The mechanism of axonal degeneration after perikaryal excitotoxic injury to the retina

Experimental Neurology 236 (2012) 34–45

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The mechanism of axonal degeneration after perikaryal excitotoxic injury to the retina Natalie D. Bull a, 1, Glyn Chidlow d, e, 1, John P.M. Wood d, e, Keith R. Martin a, b, c, Robert J. Casson d, e,⁎ a

Cambridge Centre for Brain Repair, University of Cambridge, Cambridge, UK Department of Ophthalmology, University of Cambridge, Cambridge, UK Cambridge NIHR Biomedical Research Centre, University of Cambridge, Cambridge, UK d Ophthalmic Research Laboratories, South Australian Institute of Ophthalmology, Hanson Institute Centre for Neurological Diseases, Adelaide, Australia e Department of Ophthalmology and Visual Sciences, University of Adelaide, Frome Road, Adelaide, SA 5000, Australia b c

a r t i c l e

i n f o

Article history: Received 21 December 2011 Revised 17 March 2012 Accepted 29 March 2012 Available online 5 April 2012 Keywords: Excitotoxicity Wallerian Neurodegeneration

a b s t r a c t We investigated the mechanism of secondary axonal degeneration after perikaryal excitotoxic injury to retinal ganglion cells (RGCs) by comparing pathological responses in wild-type rats and Wld(s) rats, which display delayed Wallerian degeneration. After perikaryal excitotoxic RGC injury, both types of rats exhibited a spatio-temporal pattern of axonal cytoskeletal degeneration consistent with Wallerian degeneration, which was delayed by up to 4 weeks in Wld(s) rats. Furthermore, RGC somal loss was greater in Wld(s) rats. Microglial response in the anterior visual pathway to injury was attenuated in the Wld(s) rats with lymphocytic infiltration that was relatively reduced; however, immunostaining for major histocompatibility complex class II antigens (OX6) was more pronounced in Wld(s) rats. These data indicate that perikaryal excitotoxic RGC injury causes a secondary Wallerian axonal degeneration, and support the notion of a labile, soma-derived axonal survival factor. © 2012 Elsevier Inc. All rights reserved.

Introduction A large body of evidence supports the notion that excitotoxicity plays a role in the pathogenesis of a number of neurological diseases, including central nervous system (CNS) ischemia, Alzheimer's disease, motor neuron disease, and possibly glaucoma (Casson, 2006; Doble, 1999; Hynd et al., 2004). Although the site of excitotoxic injury is principally at the level of the cell body (perikaryal) understanding the secondary effects on the neuronal axon is important because axonopathy is a documented early feature of these common neurological conditions (Fischer et al., 2004; Stokin et al., 2005). Delineation of axonal damage secondary to perikaryal excitotoxicity is, however, complicated by the fact that white matter oligodendrocytes are also highly vulnerable to glutamate-induced excitotoxicity (Matute et al., 2007). The retina and optic nerve, as approachable regions of the CNS, provide a unique anatomical substrate to investigate the effect of secondary axonal degeneration after perikaryal excitotoxic injury since the affected neuronal cell bodies (retinal ganglion cells, RGCs) which reside within the eye, are physically separated from the

⁎ Corresponding author at: Ophthalmic Research Laboratories, South Australian Institute of Ophthalmology, Hanson Institute Centre for Neurological Diseases, Frome Road, Adelaide, SA 5000, Australia. E-mail address: [email protected] (R.J. Casson). 1 These authors contributed equally to the study. 0014-4886/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2012.03.021

myelinated portion of their axons, which lie outside the eye; hence axonal degeneration is necessarily a consequence of primary somatic injury. While a substantial body of work has been performed on the mechanisms involved in excitotoxicity-induced retinal damage, remarkably little is known about the pathology of secondary axonal degeneration following such an insult. Our recent morphological studies have begun to explore this subject. Using routine light microscopy and transmission electron microscopy, we have demonstrated that excitotoxic perikaryal injury causes optic nerve damage with pathological features that resemble, to some extent, those described in classical Wallerian degeneration (Saggu et al., 2008, 2010). Notwithstanding this new information, much remains to be established; for example, the timing of injury to unmyelinated RGC axons within the retina, the involvement of glial cells, and the commonality of the overall response compared with the classical Wallerian degeneration. In contrast to the paucity of knowledge concerning optic nerve degeneration following perikaryal excitotoxicity, a wealth of literature exists regarding the mechanisms of axonal degeneration following optic nerve transection, which is a classical CNS model of Wallerian degeneration. The discovery of the spontaneous mouse mutant strain Wld s, whose transected axons survive for weeks independent of their cell body, (Perry et al., 1991) has aided this process, providing an unprecedented opportunity to unravel the molecular underpinnings of Wallerian degeneration. In exciting recent research, Coleman (2005), Gilley and Coleman (2010) and Mack et al. (2001) proposed

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a “survival factor delivery hypothesis” of axonal degeneration in which axon integrity requires continuous anterograde delivery of a labile, cell body-derived survival factor. Using superior cervical ganglia explant cultures, they convincingly demonstrated that nicotinamide mononucleotide adenylyltransferase 2 (Nmnat2) uniquely fitted the profile of that axonal survival factor and that the Wld s protein is a long half-life form of Nmnat2. The hypothesis that failure to transport a cell body-derived survival factor to axons results in Wallerian degeneration has profound implications for many neurological diseases in which axonal transport is disrupted. The major aims of the current study were to increase our understanding of the nature of axonal degeneration following perikaryal excitotoxicity in general, and the influence of the Wld s gene in particular. We hypothesized that if the survival factor delivery hypothesis is universally valid, then, following excitotoxic RGC injury: (1) despite rapid somal death, the entire length of the RGC axon, including the unmyelinated portion within the eye, should remain morphologically unchanged for the latency phase associated with Wallerian degeneration, then degenerate synchronously, and (2) optic nerve degeneration would be delayed in the Wld s rat. Such data would not only provide support for the axonal survival factor hypothesis, but also offer robust evidence for the molecular mechanism underlying axonal degeneration after perikaryal excitotoxic injury. Materials and methods Animals Adult wild-type Sprague–Dawley (SD) and adult homozygous Wld s transgenic rats, in which the Wld s cDNA transgene is driven by the β-actin promoter, (Adalbert et al., 2005, 2006) were used for this study. Rats were housed in light- and temperature-controlled conditions with constant access to food and water and maintained on a 12-hour light/dark cycle. All experimental animals were used in accordance with the UK Home Office regulations for the care and use of laboratory animals, the UK Animals (Scientific Procedures) Act (1986) and the Association for Research in Vision and Ophthalmology's Statement for the Use of Animals in Ophthalmic and Visual Research. Excitotoxic injury Rats were lightly anesthetized using isoflurane inhalation in a 2:1 O2/NO2 mix and local anesthetic was applied topically to the cornea of the left eye. The eyelids were gently retracted and 5 μl of 8 mM NMDA (40 nmol dose; Sigma-Aldrich, Gillingham, UK) dissolved in sterile Hanks' Balanced Salt Solution (Invitrogen, Paisley, UK) was injected into the vitreous of the left eye using a 30G needle attached to a 5 μl glass syringe. The injection was delivered through the superior nasal retina and the needle was left in position for about 1 min, before being withdrawn, to minimize leakage. Care was taken to ensure that the lens was not damaged and that the retinal blood supply was not affected. For characterization of the effect of NMDA on the spatio-temporal pattern of RGC degeneration, SD rats were killed at the following time points after NMDA administration: 6 h (n = 9), 1 day (n = 6), 3 days (n = 6), 7 days (n = 6). Of the 9 rats taken at 6 h, 3 were processed for TUNEL labeling, with the retinas from the remaining 6 dissected for RT-PCR analysis. Regarding the other time points, the retina, proximal ON, and proximal optic tract were taken from each rat for immunohistochemical evaluation. For assessment of the effect of Wld s on the time course of RGC degeneration after NMDA treatment, 32 SD and 32 Wld s rats were randomly assigned to one of three groups, which were killed at 1 week (SD n = 10; Wld s n = 10), 2 weeks (SD n = 10; Wld s n = 10)

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and 4 weeks (SD n = 12; Wld s n = 12) after intravitreal injection of NMDA. Tissue processing and histology Animals were perfused under terminal anesthesia with 0.1 M phosphate buffered saline (PBS) and, for those rats where tissue was not taken for RT-PCR, subsequently with 4% paraformaldehyde/ 0.1 M PBS (PFA). For immunohistochemical comparison of RGC survival in SD and Wld s retinas, tissues were post-fixed by immersion in 4% PFA at 4 °C overnight. Posterior eyecups were cryopreserved with 30% sucrose and embedded in optimal cutting temperature (OCT; Raymond A. Lamb UK, Eastbourne, UK) compound for frozen sectioning. For immunohistochemistry performed on retinal flat mounts, posterior eyecups were post-fixed by immersion in 4% PFA for 1 h. Retinas from both eyes were then dissected and prepared as flattened whole mounts by making four radial cuts. For immunohistochemistry performed on ON and optic tract sections, tissues were post-fixed by immersion in 4% PFA at 4 °C overnight. ONs and chiasma were then processed for routine paraffin-embedded sections, embedded longitudinally and 4 μm sections were cut. For assessment of RGC axonal loss, small segments of the ON 2–3 mm behind the globe (labeled ‘proximal’) and 2–3 mm adjacent to the optic chiasma (labeled ‘distal’) were immersed in 4% PFA/5% glutaraldehyde/0.1 M phosphate buffer for 7 days at 4 °C, post-fixed in 1% osmium tetroxide for 3 h, dehydrated and embedded in araldite resin for semi-thin sectioning. Semi-thin (1 μm) transverse sections were cut on an ultramicrotome, dried onto slides and stained with 1% toluidine blue. RGC soma immunohistochemistry and quantification Transverse retinal sections (14 am thick) were cut through the posterior globe at the level of the optic nerve head and collected onto treated microscope slides (Superfrost Plus; VWR International Ltd, Lutterworth, UK). At least 3 sections through the optic nerve head were collected per eye. Sections were initially washed with PBS and then blocked with PBS containing 0.2% triton (PBS-T) plus 5% normal goat serum (Invitrogen). All antibodies were diluted in this blocking solution. Sections were incubated in mouse anti-neuronal nuclei (NeuN) primary antibody overnight at room temperature. After thorough washing with PBS, an Alexa Fluor 555-conjugated goat antimouse secondary antibody (Invitrogen) was applied to the sections for 3 h at room temperature. Slides were washed, counterstained with DAPI (Invitrogen) and coverslipped with FluorSave reagent (Calbiochem/Merck Chemicals, Beeston, UK). Contiguous images along the full length of 3 transverse retinal sections cut through the optic nerve head of each eye were captured using a 10 × objective on an epifluorescent microscope (DM6000B, Leica Inc, Wetzlar, Germany). The number of NeuN-positive cells in the ganglion cell layer (GCL) along the full length of each section was counted manually and the corresponding retinal length measured. The number of NeuN-positive cells in the GCL was calculated per mm retinal length for each section, and the mean value of 3 sections per eye was calculated. The number of surviving NeuNpositive cells in the GCL in Wld s rats was compared to that in the SD control rats, at each time point, using an unpaired, two-tailed Student's t-test with p b 0.05 considered statistically significant. Optic nerve axonal quantification The number of surviving RGC axons in the ON was assessed using our previously published method (Bull et al., 2009; Johnson et al., 2010). Briefly, semi-thin ON sections were observed under light microscopy (100× magnification) and zones of approximately equal damage were identified. The total ON area was measured and the contribution of each zone to total ON area was determined. A

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representative photograph was captured at 630× magnification within each zone of homogeneous damage and the number of axons within each sample image was counted using the particle analysis/nucleus counter plug-in (from the Wright Cell Imaging Facility ImageJ plug-in bundle, University Health Network Research, Canada; http://www. uhnresearch.ca/facilities/wcif/fdownload.html) to the image analysis software ImageJ (National Institutes of Health, USA; http://rsb.info. nih.gov/ij/index.html). The number of axons within each zone of homogeneous damage was compared to the count obtained from a sample image of the uninjured companion eye to estimate the percentage of axonal survival. A weighted average calculation was then used to estimate the percentage of surviving axons in the total ON. We have previously demonstrated that this semi-quantitative method of ON axonal quantification correlates well with total ON axon counts (Johnson et al., 2010). Axonal survival between different groups was compared at each time point using a one-way ANOVA with Bonferroni's multiple comparison, where p b 0.05 was considered statistically significant.

Optic nerve immunohistochemistry For colorimetric immunohistochemistry, tissue sections were deparaffinized in xylene and rinsed in 100% ethanol, before treatment with 0.5% H2O2 for 30 min to block endogenous peroxidase activity. Antigen retrieval was achieved by microwaving the sections in 10 mM citrate buffer (pH 6.0). Tissue sections were then blocked in phosphate buffered saline containing 3% normal horse serum, incubated overnight at room temperature in primary antibody (containing 3% normal horse serum) followed by consecutive incubations with biotinylated secondary antibody (Vector, Burlingame, CA) and streptavidin–peroxidase conjugate (Pierce, Rockford, IL). Color development was achieved with 3″–3″diaminobenzidine. Sections were counterstained with hematoxylin, dehydrated and mounted. Specificity of antibody staining was confirmed by incubating adjacent sections with isotype controls for monoclonal antibodies, or normal rabbit serum for polyclonal rabbit antibodies.

Fig. 1. Localization of NMDA receptors in the rat retina and the excitotoxic effect of NMDA (A). NMDA receptors are localized to neurons in the GCL and INL (arrows) of the rat retina, as shown by immunohistochemistry for the NMDA receptor subunit, NR1 (B, C). Intravitreal injection of NMDA induced rapid death of RGC somas, as delineated by a marked reduction in the level of the RGC-specific mRNA, Thy, at 6 h after administration and by the presence of numerous TUNEL positive nuclei in the GCL and INL of the retina at the same time point. The amount of Thy1 mRNA in control (n = 6) and NMDA-treated (n = 6) retinas was determined by quantitative real-time RT-PCR. Values are expressed as mean ± SEM. An unpaired, two-tailed Student's t-test was used to compare control and NMDA groups, *p b 0.05. GCL, ganglion cell layer; INL, inner nuclear layer; RGC, retinal ganglion cell. Scale bar: 15 μm.

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Quantification of optic nerve immunohistochemistry Early axonal injury in Wld s and wild-type SD rats was quantitatively assessed by immunolabeling for SMI-32, an antibody that recognizes the non-phosphorylated chain of neurofilament heavy. SMI-32 has been consistently demonstrated to be a highly sensitive, marker of axonal cytoskeleton disruption, (Chidlow et al., 2011; Meller et al., 1994) owing to the labile nature of nonphosphorylated neurofilament heavy chain and its susceptibility to degradation by calpain. Microglial status in Wld s and wild-type SD rats was quantitatively assessed by immunolabeling for Iba1, ED1 and OX-6. Iba1 is considered to be a specific, universal marker of microglia that rapidly responds to disruption of tissue homeostasis (Ebneter et al., 2010; Ito et al., 2001). ED1 recognizes a single chain glycoprotein that is expressed predominantly on the lysosomal membrane of myeloid cells. The amount of ED1 expression can be correlated to the extent of phagocytic activity. OX-6 recognizes the major histocompatibility complex (MHC) class II RT1B chain of antigen presenting cells, which is present at low or undetectable levels within the microglia under normal physiological conditions. Quantification of immunolabeling for each marker in longitudinal sections of the intracranial ON was performed as previously described (Chidlow et al., 2011; Ebneter et al., 2010). In brief, immunostained sections, each expressing a representative level of immunoreactivity, were photographed at 200× magnification. They were then imported into NIH Image-J 1.42q software where, if counterstained, they

Fig. 3. Spatio-temporal analysis of SMI-32 immunolabeling of RGC axons after optic nerve crush. At 1 day after optic nerve crush, SMI-32 abnormalities were clearly identifiable at the site of lesion (a), but not within the distal portion of the optic nerve (b), which was uniformly, weakly labeled by SMI-32. At 2 days after optic nerve crush, RGC axons and cell bodies within the retina displayed no obvious abnormalities (c), but in the distal portion of the optic nerve (d) numerous swollen, beaded SMI-32-labeled axons were apparent. Scale bar: 30 μm. ON, optic nerve; RGC, retinal ganglion cell.

Fig. 2. Spatio-temporal analysis of SMI-32 immunolabeling of RGC axons after excitotoxic retinal injury. In control retinas, RGC axons and large RGC somas were strongly SMI-32immunopositive, while in the optic tract, RGC axons were only weakly labeled. At 1 day after NMDA administration, identical patterns of labeling were observed. By 2 days, swelling and beading of RGC axons were evident both within the retina and distally in the optic tract. After 7 days, fewer RGC axons remained, and SMI-32 abnormalities were still visible within the retina and optic tract. Scale bar: 30 μm. RGC, retinal ganglion cell; perip, peripheral; cent, central; OT, optic tract.

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underwent color deconvolution to separate diaminobenzidine reaction product from hematoxylin counterstain (Ruifrok and Johnston, 2001). Images were subsequently analyzed with regard to the specifically stained area in pixels using the in-built functions of the Image-J software. Statistical analysis was carried out using a one-way ANOVA with Bonferroni's multiple comparison, where p b 0.05 was considered statistically significant. Antibodies The following commercially available antibodies were used in the study: mouse anti-NeuN (1:500; clone A60, Millipore, Watford, UK), rabbit anti-NMDAR1 (1:500; AB9864, Millipore), mouse anti-NF-L (1:3000; clone NR4; Sigma, St Louis, MO), mouse anti-β3-tubulin (1:1000; clone TU-20; Chemicon, Temecula, CA), mouse anti-SMI-32 (1:10,000; SMI-32, Covance, Princeton, NJ), mouse anti-ED1 (1:500; MCA341; Serotec, Kidlington, Oxford, UK), mouse anti-MHC class II (1:500, OX-6, Serotec), rabbit anti-iba1 (1:50,000; 019-19741, Wako, Osaka, Japan), anti-rabbit CD3 (1:3000, AO452, Dako, Sydney, Australia).

after NMDA administration, (Fig. 1C) and by a synchronous reduction in the mRNA level of the RGC-specific marker, Thy1 (Fig. 1B). To investigate the spatio-temporal pattern of RGC axonal injury, whole mount retinas and sections from the ON and optic tract were immunolabeled for SMI-32 (Fig. 2; see Materials and methods). In normal retinas, SMI-32 delineated a subset of larger RGC somas and robustly labeled all RGC axons, while RGC axons were only lightly labeled by SMI-32 in the myelinated ON and optic tract, reflecting a much lower fraction of non-phosphorylated neurofilaments within the myelinated portion of the nerve (Fig. 2). Despite the rapid, terminal injury to the RGC soma after NMDA, no disruption to the axonal cytoskeleton of RGCs was detectable at the 6 h (data not shown) or 1 day time points. By 2 days after NMDA treatment, however, RGC axons displayed synchronous swelling and beading throughout their entire length, as highlighted proximally within the nerve fiber layer of the retina, and also within the optic tract. At 7 days after NMDA

RT-PCR Reverse-transcription polymerization chain reaction (RT-PCR) studies were carried out as described previously (Chidlow et al., 2008). In brief, retinas were dissected, total RNA was isolated and first strand cDNA was synthesized from 2 μg DNase-treated RNA. Real-time PCR reactions were carried out in 96-well optical reaction plates using the cDNA equivalent of 20 ng total RNA for each sample in a total volume of 25 μl containing 1 × SYBR Green PCR master mix (BioRad), forward and reverse primers at a final concentration of 400 nM. The thermal cycling conditions were 95 °C for 3 min and 40 cycles of amplification comprising 95 °C for 12 s, 63 °C for 30 s and 72 °C for 30 s. Primer sets used were as follows (sense primer, antisense primer, product size, accession number): GAPDH (5′-TGCACCACCAACTGCTTAGC-3′, 5′-GGCATGGACTGTGGTCATGAG-3′, 87 bp, NM_017008), Thy1.1 (5′-CAAGCTCCAATAAAACTATCAATGTG-3′, 5′GGAAGTGTTTTGAACCAGCAG-3′, 83 bp, X03150). Terminal deoxynucleotidyl transferase-mediated, dUTP nick end labeling (TUNEL) assay TUNEL assays were performed as described previously (Chidlow et al., 2009). In brief, sections from 6 h NMDA-injected retinas were treated with proteinase K. Sections were then equilibrated in reaction buffer, prior to incubation in the same buffer containing TdT (0.15 U/μl) and biotin-16-dUTP (10 μM) for 60 min at 37 °C. The reaction was terminated by two washes in saline sodium citrate solution. Nonspecific binding sites were blocked by washing in 2% bovine serum albumin. Sections were then incubated with Alexa Fluor 594-linked streptavidin–peroxidase conjugate (1:1000) for 30 min at 37 °C. Results Relationship between RGC somal and axonal degeneration after perikaryal excitotoxic injury In the rat retina, NMDA receptors are reportedly expressed exclusively by RGCs and a subset of amacrine cells (Brandstatter et al., 1994). In support of this, immunohistochemical labeling of an antibody directed against the NMDA receptor subunit, NR1, revealed a localization pattern specific to neuron cell bodies in the GCL and inner nuclear layer (Fig. 1A). Intravitreal injection of NMDA induced the rapid death of RGC somas, as evidenced by the appearance of numerous TUNEL-positive cells within the inner retina as early as 6 h

Fig. 4. Quantification of RGC soma survival after excitotoxic retinal injury. (a, b) Example photomicrographs of transverse sections of control and injured retinas immunolabeled for NeuN (arrows). (c) The number of surviving RGCs was quantified by counting the number of NeuN-positive cells per millimeter (mm) of the GCL in transverse retinal sections through the optic nerve head in uninjured control tissue (SD n = 6, Wlds n = 6), and at 1 week (SD n = 6, Wlds n = 6), 2 weeks (SD n = 6, Wlds n = 5) and 4 weeks (SD n = 7, Wlds n = 7) after intravitreal injection of NMDA. Values are expressed as mean ± SEM. An unpaired, two-tailed Student's t-test with Bonferonni's correction for multiple comparisons was used to compare SD and Wlds groups, **p b 0.01 and *p b 0.05. RGC, retinal ganglion cell; GCL, ganglion cell layer.

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treatment, SMI-32 immunolabeling demonstrated substantial damage to, and loss of, RGC axons throughout the optic pathway. In summary, NMDA-induced excitotoxicity causes prompt death of RGC somas, but no axonal degeneration is evident until after 2 days, at which point damage is visible distal to the injury throughout the anterior visual pathway, including the intraocular axons. We then compared the spatio-temporal pattern of axonal injury after perikaryal excitotoxic injury to that after intraorbital ON crush. At 1 day after the crush, SMI-32 abnormalities were evident at the site of lesion only (Fig. 3a). Apart from the crush site, no abnormal SMI-32 immunostaining was evident in any portion of the anterior visual pathway, including the retinal nerve fibers, the optic nerve (Fig. 3b) and the optic tract. However, by 2 days after the crush, the entire length of the ON and optic tract distal to the lesion site showed axonal swellings and beading analogous to those observed at the same time point after NMDA (Fig. 3d). Unlike following intravitreal injection of NMDA, intraocular axons displayed no SMI-32 abnormalities (Fig. 3c), and no TUNEL-positive somas were detectable (data not shown) in the GCL, 2 days after optic nerve crush. In summary, ON crush causes prompt injury at the site of lesion, but no axonal degeneration is evident elsewhere within RGC axons until after 2 days, at which point damage is visible only on the distal side of the lesion site. Wld s exacerbates RGC soma death after perikaryal excitotoxic injury The number of surviving RGCs in excitotoxic-injured retinas was quantified at each time point using immunohistochemical labeling of the common quantifiable RGC marker NeuN (Beirowski et al., 2008; Buckingham et al., 2008; Ebneter et al., 2011). Quantification

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of the number of NeuN-positive cells in the GCL of uninjured retinas revealed no difference in the number of RGCs in Wld s retinas (41.6 ± 2.2 cells/mm GCL, mean ± SEM), compared to wild-type tissue (40.1 ± 1.6 cells/mm GCL, mean ± SEM), prior to injury (Fig. 4). At 1 week after intravitreal injection of NMDA, approximately 65% of wild-type (14.2 ± 1.2 cells/mm GCL, mean ± SEM) and 60% of Wld s (11.9 ± 1.5 cells/mm GCL, mean ± SEM). RGCs had died, compared to uninjured control retinas. Interestingly, at 2 weeks postlesion significantly fewer RGCs remained in the Wld s retina (7.8 ± 0.8 cells/mm GCL, mean ± SEM) compared to wild-type retina (11.9 ± 0.9 cells/mm GCL, mean ± SEM). This difference in RGC survival was also maintained at 4 weeks post-excitotoxic retinal lesion, with significantly fewer cells in Wld s retina (6.1 ± 1.3 cells/mm GCL, mean ± SEM) compared to wild-type retina (12.1 ± 1.3 cells/mm GCL, mean ± SEM). Wld s delays RGC axonal degeneration after perikaryal excitotoxic injury Quantification of surviving axons in the ON of eyes subjected to an intravitreal NMDA excitotoxic insult demonstrated significant preservation of RGC axons, compared to wild-type controls (Fig. 5), but degeneration was only delayed not prevented. The number of axons was counted in ON segments proximal and distal to the globe, and the percentage of surviving axons calculated relative to the uninjured contralateral nerve. The percentage of surviving axons was approximately 3-fold greater in both the distal (Wld s 82.6 ± 7.8% vs SD 29.9 ± 1.9%, mean ± SEM) and proximal (Wld s 84.7 ± 3.5% vs SD 28.8 ± 1.9%, mean ± SEM) regions of Wld s ONs, compared to wild-type controls, at 1 week after intravitreal NMDA injection. At 2 weeks post-NMDA injection, axonal survival in Wld s ONs was 1.7

Fig. 5. Quantification of axonal survival in the optic nerve after excitotoxic retinal injury. (a, b) Example photomicrographs of transverse sections of control and injured optic nerves stained with toluidine blue. The number of axons was quantified in segments of optic nerve proximal (c) and distal (d) to the globe at 1 week (SD n = 6, Wlds n = 6), 2 weeks (SD n = 6, Wlds n = 5) and 4 weeks (SD n = 7, Wlds n = 8) after unilateral, intravitreal injection of NMDA in wild-type (SD) and Wlds rats. Percentage of optic nerve axon survival was calculated compared to the uninjured contralateral nerve. Values are expressed as mean ± SEM. A one-way ANOVA followed by a post-hoc Student's t-test with Bonferonni's correction for multiple comparisons was used to compare all groups, **p b 0.01 and *p b 0.05. ON, optic nerve.

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fold greater in the distal nerve (Wld s 66.4 ± 8.9% vs SD 38.1 ± 7.4%, mean ± SEM), and 1.5 fold greater in the proximal nerve (Wld s 61.3 ± 5.5% vs SD 42.1 ± 6.4%, mean ± SEM), compared to wild-type nerves. By 4 weeks post-lesion, the Wld s axonal neuroprotective effect was lost and there was no significant difference in axonal survival in these nerves compared to controls (Distal, Wld s 20.9 ± 5.5% vs SD 28.3 ± 2.4%; Proximal, Wld s 21.2 ± 3.6% vs SD 29.8 ± 2.2%; mean ± SEM). Statistical analysis also revealed that there was no difference in the number of surviving axons in lesioned wild-type (SD) ONs at any of the time points examined, confirming that this excitotoxic insult triggered rapid cell death after intravitreal NMDA injection in wild-type nerves, but did not lead to chronic neurodegeneration.

The toluidine blue methodology is considered the gold standard tool for assessment of gross axonal loss; however, it is less suited in demarcating the onset of axonal cytoskeletal injury. To characterize the nature of axonal degeneration further in Wld s animals compared to wild type controls, intracranial sections of the ON were immunolabeled for SMI-32, neurofilament light (NFL) and β3-tubulin. At the earliest time point analyzed, 1 week post-NMDA injection, the number of SMI-32 abnormalities in ON sections from wild-type controls was over 4-fold greater than in ONs of Wld s animals (Wld s 62 ± 12 vs SD 255 ± 42, mean ± SEM; Fig. 6a, b). These data support the conclusion that the majority of axons in Wld s ONs are morphologically undamaged at this time point (Fig. 5). The differential in axonal

Fig. 6. Analysis of axonal cytoskeleton damage in the optic nerve after excitotoxic retinal injury (a). Representative images of SMI-32 immunolabeling in control optic nerves, and injured optic nerves of wild-type (SD) and Wlds rats at 1 week after NMDA treatment (b). The number of SMI-32 abnormalities in segments of the intracranial optic nerve was quantified at 1 week (SD n = 10, Wlds n = 10) after unilateral, intravitreal injection of NMDA in SD and Wlds rats. Values are expressed as mean ± SEM. An unpaired, two-tailed Student's t-test was used to compare SD and Wlds groups, ***p b 0.001 (c). Representative images of NFL and β3-tubulin immunolabeling in control optic nerves, and injured optic nerves of SD and Wlds rats at 1 week after NMDA treatment. Scale bar: 15 μm. ON, optic nerve.

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preservation between wild-type and Wld s rats assessed by SMI-32 was greater than that measured by axon counting, suggesting that a proportion of axons in the wild-type rats had begun degenerating but remained sufficiently intact for quantification. Interestingly, visualization of NFL and β3-tubulin in both wild-type and Wld s ONs indicated that the former underwent more rapid degradation than the latter (Fig. 6c). Although, these data are only qualitative in nature there was a consistent trend of greater preservation of β3-tubulin than NFL, which was apparent both in wild-type ONs with substantial damage and Wld s ONs with minor damage. Microglial responses in wild-type and Wld s rats after perikaryal excitotoxic injury The role of microglia in optic neuropathies, such as glaucoma, is currently a topic of considerable interest. Published studies are supportive of contrasting hypotheses: (1) microglial activation occurs in response to RGC degeneration; (2) microglial activation is evident prior to overt RGC neurodegeneration, perhaps even triggering degeneration or accelerating its progression. The design of the current study provides an opportunity to shed light on this subject owing to the delay between the loss of the somal and axonal compartments of the RGC after perikaryal excitotoxic injury, and the protracted period during which affected axons degenerate in the Wld s mutant. To delineate the timing of microglial activation we immunolabeled retina and ON for expression of Iba1 (see Materials and methods). In control retinas, a relatively sparse population of ramified Iba1-positive microglia was observed, predominantly within the inner plexiform layer (Fig. 7a). At 1 day after NMDA treatment, Iba1-positive microglia were more numerous, particularly in the GCL, and showed morphological features characteristic of activation, including retraction of processes and cell body hypertrophy (Fig. 7a). A similar response was encountered at 2 days post-NMDA treatment (Fig. 7a). In control ON tissue, Iba1 was expressed by a population of quiescent microglia, characterized by small cell bodies and

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delicate processes (Fig. 7b). In contrast to the retina, there was no evidence of an altered Iba1 profile in the ON at 1 day post-NMDA (Fig. 7b). By 2 days post-NMDA treatment, however, Iba1-positive microglia frequently displayed an activated morphology, although they were not noticeably more numerous (Fig. 7b). Three markers of microglial activation were investigated in wildtype and Wld s rats: Iba1, ED1 and OX6. In control intracranial ON, Iba1 labeled a population of quiescent microglia (Fig. 8a), as described above (Fig. 7b). At 1 week post-NMDA, Iba-positive microglia in intracranial wild-type ON segments were more numerous and frequently displayed hypertrophy of the cell body (Fig. 8b). A similar phenomenon was observed in Wld s ONs, but the Iba1 response was less pronounced than in SD rats (Fig. 8c). Quantification of Iba1 expression at 1 week post-injury revealed a 3.4 fold greater increase in the Iba1 content of wild-type ONs (treated SD 7.9 ± 0.4 vs control SD 3.7 ± 0.3; mean ± SEM) compared to Wld s nerves (treated Wld s 4.8 ± 0.1 vs control Wld s 3.6 ± 0.3; mean ± SEM; Fig. 8g). At 2 weeks post-NMDA treatment, Iba1 content in wild-type ONs (treated SD 8.0 ± 1.4) was similar to that measured at 1 week. This value was 1.9 fold higher than in Wld s nerves (treated Wld s 5.9 ± 0.3), but was not found to be significantly different after statistical analysis. By 4 weeks post-lesion, Iba1 expression was markedly higher than at 2 weeks however wild-type and Wld s ONs exhibited similar expression patterns (SD 13.5 ± 1.1 vs Wld s 12.9 ± 2.5, mean ± SEM). Negligible ED1 immunolabeling was detectable in uninjured intracranial ON tissue (Fig. 8d, h). At 1 week post-NMDA injection, numerous microglia expressing ED1-positive granules were visible in ON sections from SD rats (Fig. 8e), but fewer ED1-positive microglia were observed in Wld s ONs (Fig. 8f). Quantification of ED1 expression at 1 week revealed 3.0 fold greater expression in wild-type ONs compared to Wld s nerves (SD 1.5 ± 0.2 vs Wld s 0.5 ± 0.1; mean ± SEM; Fig. 8h). At 2 weeks post-injury, ED1 content was slightly increased in both wild-type and Wld s ONs (SD 1.8 ± 0.1 vs Wld s 1.0 ± 0.2; mean ± SEM) from 1 week, with ED1 expression 1.8 fold greater in SD ONs than in Wld s nerves, a difference that was statistically

Fig. 7. Analysis of microglial activation in RGCs after excitotoxic retinal injury (a). Representative images of Iba1 expression in control retinas, and injured retinas at 1 and 2 days after NMDA treatment (b). Representative images of Iba1 expression in control optic nerves, and injured optic nerves at 1 and 2 days after NMDA treatment. Scale bar: 15 μm. ON, optic nerve.

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Fig. 8. Analysis of microglial activation in wild-type (SD) and Wlds rats after excitotoxic retinal injury. (a–f) Representative images of Iba1 and ED1 (arrows) expressions in control optic nerves, and injured optic nerves of wild-type and Wlds rats at 1 week after NMDA treatment. Scale bar: 15 μm (g, h). Iba1 and ED1 expressions in segments of the intracranial optic nerve were quantified at 1 week (SD n = 10, Wlds n = 10), 2 weeks (SD n = 10, Wlds n = 9) and 4 weeks (SD n = 11, Wlds n = 11) after unilateral, intravitreal injection of NMDA in SD and Wlds rats. Values are expressed as mean ± SEM. A one-way ANOVA followed by a post-hoc Student's t-test with Bonferonni's correction for multiple comparisons was used to compare all groups, ***p b 0.001. ON, optic nerve.

significant. By 4 weeks post-lesion, wild-type and Wld s ONs featured similar amounts of ED1 (SD 5.0 ± 0.5 vs Wld s 4.6 ± 0.4, mean ± SEM), which were substantially higher than at 2 weeks. Minimal MHC class II (OX6) expression was detectable in both wild-type and Wld s control tissues (Fig. 9A, G). Expression of OX6 in NMDA-treated ONs varied markedly between individuals at each time point analyzed in both wild-type and Wld s rats, with some rats from the same group having numerous OX6-positive microglia and others none. As a consequence, data from each individual rat are presented (Fig. 9G). Despite the variability, a clear trend emerged: in the majority of wild-type rats, there was little OX6 expression at any of the time points analyzed (Fig. 9B, G). In contrast, OX6 was more abundant at every time point in Wld s rats, with a consistently high level detectable at 4 weeks (Fig. 9C, G). Overall, the difference in OX6 expression between wild-type and Wld s ONs was statistically significant. The classical function of MHC II molecules is to present antigenic fragments to CD4 + T cells; hence, we investigated whether there was a correlation between the abundance of OX6 and the number of infiltrating T cells in degenerating ONs. In control intracranial ON, very few T cells were detected using the pan-T cell marker CD3 (Fig. 9D, H). Analysis of wild-type and Wld s ONs revealed few T cells in either group at 1 or 2 weeks (Fig. 9H). Surprisingly, at 4 weeks post-lesion (Fig. 9E, F, H), a 5-fold statistically significant increase in the number of T cells in wild-type ONs was observed, which

was not apparent in Wld s nerves (SD 21.3 ± 4.9 vs Wld s 4.6 ± 0.9; mean ± SEM) despite much greater OX6 expression in Wld s rats. Discussion Mechanism of axonal degeneration after perikaryal excitotoxicity Recently it was hypothesized (Gilley and Coleman, 2010) that discontinued neuronal soma production of a labile axonal survival factor triggers Wallerian degeneration. If this thesis is correct, then it predicts Wallerian-type degeneration of the entire length of the axon after localized somal injury and death. One difficulty in testing this postulation within the CNS is the confinement of an injury to the soma alone, without any primary excitotoxic injury to oligodendrocytes and associated axonopathy (Karadottir et al., 2005). In this study, we used the retina as an approachable region of the CNS to address this question, thereby exploiting the anatomical separation of gray and white matter delimited by the eye. The spatio-temporal pattern of axonal degeneration that we observed after localized perikaryal excitotoxic injury was entirely consistent with the notion of a labile axonal “survival factor” produced by the RGC soma in order to maintain axonal integrity. In Wallerian degeneration synchronous organized destruction of the axon distal to the site of direct injury is observed after a latent phase of approximately 40 h (Beirowski et al., 2005). This is

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Fig. 9. Analysis of microglial MHC class II expression and abundance of T cells in wild-type (SD) and Wlds rats after excitotoxic retinal injury. (A–F) Representative images of OX6 (MHC class II) and CD3 (T cells) immunoreactivity in control optic nerves, and injured optic nerves (arrows) of wild-type (SD) and Wlds rats at 4 weeks after NMDA treatment. Scale bar: 15 μm (G, H). OX6 immunoreactivity and T cell numbers in segments of the intracranial optic nerve were quantified at 1 week (SD n = 10, Wlds n = 10), 2 weeks (SD n = 10, Wlds n = 9) and 4 weeks (SD n = 11, Wlds n = 11) after unilateral, intravitreal injection of NMDA in SD and Wlds rats. For OX6, data from individual rats are shown; for CD3, values are expressed as mean ± SEM. A one-way ANOVA followed by a post-hoc Student's t-test with Bonferonni's correction for multiple comparisons was used to compare all groups, **p b 0.01. ON, optic nerve.

classically observed after axonal transection or crush. An early feature of this process is the formation of spheroids and swellings along the axon, (Beirowski et al., 2010; George et al., 1995; Zhai et al., 2003) followed by granular disintegration of the cytoskeleton. We have previously observed these features after retinal excitotoxic injury, (Saggu et al., 2008, 2010) and they were again noted in the current study. In our ON crush model, axonal degeneration distal to the site of crush had commenced by 48 h. At this time point RGC somas were viable, as evidenced by the absence of TUNEL-positive nuclei, and their axons within the retina, i.e. proximal to the lesion site, had a morphologically normal appearance. In our perikaryal excitotoxicity model, we similarly detected Wallerian-type degeneration of the RGC axons in the distal optic nerve by 48 h after the insult. Unlike ON crush, however, RGC axons within the retina also displayed degenerative features at this time point. Hence, the pathological behavior of the excitotoxic injury was as if the axonal transection had occurred at the most proximal portion of the axon, that is, immediately adjacent to the soma. If the molecular mechanism of the axonal degeneration associated with perikaryal excitotoxic injury is Wallerian in nature, then we would expect to see a delay in axonal degeneration in Wld s rats, as has been shown after ON transection/crush (Wang et al., 2006) and in experimental models of glaucoma (Beirowski et al., 2008; Howell et al., 2007). Indeed, this is exactly what we observed in the current study. The time-course of the axonal degeneration was significantly

delayed in the Wld s rats compared to the wild-type rats. However, by 4 weeks post-injury this protective effect was no longer evident, confirming the Wld s mutation only delayed degeneration but did not prevent it. This result supports the conclusion that the mutant Wld s protein is a long half-life variant of the endogenous Nmnat2 survival factor and expression of this transgene underlies the Wld s axo-protective effect, as suggested by Gilley and Coleman (2010) previously. An unexpected finding in the current study was increased loss of RGCs in the retina of Wld s rats after localized somatic injury. This pattern was evident at both the two-week and four-week time points after excitotoxic injury. The delayed axonal degeneration in the peripheral nervous system of the Wld s mouse has been associated with a reduced regenerative capacity in sensory and motor neurons (Brown et al., 1992; Chen and Bisby, 1993); but, to our knowledge, a detrimental effect on the neuronal soma has not previously been reported. The reason for this increased somal loss is unclear. The response of microglia to perikaryal excitotoxic injury Microglia are the resident immune cells of the CNS. They are rapidly activated by even minor homeostatic imbalance and have been shown to respond in numerous ways (Hanisch and Kettenmann, 2007). Two issues that have prompted much controversy are whether activated microglia play a beneficial role after injury or promote

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neurodegeneration in pathological situations, (Walter and Neumann, 2009) and whether microglial activation is evident prior to overt neuronal degeneration. In the visual system, there exists a close spatial relationship between microglia and RGCs, and microglia are ideally positioned to respond to RGC stress. Recent evidence from a chronic mouse model of glaucoma argues that microglial activation represents the earliest reported pathological alteration (Bosco et al., 2011). Hitherto, no information was available on the response of resident microglia in the ON either during secondary axonal degeneration, after perikaryal excitotoxicity, or in the Wld s mutant. First, we sought to determine whether RGC somal injury and death, and the resultant cessation of axonal transport, could trigger microglial activation prior to the appearance of axonal cytoskeletal abnormalities, i.e. during the latency phase of Wallerian degeneration. The results indicated not. Microglial activation within the retina occurred concurrent with somal injury, but no microglial activation was evident in the ON until the appearance of axonal cytoskeletal abnormalities evidenced by neurofilament beading and swelling. Second, we investigated the microglial response in the Wld s mutant in order to ascertain whether: (1) the degree of activation was simply proportional to the extent of axonal degeneration at the time points analyzed, or (2) activation occurred in advance of axonal degeneration, during the period when the somas had degenerated but the Wld s protein was protecting axonal architecture. Here, again, the pattern of the microglial response precisely mirrored the degree of axonal injury in each animal: at 1 week post-lesion there was 3-fold greater preservation of axon number in Wld s rats compared to controls, alongside 3-fold less Iba1 and ED1 expression, while at 2 weeks after injury these figures had diminished to 1.9 greater axonal survival and 1.8 fold less expression of microglial markers. By 4 weeks, microglial activation was markedly elevated in the ONs of both wild-type and Wld s rats, a finding consistent with the major role microglia play in the lengthy process of myelin clearance. The logical conclusion to draw from these data is that the delayed microglial activation in the Wld s rats is simply a consequence of the delayed axonal degeneration. It is known that the Wld s mutant protein is intrinsic to neurons and acts to stabilize the structure of the axon. Thus, while the Wld s axons remain intact, the microglia are quiescent. At the point when the Wld s protein is turned over (and not replaced owing to the death of the soma), the axon begins to degenerate and the microglia become activated. The overall results are similar to those previously observed in an acute rat model of glaucomatous optic neuropathy, (Ebneter et al., 2010) and support the view that microglia respond to axonal damage in a rapid and graded manner in order to facilitate the orderly phagocytosis of dying axonal tissue. The results offer no support to the theory that microglial activation occurs in advance of, or exacerbates, axonal degeneration. A caveat to this conclusion would be that more sensitive markers might offer a different perspective. There was, however, one curious feature to the microglial response in Wld s animals compared to controls: while MHC class II immunolabeling was more pronounced in the Wld s rat tissue, the number of infiltrating T cells was actually lower than that observed in wild-type controls. Since expression of MHC molecules by microglia is considered critical for T cell interaction, (van den Hoorn et al., 2011) the reverse might be expected. The explanation for this finding remains unclear, but may reflect changes in the immune response in Wld s animals. Conceivably, any such changes may influence axonal degradation, but seem unlikely to be responsible for the protective effect observed. Conclusion We have provided evidence that the mechanism of axonal degeneration after perikaryal excitotoxic injury is Wallerian in nature, a finding consistent with the notion that Nmnat2 is a neuronal soma-

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