Transcript No Slide Title

Protected Retinal Function by Sulforaphane on Retinal Ischemic Injury #B0156
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.
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 ischemicreperfusion 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.
RESULTS
DISCUSSION
Log cd s/m2
1.4
0.6
0.0
-0.6
-1.6
-2.4
Normal
SFN
Control
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.
(a)
(b)
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.
CONCLUSIONS
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.
METHODS
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.
Contact email: [email protected]
REFERENCES
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).
ONL
INL
IPL
GCL
Normal (non ischemic)
SFN (ischemic)
Control (ischemic)
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
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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.