Study Of The Pharmacologic Action Of Natamycin Treatment Of Experimental Fungal Keratitis In Rabbits

INTRODUCTIONFungal keratitis, which is generally caused by external injury, becomes a serious ophthalmic infection due to the high rate of and blinding. For the past few years, the rate of fungal keratitis’recurring quickly increased in china, which is mainly related to the application of broad-spectrum antibiotic, antitumor drugs, immunosuppressive agent and corticosteroid hormone, and so on.Because of the unclear pathogenic mechanisms, as well as the deficiency of effective antifungals, it is hard to treat the fungal keratitis. Moreover, it can lead to serious complications such as endophthalmitis, perforation and blindness. In ophthalmologic field, fungal keratitis continues to be a therapeutic challenge to oculists all over the world.Topical natamycin5%has been shown to be effective in fungal keratitis, and it is extensively used in china, but such drugs in china has not their own intellectual property rights. Our research is to investigate the efficacy and irritation of natamycin, produced by China Shijiazhuang Pharmaceutical Group Company, and to provide experimental basis for the application ofclinical safety.The first part A Study of Biological Character and Influence of Temperature on the Growth and Spore Production of Fusarium SolaniPurpose:To investigate the influence of temperature on the growth, sporulation quantity and pathogenicity of Fusarium Solani. Find the best way for promoting conidial production of Fusarium Solani.Methods:Separated Fusarium Solani into different groups by temperature, Group A, B, C, D and E were incubated in dark for7days at20℃,25℃,30℃,35℃, and37℃, respectively; group F were incubated in dark for3days at35℃, and from the4th to the7th day, at25℃.On24h,3d,5d,7d measure the diameter of the colonyof Fusarium solani by vernier caliper, on3d,5d,7d measure the conidial production of Fusarium solani by hematimeter.Results:Between20℃-30℃, the growing speed of Fusarium solani colony is increasing with the increase of the temperature, between30℃-37℃, the growing speed of the colony is decreasing with the increase of the temperature. The spore production of group F is1.025×109CFU/ml.Conclusion:The result showed Fusarium solani growth and conidiospore production occurred at20to35℃. Temperature affected obviously the pathogenicity of the Fusarium solani.The suitable temperature for the growth was30℃. The suitable way for sporulati on was put it into35℃incubator brood for3days first and then put it into25℃incubator brood to7days.The second part Establishment and pathological observation of Rabbit model with fungal keratitisPurpose:Duplicate the way to make animal models with fugal keratitis and observe its histopathological changes.Methods:15(20eyes) newzealand white rabbits, divided into3groups: control group, saline group and model group. Fusarium Solani was injected into the corneal stroma of the eyes of model group to establish fungal keratitis. Saline was injected into the corneal stroma of the eyes of saline group. Do not do anything to the control group. Take the culture from the lesion of cornea after inoculated fungus for2days. The rabbits were sacrificed with ear vein of air embolism at15days after inoculation. Observe the histological changes through HE, PAS and electron microscope.Results:After inoculated fungus for2days, there were corneal edema, ulceration, dryness and tightness, rough, uneven and swelling. Pathological staining showed that scattered fungal spores and hyphaewere at corneal epithelium, and hyphae grew into corneal stroma. Hematoxylin Eosin Stain and PAS Stain show that There was seprartion, round spores grew in scatter, way and hyphae and spores were seen at anterior chanber in part specimen. Transmission Electron Microscope shows that the ultrastructure features of the corneal stromal Pathological change caused by Fusarium Solani.Conclusion:Injecte the Fusarium Solani into the corneal of the eyes can be used to establish animal models with fungal keratitis successfully.The third part Study of the Pharmacologic Action of Natamycin Treatment of Experimental Fungal Keratitis in rabbits.Purpose:Evaluate the efficacy of topical natamycin in an experimental rabbit model of Fusarium Solani, to provide experimental basis for the application of clinical safety.Methods:Fungal Keratitis was induced in the eyes of30New Zealand rabbits.1.5×106CFU (colony forming unit)/mL Fusarium Solani spore suspension was injected into the central corneal stroma of the eyes of rabbits. Forty-eight hours after inoculation, the animals were divided into5groups,10rabbit eyes of each group. Group1did not receive any eye drops. Group2received normal saline eye drops everyday. Group3received vehicle eye drops. Group4received natacyn, Group5received natamycin. Normal control group neither induced Fungal Keratitis nor received any eye drops. The eyes of each group was examined clinically and respectively by slit lamp with a scoring system on day4, day10, day15, day21to observe whether the eyes were healed or not, corner vascularisation, iritis, hypopyon and macular nebula were examined and recorded on day10and day21.Results:(1)Evaluate the score on day4, day10, day15, day21:Normal saline group and vehicle eye drops group were no difference with FK group(P>0.05). Natacyn group and natamycin group were different from FK group (P<0.05). There is no difference between natacyn group and natamycin group(P>0.05).(2)Observed whether the eyes were healed or not on day10and day21: Normal saline group and vehicle eye drops group were no difference with FK group(P>0.05). Natacyn group and natamycin group were different from FK group (P<0.05). There is no difference between natacyn group and natamycin group(P>0.05).(3)Examined the corner vascularisation, iritis, hypopyon and macular nebula on day10and day21:Normal saline group and vehicle eye drops group were no difference with FK group(P>0.05). Natacyn group and natamycin group were different from FK group (P<0.05). There is no difference between natacyn group and natamycin group(P>0.05).Conclusion:Natamycin was found to be effective in fungal keratitis, as well as natacyn, and it can stop the corner vascularisation, iritis, hypopyon and macular nebula to happen. Natamyin as a kind of effective against fungal keratitis, with low toxicity on eyes is of great prospect, worthy of popularization. The fourth part Irritation of Natamycin on Rabbit Eyes Purpose:To investigate the irritation of natamycin on normal rabbit eyes, to provide experimental basis for the application of clinical safety.Methods:10rabbit eyes of each group were dropped respectively by natamycin, natacyn, normal saline and vehicle eyedrops. The irritation of eyes was double-blinded evaluated by slit-lamp examinations and scored.Results:The results showed that there was not irritation in any experimental groups, and no significant differences compared with normal saline control group.Conclusion:natamyin as a kind of effective against fungal keratitis, with low toxicity on eyes is of great prospect.Conclusionl.The result showed Fusarium Solani growth and conidiospore production occurred at20to35℃. Temperature affected obviously the pathogenicity of the Fusarium Solani.The suitable temperature for the growth was30℃. The suitable way for sporulati on was put it into35℃incubator brood for3days first and then put it into25℃incubator brood to7days.2.Injecte the Fusarium Solani into the corneal of the eyes can be used to establish animal models with fungal keratitis successfully.3. natamycin was found to be effective in fungal keratitis, as well as natacyn, and it can stop the corner vascularisation, iritis, hypopyon and macular nebula to happen. Natamyin as a kind of effective against fungal keratitis, with low toxicity on eyes is of great prospect, worthy of popularizing.4.natamyin as a kind of effective against fungal keratitis, with low toxicity on eyes is of great prospect.