AXONAL TRANSPORT IN EXPERIMENTAL GLAUCOMA Background Synopsis and Aims Glaucoma is a progressive, intraocular pressure-sensitive optic neuropathy with a poorly understood pathogenesis and limited treatment options [1]. It affects 3% of the Australian population over 49, with the prevalence increasing exponentially thereafter [2], and is the leading cause of irreversible blindness worldwide [3]. A better understanding of the pathogenesis of glaucoma will lead to novel therapeutic targets and a reduction in the disease burden at the individual, population and economic levels. The applicant has recently completed a comprehensive study in which he formed a coherent picture of the spatio-temporal pattern of injury in a validated, experimentally-induced, rat model of glaucoma [4]. He established that the earliest indication of damage was disruption to orthograde fast axonal transport within axons in the optic nerve head (ONH). Axonal cytoskeletal abnormalities were observed in the ONH a short time later, with a spatial pattern that overlapped with axonal transport disruption. Wallerian-like degeneration of injured axons then ensued. Somato-dendritic injury to the retinal ganglion cells (RGCs) occurred subsequent to axonal injury during experimental glaucoma. The objective of the current study is to shed light on the relationship between axonal transport disruption and optic nerve (ON) degeneration. The aims are as follows: (1) to delineate whether short duration axonal transport disruption is reversible; (2) to ascertain whether a continuous period of elevated IOP-induced axonal transport disruption results in greater damage than two discrete periods of elevated IOP; (3) to determine whether axonal transport disruption is associated with hypoxia/ischemia. Background Glaucoma refers to a family of ocular diseases with multifactorial etiology united by a clinically characteristic optic neuropathy. To date, treatment options for glaucoma remain limited to lowering intraocular pressure (IOP), the highest profile risk factor for the disease [5]. Although this strategy mitigates progression and prevents blinding disease in many patients [6], glaucoma is the leading cause of irreversible blindness worldwide. Pathologically, glaucoma is characterized by a loss of all RGC compartments: somata, axons and dendrites; clinically, loss of axons at the ONH heralds the diagnosis of glaucoma. This observation, together with other converging clinical evidence, has given rise to a longstanding belief that the primary site of injury is at the ONH [7], a viewpoint that is not universally accepted [8]. To date, the pathogenesis of glaucoma remains poorly understood, with relatively little known about the molecular pathways involved in the loss of RGCs and their axons. Essentially, two theories have been proposed: “the mechanical theory” and “the vascular theory” [9]. The former hypothesis contends that elevated IOP deforms the lamina cribrosa, leading to kinking and distortion of axon bundles, which disrupts orthograde and retrograde axoplasmic transport [10-12]. The latter hypothesis proposes that the ONH undergoes chronic hypoxic or ischemic injury as a result of compromised local blood flow, resulting from either increased IOP or other risk factors that lead to vascular dysregulation. The vascular hypothesis is given credibility by the considerable evidence for blood flow incompetence at the ONH in glaucoma patients [13,14], but direct evidence is lacking. Animal models of glaucoma: To facilitate a greater understanding of glaucoma, a number of rodent paradigms have been developed. These can broadly be divided into models in which elevated IOP is induced experimentally [15] and models, such as the DBA/2J strain, where IOP elevation occurs

spontaneously [16]. One key advantage of experimentally-induced glaucoma models is that the timing of the IOP increase following the surgical intervention is known. This engenders greater confidence in conclusions drawn about the chronology of pathological events. The best-characterised, most widelyaccepted and frequently-used rodent model of glaucoma was developed by Levkovitch et al [17], in which chronically elevated IOP is achieved in rats by translimbal laser photocoagulation to the trabecular meshwork. In recent years, we have gained much expertise with this paradigm [4,18,19]. Axonal transport in glaucoma: Anterograde fast axonal transport is responsible for movement of newly synthesized molecules essential for the maintenance of axonal structure and metabolism away from the cell body. Obstruction of this process rapidly compromises the integrity of the distal axon. In glaucoma, the lamina cribrosa of the ONH has for many years been considered a likely site of axonal transport failure. This hypothesis was formed after pioneering work performed in monkeys, which demonstrated that radioactive leucine accumulated within axons at the ONH during periods of moderately elevated IOP. Similar results have also been found in pigs. However, the majority of glaucoma research nowadays is carried out in rodents, and, unlike monkeys and pigs, rodents do not have a true lamina cribrosa. As such, it has been important to ascertain whether the ONH is an important site of axonal transport failure in rodents. Data obtained from the DBA/2J mouse are confusing: Howell et al [20] provided robust evidence for an insult at the lamina of the ONH, while Crish et al [8] ascertained that axonal transport dysfunction appeared first at the superior colliculus with a distal-proximal progression. Using the laser model of glaucoma, we have recently demonstrated early axonal transport disruption throughout the ONH, but not distal to this location in the myelinated ON or optic tract [4]. The time course of axonal transport failure correlated well with the early studies performed in monkeys, with detectable accumulation by 8 hours and widespread dysfunction from 24 hours. These new findings demonstrate that the ONH is the pivotal site of RGC injury following moderate elevation of IOP. However, fundamental issues remain to be resolved; for example, is axonal transport disruption a reversible process? And, if so, is there a time-window after which axon damage is irreversible? What is the effect of a repeated elevation in IOP? i.e., do axons that survive a defined period of chronic ocular hypertension necessarily continue to survive if the IOP is raised, for the same duration, a second time? Is axonal transport disruption in the ONH simply a consequence of mechanical stress placed on axons? or is there a vascular component? It can be argued that since fast axonal transport is an active process, accumulation of transported proteins within the ONH may result from energy failure owing to local hypoxia/ischemia. Detailed Research Plan Experiment 1: To investigate whether axonal transport disruption is reversible Rationale: Our recent work demonstrates that axonal transport disruption in the ONH is maximal by 1d after elevation of IOP (see Figure 1), remains widespread throughout the first week, before decreasing thereafter. In contrast, axonal degeneration is minimal at 1d and increases gradually from 3d onwards [4]. At present, it is unknown whether axonal transport disruption is reversible, and, if so, what duration of axonal transport perturbation can be tolerated by axons before irreversible injury ensues.

Figure 1: Accumulation of APP and CTB in the rat ONH (arrows) after 1d of elevated IOP. A high magnification image showing double labelling of CTB and synaptophysin is also shown. Protocol: Using the experimental glaucoma model, we will elevate the IOP in 6 groups of rats (n=8). 2 of these groups will be subjected to 1d of ocular hypertension, 2 groups to 3d of ocular hypertension, and 2 groups to 7d of ocular hypertension. At each time point (1d, 3d, 7d), one of the groups will be killed, while the other group will be subjected to pharmacological lowering of the IOP to physiological levels before being killed 24h later. All rats will be injected intravitreally with the axonal tracer AlexaFluor 594-cholera toxin β-subunit (CTB) 2d before killing. Globes will be dissected, embedded in the same orientation, sections taken through the ONH, and immunolabelled for the fast axonal transport markers APP and synaptophysin using AlexaFluor 488. Axons that remain viable at each time point will resume normal transport after lowering of IOP and display minimal disruption to axonal transport in the ONH compared with the group that did not undergo lowering of IOP. Measured Outcomes: Quantification of CTB, APP and synaptophysin abundances in ONH sections [4]. Experiment 2:

To ascertain the effect of a second period of elevated IOP

Rationale: We, and others, have shown that elevating the IOP for a period of 1 week leads to the degeneration of a proportion of RGC axons. Necessarily, the surviving RGCs are resistant to 1 week of ocular hypertension and require a longer period of elevated IOP to degenerate. We are interested in learning whether subjecting rats to a second week-long period of elevated IOP, which is preceded by an interval of normal IOP, leads to additional axonal transport disruption and cytoskeletal degeneration, or, whether all RGCs that can tolerate one defined period of ocular hypertension can similarly tolerate a second period of the same duration. This is an important issue as sporadic patient non-adherence to glaucoma medication regimes is considered a major risk factor for disease progression, yet, knowledge of the effect of intermittent, as opposed to a continuous, ocular hypertension, is negligible. Protocol: Using the experimental glaucoma model, we will elevate the IOP in 4 groups of rats for a period of 1 week. Groups 1-3 will then be allowed to recover at normal IOPs for the next 4 weeks, after which the IOP will then be re-elevated by a 2nd laser treatment. Measured Outcomes: Group 1 will be killed at 1d after the 2nd laser treatment and ONH sections analysed for axonal transport disruption [4]. Group 2 will be killed at 7d after the 2nd laser treatment and ON sections quantitatively analysed for non-phosphorylated neurofilament heavy (np-NFH), which is a sensitive marker of ongoing axonal degeneration [4]. Group 3 will be killed at 5 weeks after the 2nd laser treatment and ON counts performed in toluidine blue-stained resin sections [18]. Group 4 will be re-lasered at the end of the 1st week, killed at the same time point as group 3 and ON counts performed. Comparison of groups 3 and 4 will reveal whether greater damage occurs in rats that have 2 weeks of continuously Figure 2: Experimental design = laser application • = time of death

raised IOP compared with rats that have two discrete weeks of raised IOP (see schematic below for overview). Group 1 Group 2 Group 3 Group 4 1w

2w

3w

4w

5w

6w

7w

8w

9w

10w

Experiment 3: To determine whether axonal transport disruption is associated with hypoxia/ischemia Rationale: The vascular hypothesis of glaucoma, which proposes that the ONH undergoes chronic hypoxic or ischemic injury as a result of compromised local blood flow, is supported by a considerable amount of indirect clinical evidence [13,14]. Yet, direct evidence is lacking. We aim to identify whether glial cells in the ONH express markers indicative of hypoxia/ischemia during periods of ongoing axonal transport disruption following induction of experimental glaucoma. Protocol: Using the experimental glaucoma model, we will elevate the IOP in 4 groups of rats to be killed at 8h, 1d, 3d and 7d time points (n=8). Measured Outcomes: Hypoxyprobe-1, which allows visualisation of hypoxic cells in tissue sections, will be employed to identify any hypoxia in the ONH. Double labelling will be performed with other markers of interest, such as GFAP and iba1, which are specific to astrocytes and microglia respectively, to determine which cell types are affected. Immunohistochemical markers of oxidative stress will also be employed, including haemoxygenase-1 (HO-1); hypoxia inducible factor 1α (HIF-1α); superoxide dismutase (SOD)-1 and -2; peroxiredoxins (Prxs); 3-nitrotyrosine and 4-hydroxynonenal, indicators of protein nitration and lipid peroxidation. In addition to immunohistochemical means of detecting hypoxia/ischemia, we will utilise the rat Oxidative Stress and Antioxidant Defense PCR SuperArray (Qiagen), which allows the analysis of 84 genes related to oxidative stress by conventional real-time qPCR. Two groups of rats (n=8) will be subjected to either 24h or 7d of chronic ocular hypertension. Measured Outcomes: ONHs from controls, 24h and 7d samples will be dissected and processed for use in the real-time PCR SuperArray. Methods Experimental model of glaucoma: We have extensive experience of the rat model of glaucoma developed by Levkovitch et al [17]. A 532nm diode laser is used to deliver laser energy to the trabecular meshwork of the rat eye in order to prevent outflow of aqueous humor and, hence, cause a gradual elevation in IOP. The treatment is administered unilaterally, produces a typical increase of 25 mmHg, which is normally maintained for one week, and can be repeated on a second occasion when the IOP of the treated eye has decreased. IOPs are measured in both eyes using a rebound tonometer.

Pharmacological lowering of the IOP for 24h is achieved by intraperitoneal injection of acetazolamide at 8 hourly intervals together with a single topical application of Brinzolamide/timolol. Immunohistochemistry: Immunohistochemistry is performed according to the established protocols used routinely by CIA . In brief, tissue sections are deparaffinised, endogenous peroxidase activity blocked and antigen retrieval undertaken. Tissue sections are blocked in 3% normal horse serum, incubated overnight in primary antibody, followed by consecutive incubations with biotinylated secondary antibody and streptavidin-peroxidase conjugate. Colour development is achieved using 3’,3’-diaminobenzidine. Specificity of staining is verified by incubating adjacent sections with the correct isotype controls for monoclonal antibodies, or normal rabbit/goat serum for polyclonal antibodies. For fluorescent double labelling, one antigen is visualised using a 3-step procedure (primary antibody, biotinylated secondary antibody, streptavidin-conjugated AlexaFluor 594), while the second antigen is labelled by a 2-step procedure (primary antibody, secondary antibody conjugated to AlexaFluor 488). Hypoxyprobe-1 labelling: The Hypoxyprobe-1 Plus Kit (Millipore) will be employed as previously used in the rat retina and ON [21,22]. This kit uses pimonidazole hydrochloride as a specific marker for detecting hypoxic cells. This reagent forms covalent adducts with cells that have an oxygen partial pressure of less than 10 mmHg. Prior to killing rats, hypoxyprobe-1 will be administered into the tail vein of anaesthetised rats using an infusion pump according to kit instructions. Rats are then killed by perfuse fixation, fixed overnight and processed for routine paraffin embedding. PCR analyses: Retinas will be dissected and the RNA species extracted. A standard real-time RT-PCR methodology, used routinely by the applicant, will be employed to determine the levels of mRNAs. In brief, first strand cDNA is synthesised from DNase-treated RNA. PCR reactions are carried out in 96well optical reaction plates using the cDNA equivalent of 20ng total RNA for each sample with 1×SYBR Green PCR master mix, forward and reverse primers. For the Oxidative Stress and Antioxidant Defense PCR Array, each ONH is removed from the posterior pole of the globe using a trephine. The dissected ONH is cut to 1 mm in length, a segment that included the optic disc, all the unmyelinated ONH, and the initial 0.5 mm of the myelinated optic nerve. The ONH is frozen on dry ice and stored at -80°C. Each frozen nerve head is sonicated in TriReagent, the RNA isolated and the resultant RNA purified using QiagenTM RNeasy Mini columns. cDNA is then prepared from the RNA sample using the supplied RT2 First Strand Kit. Feasibility: Ethics approval is in place for many of the outlined studies. The chief investigator has a wealth of experience with the rat model of glaucoma, of real-time PCR and immunohistochemistry, particularly with regard to the optimisation and validation of antibodies. He routinely visualises axonal transport disruption and assesses the integrity of the axonal cytoskeleton. Acquisition of biologicallymeaningful immunohistochemistry data requires validated antibodies and an optimized methodology. We have not, to date, visualised oxidative stress immunohistochemically. Thus, we are currently optimising such antibodies using tissues from ischemic rat brain (see Figure 3).

Figure 3: Optimisation of immunohistochemical markers of hypoxia/ischemia in the brain after middle cerebral artery occlusion. Upregulation of HO-1, HIF-1α and Prx6 in astrocytes and SOD-2 in neurons. The technique of lowering of the IOP pharmacologically using acetazolamide together with topical brinzolamide/timolol has not been published previously. We have recently obtained preliminary data from 8 rats. At 1d after induction of experimental glaucoma the mean IOP was 38.4±7.1. At 1 hour after administration of IOP lowering drugs, the mean IOP was 24.9±8.0. By 2 hours, the mean IOP was 20.6±4.4. These data attest to the feasibility of the proposed study. Analysis of Data and Power calculation: For all quantitative data, if parametric assumptions are met a Student’s t-test or an ANOVA and Tukey HSD test is performed. Based on previous experience of analysing mRNA levels, axonal transport disruption, ON counts and axonal cytoskeleton abnormalities, the effect size [(largest mean-smallest mean)/SD] of the observed variable is generally 3-4; hence with 2-4 groups for an ANOVA analysis with alpha conservatively set at 0.01 to obtain a power of 90%, the ‘n’ per group is no more than 8 (using Stata 10.0 fpower command). Outcomes and Significance: The proposed series of experiments have profound significance in terms of understanding glaucoma pathogenesis and clinical application. Preliminary data indicate a high probability of success. All aspects of the work will produce data suitable for publication in leading journals and the data will prove valuable to clinicians as well as scientists. In aims 1 and 2, we investigate the relationship between raised IOP and axonal viability. These results will have immediate clinical implication in terms of IOP stability. In aim 3, we delineate whether hypoxic/ischemic events occur at the ONH during chronic ocular hypertension. These data will have important implications as regards the credibility of the vascular hypothesis of glaucoma and will highlight whether there is an urgent need for therapies to address this issue.

References 1. Chidlow G, Wood JP, Casson RJ. Pharmacological neuroprotection for glaucoma. Drugs 2007; 67:725-59. 2. Mitchell P, Smith W, Attebo K, Healey PR. Prevalence of open-angle glaucoma in Australia. The Blue Mountains Eye Study. Ophthalmology 1996; 103:1661-9. 3. Quigley HA. Number of people with glaucoma worldwide. Br J Ophthalmol 1996; 80:38993. 4. Chidlow G, Ebneter A, Wood JP, Casson RJ. The optic nerve head is the site of axonal transport disruption, axonal cytoskeleton damage and putative axonal regeneration failure in a rat model of glaucoma. Acta Neuropathol 2011; In Press. 5. Kass MA, Heuer DK, Higginbotham EJ, Johnson CA, Keltner JL, Miller JP, Parrish RK, 2nd, Wilson MR, Gordon MO. The Ocular Hypertension Treatment Study: a randomized trial determines that topical ocular hypotensive medication delays or prevents the onset of primary open-angle glaucoma. Arch Ophthalmol 2002; 120:701-13. 6. Heijl A, Leske MC, Bengtsson B, Hyman L, Hussein M. Reduction of intraocular pressure and glaucoma progression: results from the Early Manifest Glaucoma Trial. Arch Ophthalmol 2002; 120:1268-79. 7. Quigley HA, Addicks EM, Green WR, Maumenee AE. Optic nerve damage in human glaucoma. II. The site of injury and susceptibility to damage. Arch Ophthalmol 1981; 99:635-49. 8. Crish SD, Sappington RM, Inman DM, Horner PJ, Calkins DJ. Distal axonopathy with structural persistence in glaucomatous neurodegeneration. Proc Natl Acad Sci USA 2010; 107:5196-201. 9. Fechtner RD, Weinreb RN. Mechanisms of optic nerve damage in primary open angle glaucoma. Surv Ophthalmol 1994; 39:23-42. 10. Quigley HA. Neuronal death in glaucoma. Prog Retin Eye Res 1999; 18:39-57. 11. Crawford Downs J, Roberts MD, Sigal IA. Glaucomatous cupping of the lamina cribrosa: A review of the evidence for active progressive remodeling as a mechanism. Exp Eye Res 2011. 12. Burgoyne CF, Downs JC, Bellezza AJ, Suh JK, Hart RT. The optic nerve head as a biomechanical structure: a new paradigm for understanding the role of IOP-related stress and strain in the pathophysiology of glaucomatous optic nerve head damage. Prog Retin Eye Res 2005; 24:39-73. 13. Schmidl D, Garhofer G, Schmetterer L. The complex interaction between ocular perfusion pressure and ocular blood flow - Relevance for glaucoma. Exp Eye Res 2011; In Press. 14. Yanagi M, Kawasaki R, Wang JJ, Wong TY, Crowston J, Kiuchi Y. Vascular risk factors in glaucoma: a review. Clin Experiment Ophthalmol 2011; In Press. 15. Morrison JC, Johnson EC, Cepurna W, Jia L. Understanding mechanisms of pressureinduced optic nerve damage. Prog Retin Eye Res 2005; 24:217-40. 16. John SW. Mechanistic insights into glaucoma provided by experimental genetics the cogan lecture. Invest Ophthalmol Vis Sci 2005; 46:2649-61. 17. Levkovitch-Verbin H, Quigley HA, Martin KR, Valenta D, Baumrind LA, Pease ME. Translimbal laser photocoagulation to the trabecular meshwork as a model of glaucoma in rats. Invest Ophthalmol Vis Sci 2002; 43:402-10.

18. Ebneter A, Chidlow G, Wood JP, Casson RJ. Short-term Hyperglycemia Protects Retinal Ganglion Cells and the Optic Nerve in Experimental Glaucoma. Arch Ophthalmol 2011; In Press. 19. Ebneter A, Casson RJ, Wood JP, Chidlow G (2010) Microglial Activation in the Visual Pathway in Experimental Glaucoma: Spatio-Temporal Characterisation and Correlation with Axonal Injury. Invest Ophthalmol Vis Sci. 51:426-37. 20. Howell GR, Libby RT, Jakobs TC, Smith RS, Phalan FC, Barter JW, Barbay JM, Marchant JK, Mahesh N, Porciatti V, Whitmore AV, Masland RH, John SW. Axons of retinal ganglion cells are insulted in the optic nerve early in DBA/2J glaucoma. J Cell Biol 2007; 179:152337. 21. Danylkova NO, Pomeranz HD, Alcala SR, McLoon LK. Histological and morphometric evaluation of transient retinal and optic nerve ischemia in rat. Brain Res 2006; 1096:209. 22. Holcombe DJ, Lengefeld N, Gole GA, Barnett NL. The effects of acute intraocular pressure elevation on rat retinal glutamate transport. Acta Ophthalmol 2008; 86:408-14.