Granulocyte colony-stimulating factor promotes growth of processes, growth associated protein 43 and microtubule-associated protein 2 expression in cultured rat retinal ganglion cells in vitro☆

Haitao Xu, Yuying Jiang, Xiuhong Qin, Lihui Si, Jie Zhao, Lijuan Liu, Yazhen Wu

1Department of Ophthalmology, Second Clinical Hospital of Jilin University, Changchun 130041, Jilin Province, China

2Department of Ophthalmology, China-Japan Friendship Hospital, Jilin University, Changchun 130033, Jilin Province, China

3Department of Gynaecology and Obstetrics, Second Clinical Hospital of Jilin University, Changchun 130041, Jilin Province, China

4Changchun Medical College, Changchun 130013, Jilin Province, China

5Emergency Center of Changchun, Changchun 130021, Jilin Province, China

INTRODUCTION

The optic nerve primarily consists of retinal ganglion cell (RGC) axons.Ischemia,trauma, intraocular hypertension, inflammation and oncothlipsis can result in severe loss of vision or even acroisa.Since ganglion cells are not regenerative, no effective clinical treatments are currently available for optic nerve injury caused by ganglion cell injury.Therefore, ways of promoting functional recovery of injured RGCs, in particular the regeneration of axons and dendrites,has become a focus for research in the field of neuroprotection.Stimulation of axonal regeneration in RGCs has been examined using drug treatment[1-3], transplantation of substances that promote regeneration[4-5],and peripheral nerve grafts[6-8].

Granulocyte colony-stimulating factor(G-CSF) is a glycoprotein with molecular weight of 19.6 kDa.G-CSF is mainly secreted by monocytes and macrophages,and has been found to specifically regulate granulocyte proliferation and differentiation, as well as enhancing granulocyte function[9].However, increasing evidence indicates that G-CSF binds G-CSF receptors on cell membranes to regulate cell growth, inhibit apoptosis, block glutamic acid excitotoxicity, and promote collateral angiogenesis[10-12].Recent studies have revealed that G-CSF receptors are also located on RGCs[13], and that G-CSF can attenuate RGC injury through the G-CSF receptor, promoting axonal repair and production[14].

In the present study,in vitrocultured RGCs were treated with G-CSF to investigate the effects on axonal production.In addition,RGCs were exposed to CoCl2to investigate the influence of G-CSF on injured RGCs.

RESULTS

RGC culture and identification

Under an inverted microscope, primary cultured RGCs presented with round or ellipse-shaped cell bodies, with halation, and the aggregation of some cells.After 24 hours, RGCs adhered, and a few cells exhibited small, short processes (Figure 1).After 48 hours, the cell bodies were enlarged.In addition, processes were increased in number and extended in length,with the longest processes forming the axons, consistent with morphological features of RGCs.

Thy1.1 antigen is a marker of mature RGCs in mammals, and a specific marker of RGCs[15].Under a fluorescence microscope,RGC bodies and processes exhibited green fluorescence following Thy1.1 staining.This morphology was consistent with inverted microscope observations (Figure 2).RGC purity was found to be 90%-95% by dividing the number of positive RGCs from the total number of cells in the corresponding field of view.The results indicated successful isolation and culturing of RGCs.

Figure 1 Retinal ganglion cells exhibited round or ellipse-shaped cell bodies with small and short processes after culturing for 24 hours (scale bar: 50 μm).

Figure 2 Thy1.1 monoclonal antibody-positive retinal ganglion cells presented with green fluorescent cell bodies and processes (fluorescein isothiocyanate labeling; scale bar: 50 μm).

G-CSF promoted the growth of RGC processes

After 1 day in culture, a few RGCs in the control (normal culture) and G-CSF (recombinant human G-CSF, and rhG-CSF) groups exhibited small and short processes,and there were no significant differences in the number of cells with processes between two groups.After 3 days,the number of cells with processes significantly increased compared with that at 1 day (P＜0.01), and there were significantly more cells with processes in the G-CSF group compared with the control group (P＜0.01).After 5 days, the number of cells with processes was decreased compared with that at 3 days (P＜0.01), but the number of cells with processes in the G-CSF group remained significantly greater than in the control group(P＜0.05; Table 1).

Table 1 Number of retinal ganglion cells with processes(/200-fold field) in each group following granulocyte colony-stimulating factor (G-CSF) treatment

G-CSF upregulated growth associated protein 43(GAP-43) and microtubule-associated protein 2(MAP-2) mRNA expression in RGCs

GAP-43 and MAP-2 mRNA expression in RGCs was increased following hypoxia (P＜0.01,P＜0.05).In particular, GAP-43 mRNA expression was higher in G-CSF-treated RGCs compared with RGCs exposed to hypoxia (P＜0.05), but MAP-2 mRNA expression was only higher than RGCs exposed to hypoxia at 6 and 12 hours (P＜0.05; Figure 3).

Figure 3 Influence of granulocyte colony-stimulating factor (G-CSF) on growth-associated protein 43 (GAP-43)and microtubule-associated protein 2 (MAP-2) mRNA expression in retinal ganglion cells (RGCs).The absorbance of GAP-43 mRNA was increased in the hypoxia group and the G-CSF treatment group.Compared with the hypoxia group, the absorbance of GAP-43 mRNA was significantly increased in the G-CSF group (A).In addition,absorbance of MAP-2 mRNA was increased in the hypoxia group and the G-CSF group: at 6 and 12 hours, and the absorbance of MAP-2 mRNA significantly increased in the G-CSF group (B).aP＜0.05, bP＜0.01, vs.control group;cP＜0.05, vs.hypoxia group.Data were expressed as absorbance ratios of target mRNA to β-actin mRNA (mean± SD).High absorbance represents strong protein expression.Intergroup comparison was performed using analyses of variance and q-test.

G-CSF decreased RhoA/Rock expression in RGCs following hypoxia

Western blot results showed that after 24 hours of culturing, RhoA and Rock expression were increased after hypoxia compared with the control group (P＜0.01).G-CSF inhibited RhoA and Rock expression in RGCs(P＜0.05; Figure 4).

Figure 4 RhoA (A) and Rock (B) expression in retinal ganglion cells.RhoA and Rock expression were higher in hypoxia and granulocyte colony-stimulating factor(G-CSF) groups compared with the control group.RhoA and Rock expression was lower in the G-CSF group compared with the hypoxia group.High absorbance represents strong protein expression.aP＜0.01, vs.control group; bP＜0.05, vs.hypoxia group.Data were expressed as absorbance ratios of target protein to β-actin (mean ±SD).Intergroup comparisons were performed using analyses of variance and q-test.

DISCUSSION

Rat RGCs were successfully culturedin vitroby Raffet alin 1979[16], and have been widely used in studies of glaucoma, retinal ischemia, and trauma[15,17].However,this process is difficult, and RGCs culturedin vitrotypically survive for a short period of time.Based on a previously described method[18], in the present study we cultured RGCs using serum-free neurobasal medium,which prevented the influence of various components in the serum on the experimental results.The results indicated that this method prolonged thein vitrosurvival of RGCs, which began to adhere at 4-6 hours and completely adhered at 24 hours.A few cells exhibited short and small processes.At 72 hours, the processes connected and RGCs survived for more than 5 days.

G-CSF can promote cell growth, cell differentiation, and stem cell migration, as well as antagonizing inflammation and apoptosis[19-22].As such, it is used extensively in preclinical settings as a neuroprotective treatment.Previous studies in a rat experimental model of optic nerve compression reported that G-CSF protected injured RGCs against apoptosis by antagonizing the PI3K/AKT pathway, and promoted RGC axonal regeneration[13,23].The present study is the first examination of the influence of G-CSF on process growth of untreated RGCs.The current results revealed that G-CSF effectively promoted RGC process growth in an RGC culture system.However, the experimental conditions limited further investigation of the underlying mechanisms involved.We assumed that G-CSF could promote downstream axon-associated protein expression through the mitogen-activated protein kinase and extracellular regulated protein kinase pathways to improve the growth of RGC processes.

GAP-43 is used as a molecular marker of nerve plasticity during development and injury repair[24-26].A high level of GAP-43 expression in RGCs is critical for RGC axonal regeneration[27-28].MAP-2 is an important member of the MAP family, distributed in RGC bodies and dendrites,and is a crucial component for maintaining RGC structure and promoting the repair of RGCs processes[29].Following brain or spinal cord injury, G-CSF can promote neurological function by increasing GAP-43 and MAP-2 expression[30-31].In the present study, GAP-43 and MAP-2 expression were increased with prolonged periods of hypoxia.GAP-43 and MAP-2 expression were significantly increased in the G-CSF group compared with the hypoxia group at 12 hours following hypoxia,indicating that G-CSF can repair RGCs axons by increasing the expression of axon-associated protein.RhoA is a protein in the Rho GTPase family, and has been shown to play an important role in signal conduction of axonal inhibitors[32-33].A variety of injuries can activate RhoA enzymes in RGCs, and activated RhoA conducts signals through Rock activation to inhibit axonal repair[34].In the present study, western blot analysis revealed that Rho and Rock protein content in RGCs exposed to hypoxia was significantly increased compared with the control group, but G-CSF treatment significantly attenuated this increase.This finding indicates that G-CSF promoted axonal repair and regeneration,possibly through the RhoA/Rho pathway.

MATERIALS AND METHODS

Design

A randomized, controlled, cytobiological study.

Time and setting

This study was conducted at the Laboratory of Ophthalmology, Second Hospital of Jilin University, China from July to December 2010.

Materials

A total of six Sprague-Dawley rats, 1-3 days after birth,of either gender, weighing 6-7 g, were provided by the Animal Experimental Center of Jilin University (No.SCXK (Ji) 2003-0002).Rats were housed in a room that was lit between the hours of 7:00-19:00, kept at 20±2°C,with humidity of 45-55%.Experimental procedures were performed in accordance with theGuidance Suggestions for the Care and Use of Laboratory Animals, issued by the Ministry of Science and Technology of China[35].

Methods

RGCs isolation, culture and identification

According to previously described method[18], neonatal rats were disinfected with 75% alcohol solution and sacrificed.The eyeball was removed using micro-ophthalmic surgical instrument (Shanghai Fuzhong Instrument,Shanghai, China), rinsed with D-Hanks solution (Solarbio,Beijing, China) three times and placed in a culture dish.The cornea was sheared along the corneoscleral junction using microscissors under a microscope(Olympus, Tokyo, Japan), and the lens and vitreous body were removed using microforceps.The retinal tissues of the nervous layer were blunt-dissected,rinsed with D-Hanks solution three times, centrifuged at 1 000 r/min for 5 minutes, digested with 2 mL solution containing 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid after discarding supernatant, and incubated at 37°C for 30 minutes.The digestion was terminated using Dulbecco's modified Eagle's medium (DMEM; Hufeng Biotechnology,Shanghai, China) containing 10% fetal bovine serum,and the products were centrifuged at 1 000 r/min for 5 minutes, incubated in NEUROBASALTMmedium(Gibco, Carlsbad, CA, USA) in an incubator at 37°C for 4 hours.Cell concentration was adjusted to 5 ×105/mL.Cell morphology was observed at 24 and 48 hours using a phase contrast microscope (Shanghai Qixin Scientific Instrument Co., Ltd., Shanghai,China).

According to a previously described method[36], cells cultured for 3 days were harvested, washed with PBS three times (2 minutes each time) after discarding the medium, fixed with 4% paraformaldehyde for 4 hours,washed with PBS three times (2 minutes each time),incubated with goat anti-rat Thy1.1 monoclonal antibody (1: 200; Chemicon, Billerica, MA, USA) and FITC-labeled rabbit anti-goat IgG (1: 100; Yongyi Biotechnology Co., Ltd., Xi’an, Shaanxi Province, China) at 37°C for 60 minutes, washed with PBS three times (2 minutes each time), observed under a fluorescence microscope (Olympus) and photographed.Negative control samples were treated with PBS rather than antibodies.

Observation of RGC process growth

Isolated RGCs were collected, seeded in 24-well culture plate at a density of 1 × 106/mL, and randomly assigned to control and G-CSF groups.Cells in the control group were cultured in culture solution alone, and the G-CSF group was additionally treated with recombinant human G-CSF (GeneScience Pharmaceuticals Co., Ltd.,Changchun, China) to a terminal concentration of 10 ng/mL[37].Two-group cells were incubated in 5% CO2at 37°C.The medium was replaced every 24 hours.Cells were observed and photographed at 1, 3, and 5 days.Using the center of the culture plate as a central reference point, five fields of view were randomly selected from the top, bottom, left, right and central points of the center (200 ×, frame area 0.22 mm2).Two wells were randomly selected from each group in each experiment.The experiment was performed in triplicate.The number of cells with processes was quantified using a Leica QWin image analysis system (Leica, Tokyo, Japan).The mean value was calculated.

Detection of GAP-43 and MAP-2 mRNA expression in RGCs

Primary cultured RGCs were randomly assigned to the control, hypoxia and G-CSF groups, seeded in a 24-well culture plate with three parallel wells in each group.RGCs in the control group were cultured in culture medium alone.The hypoxia group was additionally treated with 50 μL CoCl2(150 μmol/L; Sigma, St.Louis, MO,USA) to establish model of hypoxia.G-CSF group was treated with rhG-CSF to a terminal concentration of 10 ng/mL.The three groups were incubated in 5% CO2at 37°C.

Cell suspension of three groups was harvested at 6, 12,24, 48 hours, centrifuged at 800 ×gfor 5 minutes, and the supernatant was discarded.PCR was performed according to a previously described method[38].Trizol(Invitrogen, Carlsbad, CA, USA) was added and total RNA was extracted.The RNA content was determined using UV-240 ultraviolet spectrophotometer (Beijing Kaiao Technology Development, Beijing, China).The absorbance ratios at 260 nm/280 nm of total RNA was between 1.80 and 2.00.The 2 μg total RNA was harvested to synthesize cDNA using a Superscript III Reverse Transcriptase kit (Invitrogen).Real-time PCR was performed using an SYBR Green PCR kit (TransGen Biotech, Beijing, China).

GAP-43 and MAP-2 specific primer reference sequences were obtained from GeneBank(http://www.ncbi.nlm.nih.gov/genbank/), designed using Primer Premier 5.0 software (Premier Biosoft, Palo Alto, CA, USA), and synthesized at the Shanghai Research Center of Bioengineering.The primers were as follows:

Primer Sequence Product size (bp)GAP-43Upstream: 5’-ATG CTG TGC TGT ATG AGA AGA ACC-3’Downstream: 5’-GGC AAC GTG GAA AGC CGT TTC TTA AAG T-3’218 MAP-2 Upstream: 5’-GCC ATG ATC TTT CCC CTC TGG CTT-3’Downstream: 5’-GTC TGG TTT TAC GGG TTG GCT GTC-3’224 β-actin Upstream: 5'-ATG CCA TCC TGC GTC TGG ACC TGG C-3'Downstream: 5’-AGC ATT TGC GGT GCA CGA TGG AGG G-3’606

A total of 1 μL specific primer (containing 0.5 μL upstream and 0.5 μL downstream primers), 12.5 μL 2 ×SYBR Green QPCR Master Mix, 2.5 μL diluted cDNA and 9 μL nuclease-free PCR-grade water was used.This mixture was subjected to denaturation at 95°C for 5 minutes, followed by 40 cycles of 95°C for 30 seconds, 60°C for 1 minute and 72°C for 30 seconds.PCR product SYBR green fluorescence was detected using an Eco Real-Time PCR System (Illumina, San Diego, CA, USA).The fluorescence intensity ratio of product to β-actin was used as a measure of expression intensity.

Determination of RhoA/Rock expression in RGCs

At 24 hours following establishment of hypoxia in the model, RGCs of three groups were collected, and cell protein was harvested.RhoA and Rock content was determined with Western blot analysis.Briefly, RGC suspension was centrifuged at 800 r/min for 5 minutes to collect cell protein, mixed with triple-detergent lysis buffer pretreated with protease inhibitor (Invitrogen) on ice for 30 minutes, and centrifuged at 12 000 r/min for 20 minutes.The total protein concentration was determined using the bicinchoninic acid method[39].Total protein (15 μg) from each sample was mixed with 4 μL 6 × loading buffer, denatured in boiling water for 5 minutes, followed by 10% sodium dodecyl sulfate polyacrylamide gel electropheresis at 80 V for 40 minutes, 110 V for 90 minutes, and transmembrane at 200 mA for 90-150 minutes.The protein was blocked with BSA (Wuhan Yide, Wuhan, China) and skimmed milk for 2 hours,eluted with Tris-Buffered Saline and Tween 20(TBST; Wuhan Yide) six times, 10 minutes each,incubated with rabbit anti-rat RhoA and Rock polyclonal antibody (R&D Systems China, Shanghai; 1:2 000) overnight at 4°C, followed by TBST eluting six times (10 minutes each time) the next day.The products were incubated with horseradish peroxidase-labeled mouse anti-rabbit IgG (R&D Systems China; 1: 200) at room temperature for 2 hours, following ECL chemiluminescence[39].The absorbance ratio of product to β-actin was used to represent the results of the analysis.

Statistical analysis

Data were analyzed using SPSS 11.5 software (SPSS,Chicago, IL, USA).Measurement data were expressed as mean±SD, and grouped data were expressed as relative number.Intergroup differences were compared using analyses of variance andq-test.A significance level ofα=0.05 was used to determine significant differences.

Author contributions:Haitao Xu and Yazhen Wu designed and evaluated this study.All authors took part in conducting the experiments.

Conflicts of interest:None declared.

Ethical approval:This study received permission from the Animal Ethics Committee of Jilin University, China.

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