Experimental Eye Research 141 (2015) 3e8
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Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer
An acute intraocular pressure challenge to assess retinal ganglion cell injury and recovery in the mouse Jonathan G. Crowston a, Yu Xiang G. Kong a, Ian A. Trounce a, Trung M. Dang a, Eamonn T. Fahy a, Bang V. Bui b, John C. Morrison c, Vicki Chrysostomou a, * a b c
Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital, East Melbourne, Victoria, Australia Department of Optometry and Vision Sciences, University of Melbourne, Parkville, Victoria, Australia Casey Eye Institute, Oregon Health and Science University, Portland, OR, United States
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 December 2014 Received in revised form 3 March 2015 Accepted in revised form 6 March 2015 Available online 7 March 2015
We describe a model of acute intraocular pressure (IOP) elevation in the mouse eye that induces reversible loss of inner retinal function associated with oxidative stress, glial cell activation and minimal loss of retinal ganglion cell (RGC) number. Young healthy mouse eyes recover inner retinal function within 7-days but more persistent functional loss is seen in older mice. Manipulation of diet and exercise further modify RGC recovery demonstrating the utility of this injury model for investigating lifestyle and therapeutic interventions. We believe that systematic investigation into the characteristics and determinants of RGC recovery following an IOP challenge will shed light on processes that govern RGC vulnerability in the early stages of glaucoma. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Retinal ganglion cell Intraocular pressure Animal models Glaucoma Neurorecovery Electroretinography
1. Introduction We adapted an acute intraocular pressure (IOP) elevation injury model to investigate how retinal ganglion cells (RGCs) respond to a titratable and highly reproducible injury. Our goal in generating this “optic nerve stress test” was to identify levels of injury that, in a healthy young mouse retina, induce selective but reversible loss of RGC function while maintaining normal outer retinal function. Additionally, we sought an injury that does not lead to manifest ischemia of the inner retina with minimal RGC death. Our interest lies in determining the parameters that influence RGC recovery over time. Glaucoma is a chronic, progressive optic neuropathy characterized by the selective death of RGCs and their axons. How then does our acute IOP injury model inform us of processes that are relevant to this disease? In the very early stages of glaucoma, the optic nerve head is repeatedly exposed to cycles of relatively minor injury (Downs et al., 2011). We propose that healthy RGCs are initially able
* Corresponding author. Centre for Eye Research Australia, Level 1, 32 Gisborne Street, East Melbourne VIC 3002, Australia. E-mail address: [email protected] (V. Chrysostomou). http://dx.doi.org/10.1016/j.exer.2015.03.006 0014-4835/© 2015 Elsevier Ltd. All rights reserved.
to withstand and recover from such minor injuries. However, over time, repeated insults or an intrinsic impairment in the capacity of RGCs to recover, will overwhelm their ability to recover resulting in induction of a cell death program. Measuring the capacity for RGC recovery following an acute IOP injury, we believe, will shed light on the very early stages of glaucoma. Furthermore, this test will enable us to investigate factors such as aging, diet and exercise that can improve or impair RGC recovery. By incrementally increasing the IOP level or duration of injury we will also be able to investigate the critical switch point at which full functional recovery is not possible.
2. Acute IOP injury model 2.1. Overview Our injury model involves acute elevation of IOP through insertion of a fine needle attached to either a column of fluid or a motorized syringe pump. RGC function is measured using the positive scotopic threshold response (pSTR) or the photopic negative response (PhNR) components of the full-field electroretinogram (ERG). A single animal can be monitored over a number of
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timepoints post-injury but to mitigate potential effects of repeat anesthesia, we leave 7 days between injury and functional assessment by the ERG. Following functional assessment, mice are euthanized and retinas and optic nerves are harvested and subsequently analyzed using a range of histological and biochemical assays. 2.2. Experimental techniques All animal procedures conform to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and with the requirements of our institutional Animal Research and Ethics Committee. 2.2.1. IOP elevation Animals are anesthetized with intraperitoneal injection of ketamine (60 mg/kg; Troy Laboratories Pty Ltd.) and xylazine (10 mg/ kg; Troy Laboratories Pty Ltd.) before resting IOP is measured using a hand-held rebound tonometer (TonoLab; Icare Finland Oy, Espoo, Finland). Pupil dilation and corneal anesthesia is achieved by topical application of one drop each of 0.5% proxymetacaine hydrochloride (Alcon Laboratories Pty Ltd., Frenchs Forest, NSW, Australia) and 0.5% tropicamide (Minims; Chauvin Pharmaceuticals, Surrey, UK). Movement of iris by mydriasis aids subsequent needle insertion. Animals are lightly secured to a platform with wire loops across the upper back and nose. Body temperature is maintained at 37e38 C with a homeothermic heating unit. The mouse eye is proptosed using curved forceps. Using a micromanipulator, the tip of a pulled glass borosilicate needle (~50 mm, 1B100-6, WPI, Sarasota, FL, USA) is inserted into the anterior chamber through the center of the cornea. After insertion, the microneedle is then repositioned to minimize corneal deformation and to ensure that the eye remains in its natural position. The cannulated needle is connected via polyethylene tubing (0.97 mm inner diameter, Microtube Extrusions, North Rocks, NSW, Australia) to a pressure transducer (Transpac IV, Abbott Critical Care Systems, Sligo, Ireland), which is in series with a sterilefiltered endotoxin-tested Hanks' balanced salt solution reservoir (HBSS, JRH Biosciences, Lenexa, KA, USA). The HBSS reservoir acts as a pressure column, the height of which determines the hydrostatic pressure delivered to the eye. Pressure transducer calibration is performed by referencing to an aneroid sphygmomanometer (Livingstone, Rosebery, NSW, Australia). HBSS is chosen as the fluid in the pressure column as its concentrations of salts and ions show a close approximation to aqueous humor. Initial cannulation of the anterior chamber is performed with the reservoir height set to eye level and the reservoir valve closed. Patency of the needle following cannulation is confirmed by anterior chamber distension observed under the microscope. IOP is raised to the desired level by altering the height of the reservoir, and monitored in real time using a PowerLab data acquisition system. The target pressure is confirmed using a hand-held rebound tonometer and pressure is maintained within 1 mmHg throughout. In each animal, pressure is elevated to 50 mmHg for 30 min in one eye (injured) while the fellow eye is cannulated and held at physiological IOP (sham, 12 mmHg). The contralateral sham-treated eye acts as an internal control to mitigate any variations induced by anesthesia, temperature etc. A variation of this approach has been developed more recently where a syringe filled with HBBS is mounted on a motorized syringe pump (PHD Ultra CP; Harvard Apparatus, Massachusetts, USA). This pump can be set to drive fluid at an adjustable rate to maintain a set pressure. Side by side comparison of the adjustable column and motorized syringe pump has revealed no difference in
RGC dysfunction (not shown). 2.2.2. Electroretinography The ERG is used to measure retinal function serially in animals, one day before (baseline) and 7 days after IOP elevation. Responses recorded after IOP elevation are normalized to baseline values to determine pressure-induced changes. After overnight dark-adaptation (minimum 12 h), animals are prepared for recording under dim red illumination. Animals are anesthetized with intraperitoneal injection of ketamine (60 mg/kg) and xylazine (10 mg/kg). Pupil dilation and corneal anesthesia are achieved by topical application of one drop each of 0.5% tropicamide (Minims; Chauvin Pharmaceuticals, Surrey, UK), 2.5% phenylephrine (Minims; Chauvin Pharmaceuticals) and 0.5% proxymetacaine hydrochloride (Alcon Laboratories Pty Ltd.). Body temperature is maintained at 37 C with a homeothermic controller and unit. Electrical signals are recorded with a platinum wire electrode contacting the central cornea while a gold pellet placed in the mouth serves as common reference. A stainless steel needle electrode (F-E2-60, Grass Technologies, West Warwick, RI, USA) inserted at the base of the tail acts as ground. Retinal responses are recorded simultaneously from both eyes with the testing protocol lasting approximately 30 min. Full-field flash ERGS are recorded using an Espion Diagnosys system (Diagnosys LLC, Littleton, MA, USA). Light stimuli are delivered via a Ganzfeld unit from white light-emitting diodes. Light energies are calibrated as luminance energy units, candela second per meter squared (cd s/m2). Scotopic threshold responses (STRs) are elicited using a 5.45 log cd s/m2 stimulus. Twenty-five flashes with an inter-stimulus interval of 1500 ms are averaged. Scotopic a-waves and b-waves are elicited with a stimulus intensity of 2.22 log cd s/m2 with no averaging required. Photopic responses to 6 different stimulus strengths between 0.34 and 2.22 log cd s/m2 are presented on a 40.0 cd s/m2 rod-saturating green background. At each intensity, twenty-five flashes with an inter-stimulus interval of 3000 ms are averaged. Response amplitudes of the scotopic a-wave, scotopic b-wave, pSTR, PhNR and photopic b-wave are measured in two ways: (1) by identifying the maximum peak/trough and obtaining the baselinetrough/peak amplitude; (2) by taking the amplitude at a fixed criterion time after the stimulus onset, again with respect to baseline. Criterion times are chosen to correspond with the mean implicit time of all data from our study. 2.2.3. Retina and optic nerve structure and biochemistry At chosen timepoints after acute IOP elevation, retina and optic nerve are harvested from injured eyes and contralateral shamtreated eyes, and subsequently processed in various ways. We routinely assess cell structure and survival by histological staining and the TUNEL technique; protein expression levels by immunohistochemistry, Western blotting and ELISA; and gene expression by quantitative real-time PCR. We have published detailed descriptions of these protocols elsewhere (Chrysostomou and Crowston, 2013; Chrysostomou et al., 2014, 2008; Kong et al., 2012) and provide brief summaries below. For immunohistochemical labeling, retinal cryosections are prepared from paraformaldehyde-fixed eyes and subsequently incubated overnight at 4 C with primary antibodies. Common antibodies used in our studies include glial fibrillary acidic protein (GFAP; 1:800, Dako, Campbellfield VIC, Australia), heme oxygenase-1 (HO-1; 1:400, Enzo Life Sciences, Ann Arbor, MI, USA) and Brn-3a (1:100, Millipore, Kilsyth VIC, Australia). After incubation with biotin-conjugated secondary antibody (1:200; SigmaeAldrich, St. Louis, MO, USA) and with Alexa Fluor Streptavidin conjugate (1:1000, Invitrogen; Invitrogen, Eugene, OR, USA),
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sections are nuclear-counterstained with Hoechst (1:10,000) before being coverslipped with a glycerol/gelatin medium. We extract RNA from retina and optic nerve stump and synthesize complementary DNA using commercial kits (Applied Biosystems, Mulgrave, Australia). Quantitative real-time PCR (qPCR) is then performed using a TaqMan®-based assay (Applied Biosystems) and probes specific for target genes such as GFAP (NM_001131020.1) and Thy-1 (NM_009382.3). Following normalization of each sample for b-actin, relative quantitation of gene expression is calculated using the comparative threshold cycle method. 2.3. Model characteristics 2.3.1. Selective loss of inner retinal function Initial work determined the level of IOP elevation that led to selective loss of inner retinal function but left outer and mid retinal function intact (Kong et al., 2009). We employed a “staircase” increment of IOP elevation in dark-adapted mouse eyes while simultaneously measuring retinal function with the ERG. Amplitudes of the pSTR component of the ERG were used as a measure of inner retinal function while amplitudes of scotopic a-wave and bwaves were used to indicate photoreceptoral (a-wave), and ONbipolar (b-wave) function. In these experiments, we monitored the full-field ERG during and in the first 2 h following IOP elevation. Here, we found that the greatest separation of inner and outer retinal function occurred at an IOP of 50 mmHg. We have since established, characterized and published protocols for recording photopic negative responses (PhNRs) from the mouse retina, as an additional measure of inner retinal function (Chrysostomou and Crowston, 2013), the amplitudes of which are also selectively reduced in response to elevation of IOP to 50 mmHg. The selective loss of inner retinal function in response to our injury model is summarized in Fig. 1. 2.3.2. Age-related susceptibility As aging is one of the greatest risk factors for the development of glaucoma, we sought an injury to which animals showed increasing vulnerability with advancing age. In defining experiments, we recorded the ERG prior to, during, and after a fixed IOP challenge (50 mmHg for 30 min) in 3, 12, and 18-month old animals. We found that by 7 days post-injury, inner retinal function had recovered fully in young 3-month mice but a persistent loss of function was seen in 12-month old and 18-month old mice (Kong et al., 2012). This negative impact of aging has also been shown to be ameliorated by diet restriction (Kong et al., 2012) or exercise (Chrysostomou et al., 2014). 2.3.3. Signs of inner retinal stress Western blot and immunohistochemistry have been used to assess retina for oxidative stress and glial cell activation at baseline, 1- and 7-days post-injury. We found that a single 30 min elevation of IOP to 50 mmHg leads to marked astrocytic gliosis, seen as upregulation of GFAP protein and mRNA expression (Fig. 2 (Chrysostomou and Crowston, 2013)). Retinal expression of HO-1, used here as an oxidative stress marker, also increases in the week following injury (Fig. 2a). Thy-1 expression, an RGC marker that is known to be reduced post-injury, was reduced following acute IOP elevation (Fig. 2b). We have also published data showing that this form of injury induces microglia activation in the retina as well as accumulation of subretinal macrophages (Chrysostomou et al., 2014; Kezic et al., 2013). 2.3.4. Limited cell loss TUNEL staining was used to identify DNA damage characteristic
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of apoptosis in the ganglion cell layer (GCL) following IOP injury. Low levels of TUNEL-positive nuclei can be detected in the GCL at 1day post injury but not at other timepoints. RGC survival was estimated by counting the number of cells immunolabeled for Brn3a on retinal cryosections. Brn-3a is a reliable marker of RGCs with previous reports showing >90% correlation between the number of Brn-3a-positive cells in the GCL and the number of RGCs visualized s et al., 2009). by retrograde labeling with flurogold (Nadal-Nicola According to this analysis, there is no significant reduction in Brn3a-positive cells in the GCL at 7-days, suggesting no substantial RGC loss following injury (Fig. 3). Furthermore, there is no loss in the number of nuclei in either the inner or outer nuclear layer at 7days, confirming the specificity of this injury to the inner retina. 2.3.5. Maintenance of inner retinal perfusion Next, we investigated whether there was loss of inner retinal perfusion during the injury using the Heidelberg flow meter to measure inner retinal capillary blood flow. Elevation of IOP to 50 mmHg for 30 min did not affect inner retinal capillary perfusion in mice up to 18-months of age. Both 3-month old and 18-month old animals did not show significant decreases in retinal blood flow at IOP levels below approximately 70 mmHg (Kong et al., 2012). 3. Discussion 3.1. Pros and Cons of this injury model An advantage of an IOP challenge is that it can be easily titrated by altering the magnitude or duration of IOP elevation (Table 1). It provides a precise and reproducible injury within and between experiments. This level of control contrasts with many of the more chronic IOP elevation models where the degree and duration of IOP elevation are much more unpredictable and often vary substantially between animals exposed within the same experiment. The discrete temporal nature of a single acute IOP challenge allows the investigator to follow the time-course of RGC recovery. A fine borosilicate needle (50 mm tip) is inserted through the cornea and an experienced operator will induce minimal trauma to the anterior segment. The ocular media remain clear and so injured animals can be assessed longitudinally for structure and functional changes. To date we have used only static levels of elevated pressure. However, tracings derived from telemetric studies in non-human primates show that IOP varies with significant IOP fluctuations accounting for up to 15% of the area under the IOP curve in non-human primates (Downs et al., 2011). As such, it may be possible to reproduce these IOP spikes with our model system and more accurately replicate physiological IOP exposure. As indicated above, glaucoma is a chronic disease usually associated with longer-term exposures to IOP elevation with resultant loss of RGCs. We are not aware of any prior systematic investigation of “injured” RGCs in glaucoma, although a number of clinical cohort studies have demonstrated reversible loss of visual function in glaucoma patients by visual field (Gandolfi, 1995), contrast sensitivity (Gandolfi et al., 2005) or electrophysiology (Ventura and Porciatti, 2005). Recently we have demonstrated significant improvement of the PhNR of the ERG in glaucoma patients following IOP lowering (Niyadurupola et al., 2013) using the same component of the ERG that we use in the mouse model (Chrysostomou and Crowston, 2013). How or why this reversal in visual function occurs in humans is still not known, but significant advantages may be derived from using similar endpoints in experimental and clinical studies. Limitations of our model include the fact that the animals need to be anesthetized for the duration of injury and re-anesthetized for
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Fig. 1. Selective loss of inner retinal function after injury. Elevation of IOP to 50 mmHg for 30 min in 12-month old mice leads to a significant reduction in ERG responses derived from the inner retina (pSTR and PhNR) at 7 days post-injury but has no appreciable effect on signals derived from the mid and outer retina (scotopic a/b-waves and photopic bwave). (A) Mean amplitudes (±SEM) of saturated ERG responses, presented as relative changes from baseline and normalized to sham-treated contralateral eyes (*P < 0.05). (B) Representative traces showing selective reduction of the inner-retinal derived PhNR and pSTR by IOP elevation. ERGs were recorded serially in individual mice, 1 day before (baseline) and 7 days after elevation of IOP to 50 mmHg for 30 min. Amplitudes were measured in response to flash stimuli of 2.22 log cd s/m2 (photopic b-wave, scotopic a-wave and scotopic b-wave), 5.45 log cd s/m2 (pSTR) and 0.92 log cd s/m2 (PhNR), with a background illumination of 40.0 cd s/m2 for photopic signals.
Fig. 2. Signs of retinal stress after injury. Elevation of IOP to 50 mmHg for 30 min activates stress responses in retinal microglia and neurons. (A) Immunohistochemical labeling of retinal cryosections showing upregulation of HO-1 and GFAP expression in Müller cell processes 1 day and 7 days after IOP elevation. (B) Gene expression measured by qPCR, showing upregulation of GFAP and downregulation of the RGC-specific marker Thy-1 in retinas 7 days after IOP elevation. mRNA levels are normalized to the housekeeping gene bactin and expressed relative to sham-treated fellow eyes. Scale bars ¼ 40 mm. Error bars ¼ SEM; n ¼ 10; *P < 0.05.
assessment of retinal function (Table 1). Although we are careful to monitor for confounding variables such as lowering of blood pressure, it is possible that other confounders also contribute to the injury. A second negative is that IOP elevation is currently performed using a fine needle, which induces some trauma to the cornea. We have recently published evidence that even minimally
traumatic needle insertion in the absence of IOP elevation can induce some microglial activation in the retina (Kezic et al., 2013). We are currently exploring an alternative approach for elevating pressure in the eye without the need to cannulate the anterior chamber and this will be the subject of a future manuscript. A further potential limitation is the possible influence of
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Fig. 3. Minimal RGC death and loss after injury. Elevation of IOP to 50 mmHg for 30 min causes a small increase in the number of TUNEL-positive nuclei in the ganglion cell layer (GCL) but this does not translate to a significant reduction in overall RGC number at 7 days. (A) Representative micrographs of immunolabeled retinal sections showing a TUNELpositive cells (green) in the GCL. (B) Quantification of TUNEL-positive nuclei in the GCL in the week following IOP elevation. (C) Quantification of retinal cell survival 7 days after IOP elevation. Retinal thickness was measured on Hoechst-labeled retinal cryosections. RGC survival was estimated by counting the number of cells immunolabeled for Brn-3a. Sections cut through the optic nerve head and ora serrate were scanned from superior to inferior edge and data are expressed as the number of Brn-3a-positive cells per 100 mm retinal length. Scale bar ¼ 25 mm. Error bars ¼ SEM; n ¼ 6. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.
Table 1 Advantages and disadvantages of an acute IOP injury model. Pros
Cons
Well defined fully recoverable and treatable injury Highly reproducible Non-ischemic Ability to measure impact of demographics (eg aging) on RGC recovery Ability to easily measure impact of interventions on RGC recovery Not a disease/glaucoma model per-se Invasive technique although minimal trauma associated with careful insertion of a 50 micron needle Need for repeat anesthesia
amacrine cell dysfunction in determining the PhNR or pSTR signals of the ERG. To address this, we have recently been patch-clamping RGCs in flatmounted retina taken from mouse eyes. Subsequent filling of these cells with Lucifer yellow or biocytin provides information on their morphology. RGCs are identified by the presence of an axon. Single cell recordings in injured RGCs confirm loss of function observed in the full field ERG (data not shown). 3.2. Relationship between acute IOP elevation and chronic glaucoma As mentioned in the Introduction, questions remain about the exact relationship of events induced by this acute model to those produced in chronic glaucoma. Recent work by other laboratories is beginning to provide some answers to this. Jakobs and colleagues, also working with mice and a similar pressure paradigm, have shown that brief exposures to mild IOP elevation can produce significant alterations in the morphology of astrocytes with the optic nerve head (ONH) (Lye-Barthel et al., 2013; Sun et al., 2013). Considering the important structural and supportive role of these cells for axons within the ONH, the likely site of initial injury in glaucoma, this work strongly supports the relevance of responses initiated by this acute pressure challenge to events occurring in glaucoma. Additional work in rats, using longer, 8 h pressure exposures, is now showing that the acute pressure paradigm can affect axons as well as cellular responses within the ONH. Abbott and colleagues have shown that 50 mmHg for 8 h results in increased nerve fiber layer thickness that recovers after 3 weeks, with no persistent
axonal transport deficits or detectable RGC loss (Abbott et al., 2014). Morrison and colleagues, primarily working with an 8 h exposure to 60 mmHg, demonstrated that this will produce mild axonal injury, as well as gene expression profiles within the ONH similar to those observed after chronic IOP elevation (Morrison et al., 2010, 2014). As these pressure levels do not produce extensive alterations in ONH or retinal perfusion (Zhi et al., 2012), these results strongly suggest that this approach offers a powerful opportunity to understand the relative importance of these IOP-induced gene expression responses and how they may contribute to axonal injury. Funding acknowledgment The Centre for Eye Research Australia receives Operational Infrastructure Support from the Victorian Government. References Abbott, C.J., Choe, T.E., Lusardi, T.A., Burgoyne, C.F., Wang, L., Fortune, B., 2014. Evaluation of retinal nerve fiber layer thickness and axonal transport 1 and 2 weeks after 8 hours of acute intraocular pressure elevation in rats. Invest. Ophthalmol. Vis. Sci. 55, 674e687. Chrysostomou, V., Crowston, J.G., 2013. The photopic negative response of the mouse electroretinogram: reduction by acute elevation of intraocular pressure. Invest. Ophthalmol. Vis. Sci. 54, 4691e4697. Chrysostomou, V., Kezic, J.M., Trounce, I.A., Crowston, J.G., 2014. Forced exercise protects the aged optic nerve against intraocular pressure injury. Neurobiol. Aging 35, 1722e1725. Chrysostomou, V., Stone, J., Stowe, S., Barnett, N.L., Valter, K., 2008. The status of cones in the rhodopsin mutant P23H-3 retina: light-regulated damage and repair in parallel with rods. Invest. Ophthalmol. Vis. Sci. 49, 1116e1125. Downs, J.C., Burgoyne, C.F., Seigfreid, W.P., Reynaud, J.F., Strouthidis, N.G., Sallee, V., 2011. 24-Hour IOP telemetry in the non-human primate: implant system performance and initial characterization of IOP at multiple timescales. Invest. Ophthalmol. Vis. Sci. 52, 7365e7375. Gandolfi, S.A., 1995. Improvement of visual field indices after surgical reduction of intraocular pressure. Ophthalmic Surg. 26, 121e126. Gandolfi, S.A., Cimino, L., Sangermani, C., Ungaro, N., Mora, P., Tardini, M.G., 2005. Improvement of spatial contrast sensitivity threshold after surgical reduction of intraocular pressure in unilateral high-tension glaucoma. Invest. Ophthalmol. Vis. Sci. 46, 197e201. Kezic, J.M., Chrysostomou, V., Trounce, I.A., McMenamin, P.G., Crowston, J.G., 2013. Effect of anterior chamber cannulation and acute IOP elevation on retinal macrophages in the adult mouse. Invest. Ophthalmol. Vis. Sci. 54, 3028e3036. Kong, Y.X., Crowston, J.G., Vingrys, A.J., Trounce, I.A., Bui, B.V., 2009. Functional changes in the retina during and after acute intraocular pressure elevation in
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mice. Invest. Ophthalmol. Vis. Sci. 50, 5732e5740. Kong, Y.X.G., van Bergen, N., Bui, B.V., Chrysostomou, V., Vingrys, A.J., Trounce, I.A., Crowston, J.G., 2012. Impact of aging and diet restriction on retinal function during and after acute intraocular pressure injury. Neurobiol. Aging 33, 1126. Lye-Barthel, M., Sun, D., Jakobs, T.C., 2013. Morphology of astrocytes in a glaucomatous optic nerve. Invest. Ophthalmol. Vis. Sci. 54, 909e917. Morrison, J.C., Cepurna, W.O., Doser, T.A., Dyck, J.A., Johnson, E.C., 2010. A short interval of controlled elevation of IOP (CEI) reproduces early chronic glaucoma model optic nerve head (ONH) gene expression responses. Invest. Ophthalmol. Vis. Sci. E-Abstr. 51, 5216. Morrison, J.C., Choe, T.E., Cepurna, W.O., Johnson, E.C., 2014. Optic nerve head (ONH) gene expression responses to elevated intraocular pressure (IOP), anesthesia and anterior chamber cannulation in the CEI (Controlled Elevation of IOP) model of IOP-induced optic nerve injury. Invest. Ophthalmol. Vis. Sci. E-Abstr. 54, 2402. s, F.M., Jime nez-Lo pez, M., Sobrado-Calvo, P., Nieto-Lo pez, L., Ca novasNadal-Nicola Martínez, I., Salinas-Navarro, M., Vidal-Sanz, M., Agudo, M., 2009. Brn3a as a
marker of retinal ganglion cells: qualitative and quantitative time course studies in naïve and optic nerveeinjured retinas. Invest. Ophthalmol. Vis. Sci. 50, 3860e3868. Niyadurupola, N., Luu, C.D., Nguyen, D.Q., Geddes, K., Tan, G.X.V., Wong, C.C.W., Tran, T., Coote, M.A., Crowston, J.G., 2013. Intraocular pressure lowering is associated with an increase in the photopic negative response (PhNR) amplitude in glaucoma and ocular hypertensive eyes. Invest. Ophthalmol. Vis. Sci. 54, 1913e1919. Sun, D., Qu, J., Jakobs, T.C., 2013. Reversible reactivity by optic nerve astrocytes. Glia 61, 1218e1235. Ventura, L.M., Porciatti, V., 2005. Restoration of retinal ganglion cell function in early glaucoma after intraocular pressure reduction: a pilot study. Ophthalmology 112, 20e27. Zhi, Z., Cepurna, W.O., Johnson, E.C., Morrison, J.C., Wang, R.K., 2012. Impact of intraocular pressure on changes of blood flow in the retina, choroid, and optic nerve head in rats investigated by optical microangiography. Biomed. Opt. Express 3, 2220e2233.
Contents lists available at ScienceDirect
Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer
An acute intraocular pressure challenge to assess retinal ganglion cell injury and recovery in the mouse Jonathan G. Crowston a, Yu Xiang G. Kong a, Ian A. Trounce a, Trung M. Dang a, Eamonn T. Fahy a, Bang V. Bui b, John C. Morrison c, Vicki Chrysostomou a, * a b c
Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital, East Melbourne, Victoria, Australia Department of Optometry and Vision Sciences, University of Melbourne, Parkville, Victoria, Australia Casey Eye Institute, Oregon Health and Science University, Portland, OR, United States
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 December 2014 Received in revised form 3 March 2015 Accepted in revised form 6 March 2015 Available online 7 March 2015
We describe a model of acute intraocular pressure (IOP) elevation in the mouse eye that induces reversible loss of inner retinal function associated with oxidative stress, glial cell activation and minimal loss of retinal ganglion cell (RGC) number. Young healthy mouse eyes recover inner retinal function within 7-days but more persistent functional loss is seen in older mice. Manipulation of diet and exercise further modify RGC recovery demonstrating the utility of this injury model for investigating lifestyle and therapeutic interventions. We believe that systematic investigation into the characteristics and determinants of RGC recovery following an IOP challenge will shed light on processes that govern RGC vulnerability in the early stages of glaucoma. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Retinal ganglion cell Intraocular pressure Animal models Glaucoma Neurorecovery Electroretinography
1. Introduction We adapted an acute intraocular pressure (IOP) elevation injury model to investigate how retinal ganglion cells (RGCs) respond to a titratable and highly reproducible injury. Our goal in generating this “optic nerve stress test” was to identify levels of injury that, in a healthy young mouse retina, induce selective but reversible loss of RGC function while maintaining normal outer retinal function. Additionally, we sought an injury that does not lead to manifest ischemia of the inner retina with minimal RGC death. Our interest lies in determining the parameters that influence RGC recovery over time. Glaucoma is a chronic, progressive optic neuropathy characterized by the selective death of RGCs and their axons. How then does our acute IOP injury model inform us of processes that are relevant to this disease? In the very early stages of glaucoma, the optic nerve head is repeatedly exposed to cycles of relatively minor injury (Downs et al., 2011). We propose that healthy RGCs are initially able
* Corresponding author. Centre for Eye Research Australia, Level 1, 32 Gisborne Street, East Melbourne VIC 3002, Australia. E-mail address: [email protected] (V. Chrysostomou). http://dx.doi.org/10.1016/j.exer.2015.03.006 0014-4835/© 2015 Elsevier Ltd. All rights reserved.
to withstand and recover from such minor injuries. However, over time, repeated insults or an intrinsic impairment in the capacity of RGCs to recover, will overwhelm their ability to recover resulting in induction of a cell death program. Measuring the capacity for RGC recovery following an acute IOP injury, we believe, will shed light on the very early stages of glaucoma. Furthermore, this test will enable us to investigate factors such as aging, diet and exercise that can improve or impair RGC recovery. By incrementally increasing the IOP level or duration of injury we will also be able to investigate the critical switch point at which full functional recovery is not possible.
2. Acute IOP injury model 2.1. Overview Our injury model involves acute elevation of IOP through insertion of a fine needle attached to either a column of fluid or a motorized syringe pump. RGC function is measured using the positive scotopic threshold response (pSTR) or the photopic negative response (PhNR) components of the full-field electroretinogram (ERG). A single animal can be monitored over a number of
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timepoints post-injury but to mitigate potential effects of repeat anesthesia, we leave 7 days between injury and functional assessment by the ERG. Following functional assessment, mice are euthanized and retinas and optic nerves are harvested and subsequently analyzed using a range of histological and biochemical assays. 2.2. Experimental techniques All animal procedures conform to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and with the requirements of our institutional Animal Research and Ethics Committee. 2.2.1. IOP elevation Animals are anesthetized with intraperitoneal injection of ketamine (60 mg/kg; Troy Laboratories Pty Ltd.) and xylazine (10 mg/ kg; Troy Laboratories Pty Ltd.) before resting IOP is measured using a hand-held rebound tonometer (TonoLab; Icare Finland Oy, Espoo, Finland). Pupil dilation and corneal anesthesia is achieved by topical application of one drop each of 0.5% proxymetacaine hydrochloride (Alcon Laboratories Pty Ltd., Frenchs Forest, NSW, Australia) and 0.5% tropicamide (Minims; Chauvin Pharmaceuticals, Surrey, UK). Movement of iris by mydriasis aids subsequent needle insertion. Animals are lightly secured to a platform with wire loops across the upper back and nose. Body temperature is maintained at 37e38 C with a homeothermic heating unit. The mouse eye is proptosed using curved forceps. Using a micromanipulator, the tip of a pulled glass borosilicate needle (~50 mm, 1B100-6, WPI, Sarasota, FL, USA) is inserted into the anterior chamber through the center of the cornea. After insertion, the microneedle is then repositioned to minimize corneal deformation and to ensure that the eye remains in its natural position. The cannulated needle is connected via polyethylene tubing (0.97 mm inner diameter, Microtube Extrusions, North Rocks, NSW, Australia) to a pressure transducer (Transpac IV, Abbott Critical Care Systems, Sligo, Ireland), which is in series with a sterilefiltered endotoxin-tested Hanks' balanced salt solution reservoir (HBSS, JRH Biosciences, Lenexa, KA, USA). The HBSS reservoir acts as a pressure column, the height of which determines the hydrostatic pressure delivered to the eye. Pressure transducer calibration is performed by referencing to an aneroid sphygmomanometer (Livingstone, Rosebery, NSW, Australia). HBSS is chosen as the fluid in the pressure column as its concentrations of salts and ions show a close approximation to aqueous humor. Initial cannulation of the anterior chamber is performed with the reservoir height set to eye level and the reservoir valve closed. Patency of the needle following cannulation is confirmed by anterior chamber distension observed under the microscope. IOP is raised to the desired level by altering the height of the reservoir, and monitored in real time using a PowerLab data acquisition system. The target pressure is confirmed using a hand-held rebound tonometer and pressure is maintained within 1 mmHg throughout. In each animal, pressure is elevated to 50 mmHg for 30 min in one eye (injured) while the fellow eye is cannulated and held at physiological IOP (sham, 12 mmHg). The contralateral sham-treated eye acts as an internal control to mitigate any variations induced by anesthesia, temperature etc. A variation of this approach has been developed more recently where a syringe filled with HBBS is mounted on a motorized syringe pump (PHD Ultra CP; Harvard Apparatus, Massachusetts, USA). This pump can be set to drive fluid at an adjustable rate to maintain a set pressure. Side by side comparison of the adjustable column and motorized syringe pump has revealed no difference in
RGC dysfunction (not shown). 2.2.2. Electroretinography The ERG is used to measure retinal function serially in animals, one day before (baseline) and 7 days after IOP elevation. Responses recorded after IOP elevation are normalized to baseline values to determine pressure-induced changes. After overnight dark-adaptation (minimum 12 h), animals are prepared for recording under dim red illumination. Animals are anesthetized with intraperitoneal injection of ketamine (60 mg/kg) and xylazine (10 mg/kg). Pupil dilation and corneal anesthesia are achieved by topical application of one drop each of 0.5% tropicamide (Minims; Chauvin Pharmaceuticals, Surrey, UK), 2.5% phenylephrine (Minims; Chauvin Pharmaceuticals) and 0.5% proxymetacaine hydrochloride (Alcon Laboratories Pty Ltd.). Body temperature is maintained at 37 C with a homeothermic controller and unit. Electrical signals are recorded with a platinum wire electrode contacting the central cornea while a gold pellet placed in the mouth serves as common reference. A stainless steel needle electrode (F-E2-60, Grass Technologies, West Warwick, RI, USA) inserted at the base of the tail acts as ground. Retinal responses are recorded simultaneously from both eyes with the testing protocol lasting approximately 30 min. Full-field flash ERGS are recorded using an Espion Diagnosys system (Diagnosys LLC, Littleton, MA, USA). Light stimuli are delivered via a Ganzfeld unit from white light-emitting diodes. Light energies are calibrated as luminance energy units, candela second per meter squared (cd s/m2). Scotopic threshold responses (STRs) are elicited using a 5.45 log cd s/m2 stimulus. Twenty-five flashes with an inter-stimulus interval of 1500 ms are averaged. Scotopic a-waves and b-waves are elicited with a stimulus intensity of 2.22 log cd s/m2 with no averaging required. Photopic responses to 6 different stimulus strengths between 0.34 and 2.22 log cd s/m2 are presented on a 40.0 cd s/m2 rod-saturating green background. At each intensity, twenty-five flashes with an inter-stimulus interval of 3000 ms are averaged. Response amplitudes of the scotopic a-wave, scotopic b-wave, pSTR, PhNR and photopic b-wave are measured in two ways: (1) by identifying the maximum peak/trough and obtaining the baselinetrough/peak amplitude; (2) by taking the amplitude at a fixed criterion time after the stimulus onset, again with respect to baseline. Criterion times are chosen to correspond with the mean implicit time of all data from our study. 2.2.3. Retina and optic nerve structure and biochemistry At chosen timepoints after acute IOP elevation, retina and optic nerve are harvested from injured eyes and contralateral shamtreated eyes, and subsequently processed in various ways. We routinely assess cell structure and survival by histological staining and the TUNEL technique; protein expression levels by immunohistochemistry, Western blotting and ELISA; and gene expression by quantitative real-time PCR. We have published detailed descriptions of these protocols elsewhere (Chrysostomou and Crowston, 2013; Chrysostomou et al., 2014, 2008; Kong et al., 2012) and provide brief summaries below. For immunohistochemical labeling, retinal cryosections are prepared from paraformaldehyde-fixed eyes and subsequently incubated overnight at 4 C with primary antibodies. Common antibodies used in our studies include glial fibrillary acidic protein (GFAP; 1:800, Dako, Campbellfield VIC, Australia), heme oxygenase-1 (HO-1; 1:400, Enzo Life Sciences, Ann Arbor, MI, USA) and Brn-3a (1:100, Millipore, Kilsyth VIC, Australia). After incubation with biotin-conjugated secondary antibody (1:200; SigmaeAldrich, St. Louis, MO, USA) and with Alexa Fluor Streptavidin conjugate (1:1000, Invitrogen; Invitrogen, Eugene, OR, USA),
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sections are nuclear-counterstained with Hoechst (1:10,000) before being coverslipped with a glycerol/gelatin medium. We extract RNA from retina and optic nerve stump and synthesize complementary DNA using commercial kits (Applied Biosystems, Mulgrave, Australia). Quantitative real-time PCR (qPCR) is then performed using a TaqMan®-based assay (Applied Biosystems) and probes specific for target genes such as GFAP (NM_001131020.1) and Thy-1 (NM_009382.3). Following normalization of each sample for b-actin, relative quantitation of gene expression is calculated using the comparative threshold cycle method. 2.3. Model characteristics 2.3.1. Selective loss of inner retinal function Initial work determined the level of IOP elevation that led to selective loss of inner retinal function but left outer and mid retinal function intact (Kong et al., 2009). We employed a “staircase” increment of IOP elevation in dark-adapted mouse eyes while simultaneously measuring retinal function with the ERG. Amplitudes of the pSTR component of the ERG were used as a measure of inner retinal function while amplitudes of scotopic a-wave and bwaves were used to indicate photoreceptoral (a-wave), and ONbipolar (b-wave) function. In these experiments, we monitored the full-field ERG during and in the first 2 h following IOP elevation. Here, we found that the greatest separation of inner and outer retinal function occurred at an IOP of 50 mmHg. We have since established, characterized and published protocols for recording photopic negative responses (PhNRs) from the mouse retina, as an additional measure of inner retinal function (Chrysostomou and Crowston, 2013), the amplitudes of which are also selectively reduced in response to elevation of IOP to 50 mmHg. The selective loss of inner retinal function in response to our injury model is summarized in Fig. 1. 2.3.2. Age-related susceptibility As aging is one of the greatest risk factors for the development of glaucoma, we sought an injury to which animals showed increasing vulnerability with advancing age. In defining experiments, we recorded the ERG prior to, during, and after a fixed IOP challenge (50 mmHg for 30 min) in 3, 12, and 18-month old animals. We found that by 7 days post-injury, inner retinal function had recovered fully in young 3-month mice but a persistent loss of function was seen in 12-month old and 18-month old mice (Kong et al., 2012). This negative impact of aging has also been shown to be ameliorated by diet restriction (Kong et al., 2012) or exercise (Chrysostomou et al., 2014). 2.3.3. Signs of inner retinal stress Western blot and immunohistochemistry have been used to assess retina for oxidative stress and glial cell activation at baseline, 1- and 7-days post-injury. We found that a single 30 min elevation of IOP to 50 mmHg leads to marked astrocytic gliosis, seen as upregulation of GFAP protein and mRNA expression (Fig. 2 (Chrysostomou and Crowston, 2013)). Retinal expression of HO-1, used here as an oxidative stress marker, also increases in the week following injury (Fig. 2a). Thy-1 expression, an RGC marker that is known to be reduced post-injury, was reduced following acute IOP elevation (Fig. 2b). We have also published data showing that this form of injury induces microglia activation in the retina as well as accumulation of subretinal macrophages (Chrysostomou et al., 2014; Kezic et al., 2013). 2.3.4. Limited cell loss TUNEL staining was used to identify DNA damage characteristic
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of apoptosis in the ganglion cell layer (GCL) following IOP injury. Low levels of TUNEL-positive nuclei can be detected in the GCL at 1day post injury but not at other timepoints. RGC survival was estimated by counting the number of cells immunolabeled for Brn3a on retinal cryosections. Brn-3a is a reliable marker of RGCs with previous reports showing >90% correlation between the number of Brn-3a-positive cells in the GCL and the number of RGCs visualized s et al., 2009). by retrograde labeling with flurogold (Nadal-Nicola According to this analysis, there is no significant reduction in Brn3a-positive cells in the GCL at 7-days, suggesting no substantial RGC loss following injury (Fig. 3). Furthermore, there is no loss in the number of nuclei in either the inner or outer nuclear layer at 7days, confirming the specificity of this injury to the inner retina. 2.3.5. Maintenance of inner retinal perfusion Next, we investigated whether there was loss of inner retinal perfusion during the injury using the Heidelberg flow meter to measure inner retinal capillary blood flow. Elevation of IOP to 50 mmHg for 30 min did not affect inner retinal capillary perfusion in mice up to 18-months of age. Both 3-month old and 18-month old animals did not show significant decreases in retinal blood flow at IOP levels below approximately 70 mmHg (Kong et al., 2012). 3. Discussion 3.1. Pros and Cons of this injury model An advantage of an IOP challenge is that it can be easily titrated by altering the magnitude or duration of IOP elevation (Table 1). It provides a precise and reproducible injury within and between experiments. This level of control contrasts with many of the more chronic IOP elevation models where the degree and duration of IOP elevation are much more unpredictable and often vary substantially between animals exposed within the same experiment. The discrete temporal nature of a single acute IOP challenge allows the investigator to follow the time-course of RGC recovery. A fine borosilicate needle (50 mm tip) is inserted through the cornea and an experienced operator will induce minimal trauma to the anterior segment. The ocular media remain clear and so injured animals can be assessed longitudinally for structure and functional changes. To date we have used only static levels of elevated pressure. However, tracings derived from telemetric studies in non-human primates show that IOP varies with significant IOP fluctuations accounting for up to 15% of the area under the IOP curve in non-human primates (Downs et al., 2011). As such, it may be possible to reproduce these IOP spikes with our model system and more accurately replicate physiological IOP exposure. As indicated above, glaucoma is a chronic disease usually associated with longer-term exposures to IOP elevation with resultant loss of RGCs. We are not aware of any prior systematic investigation of “injured” RGCs in glaucoma, although a number of clinical cohort studies have demonstrated reversible loss of visual function in glaucoma patients by visual field (Gandolfi, 1995), contrast sensitivity (Gandolfi et al., 2005) or electrophysiology (Ventura and Porciatti, 2005). Recently we have demonstrated significant improvement of the PhNR of the ERG in glaucoma patients following IOP lowering (Niyadurupola et al., 2013) using the same component of the ERG that we use in the mouse model (Chrysostomou and Crowston, 2013). How or why this reversal in visual function occurs in humans is still not known, but significant advantages may be derived from using similar endpoints in experimental and clinical studies. Limitations of our model include the fact that the animals need to be anesthetized for the duration of injury and re-anesthetized for
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Fig. 1. Selective loss of inner retinal function after injury. Elevation of IOP to 50 mmHg for 30 min in 12-month old mice leads to a significant reduction in ERG responses derived from the inner retina (pSTR and PhNR) at 7 days post-injury but has no appreciable effect on signals derived from the mid and outer retina (scotopic a/b-waves and photopic bwave). (A) Mean amplitudes (±SEM) of saturated ERG responses, presented as relative changes from baseline and normalized to sham-treated contralateral eyes (*P < 0.05). (B) Representative traces showing selective reduction of the inner-retinal derived PhNR and pSTR by IOP elevation. ERGs were recorded serially in individual mice, 1 day before (baseline) and 7 days after elevation of IOP to 50 mmHg for 30 min. Amplitudes were measured in response to flash stimuli of 2.22 log cd s/m2 (photopic b-wave, scotopic a-wave and scotopic b-wave), 5.45 log cd s/m2 (pSTR) and 0.92 log cd s/m2 (PhNR), with a background illumination of 40.0 cd s/m2 for photopic signals.
Fig. 2. Signs of retinal stress after injury. Elevation of IOP to 50 mmHg for 30 min activates stress responses in retinal microglia and neurons. (A) Immunohistochemical labeling of retinal cryosections showing upregulation of HO-1 and GFAP expression in Müller cell processes 1 day and 7 days after IOP elevation. (B) Gene expression measured by qPCR, showing upregulation of GFAP and downregulation of the RGC-specific marker Thy-1 in retinas 7 days after IOP elevation. mRNA levels are normalized to the housekeeping gene bactin and expressed relative to sham-treated fellow eyes. Scale bars ¼ 40 mm. Error bars ¼ SEM; n ¼ 10; *P < 0.05.
assessment of retinal function (Table 1). Although we are careful to monitor for confounding variables such as lowering of blood pressure, it is possible that other confounders also contribute to the injury. A second negative is that IOP elevation is currently performed using a fine needle, which induces some trauma to the cornea. We have recently published evidence that even minimally
traumatic needle insertion in the absence of IOP elevation can induce some microglial activation in the retina (Kezic et al., 2013). We are currently exploring an alternative approach for elevating pressure in the eye without the need to cannulate the anterior chamber and this will be the subject of a future manuscript. A further potential limitation is the possible influence of
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Fig. 3. Minimal RGC death and loss after injury. Elevation of IOP to 50 mmHg for 30 min causes a small increase in the number of TUNEL-positive nuclei in the ganglion cell layer (GCL) but this does not translate to a significant reduction in overall RGC number at 7 days. (A) Representative micrographs of immunolabeled retinal sections showing a TUNELpositive cells (green) in the GCL. (B) Quantification of TUNEL-positive nuclei in the GCL in the week following IOP elevation. (C) Quantification of retinal cell survival 7 days after IOP elevation. Retinal thickness was measured on Hoechst-labeled retinal cryosections. RGC survival was estimated by counting the number of cells immunolabeled for Brn-3a. Sections cut through the optic nerve head and ora serrate were scanned from superior to inferior edge and data are expressed as the number of Brn-3a-positive cells per 100 mm retinal length. Scale bar ¼ 25 mm. Error bars ¼ SEM; n ¼ 6. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.
Table 1 Advantages and disadvantages of an acute IOP injury model. Pros
Cons
Well defined fully recoverable and treatable injury Highly reproducible Non-ischemic Ability to measure impact of demographics (eg aging) on RGC recovery Ability to easily measure impact of interventions on RGC recovery Not a disease/glaucoma model per-se Invasive technique although minimal trauma associated with careful insertion of a 50 micron needle Need for repeat anesthesia
amacrine cell dysfunction in determining the PhNR or pSTR signals of the ERG. To address this, we have recently been patch-clamping RGCs in flatmounted retina taken from mouse eyes. Subsequent filling of these cells with Lucifer yellow or biocytin provides information on their morphology. RGCs are identified by the presence of an axon. Single cell recordings in injured RGCs confirm loss of function observed in the full field ERG (data not shown). 3.2. Relationship between acute IOP elevation and chronic glaucoma As mentioned in the Introduction, questions remain about the exact relationship of events induced by this acute model to those produced in chronic glaucoma. Recent work by other laboratories is beginning to provide some answers to this. Jakobs and colleagues, also working with mice and a similar pressure paradigm, have shown that brief exposures to mild IOP elevation can produce significant alterations in the morphology of astrocytes with the optic nerve head (ONH) (Lye-Barthel et al., 2013; Sun et al., 2013). Considering the important structural and supportive role of these cells for axons within the ONH, the likely site of initial injury in glaucoma, this work strongly supports the relevance of responses initiated by this acute pressure challenge to events occurring in glaucoma. Additional work in rats, using longer, 8 h pressure exposures, is now showing that the acute pressure paradigm can affect axons as well as cellular responses within the ONH. Abbott and colleagues have shown that 50 mmHg for 8 h results in increased nerve fiber layer thickness that recovers after 3 weeks, with no persistent
axonal transport deficits or detectable RGC loss (Abbott et al., 2014). Morrison and colleagues, primarily working with an 8 h exposure to 60 mmHg, demonstrated that this will produce mild axonal injury, as well as gene expression profiles within the ONH similar to those observed after chronic IOP elevation (Morrison et al., 2010, 2014). As these pressure levels do not produce extensive alterations in ONH or retinal perfusion (Zhi et al., 2012), these results strongly suggest that this approach offers a powerful opportunity to understand the relative importance of these IOP-induced gene expression responses and how they may contribute to axonal injury. Funding acknowledgment The Centre for Eye Research Australia receives Operational Infrastructure Support from the Victorian Government. References Abbott, C.J., Choe, T.E., Lusardi, T.A., Burgoyne, C.F., Wang, L., Fortune, B., 2014. Evaluation of retinal nerve fiber layer thickness and axonal transport 1 and 2 weeks after 8 hours of acute intraocular pressure elevation in rats. Invest. Ophthalmol. Vis. Sci. 55, 674e687. Chrysostomou, V., Crowston, J.G., 2013. The photopic negative response of the mouse electroretinogram: reduction by acute elevation of intraocular pressure. Invest. Ophthalmol. Vis. Sci. 54, 4691e4697. Chrysostomou, V., Kezic, J.M., Trounce, I.A., Crowston, J.G., 2014. Forced exercise protects the aged optic nerve against intraocular pressure injury. Neurobiol. Aging 35, 1722e1725. Chrysostomou, V., Stone, J., Stowe, S., Barnett, N.L., Valter, K., 2008. The status of cones in the rhodopsin mutant P23H-3 retina: light-regulated damage and repair in parallel with rods. Invest. Ophthalmol. Vis. Sci. 49, 1116e1125. Downs, J.C., Burgoyne, C.F., Seigfreid, W.P., Reynaud, J.F., Strouthidis, N.G., Sallee, V., 2011. 24-Hour IOP telemetry in the non-human primate: implant system performance and initial characterization of IOP at multiple timescales. Invest. Ophthalmol. Vis. Sci. 52, 7365e7375. Gandolfi, S.A., 1995. Improvement of visual field indices after surgical reduction of intraocular pressure. Ophthalmic Surg. 26, 121e126. Gandolfi, S.A., Cimino, L., Sangermani, C., Ungaro, N., Mora, P., Tardini, M.G., 2005. Improvement of spatial contrast sensitivity threshold after surgical reduction of intraocular pressure in unilateral high-tension glaucoma. Invest. Ophthalmol. Vis. Sci. 46, 197e201. Kezic, J.M., Chrysostomou, V., Trounce, I.A., McMenamin, P.G., Crowston, J.G., 2013. Effect of anterior chamber cannulation and acute IOP elevation on retinal macrophages in the adult mouse. Invest. Ophthalmol. Vis. Sci. 54, 3028e3036. Kong, Y.X., Crowston, J.G., Vingrys, A.J., Trounce, I.A., Bui, B.V., 2009. Functional changes in the retina during and after acute intraocular pressure elevation in
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mice. Invest. Ophthalmol. Vis. Sci. 50, 5732e5740. Kong, Y.X.G., van Bergen, N., Bui, B.V., Chrysostomou, V., Vingrys, A.J., Trounce, I.A., Crowston, J.G., 2012. Impact of aging and diet restriction on retinal function during and after acute intraocular pressure injury. Neurobiol. Aging 33, 1126. Lye-Barthel, M., Sun, D., Jakobs, T.C., 2013. Morphology of astrocytes in a glaucomatous optic nerve. Invest. Ophthalmol. Vis. Sci. 54, 909e917. Morrison, J.C., Cepurna, W.O., Doser, T.A., Dyck, J.A., Johnson, E.C., 2010. A short interval of controlled elevation of IOP (CEI) reproduces early chronic glaucoma model optic nerve head (ONH) gene expression responses. Invest. Ophthalmol. Vis. Sci. E-Abstr. 51, 5216. Morrison, J.C., Choe, T.E., Cepurna, W.O., Johnson, E.C., 2014. Optic nerve head (ONH) gene expression responses to elevated intraocular pressure (IOP), anesthesia and anterior chamber cannulation in the CEI (Controlled Elevation of IOP) model of IOP-induced optic nerve injury. Invest. Ophthalmol. Vis. Sci. E-Abstr. 54, 2402. s, F.M., Jime nez-Lo pez, M., Sobrado-Calvo, P., Nieto-Lo pez, L., Ca novasNadal-Nicola Martínez, I., Salinas-Navarro, M., Vidal-Sanz, M., Agudo, M., 2009. Brn3a as a
marker of retinal ganglion cells: qualitative and quantitative time course studies in naïve and optic nerveeinjured retinas. Invest. Ophthalmol. Vis. Sci. 50, 3860e3868. Niyadurupola, N., Luu, C.D., Nguyen, D.Q., Geddes, K., Tan, G.X.V., Wong, C.C.W., Tran, T., Coote, M.A., Crowston, J.G., 2013. Intraocular pressure lowering is associated with an increase in the photopic negative response (PhNR) amplitude in glaucoma and ocular hypertensive eyes. Invest. Ophthalmol. Vis. Sci. 54, 1913e1919. Sun, D., Qu, J., Jakobs, T.C., 2013. Reversible reactivity by optic nerve astrocytes. Glia 61, 1218e1235. Ventura, L.M., Porciatti, V., 2005. Restoration of retinal ganglion cell function in early glaucoma after intraocular pressure reduction: a pilot study. Ophthalmology 112, 20e27. Zhi, Z., Cepurna, W.O., Johnson, E.C., Morrison, J.C., Wang, R.K., 2012. Impact of intraocular pressure on changes of blood flow in the retina, choroid, and optic nerve head in rats investigated by optical microangiography. Biomed. Opt. Express 3, 2220e2233.
