Log cd s/m 2. ABSTRACT.
Purpose: Retinal ischemia is a major contributor to vision loss in multiple diseases including acute angle-closure glaucoma, primary open angle glaucoma, diabetic retinopathy, and retinal vascular occlusions. The increase in oxidative stress is widely believed to play an important role in retinal ischemic injury. Sulforaphane, an isothiocyanate, and precursor of glucosinolate in cruciferous vegetables such as broccoli, has demonstrated neuroprotective effects in several experimental models. In this study, we determined the neuroprotective effects of sulforaphane on retinal ischemia in vivo, using a mouse model of ischemic-reperfusion injury. By quantifying relative changes in electrophysiology (ERG) and retina histology, the neuroprotective effects of sulforaphane were evaluated
Methods: Two groups of C56BL/6 wild type mice (6-8 weeks old) (n= 8 per group) were used for this study. The retinal ischemic reperfusion injury was induced by elevation of intraocular pressure for 45 minutes. Following ischemic insult, vehicle (1% DMSO saline) or sulforaphane (25mg/kg/day) was administrated intraperitoneally once per day for 5 days. Retinal function was quantified by recording scotopic ERGs in dark-adapted mice prior to and one-week following ischemic insult.
Results: Scotopic ERG a- and b-wave amplitudes prior to ischemic injury were 408 ± 82 µV and 856 ± 146 µV, respectively. Following ischemic-reperfusion injury, scotopic ERG a- and b-wave amplitudes of vehicle-treated mice were 152 ± 50 µV and 332 ± 87 µV, respectively. By comparison, ERG a- and b-wave responses from sulforaphane-treated mice were 306 ± 73 µV and 664 ± 123 µV, respectively. Histologic comparison of the retinas demonstrated the preservation of retinal architecture in mice treated with sulforaphane following ischemic insult as compared to vehicle-treated mice.
Conclusions: Intraocular ischemic-reperfusion insult elicits marked deficits in retinal function as quantified by scotopic ERG. Administration of sulforaphane protects retinal function against ischemic-reperfusion injury. These preliminary findings suggest that sulforaphane may have therapeutic value in the early treatment of retinal ischemic diseases.
The major finding of this ongoing study is that sulforaphane demonstrates a generalized trend of a protective effect against retinal-ischemic injury. With in vivo experiments, there appears to be a trend towards improved a-wave and b-wave amplitudes with use of sulforaphane as compared to inert vehicle as well as preservation of retinal architecture.
SFN is an indirect antioxidant which induces antioxidant enzymes through upregulation of the nuclear factor erythroid 2-related factor 2 (Nrf2) (Kelsey et al., 2010). Ping et al (2010) reported that pretreatment with SFN reduced infarct ratio in neonatal brain hypoxia-ischemia model (Ping et al., 2010). Recently, Wu et al (2012) demonstrated that SFN protected neuron cultures against injury caused by oxygen and glucose deprivation/re-oxygenation, possibly via anti-apoptosis (Wu et al., 2012). SFN has also been shown to prevent cisplatin-induced nephropathy by modulating cell death and pro-inflammatory pathways (Guerrero-Beltran et al., 2012). SFN prevents retinal outer nuclear layer thinning and functional changes in a mouse light-induced retina injury (Tanito et al., 2005).
There is much left to discover in examining the use of sulforaphane in ischemic retinal disease. As we continue this project, we aim to include a larger sample size as well as histology and morphometry. Furthermore, the exact mechanism of action of sulforaphane remains unknown. We also plan to determine if the neuroprotective effects of sulforaphane on ischemic retinal injury depend on the presence of Nrf2 by quantifying relative changes in Nrf2 and its downstream effecter HO-1 by immunohistochemistry in wildtype mice.
Mice treated with sulforphane may demonstrate an improvement in ERG a-wave and b-wave amplitudes as well as the preservation of retinal architecture following ischemic insult as compared to vehicle-treated mice. These preliminary findings suggest that sulforaphane has therapeutic value in the management of retinal ischemic diseases.
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C56BL/6 wild type mice (6-8 weeks old) were randomly assigned to two groups: vehicle-treated retinal ischemic injury mice, and SFN-treated retinal ischemic injury mice. Retinal ischemia was induced by transient elevation of intraocular pressure as described below. Subsequently, vehicle (1% DMSO in saline) or SFN 25 mg/kg in 1% DMSO in saline was injected intraperitoneally daily for 5 days.
Induction of pressure-induced ischemia: Retinal Ischemia was induced in anesthetized mice by transient elevation of intraocular pressure (IOP) as previously described and successfully employed in our laboratory (Bu et al., 2010) (Vin et al., 2012). The anterior chamber of the right eye of each mouse was cannulated with a 30-gauge sterile needle connected to an elevated isotonic sterile saline bag. The IOP was raised to 110 mm Hg for 45 min. Retinal ischemia was confirmed by rapid blanching of the ocular fundus and the collapse of the retinal artery by indirect ophthalmoscope. The left eye used as the non-ischemic control.
Electroretinographic responses: To determine if SFN treatment can protect against acute retinal ischemic injury, retinal function was measured before inducing ischemic retinal injury and 7 days after inducing ischemic retinal injury. The amplitude of the scotopic a-waves, b-waves was quantified and compared between SFN treated mice to vehicle treated mice after ischemic insult.
Histologic documentation: The mice were sacrificed on day 7, 1 week post-ischemic injury. Each mouse underwent bilateral enucleation and the eyes were fixated in phosphate-buffered (pH=7.4) 2.5% glutaraldehyde-2% paraformaldehyde solution and embedded in epoxy resin. 1 μm sections (600-1000 μm from the optic nerve) were stained with toluidine blue.
Protected Retinal Function by Sulforaphane on Retinal Ischemic Injury #B0156
Normal (non ischemic)
Ambrecht LA1,3, Perlman JI1,2,3,4, McDonnell JF1, Bu P1,4
Department of Ophthalmology1 and Pathology2 Loyola University Chicago, Maywood, IL.
Surgery3 and Research4 Edward Hines, Jr. VA Hospital, Hines, IL.
Figure 1 . Representative dark-adapted ERGs obtained from strobe flash stimuli from normal (non-ischemic), SFN-treated (ischemic) and control-treated (ischemic) mice. Scales bar, 200 µV and 20 ms.Note preservation of ERG a- and b-waves in the SNF-treated ischemic mice compared with the control- treated ischemic mice.
Figure 2: Quantitative changes in ERG (A) a-wave and (B) b-wave amplitudes at different flash intensity. *P<0.01; One-way ANOVA analysis comparing control ischemic with SFN treatment ischemic and non ischemic and control ischemic (n=8).
Figure 3: Histological cross sections of the retina of, normal wild type mice without ischemia, sulforaphane-treated ischemic mice, and ischemic mice treated with inert vehicle
Acknowledgements: This work was supported by The Richard A. Perritt Charitable Foundation, Illinois Society for the Prevention of Blindness, and American Society of Cataract and Refractive Surgery.
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