顆粒白血球聚集刺激因子對大白鼠視神經壓碎後的神經退化的保護研究

Neuroprotective Effects of Recombinant Human Granulocyte Colony-stimulating Factor (G-CSF) in Neurodegeneration after Optic Nerve Crush in Rats

蔡榮坤(Rong-Kung Tsai)

指導教授 ： 王惠珠

高雄醫學大學/醫學院/醫學研究所/博士(2008年)

https://doi.org/10.6832/KMU.2008.00134

摘要

目的：目前針對外傷性視神經病變尚無有效的治療，而臨床已使用很久的老藥-顆粒白血球聚集刺激因子(G-CSF, Filgrastin)可以將骨髓幹細胞移至周邊血液，具神經保護效果。因此本研究目的為探討顆粒白血球聚集刺激因子的給予在大白鼠視神經壓碎模式所產生的神經退化是否有神經保護作用?
材料與方法：以92隻雄性Wistar大白鼠給予標準化的視神經壓碎實驗後，治療組給予顆粒白血球聚集刺激因子(100 μg/kg/day in 0.2 ml磷酸鹽平衡液)每天一次皮下注射，連續五天。對照組則給予磷酸鹽平衡液注射，在術後一及兩週犧牲以作相關研究。我們利用視覺誘發波來檢驗兩組的視覺功能，及利用Fluoro-Gold注射到大白鼠上視丘的逆行性標識來計算視網膜節細胞的密度及其存活率。視網膜進行凋零研究(TUNEL apoptotic assay) 及磷酸化的Akt 蛋白訊號途徑(p-Akt)的西方墨點分析，組織免疫染色研究包括視網膜切片磷酸化的Akt 蛋白訊號途徑(p-Akt)；在視神經則以ED-1(標示吞噬細胞及小膠質細胞,macrophage/microglia)染色研究發炎反應以及Fluoro-Jade B在視神經與視網膜的退化神經染色。也以反轉錄聚合酵素連鎖反應研究視網膜TNF-α mRNA的表現。
結果：在術後兩週後，視網膜節細胞(RGC)在中央視網膜於G-CSF組有60%存活率，PBS組為19.6%；週邊視網膜RGC在GCSF組有 46.5%存活率，PBS組為23.9%，具統計差異(p<0.05)。視覺誘發波也顯示在視神經壓碎兩週後，G-CSF治療組的P1波之潛伏期較對照組有顯著的受到保存(正常眼78±9 ms，G-CSF治療組98±16 ms，PBS組174±16 ms; p<0.001)。視網膜的凋零研究(TUNEL apoptotic assay)顯示G-CSF可以降低凋零死亡，而視網膜的免疫組織染色及西方墨點分析也顯示G-CSF可以加強磷酸化的Akt蛋白訊號途徑在視網膜的表現。此外，視神經免疫組織染色也證實G-CSF可以在視神經受傷後降低視神經受傷部位的發炎反應以及ED1炎性細胞的聚集。FJB免疫染色結果也支持G-CSF可以減少視神經與視網膜因視神經壓碎所產生的神經退化。而視網膜TNF-α mRNA反轉錄聚合酵素連鎖反應顯示G-CSF會抑制視網膜TNF-α mRNA的表現。
結論：本研究結果顯示G-CSF的治療對視神經壓碎動物模式可能是相當有潛力的神經保護劑。型態學上不但可以降低視網膜節細胞的死亡，也在視覺功能上保存了視覺誘發波的傳導速度。而顆粒白血球聚集刺激因子的神經保護作用可能藉由在視網膜上加強磷酸化的Akt蛋白訊號途徑以及抑制TNF-α mRNA的表現來達到抗凋零死亡以及在視神經的抗發炎雙重作用來達成。

關鍵字： 顆粒白血球聚集刺激因子、大白鼠視神經壓碎模式、眼部神經保護、閃爍視覺誘發波、視網膜節細胞密度

關鍵字

顆粒白血球聚集刺激因子 ； 大白鼠視神經壓碎模式 ； 眼部神經保護 ； 閃爍視覺誘發波 ； 視網膜節細胞密度

並列摘要

Purpose: 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.
Materials and Methods: 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 µg/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 sacrificed at one or two 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 and Fluoro-Jade B in both the retina and the ON were conducted. RT-PCR of TNF-α mRNA in the retinas was also evaluated.
Results: Two weeks after the insult, the RGC densities in the central and mid-peripheral retinas in ON crushed, G-CSF-treated rats were significantly higher than that of the corresponding ON crushed, PBS-treated 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-CSF-treated rats. P-Akt immunoreactivity was up-regulated in the retinas of the ON crushed, G-CSF-treated rats at one and two 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. Fluoro-Jade B immunoreactivity also decreased in both the retina and the ON in the ON crushed, G-CSF-treated group. The RT-PCR of TNF-α mRNA showed the expression of TNF-α mRNA in the retinas also inhibited in the G-CSF-treated group.
Conclusions: 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 and inhibited expression of TNF-α mRNA in the retinas.

Key words: granulocyte colony-stimulating factor, rat model, optic nerve crush, ocular neuroprotection, flash visual-evoked potential, RGC density.

並列關鍵字

granulocyte colony-stimulating factor ； rat model ； optic nerve crush ； ocular neuroprotection ； flash visual-evoked potential ； RGC density

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