豚鼠眶周注射曲安奈德/透明质酸酶后的病理变化

Pathological Changes after Periocular Injection of Triamcinolone and Hyaluronidase in Guinea Pigs

目的评价豚鼠眶周注射曲安奈德/透明质酸酶后病理形态学的改变。方法将20只清洁级荷兰种豚鼠随机分成A、B、C、D 4个实验组,每组5只,每只豚鼠右眼作为实验眼,左眼作为空白对照。A组实验眼眶周注射生理盐水0.2mL,B组注射曲安奈德2mg(0.2mL),C组注射透明质酸酶80IU(0.2mL),D组注射曲安奈德2mg(0.1mL)+透明质酸酶80IU(0.1mL),每周注射1次,连续注射4次。4组均以第1次注射为时间原点,在之后的第3d、7d、14d、28d、35d分次各取1只豚鼠处死,取眶周组织标本常规石蜡包埋切片,经苏木精-伊红(HE)染色及Masson三色染色后进行形态学观察,期间取各组少量第3次标本组织进行电镜观察,并筛选部分肉芽肿形成良好的标本进行转化生长因子-β1(transforming growth factor-β1,TGF-β1)免疫组织化学染色观察。结果与空白对照组相比,A组眶周组织HE染色无明显病理改变,C组眶周结缔组织较空白对照组疏松,细胞间隙增宽。B组第2到5次切片中均可见异物肉芽肿组织,D组在第3次和第5次切片中也观察到散在的肉芽肿样组织,但从HE及Masson三色染色切片的对比发现,D组肉芽肿的大小及其中类上皮细胞和成纤维细胞密度均较B组低。B组肉芽肿区域TGF-β1染色呈强阳性,D组肉芽肿区域TGF-β1染色呈弱阳性,D组染色阳性细胞数量少于B组,两组之间差异有统计学意义(P<0.05)。电镜观察发现B组成纤维细胞多为高活跃状态,细胞呈矮胖形,核卵圆状,胞浆多,细胞器发达,C组、D组成纤维细胞处于相对低活跃状态,细胞及核均呈长梭状,胞浆少,细胞器不发达。结论长期反复眶周注射曲安奈德可导致注射区域异物肉芽肿的形成,进行性纤维化,并与周围组织产生粘连。联合透明质酸酶注射能增强曲安奈德的有效扩散和吸收,减轻因药物吸收不良所引起的晚期并发症。

Objective To analyze the pathological change after periocular injection of triamcinolone and hyaluronidase in guinea pigs. Methods Twenty guinea pigs of clean grade were used in this study. The animals were randomly divided into 4 groups （n-5 each）. The right eye was used as intervention side, while the left eye was used as control side. Group A received perioeular injection of saline water 0. 2 mL at right eyes, Group 13 received periocular injection of triamcinolone 2 mg （0.2 mL）, Group C received perioeular injection of hyaluronidase 80 IU （0.2 mL）, Group D received periocular injection of triamcinolone 2 mg （0.1 mL） ＋hyaluronidase 80 IU （0.1 mL）. The injection was performed once a week for totally 4 times. One animal from each group was sacrificed at day 3, 7, 14, 28 and 35 after the first injection. The specimens of periocular tissue were obtained for microscopic examination and transmission electron microscopic examination to observe the pathological changes. The expressions of transforming growth factor-β1 （TGF-β1） in the granulomas were detected by immunohistochemistry stain method. Results Compared with the control side, group A presented no obvious change. Group C presented looser intercellular space than the control group. Foreign body granulomas was seen in 2^nd-5^th pathological section of group B and in the third and fifth pathological sections of group D. The size of the granulomas was smaller and the density of fibroblast was lower in group D than those in group B. The immunohistochemistry stain of TGF β1 showed strong positive reaction in group B, while weakly positive reaction in group D. The positive staining cells in group D were fewer than those in group B, and the difference was statistically significant （P〈0.05）. Most of the fibroblasts in group B presented as pyknic type with oval nucleus, amounts of cytoplasm and cell organs under electron microscope. Comparatively, the fibroblasts in group C and D remain inactive with spindle nucleus, less cytoplasmand cell organs. Conclusion Repeated periocular injection of triamcinolone may cause the formation of foreign body granulomas around the injection site, leading to fibrosis and adhesion at later period. The injection combined with hyaluronidase can enhance the diffusion and absorption of triamcinolone, relieving the possible fibrosis.