Neuroprotective effects of low-dose G-CSF plus meloxicam in a rat model of anterior ischemic optic neuropathy

Abstract

Non-arteritic anterior ischemic optic neuropathy (NAION) causes a sudden loss of vision and lacks effective treatment. Granulocyte colony-stimulating factor (G-CSF) provides neuroprotection against the experimental optic nerve injuries but also induce leukocytosis upon typical administration. We found synergetic neuroprotective effects of meloxicam and low dose G-CSF without leukocytosis in a rat model of anterior ischemic optic neuropathy (rAION). The WBC counts in the low-dose G-CSF-plus meloxicam-treated group were similar to the sham-operated group. Combination treatment of low-dose G-CSF plus meloxicam preserved RGCs survival and visual function, reduced RGC apoptosis and the macrophages infiltration, and promote more M2 phenotype of macrophage/microglial transition than the low-dose GCSF treatment or the meloxicam treatment. Moreover, the combination treatment induced higher serine/threonine kinase 1 (Akt1) expression. The combination treatment of low-dose G-CSF plus meloxicam lessened the leukocytotic side effect and provided neuroprotective effects via Akt1 activation in the rAION model. This approach provides crucial preclinical information for the development of alternative therapy in AION.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1

Analysis of WBC counts in the sham-operated rats, PBS-treated rats, meloxicam-treated rats, high-dose G-CSF-treated rats, low-dose G-CSF-treated rats, and low-dose G-CSF-plus-meloxicam-treated rats. Treatment with meloxicam, low-dose G-CSF, and low-dose G-CSF plus meloxicam did not induce leukocytosis in the rats, but treatment with high-dose G-CSF induced leukocytosis after a 5-day treatment. *p 

Figure 2

Evaluation of the visual function by using FVEPs in the rAION model. (A) Representative FVEP tracings at four weeks after rAION induction in the sham group, PBS-treated group, meloxicam-treated group, low-dose G-CSF-treated group, and low-dose G-CSF-plus-meloxicam-treated group. (B) Bar charts demonstrate the P1-N2 amplitude. The values of amplitude are expressed as mean ± SD in each group (n = 12 in each group). The amplitudes of the P1-N2 waves in the combination-treated group were significantly higher than meloxicam-treated and low-dose G-CSF-treated groups, respectively. *p < 0.05.

Figure 3

Survival of RGCs in rAION-induced rats with PBS treatment, meloxicam treatment, G-CSF treatment, and G-CSF plus meloxicam treatment at 28 days after rAION induction. (A) A representative of flat-mounted central retinas and the morphometry of RGCs in each group through FluoroGold retrograde labeling at four weeks after rAION induction. (B) RGC density in the central retina in each group. Data are expressed as mean ± SD for each group (n = 12). The number of RGCs in the combination-treated group was 1.58- and 1.45-fold higher than in the meloxicam-treated and low-dose G-CSF-treated groups, respectively. *p < 0.05.

Figure 4

Analysis of RGC apoptosis in the RGC layer through TUNEL assay at four weeks after rAION induction. (A) Representative images of double-stained apoptotic cells in the RGC layers in each group. The apoptotic cells (TUNEL-positive cells) in green were stained with TUNEL staining, and the nuclei of the RGCs in blue were labeled with DAPI staining. (B) Quantification of TUNEL-positive cells per high-power field. Data are expressed as mean ± SD for each group (n = 6). Treatment with low-dose G-CSF plus meloxicam significantly reduced the number of apoptotic RGC by 3.6- and 2.5-fold compared with the meloxicam-treated and low-dose G-CSF-treated groups, respectively. *p < 0.05.

Figure 5

Immunohistochemistry (IHC) of ED1 in the optic nerve at four weeks after rAION induction for evaluating the inflammatory infiltration of macrophages. (A) Representative images of ED1 staining in the longitudinal sections of the optic nerve. The ED1-positive cells in green were stained with FITC, and the nuclei in blue were labeled with DAPI. (B) Quantification of ED1-positive cells per high-power field. Data are expressed as mean ± SD in each group (n = 6). Macrophage recruitment was decreased by 6.75- and 4.1-fold in the combination-treated group compared with the meloxicam-treated and low-dose G-CSF-treated groups, respectively (C) Evaluation of M2 macrophage polarization at four weeks after rAION induction. Relative mRNA expression levels of the markers of M2 macrophages in the optic nerve are shown as histograms. Each value was normalized to CypA. The expression levels of Arg 1, CD206, and Fizz1 (markers of M2 macrophages) increased after treatment with low-dose G-CSF plus meloxicam compared with treatment with PBS-treated group, meloxicam alone, and low-dose G-CSF alone, respectively. *p < 0.05, **p < 0.01.

Figure 6

Immunoblots of the optic nerve. (A) Analysis of p-Akt1 expression by using Western blotting. (B) Quantification of the protein bands of p-Akt1. Each value was normalized to GAPDH. Data are expressed as mean ± SD in each group (n = 6 in each group). The combination treatment induced higher p-Akt1 expression than treatment with meloxicam or low-dose G-CSF in the rAION model. *p < 0.05.