Secondary degeneration of the optic nerve following partial transection: The benefits of lomerizine

Experimental Neurology 216 (2009) 219–230

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Experimental Neurology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / yex n r

Secondary degeneration of the optic nerve following partial transection: The benefits of lomerizine Melinda Fitzgerald a,b,⁎, Carole A. Bartlett a,b, Lauren Evill a,b, Jenny Rodger a,b, Alan R. Harvey a,c, Sarah A. Dunlop a,b a b c

Experimental and Regenerative Neurosciences, University of Western Australia, Crawley, 6009, WA, Australia School of Animal Biology and Western Australian Institute of Medical Research, University of Western Australia, Crawley, 6009, WA, Australia School of Anatomy and Human Biology, University of Western Australia, Crawley, 6009, WA, Australia

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Article history: Received 12 September 2008 Revised 24 November 2008 Accepted 30 November 2008 Available online 11 December 2008 Keywords: Neurotrauma Secondary injury Visual System Retinal ganglion cell Neuronal death Glia Chondroitin sulphate proteoglycans Oxidative stress

a b s t r a c t Secondary degeneration is a form of ‘bystander’ damage that can affect neural tissue both nearby and remote from an initial injury. Partial optic nerve transection is an excellent model in which to unequivocally differentiate events occurring during secondary degeneration from those resulting from primary CNS injury. We analysed the primary injury site within the optic nerve (ON) and intact areas vulnerable to secondary degeneration. Areas affected by the primary injury showed morphological disruption, loss of β-III tubulin axonal staining, reduced myelinated axon density, greater proteoglycan expression (phosphacan), increased microglia and macrophage numbers and increased oxidative stress. Similar, but less extreme, changes were seen in areas of the optic nerve undergoing secondary degeneration. The CNS-specific L- and T-type calcium channel blocker lomerizine alleviated some of the changes in areas vulnerable to secondary degeneration. Lomerizine reduced morphological disruption, oxidative stress and phosphacan expression, and limited early increases in macrophage numbers. However, lomerizine failed to prevent progressive de-myelination of ON axons. Within the retina, secondary retinal ganglion cell (RGC) death was significant in areas vulnerable to secondary degeneration. Lomerizine protected RGCs from secondary death at 4 weeks but did not fully restore behavioural function (optokinetic nystagmus). We conclude that blockade of calcium channels is neuroprotective and limits secondary degenerative changes following CNS injury. However such an approach may need to be combined with other treatments to ensure long-term maintenance of full visual function. © 2008 Elsevier Inc. All rights reserved.

Introduction Traumatic injury to the CNS results in rapid death of neurons in the injury site and delayed death of most axotomised cells (Bradbury and McMahon, 2006). In addition to cells directly affected by the primary injury, neurons and glia outside the injury are also vulnerable. Despite being physically intact, these cells may undergo delayed death due to secondary metabolic events involving mechanisms such as glutamateinduced excitotoxicity leading to calcium overload and oxidative stress, free radical formation, mitochondrial dysfunction and energy failure, enzymatic degradation, membrane instability and resultant inflammation (Khodorov, 2004; Shacka and Roth, 2005; Tezel, 2006; Farkas and Povlishock, 2007). Such secondary degeneration is a form of ‘bystander’ damage that can affect both nearby and remote tissues; the resulting loss of neurons, myelin and function has been

⁎ Corresponding author. Experimental and Regenerative Neurosciences, School of Animal Biology, University of Western Australia, Crawley, 6009, WA, Australia. Fax: +61 8 6488 7527. E-mail address: melfi[email protected] (M. Fitzgerald). 0014-4886/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2008.11.026

documented after both brain and spinal cord injuries (Crowe et al., 1997; Farooque et al., 1999; Buss et al., 2005). Rescuing intact, but vulnerable, tissue from secondary degeneration is now recognised as critical to improving long term function after traumatic CNS injury (Crowe et al., 1997; Buss et al., 2005). In the eye, similar approaches may also be important in the treatment of degenerative diseases such as glaucoma (Whitmore et al., 2005; Nickells, 2007). The optic nerve (ON) is emerging as an excellent CNS model in which to differentiate damage arising as a result of primary (direct) injury, for example crush or transection, from secondary degeneration that develops after partial injury to intact tissue (Levkovitch-Verbin et al., 2001; Levkovitch-Verbin et al., 2003; Blair et al., 2005). Responses to ON injury have been well characterised in a range of models (Kreutz et al., 1999; Schwartz, 2004; Harvey et al., 2006). However, partial ON crush models, in which the injury is distributed diffusely across the ON, do not allow spatial separation of retinal ganglion cell (RGC) axons that are axotomized by the primary injury from intact axons that may subsequently be vulnerable to secondary degeneration. By contrast, partial transection of the dorsal ON allows spatial separation of RGCs axotomised in the primary injury from initially uninjured RGCs in

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ventral regions of the ON and retina. While oxidative amplification, extracellular matrix changes and excessive microglial and macrophage activation have been associated with primary degeneration (Nguyen et al., 2003; Ahn et al., 2006; Lobsiger and Cleveland, 2007; Donnelly and Popovich, 2008), such damage is yet to be unequivocally demonstrated in tissue untouched by the primary injury but vulnerable to secondary degeneration. A number of calcium channel blockers have been tested in various models of neurotrauma (Winkler et al., 2003; Coleman, 2005; Vergouwen et al., 2006). Lomerizine dihydrochloride (KB-2796) is a relatively new L- and T-type calcium channel blocker used in the treatment of migraine (Akaike et al., 1993; Toriu et al., 2000). Its effects are localised predominantly to the CNS thereby avoiding detrimental side effects due to lowered systemic blood pressure (Hara et al., 1999; Toriu et al., 2000). Lomerizine reduces plasma extravasation (Hashimoto et al., 1997) and may be effective in preventing secondary RGC death after diffuse axonal injury (Karim et al., 2006). Using the partial ON transection model, we show here that lomerizine protects RGCs in ventral retina which, due to their topographic location, are unaffected by the initial injury but which are nevertheless vulnerable to secondary degeneration. We also demonstrate that while lomerizine alleviates gross morphological changes, excessive macrophage infiltration, oxidative stress and expression of aggregates of the chondroitin sulphate proteoglycan (CSPG) isomer phosphacan in ON regions undergoing secondary degeneration, it fails to prevent the loss of myelinated axons and did not fully restore behavioural function. Materials and methods Animals Female PVG Hooded rats (160–180 g) were bred at the Animal Resources Centre (Murdoch, WA), housed under a standard 12 hour light/dark cycle and fed standard rat chow and water ad libitum. Procedures conformed to “Principles of laboratory animal care” (NIH publication No. 86–23, revised 1985) and were approved by the University of Western Australia's Animal Ethics Committee. Rats were anaesthetized with xylazine (llium xylazil, Troy Laboratories, i.p. 10 mg/kg) in combination with ketamine (Ketamil, Troy Laboratories, 50 mg/Kg) and euthanased with Euthal (Pentobarbitone sodium 850 mg/kg; Phenytoin sodium 125 mg/kg; i.p.). Seven groups of animals were used, n = 7–12 for test groups and n = 4–6 for normal control and sham operated groups. Partial optic nerve transection The procedure was similar to that described in previous studies (Levkovitch-Verbin et al., 2001; Levkovitch-Verbin et al., 2003; Blair et al., 2005). Briefly, the skin overlying the skull was incised along the midline, retracted and the ON accessed by deflecting lachrymal tissue immediately behind the eye. The nerve parenchyma was exposed about 1 mm behind the eye by making a slit in the dura mater with ophthalmic scissors. A controlled 200 µm cut (approximately 1/4 of ON width) was made in the dorsal nerve using a diamond radial keratotomy knife; the depth was determined by the protrusion of the blade beyond a surrounding guard. Care was taken not to stretch the ON or to damage major ophthalmic blood vessels. The sheath was closed, the skin sutured (4/0 Silkam) and animals recovered on a warming blanket. Animals were treated with antibiotic and analgesic subcutaneously (Neomycin, 10 mg/kg in sterile PBS; Norocarp, 2.8 mg/ kg) and a topical antibiotic to the skin wound (Tricin, Jurox, Australia). Completely normal animals were used as controls as well as sham operated control groups in which all procedures were identical to those for the animals undergoing partial ON cut, except for the final 200 µm cut (i.e. dura was opened).

Lomerizine treatment Rats were treated orally twice daily with 30 mg/kg lomerizine dihydrochloride (LKT Labs) (Karim et al., 2006), administered in butter using a small spatula tip ensuring full delivery of the dosage with minimal stress. Electrospray-liquid chromatography-mass spectrometry analyses confirmed the stability of lomerizine dihydrochloride in this medium. Treatment commenced on the day of partial ON cut, immediately following recovery from surgery. Placebo treated animal groups (all with partial ON cut) as well as normal control and sham operated groups, received butter alone. Animals were treated for 7 or 28 days, until perfusion. Tissue preparation and analyses of optic nerve sections Following transcardial perfusion with 0.9% saline followed by 4% paraformaldehyde, ONs were dissected and divided into distal and proximal segments. Immunohistochemistry The distal segment encompassed the nerve from 0.5 mm to 2.5 mm distal to the injury and was post fixed in 4% paraformaldehyde overnight, cryoprotected by immersion in 15% sucrose in PBS overnight, embedded in OCT (Tissue-Tek), and cryosectioned transversely (16 µm) before mounting on Superfrost Plus microscope slides. Sections at 0.5–1.5 mm distal to the injury site (i.e. towards the superior colliculus, SC) were incubated overnight at 4 °C in primary antibodies detecting: β-III tubulin (TUJ1; 1:500; Covance, NJ); reactive resident microglia/macrophages (Ibal, 1:1000, Wako) (Kanazawa et al., 2002); monocytes/macrophages (ED-1, 1:500, Serotec) (Cui et al., 2007); astrocyte GFAP (GA5, 1:500, Sigma); nestin (1:1000, Sapphire); manganese superoxide dismutase (MnSOD) (1:500, Stressgen); phosphacan (mAb 3F8, 1:500, DSHB) or oligodendrocytes (olig2, 1:250, Chemicon) (Gao et al., 2006). Antibody binding was visualised following a 2 hour incubation with anti-rabbit (Alexa Fluor 488, 1:400 Molecular Probes) or anti-mouse (Alexa Fluor 546, 1:400, Molecular Probes) secondary antibodies. Slides were coverslipped using Fluromount-G (Southern Biotech) and viewed using fluorescence microscopy. The processes of degenerating neurons were visualised by histochemical staining with fluorojade C according to the manufacturer's instructions (Chemicon). Control sections stained only with secondary antibodies were included in all experiments and showed no fluorescence (data not shown). The areas of disruption of β-III tubulin positive axonal profiles were quantified using Image J analysis software and expressed as a percentage of total ON cross-sectional area. Values were statistically analysed using ANOVA and Bonferroni/Dunn post hoc tests, requiring a significance value of p ≤ 0.05. Numbers of: Iba1 positive reactive resident microglia/macrophages; ED-1 positive macrophages; olig2 positive oligodendrocytes; MnSOD and phosphacan aggregates in dorsal and ventral halves of each section were counted and statistically analysed using ANOVA and Bonferroni/Dunn post hoc tests, requiring a significance value of p ≤ 0.05. In experiments describing the longitudinal spread of secondary degeneration, the entire ON from the ON head through the injury site to the optic chiasm was post fixed and cryoprotected as described earlier. The ON was sectioned transversely or longitudinally and sections were stained immunohistochemically for β-III tubulin to assess the loss of staining along this part of the visual pathway. Assessment of the injury site and the extent of myelination The proximal segment encompassed the ON head through the injury site to 0.5 mm distal to the injury and was post-fixed in 1% osmium (ProScitech) for 90 min with shaking, and processed using a Lynx tissue processor into Araldite Procure mixture (ProScitech).

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Embedded nerves were cured for 24 h and serially sectioned (1 µm) in three sets of three sections at 50 µm intervals to ensure identification of the lesion site. The resultant semi-thin sections were deplasticised with saturated NaOH in ethanol and stained for 15 to 30 s at 95 °C in aqueous 5% toluidine blue in 1% borax (Scott Scientific). Low power photographs of entire sections were taken to assess the overall appearance of the lesion site. The number of myelinated axons within ventral and central (non-lesioned) regions of the ON from the three semi-thin sections around the injury site for each animal was counted (n = 3–6 animals per treatment group). The area of ON counted for each semi-thin section corresponded to approximately 4% of the total section area. The number of myelinated axons was expressed per unit area and adjusted for any variation in the total cross-sectional area of the nerve following injury. The number of myelinated axons obtained using this method was compared to results obtained by counting electron micrographs of adjacent sections and was found to vary by only 0.09 ± 5.36%. Myelinated axon densities were statistically analysed using ANOVA and Bonferroni/Dunn post hoc tests, requiring a significance value of p ≤ 0.05. Similar analyses were conducted assessing the density of blood vessels in ventral and central regions of the ON from the three semi-thin sections around the injury site (n = 3–6 animals per treatment group).

as described previously (Abdeljalil et al., 2005; Haustead et al., 2008). The rat was placed on a well illuminated round platform (9 cm diameter, 14 cm high) surrounded by a cylinder (29 cm diameter, 29.7 cm high). The walls outside the cylinder were covered by black and white grating stripes of 5 mm thickness. Rats were allowed 2 min inside the cylinder to adjust and acclimatise to their surroundings. Rats showed no signs of distress when placed inside the drum and often exhibited normal grooming behaviour. The grating (0.13 CPD) was rotated anticlockwise at a speed of 1.4 revolutions per minute for 2 min. A normal optokinetic nystagmus was observed when the rat turned its head in smooth pursuit to follow the grating at the same speed as the drum was turning, followed by a rapid re-alignment movement or re-setting phase (Delgado-Garcia, 2000; Cahill and Nathans, 2008; Hogie et al., 2008). A video camera above the rat was used to record responses, with smooth pursuit and re-setting phases analysed separately. Re-setting phases were further defined as a rapid re-alignment movement following a smooth pursuit or an interrupted, jerky tracking movement, not at the same speed as the drum (confirmed as an impaired pursuit by the subsequent re-setting movement). The numbers of responses were statistically analysed using ANOVA and Bonferroni/Dunn post hoc tests, requiring a significance value of p ≤ 0.05.

Retrograde labelling with fluorogold and quantitative analysis of RGCs

Results

The fluorescent dye fluorogold (FG, Fluorochrome, CO) is a highly sensitive retrograde marker giving robust and persistent labelling (Cui et al., 2003). The technique labels only those RGC somas with axons that are projecting and still connected to the SC. Briefly, 3 days before sacrifice, a small zone of cortex was aspirated to reveal the underlying SC. 5 µl of 5% FG dissolved in 10% DMSO was applied to the SC surface in a gelfoam pledget. After the 3 days allowed for retrograde FG transport, animals were perfused transcardially with 0.9% saline, followed by 4% paraformaldehyde in 0.1 M Phosphate buffer, pH 7.2. Eyes and ONs were dissected for histochemical and morphological analysis. Retinae were washed in PBS, mounted onto Superfrost plus slides, air dried and coverslipped with Fluoromount-G™. The number of FG positive RGCs in six fields of view (100 µm × 100 µm) of central and ventral retinae equally spaced across the nasal/temporal axis, was counted for each animal by two independent observers (n = 6–9 partially transected animals for each of the four test groups and 2–4 for the three normal control and sham operated groups). Results from the two observers differed by less than 5% and were averaged and statistically analysed using ANOVA and Bonferroni/Dunn post hoc tests, requiring a significance value of p ≤ 0.05. In parallel experiments, the number of β-III tubulin positive RGCs in sagittal sections of central retinae exposing the dorsal/ventral axis (three fields of view, 12 retinal sections/animal) was counted. RGCs were counted in central regions of central sections, from animals 28 days +/− partial ON transection (normal, n = 3; partially transected, n = 2). The optical fractionator technique was used during counting, to ensure RGCs were not counted twice in adjacent sections (Petersen et al., 2006). Analysis of sections avoided under-counting β-III tubulin positive somata which, in wholemounts, are obscured by overlying βIII tubulin positive RGC axons. The number of RGCs in retinal sections was expressed per mm2.

Lomerizine alleviated gross morphological secondary disturbances in the optic nerve

Optokinetic responses Animals were anaesthetized (n = 4–6 animals per treatment group) and the uninjured eye of each animal was held shut with gauge 6–0 silken suture. Closure of the normal eye was required to ensure that visual responses observed were due to sight via the experimental eye (partially transected ON) and was not confounded by the normal eye. When the animals were fully recovered from anaesthesia (N5 h), first line visual screening tests were conducted using an optokinetic drum

Partial ON transection resulted in considerable disruption to normal axonal morphology 7 days after injury (Figs. 1A, B). Not only was the primary injury (dorsal 200 µm segment) characterised by increased cellular infiltration, loss of myelinated axons and disruption of normal fascicular architecture, but the initially uninjured area of the ON, vulnerable to secondary degeneration (central region) was similarly disrupted (Fig. 1B). Gross ON morphology continued to deteriorate, with greater disruption in central and ventral regions 28 days following partial transection (Fig. 1C). The bulbous protrusion from the injury site contained glia, identified using transmission electron microscopy (Fig. 1D). The areas of disruption of β-III tubulin positive axonal profiles rose in the primary injury site as well as in areas vulnerable to secondary degeneration over the 28 days following partial transection (normal animals = no disruption; day 7 = 33.5 ± 1.7% disruption; day 28 = 34.4 ± 2.1% disruption, Figs. 1G–I). Similar axonal profiles were observed with panN immunohistochemistry (data not shown). Fluorojade staining of the primary injury demonstrated axon degeneration 7 days after partial transection (Fig. 1L). Axonal degeneration was observed at low levels in central, but seldom in ventral, regions of the partially transected nerve at this time (Figs. 1M, N). Fluorojade staining was also observed 28 days after partial transection, the intensity of staining reflecting the degree of secondary degeneration (data not shown). There were no differences in the numbers of blood vessels in central and ventral regions of the ON following partial transection (data not shown). Immediately after partial transection disruption of β-III tubulin positive axonal profiles was confined to dorsal ON at the injury site only (Fig. 2A). As secondary degeneration spread following partial transection, β-III tubulin positive axonal profiles were lost in central and ventral ON 0.5 mm proximal to the injury site and at least 3.0 mm distal towards the optic chiasm by 28 days after injury (Fig. 2B). Dorsal ON axons axotomised by the partial transection were lost along the length of the ON, as evidenced by the lack of β-III tubulin positive axonal profiles at day 28 (Fig. 2B). Lomerizine treatment alleviated morphological disruption and loss of β-III tubulin in areas of the ON vulnerable to secondary degeneration (Figs. 1E, F, J, K). Secondary morphological disruption

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Fig. 1. Morphological effects of secondary degeneration following partial transection +/− lomerizine treatment. Semi-thin sections stained with toluidine blue were taken from the injury site of partially transected optic nerves 7 (middle panel) or 28 (right panel) days after injury, from animals treated with lomerizine (E, F) or placebo (B, C). A transmission electron micrograph from the injury site 28 days after partial transection illustrated glial nuclei (D). Optic nerve sections distal to the injury site were immunohistochemically stained for β-III tubulin 7 (middle panel) or 28 (right panel) days after injury, from animals treated with lomerizine (J, K) or placebo (H, I). Normal un-operated animals were included as controls and were not different from shams (A, G). Scale bar = 100 µm, magnification ×10, all images are oriented with dorsal uppermost. Optic nerve sections distal to the injury site were histochemically stained with fluorojade 7 days after partial transection. Dorsal (L), central (M) and ventral (N) regions are shown at 100 × magnification, scale bar = 25 µm. Images shown are representative of results observed from 7–12 animals per treatment group.

was only observed in 2 of 9 animals treated with lomerizine for 7 days, but was observed in 6 of 7 placebo treated animals. The areas of disruption of β-III tubulin positive axonal profiles fell from

33.5 ± 1.7% to 18.9 ± 1.0% with lomerizine treatment at this time (p ≤ 0.05). Similarly, lomerizine reduced secondary morphological disruption 28 days after partial transection (p ≤ 0.05). Lomerizine

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Fig. 2. Spread of secondary degeneration in the optic nerve following partial transection. ON sections at and surrounding the injury site were immunohistochemically stained for β-III tubulin. ON sections at the injury site 5 min after partial transection (A, scale bar = 100 µm, magnification ×10) and the ON from the ON head to just before the optic chiasm 28 days after partial transection (B, scale bar = 0.5 mm, magnification ×10). Both images are oriented with dorsal uppermost. Images shown are representative of results observed from 3–5 animals.

Fig. 3. Resident microglia and macrophages following partial transection +/− lomerizine treatment. Optic nerve sections distal to the injury site were immunohistochemically stained for microglia/macrophages with Iba1 (A–E), or macrophages with ED-1 (F–J). Sections were taken 7 (middle panel) or 28 (right panel) days after injury, from animals treated with placebo (B, C, G, H) or lomerizine (D, E, I, J). Normal un-operated animals were included as controls and were not different from shams (A, F). Scale bar = 100 µm, magnification ×10, all images are oriented with dorsal uppermost. Images shown are representative of results observed from 3–12 animals per treatment group.

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also reduced the areas of disruption of β-III tubulin positive axonal profiles 28 days after partial transection, from 34.4 ± 2.1% to 19.0 ± 1.4% (p ≤ 0.05). No obvious reduction in fluorojade labelled axon degeneration was seen following lomerizine treatment. Lomerizine reduced numbers of macrophages but not resident microglia in the optic nerve Partial transection of the ON resulted in increases in Iba1 positive microglia/macrophages and ED-1 positive macrophages particularly at the primary injury site, but also in areas undergoing secondary degeneration (Figs. 3A–C, F–H). Areas undergoing secondary degeneration (central/ventral ON) were identified by comparison with adjacent β-III tubulin stained sections. Normal or sham operated ONs had few microglia and virtually no macrophages (Figs. 3A, F). Peripheral T-lymphocytes (R-73 immunostaining, Hunig et al., 1989) were not detected within the ON following partial transection.

Lomerizine significantly reduced number of microglia, but only in the primary injury (dorsal ON) 28 days after partial transection (p ≤ 0.05, Figs. 3 –E and Fig. 7). Lomerizine reduced the number of ED-1 positive macrophages in ventral ON 7 days after partial transection (p ≤ 0.0001), but by 28 days the effects were no longer significant (p N 0.05, Figs. 3G–J and Fig. 7). The increase in ED-1 positive macrophages at the injury site was also reduced by lomerizine, at 28 days (p ≤ 0.001, Fig. 7). Lomerizine reduced oxidative stress in the optic nerve Partial transection of the ON resulted in a significant increase in MnSOD expression, indicative of oxidative stress, in both the primary injury site and ventral ON 28 days after injury (p ≤ 0.001, Figs. 4A–C, Fig. 7). The strong spheroid MnSOD expression was similar in size and distribution to cellular debris observed in semi-thin sections of the primary injury (Figs. 4C, F). In addition, MnSOD co-localised with some β-III tubulin positive RGC axons (Fig. 4G), and occasional ED-1

Fig. 4. MnSOD expression indicating oxidative stress following partial transection +/− lomerizine treatment. Optic nerve sections distal to the injury site were immunohistochemically stained for MnSOD. Sections were taken 7 (middle panel) or 28 (right panel) days after injury, from animals treated with placebo (B, C) or lomerizine (D, E). Normal un-operated animals were included as controls and were not different from shams (A). Scale bar = 100 µm, magnification ×10, all images are oriented with dorsal uppermost. Images shown are representative of results observed from 6–10 animals per treatment group. Cellular debris in semi-thin sections stained with toluidine blue (example indicated with arrow) was of similar morphology and in similar locations as MnSOD spherical bodies (F). MnSOD co-localised with some β-III tubulin positive RGC axons (green, G) and occasional ED-1 positive macrophages (green, H) and Iba1 positive microglia/macrophages (green, I) Co-localisation indicated by orange colour and arrows. MnSOD did not co-localise with GA-5 positive astrocytes (green, J) or olig2 positive oligodendrocytes (green, K). Images F–K, scale bar = 50µm, magnification = × 40 .

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positive macrophages (Fig. 4H) and Iba1 positive microglia/macrophages (Fig. 4I) in areas undergoing secondary degeneration. MnSOD spherical bodies were not associated with astrocytes (Fig. 4J) or oligodendrocytes (Fig. 4K). Lomerizine significantly reduced spheroid MnSOD expression, in both the primary injury site and in ventral ON, 28 days after injury (p ≤ 0.05, Figs. 4C, E and Fig. 7). Similar studies were conducted using detection of 3-nitrotyrosine as an additional marker of oxidative stress; however there were no detectable 3-nitrotyrosine increases in areas undergoing secondary degeneration when compared to constitutive levels in uninjured nerves (not shown). Immunostaining of astrocytes by detection of GFAP revealed no significant differences in staining intensity or morphology following partial optic nerve transection. It has been suggested that co-expression of GFAP with nestin is a marker for activated astrocytes (Yoo et al., 2005). While the intensity of nestin staining that was co-localised with GFAP was slightly increased following partial transection, there were no significant decreases with lomerizine treatment (not shown). Lomerizine reduced phosphacan expression in the optic nerve Partial transection of the ON resulted in a marked increase in the numbers of aggregates of secreted phosphacan not associated with cells (as indicated by Hoescht staining), in both the primary injury and areas undergoing secondary degeneration 28 days after injury (p ≤ 0.001, Figs. 5A–C, Fig. 7). Lomerizine largely prevented this increase in phosphacan expression (p ≤ 0.005, Figs. 5C, E, Fig. 7). There were no discernible differences in expression of membrane associated phosphacan at high magnification under any conditions. Neurocan was also detected immunohistochemically in the primary injury, but was not detectable in areas of the ON undergoing secondary degeneration (not shown). Lomerizine did not prevent demyelination in the optic nerve While partial transection of the ON resulted in the disruption of olig2 immunostaining of oligodendrocytes in the primary injury

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site, there were no statistically significant differences in the number of oligodendrocytes in either dorsal or ventral ON following partial ON transection, with or without lomerizine treatment (Figs. 6A–E, Fig. 7). The density of myelinated axons in areas undergoing secondary degeneration fell significantly 28 days after partial transection when compared to both normal and sham operated animals (p ≤ 0.05, Fig. 6F). Differences in ON cross-sectional area were not statistically significant for any of the treatment groups (p N 0.05). Lomerizine treatment did not significantly increase the density of myelinated axons (p N 0.05, Fig. 6F). Lomerizine protected RGCs from secondary death The initial partial injury to the dorsal 200 µm of the ON results in a corresponding primary loss of retrogradely labelled RGC soma in the dorsal retina of Wistar rats, due to the topographic arrangement of axons in the ON (Levkovitch-Verbin et al., 2003; Blair et al., 2005). Surviving retrogradely labelled somata constituted intact RGCs whose axons were connected to the SC and capable of retrograde transport; in other words, they had avoided the primary injury. Death of RGCs due to secondary degeneration was therefore defined as the loss of retrogradely labelled RGC somata in central and particularly ventral retinal regions. 7 days after partial transection, RGC death was significant in central (p ≤ 0.05) but not in ventral retinae (p N 0.05, Fig. 8). By 28 days after partial transection, significant secondary RGC death had spread to ventral retinae (p ≤ 0.05, Fig. 8). Sham operated animals had no significant secondary RGC death compared to normal controls in either central or ventral retinal regions, after 7 or 28 days (p N 0.05, not shown). Lomerizine protected RGCs from secondary death in both central and ventral retinae 28 days after partial transection (p ≤ 0.05, Fig. 8). Confirmation that the reduction in FG labelled RGCs 28 days after partial transection accurately reflected a reduction in the total number of RGCs, was obtained by counting the number of β-III tubulin positive RGCs in central retinal sections (normal = 2682.9 ± 112.3 RGCs/mm2, partial transection 28 days = 1684.4 ± 148.8 RGCs/mm2, p ≤ 0.05).

Fig. 5. Phosphacan expression following partial transection +/− lomerizine treatment. Optic nerve sections distal to the injury site were immunohistochemically stained for phosphacan. Sections were taken 7 (middle panel) or 28 (right panel) days after injury, from animals treated with placebo (B, C) or lomerizine (D, E). Normal un-operated animals were included as controls and were not different from shams (A). Scale bar = 100 µm, magnification ×10, all images are oriented with dorsal uppermost. Images shown are representative of results observed from 6–10 animals per treatment group.

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Fig. 6. Oligodendrocytes and myelination following partial transection +/− lomerizine treatment. Optic nerve sections distal to the injury site were immunohistochemically stained for oligodendrocytes with olig2. Sections were taken 7 (middle panel) or 28 (right panel) days after injury, from animals treated with placebo (B, C) or lomerizine (D, E). Normal unoperated animals were included as controls and were not different from shams (A). Scale bar = 100 µm, magnification ×10, all images are oriented with dorsal uppermost. Images shown are representative of results observed from 3 animals per treatment group. The density of myelinated axons per µm2, corrected for section area was determined as detailed in the Materials and methods section and illustrated graphically (F). Statistically significant reductions in density of myelinated axons from normal controls (not different from shams), were represented by ⁎(p ≤ 0.05), NS indicated non significant differences.

Lomerizine preserved fast re-setting phase movements but not smooth pursuit responses following partial injury The optokinetic nystagmus consists of smooth pursuit in the direction of the stimulation in alternation with fast resetting phases (Delgado-Garcia, 2000). Partial transection of the ON resulted in a significant reduction in the number of smooth pursuits and fast resetting phase movements when tested 28 days after injury (p ≤ 0.05, Table 1). Sham operated animals had similar numbers of smooth pursuits and fast re-setting phase movements as normal animals (data not shown). Treatment with lomerizine preserved the number of fast re-setting phase movements (p ≤ 0.05, significantly different from placebo) but did not preserve smooth pursuit responses (p N 0.05, not significantly different from placebo, Table 1).

Discussion We have used a partial ON transection model in which secondary degeneration is spatially separated from the primary injury site to examine cellular responses in both the ON and retina. ON areas undergoing secondary degeneration were characterised by increasing morphological disruption, with loss of axons and their parent RGC somata and increased numbers of microglia and macrophages accompanied by oxidative stress and phosphacan deposition. As secondary degeneration progressed, myelin and visual function were lost. Treatment with the calcium channel blocker lomerizine successfully alleviated much of the morphological disruption, including neural loss and oxidative stress, but failed to fully prevent the loss of myelin or protect normal visual function (summary, Table 2).

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Fig. 8. RGC numbers following partial transection +/− lomerizine treatment. Optic nerves were partially transected and animals treated with lomerizine or placebo for 7 or 28 days. RGCs were retrogradely labelled with fluorogold and counted in central and ventral regions of retinal wholemounts from 4 to 9 animals per treatment group, as detailed in the Materials and methods section. Results were expressed as numbers of RGCs/mm2. Untreated normal animals were used as controls and values obtained were not different from shams. Statistically significant differences between groups were represented by ⁎(p ≤ 0.05).

Secondary degeneration occurred in defined zones adjacent to apparently unaltered tissue, reflecting similar heterogeneous patterns of alteration in post traumatic spinal cord (Wang et al., 2004). Calcium plays a fundamental role in neural degeneration (Coleman, 2005; Whitmore et al., 2005), with calcium waves propagated through the linked astrocyte network spreading local injury through multiple mechanisms (Wang et al., 2004; Lobsiger and Cleveland, 2007). A key component of calcium-induced neurodegeneration is oxidative stress, defined as a toxic over abundance of reactive oxygen species (Maher and Hanneken, 2005; Tezel, 2006). Release of these reactive oxygen species from dysfunctional glial and inflammatory cells may result in death or damage to neighbouring neurons (Block et al., 2007; Donnelly and Popovich, 2008). MnSOD is a marker of increased production of the free radical superoxide which it catalytically converts to H2O2 (Aucoin et al., 2005). While MnSOD has been shown to be protective of RGCs in experimental optic neuritis (Qi et al., 2007), it is associated with RGC death following injury induced by excess H2O2 production (Levkovitch-Verbin et al., 2000; Nguyen et al., 2003). Increased MnSOD during secondary degeneration, reduced with lomerizine treatment, reflected the degree of secondary degeneration we observed and illustrated the importance of controlling excess calcium flux. Our findings are in line with in vitro studies demonstrating that lomerizine reduces the formation of reactive oxygen species in CNS neurons (Akaike et al., 1993). Axons in the process of dying back in response to stress are characterised by accumulation of autophagic vacuoles, dense bodies and neuroaxonal spheroids (Coleman, 2005; Whitmore et al., 2005; Nixon, 2006). It is possible that some of the spheroid MnSOD expression observed in our study corresponds to similar structures. Inflammatory responses are among the mechanisms that may spread secondary degeneration (Schwartz, 2004), although studies in this area have been contradictory with some unpredictable and strainspecific outcomes (Cui et al., 2007). Macrophage activation resulted in beneficial effects shortly after induction but an unfavourable environment thereafter (Yin et al., 2003; Harvey et al., 2006). Fig. 7. Quantification of the numbers of Iba1 positive reactive resident microglia/ macrophages; ED-1 positive monocytes/macrophages; MnSOD and phosphacan aggregates and olig2 positive oligodendrocytes in dorsal and ventral halves of ON sections. Analyses were conducted on sections derived from control and test groups as described in Figs. 2–5. n = 3–12 for test groups and n = 4–6 for normal control and sham operated groups. Statistically significant differences were indicated by ⁎(p ≤ 0.05).

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Table 1 Optokinetic nystagmus following partial transection +/− lomerizine

Smooth pursuits Fast re-setting phases

Normal (n = 5)

Placebo (n = 6)

Lomerizine (n = 4)

7.8 ± 1.1 7.2 ± 0.9

3.7 ± 0.2 3.5 ± 0.2

4.8 ± 0.5 7.8 ± 1.0⁎

Smooth pursuits and fast re-setting phase movements following partial transection +/− lomerizine treatment were analysed as outlined in the Materials and methods section and compared to responses by normal animals. Significant differences following lomerizine treatment compared to placebo are indicated: ⁎ p ≤ 0.05.

Similarly, activated microglia facilitate repair through neurotrophin release but can release excess reactive oxygen species that cause neurotoxicity (Block et al., 2007). We found increased numbers of macrophages and microglia in areas undergoing secondary degeneration. Lomerizine treatment temporarily reduced the numbers of macrophages, which may have contributed to its beneficial effects on secondary degeneration. It is not clear how lomerizine reduced macrophage numbers, but it may have been a result of reduced axonal degeneration, or increased cerebral blood flow. Our results are in line with an early demonstration of reduced neurogenic inflammation with lomerizine (Hashimoto et al., 1997) and indicate greater efficacy than methylprednisolone (Ohlsson et al., 2004). While T cells have been reported to have both beneficial and damaging effects following optic nerve injury (Gonzalez et al., 2003; Schwartz, 2004; Johnson et al., 2007), we were unable to detect these cells in this model. CSPGs are part of the matrix found in glial scars in the CNS, existing in both membrane associated and secreted forms (Fawcett and Asher, 1999). They inhibit axonal growth through various mechanisms including calcium dependent EGFR activation which in turn is required for CSPG-mediated outgrowth inhibition (Koprivica et al., 2005; Harvey et al., 2006). Phosphacan is a key CSPG isomer that is significantly upregulated at some distance from the lesion site following spinal cord injury (Vitellaro-Zuccarello et al., 2007), indicating its involvement in secondary degeneration processes. Increased secreted phosphacan observed following partial transection may have been released from astrocytes (McKeon et al., 1999) and/or from oligodendrocyte precursor cells (Fawcett and Asher, 1999). Our observed reduction in the numbers of phosphacan aggregates in areas undergoing secondary degeneration with lomerizine treatment implies that lowering calcium levels not only interferes with the feedback loop of CSPG signalling, but reduces expression of a CSPG isomer. The reductions in phosphacan we have observed with a calcium channel blocker have not previously been described and may be contributing to the reduced progression of secondary degeneration seen in these animals via as yet unknown mechanisms. The morphological changes and delayed but significant loss of RGCs we observed following partial transection were similar to those seen by Levkovitch-Verbin et al. (2003). Further, we demonstrated that lomerizine protected RGC somata and axonal profiles vulnerable to secondary degeneration. The neuroprotective effects of lomerizine may have been due to reductions in oxidative stress amplification and/ or modulation of unfavourable macrophage responses, but also to direct effects on calcium flux in the RGCs. Direct modulation of pathological calcium fluxes with calcium channel antagonists have been postulated to be neuroprotective for RGCs (Whitmore et al., 2005; Yamada et al., 2006). Moderation of calcium increases in injured RGCs is a molecular marker of recovery (Stys and Jiang, 2002; Prilloff et al., 2007). The RGC protection we observed with lomerizine treatment is likely to be due to similar processes and confirms and extends previous studies assessing lomerizine using the ON crush model (Karim et al., 2006). We speculate that the primary neuroprotective effect of lomerizine is due to control of calcium fluxes in RGCs, which may have been amplified by glial networks and/or macrophages in the early phase of injury (Wang et al., 2004; Lobsiger and Cleveland, 2007). It is not, at this stage, clear whether the

neuroprotective effects of lomerizine are due to the control of excitotoxicity within the retina or whether this was secondary to protection of axons within the ON. Despite the fact that lomerizine protected RGCs from death, prevented excess macrophage involvement, reduced oxidative stress and reduced phosphacan expression, it did not prevent de-myelination or fully preserve visual function. Our observation of reduced numbers of myelinated axons in both the primary injury site and areas of secondary degeneration indicates spreading demyelination, which may explain the loss of vision after 28 days in this model. It is possible that re-myelination may occur with time, resulting in return of visual function. Lomerizine was unable to significantly improve the density of myelinated axons, a finding in accordance with earlier nonquantitative studies (Karim et al., 2006). In order to preserve myelin and/or promote re-myelination, optimisation of the lomerizine dosage regimen and/or combinatorial approaches with therapies known to target de-myelination may be useful. Semaphorin 3F has recently been shown to attract oligodendrocyte precursor cells and promote myelination (Williams et al., 2007), and LINGO-1 antagonists delivered intrathecally or systemically have been shown to increase myelination competence following SCI as well as promote RGC survival after ocular hypertension (Mi et al., 2005; Fu et al., 2008). One of these agents together with lomerizine is likely to be a useful combinatorial approach to prevent secondary degeneration. The optokinetic nystagmus consists of regular slow pursuit phases in the direction of the stimulation in alternation with quick resetting phases (Delgado-Garcia, 2000; Cahill and Nathans, 2008; Hogie et al., 2008). Due to the small binocular overlap in rodents, visual responses rely predominantly on head rather than eye movements (Walls, 1942), mediated through the pretectum and SC (Haustead et al., 2008). Signals are then transferred to motor commands via multiple circuits including visual areas of the lateral geniculate nucleus, parietal cortex and cerebellum (Delgado-Garcia, 2000). Partial ON transection is unlikely to selectively inhibit specific circuits; non-specific reductions in visual input from the ON are more likely and were implied by our observed reductions in both smooth pursuits and fast re-setting phases. Our demonstration of beneficial effects of a calcium channel blocker on a specific component of nystagmus is novel and the mechanism of selectivity is as yet unclear. Maintenance of connectivity in 10% of RGCs was considered sufficient to maintain visual function on orienting brightness and pattern discrimination tasks following ON crush (reviewed in Sabel, 1999). However, we have shown that although lomerizine treatment results in significant reductions in various cellular aspects of secondary degeneration and in approximately 75% of RGCs maintaining their connectivity, it does not restore full visual behaviour. We conclude that even in the face of substantial rescue of RGCs by Table 2 Summary of the benefits of lomerizine on secondary degeneration events Parameter Optic nerve Morphological disruption Resident microglia Macrophages Oxidative stress Phosphacan Oligodendrocytes Myelination Retina RGCs Function

Normal

Day 7

Day 28

Placebo

Lomerizine

Placebo

Lomerizine

− + − − − + ++

+ ++ ++ +/− − + ++

↓ ++ ↓ +/− − + ++

++ ++ ++ + + + +

↓ ++ ++ ↓ ↓ + +

++ +

++/+ NA

++ NA

+ −

↑ +/−

Parameters in areas vulnerable to secondary degeneration were analysed in animals following partial transection +/− lomerizine treatment and compared to completely normal animals (left column). Parameters were scored as: not present = −; weakly positive = +/−; positive = +; strongly positive = ++; reduced by lomerizine = ↓; increased by lomerizine = ↑; not analysed = NA.

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lomerizine, additional treatments will be necessary to maintain full visual function. Acknowledgments This work was supported by the Neurotrauma Research Program (Western Australia) and NH&MRC. Prof. Dunlop is a Professorial Fellow (Research) and Senior Research Fellow of the NH&MRC, Australia (Grant ID: 254670). We thank Woodside Australia for supporting our research by naming us as beneficiaries of the Woodside Sustainability Award 2007. We thank Michael Archer, Marissa Penrose, Vince Clark, Daniel Haustead and Sherralee Lukehurst for assistance with myelination analyses and technical procedures. The phosphocan monoclonal antibody developed by Dr. R.U. Margolis and Dr. R.K Margolis, was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. The authors acknowledge the facilities, scientific and technical assistance of the Australian Microscopy and Microanalysis Research Facility at the Centre for Microscopy, Characterization and Analysis, the University of Western Australia, a facility funded by the University, State and Commonwealth Governments. References Abdeljalil, J., Hamid, M., Abdel-Mouttalib, O., Stephane, R., Raymond, R., Johan, A., Jose, S., Pierre, C., Serge, P., 2005. The optomotor response: a robust first-line visual screening method for mice. Vision Res. 45, 1439–1446. Ahn, Y.H., Lee, G., Kang, S.K., 2006. Molecular insights of the injured lesions of rat spinal cords: inflammation, apoptosis, and cell survival. Biochem. Biophys. Res. Commun. 348, 560–570. Akaike, N., Ishibashi, H., Hara, H., Oyama, Y., Ueha, T., 1993. Effect of KB-2796, a new diphenylpiperazine Ca2+ antagonist, on voltage-dependent Ca2+ currents and oxidative metabolism in dissociated mammalian CNS neurons. Brain Res. 619, 263–270. Aucoin, J.S., Jiang, P., Aznavour, N., Tong, X.K., Buttini, M., Descarries, L., Hamel, E., 2005. Selective cholinergic denervation, independent from oxidative stress, in a mouse model of Alzheimer's disease. Neuroscience 132, 73–86. Blair, M., Pease, M.E., Hammond, J., Valenta, D., Kielczewski, J., Levkovitch-Verbin, H., Quigley, H., 2005. Effect of glatiramer acetate on primary and secondary degeneration of retinal ganglion cells in the rat. Invest. Ophthalmol. Vis. Sci. 46, 884–890. Block, M.L., Zecca, L., Hong, J.S., 2007. Microglia-mediated neurotoxicity: uncovering the molecular mechanisms. Nat. Rev. Neurosci. 8, 57–69. Bradbury, E.J., McMahon, S.B., 2006. Spinal cord repair strategies: why do they work? Nat. Rev. Neurosci. 7, 644–653. Buss, A., Pech, K., Merkler, D., Kakulas, B.A., Martin, D., Schoenen, J., Noth, J., Schwab, M.E., Brook, G.A., 2005. Sequential loss of myelin proteins during Wallerian degeneration in the human spinal cord. Brain 128, 356–364. Cahill, H., Nathans, J., 2008. The optokinetic reflex as a tool for quantitative analyses of nervous system function in mice: application to genetic and drug-induced variation. PLoS ONE 3, e2055. Coleman, M., 2005. Axon degeneration mechanisms: commonality amid diversity. Nat. Rev. Neurosci. 6, 889–898. Crowe, M.J., Bresnahan, J.C., Shuman, S.L., Masters, J.N., Beattie, M.S., 1997. Apoptosis and delayed degeneration after spinal cord injury in rats and monkeys. Nat. Med. 3, 73–76. Cui, Q., Hodgetts, S.I., Hu, Y., Luo, J.M., Harvey, A.R., 2007. Strain-specific differences in the effects of cyclosporin A and FK506 on the survival and regeneration of axotomized retinal ganglion cells in adult rats. Neuroscience 146, 986–999. Cui, Q., Yip, H.K., Zhao, R.C., So, K.F., Harvey, A.R., 2003. Intraocular elevation of cyclic AMP potentiates ciliary neurotrophic factor-induced regeneration of adult rat retinal ganglion cell axons. Mol. Cell Neurosci. 22, 49–61. Delgado-Garcia, J.M., 2000. Why move the eyes if we can move the head? Brain Res. Bull. 52, 475–482. Donnelly, D.J., Popovich, P.G., 2008. Inflammation and its role in neuroprotection, axonal regeneration and functional recovery after spinal cord injury. Exp. Neurol. 209, 378–388. Farkas, O., Povlishock, J.T., 2007. Cellular and subcellular change evoked by diffuse traumatic brain injury: a complex web of change extending far beyond focal damage. Prog. Brain Res. 161, 43–59. Farooque, M., Isaksson, J., Jackson, D.M., Olsson, Y., 1999. Clomethiazole (ZENDRA, CMZ) improves hind limb motor function and reduces neuronal damage after severe spinal cord injury in rat. Acta Neuropathol. 98, 22–30. Fawcett, J.W., Asher, R.A., 1999. The glial scar and central nervous system repair. Brain Res. Bull. 49, 377–391. Fu, Q.L., Hu, B., Wu, W., Pepinsky, R.B., Mi, S., So, K.F., 2008. Blocking LINGO-1 function promotes retinal ganglion cell survival following ocular hypertension and optic nerve transection. Invest. Ophthalmol. Vis. Sci. 49, 975–985.

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