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Granulocyte-colony stimulating factor protects retinal neurons from
ischemic reperfusion injury
P. Bu1,3, B. Basith3, Gabriel Jacob1,, E.B. Stubbs, Jr.1,3, J.I. Perlman1,2,3,4, Department of Ophthalmology1, Pathology2
Loyola University Health System, Maywood, IL. Research3 and Surgery4 , Edward Hines, Jr. VA Hospital,
Hines, IL.
RESULTS
INTRODUCTION
SUMMARY
G-CSF
Vehicle
Granulocyte-colony-stimulating factor (G-CSF) is a powerful and specific growth factor for
neutrophilic granulocytes. G-CSF is currently used clinically for the treatment of neutropenia. GCSF promotes the proliferation, survival, and maturation of cells committed to the neutrophilic
granulocyte (NG) lineage through the specific G-CSF receptor (G-CSFR) (1).
Recently, G-CSF has been reported to exhibit novel anti-inflammatory and neuroprotective
properties. Subcutaneous administration of G-CSF or vitreous body injection of G-CSF reduced
retinal ganglion cell death in optic nerve crush animal model, it may work by being anti-apoptotic
involving the p-AKT signaling pathway as well as by attenuation of the inflammatory response at
the injury sites (2, 3).
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Acute Ischemia and G-CSF treatment: Two groups of anesthetized adult male Lewis rats (n=8 per
group) were subjected to an acute (45 min) episode of retinal ischemic injury (4). Retinal ischemia
was confirmed by blanching of the ocular fundus and the collapse of the retinal artery by indirect
ophthalmoscopy. G-CSF (0.1 mg/kg/day) (Neupogen, Amgen Inc) or vehicle (5% dextrose) were
subcutaneously administrated once per day x 5 days followed retinal ischemia injury.
Electroretinography (ERG): Retinal function was evaluated in rats before and 1 week after retinal
ischemia-reperfusion injury. Rats were dark-adapted overnight, anesthetized with ketamine
(100mg/kg) and xylazine (5mg/kg) and their pupils dilated. ERG’s were recorded with a stainless
steel wire electrode loop (LKC UTAS-E 3000) contacting the corneal surface through a layer of 1%
methylcellulose, as we have previously reported (5). Platinum needle electrodes were placed in the
cheek and tail served as reference and ground leads, respectively. A single-flash stimulus was
presented in order of increasing luminance across a 4-log unit stimulus range (-3.8 to +1 log cd
sec/m2) with an interstimulus interval of 1 minute in duration. The body temperature of anesthetized
rats was maintained at 37 ºC with a heating pad.
Histology and Morphometry: One week after transient retinal ischemic injury anesthetized rats
were sacrificed. Enucleated eyes were fixed in phosphate-buffered (pH 7.4) 2.5% glutaraldehyde-2%
paraformaldehyde solution and embedded in epoxy resin. Sections (1 µm), cut along the vertical
meridian of the eye and passed through the optic nerve head, were stained with toluidine blue. The
inner retinal thickness (between the inner limiting membrane and the boundary of the ONL and
OPL) and whole retinal thickness were measured as previously described (6). For each retinal
section, four separate measurements were performed.
Immunostaining: The eyes were enucleated from rats without retinal ischemia. The eye cups fixed
without cornea and lens for 1 hour in 4% paraformaldehyde in PBS at 4 ºC. Then immersed in 30%
sucrose in PBS overnight at 4 ºC and frozen in cutting compound in - 80 ºC. Cryosections ((15 µm)
were incubated in 10% normal goat serum in PBS containing 0.3% triton X-100 at room temperature
for 1 hour. Sections were then washed and incubated with anti-G-CSF receptor antibody(1: 500,
Santa Cruz Biotechnology) at 4 ºC overnight. Immunoreactivity was visualized by incubating the
section with Goat anti-rabbit Cy2-conjugated IgG (1:200, Jackson ImmunoResearch).
Immunofluorescence was imaged with an fluorescence microscope.
Post-ischemia
DISCUSSION
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METHODS
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In this study we investigated whether prophylactically-administered G-CSF can protect retinal
neurons against acute ischemic reperfusion injury.
G-CSFR was expressed in retinal neurons in ganglion cell layer and inner nuclear cell layer
Transient elevation of intraocular pressure produced marked thinning of inner retinal
layers, Inner retinal layer was partially preserved with G-CSF treatment
G-CSF treatment significantly protected against ischemia-induced loss of retinal function
Pre-ischemia
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The major finding of this preliminary study is that a short-term application of G-CSF
preserves retinal function and morphology following ischemic-reperfusion injury compared
with vehicle-treated controls.
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Recent studies strongly suggest that G-CSF play a crucial role in protecting cerebral neurons
from stroke (7). G-CSF as neuroprotective agents following retinal ischemic injury secondary
to optic nerve ligation has been reported (2, 3). G-CSF has not been evaluated in
neuroprotection of acute retinal ischemia-reperfusion injury.
Here, we found that retinal function and morphology following ischemic-reperfusion injury
was partially preserved with G-CSF treatment. G-CSFR was expressed in retinal cells in
ganglion cell layer and inner nuclear cell layer.
These preliminary findings raise awareness for the potential of G-CSF to protect against
ischemic-reperfusion retinal injuries.
CONCLUSIONS
Figure 2. Effect of G-CSF treatment on retinal function following ischemic insult. (Upper panels) ERG recordings from
Lewis rats treated with vehicle (5 % dextrose) or G-CSF (0.1 mg/kg) as indicated. Tracings shown were obtained prior to
(pre-ischemia) and 1-week (post-ischemia) following ischemic insult. (Lower panels) Quantitative changes in ERG a- and bwave amplitudes as indicated. Pre, prior to ischemic insult; Post, 1-week following ischemic insult. * p<0.05, Student’s ttest (n = 8 per group) comparing post-ischemic insult data between vehicle- and G-CSF treated rats.
G-CSF-treatment protects against acute ischemia-mediated changes in retinal morphology and
function. These findings suggest that G-CSF may be of therapeutic value in treatment of retinal
ischemic disorders.
Subcutaneously administration of G-CSF preserves retinal function following ischemic insult.
REFERENCES
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Naïve, non-ischemic retina
Vehicle-treated ischemic retina
G -CSF treated ischemic retina
Morphological changes in retina following ischemic-reperfusion injury
Figure 1. G-CSFR Expression in Normal Retina. Immunohistochemistry of normal rat retina shown that GCSFR was expressed in ganglion cell layer and inner nuclear cell layer (left). The isotype control of rabbit IgG
was used as negative control (right).
Group
IRL (µm)
Whole retina (µm)
Control (non-ischemic, n = 8)
57.7 ± 2.2
125.7 ± 3.2
Vehicle-treated (ischemic, n = 8)
29.1 ± 3.5*
85.6 ± 3.7**
G-CSF treated (ischemic, n = 8)
46.2 ± 0.6*
107.9 ± 2.1**
Figure 3. Effect of G-CSF on preservation of retinal morphology following ischemic insult. Shown are representative toluidine bluestained retinal sections from naïve, vehicle-, and G-CSF treated (0.1mg/kg/day X 5day) Lewis rats. IRL, Inner Retinal Thickness
(between the inner limiting membrane and the boundary of the ONL and OPL). *p < 0.05, **p < 0.05 Student’s t-test comparing
vehicle- to G-CSF treated groups.
Ischemic-reperfusion injury produced a marked thinning of the inner retinal layers and whole retina. G-CSF treatment
preserved retinal morphology following ischemic insult.
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colony-stimulating factor in neurodegeneration after optic nerve crush in rats. Experimental Eye
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in Experimental Diabetic Retinopathy. 2006 Annual Meeting Abstract and Program Planner
accessed at www.arvo.org. Association for Research in Vision and Ophthalmology. Abstract
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Acknowledgements:
work was supported
by Department
of Veterans Affairs
The
ischemia
2003; Foundation.
34:745-751
Richard A.
PerrittStroke
Charitable