- Email: [email protected]
Neuroprotective effect of resveratrol prophylaxis on experimental retinal ischemic injury
Experimental Eye Research 108 (2013) 72e75
Contents lists available at SciVerse ScienceDirect
Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer
Short communication
Neuroprotective effect of resveratrol prophylaxis on experimental retinal ischemic injury Anita P. Vin a, Hanbo Hu f, Yougang Zhai c, Cynthia L. Von Zee a, d, Andrew Logeman a, Evan B. Stubbs Jr. a, d, Jay I. Perlman a, b, e, Ping Bu a, d, * a
Department of Ophthalmology, Loyola University Chicago, Maywood, IL 60153, USA Department of Pathology, Loyola University Chicago, Maywood, IL 60153, USA Department of Microbiology and Immunology, Loyola University Chicago, Maywood, IL 60153, USA d Research Service, Edward Hines, Jr. VA Hospital, Hines, IL 60141, USA e Surgery Service, Edward Hines, Jr. VA Hospital, Hines, IL 60141, USA f Department of Medicine, University of Florida, Malcolm Randall VA Medical Center, Gainesville, FL 32610, USA b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 September 2012 Accepted in revised form 28 November 2012 Available online 31 December 2012
The purpose of the present study was to investigate whether systemically administered resveratrol can protect against acute retinal ischemic reperfusion injury. Two groups of adult male Sprague Dawley rats (n ¼ 6 per group) were used for this study. Resveratrol (30 mg/kg) or an equal volume of vehicle (30% Solutol HS 15 in 0.9% saline) was administered daily for 5 days via intraperitoneal injection. On the third day of treatment, retinal ischemic injury was induced by elevation of intraocular pressure for 45 min. Prior to resveratrol administration and one-week following ischemic insult, retinal function was measured by scotopic electroretinography (ERG). Retinas were harvested and morphologically analyzed one week after ischemic insult. ERG a- and b-wave amplitudes were significantly reduced following ischemic reperfusion injury. Resveratrol treatment attenuated ischemic-induced loss of retinal function. In control vehicle-treated rats, ischemic reperfusion injury elicited marked thinning of inner retinal layers. Resveratrol prophylactic treatment reduced ischemia-mediated thinning of the whole retina and in particular the inner retinal layers. Therefore, resveratrol may have therapeutic value for the management of retinal ischemic disorders. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: resveratrol retinal ischemic reperfusion injury electroretinography neuroprotection
Resveratrol (trans-3, 5, 40 -trihydroxystilbene) is a nontoxic polyphenol found in a wide variety of plant species, and constitutes one of the components of red wine. Resveratrol has been reported to have anti-inflammatory and anti-aging (de la Lastra and Villegas, 2005), anti-oxidant (Ray et al., 1999), and anti-tumor activities (Atten et al., 2005). Studies have demonstrated protective effects in the spinal cord, kidneys, heart, and brain from ischemiareperfusion injury (Giovannini et al., 2001; Huang et al., 2001; Mokni et al., 2007). Several studies have aimed at elucidating the mechanism of resveratrol’s anti-apoptotic effect. In a rat model of focal cerebral ischemia, in the presence of resveratrol, protein and mRNA expression for iNOS was downregulated and protein and mRNA expression for eNOS in ischemic brain tissue was upregulated (Tsai et al., 2007). Li et al. (2010) further reported a resveratrol-mediated * Corresponding author. Department of Ophthalmology, Loyola University Chicago, 2160 S. First Avenue, Maywood, IL 60153, USA. Tel.: þ1 708 216 6262; fax: þ1 708 216 3557. E-mail address: [email protected] (P. Bu). 0014-4835/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.exer.2012.11.022
decrease in the release of excitatory neurotransmitters and increase in inhibitory neurotransmitters, thereby preventing excitotoxicity, while Anekonda and Adamus (2008) found that resveratrol prevents antibody-induced apoptotic death of retinal cells. A recent study also revealed that resveratrol application to light-exposed mice ameliorated apoptotic cell death, retinal structural damage, and functional damage induced by light damage (Kubota et al., 2010). Resveratrol suppressed retinal vascular degeneration in mice with retinal ischemic injury by inhibiting endoplasmic reticulum stress (Li et al., 2012). Retina ischemia is a common cause of visual impairment and blindness. Ischemic insult to the mammalian retina is observed in open-angle glaucoma, diabetic and hypertensive retinopathies. Ischemic retinal injury leads to energy dependent dysfunction, tissue edema, and eventual retinal ganglion cell death (Hughes, 1991). The mechanism of cell death induced by retinal ischemia is not completely understood but neuronal injury is associated with enhanced production of endogenous substances such as glutamate, oxygen free radicals, nitric oxide (NO) and calcium (Choi, 1995; Dreyer, 1998; Louzada-Junior et al., 1992).
A.P. Vin et al. / Experimental Eye Research 108 (2013) 72e75
A thorough review of the literature indicates the absence of any studies examining the retinal function and histologic changes of resveratrol in retinal ischemia induced by elevated intraocular pressure. The purpose of present study is to communicate our early results indicating neuroprotective effects of resveratrol on retinal ischemia in vivo using an established model of high pressureinduced retinal ischemia. Animal care and experimental procedures were performed in accordance with the ARVO statement for the use of Animal in Ophthalmic and Vision Research, and were approved by the Institutional Animal Care and Use Committee, Edward Hines, Jr. VA Hospital. All procedures were performed on animals anesthetized with ketamine (100 mg/kg body weight) and xylazine (5 mg/kg body weight). Topical 0.5% proparacaine hydrochloride (Alcaine, Alcon) was applied to the eye prior to ischemic insult. Two groups of adult (200 g) male Sprague Dawley rats (n ¼ 6 per group) were used for this study. Resveratrol (30 mg/kg; Sigma) or an equal volume of vehicle (30% Solutol HS 15 BASF Ladwigshafen, Germany in 0.9% saline) (Singleton et al., 2010) was intraperitoneally administered daily for 5 days. On the third day of treatment, retinal ischemia was induced by elevation of intraocular pressure for 45 min as previously described (Bu et al., 2010). Briefly, the anterior chamber of the right eye of each rat was cannulated with 30-gauge sterile needle connected to an elevated isotonic sterile saline bag. The intraocular pressure was raised to 70e80 mm Hg for duration of 45 min. IOP was measured with a hand-held
73
TonoLab tonometer. Retinal ischemia was confirmed by rapid blanching of the ocular fundus and the collapse of the retinal arteries by indirect ophthalmoscope. The left eye was used as a nonischemic control. Retinal function was evaluated in rats prior to resveratrol or vehicle administration and 1 week following retinal ischemic reperfusion injury. Rats were dark-adapted overnight, anesthetized and pupils dilated with 1% tropicamide and 2.5% phenylephrine hydrochloride (Bausch & Lomb Inc). The ERG was recorded from the corneal surface using a stainless steel electrode that was wetted with 1% methylcellulose. Needle electrodes placed in the cheek and tail served as reference and ground leads, respectively. Responses were differentially amplified (0.3e1500 Hz), averaged, and stored using a UTAS E-3000 signal averaging system (LKC Technologies, Gaithersburg, MD). Flash stimuli were presented in darkness within a Ganzfeld bowl and ranged in luminance from 3.6 to 2.1 log cd s/ m2; inter-stimulus intervals increased from 4 s at the lowest flash luminance to 61 s at the highest stimulus levels. Stimuli were present in order of increased luminance and at least two successive responses were averaged for each stimulus condition. The body temperature of anesthetized rats was maintained at 37 C with a heating pad. The amplitude of a-wave was measured 6.5 ms after flash onset from the pre-stimulus baseline. The amplitude of bwave was measured from the a-wave trough to the peak of the bwave or if no a-wave was present, from the pre-stimulus baseline (Yu et al., 2012).
Fig. 1. Protective effect of resveratrol on retinal function following ischemic reperfusion injury. (A) Representative dark-adapted ERGs obtained from strobe flash stimuli from normal (non-ischemic), resveratrol-treated (ischemic) and vehicle-treated (ischemic) rats. Scales bar, 250 mV and 20 ms. Luminance-response functions for (B) a-wave and (C) b-wave components of the ERG. *p < 0.01; two-way ANOVA with Bonferroni post hoc analysis comparing vehicle e with resveratrol treatment (n ¼ 6).
74
A.P. Vin et al. / Experimental Eye Research 108 (2013) 72e75
One week after transient retinal ischemic injury, the rats were euthanized immediately after ERG recording by pentobarbital overdose (200 mg/kg, IP injection). Enucleated eyes were fixed in phosphate-buffered (pH 7.4) 2.5% glutaraldehyde-2% paraformaldehyde solution and embedded in epoxy resin. Sections (1 mm) were cut along the vertical meridian of each eye and passed through the optic nerve head and stained with toluidine blue. The average of two separate morphometric measurements of the superior and inferior retina at 250 mm, 500 mm, and 750 mm from the optic disc (Bu et al., 2010) were performed. The inner retina was defined as the margins between the inner limiting membrane and the boundary of the ONL and OPL. Prior to induction of retinal ischemic injury, scotopic ERG responses increased with increasing light luminance (Fig. 1A, ‘Normal’ non-ischemic). Scotopic ERG a- and b-waves were significantly attenuated after ischemic injury in vehicle-treated control rats (Fig. 1A, ‘Vehicle’). In contrast, rats treated with resveratrol showed measurable functional responses including oscillatory potentials with increasing light luminance (Fig. 1A, ‘Resveratrol’). ERG a- and b-wave amplitudes recorded between vehicle and resveratrol treated rats were significantly different (p < 0.01), suggesting measurable protection of retinal functional integrity elicited by resveratrol (Fig. 1B and C). Experimental retinal ischemic injury resulted in prominent histopathologic changes throughout the retina. Compared with the normal non-ischemic retina, retinal ischemic injury produced marked thinning of the whole retina with disorganization of the
various layers. However, there was clear preservation of structure in the resveratrol-treated ischemic group as compared to the vehicle-treated ischemic group (Fig. 2A). Furthermore, morphometric differences in mean thickness of the whole retina and inner retinal layers between vehicle- and resveratrol-treated ischemic groups were statistically significant (p < 0.05), with uniformly thinner layers of retina observed in the vehicle-treated ischemic group (Fig. 2B and C). In the present study, we report the novel finding that prophylactic intraperitoneal administration of resveratrol can modestly protect against retinal ischemic injury. This effect was confirmed both electroretinographically and by histologic/morphometric studies. Resveratrol has long been of therapeutic interest due to its demonstrated anti-inflammatory, anti-apoptotic, and anti-oxidant effects (Baur and Sinclair, 2006). Multiple studies have raised the possibility that resveratrol might be useful in protecting against brain damage following cerebral ischemia (Huang et al., 2001; Tsai et al., 2007). The retina is a neural structure that is exquisitely sensitive to the damaging effects of free radicals and inflammation following ischemia-reperfusion injury. Our findings support the thesis that resveratrol is capable of crossing the blooderetina barrier and exerting neuroprotective effects, including protection from retinal ischemic insult and subsequent ischemia-reperfusion injury. The present findings strongly support a therapeutic role for resveratrol in the management of ischemic retinal degenerative diseases, including glaucoma and perhaps age-related macular degeneration.
Fig. 2. Effect of resveratrol on retinal histology following ischemic reperfusion injury. (A) Shown are representative toluidine blue-stained retinal sections (650e1000 mm from optic disc) from normal (non-ischemic) or resveratrol-treated (ischemic) or vehicle-treated (ischemic) retina. Morphometry of (B) whole retina and of (C) inner retinal layers. Data shown are the means SEM (n ¼ 6). Superior retina, left; inferior retina, right of optic disc. *p < 0.05, **p < 0.01; two-way ANOVA with Bonferroni post hoc analysis comparing vehicle e with resveratrol treatment (n ¼ 6).
A.P. Vin et al. / Experimental Eye Research 108 (2013) 72e75
Acknowledgments This work was supported in part by The Richard A. Perritt Charitable Foundation and Illinois Society for the Prevention of Blindness (ISPB). References Anekonda, T.S., Adamus, G., 2008. Resveratrol prevents antibody-induced apoptotic death of retinal cells through upregulation of Sirt1 and Ku70. BMC Res. Notes 1, 122. Atten, M.J., Godoy-Romero, E., Attar, B.M., Milson, T., Zopel, M., Holian, O., 2005. Resveratrol regulates cellular PKC alpha and delta to inhibit growth and induce apoptosis in gastric cancer cells. Invest. New Drugs 23, 111e119. Baur, J.A., Sinclair, D.A., 2006. Therapeutic potential of resveratrol: the in vivo evidence. Nat. Rev. Drug Discov. 5, 493e506. Bu, P., Basith, B., Stubbs Jr., E.B., Perlman, J.I., 2010. Granulocyte colony-stimulating factor facilitates recovery of retinal function following retinal ischemic injury. Exp. Eye Res. 91, 104e106. Choi, D.W., 1995. Calcium: still center-stage in hypoxic-ischemic neuronal death. Trends Neurosci. 18, 58e60. de la Lastra, C.A., Villegas, I., 2005. Resveratrol as an anti-inflammatory and antiaging agent: mechanisms and clinical implications. Mol. Nutr. Food Res. 49, 405e430. Dreyer, E.B.,1998. A proposed role for excitotoxicity in glaucoma. J. Glaucoma 7, 62e67. Giovannini, L., Migliori, M., Longoni, B.M., Das, D.K., Bertelli, A.A., Panichi, V., Filippi, C., Bertelli, A., 2001. Resveratrol, a polyphenol found in wine, reduces ischemia reperfusion injury in rat kidneys. J. Cardiovasc. Pharmacol. 37, 262e270. Huang, S.S., Tsai, M.C., Chih, C.L., Hung, L.M., Tsai, S.K., 2001. Resveratrol reduction of infarct size in LongeEvans rats subjected to focal cerebral ischemia. Life Sci. 69, 1057e1065.
75
Hughes, W.F., 1991. Quantitation of ischemic damage in the rat retina. Exp. Eye Res. 53, 573e582. Kubota, S., Kurihara, T., Ebinuma, M., Kubota, M., Yuki, K., Sasaki, M., Noda, K., Ozawa, Y., Oike, Y., Ishida, S., et al., 2010. Resveratrol prevents light-induced retinal degeneration via suppressing activator protein-1 activation. Am. J. Pathol. 177, 1725e1731. Li, C., Wang, L., Huang, K., Zheng, L., 2012. Endoplasmic reticulum stress in retinal vascular degeneration: protective role of resveratrol. Invest. Ophthalmol. Vis. Sci. 53, 3241e3249. Li, C., Yan, Z., Yang, J., Chen, H., Li, H., Jiang, Y., Zhang, Z., 2010. Neuroprotective effects of resveratrol on ischemic injury mediated by modulating the release of neurotransmitter and neuromodulator in rats. Neurochem. Int. 56, 495e500. Louzada-Junior, P., Dias, J.J., Santos, W.F., Lachat, J.J., Bradford, H.F., CoutinhoNetto, J., 1992. Glutamate release in experimental ischaemia of the retina: an approach using microdialysis. J. Neurochem. 59, 358e363. Mokni, M., Limam, F., Elkahoui, S., Amri, M., Aouani, E., 2007. Strong cardioprotective effect of resveratrol, a red wine polyphenol, on isolated rat hearts after ischemia/reperfusion injury. Arch. Biochem. Biophys. 457, 1e6. Ray, P.S., Maulik, G., Cordis, G.A., Bertelli, A.A., Bertelli, A., Das, D.K., 1999. The red wine antioxidant resveratrol protects isolated rat hearts from ischemia reperfusion injury. Free Radic. Biol. Med. 27, 160e169. Singleton, R.H., Yan, H.Q., Fellows-Mayle, W., Dixon, C.E., 2010. Resveratrol attenuates behavioral impairments and reduces cortical and hippocampal loss in a rat controlled cortical impact model of traumatic brain injury. J. Neurotrauma 27, 1091e1099. Tsai, S.K., Hung, L.M., Fu, Y.T., Cheng, H., Nien, M.W., Liu, H.Y., Zhang, F.B., Huang, S.S., 2007. Resveratrol neuroprotective effects during focal cerebral ischemia injury via nitric oxide mechanism in rats. J. Vasc. Surg. 46, 346e353. Yu, M., Sturgill-Short, G., Ganapathy, P., Tawfik, A., Peachey, N.S., Smith, S.B., 2012. Age-related changes in visual function in cystathionine-betasynthase mutant mice, a model of hyperhomocysteinemia. Exp. Eye Res. 96, 124e131.
Contents lists available at SciVerse ScienceDirect
Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer
Short communication
Neuroprotective effect of resveratrol prophylaxis on experimental retinal ischemic injury Anita P. Vin a, Hanbo Hu f, Yougang Zhai c, Cynthia L. Von Zee a, d, Andrew Logeman a, Evan B. Stubbs Jr. a, d, Jay I. Perlman a, b, e, Ping Bu a, d, * a
Department of Ophthalmology, Loyola University Chicago, Maywood, IL 60153, USA Department of Pathology, Loyola University Chicago, Maywood, IL 60153, USA Department of Microbiology and Immunology, Loyola University Chicago, Maywood, IL 60153, USA d Research Service, Edward Hines, Jr. VA Hospital, Hines, IL 60141, USA e Surgery Service, Edward Hines, Jr. VA Hospital, Hines, IL 60141, USA f Department of Medicine, University of Florida, Malcolm Randall VA Medical Center, Gainesville, FL 32610, USA b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 September 2012 Accepted in revised form 28 November 2012 Available online 31 December 2012
The purpose of the present study was to investigate whether systemically administered resveratrol can protect against acute retinal ischemic reperfusion injury. Two groups of adult male Sprague Dawley rats (n ¼ 6 per group) were used for this study. Resveratrol (30 mg/kg) or an equal volume of vehicle (30% Solutol HS 15 in 0.9% saline) was administered daily for 5 days via intraperitoneal injection. On the third day of treatment, retinal ischemic injury was induced by elevation of intraocular pressure for 45 min. Prior to resveratrol administration and one-week following ischemic insult, retinal function was measured by scotopic electroretinography (ERG). Retinas were harvested and morphologically analyzed one week after ischemic insult. ERG a- and b-wave amplitudes were significantly reduced following ischemic reperfusion injury. Resveratrol treatment attenuated ischemic-induced loss of retinal function. In control vehicle-treated rats, ischemic reperfusion injury elicited marked thinning of inner retinal layers. Resveratrol prophylactic treatment reduced ischemia-mediated thinning of the whole retina and in particular the inner retinal layers. Therefore, resveratrol may have therapeutic value for the management of retinal ischemic disorders. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: resveratrol retinal ischemic reperfusion injury electroretinography neuroprotection
Resveratrol (trans-3, 5, 40 -trihydroxystilbene) is a nontoxic polyphenol found in a wide variety of plant species, and constitutes one of the components of red wine. Resveratrol has been reported to have anti-inflammatory and anti-aging (de la Lastra and Villegas, 2005), anti-oxidant (Ray et al., 1999), and anti-tumor activities (Atten et al., 2005). Studies have demonstrated protective effects in the spinal cord, kidneys, heart, and brain from ischemiareperfusion injury (Giovannini et al., 2001; Huang et al., 2001; Mokni et al., 2007). Several studies have aimed at elucidating the mechanism of resveratrol’s anti-apoptotic effect. In a rat model of focal cerebral ischemia, in the presence of resveratrol, protein and mRNA expression for iNOS was downregulated and protein and mRNA expression for eNOS in ischemic brain tissue was upregulated (Tsai et al., 2007). Li et al. (2010) further reported a resveratrol-mediated * Corresponding author. Department of Ophthalmology, Loyola University Chicago, 2160 S. First Avenue, Maywood, IL 60153, USA. Tel.: þ1 708 216 6262; fax: þ1 708 216 3557. E-mail address: [email protected] (P. Bu). 0014-4835/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.exer.2012.11.022
decrease in the release of excitatory neurotransmitters and increase in inhibitory neurotransmitters, thereby preventing excitotoxicity, while Anekonda and Adamus (2008) found that resveratrol prevents antibody-induced apoptotic death of retinal cells. A recent study also revealed that resveratrol application to light-exposed mice ameliorated apoptotic cell death, retinal structural damage, and functional damage induced by light damage (Kubota et al., 2010). Resveratrol suppressed retinal vascular degeneration in mice with retinal ischemic injury by inhibiting endoplasmic reticulum stress (Li et al., 2012). Retina ischemia is a common cause of visual impairment and blindness. Ischemic insult to the mammalian retina is observed in open-angle glaucoma, diabetic and hypertensive retinopathies. Ischemic retinal injury leads to energy dependent dysfunction, tissue edema, and eventual retinal ganglion cell death (Hughes, 1991). The mechanism of cell death induced by retinal ischemia is not completely understood but neuronal injury is associated with enhanced production of endogenous substances such as glutamate, oxygen free radicals, nitric oxide (NO) and calcium (Choi, 1995; Dreyer, 1998; Louzada-Junior et al., 1992).
A.P. Vin et al. / Experimental Eye Research 108 (2013) 72e75
A thorough review of the literature indicates the absence of any studies examining the retinal function and histologic changes of resveratrol in retinal ischemia induced by elevated intraocular pressure. The purpose of present study is to communicate our early results indicating neuroprotective effects of resveratrol on retinal ischemia in vivo using an established model of high pressureinduced retinal ischemia. Animal care and experimental procedures were performed in accordance with the ARVO statement for the use of Animal in Ophthalmic and Vision Research, and were approved by the Institutional Animal Care and Use Committee, Edward Hines, Jr. VA Hospital. All procedures were performed on animals anesthetized with ketamine (100 mg/kg body weight) and xylazine (5 mg/kg body weight). Topical 0.5% proparacaine hydrochloride (Alcaine, Alcon) was applied to the eye prior to ischemic insult. Two groups of adult (200 g) male Sprague Dawley rats (n ¼ 6 per group) were used for this study. Resveratrol (30 mg/kg; Sigma) or an equal volume of vehicle (30% Solutol HS 15 BASF Ladwigshafen, Germany in 0.9% saline) (Singleton et al., 2010) was intraperitoneally administered daily for 5 days. On the third day of treatment, retinal ischemia was induced by elevation of intraocular pressure for 45 min as previously described (Bu et al., 2010). Briefly, the anterior chamber of the right eye of each rat was cannulated with 30-gauge sterile needle connected to an elevated isotonic sterile saline bag. The intraocular pressure was raised to 70e80 mm Hg for duration of 45 min. IOP was measured with a hand-held
73
TonoLab tonometer. Retinal ischemia was confirmed by rapid blanching of the ocular fundus and the collapse of the retinal arteries by indirect ophthalmoscope. The left eye was used as a nonischemic control. Retinal function was evaluated in rats prior to resveratrol or vehicle administration and 1 week following retinal ischemic reperfusion injury. Rats were dark-adapted overnight, anesthetized and pupils dilated with 1% tropicamide and 2.5% phenylephrine hydrochloride (Bausch & Lomb Inc). The ERG was recorded from the corneal surface using a stainless steel electrode that was wetted with 1% methylcellulose. Needle electrodes placed in the cheek and tail served as reference and ground leads, respectively. Responses were differentially amplified (0.3e1500 Hz), averaged, and stored using a UTAS E-3000 signal averaging system (LKC Technologies, Gaithersburg, MD). Flash stimuli were presented in darkness within a Ganzfeld bowl and ranged in luminance from 3.6 to 2.1 log cd s/ m2; inter-stimulus intervals increased from 4 s at the lowest flash luminance to 61 s at the highest stimulus levels. Stimuli were present in order of increased luminance and at least two successive responses were averaged for each stimulus condition. The body temperature of anesthetized rats was maintained at 37 C with a heating pad. The amplitude of a-wave was measured 6.5 ms after flash onset from the pre-stimulus baseline. The amplitude of bwave was measured from the a-wave trough to the peak of the bwave or if no a-wave was present, from the pre-stimulus baseline (Yu et al., 2012).
Fig. 1. Protective effect of resveratrol on retinal function following ischemic reperfusion injury. (A) Representative dark-adapted ERGs obtained from strobe flash stimuli from normal (non-ischemic), resveratrol-treated (ischemic) and vehicle-treated (ischemic) rats. Scales bar, 250 mV and 20 ms. Luminance-response functions for (B) a-wave and (C) b-wave components of the ERG. *p < 0.01; two-way ANOVA with Bonferroni post hoc analysis comparing vehicle e with resveratrol treatment (n ¼ 6).
74
A.P. Vin et al. / Experimental Eye Research 108 (2013) 72e75
One week after transient retinal ischemic injury, the rats were euthanized immediately after ERG recording by pentobarbital overdose (200 mg/kg, IP injection). Enucleated eyes were fixed in phosphate-buffered (pH 7.4) 2.5% glutaraldehyde-2% paraformaldehyde solution and embedded in epoxy resin. Sections (1 mm) were cut along the vertical meridian of each eye and passed through the optic nerve head and stained with toluidine blue. The average of two separate morphometric measurements of the superior and inferior retina at 250 mm, 500 mm, and 750 mm from the optic disc (Bu et al., 2010) were performed. The inner retina was defined as the margins between the inner limiting membrane and the boundary of the ONL and OPL. Prior to induction of retinal ischemic injury, scotopic ERG responses increased with increasing light luminance (Fig. 1A, ‘Normal’ non-ischemic). Scotopic ERG a- and b-waves were significantly attenuated after ischemic injury in vehicle-treated control rats (Fig. 1A, ‘Vehicle’). In contrast, rats treated with resveratrol showed measurable functional responses including oscillatory potentials with increasing light luminance (Fig. 1A, ‘Resveratrol’). ERG a- and b-wave amplitudes recorded between vehicle and resveratrol treated rats were significantly different (p < 0.01), suggesting measurable protection of retinal functional integrity elicited by resveratrol (Fig. 1B and C). Experimental retinal ischemic injury resulted in prominent histopathologic changes throughout the retina. Compared with the normal non-ischemic retina, retinal ischemic injury produced marked thinning of the whole retina with disorganization of the
various layers. However, there was clear preservation of structure in the resveratrol-treated ischemic group as compared to the vehicle-treated ischemic group (Fig. 2A). Furthermore, morphometric differences in mean thickness of the whole retina and inner retinal layers between vehicle- and resveratrol-treated ischemic groups were statistically significant (p < 0.05), with uniformly thinner layers of retina observed in the vehicle-treated ischemic group (Fig. 2B and C). In the present study, we report the novel finding that prophylactic intraperitoneal administration of resveratrol can modestly protect against retinal ischemic injury. This effect was confirmed both electroretinographically and by histologic/morphometric studies. Resveratrol has long been of therapeutic interest due to its demonstrated anti-inflammatory, anti-apoptotic, and anti-oxidant effects (Baur and Sinclair, 2006). Multiple studies have raised the possibility that resveratrol might be useful in protecting against brain damage following cerebral ischemia (Huang et al., 2001; Tsai et al., 2007). The retina is a neural structure that is exquisitely sensitive to the damaging effects of free radicals and inflammation following ischemia-reperfusion injury. Our findings support the thesis that resveratrol is capable of crossing the blooderetina barrier and exerting neuroprotective effects, including protection from retinal ischemic insult and subsequent ischemia-reperfusion injury. The present findings strongly support a therapeutic role for resveratrol in the management of ischemic retinal degenerative diseases, including glaucoma and perhaps age-related macular degeneration.
Fig. 2. Effect of resveratrol on retinal histology following ischemic reperfusion injury. (A) Shown are representative toluidine blue-stained retinal sections (650e1000 mm from optic disc) from normal (non-ischemic) or resveratrol-treated (ischemic) or vehicle-treated (ischemic) retina. Morphometry of (B) whole retina and of (C) inner retinal layers. Data shown are the means SEM (n ¼ 6). Superior retina, left; inferior retina, right of optic disc. *p < 0.05, **p < 0.01; two-way ANOVA with Bonferroni post hoc analysis comparing vehicle e with resveratrol treatment (n ¼ 6).
A.P. Vin et al. / Experimental Eye Research 108 (2013) 72e75
Acknowledgments This work was supported in part by The Richard A. Perritt Charitable Foundation and Illinois Society for the Prevention of Blindness (ISPB). References Anekonda, T.S., Adamus, G., 2008. Resveratrol prevents antibody-induced apoptotic death of retinal cells through upregulation of Sirt1 and Ku70. BMC Res. Notes 1, 122. Atten, M.J., Godoy-Romero, E., Attar, B.M., Milson, T., Zopel, M., Holian, O., 2005. Resveratrol regulates cellular PKC alpha and delta to inhibit growth and induce apoptosis in gastric cancer cells. Invest. New Drugs 23, 111e119. Baur, J.A., Sinclair, D.A., 2006. Therapeutic potential of resveratrol: the in vivo evidence. Nat. Rev. Drug Discov. 5, 493e506. Bu, P., Basith, B., Stubbs Jr., E.B., Perlman, J.I., 2010. Granulocyte colony-stimulating factor facilitates recovery of retinal function following retinal ischemic injury. Exp. Eye Res. 91, 104e106. Choi, D.W., 1995. Calcium: still center-stage in hypoxic-ischemic neuronal death. Trends Neurosci. 18, 58e60. de la Lastra, C.A., Villegas, I., 2005. Resveratrol as an anti-inflammatory and antiaging agent: mechanisms and clinical implications. Mol. Nutr. Food Res. 49, 405e430. Dreyer, E.B.,1998. A proposed role for excitotoxicity in glaucoma. J. Glaucoma 7, 62e67. Giovannini, L., Migliori, M., Longoni, B.M., Das, D.K., Bertelli, A.A., Panichi, V., Filippi, C., Bertelli, A., 2001. Resveratrol, a polyphenol found in wine, reduces ischemia reperfusion injury in rat kidneys. J. Cardiovasc. Pharmacol. 37, 262e270. Huang, S.S., Tsai, M.C., Chih, C.L., Hung, L.M., Tsai, S.K., 2001. Resveratrol reduction of infarct size in LongeEvans rats subjected to focal cerebral ischemia. Life Sci. 69, 1057e1065.
75
Hughes, W.F., 1991. Quantitation of ischemic damage in the rat retina. Exp. Eye Res. 53, 573e582. Kubota, S., Kurihara, T., Ebinuma, M., Kubota, M., Yuki, K., Sasaki, M., Noda, K., Ozawa, Y., Oike, Y., Ishida, S., et al., 2010. Resveratrol prevents light-induced retinal degeneration via suppressing activator protein-1 activation. Am. J. Pathol. 177, 1725e1731. Li, C., Wang, L., Huang, K., Zheng, L., 2012. Endoplasmic reticulum stress in retinal vascular degeneration: protective role of resveratrol. Invest. Ophthalmol. Vis. Sci. 53, 3241e3249. Li, C., Yan, Z., Yang, J., Chen, H., Li, H., Jiang, Y., Zhang, Z., 2010. Neuroprotective effects of resveratrol on ischemic injury mediated by modulating the release of neurotransmitter and neuromodulator in rats. Neurochem. Int. 56, 495e500. Louzada-Junior, P., Dias, J.J., Santos, W.F., Lachat, J.J., Bradford, H.F., CoutinhoNetto, J., 1992. Glutamate release in experimental ischaemia of the retina: an approach using microdialysis. J. Neurochem. 59, 358e363. Mokni, M., Limam, F., Elkahoui, S., Amri, M., Aouani, E., 2007. Strong cardioprotective effect of resveratrol, a red wine polyphenol, on isolated rat hearts after ischemia/reperfusion injury. Arch. Biochem. Biophys. 457, 1e6. Ray, P.S., Maulik, G., Cordis, G.A., Bertelli, A.A., Bertelli, A., Das, D.K., 1999. The red wine antioxidant resveratrol protects isolated rat hearts from ischemia reperfusion injury. Free Radic. Biol. Med. 27, 160e169. Singleton, R.H., Yan, H.Q., Fellows-Mayle, W., Dixon, C.E., 2010. Resveratrol attenuates behavioral impairments and reduces cortical and hippocampal loss in a rat controlled cortical impact model of traumatic brain injury. J. Neurotrauma 27, 1091e1099. Tsai, S.K., Hung, L.M., Fu, Y.T., Cheng, H., Nien, M.W., Liu, H.Y., Zhang, F.B., Huang, S.S., 2007. Resveratrol neuroprotective effects during focal cerebral ischemia injury via nitric oxide mechanism in rats. J. Vasc. Surg. 46, 346e353. Yu, M., Sturgill-Short, G., Ganapathy, P., Tawfik, A., Peachey, N.S., Smith, S.B., 2012. Age-related changes in visual function in cystathionine-betasynthase mutant mice, a model of hyperhomocysteinemia. Exp. Eye Res. 96, 124e131.