Neuroprotective effect of transcorneal electrical stimulation on light-induced photoreceptor degeneration

Experimental Neurology 219 (2009) 439–452

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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 e v i e r. c o m / l o c a t e / y e x n r

Neuroprotective effect of transcorneal electrical stimulation on light-induced photoreceptor degeneration☆ Ying-qin Ni a, De-kang Gan a, Hai-dong Xu b, Ge-zhi Xu a,⁎, Cui-di Da a a b

Department of Ophthalmology, Eye and ENT Hospital of Fudan University, 83 Fen Yang Road, Shanghai 200031, People's Republic of China Institute of Acoustics, Chinese Academy of Science, Shanghai, People's Republic of China

a r t i c l e

i n f o

Article history: Received 2 January 2009 Revised 17 June 2009 Accepted 20 June 2009 Available online 2 July 2009 Keywords: Electrical stimulation Light-induced Photoreceptor degeneration Retina Apoptosis Neurotrophic factor

a b s t r a c t Direct electrical stimulation of neural tissues is a strategic approach to treat injured axons by accelerating their outgrowth [Al-Majed, A.A., Neumann, C.M., Brushart, T.M., Gordon, T., 2000. Brief electrical stimulation promotes the speed and accuracy of motor axonal regeneration. J. Neurosci. 20, 2602–2608] and promoting their regeneration [Geremia, N.M., Gordon, T., Brushart, T.M., Al-Majed, A.A., Verge, V.M.K., 2007. Electrical stimulation promotes sensory neuron regeneration and growth-associated gene expression. Exp. Neurol. 205, 347–359]. Recently, transcorneal electrical stimulation (TCES), a novel less invasive method, has been shown to rescue axotomized and damaged retinal ganglion cells [Morimoto, T., Miyoshi, T., Matsuda, S., Tano, Y., Fujikado, T., Fukuda, Y., 2005. Transcorneal electrical stimulation rescues axotomized retinal ganglion cells by activating endogenous retinal IGF-1 system. Invest. Ophthalmol. Vis. Sci. 46(6), 2147–2155]. Here, we investigated the neuroprotection of TCES on light-induced photoreceptor degeneration and the underlying mechanism. Adult male Sprague–Dawley (SD) rats received TCES before (pre-TCES) or after (post-TCES) intense light exposure. After fourteen days of light exposure, retinal histology and electroretinography were performed to evaluate the neuroprotective effect of TCES. The mRNA and protein levels of apoptoticassociated genes including Bcl-2, Bax, Caspase-3 as well as ciliary neurotrophic factor (CNTF) and brainderived neurotrophic factor (BDNF) in the retinas were determined by real-time PCR and Western blot analysis. The localization of these gene products in the retinas was examined by immunohistochemistry. Both pre- and post-TCES ameliorated the progressive photoreceptor degeneration. The degree of rescue depended on the strength of the electric charge. Post-TCES showed a relatively better and longer-term protective effect than pre-TCES. Real-time PCR and Western blot analysis revealed an upregulation of Bcl-2, CNTF, and BDNF and a downregulation of Bax in the retinas after TCES. Immunohistochemical studies showed that Bcl-2 and CNTF were selectively upregulated in Müller cells. These findings provide a new therapeutic method to prevent or delay photoreceptor degeneration through activating the intrinsic survival system. © 2009 Elsevier Inc. All rights reserved.

Introduction Excessive exposure to light induces irreversible photoreceptor degeneration and visual function loss due to the inability of photoreceptors to regenerate (Noell et al., 1966; Shahinfar et al., 1991; Abler et al., 1996; Hafezi et al., 1999). Studies have been performed on potential therapeutic strategies to promote photoreceptor survival, but a complete functional recovery has not been achieved and remains a major goal of this research area. Electrical stimulation has been associated with neuroprotection in several different neural tissues. In motor and sensory neurons, electrical stimulation accelerated axonal regeneration and restored

☆ This work was supported by research grants from the National Basic Research Program of China (No. 2007CB512205) and the National Natural Science Foundation for the Young Scholars of China (No. 30801267). ⁎ Corresponding author. Fax: +21 64376491. E-mail address: [email protected] (G. Xu). 0014-4886/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2009.06.016

the specificity of regeneration by enhancing expression of regeneration- and growth-associated genes (Al-Majed et al., 2000; Geremia et al., 2007; Brushart et al., 2005). In the auditory system, chronic electrical stimulation promoted the survival of spiral ganglion cells that otherwise would have degenerated from the drug ototoxic effect (Lousteau, 1987; Leake et al., 1999; Miller and Altschuler, 1995). In the visual system, subretinal electrical stimulation rescued photoreceptor degeneration in the Royal College of Surgeon's (RCS) rat (Pardue et al., 2005a), and direct electrical stimulation of the transected optic nerve (ON) stump promoted the survival of axotomized retinal ganglion cells (RGCs) in vivo (Morimoto et al., 2002). As a less invasive method, TCES presents a potentially a new clinical therapeutic option. Recently, TCES was observed to rescue axotomized RGCs (Morimoto et al., 2005) and improve the visual function of patients with traumatic optic neuropathy (TON) or nonarteritic ischemic optic neuropathy (NION) (Fujikado et al., 2006). In contrast to the research investigating RGCs damage, little is known about the effect of TCES on light-induced photoreceptor degeneration. Based on previous research, we

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hypothesized that TCES would also have a neuroprotective effect on photoreceptor apoptosis, which was recognized as the final common death pathway in many human retinal diseases such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD) (Wenzel et al., 2005). Neuronal survival appears to depend on the balance between cell survival and cell death signaling. Some in vivo and in vitro studies have shown that neutralizing the imbalance between survival and cell death signals can block crucial steps in apoptosis and prevent cell death (Caprioli, 1997; Yoles and Schwartz, 1998). In the visual system, neurotrophins including CNTF and BDNF have been identified as potent survival factors for the injured photoreceptors (Huang et al., 2004; Tao et al., 2002; Kano et al., 2002; Caffé et al., 2001). Additionally, apoptosis-associated genes such as Bcl-2, Bax and Caspase-3 also played important roles in the survival response for light-induced photoreceptor degeneration (Hahn et al., 2003; Chen et al., 1996). It has been reported that TCES can regulate the expression of neurotrophic factors in neural tissues such as insulin-like growth factor-1 (IGF-1) (Morimoto et al., 2005). However, whether TCES regulates the expression of apoptosis-associated genes and whether it alters the intrinsic survival microenvironment of photoreceptors have not been examined. Excessive exposure to light directly induces photoreceptor apoptosis in albino rats and mice (Noell et al., 1966; Shahinfar et al., 1991; LaVail et al., 1999). Thus, these animals are the classic models to study the photoreceptor degeneration in human retinal diseases such as RP and AMD. The purpose of this study was to evaluate the effect of TCES on the survival of photoreceptors after exposure to intense light in vivo and to determine the underlying mechanisms. The morphologic and electrophysiological analyses in the present study showed that TCES delayed the progression of light-induced photoreceptor degeneration and that this effect was associated with increased levels of Bcl2,CNTF and BDNF and a decreased level of Bax, suggesting the important role of intrinsic survival microenvironment, activated by TCES, in photoreceptor protection. Materials and methods Animals All procedures were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All experimental procedures were approved by the Institutional Review Board at Fudan University Graduate School of Medicine. Adult male SD rats weighing 225–250 g were housed in a 12/12-h light/dark cycle. Commercial rat diet and water were provided ad libitum. Animals were anesthetized by intraperitoneal injection of ketamine (120 mg/kg) and xylazine (12.5 mg/kg) during the examination and euthanized by an overdose of pentobarbitone at the end of the experiment. For histological and functional evaluation, 120 rats were randomly divided into 3 groups: the control group (rats only receiving light exposure), the pre-TCES group (rats receiving TCES before light exposure) and the post-TCES group (rats receiving TCES after light exposure) (n = 6/parameter/time point). Another 112 rats were used at different time points after 1-hour TCES (300 μA, 20 Hz, 3 ms/phase) for immunohistochemistry (n = 2/time point), real-time PCR (n = 6/ time point) and Western blot analyses (n = 6/time point). Light exposure Albino rats were placed separately in cages, and both eyes were exposed to evenly-distributed bright blue light. The floor of each cage was illuminated by approximately 2500 lx. The temperature inside each cage was maintained at 24 °C. The rats were placed under these conditions at the same time of day for each experiment and

returned to their normal light/dark cycle after 24 h of intense blue light exposure. Transcorneal electrical stimulation For electrical stimulation, a noninvasive contact lens electrode with a golden wire ring attached to the inner surface was used as the stimulating electrode, and a needle electrode through the ipsilateral subcutaneous tissue served as a reference electrode. The contact lens electrode was placed on the cornea after surface anesthesia by 0.4% oxybuprocaine HCL and corneal protection with 1.3% hydroxyethylcellulose gel. The electrical stimuli consisting of biphasic rectangular wave pulses was generated by a stimulator (JL-E; Shanghai, China) and delivered from a constant current isolator (JL-G; Shanghai, China). In the pre-TCES group, rats received 1.5 h TCES (current intensity: 100–500 μA; pulse duration: 3 ms; frequency: 20–100 Hz) before exposure to intense light. Fundus examination was carried out before and after light exposure to ensure that rats with any opacity of media would not be used any further in the experiment. In the post-TCES group, rats received 1 h chronic and low-level TCES (current intensity: 200 μA, 300 μA; pulse duration: 3 ms; frequency: 20 Hz) every 3 d after exposure to light for up to 14 d. Electroretinography Electroretinograms (ERGs) were recorded at 14 d after light exposure (UTAS-E3000 Visual Electrodiagnose System; LKC Technologies, Gaithersburg, MD). The scotopic flash electroretinograms were recorded from dark-adapted rats by placing a golden-ring electrode in contact with the cornea (Hansen Ophthalmic Development Lab, USA) and a reference electrode through the tongue. A grounding electrode was attached to the scruff of the neck. The pupils were dilated with 1% tropicamide and 2.5% phenylephrine, and the corneas were kept moist with application of 1% carboxymethylcellulose as needed. All procedures were performed in dim red light and the rats were kept warm during and after the procedures. Stimuli were brief white flashes delivered via a Ganzfield integrating sphere, and signals were recorded with band-pass settings of 0.3 to 500 Hz. After a 10-minute stabilization in the dark, a scotopic intensity-response series (6 recordings, from −32 dB to 2.5 dB) was given which included rod-specific and bright-flash responses. The a-wave was measured as the difference in amplitude between the recording at 5 ms and the trough of the negative deflection. The b-wave amplitude was measured from the trough of the a-wave to the peak of the b-wave. The baseline recordings were taken at least 7 d before treatment. Histopathology and immunohistochemistry Anesthetized rats were transcardially perfused with 200 ml of 0.9% saline, followed by 4% paraformaldehyde (PFA) solution in 0. 1 M PB (pH = 7.4). The eyes were then enucleated and immersion fixed in 4% PFA for 1 h, transferred to 10% neutral-buffered formalin overnight, and processed for routine paraffin-embedded sectioning using an automated tissue processor (Shandon Pathcentre, Thermo Shandon Inc., Pittsburgh, PA, USA). The eyes were embedded sagittally and 5μm serial sections were cut with a rotary microtome (Microm HM 330, McBain Instruments, Chatsworth, CA). All eyes were cut vertically, and only sections through optic nerves were collected for subsequent studies. Hematoxylin and eosin staining were used. The thickness of the outer nuclear layer (ONL) was determined by an image-analysis system (Leica Qwin QG2-32, Bensheim, Germany). In total, 18 locations of each retinal section (superior quadrant: S1–S9; inferior quadrant: I1–I9; Fig. 1A) were measured starting from either side of the optic nerve with each segment at 0.5 mm apart. For immunohistochemical studies of retinal sections, eyecups were fixed in 4% PFA for 2 h after removal of the cornea and lens. The

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Fig. 1. Hematoxylin–eosin staining of rat retinas showed light-induced photoreceptor degeneration and the protective effects of TCES. (A) Schematic diagram of retinal vertical sections through the optic nerve. S1–S9 and I1–I9 indicated nine measured regions with each 0.5 mm wide in the superior and inferior quadrant respectively. All the pictures were taken in the S3 area which was susceptible to intense light. (B) 10–12 rows of photoreceptors were in the ONL of normal retina. The ONL thickness decreased remarkably with only 2– 3 rows of photoreceptor remaining at 7 d after light exposure (C) and photoreceptors almost disappeared at 14 d (D) in the control retinas. The ONL (3–5 rows of photoreceptors), inner and outer segments were relatively better preserved in both 20 Hz, 300 μA (E) and 20 Hz, 400 μA (F) pre-TCES retinas at 7 d after light exposure. 20 Hz, 200 μA (G) and 20 Hz, 300 μA (H) post-TCES treatments significantly increased the ONL thickness even at 14 d when compared with the control group (D). Arrows indicate the ONL. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; ONH, optic nerve head. Bar = 75 μm.

eyecups were cryoprotected in graded sucrose solutions (20%–30% in PB) at 4 °C, embedded in optimal cutting temperature (OCT) compound (Tissue-Tek; Ted Pella, Inc., Redding, CA) and frozen in liquid nitrogen. 10-μm sections were cut using a cryostat (Bright Instruments Ltd., Huntingdon, UK). Sections within approximately 1 mm of the optic nerve head were recovered onto gelatin-coated slides. Air-dried slides were incubated with blocking buffer (PBS

containing 5% goat serum) at room temperature for 1 h. After three washes with 0.01 M PBS, the sections were incubated with the following primary antibodies: monoclonal mouse anti-rat Bcl-2 antibody (1:500; Sigma-Aldrich, St. Louis, MO, USA) and/or polyclonal rabbit anti-rat GFAP antibody (1:400; Sigma-Aldrich, St. Louis, MO, USA); mouse anti-rat CNTF antibody (1:200; Chemicon, Temecula, CA) and/or polyclonal rabbit anti-rat GS antibody (1:800; Sigma-

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Aldrich, St. Louis, MO, USA); polyclonal rabbit anti-rat BDNF antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA); monoclonal rabbit anti-rat Bax antibody (1:200; Epitomics, California, USA). The sections were then rinsed three times in 0.01 M PBS and incubated with the secondary antibody conjugated to CY3 (1:200; Sigma-Aldrich, St. Louis, MO, USA) and/or secondary antibody conjugated to FITC (1:200; Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 45 min. Followed by three rinses with 0.01 M PBS, the sections were mounted with an antifade mounting medium (Vectashield; Vector Laboratories, Burlingame, CA) and examined under a confocal laser microscope (TCS SP2; Leica Microsystems, Bensheim, Germany). Quantitative real-time PCR Eyes that underwent 1-hour TCES (300 μA, 20 Hz, 3 ms/phase) were removed at selected time points from 2 h to 14 d. The retinas were dissected in a shallow bath of cold phosphate buffered saline (PBS) and were stored at liquid nitrogen until use. Total RNA was extracted by RNeasy Mini Kit (Qiagen, Hilden, Germany) from pooled retinas and quantified spectrophotometrically (Gene Quant II; Amersham Pharmacia Biotech, Piscataway, NJ). First-strand cDNA was synthesized from 1 μg total RNA by reverse transcription using oligo-dT primers and reverse transcriptase (superscript II; Invitrogen) according to the manufacturer's instructions. Real-time PCR reactions were performed in a 20 μl mixture, containing 1 μl of the cDNA preparation, 10 × PCR mix (iQ SYBR Green Supermix; Bio-Rad, Hercules, CA) and 500 nm of each primer, in a thermocycler (iCycler iQ system; Bio-Rad) using the following PCR parameters: 95 °C for 5 min followed by 50 cycles at 95 °C for 15 s, 65 °C for 15 s, and 72 °C for 15 s. The fluorescence threshold (Ct) was calculated with the system software. The absence of nonspecific products was confirmed by both the analysis of the melting-point curves and by electrophoresis in 3% agarose gels. GAPDH served as an internal standard of mRNA expression. The primer sequences were: Bcl-2 forward, 5′-GGATTGTGGCCTTCTTTGAGTTC-3′, reverse,5′-AGGTATGCACCCAGAGTGATGC-3′; Bax forward, 5′-CTGACATGTTTGCTGATGGCA-3′, reverse, 5′-TGAGGACTCCAGCCACAAAGA-3′; Caspase-3 5′-AATTC AAGGGACGGGTCATG-3′, reverse, 5′-GCTTGTGCGCGTACAGTTTC-3′; CNTF forward, 5′-TGTACCAGTGGCAAGCACTGA-3′, reverse, 5′-TAGCT GGTAGGCAAAGGCAGA-3′; BDNF forward, 5′-CTGACACTTTTGAGCAC GTGATC-3′, reverse, 5′AGGCTCCAAAGGCACTTGACT-3′; GAPDH forward, 5′-TGAACGGGAAGCT-3′, reverse, 5′-TCCACCACCCTGTTGC TGTA-3′. Western blot analysis The retinas were homogenized in RIPA buffer containing 1% Triton X-100, 5% SDS, 5% deoxycholic acid, 0.5 M Tris–HCl pH 7.5, 10% glycerol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 × 10− 12 μg/ml aprotinin, 1 × 10− 12 μg/ml leupeptin, 1 × 10− 12 μg/ml pepstatin, 200 mM sodium orthovanadate and 200 mM sodium fluoride. Tissue extracts were incubated on ice for 10 min and centrifuged at 10,000 ×g for 25 min at 4 °C. Total protein in retinal extracts was measured using a standard BCA assay (Pierce, Rockford, IL, USA), and the protein concentration was determined by the Lowry method (Bio-Rad Life Science, Mississauga, ON, Canada). Retinal extracts were resuspended in 5× sample buffer (60 mM Tris–HCl pH 7.4, 25% glycerol, 2% SDS, 14.4 mM 2-mercaptoethanol, 0.1% bromophenol blue), boiled for 5 min, and resolved on a 12% SDS-PAGE gel. Proteins were transferred onto a nitrocellulose membrane (Hybond-C, Amersham Pharmacia Biotech, Germany), and blots were stained with Ponceau S (Sigma-Aldrich, St. Louis, MO, USA) to visualize the protein bands, in order to ensure equal protein loading and a uniform transfer. The blots were then washed with TBST buffer (20 mM Tris–HCl pH 7.6, 137 mM NaCl, and 0.1% Tween 20) and blocked with 5% non-fat dry milk in TBST buffer for 45 min. The blots were probed with primary antibody at 4 °C for 24 h, followed by incubating with the horseradish-

peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:10,000 dilution) at room temperature for 1 h. The primary antibodies used in this study were monoclonal mouse anti-rat Bcl-2 antibody (1:1000, Sigma-Aldrich, St. Louis, MO, USA); monoclonal mouse anti-rat Bax antibody (1:1000, Sigma-Aldrich, St. Louis, MO, USA); monoclonal mouse anti-rat CNTF antibody (1:800, Chemicon; Temecula, CA); polyclonal rabbit anti-rat BDNF antibody (1:600, Santa Cruz Biotechnology, Santa Cruz, CA). The bound antibodies were detected using an enhanced chemiluminescence system (ECL, Amersham, MA, USA) and X-ray film. Signal intensity was measured by using an ImageMasterRVDS (Pharmacia Biotech, CA, USA), and the optical densities (mean ± SD) for each sample were obtained from three measurements on three separate blots. Data analysis The data on the average ONL thickness of the whole retina and from each defined location across the retina from various groups were compared with a one-way ANOVA or Student's t-test. The amplitudes of intensity-related and dark-adapted ERG a- and b-waves from various groups were compared by the one-way ANOVA. Results Survival-promoting effect of TCES on injured photoreceptors after intense light exposure Retinal morphology Albino rats exposed to intense blue light showed significant photoreceptor cell loss and progressive decrease of the ONL thickness. At 7 d after light exposure (Fig. 1C), the average ONL thickness of the whole retina was significantly decreased (16.77 ± 1.10 μm, 43.57% of normal) with disorganization of the inner and outer segments when compared with normal rats (38.49 ± 4.62 μm) (Fig. 1B). At 14 d after light exposure (Fig. 1D), the photoreceptors in the posterior retina of the superior hemisphere (S2–S5 region), which was most susceptible to light damage, almost completely disappeared with only one row of cells remaining and the average ONL thickness was significantly decreased (13.18 ± 0.65 μm, 34.24% of normal). In rats receiving 1.5-hour, 20 Hz, 300 μA (Fig. 1E) or 20 Hz, 400 μA (Fig. 1F) pre-TCES, 4 to 6 layers of photoreceptors remained intact in the posterior region, with some inner and outer segments present at 7 d after light exposure. However, only 2 to 3 layers of photoreceptors remained intact in the similar area in the control group (Fig. 1C). Morphometric analysis also showed that the average ONL thickness in the moderate currents (300 μA, 400 μA, 500 μA) pre-TCES group was significantly increased compared with the control at 7 d, however no difference at 14 d (Fig. 2A). This suggested that preTCES provided a transient neuroprotective effect on light-induced photoreceptor degeneration. The transient neuroprotective effect of pre-TCES was also intensity and frequency-related. Pre-TCES with 300 μA, 400 μA and 500 μA increased the ONL thickness from 43.57% of normal to 51.48%, 60.87% and 59.87% respectively (Fig. 2A). Regional analysis showed that 300 μA pre-TCES provided better preservation in the central retinas than the peripheral retinas, while 400 μA and 500 μA provided similar preservation in both the central and peripheral retinas at 7 d (Fig. 2B). The average ONL thickness in the 50 Hz pre-TCES group (26.34 ± 1.45 μm, 200 μA; 25.89 ± 3.05 μm, 300 μA) was significantly increased compared with 20 Hz group (17.65 ± 1.59 μm, 200 μA; 19.04 ± 0.84 μm, 300 μA) (P b 0.01 respectively) at 7 d (Fig. 2C). Post-TCES with chronic and low-level repetitive stimulation showed a relatively better and longer-term neuroprotective effect on light-induced photoreceptor degeneration than pre-TCES (Figs. 1G, H and 3). In contrast to the severe loss and damage of the photoreceptors of the control group, the ONL and outer segments were remarkably

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with 200 μA (23.31 ± 0.33 μm, 7 d; 23.16 ± 0.54 μm, 14 d) and 300 μA (25.85 ± 0.58 μm, 7 d; 24.53 ± 1.52 μm, 14 d) were significantly increased compared with the control group at both 7 d (16.78 ± 1.15 μm) and 14 d (13.03 ± 0.78 μm) (P b 0.01 respectively) (Fig. 3A). The average ONL thicknesses at 14 d after light exposure in both the post-TCES and the control groups were significantly less than at 7 d. Furthermore, post-TCES showed a neuroprotective effect in the peripheral retinas as well as the superior and inferior central retinas (Fig. 3B). Retinal function The effect of TCES on light-induced photoreceptor degeneration was further examined electrophysiologically by dark-adapted ERG. Post-TCES (20 Hz, 200 μA; 20 Hz, 300 μA) groups showed significantly higher responses in the rod photoreceptor a-wave amplitudes with stimulation intensities ranging from − 8 dB to 2.5 dB when compared with the control group at 14 d after light exposure (P b 0.01, respectively, Figs. 4A, B). For the b-wave, post-TCES groups also showed higher responses with stimulation intensities ranging from −24 dB to 2.5 dB (P b 0.01, respectively, Figs. 4A, C). At the same time point, both of the a-wave and b-wave amplitudes in the pre-TCES groups (20 Hz, 300 μA; 20 Hz, 400 μA) showed no significant difference compared with the control group (Fig. 4). Retinal levels of Bcl-2, Bax, Caspase-3, CNTF and BDNF after TCES Rats without light exposure were used for real-time PCR and Western blot analysis after 1-hour 300 μA, 20 Hz, 3 ms/phase TCES

Fig. 2. Pre-TCES showed a relatively transient neuroprotective effect on light-induced photoreceptor degeneration which was current intensity and frequency-dependent. (A) In the control retinas, the average ONL thickness decreased to 43.57% of normal at 7 d and 34.24% at 14 d after light exposure. In the moderate current (300 μA, 400 μA, 500 μA) preTCES groups, the ONL thickness was significantly increased compared with the control at 7 d, however no difference at 14 d. (B) In the control retina, the photoreceptors were more severely damaged in the superior retinas than those in the inferior retinas and the ONL thickness was less at the posterior retinas than that in the peripheral. Significant protection of pre-TCES with different current intensities was noted in both the superior and inferior central quadrants of the retinas at 7 d. (C) The neuroprotective effect of preTCES was also frequency-dependent. The average ONL thickness of the 50 Hz groups was significantly better preserved than the 20 Hz groups at 7 d. A, B: One-way ANOVA; C: Student's t-test for two groups; mean ± SD, (n = 6), ⁎, P b 0.05, ⁎⁎, P b 0.01.

well preserved in the posterior region of the 200 μA (Fig. 1G) and 300 μA (Fig. 1H) post-TCES groups. Morphometric analysis also showed that the average ONL thicknesses of the post-TCES groups

Fig. 3. Post-TCES showed a relatively better and longer-term neuroprotective effect on light-induced photoreceptor degeneration. (A) The average ONL thickness in 200 μA and 300 μA post-TCES groups significantly increased compared to the control group at both 7 d and 14 d after light exposure. (B) The significant ONL thickness preservation of the post-TCES groups was noted in the peripheral retinas as well as in the superior and inferior central retinas at 14 d. One-way ANOVA; mean ± SD, (n = 6), ⁎⁎, P b 0.01.

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Fig. 4. Measurement of the dark-adapted ERG amplitudes at 14 d after exposure to light. (A) Dark-adapted ERG responses measured at 14 d after exposure to light. Stimulus flash intensity is indicated along the left side. (B, C) ERG amplitude versus flash intensity curves. Post-TCES groups preserved better dark-adapted a-wave (B) and b-wave (C) than the preTCES groups and the control. One-way ANOVA; mean ± SD, (n = 6), ⁎⁎, P b 0.01.

treatment. Real-time PCR was used to survey the changes in the mRNA expression of the following apoptotic-associated genes: Bcl-2, Bax and Caspase-3. A quantitative analysis showed that the mRNA level of Bcl2 increased sharply from 2 h, to peak at 6 h (31-fold above the level of intact normal retina) and remained elevated even at 14 d after TCES (Fig. 5A). However, the mRNA level of Bax was significantly downregulated from 2 h, reached a trough at 12 h (10% of normal retina), remained decreased at 3 d, and came back to the baseline at 7 d (Fig. 5B). The mRNA expression of Caspase-3 did not change significantly (Fig. 5C). Western blot analysis showed that the Bcl-2 protein increased significantly at 2 h, reached its peak at 7 d (7.6-fold above the normal) and remained elevated at 14 d (Figs. 6A, B), while the Bax

protein was downregulated at 2 h and continuously decreased at 14 d (Figs. 6A, C), confirming the results obtained from the real-time PCR. However, the Bcl-2 and Bax proteins changed relatively slower and smoother throughout the timecourse, and remained elevated or decreased even as mRNA returned to normal baseline levels. A quantitative analysis of CNTF and BDNF mRNA and protein expression was also performed by real-time PCR and Western blot analysis. The mRNA level of the two neurotrophic factors were both significantly upregulated at 1 d with CNTF peaking at 7 d (5.94-fold above the normal) and BDNF peaking at 3 d (10.72-fold above the normal) (Figs. 5D, E). At 14 d, both of the CNTF and BDNF mRNA came down to the baseline level (Figs. 5D, E). Western blot analysis showed

Y. Ni et al. / Experimental Neurology 219 (2009) 439–452 Fig. 5. Real-time PCR analysis for Bcl-2, Bax, Caspase-3, CNTF and BDNF mRNA in the retinas at different time points ranging from 2 h to 14 d after 1-hour TCES (300 μA, 20 Hz, 3 ms). (A) The Bcl-2 mRNA was upregulated as early as 2 h after TCES, reached the peak at 6 h and slowly came back toward the baseline at 14 d. (B) The Bax mRNA was downregulated to reach the trough at 12 h and come back to the baseline at 7 d. (C) In contrast, the levels of Caspase-3 mRNA in retinas after different time points of TCES treatment didn't show statistical significantly change when compared with normal retinas. (D, E) The CNTF and BDNF mRNA were upregulated at 1 d with CNTF peaking at 7 d and BDNF at 3 d. The GAPDH served as the loading control. The mean ± SD of data from three independent experiments was shown.

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Fig. 6. Western blot analysis for Bcl-2, Bax, CNTF, BDNF protein expression in the retinas after 1-hour TCES (300 μA, 20 Hz, 3 ms). (A).The gel was shown as one representative of three independent experiments. Densitometric analysis showed the upregulation of Bcl-2 (B), CNTF (D), BDNF (E) and a downregulation of Bax (C) protein level after TCES. β-actin is served as the loading control. The mean ± SD of data from three independent experiments is shown.

that the CNTF and BDNF proteins were gradually upregulated from 1 d to 7 d and remained elevated at 14 d (Figs. 6A, D, E). Immunohistochemical analysis of Bcl-2, Bax, CNTF and BDNF in the retinas after TCES Immunohistochemical studies were performed with Bcl-2, Bax, CNTF and BDNF antibody to determine the distribution of those differentially expressed proteins in the retinas from 1 d to 14 d after TCES. In the normal retinas, Bcl-2 immunoreactivity was weak and restricted primarily to the ganglion cell layer (GCL) (Fig. 7A). At 1 d

after TCES, intense immunoreactivity of Bcl-2 appeared from the inner limiting membrane (ILM) to the GCL (Fig. 7B). At 3 d, Bcl-2 immunoreactivity further expanded, and the radial elements extending from the ILM to the inner plexiform layer (IPL) were detected (Fig. 7C). At 7 d, the staining for Bcl-2 was strongest within the inner retina, and intense staining for Bcl-2 was seen in the radial processes of the ILM through the inner nuclear layer (INL) (Fig. 7D). At 14 d, the immunoreactivity for Bcl-2 in the inner retina decreased but its signals in the radial processes remained within the nerve fiber layer (NFL) and GCL (Fig. 7E). For Bax, very weak positive staining was detected in the cytoplasm of the RGCs in the normal retina while

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Fig. 7. Immunohistochemical analysis of Bcl-2 in the retinas after 1-hour TCES (300 μA, 20 Hz, 3 ms). (A–E) Localization of Bcl-2 in the retinas at different time points after TCES. (A) Bcl-2 immunoreactivity in the GCL of the normal retina. Bcl-2 staining increased at 1 d after TCES (B) and increased more and spread from the ILM to the GCL at 3 d after TCES (C). Bcl2 immunoreactivity reached a peak at 7 d (D) and decreased at 14 d (E). (F–N) Double staining for Bcl-2 (red) and GFAP (green), a marker of activated Müller cells in the retina. In the normal retinas, weak Bcl-2 immunoreactivity was detected only in the GCL (F) and GFAP immunoreactivity was present in the astrocytes (G). The merged image (H) showed no colocalization. At 7 d after TCES, strong immunoreactivity for Bcl-2 appeared from the ILM to the IPL (I), and the merged image showed that Bcl-2 immunoreactivity appeared in the endfeet and processes of Müller cells (K). A high-magnification view of Bcl-2 and GFAP colocalization (N) strongly suggested that Bcl-2 was localized in the endfeet and processes of Müller cells toward the outer retina. Scale bar: (A–K) 50 μm; (L–N) 10 μm.

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Fig. 8. Immunohistochemical analysis of CNTF in the retinas after 1-hour TCES (300 μA, 20 Hz, 3 ms). (A–D) Localization of CNTF in the retinas at different time points after TCES. (A) CNTF immunoreactivity in the normal retina. CNTF staining increased at 1 d after TCES (B) and increased more in the nucleus of INL at 3 d (C). At 7 d after TCES, strong immunoreactivity for CNTF appeared from the ILM to the OLM (D). (D–F) Double staining for CNTF (red) and GS (green), a specific marker for Müller cells in the retina. The merged image showed that CNTF immunoreactivity selectively expressed in the endfeet and processes of Müller cells (F) which was similar to Bcl-2. Bar = 75 μm.

undetectable staining in the retinas at all measured time points after TCES (data not shown). Similar to Bcl-2, the expression of CNTF after TCES showed a time-dependent and radial expanding pattern from the GCL to the outer retina (Figs. 8A–D). Cytoplasmic immunoreactivity of BDNF was noted in the GCL of the normal retina. After TCES, the overall staining in the INL was much weaker than that in the GCL at 1 d, but a number of intensely labeled cells were observed in the inner most region of the INL at 3 d and 7 d (Figs. 9A–D).

the retinas at higher magnification at 7 d after TCES demonstrated that the strong Bcl-2 immunoreactivity appeared in the endfeet and processes of Müller cells (Figs. 7L–N), confirming that Bcl-2 was localized in Müller cells. Similar to Bcl-2, at 7 d after TCES, there was strong coimmunolocalization of CNTF and GS in the retina indicating the expression of CNTF in Müller cell processes (Figs. 8D–F). Preliminary safety analysis of TCES

Bcl-2 and CNTF in Müller cells Based on the radial distribution of Bcl-2 and CNTF signals in the retina assayed by immunohistochemical studies, we speculated that Müller cells would be the major origin of those factors. To test this hypothesis, further double-labeling immunofluorescence was performed on the retina with antibodies to Bcl-2, CNTF and GFAP which is expressed in activated Müller cells or GS which is known as the Müller cell marker. No colocalization of Bcl-2 and GFAP was observed in the normal retina (Figs. 7F–H). At 7 d after TCES, strong immunoreactivity for Bcl-2 appeared from the ILM to the IPL which was colocalized with GFAP (Figs. 7I–K). Examination of

In the 400 μA, 50 Hz pre-TCES group, 2 animals (2.38%) of the 84 pre-TCES rats were observed with slight corneal epithelium proliferation and retinal damage by histopathologic analysis (Fig. 10). Compared with the normal control (Fig. 10A), the two rats showed similar sparsely and disorderly distribution of the corneal basal cells and proliferation of squamous cells (Fig. 10B). In the retina, one rat showed disorganized photoreceptors and obvious proliferation of INL cells (Fig. 10C). The other showed scleral penetration (Fig. 10D), which we have never observed in the natural degeneration of photic injury model. None of the above histopathologic changes was observed in post-TCES rats.

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Fig. 9. Immunohistochemical analysis of BDNF in the retinas after 1-hour TCES (300 μA, 20 Hz, 3 ms). (A–D) Localization of BDNF in the retinas at different time points after TCES. (A) Weak cytoplasmic immunoreactivity of BDNF in the GCL of the normal retina. BDNF immunoreactivity increased slightly in the inner most region of the INL at 1 d after TCES (B), more at 3 d (C) and reached the peak at 7 d (D). Bar = 50 μm.

Discussion Our present study demonstrated that TCES prolonged the survival of photoreceptors and delayed the decrease of retinal function after intense light exposure in vivo. The degree of protection was dependent on the starting time and strength of TCES: chronic and low-intense

post-TCES significantly promoted photoreceptor survival up to 14 d after light exposure, while acute pre-TCES only had a transient protection against photoreceptor degeneration at 7 d. Our results also showed that the mRNA and protein levels of Bcl-2, CNTF and BDNF were time-dependently upregulated and Bax was downregulated in the retinas after TCES. Immunohistochemical analyses showed that

Fig. 10. Histopathologic changes of the cornea and retina after 1.5 h, 400 μA, 50 Hz TCES. The corneal basal cells were sparsely and disorderly distributed and the squamous cells demonstrated significant proliferation (black arrow) (B) when compared to the normal control (A). (C) The photoreceptors were disorganized with rosette formation. (D) The sclera was penetrated with the infiltration of some large nuclear cells as the black arrow indicated. Bar = 75 μm.

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Bcl-2 and CNTF were specifically located in Müller cells, and TCES led to a spread of those factors from the inner retina to the outer layers. These results support our hypothesis that TCES activated the intrinsic survival system, especially the activation of Müller cells, and then protected the light-injured photoreceptors. TCES promotes photoreceptor survival after light injury In a Food and Drug Administration-approved Phase I Study, scientists accidentally discovered that low-level electrical stimulation, produced by a subretinal implant (artificial silicon retina, ASR), could provide a neuroprotective effect on RP patients: 10 RP patients accepted ASR implantation in the temporal periphery of the retina reported improvement in visual function in areas that were distinctly different from the implant sites (Chow et al., 2004). Based on these clinical discoveries, the effect of ASR in RCS rats was investigated and the results indicated that the subretinal electrical stimulation provided temporary retinal function and photoreceptor preservation (Pardue et al., 2005a). However, further study showed that sham surgery or an inactive implant were also effective in protecting photoreceptors indicating that mechanical injury or a chronic foreign body may provide similar protective effect as subretinal electrical stimulation (Pardue et al., 2005b). Additionally, subretinal electrical stimulation is too invasive to be clinically applicable. Researchers then investigated the less invasive TCES, which is known to evoke electrical responses in the superior colliculus and activate the entire retina. TCES consistently showed the survival-promoting effect on the axotomized RGCs in animal models (Morimoto et al., 2005) and on the injured RGCs in patients with optic nerve diseases (Fujikado et al., 2006). In addition, Morimoto et al. (2007) demonstrated that 100 μA TCES once a week significantly prolonged the survival of photoreceptors and delayed the decrease of retinal function in RCS rats. Their findings indicated that noninvasive TCES could be used to delay the degenerating progress of patients with inherited photoreceptor degeneration. However, the RCS rat model is a counterpart of only one type of human RP, and thus more work is needed to confirm the protective effect of TCES on different photoreceptor degeneration animal models. In this regard, it is our interest to determine if TCES also has potential neuroprotective effect on the injured photoreceptors exposed to intense light in which the retinal degeneration proceeds faster and in a more synchronized way than degeneration in most inherited animal models including RCS rats (Wenzel et al., 2005). Our morphologic and electrophysiological analyses demonstrated that TCES with optimum parameters prolonged the survival of photoreceptors and preserved the retinal function against the light-induced photoreceptor degeneration. The neuroprotective effect of TCES on photoreceptor degeneration was dependent on the current intensity, the frequency of current pulses, and the starting time. It was consistent with previous research investigating the parameters of electrical stimulation on RGCs survival (Okazaki et al., 2008). In our examination, 1.5 h pre-TCES only had a temporary effect in promoting photoreceptor survival at 7 d after exposure to light, but the chronic and low-level post-TCES showed a relatively long-term survival-promoting effect at 14 d. It is conceivable that the acutely injured and progressively degenerated photoreceptors need repetitive and mild electrical stimulation to induce sustained neuroprotective effect in the microenvironment against light damage for long-term survival. It is interesting that 300 μA pre-TCES provided better preservation in the central retinas than the peripheral retinas. We speculated that this regional difference may be caused by the asymmetrical distribution of the relative low-density current as it preferred to go through the vitreous via a low-impedance path such as the optic nerve, which is located in the central retina. Additional experiments are needed to further investigate this probability. It has been previously reported that 100 μA 1 ms/phase TCES was able to evoke electrical responses in the superior colliculus (Potts et al., 1968; Potts and Inoue, 1970; Shimazu et al., 1999). Thus, we cannot

rule out the possibility that the electrical activation of the photoreceptors may have contributed to the effect of TCES. However, unlike RGCs and dorsal root ganglion cells (DRGs) which can be antidromically activated by a specific frequency (20 Hz) of electrical stimulation (Okazaki et al., 2008; Udina et al., 2008), photoreceptors seemed insensitive to a specific parameter in our study because both of the pre- and post-TCES showed a frequency or intensity-dependent survival-promoting effect. On the other hand, as a photosensitive cell with the ability of phototransduction, whether the photoreceptor can be directly depolarizated or hyperpolarizated by electrical stimulation is still under investigation. Therefore, we speculate that the activation of intrinsic survival microenvironment is important for TCES-induced neuroprotection, even though some other mechanisms may contribute to the effect of TCES. Intrinsic retinal survival microenvironment as a key system for TCES-induced neuroprotection Following injury, two major events including programmed cell death and survival pathways occurred simultaneously to maintain the cellular homeostasis in the retina (Kim and Park, 2005). In lightinduced photoreceptor degeneration, photoreceptor death predominates when the intrinsic cell survival machinery loses the capacity to resist extrinsic injury caused by intense blue light exposure (Grimm et al., 2000a; Grimm et al., 2000b; Hao et al., 2002). In this case, an intrinsic cell survival mechanism triggered at the early stage of photic injury may play a critical role in control of the retinal photoreceptor apoptotic or survival decision. Recently, electrical stimulation of the cerebellar fastigial nucleus (FNS) has been shown to activate the intrinsic survival pathways in the brain and protect the organ from ischemic or anoxic injury, a phenomenon termed as “conditioned central neurogenic neuroprotection” (Nakai et al., 1983; Reis et al., 1991, 1998; Zhang and Iadecola, 1992). Based on these results, the hypothesis that TCES protects light-injured photoreceptors by modulating the intrinsic survival microenvironment was investigated by analyzing the expression changes of retinal neurotrophic factors and apoptosis-associated genes after TCES. Our results demonstrated that TCES time-dependently upregulated the expression of BDNF, CNTF and Bcl-2, and downregulated the expression of Bax in the retina. In accordance with the previous reports (Fujieda and Sasaki, 2008; Seki et al., 2003; Vecino et al., 1998), the positive staining of BDNF was mostly localized in the cytoplasm of RGCs in the normal retinas and significantly increased in the inner most region of the INL after TCES. The Bcl-2 expression after TCES was restricted in the Müller cell radial processes similar with its expression during the development and degeneration in RCS rat (Sharma, 2001). Our results showed that CNTF was expressed weakly in the normal GCL. It was differing from Walsh et al.'s (2001) report that CNTF was prominent in normal retinal macroglial cells (Müller cells and astrocytes). However, similar with Bcl-2, the expression of CNTF after TCES was also distributed within the processes of Müller cells confirming a number of previous reports about the CNTF expression pattern during stress (Walsh et al., 2001; Kirsch et al., 1997; Liu et al., 1998). As we have known, the communication between photoreceptors and Müller cells indicated that a relatively modest benefit to the Müller cells may sufficiently reduce the chances of photoreceptor death (Bringmann et al., 2006). For example, Müller cells can alter bFGF production in response to exogenous NGF and NT-3 in vitro, which can directly rescue light-induced photoreceptor apoptosis (Harada et al., 2000). Additionally, activated microglia indirectly influenced photoreceptor survival by modulating secondary trophic factor expression in Müller glial (Harada et al., 2002). As Müller glial cells play an important role to maintain the microenvironment homeostasis through the glianeuron network, we speculate that TCES activated the intrinsic survival system, especially the Müller cells, and subsequently rescued the light-injured photoreceptors.

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Neurotrophic factors clearly provide a survival-promoting effect on photoreceptors (Chaum, 2003). A simple piercing wound through the eyeball causes the upregulation of basic fibroblast growth factor (FGF2), BDNF and CNTF in a localized region, which temporarily protects the retina from degeneration due to light-induced damage (Wen et al., 1995; Ikeda et al., 2003). The use of electrical stimulation to induce the upregulation of endogenous growth factors in the retina is an obvious step. In our present study, electrical stimulation was applied through the cornea and resulted in the upregulation of neurotrophins (BDNF and CNTF) in the retina which enhanced the survival of light-injured photoreceptors. However, in contrast to our study, Morimoto et al.'s experiments on axotomized RGCs demonstrated that neurotrophic factors such as CNTF and BDNF did not change significantly after TCES except for an upregulation of IGF-1 which they considered as the key molecule for TCES-induced neuroprotection (Morimoto et al., 2005). One of the possible reasons is that our present density for photoreceptor survival (300 μA, 3 ms/phase, 20 Hz) was higher than theirs for RGCs survival (100 μA, 1 ms/phase, 20 Hz). The other possible reason is that the position of reference electrode was quite different, although we share the common electrical stimulation principles as TCES. Their reference electrode was on the cornea (bipolar contact lens electrode) while ours was through the ipsilateral subcutaneous, allowing the current density to be more sufficiently dissipated in the retina as the current was shorted through the vitreous via low-impedance paths. Cell survival and apoptotic death have been shown to be regulated by genes of the bcl-2 family. Thus, several studies demonstrated a protective, anti-apoptotic effect of Bcl-2 protein in neural cells both in vitro and in vivo (Martinou et al., 1994; Cenni et al., 1996). Several proteins sharing homology with Bcl-2, such as Bax, render cells more susceptible to apoptotic stimuli (Oltvai et al., 1993; Deckwerth et al., 1996). Our present study is the first report to demonstrate that noninvasive electrical stimulation through the cornea upregulated Bcl-2 expression and downregulated Bax expression in the retina. Upregulation of Bcl-2 immunoreactivity was noted in GFAP-positive cells in the nerve fiber and ganglion cell layers, and Bcl-2 induction was noted in Müller cell processes. The increased expression of the anti-apoptotic Bcl-2 in retinal glial cells after TCES may contribute to prevent themselves from undergoing apoptosis, suggesting a possible role of glial cells in activation of a competing survival mechanism to maintain cellular homeostasis. Benefits of TCES: potential clinical technique Our findings indicated that TCES remarkably ameliorated the loss of photoreceptors due to intense light exposure by activating the intrinsic neuroprotective system. This electrical stimulation therapy is simple and less invasive compared to alternative therapies, and severe ocular side effects such as bleeding and cataract were not observed even during the course of repetitive stimulation. Therefore, we suggest that the manipulation of this commitment phase by TCES be considered as one of the therapeutic methods for photoreceptor degeneration. In fact, TCES has been clinically applied on patients with optic neuropathies that are difficult to treat by present methods such as TON and NAION and has been found to improve visual function (Fujikado et al., 2006). However, in our study, two rats with parameters of 400 μA, 50 Hz showed the corneal epithelium proliferation and retinal perforation by the histopathologic analysis which haven't been reported in other TCES studies. It has been reported that electrical stimulation damaged the retina when it was directly contact with the electrode (Majji et al., 1999; Walter et al., 1999) and the histopathologic changes were similar with our findings that photoreceptors and RPE exhibited the most severe response to direct electrical stimulation (Colodetti et al., 2007). The damage could be the result of an excitotoxic effect (Agnew et al., 1993) or mechanical pressure (Colodetti et al., 2007). Although TCES is a noninvasive

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method, considering the high charge density stimulation (400 μA, 50 Hz), it is possible that some of the current density could not be sufficiently dissipated at the retina and cause damage. Therefore, we do not advocate applying the high stimulus levels chronically. For photoreceptor degeneration, before consideration for clinical therapy, the potential damage to retinas or corneas should be excluded by histological or electrophysiological methods and the neuroprotective effect of TCES should be further evaluated in other animal models with genetic mutations. In conclusion, our results showed that TCES led to the activation of an intrinsic survival system with the upregulation of anti-apoptotic gene Bcl-2, neurotrophic CNTF and BDNF, and downregulation of proapoptotic gene Bax, consequently protected the photoreceptors from light-induced degeneration. 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