Regeneration of retinal ganglion cell axons in organ culture is increased in rats with hereditary buphthalmos

Regeneration of retinal ganglion cell axons in organ culture is increased in rats with hereditary buphthalmos

Experimental Eye Research 85 (2007) 90e104 www.elsevier.com/locate/yexer Regeneration of retinal ganglion cell axons in organ culture is increased in...

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Experimental Eye Research 85 (2007) 90e104 www.elsevier.com/locate/yexer

Regeneration of retinal ganglion cell axons in organ culture is increased in rats with hereditary buphthalmos Julia Lasseck a,b, Uwe Schro¨er a, Simone Koenig b, Solon Thanos a,b,* a

Department of Experimental Ophthalmology, School of Medicine, University Eye Hospital Muenster, Domagkstrasse 15, 48149 Muenster, Germany b Integrated Functional Genomics, IZKF Muenster, Roentgenstrasse 21, 48149 Muenster, Germany Received 10 May 2006; accepted in revised form 9 March 2007 Available online 24 March 2007

Abstract This study used organ cultures to examine whether retinal ganglion cells (RGCs) retain their ability to regenerate axons in buphthalmos. A rat mutant with hereditary buphthalmos was used to (1) determine whether the extent of RGC loss corresponds to the severity and duration of elevated intraocular pressure (IOP), (2) examine whether RGCs exposed to an elevated IOP are able to regenerate their axons in a retina culture model, and (3) analyze the proteome of the regenerating retina in order to identify putative regeneration-associated proteins. Retrograde labeling of RGCs revealed a decrease in their numbers in the retinas of buphthalmic eyes that increased with age. Quantification of axons growing out of retinal explants taken at different stages of the disease demonstrated that buphthalmic RGCs possess a remarkable potential to regrow axons. As expected, immunohistochemistry and immunoblotting revealed that elevated IOP was associated with upregulation of certain known proteins, such as growth-associated protein 43, glial fibrillary acidic protein, and endothelin-1. In addition, two-dimensional polyacrylamide gel electrophoresis and mass spectrometry revealed several spots corresponding to proteins that were specifically regulated when buphthalmic RGCs were permitted to regrow their axons. Out of the proteins identified, heat-shock protein (HSP)-60 was constantly expressed during axonal growth at all stages of the disease. Antibodies against HSP-60 reduced axonal growth, indicating the involvement of this protein in regenerative axonal growth. These data are the first to show that diseased retinal neurons can grow their axons, and that HSP-60 supports neuritogenesis. This model may help to elucidate the fundamental mechanisms of optic neuropathy at stages preceding death caused by chronic injury, and aid in the development of neuroprotective strategies. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: rat; buphthalmos; regeneration; organ culture; mass spectrometry

Abbreviations: ANOVA, analysis of variance; BP, blocking peptide; CNS, central nervous system; DAPI, 40 ,6-diamino-2-phenylindole dihydrochloride hydrate; ET-1, endothelin-1; GAP, growth-associated protein; GCL, ganglion cell layer; GFAP, glial fibrillary acidic protein; GLR, mutant glaucoma rat; HBSS, Hanks’ balanced salt solution; HSP, heat-shock protein; IOP, intraocular pressure; IPG, immobilized pH gradient; IPL, inner plexiform layer; LI, lens injury; MS, mass spectrometry; NCBI, National Center for Biotechnology Information; NFL, nerve fiber layer; ON, optic nerve; ONC, optic nerve crush; PBS, phosphate-buffered saline; RCS, Royal College of Surgeons; RGCs, retinal ganglion cells; SDR, SpragueeDawley rat; SDS, sodium dodecyl sulfate; 2D-PAGE, two-dimensional polyacrylamide gel electrophoresis; 4-Di-10 ASP, 4-(4-dimethylaminostyryl)-N-methylpyridium-iodide. * Corresponding author. Department of Experimental Ophthalmology, University Eye Hospital Muenster, School of Medicine, Domagkstrasse 15, 48149 Muenster, Germany. Tel.: þ49 251 83 56915/56033; fax: þ49 251 83 56916. E-mail address: [email protected] (S. Thanos). 0014-4835/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2007.03.005

1. Introduction In addition to acute traumatic injuries to central nervous system (CNS) pathways, chronic injuries represent a major group of neurodegenerative diseases. Buphthalmos is a hereditary form of the chronic glaucomas that affect about 70 million individuals worldwide (Hiller and Kahn, 1975). The characteristic features of the different forms of glaucoma include abnormally elevated intraocular pressure (IOP) accompanied by progressive changes in the optic disc and retinal nerve fiber layer (NFL), as well as visual-field defects. Understanding the mechanisms that determine the susceptibility of retinal ganglion cells (RGCs) and the optic nerve (ON) to elevated

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IOP also requires suitable animal models of induced or spontaneous diseases that permit extensive and invasive investigations that are not possible in humans (Gelatt, 1977). Usually non-human primates, dogs, and rabbits are utilized. In some of the best rodent models developed in recent years, an increase in IOP is induced by injecting hypertonic saline into aqueous-humor collecting veins (Moore et al., 1993; Morrison et al., 1997), cauterizing two or three episcleral veins (Mittag et al., 2000; Naskar et al., 2002; Shareef et al., 1995), blocking aqueous outflow pathways by photocoagulation, or injecting India ink into the anterior chamber of the eye (Ueda et al., 1998). However, these models do not represent the endogenous diseases observed in most human forms of glaucoma. Hereditary animal models of glaucoma with slowly but chronically developing elevated IOP offer the opportunity to analyze RGCs at different stages of the disease. Models of chronic glaucoma include pigment dispersion glaucoma in the DBA/2 mouse (Chang et al., 1999; John et al., 1998), and buphthalmic rabbits (Bunt-Milam et al., 1987), beagles (Gelatt, 1977), and rats (Addison and How, 1926; Heywood, 1975; Young et al., 1974). However, no rat strain with a high incidence of buphthalmos has yet been bred for use in biomedical research (Goldblum and Mittag, 2002). A breeding mutant of the Royal College of Surgeons (RCS) rat strain with hereditary buphthalmos and signs of neuropathy (called the glaucoma rat, GLR) was established recently by Thanos and Naskar (2004). This rat strain offers the advantage that different stages of the disease can be studied throughout the lifetime of an individual animal. The main reason for glaucomatous impairment is the selective death of RGCs. Identification of intrinsic cellular mechanisms of resistance and perhaps of axonal regrowth might lead to methods to prevent exposure to elevated IOP resulting in the death of RGCs. The intrinsic regenerative ability of RGCs to survive injuries and regrow axons has been demonstrated in acute ON neuropathies such as ON crush (ONC) or cutting (Bahr et al., 1988; Thanos et al., 1989). This regenerative ability was attributed to injury-induced upregulation of growthassociated proteins (GAPs) such as GAP-43 (Benowitz and Routtenberg, 1997) and molecules assembled to form growth cones and axons (Fournier et al., 1997). Such a regenerative potential has not been attributed to RGCs in glaucomatous retinas, although recently it has been shown that some RGCs are present within the human retina even at very advanced stages of the disease (Pavlidis et al., 2003). The present study examined whether prior to their death and disappearance, RGCs from buphthalmic retinas possess a regenerative propensity in vitro. Retinal explants subject to buphthalmos were maintained in organ cultures, and their regeneration efficacy was quantified by counting the numbers of axons and comparing them with controls obtained from either normal retinas or from acutely injured retinas exposed to ONC and lens injury (LI). The proteome of the regenerating retinal stripes was then analyzed with two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and peptide mapping and sequencing in order to detect putative proteins specifically expressed under regenerative conditions. To

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further characterize the main features of buphthalmic neuropathy, histological investigations were performed to complement our previous results (Thanos and Naskar, 2004). 2. Methods 2.1. Rats All experiments were conducted in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Animals were obtained from the colony established in the laboratory that was derived from the rdy(/) strain (RCS rats) that develops glaucoma (Thanos and Naskar, 2004). The rats were housed in a standard animal room with food and water provided ad libitum and a 12-h/12-h light/ dark cycle. All surgical procedures on the rats were carried out under general anesthesia induced by the intraperitoneal administration of a mixture of 50 mg/kg ketamine (Ceva-Sanofi, Duesseldorf, Germany) and 2 mg/kg xylazine (Ceva-Sanofi). A topical antibiotic containing gentamicin (Gentamytrex, Dr. Mann Pharma, Berlin, Germany) was applied after ocular surgery. All experiments were carried out on rats in three age groups: group 1, 2 months old; group 2, 6 months old; and group 3, 12 months old. The IOP was measured using a tonometer (Tono-Pen XL-2, Mentor, Norwell, MA) (Krupin et al., 1980; Mirakhur et al., 1990; Moore et al., 1993; Naskar et al., 2002) while the GLRs were lightly anesthetized by ether inhalation. On any given eye, 10 readings taken directly from the display of the tonometer were recorded and averaged. Elevated IOPs in glaucomatous eyes were defined as values >18 mmHg (eyes with an IOP < 18 mmHg served as controls). For histological investigations of the retina and ON, GLRs with unilateral glaucoma were killed with an overdose of anesthetic, after which the eyes were enucleated, immersion fixed in 4% phosphate-buffered formalin, and processed for paraffin embedding. Paraffin sections (5 mm thick) were cut on a microtome, deparaffinated in xylene, and processed for hematoxylineeosin staining. 2.2. Retrograde labeling of RGCs for quantification and morphology RGCs of GLRs were retrogradely labeled with 4-(4-dimethylaminostyryl)-N-methylpyridium-iodide (4-Di-10 ASP; Molecular Probes, Eugene, OR) as described previously (Naskar et al., 2002). The aims of the data analysis were to (1) determine the central-to-peripheral distribution of RGCs by counting six eccentrically randomized areas (three times), each corresponding to an area of 0.096 mm2, in the same retinal quadrant per retina for all animals, and (2) determine the RGC population in each retina whole-mount preparation by evaluating a further 20 randomly chosen areas (a total of 38 areas), distributed in the three remaining retinal quadrants in all eccentricities, at a final magnification of 400 (Fig. 2A). For each retina the total number of RGCs was divided by the area analyzed, and this density was multiplied by the

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Fig. 1. Phenotype of the mutant with unilateral buphthalmos showing characteristic features of the disease. (A, B) Physiological appearance of the ON head and retina of the contralateral control eye without elevated IOP. (C) Buphthalmic ON head with cupping and atrophy (arrow) resulting from elevated IOP. (D) Loss of RGCs (arrow) was marked in the GCL and NFL in diseased eyes, and associated with substantial thinning of the retina. Scale bars: 100 mm (A, C) and 20 mm (B, D). Abbreviations are defined in the separate list.

area of each retina to produce the total number of RGCs in the retina. The mean density of RGCs was compared between buphthalmic and control retinas. Equivalent randomized fields from the same quadrants in all retinas were examined and compared. 2.3. Immunohistochemistry and Western blot The eyes of GLRs with and without elevated IOP were enucleated, embedded in Tissue-Tek (Sakura-Finetek, Torrance, CA), and frozen in liquid nitrogen. Frozen sections (12 mm) were cut through the middle one-third of the eye and collected on gelatinized slides. Immunohistochemistry was performed according to standardized protocols. The following monoclonal antibodies and antisera were used to examine the morphology of the retina and determine the expression pattern of

proteins known to be associated with regeneration and degeneration: anti-GAP-43 (dilution 1:400; Sigma, MO) and antiGAP-43-phosphoSer41 (dilution 1:100; Chemicon, Temecula, CA), which are major proteins expressed during regeneration in axonal growth cones (Benowitz and Routtenberg, 1997); anti-GFAP (glial fibrillary acidic protein; dilution 1:400; Sigma), which is upregulated in astrocytes and Mueller cells under various environmental stress conditions (Lam et al., 2003); anti-endothelin-1 (ET-1; dilution 1:1000; Sigma), which is found in the CNS in capillary endothelial and glial cells in association with glaucoma (Yorio et al., 2002); antirhodopsin (dilution 1:100; Chemicon), which labels photoreceptors; and anti-OX-42 (dilution 1:50; Serotec, Duesseldorf, Germany), which recognizes most microglia and macrophages in the CNS (Wang et al., 2000). Primary antibodies were then conjugated with the corresponding Cy2Ô secondary

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Fig. 2. Retrograde labeling of RGCs revealed a decrease in RGCs in the retina that increased with age. (A) Topographical location of areas on flat-mounted retinas selected for cell counting to determine the RGC densities. Each area measures 0.096 mm2. (BeD) Retrograde labeling of RGCs in a montage of low-magnification (100; scale bar, 200 mm) images of the fixed, flat-mounted retinas of rat nos. 1, 3, and 5 with unilaterally elevated IOP (left eyes) and the control right eyes. Enlarged images from a laser-scanning microscope (magnification, 400; scale bar, 50 mm) from the same retinas in the central (left margin) and peripheral (right margin) regions clearly illustrate the significant loss of all ganglion cell types in the diseased retina. (E) IOPs in five GLRs at the time of labeling (rat nos. 1 and 2, 2 months old; rat nos. 3 and 4, 6 months old; rat no. 5, 12 months old) with unilateral glaucoma. (F) Intraindividual comparison of the ganglion cell population with individual IOPs corroborates the continual decrease in RGC density with progression of the disease. (G) Interindividual comparison of the central-to-peripheral RGC distribution showing the homogeneous loss of ganglion cells in all retinal quadrants and eccentricities.

antibodies (dilution 1:200; Jackson ImmunoResearch, West Grove, PA). The nuclei of retinal cells were stained by adding 40 ,6-diamino-2-phenylindole dihydrochloride hydrate (DAPI; Sigma) to the Moviol embedding medium. Slides were examined under a fluorescence microscope (Axiophot, Carl Zeiss). Western blot analyses were performed as described previously (Fischer et al., 2000). Six retinas were freshly dissected from GLRs at different stages of the disease (divided into groups 1e 3 on the basis of age) with and without elevated IOP, and prepared and analyzed for the detection of GAP-43, GAP-43p, GFAP, ET-1, and rhodopsin.

2.4. Surgical procedures ONC and LI have been shown to promote neuronal regeneration and axonal outgrowth in vitro (Bahr et al., 1988; Fischer et al., 2000). We performed ONC and LI on the left eyes of 15 SpragueeDawley rats (SDRs) of both sexes to compare the regenerative capacity of RGCs after acute injury with that of buphthalmic RGCs injured by chronically elevated IOP. All surgical manipulations were unilateral, with the contralateral eyes serving as negative controls for axonal regeneration. Following surgery the health and behavior of the animals were

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monitored continuously, and 5 days after surgery both eyes were removed for the in vitro experiments. 2.5. Explantation of the retina The in vitro experiments were performed with retinas explanted and cultured according to the protocol of Bahr et al. (1988). The model consists of removing the eye, dissecting the retina, cutting it into eight wedge-like pieces, and placing the stripes with the ganglion cell layer (GCL) on a substrate consisting of polylysine and laminin (Bahr et al., 1988). The following four groups were formed to compare the extent of axonal regeneration: retinas of the mutant GLR strain with (group I) and without (group II) an elevated IOP, and retinas of the SDR strain following acute ONC and LI (group III) and their untouched contralateral control eyes (group IV). After preparing retinal explants, 4 ml of S4 growth medium was added (Bahr et al., 1988; Ford-Holevinski et al., 1986) (Astrocyte Microglia Growth Medium, Promocell, Heidelberg, Germany) and the explants were cultured in an incubator at 37  C for 4 days. The number of regrowing axons from retinal explants was counted after 24, 48, 72, and 96 h in vitro under an inverted phase-contrast microscope (Axiovert 135, Carl Zeiss) at a final magnification of 200. As a measure of axonal growth, we counted all axons that crossed an imaginary line 0.25 mm from the margin of the explants. The presence of fasciculation and branching when the explant contained many axons increased the difficulty of the evaluation. In such cases counts were made at a higher final microscope magnification (400), which allowed individual axons to be estimated even within fasciculi. 2.6. Two-dimensional gel electrophoresis and proteomics When axon regeneration occurred, the explants of retinas after regeneration (elevated IOP) and retinas from buphthalmic eyes without regeneration (normal IOP) were harvested and used for proteomic analysis using 2D-PAGE and mass spectrometry (MS) peptide mapping and sequencing. 2DPAGE was performed according to the method first described by O’Farrell (1975). In detail, the explants of each retina were boiled in 10% sodium dodecyl sulfate (SDS; Sigma, Taufkirchen, Germany) and homogenized in 2D lysis buffer (7 M urea, 2 M thiourea; Merck, Darmstadt, Germany), 4% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (USB, Cleveland, OH), 40 mM Trisbase (Roth, Karlsruhe, Germany), 1 mM PMSF (Sigma), and 10 mM dithiothreitol (Roche, Mannheim, Germany). The final SDS concentration was 0.25%. Soluble protein (200 mg, according to the Bradford test) together with a 2% immobilized pH gradient (IPG) buffer (pH 3e10, Amersham Biosciences, Freiburg, Germany) and 20 mM dithiothreitol were loaded on Immobiline Drystrips (pH 4e7, 18 cm; Amersham Biosciences) and rehydrated overnight. The rehydrated strips were focused on a Multiphor II (Amersham Biosciences) electrophoresis system for w80 kVh.

Focused IPG strips were incubated twice for 15 min in equilibration solution [50 mM TriseHCl (pH 8.8), 6 M urea, 30% glycerol, and 2% w/v SDS] and a trace of bromophenol blue (Merck), with 1% b-mercaptoethanol and 2.5% iodoacetamide (Sigma) added to the first and second equilibration steps, respectively. For the second dimension, the equilibrated IPG strips were fixed with 0.5% w/v melted agarose (Merck) on homogeneous 12.5% SDS gels (rotiphorese Gel 30, Roth). Proteins were separated by vertical SDSePAGE (BioRad, Munich, Germany) according to Laemmli (1970). Protein spots were initially labeled with colloidal Coomassie Brilliant Blue G250 (Merck). Spots were manually excised from the gel, tryptically digested in the gel, extracted, purified using Ziptips (microbed C18; Millipore, Bedford, MA), and subjected to MS analysis. Peptide maps were generated using a TOF-Spec-2E device (Micromass, Manchester, UK), and selected retinal peptides were sequenced using a nanoHPLC-MS/MS device (Ultimate, LC Packings, Amsterdam, The Netherlands; Esquire3000, Bruker Daltonics, Bremen, Germany). Six gel replicates were compared. National Center for Biotechnology Information (NCBI) and SWISS-PROT databases were searched using Mascot software (Matrix Science, London, UK). Three gels stained with silver nitrate were subjected to additional image analysis (Shevchenko et al., 1996). 2.7. Blocking experiments Anti-heat-shock protein (HSP)-60 antibody was added to the culture medium to determine whether HSP-60 modulated the axon regeneration of injured RGCs in vitro. Retinas were removed and explanted as described above. Explants of experimental eyes were allotted to petri dishes containing different supplements. The first sample contained 20 ml of polyclonal anti-HSP-60 (Santa Cruz Biotechnology, Santa Cruz, CA) and 125 ml of Hanks’ balanced salt solution (HBSS; PAA Laboratories, Pasching, Austria) in 1855 ml of S4 growth medium. The antibody was neutralized using controls containing 20 ml of anti-HSP-60 and 125 ml of antibody-blocking peptide (BP) in fivefold excess (Santa Cruz Biotechnology) in 1855 ml of S4 growth medium. Further controls contained 20 ml of anti-GFAP (Sigma) or 20 ml of anti-HSP-90 (Santa Cruz Biotechnology) in 125 ml of HBSS with 1855 ml of S4 growth medium or in only 2 ml of S4 growth medium. Dialysis was performed with Mini Dialysis Units (Slide-ALyzer, Pierce, Rockford, IL) the day before explantation against HBSS overnight at 4  C to remove sodium azide and other eventually toxic or interfering substances and preservatives from the samples. The coculturing of explants with anti-GFAP was used to confirm that (1) sufficient amounts of the antibody remained after dialysis, (2) the antibody was taken up into cells during culturing, persisted unaltered, and acted with the antigen, and (3) the primary antibody could be detected through immunohistochemistry with a second antibody (Cy2Ô). The number of regrowing axons from retinal explants was determined after 24, 48, and 72 h in culture and compared

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among the experimental groups. To perform immunohistochemistry with cocultured explants, the tissue was fixed in 4% paraformaldehyde for 3 days. After removing the nitrocellulose membrane, the explants were treated for 5 min with methanol at 20  C and then washed in phosphate-buffered saline (PBS) and blocked in fetal calf serum. The explants were then incubated with the second antibody for 2 h and then again washed with PBS and fixed in Moviol embedding medium containing DAPI. All axon quantifications are expressed as mean  S.D. values. The protein levels of HSP60 and HSP-90 in glaucomatous eyes were further analyzed using Western blotting. 2.8. Statistical analysis Results were expressed as means and standard deviation. Statistical analysis was performed using two flanked, unpaired t-test. A p value of <0.05 was considered statistically significant. The mean numbers of axons per group were determined in the regenerating explants. The number of axons from each group was also analyzed by ANOVA testing, with significance assumed at the 95% confidence level. 3. Results 3.1. Clinical and histological findings In the GLR strain the first signs of asymmetric eye size can be detected at 2e3 months of life, after which the eyes affected continuously increase in size to become obviously buphthalmic (Thanos and Naskar, 2004). Mutant rats displayed uni- or bilateral elevated IOPs of 19e45 mmHg, as compared to control values of 12e18 mmHg. Relative to control eyes (Fig. 1A,B), changes suggestive of ON-head cupping (Fig. 1C) and progressive RGC loss accompanied with substantial thinning of all retinal layers (Fig. 1D) were found in buphthalmic eyes. 3.2. Retinal ganglion cell loss Buphthalmos-induced RGC degeneration was detected by retrograde labeling of cells by injecting the fluorescent dye 4-Di-10 ASP into both superior colliculi. Fig. 2A shows the topographical locations in flat-mounted retinas from the five animals with unilaterally elevated IOP in which the cell densities were determined. Fig. 2BeD shows three of these retinal

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pairs obtained from rat nos. 1, 3, and 5. Intraindividual comparison of the animals and their IOPs in the context to their RGC populations suggested a pressure-dependent decrease in ganglion cells according to the severity and progression of the disease (Fig. 2E,F). The RGC densities and the IOPs in the buphthalmic and nonbuphthalmic eyes are given in Table 1 and Fig. 2E, which indicates that they were not strongly correlated. The RGC density was defined with n ¼ 10 areas of counting in each retina. When the cells were counted after retrograde staining, the youngest animals (nos. 1 and 2) showed very similar RGC densities in both retinas (Fig. 2F). There was a slight difference between the retinas of animal 1 (Fig. 2F, 2511  72 cells/mm2 in left eye and 2681  76 cells/mm2 in right eye, n ¼ 10), and the retinas of animal 2 (with similar IOP) had almost identical RGC densities (2806  71 cells/mm2 in the left eye and 2823  74 cells/mm2 in the right eye, n ¼ 10). At 6 and 12 months of age (animals 3e5), the IOP was clearly lower in the non-buphthalmic eyes than in affected eyes (Fig. 2E,F). To examine whether there was any regional selectivity in the disappearance of RGCs, we counted cells along the central-to-peripheral eccentricity of the retina (Fig. 2A). In the normal retinas (IOP < 18 mm, n ¼ 5) there was a clear central-to-peripheral decrease in the RGC densities (Fig. 2G). In the buphthalmic eyes (IOP > 18 mmHg, n ¼ 5), the RGC densities were reduced in all eccentricities, and there were significant differences ( p < 0.01) in all corresponding areas between buphthalmic and control retinas (Fig. 2G). In addition to the uniformly reduced RGCs with respect to eccentricity, different types and sizes of RGCs could be discerned both in the central and peripheral retina at all stages of the disease (enlarged areas in Fig. 2BeD). 3.3. Immunohistochemistry and Western blot analysis Immunohistochemistry was performed to compare the expressions of various proteins in buphthalmic retinas as the disease progressed. All observations were compared with control retinas. To confirm the data, Western blots were additionally prepared with retinas of rats with unilaterally elevated IOP. In control animals (SDRs, or juvenile, buphthalmic rats) rhodopsin-positive photoreceptors that were immunohistochemically visible at juvenile stages (Fig. 3A) continually disappeared in older (>2 months old) GLRs and RCS rats (Fig. 3B,C), as expected from the ongoing photoreceptor

Table 1 RGC densities and the IOPs in the buphthalmic and nonbuphthalmic eyes Rat no.

Age (months)

IOP (mmHg) left/right

RGC densities (cells/mm2) left/right

p-value RGC densities intraindividual left/right

p-value RGC densities interindividual b*/nb*

1 2 3 4 5

2 2 6 6 12

24/17 15/17 23/18 16/24 26/20

2511  72/2681  76 2806  71/2823  74 737  24/1927  46 2139  58/1913  55 694  24/1762  44

<0.001 0.607 <0.001 <0.001 <0.001

n.a. n.a. n¼3 p ¼ 0.167

*Buphthalmic, *non-buphtalmic. n.a., not available.

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Fig. 3. Immunohistochemistry and Western blotting. Rhodopsin expression decreases with age in both the non-buphthalmic RCS- and the buphthalmic GLR-retinas (AeD). GFAP is upregulated in glial cells of both strains of rats (EeH). ET-1 shows a similar pattern with GFAP (IeL), whereas GAP-43 was expressed in the ganglion cell layer and optic fiber layer with increasing age in both strains (M-P). OX-42 staining was positive in microglial cells of RCS- and GLR-retinas (QeS). Images in A, E, I, M, Q, show 6-month-old non-buphthalmic RCS-retinas, images B, F, J, N, R show 6-month-old buphthalmic retinas, and images C, G, K, O and S. show 12-month-old buphthalmic retinas. The images D, H, L, and P show immunoblots of the same stages used for immunocytochemistry. Note that the OX-42 antibody does not work in Western blots. Blue DAPI staining indicates cell nuclei. Scale bars: 100 mm (A, B, C, E, F, G, I, J, S) and 50 mm (K, M, N, O, Q, R). G, buphthalmos; C, control; RB, rat brain; 1, 2 months old; 2, 6 months old; 3, 12 months old. Molecular masses are given in kilodaltons in the right margin.

dystrophy that is present in this strain. In Western blots, rhodopsin that was detectable in controls and juvenile buphthalmic rats disappeared completely after the 3rd month (Fig. 3D). Consistent with the rhodopsin findings, the number of GFAP-positive astrocytes and Mueller cells was minimal in control retinas (Fig. 3E) but increased in an age-dependent manner in buphthalmic retinas (Fig. 3F,G). Western blotting revealed that GFAP increased monotonically with age (Fig. 3H), thus confirming the immunohistochemistry results. A similar pattern of expression was observed for ET-1 (Fig. 3IeK), indicating that the response in glial and capillary endothelial cells to the degenerative disease involves the upregulation of this protein. The ET-1 staining (Fig. 3J) and Western immunoblotting (Fig. 3L) were both strongest at 6 months of age. Faint GAP-43 staining was seen in the GCL of juvenile rats (Fig. 3M), which increased slightly in the retinas of 6month-old buphthalmic eyes (Fig. 3N), and in retinas older than 12 months in the GCL and NFL (Fig. 3O). As expected from this immunohistochemical pattern, the expression of GAP-43 in Western blotting was relatively stable at all stages (Fig. 3P). Finally, OX-42, a marker for resident microglial

cells and immigrating macrophages (Fig. 3QeS), was not expressed in the control retina (Fig. 3Q) but was strongly expressed in the GCL of glaucomatous eyes (Fig. 3R,S). Microglial cells were activated throughout the progression of the disease. This antibody was not stained in Western blots. 3.4. Regeneration of retinal ganglion cell axons in vitro The regenerative potential of buphthalmic retinas was compared with those of control retinas and retinas subjected to acute ON injury by studying axonal regrowth in culture (Bahr et al., 1988; Thanos et al., 1989). Those retinal stripes that had remained attached to the substrate (80%) were evaluated for axonal outgrowth after 24, 48, 72, and 96 h in culture. Axons grew out of the explants within the first few hours after culturing in the ONCþLI group and in the buphthalmos retinas, and increased both in length and number over time (Fig. 4A,B). In contrast, very few RGCs extended axons in the control RCS-rat retinas (Fig. 4C). The numbers of axons in all experimental retinas was measured from 24 h in culture; a quantitative analysis of the axons that extended under the different experimental

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Fig. 4. Axon regeneration in retinal explants. (AeC) Regenerating RGC axons extending from retinal explants (white areas) mounted on nitrocellulose filters (black) after 96 h in culture: (A) retinal explant following ONCþLI, (B) explant from a 6-month-old buphthalmos retina, and (C) untreated control explant of an RCS-rat/GLR without buphthalmos. Scale bars: 0.5 mm (magnification, 20; upper panels) and 50 mm (magnification, 40; lower panels). (D) Quantitative representation of the number of axons/retina that extend from the ONCþLI group, the sham controls without ONCþLI [C (S.D.)], and buphthalmic (G) and control [C (RCS-rat)] explants.

conditions is shown in Fig. 4D. After 96 h in culture, ONCþLI retinal explants exhibited 7584  867 axons/retina (n ¼ 13 retinas), compared to only 521  85 axons/retina (n ¼ 14 retinas; p < 0.001 compared to ONCþLI group) in control explants from untreated SDRs. Buphthalmic retinal explants extended 954  163 axons/retina (n ¼ 17 retinas, p < 0.001 compared to ONCþLI group and p < 0.001 compared to the control group) and RCS-rat control retinas extended 297  64 axons/ retina (n ¼ 12 retinas; p < 0.001 compared to buphthalmic retinas and p < 0.001 compared to ONCþLI group; Fig. 4D). The numbers of RGC axons grown in culture from both buphthalmic and control retinal explants increased over time, but was

typically threefold higher in buphthalmic retinas than in controls at all measurement times (Fig. 5A): after 24, 48, 72, and 96 h in culture there were 10  3, 126  15, 464  73, and 954  163 axons/retina in buphthalmic retinas, and 4  1, 60  13, 172  35, and 297  64 axons/retina in control retinas, respectively ( p < 0.001 at each time in culture, Fig. 5A). The ability of buphthalmic RGCs to extend axons in culture depended on the age of the animal, thus reflecting different stages of the disease, and hence different durations of exposure to an elevated IOP. Retinas aged 2 months (i.e., around the beginning of the disease) exhibited the highest regenerative capacity, which gradually decreased with age but was still

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Fig. 5. Quantification of axonal regrowth. (A) Quantitative comparison of the regenerative capacity between buphthalmic and control explants for 24- to 96-h cultures. (B) Quantitative representation of the regenerative capacity of buphthalmic RGC axons according to age, between 24 and 96 h. (C) Comparison between the regenerating axons per explant after 96 h in culture (right axis) and the corresponding total number of RGCs per retina in buphthalmic (G) and control [C (RCSrat)] eyes (left axis). (D) Comparisons of relative numbers of regenerating axons and RGCs per retina in the ONCþLI group, buphthalmic, and control explants.

evident in animals older than 12 months (Fig. 5B): after 24, 48, 72, and 96 h of culture there were 14  4, 174  26, 747  138, and 1503  349 axons/retina in 2-month-old animals (n ¼ 6); 7  1, 105  12, 337  38, and 721  110 axons/retina in 6-month-old animals (n ¼ 6); and 10  7, 93  27, 278  67, and 574  127 axons/retina in 12month-old animals (n ¼ 5), respectively. The decrease in the numbers of axons with increasing age differed significantly between the three age groups ( p < 0.001). This decrease from the 2nd to the 12th month of life may reflect a decrease in recruitable RGCs within the retina, because many of them had died due to the buphthalmos. In order to correlate the numbers of growing axons and the total populations of RGCs available for such growth, the RGCs were retrogradely labeled with 4-Di-10 ASP. The left half of Fig. 5C shows that there were 99017  7413 RGCs/retina (n ¼ 5) and 60940  14108 RGCs/retina (n ¼ 5) in the control and buphthalmic rats, respectively. The total numbers of regenerating axons after 96 h in culture are shown in the right half of Fig. 5C. The buphthalmic retina extended 954  163 axons/retina (n ¼ 17 retinas), although the actual number of RGCs was reduced. In contrast, the control retina with a normal population of RGCs extended only 297  64 axons/retina (n ¼ 12 retinas; Fig. 5C, left half). A total of 1.6  0.5% of buphthalmic RGCs were able to regenerate axons in organ culture, compared to 0.3  0.1% in control eyes which is a fivefold difference ( p < 0.001),. On the other hand, after acute ONCþLI, 7.6  1.4% of the RGCs regenerated their axons (out of a total ganglion cell

population of 100,000 cells/retina; Fig. 5D, p < 0.001 compared to either group), indicating that the induction of regeneration is stronger for acute injury than for buphthalmos. 3.5. Two-dimensional electrophoresis and peptide analysis MS-assisted peptide analyses of the regenerated buphthalmic retinas and the controls revealed that some proteins were differentially expressed within the buphthalmic retinal explants. Furthermore, the expression of similarly distributed proteins was examined and used to define the accuracy of the procedure. Fig. 6A shows the topography of the proteins in a gel of a juvenile control retina, which were equally expressed in both experimental groups and were continuously present without any age dependence in all experimental retinas. The gel in Fig. 6B is an example of a buphthalmic retina that shows that proteins were differentially expressed within the two groups. Table 2 lists the spot number, NCBI number, potential function, and molecular weight of each expressed protein. HSP-60 (spot 13) was consistently upregulated in buphthalmic eyes in all examined gels, as was enolase 2 (spot 2b) (n ¼ 6); however, recoverin (spot 27) was only expressed in juvenile buphthalmic retinas. In contrast, retinoic acid receptor beta (spot 26), ATP synthase beta subunit (spot 2a), and tropomyosin (spot 16) were commonly expressed in all control samples, whereas retinaldehyde-binding protein (spot 6a) was only evident in gels of the older control animals. Furthermore, adaptor-related protein complex 3, sigma 1 (spot

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Fig. 6. Peptide mapping of cultured rat retinal explants. (A) Gel of a juvenile control retina with normal IOPs with less regeneration of axons, identifying 27 repeatedly analyzed spots that were found in all control and buphthalmic-plus-regeneration retinas. (B) Gel of a juvenile buphthalmic retina after regeneration of axons, representing the localization of differentially expressed proteins. The proteins identified are listed in Table 2. Proteins that were regulated after the regeneration of axons are in boldface. All gels were performed in triplicate.

19), and kinase-associated HSP-90 (spot 6b) were expressed in younger animals of both experimental groups. Calreticulin (spot 3) and craniofacial developmental protein 1 (spot 17) were more pronounced in older animals. 3.6. Blocking experiments HSP-60 was found to be markedly upregulated in regenerated buphthalmic RGC axons at all stages of the disease,

suggesting that this protein plays an important role in modulating their intrinsic regenerative potential. Because of this, we functionally analyzed the role of HSP-60 by introducing antibodies directed at this protein. Antibodies against HSP-60 prevented axon growth and resulted in them degenerating (Fig. 7A): 98  32, 348  81 and 518  100 axons (n ¼ 8) grew out of control retinal explants cultivated in 2 ml of S4 growth medium after 24, 48, and 72 h, respectively, whereas only 38  13, 106  39 and 168  70

Table 2 Peptide mapping showing the 27 spots excised and analyzed from gels (performed in triplicate for each gel) Spot

NCBI acc. no.

Protein

Potential function

MW (Da)

1 2a 2b 3 4 5 6a 6b 7 8 9 10 11 12 13* 14 15 16 17 18 19 20 21 22 23 24 25 26 27

24234686 92350 26023949 11693172 71620 16758840 10181110 27531723 27691430 27731121 27684483 2507440 8393910 2119726 11560024 26023949 22096350 7441398 45501320 27720565 27658962 15100179 1352495 16758348 9506411 127984 14134107 27704612 18266710

HSP-70 8 isoform 2 ATP synthase beta subunit Enolase 2 Calreticulin Beta actin Crystallin mu Retinaldehyde-binding protein Kinase-associated HSP-90 Retinoic acid receptor responder protein 1 Glucose-6-phosphate isomerase Class I beta tubulin Syntaxin 2 Phosphatidylethanolamine-binding protein Glucose-related protein 75 HSP-60 1 Enolase 2 Enolase 1 Tropomyosin Craniofacial dev. protein 1, cyclin G1 Calmodulin Adaptor-related protein complex 3 Malate dehydrogenase 1 Beta nerve growth factor Peroxiredoxin 6 ATP synthase subunit d Nucleoside diphosphate kinase B Tropomyosin alpha isoform Retinoic acid receptor beta Recoverin

Protein folding, ATPase activity ATP biosynthesis Carbohydrate transport Calcium-binding protein Structural protein, cytoskeleton Amino acid transport and metabolism Transport of retinaldehyde and retinol Molecular chaperone Type 2 membrane protein Carbohydrate transport and metabolism Tubulin, cytoskeleton Epithelial morphogenesis Lipid and ATP binding Molecular chaperone Chaperonin, protein turnover Carbohydrate transport and metabolism Glycolysis, lyase Actin-myosin binding and interaction Cytochrome, craniofacial development Calcium-binding protein Intracellular trafficking and secretion Energy production and conversion Development of the nervous system Thiol-specific antioxidant protein ATP biosynthesis Synthesis of nucleoside triphosphates Actin-myosin binding and interaction Nuclear receptor protein Regulation of rhodopsin

53484 50738 47111 47966 41724 33533 44482 44482 28811 28896 45097 33338 20788 73699 60927 47111 47171 28292 34000 35066 34345 36460 26993 24803 18752 18000 32836 36741 23319

Boldface proteins were differentially expressed or upregulated in the regenerating retinas. The function of HSP-60 (spot 13*) was further analyzed.

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Fig. 7. Role of HSP-60 in hereditary buphthalmos. (A) Antibodies against HSP-60 prevented axon growth after 72 h in organ culture. In contrast, antibodies against HSP-90, GFAP, and HSP-60 with BP had no effect on axon regeneration, as also seen in control studies with 2 ml of S4 growth medium. (B) In Western blotting, HSP-60 was continually and highly expressed in all buphthalmic retinas whereas HSP-90 showed only a moderate expression in older animals. (C) Immunohistochemistry of retinal explants cultured in the presence of anti-HSP-60 resulted in the detection of HSP-60-positive cells in the GCL (arrows) with the second antibody. (D) Immunohistochemistry of retinal explants cultured in the presence of anti-GFAP resulted in the detection of immunopositive microglia cells (arrow) with the second antibody, confirming the in vitro uptake of antibodies.

axons (n ¼ 10) were present in retinal explants cultivated with anti-HSP-60 (Fig. 7A). Further control experiments were performed with organ cultures containing anti-HSP-90 (n ¼ 3), anti-GFAP (n ¼ 2), and anti-HSP-60 plus anti-HSP-60 BP (n ¼ 4; Fig. 7A). In the presence of anti-HSP-90, 112  36, 442  121, and 681  115 axons grew out of retinal explants, which was similar to the numbers of axons growing out of retinal explants cultivated with anti-GFAP (70  57, 394  290, and 557  360) and anti-HSP-60þBP (121  66, 429  161, and 653  182). The difference between blocked explants and all controls was significant at p < 0.001 for each time in culture. Western blotting revealed a high content of HSP-60 and a moderate elevation in HSP-90 at all stages of buphthalmos (Fig. 7B), thus demonstrating that although both HSPs are expressed, only HSP-60 is involved in the process of regenerative growth. The uptake of the antibody and the binding capacity to its specific antigen in organ culture was confirmed by applying immunohistochemistry to retinal explants after culturing using only the second antibody: cells in the GCL became HSP-60 positive (Fig. 7C), and Mueller cells encompassing the entire retinal depth became GFAP immunopositive (Fig. 7D). 4. Discussion The present study examined whether elevated IOP is associated with RGC responses that culminate in their regeneration.

The principal findings are as follows: (1) there is a RGC loss and upregulation of GAP-43, GFAP, and ET-1 in buphthalmic rats, (2) buphthalmos induces a regenerative response that is significantly higher than that in normal retinas and significantly lower than that in retinas after ONC and LI; and (3) several proteins are expressed in regenerating retinal explants, among which HSP-60 differentially appears in the regenerating tissue obtained from buphthalmic retinas. These findings indicate that buphthalmic injury induces regenerative processes similar to those induced by axotomy, but with lower efficacy. Elevated IOP is a characteristic feature of many forms of glaucoma, including hereditary buphthalmos in rats (Thanos and Naskar, 2004), and is considered to be a major risk factor for RGC degeneration in this disease. The GLR strain used in the present study expresses a buphthalmic phenotype at 2e 3 months of life, thus indicating a slow development of the disease during the first few postnatal weeks. Although IOP measurements are difficult with a tonometer within this period of IOP establishment, the large-eyeball phenotype at the 3rd month of life precedes the IOP elevation. The onset of IOP elevation is accompanied by typical glial (e.g., GFAP upregulation), capillary endothelial cellular (e.g., ET upregulation), and neuronal (e.g., GAP-43 upregulation) responses in the retina. Retrograde staining revealed that the gradual loss of RGCs in the GLR strain is correlated with the age of animals. Retrograde labeling of RGCs from the superior colliculus with fluorescent dyes enables the simultaneous visualization

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and quantification of RGCs in retinal flat mounts. This method revealed that significantly more RGCs were labeled in control RCS-rat eyes than in buphthalmic eyes with elevated IOP. The number of RGCs in buphthalmic eyes was uniformly lower in all retinal quadrants and eccentricities, which is attributable to the pressure being equally distributed within the eyeball. This observation is different from buphthalmos in humans, which is characterized by an arcuate pattern of RGC loss. ON-head atrophy and excavation paralleled the RGC loss. The fact that no RGCs died in control retinas of the wild-type rats indicates that, as assumed in the human retina, the major risk factor for cell death is intraocular hypertension. In addition to the primary consequences of elevated IOP, secondary degeneration occurs due to a deficiency of growth factors (Schwartz et al., 1996), lack of endogenous neurotrophins (Johnson et al., 2000), and excitotoxic mechanisms (Dreyer, 1998). This indicates that buphthalmos can be considered a neurodegenerative disease, especially with regard to RGCs. However, the mechanisms underlying ON damage and RGC death remain to be elucidated (Neufeld, 1999). Using immunohistochemistry and Western blotting to examine the expression of various proteins assumed to be involved in retinal degeneration and regeneration could provide insight into the mechanisms underlying buphthalmic retinopathy. There are many reports of the alteration of gene products in the retina following ischemia, retinal detachment, laser photocoagulation, and retinal degeneration after photic or mechanical damage (Grosche et al., 1995; Hangai et al., 1995; Osborne et al., 1991; Yoshida et al., 1993). Molecules that respond to ocular injuries include cytokines (Hangai et al., 1995), growth factors (Tanihara et al., 1997a), proto-oncogenes (Grosche et al., 1995), and intermediate filaments (Grosche et al., 1995; Osborne et al., 1991; Yoshida et al., 1993), and these may be related to damaging, protective, and regenerative processes of nervous tissue (Tanihara et al., 1997b). In the present study we found that GAP-43, GFAP, and ET-1 exhibit age-dependent upregulation, which suggests that both RGCsdwhich specifically express GAP-43 (Benowitz and Routtenberg, 1997)dand glial components of the retina are involved in or respond to retinal degeneration in buphthalmic eyes. GFAP is also upregulated in Mueller cells of the RCS-rat strain due to photoreceptor dystrophy. Under physiological conditions, the level of GFAP is usually minimal in retinal astrocytes in the GCL and NFL (Wang et al., 2000). It is upregulated in astrocytes and Mueller cells following retinal detachment (Okada et al., 1990), retinal degeneration (Ekstrom et al., 1988), and buphthalmos (Naskar et al., 2002; Tanihara et al., 1997b; Wang et al., 2000). This generalized GFAP upregulation points to a gliotic response, but it is not known whether this is a neuroprotective molecular response to stress or injury in the retina. ET-1 is mainly produced in endothelial cells and is one of the most potent known physiological vasoconstrictors (Haefliger et al., 1999). It is synthesized and released from the ciliary process, and its G-protein-coupled ETA and ETB receptors are widely distributed in ocular tissues including the retina and ON (Prasanna

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et al., 2002). ET-1 levels are reportedly significantly higher in the aqueous humor of primary open-angle buphthalmos patients (Noske et al., 1997), in dogs with glaucoma (Kallberg et al., 2002), and in rats with induced buphthalmos (Prasanna et al., 2005) than in the corresponding age-matched controls. ET-1 has been considered to be an important contributing factor in glaucomatous optic neuropathy (Prasanna et al., 2002), although it is postulated that ET-1 plays a dual role in buphthalmos depending on whether it is released in the anterior or posterior chamber of the eye (Yorio et al., 2002). In situ hybridization studies showed that ET-1 mRNA is localized within the innermost layers of the retina as well as in astrocytes (Haefliger et al., 1999; Noske et al., 1997; Prasanna et al., 2002). OX-42 is an indicator of complement type 3 receptor on activated microglial cells (Wang et al., 2000), and is upregulated in buphthalmic retinas. The OX-42-positive cells are mainly located in the GCL, inner plexiform layer (IPL), and inner nuclear layer, whereas in control retinas OX-42 is restricted to retinal cells in the GCL and IPL. As expected, its expression does not change with age in hereditary glaucoma, whereas OX-42-positive microglial cells in experimental buphthalmos dropped to almost control levels at months after the manipulation (Wang et al., 2000). What are the mechanisms of cell resistance to buphthalmos? Pavlidis et al. (2003) demonstrated for the first time that some RGCs, although morphologically altered, can survive abnormally elevated IOP in extremely advanced stages of human buphthalmos with functional blindness. This suggests that RGCs possess intrinsic mechanisms for surviving elevated IOP. This was also demonstrated in the present mutant, which gradually lost RGCs with age. Since the early stages of human RGC loss cannot be examined in buphthalmos patients, it is essential to develop animal surrogates aimed at understanding the molecular biology of the population of RGCs that have not yet succumbed to the disease or have the ability to resist hypertension. This led to an examination of whether RGCs that do not immediately degenerate due to the buphthamos can be induced to extend axons under in vitro conditions (Bodeutsch et al., 1999). It is indeed the case that buphthalmic retinal explants are endowed with a regenerative capacity that gradually decreases with age, but which is still evident in animals with elevated IOP that are older than 1 year. The decrease with increasing age may reflect the elevated IOP affecting axonal growth either via the gradual depletion of RGCs or an age-related increase in the severity of trauma. On the other hand, retinas from non-buphthalmic eyes were unable to extend axons in culture. As an example, RGCs regenerate axons 100-fold in vitro following ONC in vivo (Bodeutsch et al., 1999). It is known that such axonal growth is associated with a dramatic increase in mRNA-encoding transcription factors such as c-Jun, KROX, and CREB (Bodeutsch et al., 1999; Fournier et al., 1997; Herdegen et al., 1993), as well as proteins associated with axonal growth (Fournier et al., 1997; Leon et al., 2000). Our proteomic analysis of buphthalmic retinal explants after axon regeneration revealed that several proteins were differentially expressed. Among these, HSP-60 was the most

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consistently observed in regenerating tissue and was therefore selected for further functional testing in order to elucidate the proteins associated with regenerative growth in glaucoma. HSPs function mainly as molecular chaperones that assist other proteins in intracellular folding and the assembly of polypeptides. Many HSPs are expressed constitutively, but there are also several stressors that increase their expression: heat, viral infection, anoxia, and exposure to certain cytokines such as tumor necrosis factor-a and interferon-g (Ellis, 1990; Hightower, 1991; Tezel et al., 2004). The events of cell stress and cell death are linked, and the HSPs induced in response to stress appear to function as key regulatory factors in the control of apoptosis (Garrido et al., 2001). The accumulation of HSPs in various neurons during acutely toxic metabolic states and in a variety of degenerative, inflammatory, and neoplastic neurological diseases (Plumier et al., 1996, 1997; Satoh and Kim, 1995) suggests that HSPs play a fundamental role in neuronal survival. HSP-60 appeared to be differentially expressed in buphthalmic retinal explants after axon regeneration, thus suggesting that it plays a crucial role in neuritogenesis. The role of HSP-60 in apoptosis has been controversial. Two groups (Samali et al., 1999; Xanthoudakis et al., 1999) have independently shown in HeLa and Jurkat cells that activation of caspase-3 by camptothecin occurs simultaneously with HSP-60 and HSP-10 release from the mitochondria, and demonstrated a proapoptotic role of HSP-60. In contrast with these results, it has been shown that overexpression of HSP-60 and/ or HSP-10 by stable transfection in cardiac myocytes increases their survival rate during ischemia/reperfusion injury (Lin et al., 2001). Confirming previous reports of increased HSP60 expression in human buphthalmic retinas and ON heads (Tezel et al., 2000), our proteomic analyses revealed the upregulation and differential expression of HSP-60 in regenerated RGCs of buphthalmic rats. A positive correlation between HSP-27 expression and axonal regeneration in mature RGCs was also reported recently (Hebb et al., 2006). However, this is the first report of an analogous role of HSP-60 promoting axonal growth of RGCs. We found that the addition of anti-HSP-60 to cultured retinal explants reduced the amount of axonal growth. Previous studies have shown that exogenous HSP antibodies can enter retinal cells via classic receptor-mediated endocytosis within 30 min in culture and activate a proteolytic cascade that includes the activation of caspase-8 and caspase-3 and the cleavage of poly-(ADP ribose) polymerase (Tezel and Wax, 1999, 2000). Using immunogold staining, anti-HSP-27 was detected intracellularly from 6 to 12 h (Tezel and Wax, 2000). This may explain the weak immunohistochemistry fluorescence we observed after 3 days of incubation with HSP-60 antibodies in the present study. Axons were not impaired by the presence of antibodies to HSP-90, GFAP, or anti-HSP-60 attached to BP, thus indicating the specific blockage of HSP-60 by antibodies. Although the downstream regulation of signaling cascades remains to be analyzed, we hypothesize that a proteomic or genomic analysis of regenerated retinas subjected to HSP60 blockage will reveal the specific regulation of such proteins (we are currently investigating this). The mechanisms of

HSP-60-mediated cell survival remain unclear. However, Tezel and colleagues (Tezel and Wax, 1999; Tezel et al., 1998) have shown that direct application of antibodies against small HSPs resulted in neuronal apoptosis, thus supporting the present observation that HSPs involved in cell survival are a prerequisite for axonal growth. In summary, we have studied the regenerative capacity of RGC axons in a rat strain with buphthalmos. In these rats the IOP is elevated, and the ganglion cells die with age. The ON head is excavated and atrophied, and certain proteins are specifically upregulated. We have shown for the first time that some of the ganglion cells, probably those primed by the buphthalmic injury, are able to regenerate their axons under in vitro conditions. These findings indicate that a window of therapeutic opportunity exists before RGCs succumb to a buphthalmos-induced demise, and future studies should investigate the signaling cascades involved in the regenerative ability of buphthalmic RGCs. One putative therapy target is interfering with signaling pathways involving HSPs, as shown in the present study with HSP-60. This model could therefore be useful in the testing of drugs at different stages of the disease, and it offers the opportunity to obtain tissue at various stages of buphthalmos and to analyze the molecular mechanisms of neuronal cell death and survival in the retina. Acknowledgments The authors thank Mechthild-Langkamp Flock and Helene Seifried for their excellent technical assistance, Rita Naskar for introducing us to IOP measurements, and Karin Rose for help with 2D-PAGE. The work was supported by the Interdisziplina¨res Zentrum fu¨r Klinische Forschung (IZKF, Project F5 to S.T.) and the Deutsche Forschungsgemeinschaft (Project Th 386 16-1 and 16-2 to S.T.). References Addison, W.H.F., How, H.W., 1926. Congenital hypertrophy of the eye of an albino rat. Anat. Rec., 271e274. Bahr, M., Vanselow, J., Thanos, S., 1988. In vitro regeneration of adult rat ganglion cell axons from retinal explants. Exp. Brain Res. 2, 393e401. Benowitz, L.I., Routtenberg, A., 1997. GAP-43: an intrinsic determinant of neuronal development and plasticity. Trends Neurosci. 2, 84e91. Bodeutsch, N., Siebert, H., Dermon, C., Thanos, S., 1999. Unilateral injury to the adult rat optic nerve causes multiple cellular responses in the contralateral site. J. Neurobiol. 1, 116e128. Bunt-Milam, A.H., Dennis Jr., M.B., Bensinger, R.E., 1987. Optic nerve head axonal transport in rabbits with hereditary glaucoma. Exp. Eye Res. 4, 537e551. Chang, B., Smith, R.S., Hawes, N.L., Anderson, M.G., Zabaleta, A., Savinova, O., Roderick, T.H., Heckenlively, J.R., Davisson, M.T., John, S.W., 1999. Interacting loci cause severe iris atrophy and glaucoma in DBA/2J mice. Nat. Genet. 4, 405e409. Dreyer, E.B., 1998. A proposed role for excitotoxicity in glaucoma. J. Glaucoma 1, 62e67. Ekstrom, P., Sanyal, S., Narfstrom, K., Chader, G.J., van Veen, T., 1988. Accumulation of glial fibrillary acidic protein in Muller radial glia during retinal degeneration. Invest. Ophthalmol. Vis. Sci. 9, 1363e1371. Ellis, R.J., 1990. The molecular chaperone concept. Semin. Cell Biol. 1, 1e9.

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