1. P. Bu 1,3, B. Basith 3, Gabriel Jacob 1,, E.B. Stubbs, Jr. 1,3, J.I. Perlman 1,2,3,4, Department of Ophthalmology 1, Pathology 2 Loyola University Health System, Maywood, IL. Research 3 and Surgery 4, Edward Hines, Jr. VA Hospital, Hines, IL. 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. G- CSF 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). In this study we investigated whether prophylactically-administered G-CSF can protect retinal neurons against acute ischemic reperfusion injury. INTRODUCTION 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/m 2 ) with an interstimulus interval of 1 minute in duration. The body temperature of anesthetized rats was maintained at 37 º C 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 º C. Then immersed in 30% sucrose in PBS overnight at 4 º C and frozen in cutting compound in - 80 º C. 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 º C 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. METHODS 1. Hartung T Anti-inflammatory effects of granulocyte colony-stimulating factor. Curr opin Hematol 1998; 5:221-225 2. Tsai R.K., Chang C.H., Wang H.Z. Neuroprotective effects of recombinant human granulocyte colony-stimulating factor in neurodegeneration after optic nerve crush in rats. Experimental Eye Resrarch 2008; 87:242-250 3. Frank T, Schlachetzki JC, Göricke B, Meuer K, Rohde G, Dietz GP, Both systemic and local application of granulocyte-colony stimulating factor is neuroprotective after retinal ganglion cell axotomy BMC Neuroscirnce 2009; 10:49 4. Perlman JI, McCole SM, Pulluru P, Chang J, Lam TT, Tao MOM. Disturbances in the distribution of neurotransmitters in the rat retina after ischemia. Cur Eye Res 1996;15: 589 – 596. 5. Bu P, Von Zee CL, Basith B, Stubbs EB, Perlman JI. ERG Changes and D-Aspartate Uptake in Experimental Diabetic Retinopathy. 2006 Annual Meeting Abstract and Program Planner accessed at www.arvo.org. Association for Research in Vision and Ophthalmology. Abstract 4242. 6. Shibuki H, Katai N, Kuroiwa S, Kurokawa T, Arai J, Matsumoto K, Nakamura T, Yoshimura N. Expression and neuroprotective effect of hepatocyte growth factor in retinal ischemia- reperfusion injury. Invest Ophthalmol Vis Sci 2002;43:528-536. 7. Schäbitz W.R, Kollmar R, Schwaninger M, Juettler, E, Bardutzky J, Schölzke, M.N, Sommer C, Schwab S Neuroprotective effect of granulocyte colony-stimulating factor after focal cerebral ischemia Stroke 2003; 34:745-751 REFERENCES Granulocyte-colony stimulating factor protects retinal neurons from ischemic reperfusion injury Acknowledgements: This work was supported by Department of Veterans Affairs (C3638R) and The Richard A. Perritt Charitable Foundation.  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 SUMMARY Morphological changes in retina following ischemic-reperfusion injury GroupIRL (µm)Whole retina (µm) Control (non-ischemic, n = 8)57.7 ± 2.2125.7 ± 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 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 b- wave amplitudes as indicated. Pre, prior to ischemic insult; Post, 1-week following ischemic insult. * p<0.05, Student’s t- test (n = 8 per group) comparing post-ischemic insult data between vehicle- and G-CSF treated rats. Subcutaneously administration of G-CSF preserves retinal function following ischemic insult. Figure 3. Effect of G-CSF on preservation of retinal morphology following ischemic insult. Shown are representative toluidine blue- stained 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. RESULTS Figure 1. G-CSFR Expression in Normal Retina. Immunohistochemistry of normal rat retina shown that G- CSFR was expressed in ganglion cell layer and inner nuclear cell layer (left). The isotype control of rabbit IgG was used as negative control (right). GCLGCL IPLIPL INLINL ONLONL ISIS OSOS G -CSF treated ischemic retinaVehicle-treated ischemic retinaNaïve, non-ischemic retina GCLGCL ONLONL INLINL ISIS OSOS IPLIPL 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. CONCLUSIONS 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. 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. DISCUSSION Scotopic +5 dB Flash -1500 -1000 -500 0 500 1000 1500 -1500 -1000 -500 0 500 1000 1500 -150-100-50050100150200250300 Vehicle Amplitude (µV) Time (ms) Pre-ischemia Post-ischemia Scotopic +5 dB Flash -1500 -1000 -500 0 500 1000 1500 -1500 -1000 -500 0 500 1000 1500 -150-100-50050100150200250300 G-CSF Amplitude (µV) Time (ms) Pre-ischemia Post-ischemia