A method describing the microdissection of trabecular meshwork tissue from Brown Norway rat eyes.

Glaucoma is often associated with elevated intraocular pressure (IOP), generally due to obstruction of aqueous humor outflow within the trabecular meshwork (TM). Despite many decades of research, the molecular cause of this obstruction remains elusive. To study IOP regulation, several in vitro models, such as perfusion of anterior segments or mechanical stretching of TM cells, have identified several IOP-responsive genes and proteins. While these studies have proved informative, they do not fully recapitulate the in vivo environment where IOP is subject to additional factors, such as circadian rhythms. Thus, rodent animal models are now commonly used to study IOP-responsive genes in vivo. Several single-cell RNAseq studies have been performed where angle tissue, containing cornea, iris, ciliary body tissue in addition to TM, is dissected. However, it is advantageous to physically separate TM from other tissues because the ratio of TM cells is relatively low compared to the other cell types. In this report, we describe a new technique for rat TM microdissection. Evaluating tissue post-dissection by histology and immunostaining clearly shows successful removal of the TM. In addition, TaqMan PCR primers targeting biomarkers of trabecular meshwork (Myoc, Mgp, Chi3l1) or ciliary body (Myh11, Des) genes showed little contamination of TM tissue by the ciliary body. Finally, pitfalls encountered during TM microdissection are discussed to enable others to successfully perform this microsurgical technique in the rat eye.

Expansions of the neurovascular scleral canal and contained optic nerve occur early in the hypertonic saline rat experimental glaucoma model.

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.

Modeling glaucoma in rats by sclerosing aqueous outflow pathways to elevate intraocular pressure.

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.

Rat optic nerve head anatomy within 3D histomorphometric reconstructions of normal control eyes.

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.

Profiling IOP-responsive genes in anterior and posterior ocular tissues in the rat CEI glaucoma model.

Purpose: The rat Controlled Elevation of Intraocular pressure (CEI) model allows study of in vivo responses to defined intraocular pressures (IOP). In this study, we use Nanostring technology to investigate in vivo IOP-related gene responses in the trabecular meshwork (TM) and optic nerve head (ONH) simultaneously from the same animals. Methods: Male and female rats (N=35) were subject to CEI for 8-hours at pressures simulating mean, daytime normotensive rat IOP (CEI-20), or 2.5x IOP (CEI-50). Naïve animals, receiving no anesthesia or surgical interventions, served as controls. Immediately after CEI, TM and ONH tissues were dissected, RNA isolated, and samples were analyzed with a Nanostring panel containing 770 genes. Post-processing, raw count data were uploaded to Rosalind® for differential gene expression analyses. Results: For the TM, 45 IOP-related genes were significant in the "CEI-50 vs. CEI-20" and "CEI-50 vs. naïve" comparisons, with 15 genes common to both comparisons. Bioinformatics analysis identified Notch and TGFß pathways to be the most up- and down-regulated KEGG pathways, respectively. For ONH, 22 significantly regulated genes were identified in the "CEI-50 vs. naïve" comparison. Pathway analysis identified 'defense response' and 'immune response' as two significantly upregulated biological process pathways. Conclusions: This study demonstrates the ability to assay IOP-responsive genes in both TM and ONH tissues simultaneously. In the TM, downregulation of TGFß pathway genes suggest that TM responses may prevent TGFß-induced extracellular matrix synthesis. For ONH, the initial response to elevated IOP may be protective, with astrocytes playing a key role in these gene responses.

Profiling IOP-Responsive Genes in the Trabecular Meshwork and Optic Nerve Head in a Rat Model of Controlled Elevation of Intraocular Pressure.

Purpose: The rat controlled elevation of intraocular pressure (CEI) model allows study of in vivo responses to short-term exposure to defined intraocular pressures (IOP). In this study, we used NanoString technology to investigate in vivo IOP-related gene responses in the trabecular meshwork (TM) and optic nerve head (ONH) simultaneously from the same animals. Methods: Male and female rats (N = 35) were subjected to CEI for 8 hours at pressures simulating mean, daytime normotensive rat IOP (CEI-20), or 2.5× IOP (CEI-50). Naïve animals that received no anesthesia or surgical interventions served as controls. Immediately after CEI, TM and ONH tissues were dissected, RNA was isolated, and samples were analyzed with a NanoString panel containing 770 genes. Postprocessing, raw count data were uploaded to ROSALIND for differential gene expression analyses. Results: For the TM, 45 IOP-related genes were significant in the CEI-50 versus CEI-20 and CEI-50 versus naïve comparisons, with 15 genes common to both comparisons. Bioinformatics analysis identified Notch and transforming growth factor beta (TGFß) pathways to be the most up- and downregulated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, respectively. For ONH, 22 significantly differentially regulated genes were identified in the CEI-50 versus naïve comparison. Pathway analysis identified defense response and immune response as two significantly upregulated biological process pathways. Conclusions: This study demonstrated the ability to assay short-term IOP-responsive genes in both TM and ONH tissues simultaneously. In the TM, downregulation of TGFß pathway genes suggests that TM responses may reduce TGFß-induced extracellular matrix synthesis. For ONH, the initial response to short-term elevated IOP may be protective.

Optic Nerve Head Gene Transcription Sequelae to a Single Elevated IOP Exposure Provides Insights Into Known Responses to Chronically Elevated IOP.

Purpose: To clarify the optic nerve head (ONH) gene expression responses associated with a single, axon-damaging exposure to elevated IOP in relation to the composite cellular events previously identified in models of chronically elevated IOP. Methods: Anesthetized rats were exposed unilaterally to an 8-hour pulse-train controlled elevation of IOP (PT-CEI) at 60 mm Hg, while others received normotensive CEI at 20 mm Hg. ONH RNA was harvested at 0 hours and 1, 2, 3, 7, and 10 days after either CEI and from naïve animals. RNA sequencing was performed to analyze ONH gene expression. DAVID Bioinformatics tools were used to identify significant functional annotation clusters. Gene function was compared between PT-CEI and two models of chronic ocular hypertension from the literature. Results: The number of significantly changed genes peaked immediately (n = 1354) after PT-CEI (0 hours). This was followed by a lull (<4 genes per time point) at 1 and 2 days after PT-CEI. Gene activity increased again at 3 days (136 genes) and persisted at 7 (78 genes) and 10 (339 genes) days. Significant gene functional categories included an immediate upregulation of Defense Response at 0 hours, followed by upregulation in Cell Cycle, a reduction in Axonal-related genes at 3 to 10 days, and upregulation of Immune Response-related genes at 10 days following PT-CEI. The most commonly upregulated gene expression across our PT-CEI study and two chronic models of ocular hypertension were cell cycle related. Conclusions: The PT-CEI model places in sequence ONH gene expression responses previously reported in models with chronically elevated IOP and may provide insights into their role in optic nerve damage.

Neurotrophin roles in retinal ganglion cell survival: lessons from rat glaucoma models.

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.

Early Optic Nerve Head Glial Proliferation and Jak-Stat Pathway Activation in Chronic Experimental Glaucoma.

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.

Optic Nerve Head Astrocytes Display Axon-Dependent and -Independent Reactivity in Response to Acutely Elevated Intraocular Pressure.

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 ß3 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.

Comparison of anterior segment structures in two rat glaucoma models: an ultrasound biomicroscopic study.

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 microm 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 approximately 71% larger ACDs and approximately 32% smaller CBAs than did the contralateral control eyes (P < 0.001). ACD and CBA correlated well (R2 = 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.

Hypertonic Saline Injection Model of Experimental Glaucoma in Rats.

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.

In Vivo Small Molecule Delivery to the Optic Nerve in a Rodent Model.

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.

Global changes in optic nerve head gene expression after exposure to elevated intraocular pressure in a rat glaucoma model.

PURPOSE: In glaucoma, the optic nerve head (ONH) is the likely site of initial injury and elevated intraocular pressure (IOP) is the best-known risk factor. This study determines global gene expression changes in the pressure-injured ONH. METHODS: Unilateral sustained IOP elevation (glaucoma, n = 46) or optic nerve transection (n = 10) was produced in rats. ONHs were removed, and the retrobulbar optic nerves were graded for degeneration. Gene expression in the glaucomatous ONH with extensive injury was compared with that in the fellow ONH (n = 6/group), by using cDNA microarrays. Data from 12 arrays were normalized, significant differences in gene expression determined, and significantly affected gene classes identified. For the remaining ONH, grouped by experimental condition and degree of injury, quantitative reverse transcriptase-PCR (qPCR) and ANOVA were used to compare selected message levels. RESULTS: Microarray analysis identified more than 2000 significantly regulated genes. For 225 of these genes, the changes were greater than twofold. The most significantly affected gene classes were cell proliferation, immune response, lysosome, cytoskeleton, extracellular matrix, and ribosome. A 2.7-fold increase in ONH cellularity confirmed glaucoma model cell proliferation. By qPCR, increases in levels of periostin, collagen VI, and transforming growth factor beta1 were linearly correlated to the degree of IOP-induced injury. For cyclinD1, fibulin 2, tenascin C, TIMP1, and aquaporin-4, correlations were significantly nonlinear, displaying maximum change with focal injury. CONCLUSIONS: In the ONH, pressure-induced injury results in cell proliferation and dramatically altered gene expression. For specific genes, expression levels were most altered by focal injury, suggesting that further array studies may identify initial, and potentially injurious, altered processes.

Comparison of MicroRNA Expression in Aqueous Humor of Normal and Primary Open-Angle Glaucoma Patients Using PCR Arrays: A Pilot Study.

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.

Astrocyte Structural and Molecular Response to Elevated Intraocular Pressure Occurs Rapidly and Precedes Axonal Tubulin Rearrangement within the Optic Nerve Head in a Rat Model.

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<0.05), and re-orientated back to baseline orientation 1 day post IOP normalization. ONH axonal microtubule filament label intensity was significantly reduced 1 and 3 days post IOP normalization, and returned to control levels on day 5. Phosphorylated focal adhesion kinase (FAK) levels steadily decreased after IOP normalization, while levels of phosphorylated paxillin (a downstream target of FAK involved in focal adhesion dynamics) were significantly elevated 5 days post IOP normalization. The levels of phosphorylated cortactin (a downstream target of Src kinase involved in actin polymerization) were significantly elevated 1 and 3 days post IOP normalization and returned to control levels by day 5. No significant axon degeneration was noted by morphologic assessment up to 5 days post IOP normalization. Actin-based astrocyte structure and signaling within the ONH are significantly altered within hours after IOP elevation and prior to axonal cytoskeletal rearrangement, producing some responses that recover rapidly and others that persist for days despite IOP normalization.

A Period of Controlled Elevation of IOP (CEI) Produces the Specific Gene Expression Responses and Focal Injury Pattern of Experimental Rat Glaucoma.

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.

Evaluation of inducible nitric oxide synthase in glaucomatous optic neuropathy and pressure-induced optic nerve 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.

MicroRNA Expression in the Glaucomatous Retina.

PURPOSE: MicroRNAs are small, endogenous noncoding RNAs that modulate posttranscriptional gene expression. Although the contribution of microRNAs to the pathogenesis of glaucomatous damage is unknown, supporting evidence from central nervous system (CNS) research suggests they may play a role. It was therefore hypothesized that microRNAs known to be altered in CNS injury are also altered in experimental glaucoma. METHODS: Intraocular pressure (IOP) was elevated in rats by unilateral injection of hypertonic saline and IOP monitored for 5 weeks. After rats were killed, retrobulbar optic nerve sections were graded for damage. MicroRNA was extracted from whole retinae of eyes with advanced nerve damage (n = 8) and from normal, noninjected control eyes (n = 8). Quantitative PCRs were performed using a panel of 17 microRNAs, reported from CNS research to be implicated in mechanisms also linked to glaucomatous damage. Computationally and experimentally derived gene targets were identified for the differentially expressed microRNAs. These were then integrated with existing gene array data. Functional interpretation was performed using the Molecular Signatures Database and DAVID Functional Annotation Clustering. RESULTS: Eight microRNAs were significantly downregulated in glaucomatous retinae compared with controls (miR-181c, miR-497, miR-204, let-7a, miR-29b, miR-16, miR106b, and miR-25); miR-27a was significantly upregulated. Enrichment of targets associated with extracellular matrix/cell proliferation, immune system, and regulation of apoptosis were observed. Cholesterol homeostasis and mTORC-1 pathways showed reduced expression. CONCLUSIONS: MicroRNAs are differentially expressed in retinae of eyes with advanced glaucomatous damage compared with normal controls. Integrating microRNA with gene expression data may improve understanding of the complex biological responses produced by chronically elevated IOP.

Selective ganglion cell functional loss in rats with experimental glaucoma.

PURPOSE: To characterize retinal functional consequences of elevated intraocular pressure (IOP) in a rat model of experimental glaucoma. METHODS: 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). RESULTS: 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, P < 0.0001). The average ONIG for experimental and control eye groups, respectively, was 3.4 +/- 1.7 and 1.0 +/- 0.02 (t = 6.3, P < 0.0001). The ONIG increased with mean IOP in experimental eyes (r(2) = 0.78, P < 0.0001) 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 IOP < 31 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; P < 0.001), 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. CONCLUSIONS: 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.