Neuroprotective Effect of Sulforaphane on Retinal Ischemic Injury
Ambrecht LA1,3, Perlman JI1,2,3,4, McDonnell JF1, Gaynes J4, Bu P1,4
Department of Ophthalmology1 and Pathology2 Loyola University Chicago, Maywood, IL.
Surgery3 and Research4 Edward Hines, Jr. VA Hospital, Hines, IL.
INTRODUCTION
Retina ischemia is a common cause of visual impairment and blindness. It is a major
contributor to tissue damage in diseases including acute angle-closure glaucoma,
diabetic retinopathy, retinal vascular occlusions and retinopathy of prematurity
(Osborne et al., 2004). One key element of the pathologic alteration in retinal
ischemia-reperfusion injury is the generation of reactive oxygen species (ROS) during
the reperfusion (Osborne et al., 2004). ROS created during normal cellular respiration
must be neutralized by cellular antioxidant defenses before these free radicals have the
opportunity to damage the cell. Modulation of oxidative stress reduces histopathologic
changes in retinal ischemia-reperfusion injury (Szabo et al., 1991, Chen et al., 2009).
Sulforaphane (SFN), an isothiocyanate, is a precursor of glucosinolate in cruciferous
vegetables such as broccoli. (Zhang et al., 1992). SFN has been shown to protect brain
neurons undergoing hydrogen peroxide-induced oxidative stress in a mixed neuronastrocyte culture system (Kraft et al., 2004).
A thorough review of the literature indicates the absence of any studies examining the
neuroprotective role of SFN in retinal ischemia-reperfusion injury. The goal of this
project is to determine the neuroprotective effects of SFN on retinal ischemia in
vivo by using an established animal model. The proposed study will provide novel
insights into the development of therapeutic intervention for the treatment of patients
with retinal ischemic disease.
RESULTS
DISCUSSION
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.
Non-ischemia
SFN Treated
Ischemia
Control Treated
Ischemia
Log CD s/m2
-2.4
-1.6
-0.6
0.0
0.6
1.4
Figure 1 . Representative dark-adapted ERGs. Note preservation of ERG a- and b-waves in the SNF-treated
ischemic mice compared with the control- treated ischemic mice.
a-wave
Amplitude 1.4 Log cds/mm (+10db)
250
METHODS
A
200
150
Our preliminary findings raise awareness for the potential of sulforaphane to protect against
ischemic retinal disease-related retinal ganglion cell death.
CONCLUSIONS
100
REFERENCES
50
Non-ischemic
Control -treated
SFN-treated
b-wave
700
B
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 following ischemic insult as compared to vehicle-treated mice. These preliminary
findings suggest that resveratrol has therapeutic value in the management of retinal ischemic
diseases.
0
Amplitude 1.4 Log cds/mm (+10db)
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.
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).
600
500
400
300
200
100
0
Non-ischemic Control-treated
SFN-treated
Figure 2. Effect of sulforaphane treatment on retinal function following ischemic insult. Quantitative changes in
ERG (A) a-wave and (B) b-wave amplitudes at 1.4 log cds/mm flash intensity. (n=2-4 per group)
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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.