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Neuroprotective effects of recombinant human granulocyte colony-stimulating factor (G-CSF) in neurodegeneration after optic nerve crush in rats
Experimental Eye Research 87 (2008) 242–250
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Neuroprotective effects of recombinant human granulocyte colony-stimulating factor (G-CSF) in neurodegeneration after optic nerve crush in rats Rong Kung Tsai a, c, e, *, Chung Hsing Chang b, d, Hwei Zu Wang e, * a
Department of Ophthalmology, Buddhist Tzu Chi General Hospital, Hualien, Taiwan Department of Dermatology, Buddhist Tzu Chi General Hospital, Hualien, Taiwan c Department of Ophthalmology and Visual Science, Tzu Chi University, Hualien, Taiwan d Institute of Medical Science, Tzu Chi University, Hualien, Taiwan e Graduate Institute of Medicine and Department of Ophthalmology, Kaohsiung Medical University, Kaohsiung, Taiwan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 March 2008 Accepted in revised form 9 June 2008 Available online 17 June 2008
The purpose of the present study was to investigate the effects of granulocyte colony-stimulating factor (G-CSF) on neurodegeneration of optic nerve (ON) and retinal ganglion cells (RGCs) in a rat model of ON crush. The ONs of adult male Wistar rats (150–180 g) were crushed by a standardized method. The control eyes received a sham operation. G-CSF (100 mg/kg/day in 0.2 ml phosphate-buffered saline) or phosphate-buffered saline (PBS control) was immediately administered after ON crush for 5 days by subcutaneous injection. Rats were euthanized at 1 or 2 weeks after the crush injury. RGC density was counted by retrograde labeling with FluoroGold application to the superior colliculus, and visual function was assessed by flash visual evoked potentials (FVEP). TUNEL assay, Western blot analysis and immunohistochemistry of p-AKT in the retina and ED1 (marker of macrophage/microglia) in the ON were conducted. 2 weeks after the insult, the RGC densities in the central and mid-peripheral retinas in ONcrushed, G-CSF-treated rats were significantly higher than that of the corresponding ON-crushed, PBStreated rats (survival rate was 60% vs. 19.6% in the central retina; 46.5% vs. 23.9% in mid-peripheral retina, respectively; p < 0.001). FVEP measurements showed a significantly better preserved latency of the p1 wave in the ON-crushed, G-CSF-treated rats than the ON-crushed, PBS-treated rats (78 9 ms in the sham operation group, 98 16 ms in the G-CSF-treated group, and 174 16 ms in the PBS-treated group; p < 0.001). TUNEL assays showed fewer apoptotic cells in the retinal sections in the ON-crushed, G-CSFtreated rats. p-AKT immunoreactivity was up-regulated in the retinas of the ON-crushed, G-CSF-treated rats at 1 and 2 weeks. In addition, the number of ED1-positive cells was attenuated at the lesion site of the optic nerve in the ON-crushed, G-CSF-treated group. From these results, we gather that administration of G-CSF is neuroprotective in the rat model of optic nerve crush, as demonstrated both structurally by RGC density and functionally by FVEP. G-CSF may work by being anti-apoptotic involving the p-AKT signaling pathway as well as by attenuation of the inflammatory responses at the injury site, as evidenced by less ED1-positive cell infiltration in the optic nerve. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: granulocyte colony-stimulating factor rat model optic nerve crush ocular neuroprotection flash visual-evoked potential RGC density
1. Introduction Retinal ganglion cells (RGCs) are well-characterized CNS neurons, with cell bodies located in the inner part of the retina and axonal processes along the optic nerve (ON) that reach specific targets in the brain. ON injury triggers a process of degeneration in the damaged fibers as well as in fibers outside of the primary lesion,
* Corresponding authors. Department of Ophthalmology, Buddhist Tzu Chi General Hospital & Department of Ophthalmology and Visual Science, Tzu Chi University, 707 Sec.3, Chung Yung Road, Hualien 970, Taiwan. Tel.: þ886 3 856 1825x2112; fax: þ886 3 857 7161. E-mail address: [email protected] (R.K. Tsai). 0014-4835/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2008.06.004
i.e., secondary degeneration (Yoles and Schwartz, 1998). In addition, the associated retrograde degeneration will cause the loss of RGCs. Unfortunately, there is no established treatment modality to preserve the RGCs and the axons for acute traumatic optic neuropathy (TON). Although frequently used, pulse steroid therapy in TON remains controversial. Recent reports suggest that there are no convincing data to support that steroid therapy is having any additional benefit over observation alone in the treatment of TON (Ohlsson et al., 2004b; Entezari et al., 2007; Yu-Wei-Man and Griffiths, 2007). These observations prompt renewed efforts in the search for effective treatments. Pathophysiology of cerebral ischemic injury has implicated important roles of inflammatory processes in CNS injuries (Barone and Feuerstein, 1999). Activated neutrophils have been shown to aggravate cerebral ischemia injury
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by microvascular plugging and production of cytotoxic substances (Wang et al., 1993). Similar to cerebral ischemic injury, ON crush induces a retrograde degeneration of the RGCs after the injury. Prior to that, the myelin sheath of the axons degenerates, and the ED1positive phagocytes (including macrophages and microglia) infiltrate and remove the myelin debris. It has been shown that macrophage and microglia accumulation in the ON contributes to glial scar formation (Ohlsson et al., 2004a,b), resulting in apoptosis of RGCs (Ohlsson et al., 2004b; Stoll et al., 1989). It is believed/hypothesized that by preserving axons or reversing axonal degeneration, the RGCs may be preserved for regeneration. Attempts to repair the injured ON have identified two necessary objectives: (i) to prevent or delay the loss of neurons following injury and (ii) to overcome the failure of axons to extend towards appropriate targets (Bray et al., 1991; Doster et al., 1991). Therapies that stimulate both neuronal viability and axon growth may prove beneficial after ON lesion (Levin, 2007). Since ON crush injury is primarily an axogenic disease, as such, the process of secondary neuronal degeneration is much slower than somatogenic injury; the window of opportunity for neuroprotective therapy is therefore much greater in axogenic optic neuropathy than somagenic optic neuropathy (Levin, 2007). Although several compounds, such as brain-derived neurotrophic factor (BDNF; Klo¨cker et al., 1998; Ko et al., 2000; Pernet and Di Polo, 2006), ciliary neurotrophic factor (CNTF; Leaver et al., 2006), and free radical scavengers (Klo¨cker et al., 1998; Ko et al., 2000), as well as lens-injury associated macrophage activation (Yin et al., 2003), have demonstrated a neuroprotective effect in ON injury, their effects are limited due to axonal scar formation. Administration of granulocyte colony-stimulating factor (G-CSF) results in the mobilization of hematopoietic stem cells (HSCs) from bone marrow into peripheral blood (PB) (Demetri and Griffin, 1991). G-CSF has been already used extensively for treating chemotherapy-induced neutropenia, bone marrow reconstitution and stem cell mobilization (Weaver et al., 1993). Recently, PB-derived HSCs have been used in transplantation in place of bone marrow cells and for the regeneration of non-hematopoietic tissues such as skeletal muscle (Stratos et al., 2007) and heart (Harada et al., 2005; Li et al., 2006; Orlic et al., 2001). It facilitates a functional recovery effect in rats after stroke (Schabitz et al., 2003; Shyu et al., 2004), restores memory function in Alzheimer’s diseases in mice (Tsai et al., 2007), and has anti-apoptotic effect through activating a variety of intracellular signaling pathways, including PI3K/Akt (Dong and Larner, 2000; Komine-Kobayashi et al., 2006). G-CSF has also been employed as an anti-inflammatory agent in murine endotoxemia (Go¨rgen et al., 1992). In a pilot clinical study, acute stroke patients who received G-CSF therapy showed greater improvement in neurological functions after six months than patients who received standard care (Shyu et al., 2006). To the best of our knowledge, G-CSF has not been evaluated in neuroprotection of ocular neurons. In this study, we examined the effect of G-CSF treatment on neurodegeneration of ON and RGCs after ON crush in rats.
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body weight; Sigma, St. Louis, MO, USA), and using topical 0.5% Alcaine eye drops (Alcon, Puurs, Belgium). The rats had free access to food and water, and were maintained in cages in an environmentally controlled room with temperature of 23 1 C, humidity of 55 5%, and a 12-h light/dark cycle (light period: 07:00 to 19:00). 2.2. Study design Sixty rats were divided equally into two groups. All right eyes had ON crush and all left eyes had sham operations except the additional 18 animals in the flash visual-evoked potential (FVEP) study, in which both eyes of 18 rats underwent the same operation under special considerations with approval from the IACUC. The number of rats used in each experiment was summarized in Table 1. After ON crush surgery, the rats received once daily subcutaneous injection of recombinant human G-CSF (100 mg/kg/day in 0.2 ml of saline, 30 rats) (Takasaki pharmaceutical Plant, Tokyo, Japan) or PBS (0.2 ml, 30 rats) immediately after the crush procedure for 5 days thereafter (as recommended by the manufacturer, Shyu et al., 2004). Groups of rats were euthanized at 1 (20 rats) and 2(40 rats) weeks after surgery by CO2 insufflation. RGC density was measured by retrograde labeling with FluoroGold, and visual function was assessed by use of flash visualevoked potential (FVEP). TUNEL assay and Western blot analysis of p-AKT in the retina were conducted. Immunohistochemical studies included phosphorylated Akt (p-AKT) expression in the retina, and ED1 (macrophage/microglia) expression in the optic nerve were performed. 2.3. Experiment of optic nerve crush injury Sixty rats were enrolled in the treatment groups. After general anesthesia and topical Alcaine eye drop application, an incision was made on the temporal conjunctiva of a Wistar rat on the right eye with a conjunctival scissors, and the lateral rectus muscle was detached under an operating microscope. The ON was exposed and isolated. Care was taken to avoid damaging the small vessels around the ON. A vascular clip (60-g micro-vascular clip, World Precision Instruments, FL, USA) was then applied to the ON at a distance of 2 mm posterior to the globe for 30 s to ensure a reproducible injury on each animal (Maeda et al., 2004; Sarikcioglu et al., 2007). To confirm the patency of the retinal vessels after the insult, the retina was examined under an operating microscope immediately after injury. The dissected conjunctiva was pushed back, and Tobradex eye ointment (Alcon, Puurs, Belgium) was administered. To protect the eyeball, the eyelid was sutured at 2 mm from the temporal canthus with one stitch of 4-0 silk. Though not interfering with the eyelid opening after surgery, the stitch was removed 3 days later. After the surgery, the rats were kept on electronic heating pads at 37 C for recovery. The left eyes received
2. Material and methods 2.1. Animals
Table 1 Summary of rats used in study design
Ninety-two adult male Wistar rats weighing 150–180 g (7–8 weeks old) were used in this study. Rats were obtained from the breeding colony of BioLASCO Co., Taiwan. Animal care and experimental procedures were performed in accordance with the ARVO statement for the use of Animals in Ophthalmic and Vision Research. The Institutional Animal Care and Use Committee (IACUC) at Tzu Chi Medical Center approved all animal experiments. All manipulations were performed with animals under general anesthesia, brought about by intramuscular injection of a mixture of ketamine (40 mg/kg body weight) and xylazine (4 mg/kg
Experiments
No. of rats
ON crush injury experiment G-CSF-treated and ON-crushed PBS-treated and ON-crushed FVEP Bilateral ON-crushed and G-CSF-treated Bilateral ON-crushed and PBS-treated Bilateral sham-operated Normal control Western blot analysis of p-AKT Total
60 30 30 20 6 6 6 2 12 92
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the sham operation with optic nerve exposure without the crush, and the eyelids were sutured after the surgery.
divided by the number of RGCs in the normal retina and multiplying by 100.
2.4. Peripheral white blood cell count
2.7. Flash visual-evoked potentials (FVEPs)
To ensure the induction of leukocytosis by G-CSF, 2 ml of peripheral blood was drawn from the rat tails at 1 week after the surgery in both treatment groups, and the white cell counts were obtained (Tsai et al., 2007) (n ¼ 8 in each group).
For functional evaluation of the ON, flash visual-evoked potential (FVEP) was measured at 1 and 2 weeks after surgery in 18 experimental rats and 2 normal controls. The distance between two eyes of the rat was close and there was a concern regarding interference from the other eye during FVEP measurements. To exclude the possibility that the contralateral fellow eye contaminated the FVEP test in the other eye during light stimulation, we did the same surgery in both eyes. This special consideration was approved by the IACUC. This was performed as a sham operation in both eyes of rats in the control group (n ¼ 6) and bilateral optic nerve crushes in the PBS-treated (n ¼ 6), and G-CSF-treated groups (n ¼ 6). We used the visual electrodiagnostic system (UTAS-E3000, LKC Technologies, Gaithersburg, MD, USA) to measure FVEP. The system has built-in programs for clinical measurements of FVEP. In short, under general anesthesia, hairs of rats around the visual cortex (2 mm anterior to the lambda) and frontal cortex were shaved. The gold recording electrodes in the occipital area and a reference electrode in the frontal area were separately pasted with electrode gel. The ear clip was put firmly on the ear lobe of the rat. The settings were background illumination off, a flash intensity of Ganzfeld 0 db, single flash with flash rate on 1.9 Hz, the test average at 80 sweeps, the threshold for rejecting artifacts at 50 mV, and a sample rate of 2000 Hz. Only the latency of the first positive going wavelet (P1) of FVEP in the three groups was compared. The amplitude was not used because the interpretation of amplitude measurement was highly complicated by fixation, refraction and individual factors. When the wave was non-recordable, the latency of P1 was set at 200 ms for comparison.
2.5. Optic nerve and retinal sample preparations 2.5.1. Optic nerve A segment of the ON about 5–7 mm in length between the optic chiasm and the eyeball was harvested upon sacrifice at 2 weeks. The nerve was immediately frozen at 70 C for later histologic and immunohistochemical studies. Rats that showed axotomy or massive hemorrhage upon examination of the optic nerve histologic sections were excluded from the study. 2.5.2. Retina sample preparations After the cornea, lens, and vitreous body were removed, the remaining eye cups containing the sclera and the retina were fixed in 4% paraformaldehyde for 2 h at room temperature. The tissues were then dehydrated in 30% sucrose overnight and kept at 20 C until further processing for paraffin embedding and sectioning. 2.6. Retrograde labeling of RGCs with FluoroGold and morphometry of RGCs To avoid over-counting the RGCs by mixing labeled RGCs with dye engulfing macrophage and microglia, we performed the retrograde labeling of RGCs 1 week before the rats were euthanized (Levkovitch-Verbin et al., 2003; Maeda et al, 2004). Rats were anesthetized by ketamine and xylazine mixture, and placed in a stereotactic apparatus (Stoelting, Wood Dale, IL, USA), and the skin covering the skull of rats was opened for a length of 2 cm. The brain surface was exposed by perforating the parietal bone with a dental drill to facilitate dye injection. 1.5 ml of 5% of FluoroGold (Fluorochrome, Denver, CO, USA) was injected into the superior colliculus on each side through a Hamilton syringe (Ko et al., 2000). The dye was injected at a point 5.5 mm caudal to the bregma and 1.5 mm lateral to the midline on both sides at a depth of 4.5 mm from the surface of the skull. After surgery, holes in the skull were filled with bone wax and the skin was sutured. The rats were put on electronic heating pads at 37 C for recovery. One week after the labeling, the eyeballs were harvested after euthanasia of the animals. The eyeballs were placed in 10% formalin for 1 h; the whole retina was then carefully dissected, flattened by four radial cuts, and mounted with the vitreous side up on a microscopic slide. The retina was examined with a 400 epi-fluorescence microscope (Axioskop; Carl Zeiss Meditech Inc., Thornwood, NY, USA) equipped with a filter set (excitation filter, 350–400 nm; barrier filter, 515 nm) and a digital imaging system. The retinas were examined for RGCs at a distance of 1 or 3 mm from the center to provide the central and mid-peripheral RGC densities respectively. Microglia and macrophages, which may have incorporated FluoroGold after phagocytosis of dying RGCs, were excluded based on their suggested morphology (Levkovitch-Verbin et al., 2003). We counted at least five randomly chosen areas in the central (about 40% of central area) and mid-peripheral (about 30% of midperiphery) area of each retina and their averages were taken as the mean density of RGCs per retina. Each group contained at least eight rats for obtaining the mean density. RGC survival percentage was defined as the number of RGCs in each treatment group
2.8. In situ nick end-labeling (TUNEL) assay To ensure the use of equivalent fields for comparison, all paraffin sections of retinas were prepared with retinas at 1–2 mm distance from the optic nerve head. TUNEL reaction (TdT-FragELÔ DNA Fragmentation Detection Kit, Calbiochem, Darmstadt, Germany) was performed to detect retinal cell death according to the manufacturer’s protocol. Color was developed with DAB (DAKO, Santa Barbara, CA, USA) and counterstained with methyl green. To compare the TUNEL-positive cells in each group, the TUNEL-positive cells in the RGC layer of each sample were counted in ten highpowered fields (HPF, 400); three sections per eye were averaged and there were six rats in each group. 2.9. Immunohistochemistry 2.9.1. Immunohistochemistry of p-AKT in retina Retinal sections were first deparaffinized, then boiled for 20 min in citrate buffer (pH 6.0) for antigen retrieval, and incubated with 3% hydrogen peroxide solution in methanol for 10 min at room temperature to inhibit endogenous peroxidase activity. Primary antibody of p-AKT (1:25, Cell Signaling Inc., Danvers, MA, USA) was added and the preparations were incubated at 4 C overnight. The preparations were then washed twice with PBS and exposed to biotin-SP-conjugated anti-rabbit IgG (1:1000, Jackson ImmunoResearch, West Grove, PA, USA) for 2 h at room temperature. Finally, streptavidin/HRP (1:500, Dako, Glostrup, Denmark) was applied for 1 h and visualized by the use of DAB. Hematoxylin was used for counterstaining. We also examined the p-AKT in the retina of sham-operated, G-CSF-treated eyes. Three sections per eye were examined and there were six rats in each group.
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performed. Briefly, the frozen ON sections were fixed with acetone at 20 C for 30 min, and blocked with 5% FBS containing 1% BSA for 15 min. Primary antibody was applied, and incubated overnight at 4 C. The secondary antibody conjugated with FITC (1:100, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was applied at room temperature for 1 h. Counterstaining was performed using DAPI (1:1000, Sigma, St Louis, MO, USA). For comparison, ED1-positive cells were counted in six HPF at the lesion site of optic nerve (six rats in each group). 2.10. Western blot analysis of p-AKT
Fig. 1. White blood cell counts of peripheral blood in rats after daily administration of G-CSF or PBS for 5 days. The WBC counts were analyzed at 1 week after the beginning of treatment. Note a mean increase of approximately 50% in the G-CSF-treated group (n ¼ 8 in each group). *p < 0.05, p ¼ 0.008.
2.9.2. Immunohistochemistry of ED-1 (CD68) in the optic nerve Longitudinal sections of optic nerve were stained with hematoxylin–eosin for morphologic evaluation. Immunohistochemistry of ED1 (CD68, a marker of macrophage/microglia) using a monoclonal antibody (1:50, AbD Serotec, Oxford, UK) was also
Western blot analysis of p-AKT was performed with modifications as described (Nakazawa et al., 2003). Protein samples containing 50 mg of protein were separated on 12% sodium dodecyl sulfate–polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes (PerkinElmer, Waltham, MA, USA). The membranes were incubated in TBST buffer (0.02 M Tris-base, pH 7.6, 0.8% NaCl, 0.1% Tween 20) supplemented with 5% dry skim milk for 30 min to block nonspecific binding. p-AkT and AKT antibodies (1:1000, Cell Signaling Inc., Danvers, MA, USA) were added and the preparations were incubated at 4 C overnight. The membranes were washed twice with TBST buffer followed by incubation with Biotin-SP-conjugated appropriate goat anti-rabbit IgG secondary antibodies (1:1000, Jackson Immunoresearch, PA, USA) at
Fig. 2. Flat preparations of retinas and morphometry of RGCs at 1 week after retrograde FluoroGold labeling. (A–H) Representative flat preparations of central and mid-peripheral retinas. (A, E) Sham operation; (B, F) PBS-treated at 1 week after ON crush; C&G: PBS-treated at 2 weeks after ON crush; D&H: G-CSF-treated at 2 weeks after ON crush). (I, J) Morphometry of RGCs in the central and mid-peripheral retinas. RGC densities in the un-crushed group were 2550 340/mm2 and 1550 320/mm2, respectively. One week after ON crush, the RGC densities decreased to 2070 410/mm2 and 1040 210/mm2, respectively. At 2 weeks, the densities of RGCs in the central retina of the G-CSF treatment group and the PBS treatment group were 1530 490/mm2 (60.0% survival) and 500 210/mm2 (19.6% survival) respectively, and in mid-peripheral retina were 720 260/mm2 (46.5% survival) and 370 150/mm2 (23.9% survival) respectively, showing a significant preservative effect by G-CSF (p < 0.001) (n ¼ 8 in each group, 24 rats in total). C, crush injury. *p < 0.05; **p < 0.01.
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Fig. 3. VEPs. (A, B) Representative flash VEP tracings at 1 and 2 weeks after ON crush. (C) Latency of P1 wave. Note that 1 week after ON crush, the latency of the P1 wave in the sham-operated group (80 3 ms), was similar to the G-CSF-treated and ON-crushed group (84 3 ms), while the PBS-treated and ON-crushed group (98 6 ms [p ¼ 0.001 vs. control]) had a prolonged latency. By 2 weeks, the latency of the P1 wave in the G-CSF-treated group (99 16 ms) was also prolonged (p ¼ 0.05 vs. control [78 9 ms]), yet significantly less than the PBS-treated group (174 16 ms [p < 0.001]). There were two rats in the PBS-treated group without a recordable wavelength (n ¼ 6 in each group, 18 in total). *p < 0.05; **p < 0.01.
room temperature for 2 h. The blot was then washed with TBST and incubated with streptavidin/AP (1:500, DakoCytomation, Glostrup, Denmark) at room temperature for 1 h. The specific immune complexes were detected by BCIP/NBT solution (B6404, Sigma, Steinheim, Germany). Quantification was performed on computer (ImageJ software; NIH, MD, USA). To determine the activated AKT, the percentage of activated AKT was defined as phosphorylated AKT/total AKT. 2.11. Statistical analysis All measurements in this study were performed in a masked fashion; mean values with standard deviations (S.D.) were obtained and presented here. Student’s t-test was used to evaluate the differences between both treated groups in terms of cell number and milliseconds in FVEP tests. Statistical significance was declared if a p value was <0.05.
3.2. RGC density after ON crush The densities of RGCs in the central and mid-peripheral retina in the sham-operated eyes were 2550 340/mm2 and 1550 320/ mm2, respectively. One week after ON crush and with PBS treatment, the central and mid-peripheral RGC densities decreased to 2070 410/mm2 and 1040 210/mm2, respectively (p < 0.001). At 2 weeks after the insult, the densities of RGC in the central retina of the G-CSF and PBS treatment groups were reduced to 1530 490/ mm2 and 500 210/mm2, respectively (p < 0.001), and in the midperipheral retina were 720 260/mm2 and 370 150/mm2, respectively (p < 0.001) (Fig. 2). In other words, central RGC density showed a 60.0% survival rate in the G-CSF-treated rats and 19.6% in the PBS-treated rats; mid-peripheral RGC density showed a 46.5% survival rate in the G-CSF-treated rats and 23.9% in the PBS-treated rats at 2 weeks (p < 0.001). The results demonstrate that RGC survival rate increases by approximately 40.4% in central retina and 22.6% in the mid-peripheral retina in the G-CSF-treated group as compared to the PBS-treated group.
3. Results 3.3. FVEP 3.1. Administration of G-CSF and peripheral white blood cells counts One week after the administration of G-CSF, the white cell count was significantly increased (11,650 690/mm2 vs. 7810 790/ mm2; G-CSF vs. control; p ¼ 0.008). Hence, under our experimental condition, the administration of G-CSF for 5 days had a leukocytotic effect (Fig. 1).
Consistent with a previous report, FVEP responses in shamoperated rats were identical to those of unoperated rats (Ohlsson et al., 2004b), and we therefore only present the results of shamoperated rats as a control. The latency of the P1 wave at the first week was 80 3 ms, 84 3 ms, and 98 6 ms for the control, ON-crushed and G-CSF-treated (p ¼ 0.067 vs. control), and
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Fig. 4. TUNEL in retinal sections. (A–F) Representative of the TUNEL in retinal sections at high (A–C) and low (D–F) magnification. (A, D) Control sham-operated. (B, E) PBS-treated and (C, F) G-CSF-treated at 2 weeks after ON crush. (G) TUNEL-positive cells in the RGC layer at 2 weeks after ON crush (arrowhead). Note a significant increase (p < 0.001) in the PBS-treated and ON-crushed, when compared to the sham-operated group (14.9 6.3 cells/HPF vs. 2.1 1.6 cells/HPF). G-CSF ameliorated this increase (5.7 1.4 cells/HPF) (p ¼ 0.03 vs. PBS-treated group; n ¼ 6 in each group).
ON-crushed and PBS-treated groups (p ¼ 0.001 vs. control). At 2 weeks after surgery, the latency of the P1 wave was 78 9 ms, 99 16 ms, and 174 16 ms for the control, ON-crushed and GCSF-treated (p ¼ 0.05 vs. control; p < 0.001 vs. PBS-treated), and
ON-crushed and PBS-treated groups (p < 0.001 vs. control) (Fig. 3). The FVEP results demonstrate that the G-CSF-treated group had significantly improved visual function as compared to the PBS-treated group, at both 1 and 2 weeks after ON crush.
Fig. 5. Immunohistochemistry of p-AKT in the retina. (A) Sham-operated eyes. PBS-treated at 1 (B) and 2 (C) weeks after ON crush. (D) G-CSF-treated, 1 week after sham operation. G-CSF-treated, 1 (E) and (F) 2 weeks after ON crush. Note little immunoreactivity in the sham-operated eyes (A), PBS-treated and 1 (B) and 2 (C) weeks after ON crush, and G-CSFtreated and sham-operated (D), but high reactivity (arrowhead) in G-CSF-treated and 1 week after ON crush (E) and in G-CSF-treated at 2 weeks after ON crush (F). Note the p-AKT was also detected in the inner plexiform, inner nuclear layer, outer plexiform layers and outer nuclear layer (n ¼ 6 in each group). C, crush; S, sham-operated; P, PBS; G, G-CSF.
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Two rats had non-recordable waves in the PBS-treated group at 2 weeks. 3.4. In situ nick end-labeling (TUNEL) assay TUNEL assay demonstrated that TUNEL-positive cells/HPF (high-powered field) of 2.1 1.6 cells in the sham-operated group, 14.9 6.3 cells in the PBS-treated group and 5.7 1.4 cells in the GCSF-treated group (p ¼ 0.03 vs. PBS-treated group) in the RGC layer (Fig. 4). The results demonstrated that administration of G-CSF had a significant anti-apoptotic effect on RGCs after ON crush. 3.4.1. Immunohistochemistry of p-AKT in the retina There was little or no p-AKT immunoreactivity in the shamoperated eyes treated with PBS, and in ON-crushed and PBS-treated rats at 1 or 2 weeks. However, there was some reactivity in G-CSFtreated, sham-operated eye, and predominant expression of p-AKT in the RGC layers of ON-crushed and G-CSF-treated rats at 1 and 2 weeks (Fig. 5). The p-AKT was also detected in the inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL) and outer nuclear layer (ONL). This may indicate that the G-CSF administration for 5 days can activate the signaling pathway of pAKT in the retinal ganglion cell layer of rats, which further contributes to the anti-apoptotic effect on RGCs. 3.4.2. Western blot analysis of p-AKT The activated AKT was not altered in the sham-operated retinas and the retinas of ON-crushed and PBS-treated rats. In contrast, the activated AKT increased significantly in the retinas of ON-crushed and G-CSF-treated rats at 1 and 2 weeks about 2- and 1.7-fold respectively (p ¼ 0.016 at 1 week and p ¼ 0.025 at 2 weeks) (Fig. 6). These results of Western protein analysis of p-AKT were compatible with that of IHC of p-AKT in the retinas. 3.5. ED1 in the ON At 2 weeks after the insult, the injured site of the ON showed edema, disruption of the axons and inflammatory cell infiltration in the PBS-treated group, while less edema and less inflammatory cell
infiltration at the crush site of the optic nerve was observed in the G-CSF-treated group (Fig. 7A). Immunohistochemistry of ED1 for macrophage/microglia showed few ED1-positive cells in the shamoperated group (10 4 cells/HPF), prominent ED1-positive cells at the lesion area (93 13 cells/HPF) in the PBS-treated group, but few in the G-CSF-treated group (21 7 cells/HPF). The difference between treatment groups was statistically significant (p < 0.001) (Fig. 7B and C).
4. Discussion In the present study, our morphologic results demonstrate that G-CSF has a neuroprotective effect on ON as well as in RGCs after ON crush in adult rats. In addition, the visual function as demonstrated by FVEP was also better preserved in the G-CSF-treated eyes compared to the vehicle-treated ones, confirming the beneficial effect on the ocular structures. Our TUNEL assay results and immunohistochemistry of p-AKT showed that the administration of G-CSF was anti-apoptotic on RGCs after ON crush injury and suggested an involvement of p-AKT pathway. This finding is consistent with earlier studies showing a pivotal role of p-AKT in neuronal survival after injury, and that the signaling pathway can be activated by G-CSF (Avalos, 1996; Dong. and Larner., 2000; Kilic et al., 2002). Nakazawa et al. (2003) have demonstrated that the activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway has a neuroprotective effect on injured RGCs. They reported that optic nerve clamping injury induced intrinsic activation of p-AKT in the retina within 1 h and reached a maximum at 6 h, then returned to baseline at about 72 h and the inhibition of p-AKT significantly decreased the number of surviving RGCs. Our results of Western blot analysis and IHC of p-AKT demonstrate that G-CSF can activate the p-AKT signaling pathway in the retinas of the injured RGCs at 1 and 2 weeks after ON crush. These observations may also suggest that expression of p-AKT at certain window of time after ON crush may contribute to the anti-apoptotic effect and the rescue of the RGCs from death. The mechanism of G-CSF activation of the AKT pathways in the RGCs remains to be dissected.
Fig. 6. Western blot analysis of activated p-AKT expression of the rat retinas after ON crush. (A) A representative photograph of the Western blot analysis. p-Akt, anti-phosphorylated Akt antibody; Akt, anti-AKT antibody; S, sham operation; C, crush; P, PBS; G, G-CSF. (B) The optical density of activated p-AKT in the Western blot is expressed as a ratio to that of AKT. Note the p-AKT protein expression in the retinas was up-regulated in G-CSF-treated rats at 1 and 2 weeks after ON crush. There was no significant change in that of PBS-treated rats at 1 and 2 weeks after ON crush (n ¼ 3 each). *p < 0.05, CþP 1 week vs. CþG 1 week p ¼ 0.016; CþP 2 weeks vs. CþG 2 weeks p ¼ 0.025.
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Fig. 7. Histology of ONs and immunohistochemistry of ED1 in ONs at 2 weeks after ON crush. (A) Hematoxylin–eosin staining of longitudinal sections of ONs. Note prominent axonal disruption and inflammatory cells infiltrate at the crush site in the PBS-treated group (middle panel) when compared to the control (left panel), but not in the G-CSF-treated group (right panel). The right side of each panel is the ON near the globe. (B) Immunohistochemistry of ED1. ED1-positive cells were prominent at the lesion site of ONs in the PBStreated group, and less so in the sham-operated group and the G-CSF-treated group. (C) Morphometry of ED1-positive cells in the ON. The difference of ED1-positive cells between the two treatment groups was statistically significant (n ¼ 6 in each group). p < 0.001.
After ON crush, ED1-labeled macrophages/microglia accumulated at the site of injury. Ohlsson et al. (2004b) reported that ED1positive cells continued to increase at the site of injury for 28 days after ON crush injury. Macrophages seem to be responsible for the removal of most of the myelin debris in CNS injury; however, these cells lack the capacity to remove axonal and myelin debris as fast as in the peripheral system, and contributed to glial scar formation, which is an obstacle for regeneration (Ohlsson et al., 2004a,b). ED1positive phagocytes found in ON injury include monocytes/macrophages of hematogenous origin as well as microglia. A portion of ED1-positive cells concomitantly express Ia antigen during Wallerian degeneration in the ON (Stoll et al., 1989). Our results show that ED1-positive macrophage/microglia accumulation at the ON lesion site was attenuated in the G-CSF-treated rats, implicating that immediate administration of G-CSF may have an anti-
inflammatory effect on the injured ON. In animal studies, neuroprotective effects of G-CSF have been ascribed to anti-inflammatory actions mediated by inhibition of TNF-a, and inducible nitric oxide synthase (iNOS) activity (Go¨rgen et al., 1992), and by a reduction of interleukin-1b expression (Avalos, 1996; Gibson et al., 2005; Go¨rgen et al., 1992). Macrophage/microglia accumulation can activate TNF-a, iNOS and cytokines (such as interleukins) expression. Our histopathological studies of the optic nerve also demonstrated less edema and inflammatory cell infiltration in the G-CSF-treated group. Since ON injury is axogenic, it is rational to hypothesize that if drug intervention can decrease the inflammatory response at the optic nerve, it may protect the RGCs from secondary degeneration. G-CSF recruitment of neutrophils combines with a simultaneous limitation of the harmful inflammatory reaction by inhibition of the above inflammatory mediators (Hartung, 1998). In addition,
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administration of G-CSF increases the mobilization of HSCs from bone marrow to PB; it is possible that the interactions between the increased HSCs and injured neural tissue will lead to the production of trophic factors that contribute to the anti-inflammation and survival of damaged neural tissue (Lachmund et al., 1994; Majka et al., 2001). In conclusion, we demonstrated that immediate administration of G-CSF provides ocular neuroprotection, as indicated by both RGC density counts and by functional assessment using FVEP, after ON crush in rats. The neuroprotective effects of G-CSF administration may involve both anti-apoptotic and anti-inflammatory processes. Acknowledgments The authors thank Dr. Tim T. Lam for his critical reading of the manuscript and Ms. Su Zen Chen for her excellent figure assistance. A National Science Council Grant from the Taiwanese government NSC96-2314-B-303-005, and Tzu Chi General Hospital research grant TCRD-I9606-01 supported this study. References Avalos, B.R., 1996. Molecular analysis of the granulocyte colony-stimulating factor receptor. Blood 88, 761–777. Barone, F.C., Feuerstein, G.Z., 1999. Inflammatory mediators and stroke: new opportunities for novel therapeutics. J. Cereb. Blood Flow Metab. 19, 819–834. Bray, G.M., Villegas-Pe´rez, M.P., Vidal-Sanz, M., Carter, D.A., Aguayo, A.J., 1991. Neuronal and non-neuronal influences on retinal ganglion cell survival, axonal regrowth, and connectivity after axotomy. Ann. N.Y. Acad. Sci. 633, 214–228. Demetri, G.D., Griffin, J.D., 1991. Granulocyte colony-stimulating factor and its receptor. Blood 78, 2791–2808. Dong, F., Larner, A.C., 2000. Activation of Akt kinase by granulocyte colony-stimulating factor (G-CSF); evidence of the role of a tyrosine kinase activity distinct from the janus kinases. Blood 95, 656–1662. Doster, S.K., Lozano, A.M., Aguayo, A.J., Willard, M.B., 1991. Expression of the growth associated protein GAP-43 in adult rat retinal ganglion cells following axon injury. Neuron 6, 635–647. Entezari, M., Rajavi, Z., Sedighi, N., Daftarian, N., Sanagoo, M., 2007. High-dose intravenous Methylprednisolone in recent traumatic optic neuropathy; a randomized double-masked placebo-controlled clinical trial. Graefe’s Arch. Clin. Exp. Ophthalmol. 245, 1267–1271. Gibson, C.L., Jones, N.C., Prior, M.J., Bath, P.M., Murphy, S.P., 2005. G-CSF suppresses edema formation and reduces interleukin1beta expression after cerebral ischemia in mice. J. Neuropathol. Exp. Neurol. 64, 763–767. Go¨rgen, I., Hartung, T., Leist, M., Nieho¨rster, M., Tiegs, G., Uhlig, S., Weitzel, F., Wendel, A., 1992. Granulocyte colony-stimulating factor treatment protects rodents against lipopolysaccharide-induced toxicity via suppression of systemic tumor necrosis factor-alpha. J. Immunol. 149, 918–924. Harada, M., Qin, Y., Takano, H., Minamino, T., Zou, Y., Toko, H., Ohtsuka, M., Matsuura, K., Sano, M., Nishi, J., Iwanaga, K., Akazawa, H., Kunieda, T., Zhu, W., Hasegawa, H., Kunisada, K., Nagai, T., Nakaya, H., Yamauchi-Takihara, K., Komuro, I., 2005. G-CSF prevents cardiac remodeling after myocardial infarction by activating the Jak-Stat pathway in cardiomyocytes. Nat. Med. 11, 305–311. Hartung, T., 1998. Anti-inflammatory effect of granulocyte colony-stimulating factor. Curr. Opin. Hematol. 5, 221–225. Kilic, E., Dietz, G.P., Hermann, D.M., Ba¨hr, M., 2002. Intravenous TAT-Bcl-XL is protective after middle cerebral artery occlusion in mice. Ann. Neurol. 52, 617–622. Klo¨cker, N., Cellerino, A., Ba¨hr, M., 1998. Free radical scavenging and inhibition of nitric oxide synthase potentiates the neurotrophic effects of brain-derived neurotrophic factor on axotomized retinal ganglion cells in vivo. J. Neurosci. 18, 1038–1046. Ko, M.L., Hu, D.N., Ritch, R., Sharma, S.C., 2000. The combined effect of brain-derived neurotrophic factor and a free radical scavenger in experimental glaucoma. Invest. Ophthalmol. Vis. Sci. 41, 2967–2971. Komine-Kobayashi, M., Zhang, N., Tanak, R., Hara, H., Osaka, A., Mochizuki, H., Mizuno, Y., Urabe, T., 2006. Neuroprotective effect of recombinant human granulocyte colony-stimulating factor in transient focal ischemia of mice. J. Cereb. Blood Flow Metab. 26, 402–413.
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Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer
Neuroprotective effects of recombinant human granulocyte colony-stimulating factor (G-CSF) in neurodegeneration after optic nerve crush in rats Rong Kung Tsai a, c, e, *, Chung Hsing Chang b, d, Hwei Zu Wang e, * a
Department of Ophthalmology, Buddhist Tzu Chi General Hospital, Hualien, Taiwan Department of Dermatology, Buddhist Tzu Chi General Hospital, Hualien, Taiwan c Department of Ophthalmology and Visual Science, Tzu Chi University, Hualien, Taiwan d Institute of Medical Science, Tzu Chi University, Hualien, Taiwan e Graduate Institute of Medicine and Department of Ophthalmology, Kaohsiung Medical University, Kaohsiung, Taiwan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 March 2008 Accepted in revised form 9 June 2008 Available online 17 June 2008
The purpose of the present study was to investigate the effects of granulocyte colony-stimulating factor (G-CSF) on neurodegeneration of optic nerve (ON) and retinal ganglion cells (RGCs) in a rat model of ON crush. The ONs of adult male Wistar rats (150–180 g) were crushed by a standardized method. The control eyes received a sham operation. G-CSF (100 mg/kg/day in 0.2 ml phosphate-buffered saline) or phosphate-buffered saline (PBS control) was immediately administered after ON crush for 5 days by subcutaneous injection. Rats were euthanized at 1 or 2 weeks after the crush injury. RGC density was counted by retrograde labeling with FluoroGold application to the superior colliculus, and visual function was assessed by flash visual evoked potentials (FVEP). TUNEL assay, Western blot analysis and immunohistochemistry of p-AKT in the retina and ED1 (marker of macrophage/microglia) in the ON were conducted. 2 weeks after the insult, the RGC densities in the central and mid-peripheral retinas in ONcrushed, G-CSF-treated rats were significantly higher than that of the corresponding ON-crushed, PBStreated rats (survival rate was 60% vs. 19.6% in the central retina; 46.5% vs. 23.9% in mid-peripheral retina, respectively; p < 0.001). FVEP measurements showed a significantly better preserved latency of the p1 wave in the ON-crushed, G-CSF-treated rats than the ON-crushed, PBS-treated rats (78 9 ms in the sham operation group, 98 16 ms in the G-CSF-treated group, and 174 16 ms in the PBS-treated group; p < 0.001). TUNEL assays showed fewer apoptotic cells in the retinal sections in the ON-crushed, G-CSFtreated rats. p-AKT immunoreactivity was up-regulated in the retinas of the ON-crushed, G-CSF-treated rats at 1 and 2 weeks. In addition, the number of ED1-positive cells was attenuated at the lesion site of the optic nerve in the ON-crushed, G-CSF-treated group. From these results, we gather that administration of G-CSF is neuroprotective in the rat model of optic nerve crush, as demonstrated both structurally by RGC density and functionally by FVEP. G-CSF may work by being anti-apoptotic involving the p-AKT signaling pathway as well as by attenuation of the inflammatory responses at the injury site, as evidenced by less ED1-positive cell infiltration in the optic nerve. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: granulocyte colony-stimulating factor rat model optic nerve crush ocular neuroprotection flash visual-evoked potential RGC density
1. Introduction Retinal ganglion cells (RGCs) are well-characterized CNS neurons, with cell bodies located in the inner part of the retina and axonal processes along the optic nerve (ON) that reach specific targets in the brain. ON injury triggers a process of degeneration in the damaged fibers as well as in fibers outside of the primary lesion,
* Corresponding authors. Department of Ophthalmology, Buddhist Tzu Chi General Hospital & Department of Ophthalmology and Visual Science, Tzu Chi University, 707 Sec.3, Chung Yung Road, Hualien 970, Taiwan. Tel.: þ886 3 856 1825x2112; fax: þ886 3 857 7161. E-mail address: [email protected] (R.K. Tsai). 0014-4835/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2008.06.004
i.e., secondary degeneration (Yoles and Schwartz, 1998). In addition, the associated retrograde degeneration will cause the loss of RGCs. Unfortunately, there is no established treatment modality to preserve the RGCs and the axons for acute traumatic optic neuropathy (TON). Although frequently used, pulse steroid therapy in TON remains controversial. Recent reports suggest that there are no convincing data to support that steroid therapy is having any additional benefit over observation alone in the treatment of TON (Ohlsson et al., 2004b; Entezari et al., 2007; Yu-Wei-Man and Griffiths, 2007). These observations prompt renewed efforts in the search for effective treatments. Pathophysiology of cerebral ischemic injury has implicated important roles of inflammatory processes in CNS injuries (Barone and Feuerstein, 1999). Activated neutrophils have been shown to aggravate cerebral ischemia injury
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by microvascular plugging and production of cytotoxic substances (Wang et al., 1993). Similar to cerebral ischemic injury, ON crush induces a retrograde degeneration of the RGCs after the injury. Prior to that, the myelin sheath of the axons degenerates, and the ED1positive phagocytes (including macrophages and microglia) infiltrate and remove the myelin debris. It has been shown that macrophage and microglia accumulation in the ON contributes to glial scar formation (Ohlsson et al., 2004a,b), resulting in apoptosis of RGCs (Ohlsson et al., 2004b; Stoll et al., 1989). It is believed/hypothesized that by preserving axons or reversing axonal degeneration, the RGCs may be preserved for regeneration. Attempts to repair the injured ON have identified two necessary objectives: (i) to prevent or delay the loss of neurons following injury and (ii) to overcome the failure of axons to extend towards appropriate targets (Bray et al., 1991; Doster et al., 1991). Therapies that stimulate both neuronal viability and axon growth may prove beneficial after ON lesion (Levin, 2007). Since ON crush injury is primarily an axogenic disease, as such, the process of secondary neuronal degeneration is much slower than somatogenic injury; the window of opportunity for neuroprotective therapy is therefore much greater in axogenic optic neuropathy than somagenic optic neuropathy (Levin, 2007). Although several compounds, such as brain-derived neurotrophic factor (BDNF; Klo¨cker et al., 1998; Ko et al., 2000; Pernet and Di Polo, 2006), ciliary neurotrophic factor (CNTF; Leaver et al., 2006), and free radical scavengers (Klo¨cker et al., 1998; Ko et al., 2000), as well as lens-injury associated macrophage activation (Yin et al., 2003), have demonstrated a neuroprotective effect in ON injury, their effects are limited due to axonal scar formation. Administration of granulocyte colony-stimulating factor (G-CSF) results in the mobilization of hematopoietic stem cells (HSCs) from bone marrow into peripheral blood (PB) (Demetri and Griffin, 1991). G-CSF has been already used extensively for treating chemotherapy-induced neutropenia, bone marrow reconstitution and stem cell mobilization (Weaver et al., 1993). Recently, PB-derived HSCs have been used in transplantation in place of bone marrow cells and for the regeneration of non-hematopoietic tissues such as skeletal muscle (Stratos et al., 2007) and heart (Harada et al., 2005; Li et al., 2006; Orlic et al., 2001). It facilitates a functional recovery effect in rats after stroke (Schabitz et al., 2003; Shyu et al., 2004), restores memory function in Alzheimer’s diseases in mice (Tsai et al., 2007), and has anti-apoptotic effect through activating a variety of intracellular signaling pathways, including PI3K/Akt (Dong and Larner, 2000; Komine-Kobayashi et al., 2006). G-CSF has also been employed as an anti-inflammatory agent in murine endotoxemia (Go¨rgen et al., 1992). In a pilot clinical study, acute stroke patients who received G-CSF therapy showed greater improvement in neurological functions after six months than patients who received standard care (Shyu et al., 2006). To the best of our knowledge, G-CSF has not been evaluated in neuroprotection of ocular neurons. In this study, we examined the effect of G-CSF treatment on neurodegeneration of ON and RGCs after ON crush in rats.
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body weight; Sigma, St. Louis, MO, USA), and using topical 0.5% Alcaine eye drops (Alcon, Puurs, Belgium). The rats had free access to food and water, and were maintained in cages in an environmentally controlled room with temperature of 23 1 C, humidity of 55 5%, and a 12-h light/dark cycle (light period: 07:00 to 19:00). 2.2. Study design Sixty rats were divided equally into two groups. All right eyes had ON crush and all left eyes had sham operations except the additional 18 animals in the flash visual-evoked potential (FVEP) study, in which both eyes of 18 rats underwent the same operation under special considerations with approval from the IACUC. The number of rats used in each experiment was summarized in Table 1. After ON crush surgery, the rats received once daily subcutaneous injection of recombinant human G-CSF (100 mg/kg/day in 0.2 ml of saline, 30 rats) (Takasaki pharmaceutical Plant, Tokyo, Japan) or PBS (0.2 ml, 30 rats) immediately after the crush procedure for 5 days thereafter (as recommended by the manufacturer, Shyu et al., 2004). Groups of rats were euthanized at 1 (20 rats) and 2(40 rats) weeks after surgery by CO2 insufflation. RGC density was measured by retrograde labeling with FluoroGold, and visual function was assessed by use of flash visualevoked potential (FVEP). TUNEL assay and Western blot analysis of p-AKT in the retina were conducted. Immunohistochemical studies included phosphorylated Akt (p-AKT) expression in the retina, and ED1 (macrophage/microglia) expression in the optic nerve were performed. 2.3. Experiment of optic nerve crush injury Sixty rats were enrolled in the treatment groups. After general anesthesia and topical Alcaine eye drop application, an incision was made on the temporal conjunctiva of a Wistar rat on the right eye with a conjunctival scissors, and the lateral rectus muscle was detached under an operating microscope. The ON was exposed and isolated. Care was taken to avoid damaging the small vessels around the ON. A vascular clip (60-g micro-vascular clip, World Precision Instruments, FL, USA) was then applied to the ON at a distance of 2 mm posterior to the globe for 30 s to ensure a reproducible injury on each animal (Maeda et al., 2004; Sarikcioglu et al., 2007). To confirm the patency of the retinal vessels after the insult, the retina was examined under an operating microscope immediately after injury. The dissected conjunctiva was pushed back, and Tobradex eye ointment (Alcon, Puurs, Belgium) was administered. To protect the eyeball, the eyelid was sutured at 2 mm from the temporal canthus with one stitch of 4-0 silk. Though not interfering with the eyelid opening after surgery, the stitch was removed 3 days later. After the surgery, the rats were kept on electronic heating pads at 37 C for recovery. The left eyes received
2. Material and methods 2.1. Animals
Table 1 Summary of rats used in study design
Ninety-two adult male Wistar rats weighing 150–180 g (7–8 weeks old) were used in this study. Rats were obtained from the breeding colony of BioLASCO Co., Taiwan. Animal care and experimental procedures were performed in accordance with the ARVO statement for the use of Animals in Ophthalmic and Vision Research. The Institutional Animal Care and Use Committee (IACUC) at Tzu Chi Medical Center approved all animal experiments. All manipulations were performed with animals under general anesthesia, brought about by intramuscular injection of a mixture of ketamine (40 mg/kg body weight) and xylazine (4 mg/kg
Experiments
No. of rats
ON crush injury experiment G-CSF-treated and ON-crushed PBS-treated and ON-crushed FVEP Bilateral ON-crushed and G-CSF-treated Bilateral ON-crushed and PBS-treated Bilateral sham-operated Normal control Western blot analysis of p-AKT Total
60 30 30 20 6 6 6 2 12 92
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the sham operation with optic nerve exposure without the crush, and the eyelids were sutured after the surgery.
divided by the number of RGCs in the normal retina and multiplying by 100.
2.4. Peripheral white blood cell count
2.7. Flash visual-evoked potentials (FVEPs)
To ensure the induction of leukocytosis by G-CSF, 2 ml of peripheral blood was drawn from the rat tails at 1 week after the surgery in both treatment groups, and the white cell counts were obtained (Tsai et al., 2007) (n ¼ 8 in each group).
For functional evaluation of the ON, flash visual-evoked potential (FVEP) was measured at 1 and 2 weeks after surgery in 18 experimental rats and 2 normal controls. The distance between two eyes of the rat was close and there was a concern regarding interference from the other eye during FVEP measurements. To exclude the possibility that the contralateral fellow eye contaminated the FVEP test in the other eye during light stimulation, we did the same surgery in both eyes. This special consideration was approved by the IACUC. This was performed as a sham operation in both eyes of rats in the control group (n ¼ 6) and bilateral optic nerve crushes in the PBS-treated (n ¼ 6), and G-CSF-treated groups (n ¼ 6). We used the visual electrodiagnostic system (UTAS-E3000, LKC Technologies, Gaithersburg, MD, USA) to measure FVEP. The system has built-in programs for clinical measurements of FVEP. In short, under general anesthesia, hairs of rats around the visual cortex (2 mm anterior to the lambda) and frontal cortex were shaved. The gold recording electrodes in the occipital area and a reference electrode in the frontal area were separately pasted with electrode gel. The ear clip was put firmly on the ear lobe of the rat. The settings were background illumination off, a flash intensity of Ganzfeld 0 db, single flash with flash rate on 1.9 Hz, the test average at 80 sweeps, the threshold for rejecting artifacts at 50 mV, and a sample rate of 2000 Hz. Only the latency of the first positive going wavelet (P1) of FVEP in the three groups was compared. The amplitude was not used because the interpretation of amplitude measurement was highly complicated by fixation, refraction and individual factors. When the wave was non-recordable, the latency of P1 was set at 200 ms for comparison.
2.5. Optic nerve and retinal sample preparations 2.5.1. Optic nerve A segment of the ON about 5–7 mm in length between the optic chiasm and the eyeball was harvested upon sacrifice at 2 weeks. The nerve was immediately frozen at 70 C for later histologic and immunohistochemical studies. Rats that showed axotomy or massive hemorrhage upon examination of the optic nerve histologic sections were excluded from the study. 2.5.2. Retina sample preparations After the cornea, lens, and vitreous body were removed, the remaining eye cups containing the sclera and the retina were fixed in 4% paraformaldehyde for 2 h at room temperature. The tissues were then dehydrated in 30% sucrose overnight and kept at 20 C until further processing for paraffin embedding and sectioning. 2.6. Retrograde labeling of RGCs with FluoroGold and morphometry of RGCs To avoid over-counting the RGCs by mixing labeled RGCs with dye engulfing macrophage and microglia, we performed the retrograde labeling of RGCs 1 week before the rats were euthanized (Levkovitch-Verbin et al., 2003; Maeda et al, 2004). Rats were anesthetized by ketamine and xylazine mixture, and placed in a stereotactic apparatus (Stoelting, Wood Dale, IL, USA), and the skin covering the skull of rats was opened for a length of 2 cm. The brain surface was exposed by perforating the parietal bone with a dental drill to facilitate dye injection. 1.5 ml of 5% of FluoroGold (Fluorochrome, Denver, CO, USA) was injected into the superior colliculus on each side through a Hamilton syringe (Ko et al., 2000). The dye was injected at a point 5.5 mm caudal to the bregma and 1.5 mm lateral to the midline on both sides at a depth of 4.5 mm from the surface of the skull. After surgery, holes in the skull were filled with bone wax and the skin was sutured. The rats were put on electronic heating pads at 37 C for recovery. One week after the labeling, the eyeballs were harvested after euthanasia of the animals. The eyeballs were placed in 10% formalin for 1 h; the whole retina was then carefully dissected, flattened by four radial cuts, and mounted with the vitreous side up on a microscopic slide. The retina was examined with a 400 epi-fluorescence microscope (Axioskop; Carl Zeiss Meditech Inc., Thornwood, NY, USA) equipped with a filter set (excitation filter, 350–400 nm; barrier filter, 515 nm) and a digital imaging system. The retinas were examined for RGCs at a distance of 1 or 3 mm from the center to provide the central and mid-peripheral RGC densities respectively. Microglia and macrophages, which may have incorporated FluoroGold after phagocytosis of dying RGCs, were excluded based on their suggested morphology (Levkovitch-Verbin et al., 2003). We counted at least five randomly chosen areas in the central (about 40% of central area) and mid-peripheral (about 30% of midperiphery) area of each retina and their averages were taken as the mean density of RGCs per retina. Each group contained at least eight rats for obtaining the mean density. RGC survival percentage was defined as the number of RGCs in each treatment group
2.8. In situ nick end-labeling (TUNEL) assay To ensure the use of equivalent fields for comparison, all paraffin sections of retinas were prepared with retinas at 1–2 mm distance from the optic nerve head. TUNEL reaction (TdT-FragELÔ DNA Fragmentation Detection Kit, Calbiochem, Darmstadt, Germany) was performed to detect retinal cell death according to the manufacturer’s protocol. Color was developed with DAB (DAKO, Santa Barbara, CA, USA) and counterstained with methyl green. To compare the TUNEL-positive cells in each group, the TUNEL-positive cells in the RGC layer of each sample were counted in ten highpowered fields (HPF, 400); three sections per eye were averaged and there were six rats in each group. 2.9. Immunohistochemistry 2.9.1. Immunohistochemistry of p-AKT in retina Retinal sections were first deparaffinized, then boiled for 20 min in citrate buffer (pH 6.0) for antigen retrieval, and incubated with 3% hydrogen peroxide solution in methanol for 10 min at room temperature to inhibit endogenous peroxidase activity. Primary antibody of p-AKT (1:25, Cell Signaling Inc., Danvers, MA, USA) was added and the preparations were incubated at 4 C overnight. The preparations were then washed twice with PBS and exposed to biotin-SP-conjugated anti-rabbit IgG (1:1000, Jackson ImmunoResearch, West Grove, PA, USA) for 2 h at room temperature. Finally, streptavidin/HRP (1:500, Dako, Glostrup, Denmark) was applied for 1 h and visualized by the use of DAB. Hematoxylin was used for counterstaining. We also examined the p-AKT in the retina of sham-operated, G-CSF-treated eyes. Three sections per eye were examined and there were six rats in each group.
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performed. Briefly, the frozen ON sections were fixed with acetone at 20 C for 30 min, and blocked with 5% FBS containing 1% BSA for 15 min. Primary antibody was applied, and incubated overnight at 4 C. The secondary antibody conjugated with FITC (1:100, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was applied at room temperature for 1 h. Counterstaining was performed using DAPI (1:1000, Sigma, St Louis, MO, USA). For comparison, ED1-positive cells were counted in six HPF at the lesion site of optic nerve (six rats in each group). 2.10. Western blot analysis of p-AKT
Fig. 1. White blood cell counts of peripheral blood in rats after daily administration of G-CSF or PBS for 5 days. The WBC counts were analyzed at 1 week after the beginning of treatment. Note a mean increase of approximately 50% in the G-CSF-treated group (n ¼ 8 in each group). *p < 0.05, p ¼ 0.008.
2.9.2. Immunohistochemistry of ED-1 (CD68) in the optic nerve Longitudinal sections of optic nerve were stained with hematoxylin–eosin for morphologic evaluation. Immunohistochemistry of ED1 (CD68, a marker of macrophage/microglia) using a monoclonal antibody (1:50, AbD Serotec, Oxford, UK) was also
Western blot analysis of p-AKT was performed with modifications as described (Nakazawa et al., 2003). Protein samples containing 50 mg of protein were separated on 12% sodium dodecyl sulfate–polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes (PerkinElmer, Waltham, MA, USA). The membranes were incubated in TBST buffer (0.02 M Tris-base, pH 7.6, 0.8% NaCl, 0.1% Tween 20) supplemented with 5% dry skim milk for 30 min to block nonspecific binding. p-AkT and AKT antibodies (1:1000, Cell Signaling Inc., Danvers, MA, USA) were added and the preparations were incubated at 4 C overnight. The membranes were washed twice with TBST buffer followed by incubation with Biotin-SP-conjugated appropriate goat anti-rabbit IgG secondary antibodies (1:1000, Jackson Immunoresearch, PA, USA) at
Fig. 2. Flat preparations of retinas and morphometry of RGCs at 1 week after retrograde FluoroGold labeling. (A–H) Representative flat preparations of central and mid-peripheral retinas. (A, E) Sham operation; (B, F) PBS-treated at 1 week after ON crush; C&G: PBS-treated at 2 weeks after ON crush; D&H: G-CSF-treated at 2 weeks after ON crush). (I, J) Morphometry of RGCs in the central and mid-peripheral retinas. RGC densities in the un-crushed group were 2550 340/mm2 and 1550 320/mm2, respectively. One week after ON crush, the RGC densities decreased to 2070 410/mm2 and 1040 210/mm2, respectively. At 2 weeks, the densities of RGCs in the central retina of the G-CSF treatment group and the PBS treatment group were 1530 490/mm2 (60.0% survival) and 500 210/mm2 (19.6% survival) respectively, and in mid-peripheral retina were 720 260/mm2 (46.5% survival) and 370 150/mm2 (23.9% survival) respectively, showing a significant preservative effect by G-CSF (p < 0.001) (n ¼ 8 in each group, 24 rats in total). C, crush injury. *p < 0.05; **p < 0.01.
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Fig. 3. VEPs. (A, B) Representative flash VEP tracings at 1 and 2 weeks after ON crush. (C) Latency of P1 wave. Note that 1 week after ON crush, the latency of the P1 wave in the sham-operated group (80 3 ms), was similar to the G-CSF-treated and ON-crushed group (84 3 ms), while the PBS-treated and ON-crushed group (98 6 ms [p ¼ 0.001 vs. control]) had a prolonged latency. By 2 weeks, the latency of the P1 wave in the G-CSF-treated group (99 16 ms) was also prolonged (p ¼ 0.05 vs. control [78 9 ms]), yet significantly less than the PBS-treated group (174 16 ms [p < 0.001]). There were two rats in the PBS-treated group without a recordable wavelength (n ¼ 6 in each group, 18 in total). *p < 0.05; **p < 0.01.
room temperature for 2 h. The blot was then washed with TBST and incubated with streptavidin/AP (1:500, DakoCytomation, Glostrup, Denmark) at room temperature for 1 h. The specific immune complexes were detected by BCIP/NBT solution (B6404, Sigma, Steinheim, Germany). Quantification was performed on computer (ImageJ software; NIH, MD, USA). To determine the activated AKT, the percentage of activated AKT was defined as phosphorylated AKT/total AKT. 2.11. Statistical analysis All measurements in this study were performed in a masked fashion; mean values with standard deviations (S.D.) were obtained and presented here. Student’s t-test was used to evaluate the differences between both treated groups in terms of cell number and milliseconds in FVEP tests. Statistical significance was declared if a p value was <0.05.
3.2. RGC density after ON crush The densities of RGCs in the central and mid-peripheral retina in the sham-operated eyes were 2550 340/mm2 and 1550 320/ mm2, respectively. One week after ON crush and with PBS treatment, the central and mid-peripheral RGC densities decreased to 2070 410/mm2 and 1040 210/mm2, respectively (p < 0.001). At 2 weeks after the insult, the densities of RGC in the central retina of the G-CSF and PBS treatment groups were reduced to 1530 490/ mm2 and 500 210/mm2, respectively (p < 0.001), and in the midperipheral retina were 720 260/mm2 and 370 150/mm2, respectively (p < 0.001) (Fig. 2). In other words, central RGC density showed a 60.0% survival rate in the G-CSF-treated rats and 19.6% in the PBS-treated rats; mid-peripheral RGC density showed a 46.5% survival rate in the G-CSF-treated rats and 23.9% in the PBS-treated rats at 2 weeks (p < 0.001). The results demonstrate that RGC survival rate increases by approximately 40.4% in central retina and 22.6% in the mid-peripheral retina in the G-CSF-treated group as compared to the PBS-treated group.
3. Results 3.3. FVEP 3.1. Administration of G-CSF and peripheral white blood cells counts One week after the administration of G-CSF, the white cell count was significantly increased (11,650 690/mm2 vs. 7810 790/ mm2; G-CSF vs. control; p ¼ 0.008). Hence, under our experimental condition, the administration of G-CSF for 5 days had a leukocytotic effect (Fig. 1).
Consistent with a previous report, FVEP responses in shamoperated rats were identical to those of unoperated rats (Ohlsson et al., 2004b), and we therefore only present the results of shamoperated rats as a control. The latency of the P1 wave at the first week was 80 3 ms, 84 3 ms, and 98 6 ms for the control, ON-crushed and G-CSF-treated (p ¼ 0.067 vs. control), and
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Fig. 4. TUNEL in retinal sections. (A–F) Representative of the TUNEL in retinal sections at high (A–C) and low (D–F) magnification. (A, D) Control sham-operated. (B, E) PBS-treated and (C, F) G-CSF-treated at 2 weeks after ON crush. (G) TUNEL-positive cells in the RGC layer at 2 weeks after ON crush (arrowhead). Note a significant increase (p < 0.001) in the PBS-treated and ON-crushed, when compared to the sham-operated group (14.9 6.3 cells/HPF vs. 2.1 1.6 cells/HPF). G-CSF ameliorated this increase (5.7 1.4 cells/HPF) (p ¼ 0.03 vs. PBS-treated group; n ¼ 6 in each group).
ON-crushed and PBS-treated groups (p ¼ 0.001 vs. control). At 2 weeks after surgery, the latency of the P1 wave was 78 9 ms, 99 16 ms, and 174 16 ms for the control, ON-crushed and GCSF-treated (p ¼ 0.05 vs. control; p < 0.001 vs. PBS-treated), and
ON-crushed and PBS-treated groups (p < 0.001 vs. control) (Fig. 3). The FVEP results demonstrate that the G-CSF-treated group had significantly improved visual function as compared to the PBS-treated group, at both 1 and 2 weeks after ON crush.
Fig. 5. Immunohistochemistry of p-AKT in the retina. (A) Sham-operated eyes. PBS-treated at 1 (B) and 2 (C) weeks after ON crush. (D) G-CSF-treated, 1 week after sham operation. G-CSF-treated, 1 (E) and (F) 2 weeks after ON crush. Note little immunoreactivity in the sham-operated eyes (A), PBS-treated and 1 (B) and 2 (C) weeks after ON crush, and G-CSFtreated and sham-operated (D), but high reactivity (arrowhead) in G-CSF-treated and 1 week after ON crush (E) and in G-CSF-treated at 2 weeks after ON crush (F). Note the p-AKT was also detected in the inner plexiform, inner nuclear layer, outer plexiform layers and outer nuclear layer (n ¼ 6 in each group). C, crush; S, sham-operated; P, PBS; G, G-CSF.
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Two rats had non-recordable waves in the PBS-treated group at 2 weeks. 3.4. In situ nick end-labeling (TUNEL) assay TUNEL assay demonstrated that TUNEL-positive cells/HPF (high-powered field) of 2.1 1.6 cells in the sham-operated group, 14.9 6.3 cells in the PBS-treated group and 5.7 1.4 cells in the GCSF-treated group (p ¼ 0.03 vs. PBS-treated group) in the RGC layer (Fig. 4). The results demonstrated that administration of G-CSF had a significant anti-apoptotic effect on RGCs after ON crush. 3.4.1. Immunohistochemistry of p-AKT in the retina There was little or no p-AKT immunoreactivity in the shamoperated eyes treated with PBS, and in ON-crushed and PBS-treated rats at 1 or 2 weeks. However, there was some reactivity in G-CSFtreated, sham-operated eye, and predominant expression of p-AKT in the RGC layers of ON-crushed and G-CSF-treated rats at 1 and 2 weeks (Fig. 5). The p-AKT was also detected in the inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL) and outer nuclear layer (ONL). This may indicate that the G-CSF administration for 5 days can activate the signaling pathway of pAKT in the retinal ganglion cell layer of rats, which further contributes to the anti-apoptotic effect on RGCs. 3.4.2. Western blot analysis of p-AKT The activated AKT was not altered in the sham-operated retinas and the retinas of ON-crushed and PBS-treated rats. In contrast, the activated AKT increased significantly in the retinas of ON-crushed and G-CSF-treated rats at 1 and 2 weeks about 2- and 1.7-fold respectively (p ¼ 0.016 at 1 week and p ¼ 0.025 at 2 weeks) (Fig. 6). These results of Western protein analysis of p-AKT were compatible with that of IHC of p-AKT in the retinas. 3.5. ED1 in the ON At 2 weeks after the insult, the injured site of the ON showed edema, disruption of the axons and inflammatory cell infiltration in the PBS-treated group, while less edema and less inflammatory cell
infiltration at the crush site of the optic nerve was observed in the G-CSF-treated group (Fig. 7A). Immunohistochemistry of ED1 for macrophage/microglia showed few ED1-positive cells in the shamoperated group (10 4 cells/HPF), prominent ED1-positive cells at the lesion area (93 13 cells/HPF) in the PBS-treated group, but few in the G-CSF-treated group (21 7 cells/HPF). The difference between treatment groups was statistically significant (p < 0.001) (Fig. 7B and C).
4. Discussion In the present study, our morphologic results demonstrate that G-CSF has a neuroprotective effect on ON as well as in RGCs after ON crush in adult rats. In addition, the visual function as demonstrated by FVEP was also better preserved in the G-CSF-treated eyes compared to the vehicle-treated ones, confirming the beneficial effect on the ocular structures. Our TUNEL assay results and immunohistochemistry of p-AKT showed that the administration of G-CSF was anti-apoptotic on RGCs after ON crush injury and suggested an involvement of p-AKT pathway. This finding is consistent with earlier studies showing a pivotal role of p-AKT in neuronal survival after injury, and that the signaling pathway can be activated by G-CSF (Avalos, 1996; Dong. and Larner., 2000; Kilic et al., 2002). Nakazawa et al. (2003) have demonstrated that the activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway has a neuroprotective effect on injured RGCs. They reported that optic nerve clamping injury induced intrinsic activation of p-AKT in the retina within 1 h and reached a maximum at 6 h, then returned to baseline at about 72 h and the inhibition of p-AKT significantly decreased the number of surviving RGCs. Our results of Western blot analysis and IHC of p-AKT demonstrate that G-CSF can activate the p-AKT signaling pathway in the retinas of the injured RGCs at 1 and 2 weeks after ON crush. These observations may also suggest that expression of p-AKT at certain window of time after ON crush may contribute to the anti-apoptotic effect and the rescue of the RGCs from death. The mechanism of G-CSF activation of the AKT pathways in the RGCs remains to be dissected.
Fig. 6. Western blot analysis of activated p-AKT expression of the rat retinas after ON crush. (A) A representative photograph of the Western blot analysis. p-Akt, anti-phosphorylated Akt antibody; Akt, anti-AKT antibody; S, sham operation; C, crush; P, PBS; G, G-CSF. (B) The optical density of activated p-AKT in the Western blot is expressed as a ratio to that of AKT. Note the p-AKT protein expression in the retinas was up-regulated in G-CSF-treated rats at 1 and 2 weeks after ON crush. There was no significant change in that of PBS-treated rats at 1 and 2 weeks after ON crush (n ¼ 3 each). *p < 0.05, CþP 1 week vs. CþG 1 week p ¼ 0.016; CþP 2 weeks vs. CþG 2 weeks p ¼ 0.025.
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Fig. 7. Histology of ONs and immunohistochemistry of ED1 in ONs at 2 weeks after ON crush. (A) Hematoxylin–eosin staining of longitudinal sections of ONs. Note prominent axonal disruption and inflammatory cells infiltrate at the crush site in the PBS-treated group (middle panel) when compared to the control (left panel), but not in the G-CSF-treated group (right panel). The right side of each panel is the ON near the globe. (B) Immunohistochemistry of ED1. ED1-positive cells were prominent at the lesion site of ONs in the PBStreated group, and less so in the sham-operated group and the G-CSF-treated group. (C) Morphometry of ED1-positive cells in the ON. The difference of ED1-positive cells between the two treatment groups was statistically significant (n ¼ 6 in each group). p < 0.001.
After ON crush, ED1-labeled macrophages/microglia accumulated at the site of injury. Ohlsson et al. (2004b) reported that ED1positive cells continued to increase at the site of injury for 28 days after ON crush injury. Macrophages seem to be responsible for the removal of most of the myelin debris in CNS injury; however, these cells lack the capacity to remove axonal and myelin debris as fast as in the peripheral system, and contributed to glial scar formation, which is an obstacle for regeneration (Ohlsson et al., 2004a,b). ED1positive phagocytes found in ON injury include monocytes/macrophages of hematogenous origin as well as microglia. A portion of ED1-positive cells concomitantly express Ia antigen during Wallerian degeneration in the ON (Stoll et al., 1989). Our results show that ED1-positive macrophage/microglia accumulation at the ON lesion site was attenuated in the G-CSF-treated rats, implicating that immediate administration of G-CSF may have an anti-
inflammatory effect on the injured ON. In animal studies, neuroprotective effects of G-CSF have been ascribed to anti-inflammatory actions mediated by inhibition of TNF-a, and inducible nitric oxide synthase (iNOS) activity (Go¨rgen et al., 1992), and by a reduction of interleukin-1b expression (Avalos, 1996; Gibson et al., 2005; Go¨rgen et al., 1992). Macrophage/microglia accumulation can activate TNF-a, iNOS and cytokines (such as interleukins) expression. Our histopathological studies of the optic nerve also demonstrated less edema and inflammatory cell infiltration in the G-CSF-treated group. Since ON injury is axogenic, it is rational to hypothesize that if drug intervention can decrease the inflammatory response at the optic nerve, it may protect the RGCs from secondary degeneration. G-CSF recruitment of neutrophils combines with a simultaneous limitation of the harmful inflammatory reaction by inhibition of the above inflammatory mediators (Hartung, 1998). In addition,
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