Nagano Hiroshi, Wei Pan Zhen, Wen Chen Qing, Jomori Takahito, Oku Hidehiro, Ikeda Tsunehiko, Saito Yoshihiro, Tano Yasuo
Pharmaceutical Laboratory, Sanwa Kagaku Kenkyusho Co. Ltd., 363 Shiosaki, Hokusei, Inabe, Mie 511-0406, Japan.
Curr Eye Res. 2007 Feb;32(2):113-22. doi: 10.1080/02713680601160602.
Repeated intravitreal injections of endothelin-1 (ET-1) lead to alterations in the visually evoked potentials (VEPs) and loss of retinal ganglion cells (RGCs) in rabbits. The purpose of this study was to determine whether kallidinogenase can offset the alterations induced by ET-1.
ET-1 (2.5 x 10(-7) M, 20 microL) was injected into the vitreous of the right eye of rabbits (ET-1-treated eyes, n = 30) twice a week for 4 weeks. The vehicle for ET-1 was injected into the left eye on the same schedule (vehicle treated eyes, n = 30). During this 4 weeks period, kallidinogenase (1.0 unit/kg/day, kallidinogenase-treated group) or saline (saline-injected control group) was continuously delivered intravenously by an implanted osmotic pump. VEPs were recorded before, and 2 weeks and 4 weeks after, the first ET-1 injection, and all rabbits were sacrificed at 4 weeks. The number of RGC cells was counted in hematoxylin- and eosin-stained retinal sections. In the analyses, the ET-1 induced alterations were normalized to the values in the vehicle treated control eyes, i.e., kallidinogenase (K) + ET-1/K+ vehicle or saline (S) +ET-1/S + vehicle. Retinal sections were also examined by immunohistochemistry with antibodies to single-stranded DNA (ssDNA) or to glial fibrillary acidic protein (GFAP). The effect of kallidinogenase on the ONH blood flow was determined by a hydrogen gas clearance flowmeter.
The significant prolongation of the relative VEP implicit times (ITs) 4 weeks after the ET-1 injection (P < 0.01, paired t test; post-ET-1 vs. pre-ET-1) was significantly decreased by kallidinogenase (P < 0.001, t test, K + ET-1/K+ vehicle vs. S +ET-1/S + vehicle). The relative number of RGCs was decreased in the saline-injected group, and this decrease was also decreased by kallidinogenase (P < 0.05, t test, K + ET-1/K+ vehicle vs. S +ET-1/S + vehicle). ssDNA staining showed fewer apoptotic cells in the retina of kallidinogenase-treated rabbits. Intravitreal injection of ET-1 also decreased the blood flow in the optic nerve head and increased the GFAP immunostaining and axonal degeneration. These changes were also counteracted by kallidinogenase.
These results indicate that kallidinogenase can counter the effects of ET-1 and should be considered for the treatment of ischemic retinal and optic nerve disorders related to abnormal ET-1 production.
兔眼反复玻璃体内注射内皮素 -1（ET-1）会导致视觉诱发电位（VEP）改变及视网膜神经节细胞（RGC）丢失。本研究旨在确定激肽释放酶原酶是否能抵消ET-1诱导的这些改变。
将ET-1（2.5×10⁻⁷ M，20 μL）每周两次注射到兔右眼玻璃体内（ET-1处理组，n = 30），共4周。按相同时间表将ET-1的溶媒注射到左眼（溶媒处理组，n = 30）。在这4周期间，通过植入式渗透泵持续静脉输注激肽释放酶原酶（1.0单位/千克/天，激肽释放酶原酶处理组）或生理盐水（生理盐水注射对照组）。在首次注射ET-1前、注射后2周和4周记录VEP，所有兔子在4周时处死。在苏木精 - 伊红染色的视网膜切片中计数RGC细胞数量。在分析中，将ET-1诱导的改变标准化为溶媒处理对照组眼中的值，即激肽释放酶原酶（K）+ET-1/K +溶媒或生理盐水（S）+ET-1/S +溶媒。还用抗单链DNA（ssDNA）或抗胶质纤维酸性蛋白（GFAP）抗体进行免疫组织化学检查视网膜切片。用氢气清除流量计测定激肽释放酶原酶对视神经乳头血流的影响。
ET-1注射4周后相对VEP潜伏期（ITs）显著延长（P < 0.01，配对t检验；ET-1注射后与注射前相比），激肽释放酶原酶使其显著缩短（P < 0.001，t检验，K + ET-1/K +溶媒组与S + ET-1/S +溶媒组相比）。生理盐水注射组RGC的相对数量减少，激肽释放酶原酶也减少了这种减少（P < 0.05，t检验，K + ET-1/K +溶媒组与S + ET-1/S +溶媒组相比）。ssDNA染色显示激肽释放酶原酶处理的兔子视网膜中凋亡细胞较少。玻璃体内注射ET-1还降低了视神经乳头的血流，增加了GFAP免疫染色和轴突变性。这些变化也被激肽释放酶原酶抵消。
这些结果表明激肽释放酶原酶可对抗ET-1的作用，对于治疗与ET-1产生异常相关的缺血性视网膜和视神经疾病应予以考虑。
