Chu Y, Humphrey M F, Alder V V, Constable I J
Centre for Ophthamology and Visual Science, Lions Eye Institute, University of Western Australia, Nedlands, Australia.
Aust N Z J Ophthalmol. 1998 Feb;26(1):87-96.
Argon laser photocoagulation slows photoreceptor degeneration in the Royal College of Surgeons (RCS) rat, as does intravitreal injection of basic fibroblast growth factor (bFGF). We hypothesize that up-regulation of retinal bFGF is a consequence of laser lesioning in RCS rats. Therefore, we examined the localization of bFGF after laser and correlated this with Mailer cell glial fibrillary acidic protein (GFAP) expression, which is known to increase after injury.
A total of 34 RCS rats at postnatal day 23 were anaesthetized (ketamine 40 mg/kg) and their retinas were irradiated with a grid pattern of 40 non-overlapping argon green lesions with a power of 120 mW for 0.2 s using a 50 microm spot size. At 0, 6, 12, 24 and 48 h and 7, 14 and 21 days post-lesion, rats were anaesthetized and their eyes were enucleated and cryostat sectioned and the sections were processed using either an antibody to bFGF or GFAP using the standard avidin-biotinylated peroxidase complex method. Five age-matched RCS rats without laser lesions served as controls.
Basic fibroblast growth factor immunoreactivity (IR) was normally located within cells in the ganglion cell layer inner nuclear layer and in retinal pigment epithelium cells and in the extracellular matrix/cell membranes of the outer nuclear layer (ONL). In lasered retinas, there was elevated bFGF-IR in the coagulated outer segments for the first 24 h. Retinal blood vessels/Müller cells/astrocytes were moderately labelled in and near each lesion immediately after lesion and became more intense after 48 h and persisted for at least 21 days. There was an elevation of bFGF-IR in the ONL on the lesion flanks at 14 days. Muller cell GFAP-IR was first detected at 6 h post-lesion and spread for a considerable distance beyond the lesion site. At 7 and 14 days, Müller cells at the lesion site had sprouted, while those on the flanks were still GFAP-IR.
Following laser lesion there was an increase in bFGF at the lesion core only for the first 24 h. However, elevated levels of bFGF were observed in the ONL at 14 days, which extended into the lesion flanks for a similar distance to that over which increased photoreceptor survival is found. These results provide support for the hypothesis that laser lesions induce bFGF and this may be the mechanism whereby photoreceptors are spared. Müller cell activation is consistent with growth factor stimulation, but was more widespread than the bFGF changes in ONL. However, blood vessel labelling was similarly widespread and so the responses may be linked between Müller cell GFAP reaction and blood vessel bFGF localization after laser lesions.
氩激光光凝可减缓皇家外科学院（RCS）大鼠的光感受器退变，玻璃体内注射碱性成纤维细胞生长因子（bFGF）也有同样效果。我们推测RCS大鼠视网膜bFGF的上调是激光损伤的结果。因此，我们检测了激光照射后bFGF的定位，并将其与已知在损伤后会增加的米勒细胞胶质纤维酸性蛋白（GFAP）表达相关联。
对34只出生后第23天的RCS大鼠进行麻醉（氯胺酮40mg/kg），使用50微米光斑大小，以120mW的功率对其视网膜进行40个不重叠的氩绿损伤的网格照射，持续0.2秒。在损伤后0、6、12、24和48小时以及7、14和21天，将大鼠麻醉，摘除眼球，制成冰冻切片，使用标准抗生物素蛋白-生物素化过氧化物酶复合物法，用抗bFGF或GFAP抗体对切片进行处理。5只年龄匹配未接受激光损伤的RCS大鼠作为对照。
碱性成纤维细胞生长因子免疫反应性（IR）通常位于神经节细胞层、内核层的细胞内，视网膜色素上皮细胞以及外核层（ONL）的细胞外基质/细胞膜中。在接受激光照射的视网膜中，在最初24小时内，凝固的外段中bFGF-IR升高。损伤后立即，每个损伤处及其附近的视网膜血管/米勒细胞/星形胶质细胞被中度标记，48小时后变得更强烈，并持续至少21天。在损伤后14天，损伤侧翼的ONL中bFGF-IR升高。米勒细胞GFAP-IR在损伤后6小时首次检测到，并扩散到损伤部位以外相当远的距离。在7天和14天时，损伤部位的米勒细胞已发芽，而侧翼的米勒细胞仍有GFAP-IR。
激光损伤后，仅在最初24小时损伤核心处的bFGF增加。然而，在14天时在ONL中观察到bFGF水平升高，其延伸到损伤侧翼的距离与发现光感受器存活率增加的距离相似。这些结果支持了激光损伤诱导bFGF的假说，这可能是光感受器得以保留的机制。米勒细胞的激活与生长因子刺激一致，但比ONL中bFGF的变化更广泛。然而，血管标记同样广泛，因此激光损伤后米勒细胞GFAP反应和血管bFGF定位之间的反应可能是相关联的。
