerve head (ONH) in a rat glaucoma model.Chronic intraocular pressure (IOP) elevation was produced by episcleral hypertonic saline injection and tissues were collected after 5 weeks. For comparison, eyes with optic nerve transection were collected at 2 weeks. Fellow eyes served as controls. Axonal degeneration in retrobulbar optic nerves was graded on a scale of 1 to 5. Optic nerve head sections (n ≥ 4 eyes per group) were colabeled with phalloidin (actin marker) and antibodies to astrocytic glial fibrillary acidic protein and aquaporin 4, or axonal tubulin βIII. Confocal microscopy and FIJI software were used to quantify the orientation of actin bundles.Control ONHs showed stereotypically arranged actin bundles within astrocyte processes. Optic nerve head actin bundle orientation was nearly perpendicular to axons (82.9° ± 6.3° relative to axonal axis), unlike the retrobulbar optic nerve (45.4° ± 28.7°, P0.05). With IOP elevation, ONH actin bundle orientation became less perpendicular to axons, even in eyes with no perceivable axonal injury (i.e., 38.8° ± 15.1° in grade 1, P0.05 in comparison to control ONHs). With severe injury, ONH actin bundle orientation became more parallel to the axonal axis (24.1° ± 28.4°, P0.05 in comparison to control ONHs). Optic nerve head actin bundle orientation in transected optic nerves was unchanged.Actin labeling identifies fine astrocyte processes within the ONH. Optic nerve head astrocyte process reorientation occurs early in response to elevated IOP.

Purpose We determine if several hours of controlled elevation of IOP (CEI) will produce the optic nerve head (ONH) gene expression changes and optic nerve (ON) damage pattern associated with early experimental glaucoma in rats. Methods The anterior chambers of anesthetized rats were cannulated and connected to a reservoir to elevate IOP. Physiologic parameters were monitored. Following CEI at various recovery times, ON cross-sections were graded for axonal injury. Anterior ONHs were collected at 0 hours to 10 days following CEI and RNA extracted for quantitative PCR measurement of selected messages. The functional impact of CEI was assessed by electroretinography (ERG). Results During CEI, mean arterial pressure (99 ± 6 mm Hg) and other physiologic parameters remained stable. An 8-hour CEI at 60 mm Hg produced significant focal axonal degeneration 10 days after exposure, with superior lesions in 83% of ON. Message analysis in CEI ONH demonstrated expression responses previously identified in minimally injured ONH following chronic IOP elevation, as well as their sequential patterns. Anesthesia with cannulation at 20 mm Hg did not alter these message levels. Electroretinographic A- and B-waves, following a significant reduction at 2 days after CEI, were fully recovered at 2 weeks, while peak scotopic threshold response (pSTR) remained mildly but significantly depressed. Conclusions A single CEI reproduces ONH message changes and patterns of ON injury previously observed with chronic IOP elevation. Controlled elevation of IOP can allow detailed determination of ONH cellular and functional responses to an injurious IOP insult and provide a platform for developing future therapeutic interventions.

Experimentally elevated IOP in animals produces glaucoma-like retinal ganglion cell (RGC) and optic nerve axonal loss and a characteristic remodeling of the optic nerve head (ONH) that includes the rearrangement of glial cells and the deposition of extracellular matrix (ECM) proteins. Although experimental glaucoma models have been produced in a number of species, primate and rodent models are the most extensively used. In both human and experimental glaucoma, there is a general consensus that the ONH is the primary site of initial glaucomatous injury to RGC axons.1,2 Recently, sophisticated 3-D histomorphometric and biomechanical studies have been used to examine some of the earliest morphologic and structural ONH responses in experimental glaucoma attributed to elevated IOP in primates.3–11 However, relatively few studies have examined the early cellular responses of the ONH to elevated IOP exposure. Such studies should help us understand how the ONH cells respond to an environment altered by elevated IOP and how these responses might affect axonal integrity. To examine the response to acute IOP elevation, the anterior chamber can be cannulated to allow IOP to be elevated for up to a few hours without altering retinal perfusion. After these acute elevations, abnormalities in axonal transport, including the accumulation of mitochondria, dynein, neurotrophins and their receptors, and the distribution of axonal and astrocytic proteins have been observed.12–17 For longer IOP elevation, a number of techniques are available to obstruct aqueous outflow and produce glaucoma-like optic nerve degeneration.18–23 In initial studies using our chronic, hypertonic, saline-induced rat glaucoma model and immunohistochemistry, we found characteristic glaucoma-like changes in the rat ONH ECM. As early as 11 days after IOP elevation, we observed depositions of collagen IV, collagen VI, and laminin.24 Next, we used immunohistochemistry to study the chronology of morphologic and protein distribution changes in the ONH. At 3 days after IOP elevation, we observed decreased labeling in ONH glial columns for gap junction protein alpha 1 (connexin 43, Gja1) and increases in a cell proliferation marker.25 These changes were followed at 1 week by decreased labeling for neurotrophins and Gfap, increased vascular labeling of collagen VI, and the appearance of swollen, degenerating axons in the ONH and focal lesions in optic nerve cross-sections. In a separate study using laser-induced IOP elevation, an accumulation of the retrograde transport motor dynein was observed in the ONH at approximately the same duration of IOP elevation.26 More recently, we have used our glaucoma model to determine gene expression changes in extensively injured ONH.27 As part of that study, we also reported expression changes in a few specific genes in ONH from eyes with focal optic nerve injuries (

To determine and compare gene expression patterns in the whole retina and retinal ganglion cell layer (RGCL) in a rodent glaucoma model.IOP was unilaterally elevated in Brown Norway rats (N = 26) by injection of hypertonic saline and monitored for 5 weeks. A cDNA microarray was used on whole retinas from one group of eyes with extensive optic nerve injury and on RGCL isolated by laser capture microdissection (LCM) from another group with comparable injury, to determine the significantly up- or downregulated genes and gene categories in both groups. Expression changes of selected genes were examined by quantitative reverse transcription-PCR (qPCR) to verify microarray results.Microarray analysis of the whole retina identified 632 genes with significantly changed expression (335 up, 297 down), associated with 9 upregulated and 3 downregulated biological processes. In contrast, the RGCL microarray yielded 3726 genes with significantly changed expression (2003 up, 1723 down), including 60% of those found in whole retina. Thirteen distinct upregulated biological processes were identified in the RGCL, dominated by protein synthesis. Among 11 downregulated processes, axon extension and dendrite morphogenesis and generation of precursor metabolism and energy were uniquely identified in the RGCL. qPCR confirmed significant changes in 6 selected messages in whole retina and 11 in RGCL. Increased Atf3, the most upregulated gene in the RGCL, was confirmed by immunohistochemistry of RGCs.Isolation of RGCL by LCM allows a more refined detection of gene response to elevated pressure and improves the potential of determining cellular mechanisms in RGCs and their supporting cells that could be targets for enhancing RGC survival.

Glaucomatous vision loss results from the progressive degeneration of optic nerve axons and the death of retinal ganglion cells. This process is accompanied by dramatic alterations in the functional properties and distribution of glial cells in both the retina and the optic nerve head in a reaction commonly referred to as glial activation. The recent availability of rodent and cell culture glaucoma models has substantially contributed to our knowledge of glial activation under glaucomatous conditions. Conclusions drawn from these studies have led to the refinement of existing hypotheses and the generation of new ones. Because these hypotheses encompass both protective and injurious roles for glia, the impact of specific aspects of glial activation are current topics of intensive research, speculation, and debate in the field. With these unresolved issues in mind, this review will summarize recent progress in our understanding of the process of glial activation in the glaucomatous optic nerve head and retina.

To compare the sensitivity of the TonoLab rebound tonometer with the Tono-Pen in awake Brown Norway rats and to compare their ability to predict optic nerve damage induced by experimental IOP elevation.TonoLab and Tono-Pen tonometers were calibrated in cannulated rat eyes connected to a pressure transducer. The TonoLab was used in awake animals housed in standard lighting to measure IOP during light and dark phases. Both instruments were used to monitor chronically elevated IOP produced by episcleral vein injection of hypertonic saline. Measured IOPs were correlated with quantified optic nerve damage in injected eyes.Although they were lower than transducer and Tono-Pen measurements at all levels, TonoLab readings showed an excellent linear fit with transducer readings from 20 to 80 mm Hg (R(2) = 0.99) in cannulated eyes. In awake animals housed in standard lighting, the TonoLab documented significantly higher pressures during the dark phase (27.9 +/- 1.7 mm Hg) than during the light phase (16.7 +/- 2.3 mm Hg). With elevated IOP, correlation between TonoLab and Tono-Pen readings (R(2) = 0.86, P0.0001) was similar to that in cannulated eyes. Although both instruments provided measurements that correlated well with optic nerve injury grade, only the Tono-Pen documented significant IOP elevation in eyes with the least amount of injury (P0.05).The TonoLab is sensitive enough to be used in awake Brown Norway rats, though instrument fluctuation may limit its ability to identify significant pressure elevations in eyes with minimal optic nerve damage.

Author(s): Tezel, Gulgun; Fourth ARVO/Pfizer Ophthalmics Research Institute Conference Working Group

The neurotrophin (NT) hypothesis proposes that the obstruction of retrograde transport at the optic nerve head results in the deprivation of neurotrophic support to retinal ganglion cells (RGC) leading to apoptotic cell death in glaucoma. An important corollary to this concept is the implication that appropriate enhancement of neurotrophic support will prolong the survival of injured RGC indefinitely. This hypothesis is, perhaps, the most widely recognized theory to explain RGC loss resulting from exposure of the eye to elevated intraocular pressure (IOP). Recent studies of NT signaling using rat glaucoma models, have examined the endogenous responses of the retina to pressure exposure as well as studies designed to augment NT signaling in order to rescue RGC from apoptosis following pressure-induced injury. The examination of these studies in this review reveals a number of consistent observations and provides direction for further investigations of this hypothesis.

Purpose—Optic nerve disease in chronic IOP elevation rat glaucoma models develops at different rates. This study was undertaken to investigate whether anterior chamber (AC) changes develop in two popular models in vivo and whether the changes are related to IOP. Methods—Ten female Wistar rats and 12 male Brown-Norway rats were subjected to episcleral vein cauterization (EVC) and hypertonic saline episcleral vein sclerosis (HSEVS), respectively. Contralateral untreated eyes served as controls. IOP was recorded for a period of 5 to 6 weeks, and with the rats under anesthesia, the eyes were imaged with an ultrasound biomicroscope. Measurements of the AC depth (ACD), trabecular–iris angle (TIA), iris thickness at the thickest point near the pupillary margin (IT), angle opening distance (AOD; at 200 μm from the scleral spur), and ciliary body area (CBA) were compared between control eyes of the two strains and between experimental and control eyes within each strain. The differences were correlated with IOP history. Results—Eyes subjected to EVC demonstrated greater increases in IOP than eyes subjected to HSEVS. Between rat strains, control eyes differed significantly in all the parameters studied, except for ACD. No difference was detected between experimental and control eyes in the EVC group. In contrast, experimental eyes in the HSEVS group had ~71% larger ACDs and ~32% smaller CBAs than did the contralateral control eyes (P < 0.001). ACD and CBA correlated well (R 2 = 0.80 and 0.51, respectively) with IOP in the HSEVS group. Two of the experimental eyes in this group showed the presence of ultrasound-scattering material in the AC. Conclusions—Despite apparently higher IOP exposure, eyes in the EVC rat model of glaucoma do not undergo changes in the AC. In contrast, eyes subjected to HSEVS display deepening of the AC and reduction in size of the ciliary body within 5 to 6 weeks. These changes correlate to IOP exposure and may be the result of specific changes induced by the experimental intervention. These models are likely to rely on different mechanisms of pressure elevation and cannot be used interchangeably. Rat models of glaucoma that involve chronic elevation of IOP are often used to study the retinal and optic nerve pathophysiology of the disease 1 as well as for screening potential neuroprotective agents. 2 These models presumably rely on occlusion of the outflow pathways

Rats are becoming an increasingly popular model system for understanding mechanisms of optic nerve injury in primary open-angle glaucoma (POAG). Although the anatomy of the rat optic nerve head (ONH) is different from the human, the ultrastructural relationships between astrocytes and axons are quite similar, making it likely that cellular processes of axonal damage in these models will be relevant to human glaucoma. All of these models rely on elevating intraocular pressure (IOP), a major risk factor for glaucoma. Methods that produce increased resistance to aqueous humor outflow at the anterior chamber angle, specifically hypertonic saline injection of aqueous outflow pathways and laser treatment of the limbal tissues, appear to produce a specific regional pattern of injury that may have a particular relevance to understanding regional injury in human glaucoma. Because increased pressure fluctuations are a characteristic of such models and the rodent ONH appears to have high susceptibility to elevated IOP, special instrumentation and measurement techniques are required to document pressure exposure in these eyes and understand the pressure levels that the eyes and the optic nerve are exposed to. With these techniques, it is possible to obtain an excellent correlation between pressure and the extent of nerve damage. Continued use of these models will lead to a better understanding of cellular mechanisms of pressure-induced optic nerve damage and POAG.

Glaucoma is the second leading cause of blindness worldwide, affecting approximately 60 million people.1 Although many risk factors are associated with glaucoma, IOP is the most widely recognized, and lowering IOP is the goal of current glaucoma therapies. When IOP is experimentally elevated in nonhuman primates, the pattern of optic nerve head (ONH) cupping, optic nerve axon degeneration, and selective loss of retinal ganglion cells replicates the pathologic features of human glaucoma. Clinically, human glaucomatous optic neuropathy is characterized by optic disc cupping and a pattern of visual field loss.2 Cupping results from the loss of optic nerve axons and posterior bowing and remodeling of the support structures of the ONH.3,4 Often, these changes are most pronounced in the superior and inferior parts of the nerve head. The most characteristic visual field defect is the arcuate scotoma, which arches above or below central fixation and follows the pathways of the nerve fiber bundles as they converge on the superior and inferior poles of the ONH.2,5,6 Regional variation in laminar structure suggests less support and protection for axons, possibly explaining the apparent increased susceptibility of axons that pass through the superior and inferior ONH. In the face of increased or fluctuating IOP, movement of the lamina may result in preferential mechanical injury to neural tissues in these regions.7 In addition, because the vascular supply to the ONH tissue lies within the laminar beams, this pattern could result in regionally compromised blood flow.8 Current experimental evidence supporting either of these mechanisms is limited, and the cellular events that connect the regional pattern of glaucomatous optic nerve damage with the known structural anatomy of the ONH are still largely unknown. Regardless of mechanism, the variation in the structure of the ONH provides the only anatomic correlation with the characteristic pattern of glaucomatous optic nerve axon loss. The apparent vulnerability of the ONH to pressure-induced axonal injury has led many investigators to examine changes in the composition of glaucomatous human and experimental monkey ONH tissues. These ONHs are characterized by axon loss, gliotic scaring, increased expression of matrix metalloproteinases, and abnormal deposition of extracellular matrix (ECM) materials, including collagens, tropoelastin, tenascin, and proteoglycans.9–20 In the rat, glaucoma can be modeled by sclerosing aqueous outflow pathways to produce a sustained elevation of IOP.21 In this model, pressure causes a selective loss of retinal ganglion cells and a characteristic pattern of axon degeneration that begins in the superior quadrant of the optic nerve.21,22 In addition, immunohistochemical studies of ONH reveal that pressure-induced injury is accompanied by deposition of collagens and other ECM components, similar to that in human glaucoma.23 This deposition of ECM is preceded by a loss of gap junctional connexin 43 immunolabeling and evidence of astrocytic proliferation.24 By immunohistochemical analysis, the initial evidence of ECM deposition coincided with a decrease in ONH labeling for neurotrophins and astrocytic GFAP.24 Although the ONH is recognized as the likely site of initial injury in human glaucoma and in experimental IOP elevation glaucoma models, very little is known about the changes in gene expression that accompany this injury. In this study, we used microarray analysis to identify the genes and functional gene classes most altered in expression in the rat ONH after exposure to experimentally elevated pressure that results in extensive and ongoing optic nerve degeneration. Then, we used real-time quantitative (q)PCR to verify selected changes in gene expression initially identified by microarray analysis and to examine several genes not included on the arrays. To expand this study, we included qPCR analyses of ONHs from eyes with focal regions of degeneration in optic nerve cross-sections. Focal injury occurs in eyes with more mild pressure elevations or those of shorter duration and probably reflects earlier responses to pressure-induced nerve injury. We compared injury in these ONHs to those in ONHs with more extensive injury due to elevated IOP and to those after optic nerve transection, to evaluate ONH responses to simple loss of axons. The expansion of the qPCR study to nerves with focal injuries allows us to explore the potential of discovering, by future microarray analyses, unique or more dramatic alterations in gene expression that occur early in the injury process. To our knowledge, this is the first genome-wide analysis of expression changes in the ONH in response to elevated IOP.

Glaucoma is a leading cause of irreversible worldwide blindness, characterized by progressive loss of retinal ganglion cells (RGCs) and optic nerve degeneration that is usually secondary to elevated IOP.1–3 Current treatments for glaucoma are predominantly restricted to the modulation of IOP through pharmacological and surgical approaches. However, the effectiveness of this strategy can be limited in many patients by poor medication efficacy, intolerance, or surgical failure. The primary insult leading to glaucomatous injury is thought to occur to RGC axons at the optic nerve head (ONH).4–7 In addition, patients with established glaucomatous damage may still suffer progressive loss of vision despite maximal IOP lowering. This progressive susceptibility of glaucomatous eyes to further damage may occur as a consequence of the molecular and structural mechanisms in the ONH and/or retina that occur during the disease process. An improved understanding of the biological processes in both locations may help direct future research toward the development of novel interventions to attenuate further loss of visual function in patients with glaucoma. Experimental paradigms to model glaucomatous damage have been developed in a variety of species. In models ranging from rodents to primates, chronic elevation of IOP leads to loss of RGCs in a similar manner to that observed in human glaucoma,8 with secondary cell death thought to ultimately occur due to apoptosis.9–12 Studies of gene expression within the whole retina in experimental glaucoma highlight the complex and dynamic nature of the changes that occur following the induction of glaucomatous damage, which serve to modulate both positive and negative regulatory pathways.13–19 These studies have reported common themes, including upregulation of genes associated with cell death, cell proliferation, glial activation, TNF-α signaling, immune and inflammatory responses, and downregulation of genes associated with lipid biosynthesis and cytoskeleton and light sensation.13–19 However, with the initial IOP-related insult affecting RGC axons at the ONH, the impact of the primary injury would be expected to predominantly affect RGCs within the inner ganglion cell layer of the retina. Laser capture microdissection (LCM) has been used to isolate gene expression changes within the RGC layer following induction of experimental glaucoma,16,20 allowing further refinement of both the altered functional gene classes and the magnitude of any change, by reducing the potential dilution effect from gene responses in other retinal layers. Understanding the contribution of these various responses to the development of glaucomatous damage requires a detailed understanding of the variety of signals that may further impinge on their function. MicroRNAs are small (∼18–22 nt), endogenous noncoding oligoribonucleotides that are highly conserved across species and modulate the posttranscriptional silencing of gene expression.21 They function through recognition of specific sequences in their target mRNAs and predominantly act to reduce expression of their mRNA targets.22 Understanding how these molecules modulate the mechanisms associated with glaucomatous damage may facilitate future targeting of microRNAs to attenuate glaucomatous injury. Although the influence of microRNAs on the biological processes that occur within the glaucomatous ONH and retina is not currently known, there is an emerging body of evidence in central nervous system (CNS) research that suggests they may play an important role. Experimental studies of brain and spinal cord injury have demonstrated that microRNAs influence several pathways that are also known to be modulated within the glaucomatous retina. These include apoptotic cell death,23–30 ischemia,31–39 inflammatory and immune responses,30,40–45 extracellular matrix (ECM) remodeling27,46–48 and TGF-β signaling.43,49,50 The interactions of these biological responses are highly complex. In isolation, interpretation and simplification should be performed with great caution. However, examining the pattern of microRNA expression in conjunction with gene expression changes may help in our interpretation and understanding of the mechanisms that develop following glaucomatous injury. Eyes with established severe injury are likely to demonstrate maximal responses to the ongoing primary IOP-related stimulus, in addition to the secondary mechanisms that will develop throughout the whole retina and not just restricted to the RGCs. In this study, we therefore tested the hypothesis that microRNAs altered in models of CNS injury are also altered within the glaucomatous retina with advanced damage.

Purpose To determine whether inducible nitric oxide synthase (NOS-2) is involved in glaucomatous optic neuropathy. Methods 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. Results 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. Conclusions 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.

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.

The effect of age on the number and morphology of optic nerve axons in adult Brown Norway rats (5-31 months old) (n=29) was examined using transmission electron microscopy (TEM). By manually counting every axon in areas representing 60% of the optic nerve cross-section, we found a significant negative correlation between age and axon count (R(2)=0.18, P

Extracellular matrix homeostasis is dependent in part upon a family of matrix metalloproteinases and their specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Recently, gene defects in TIMP-3 have been identified in the affected individuals of several families with Sorsby's fundus dystrophy (SFD). Very little information is available regarding TIMP-3 function or even its existence in the retina or choroid.We used reverse transcription-polymerase chain reaction and Northern analysis to evaluate the expression of TIMP mRNA and Western immunoblots to evaluate TIMP protein produced by select cells of the human retina and choroid. We also used these methods and immunohistochemistry to localize the TIMPs in the retina and choroid.TIMP-3 transcripts are found in cultured human retinal pigment epithelium (RPE), choroidal microcapillary endothelium and pericytes. RPE cells also express and secrete TIMP-3 protein, which is localized to the extracellular matrix and is not found in culture medium; TIMP-1 and -2 are found almost exclusively in the medium. Immunohistochemistry of human retina/choroid sections shows pronounced TIMP-3 immunostaining in Bruch's membrane, particularly near the surface of the RPE and endothelial cells, presumably in their basement membranes, with minimal staining in other portions of the retina. Immunostaining for TIMP-1 is absent and for TIMP-2 is much less prevalent, but detectable in Bruch's membrane.TIMP-1, -2 and -3 exhibit distinctive expression patterns in the retina and choroid. This distribution and expression pattern partially explains why TIMP-3 mutations result in SFD, rather than other retinal pathologies, such as those associated with proliferative diabetic retinopathy.

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 neurla 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.

The extracellular matrix of the optic nerve head is altered in both human glaucoma and in experimental primate models of this disease. However, the relationship of this change to glaucomatous optic nerve degeneration is unknown. This report describes similar matrix alterations in rats with unilateral elevated intraocular pressure. Brown Norway rats received episcleral vein injections of hypertonic saline to produce prolonged elevations of intraocular pressure. After up to 6 months of pressure elevation, optic nerve head sections from the rats were evaluated by light microscopic immunohistochemistry using antibodies to collagens I, III, IV and VI, laminin, elastin and chondroitin and dermatan sulfate proteoglycans. In experimental eyes with 11 days or more of pressure elevation, depositions of collagen IV, collagen VI and laminin were found within regions of the optic nerve head that, in normal eyes, are occupied solely by nerve bundles. Collagen I and III deposition appeared to be more dependent on the level and duration of the pressure rise. Eyes with lower mean intraocular pressures showed deposits of interstitial collagens primarily at the level of the sclera, while eyes with higher mean pressure elevations had depositions in the neck regions as well. Chondroitin and dermatan sulfate proteoglycans were deposited in a pattern similar to that of collagen I. No extracellular matrix deposition was seen in the orbital optic nerve in any experimental eye. These extracellular matrix changes in rats replicate previous findings in human glaucomatous eyes and monkey eyes with experimentally elevated pressures. They also suggest a sequence of extracellular matrix protein deposition in response to pressure elevation. The optic nerve head deposition of matrix materials in response to elevated intraocular pressures may affect the susceptibility of remaining axons to pressure by changing the physical properties of their support tissues, by affecting the support functions of astrocytes and by changing the microenvironment of injured axons. This model may be useful for studying these and other aspects of the process of axonal injury resulting from elevated intraocular pressure.

To define the characteristics of the diurnal variation of intraocular pressure (IOP) in eyes of awake rats, ten male brown Norway rats were entrained to a 12-hour light:12-hour dark (12L:12D) lighting schedule and were conditioned to IOP measurement with the TonoPen XL tonometer while awake, using only 0.5% proparacaine HCl anesthesia. The IOP measurements were performed in 4 experiments: Preliminary-IOP was measured at 6-hour intervals in both eyes of each animal, to determine correlation between right and left eyes; Light:Dark-lighting remained the same as in the preliminary experiment, but the measurement schedule was altered so that measurements were obtained at 4-hour intervals in alternating eyes, over two 24-hour light cycles; Dark:Dark-animals were placed in constant dark (0L:24D) and, after 72 h, measurements were obtained at 4-hour intervals in alternating eyes. Animals were then re-entrained to the previous 12L:12D schedule for 7 days, after which they were returned to constant dark and the experiment was repeated; and Dark:Light-animals were entrained to a reversed light:dark cycle (12D:12L) for 28 days, after which measurements were obtained in the same fashion as in the Light:Dark experiment. Close agreement was found between right- and left-eye IOPs. Animals on a 12L:12D schedule exhibited lowest IOP while the lights were on (19.3 +/- 1.9 mm Hg), and highest (31.3 +/- 1.3 mm Hg) while the lights were off. Pressure changes anticipated the change from light to dark and dark to light. This pattern persisted in constant dark, and was reversed when the cycle was changed to 12D:12L. Brown Norway rats possess a regular rhythm of IOP that is entrained by the cycle of light and dark, and persistence of this rhythm in constant dark establishes it as a circadian rhythm. Furthermore, our results indicate that reliable and physiologically meaningful IOP measurements can be obtained in awake rats using the TonoPen XL tonometer.

To define the architecture and extracellular matrix composition of the lamina cribrosa in rodents, normal, adult pigmented rat and guinea pig eyes were frozen and sectioned for light microscopic immunohistochemistry. Antibodies specific for collagens I, III, IV and VI, laminin, elastin, and chondroitin and dermatan sulfate proteoglycans were exposed to longitudinal and cross-sections of optic nerve heads and their binding distributions observed with the avidin-biotin-peroxidase complex technique. Cross-sections of the intraocular portion of the rat optic nerve head revealed a horizontally oval shape with distinct, vertically oriented, laminar beams. The guinea pig optic nervehead cross-section was circular, with randomly oriented beams. In both animals, collagens I, III and VI were found throughout the laminar beams, along with elastin fibrils. Collagen IV and laminin antibodies deposited along laminar beam margins and within the beams, representing astrocytic and vascular endothelial cell basement membranes. Both animals showed evidence for dermatan and chondroitin sulfate-containing proteoglycans in all connective tissue structures of the nerve head. In the rat, chondroitin-4 sulfate proteoglycans appeared localized to the sclera and laminar beams. The rat and the guinea pig optic nerve head possess an identifiable lamina cribrosa with structural proteins nearly identical to that of the primate. Both animals may provide affordable alternative animal models for in vivo studies on the role of the lamina cribrosa in glaucomatous optic nerve damage.

The progressive loss of vision in glaucoma patients has been attributed to degeneration of retinal ganglion cells (RGCs) and their axons and the eventual death of these cells. As intraocular pressure (IOP) remains the best established risk factor for glaucoma, therapy for this blinding disease continues to rely heavily on lowering pressure by medical and surgical measures. Although pressure reduction has been effective in preventing onset of glaucoma and delaying its progression,1,2 some patients do not respond well or are intolerant of these treatments, whereas others continue to experience vision loss despite significant reduction in IOP. Recently, neuroprotective strategies other than pressure lowering, including supplementation of neurotrophins and prevention of caspase activation and apoptosis, have been proposed and are being investigated as new goals for glaucoma therapy.3,4 Effective neuroprotection, aimed at salvaging functional RGCs and their axons before they are committed to die, requires early intervention and targeting of upstream events. These early changes may involve increased susceptibility of RGCs to elevated IOP, which could explain progressive vision loss in patients despite lowered eye pressure. Conversely, they may be protective mechanisms that could be therapeutically enhanced. Identification of the early critical molecular events in RGCs would add to our understanding of the nature of glaucomatous injury and provide potential targets for neuroprotective strategies. The primary injury in glaucoma is now widely believed to be axonal at the lamina in the optic nerve head.5–7 However, the nature of the injury and the initial molecular events occurring in injured RGCs are largely unknown. Studies have described early morphologic changes in RGCs,8–10 suggesting that structural and possibly functional changes occur well in advance of their death. Also, pathways involved in RGC soma degeneration, although not well characterized, have been suggested to be distinct from those in axon degeneration in glaucoma.11 To examine global changes in gene expression, several studies have applied genomewide analysis of microarrays to the retina of rodent and primate glaucoma models as well as the DBA/2J mice.12–17 These studies have found significant regulation of gene expression in the retina and implicate multiple processes in glaucomatous injury, including neuroinflammation, glia activation, apoptosis, and cytoskeleton-based processes. However, because RGCs represent only a small fraction of the total retinal cell population, in these studies performed on whole retina samples, many responses specific to RGCs may have been masked. In an effort to identify changes more specific to RGCs, Wang et al.18 studied global gene expressions in individual RGCs captured by laser in experimental glaucoma and reported extensive expression changes in genes associated with multiple signaling pathways and apoptosis. Like the whole-retina studies, however, this study did not specifically target the events of early injury. Using a rodent glaucoma model in which chronic IOP elevation is achieved by injection of hypertonic saline into episcleral veins,19 we have recently demonstrated that detection of RGC responses can be greatly enhanced by analyzing just the RGC layer (GCL) compared with the whole retina.20 Successfully isolating the GCL from the whole retina by laser capture microdissection (LCM) and applying cDNA microarray analysis to the GCL mRNA, we identified a significantly larger number of genes with altered expression in the GCL compared with those in whole retinas of eyes with comparable injury. That report, which focused on responses in eyes with extensive nerve injury, represents only part of a larger study in which we examined the GCL in eyes with all grades of optic nerve injury. Here, we present the complete analysis, but mainly concentrate on early-injury responses that, as mentioned previously, are likely to be important for understanding the cellular mechanisms of glaucoma and identifying their therapeutic implications.

Elevated intraocular pressure (IOP) is the most widely recognized risk factor for glaucoma and the target of nearly all current glaucoma therapy. The goal of our laboratory is to understand mechanism by which elevated IOP results in axonal degeneration in the optic nerve.In order to study the effects of IOP level on the optic nerve, we have developed a number of methods to modulate IOP in the rat. We sclerose the aqueous humor outflow pathways to elevate IOP for a period of weeks to produce focal optic nerve lesions. This method reproduces many of the pathological characteristics of human glaucoma. A modification of this method uses constant light (CL) housing to stabilize the normally wide circadian IOP oscillations, simplifying the correlation of IOP history with lesion size. However, the CL-stabilized IOP in untreated, fellow eyes is above the normal light phase IOP of 20 mm Hg and close to the normal dark, phase value of 30 mm Hg.

To characterize retinal functional consequences of elevated intraocular pressure (IOP) in a rat model of experimental glaucoma.Unilateral elevation of IOP was produced by hypertonic saline injection into an episcleral vein in 20 adult male Brown-Norway rats. IOP was measured in both eyes of awake animals four to five times per week. After 5 weeks, animals were dark adapted overnight (12 hours) and full-field electroretinograms (ERGs) were obtained simultaneously from both eyes. Scotopic ERG stimuli were brief white flashes (-6.64-2.72 log cd-s/m(2)). Photopic responses were also obtained (0.97-2.72 log cd-s/m(2)) after 15 minutes of light adaptation (150 cd/m(2)). Eyes were processed the following day for histologic evaluation by light microscopy, including masked determination of optic nerve injury grade (ONIG; 1, normal; 5, severe, diffuse damage).Among experimental eyes, the group average IOP (+/-SD) was 34.5 +/- 4.1 mm Hg, whereas the average for control eyes was 28.1 +/- 0.5 mm Hg (t = 7.1, P0.0001). The average ONIG for experimental and control eye groups, respectively, was 3.4 +/- 1.7 and 1.0 +/- 0.02 (t = 6.3, P0.0001). The ONIG increased with mean IOP in experimental eyes (r(2) = 0.78, P0.0001) and was unrelated to mean IOP in control eyes (r(2) = 0.09, P = 0.18). In experimental eyes with relatively mild IOP elevation (mean IOP31 mm Hg) and no structural (histologic) damage to the optic nerve evident by light microscopy (ONIG = 1.1 +/- 0.2, n = 5), there was a selective reduction of the positive scotopic threshold response (pSTR; P0.001), whereas other ERG components remained unaltered. In four of the five eyes, pSTR amplitude was reduced by more than 50%, whereas all five had normal scotopic a-wave, b-wave, and OP amplitudes. Eyes with mean IOP of more than 35 mm Hg had reduced a-wave, b-wave, and oscillatory potential (OP) amplitudes.As demonstrated by prior studies, selective loss of the pSTR is indicative of selective retinal ganglion cell (RGC) injury. In this rat model of experimental glaucoma, selective RGC functional injury occurred before the onset of structural damage, as assessed by light microscopy of optic nerve tissue. The highest IOP levels resulted in nonselective functional loss. Thus, in rodent models of experimental glaucoma, lower levels of chronically elevated IOP may be more relevant to human primary chronic glaucoma.

PURPOSE. The goal of this study was to identify altered patterns of retinal mRNA expression after experimental elevation of intraocular pressure (IOP) in a rat glaucoma model. METHODS. Brown Norway rats (N 16) received unilateral episcleral vein injection of hypertonic saline to elevate IOP. IOP was monitored daily by handheld tonometer, and retinas were collected 8 days and 5 weeks after surgery. Comparison of mRNA levels between experimental and fellow retinas was made using gene microarrays (rat U34A rat arrays; Affymetrix, Santa Clara, CA). Semiquantitative RT-PCR was used to confirm selected results from array analysis and to compare with alterations after optic nerve transection. RESULTS. IOP elevation for 5 weeks resulted in reproducible changes in levels of 81 mRNAs. Of these, 74 increased, whereas only 7 decreased. The expression levels of 27 of these same messages were changed after 8 days of IOP elevation. In addition, four other genes demonstrated altered expression after the shorter period of elevated IOP exposure. Approximately half of the mRNAs with altered expression were associated with either neuroinflammatory responses or apoptosis. For 25 of the selected functionally relevant messages altered by array analysis, the alterations were confirmed by semiquantitative RT-PCR. The levels of 24 of 25 selected messages were also changed after optic nerve transection. CONCLUSIONS. The activation of glia and the complement system after IOP elevation, which is similar to that described in several neurodegenerative diseases and after optic nerve transection, suggests that this rat glaucoma model could be used to evaluate the neuroprotective potential of therapeutic agents that target these processes. (Invest Ophthalmol Vis Sci. 2004;45: 1247‐1258) DOI:10.1167/iovs.03-1123

True daily zinc absorption was determined in rats fed a high iron diet (220 ppm Fe; 16.5 ppm Zn), and adequate iron diet (30 ppm Fe) and a high iron diet with varying levels of pyridoxine-HCl (2-40 ppm). Zinc absorption in rats fed the high iron diet was significantly less than in rats fed the adequate iron diet. Zinc absorption in rats fed the high iron diet supplemented with picolinic acid (200 ppm) was markedly increased and did not differ from that in rats fed the adequate iron diet. True, daily zinc absorption increased as the level of dietary vitamin B-6 was increased. Zinc absorption was the least in rats fed 2 ppm vitamin B-6 and was greatest in rats fed 40 ppm vitamin B-6. The concentration of picolinic acid in the pancreas increased as the level of dietary vitamin B-6 was increased. Zinc absorption was significantly elevated in rats fed the high iron diet that contained either 4 or 10 ppm vitamin B-6 and 200 ppm picolinic acid. The turnover rate of 65Zn was determined in rats fed an adequate iron, marginal zinc diet (8.5 ppm Zn) with varying levels of vitamin B-6. The turnover of 65Zn was greatest in rats fed 2 ppm vitamin B-6 and least in rats fed 40 ppm vitamin B-6 or 2 ppm vitamin B-6 + 200 ppm picolinic acid. The results suggest that high levels of dietary iron inhibit zinc absorption via competition for binding with endogenous picolinic acid. The results provide further evidence to support the hypothesis that picolinic acid facilitates absorption of dietary zinc.

The zinc concentration of liver and kidneys was determined in rat pups nursing dams given 10 micrograms Zn/ml as either zinc dipicolinate or zinc acetate in water solution during the last week of gestation and for 5 days of lactation. Dams were fed a casein-based diet that contained 8.5 micrograms Zn/g and 2.0 micrograms pyridoxine-HCl/g during this period. The zinc concentrations of both liver and kidneys from the 5-day-old pups nursing dams fed the zinc dipicolinate solution were significantly greater than the zinc concentrations of these tissues from pups nursing dams given a solution of zinc acetate. The results demonstrate that a greater quantity of dietary zinc is transferred from the intestine of the lactating female rat to the pups when zinc is fed in the form of zinc dipicolinate.