丙烯醛誘導大鼠脈絡膜新生血管的形成

王觀峰，鄒秀蘭，李東豪，王　琛，李文立，皮榮標

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丙烯醛誘導大鼠脈絡膜新生血管的形成

王觀峰，鄒秀蘭，李東豪，王琛，李文立，皮榮標

摘要

關鍵詞：丙烯醛；脈絡膜新生血管；大鼠

Abstract

?AIM:To investigate the formation of rats’ choroidal neovascularization (CNV) induced by acrolein.

?METHODS:Twelve Sprague-Dawley rats were randomly divided into three groups. Acrolein 200μL (2.5 mg/kg/d) was poured into the rats’ stomach for 4wk as acrolein 4wk and for 8wk as acrolein 8wk group. The same volume of fresh water was also done to the rats as the control group. Remove all eye balls and embed into paraffin with HE staining.

?RESLUTS:The RPE-Bruch membrane was intact with no obvious abnormality in the control group and acrolein 4wk group. Lost in the continuity of RPE and the movement of choroidal neovascularization were found in the acrolein 8wk.

?CONCLUSION:The long time use of acrolein can induce the formation of choroial neovascularization in rats.

KEYWORDS:?acrolein; choroidal neovascularization; rat

Citation:Wang GF, Zou XL, Li DH, Wang C, Li WL, Pi RB. The formation of rats’ choroidal neovascularization induced by acrolein.GuojiYankeZazhi(IntEyeSci) 2016;16(4):591-593

INTRODUCTION

Choroidal neovascularization (CNV) which lead to the average of millions patients with low vision and hundreds of thousands blind people in progress has become the main cause of blindness in our country now. Patients often have the performance of both eyes at the same time or successively irreversible, progressive vision decline[1-2]. But the pathogenesis of the disease lack of efficacy and side effects of the treatment in clinic is not clear. The disease is also easy to relapse. Thus, to study the occurrence and development of the disease have great clinical and social significance in exploration of prevention and treatments.

The cause of CNV is not clear at present, but smoking has been recognized as the most important risk factors by the domestic and foreign scholars. The relative risk (RR) of CNV in smokers is 2.4 times than that of non-smokers, and the RR of complement 402H CC genome is 3.4 times proved by statistical data[3-4]. Aldehydes, nitrogen oxides and other toxic substances which were founded in the cigarettes can not only significantly reduce the levels of ascorbic acid and hydrogenated sulfur protein in retinal pigment epithelium (RPE), but also cause the oxidation of fat and protein[5-8]. However, the toxicity of acrolein (AC) is 10 times to that of formaldehyde and 100 times to the acetaldehyde. The AC, meanwhile, can be directly reacted with the hydrogen sulfideFigure 1HE staining (10×20)A: Control group; B: Acrolein 4wk group; C: Acrolein 8wk group; D: Acrolein 8wk group.

protein to have a higher risk index than other five kinds of toxic aldehydes in cigarettes. In earlier years, we succeeded in replicating the damage process in RPE cell research by AC. Thus, we will explore the induction of CNV rats to provide more experimental data for studying the formation and development of CNV disease.

SUBJECTS AND METHODS

Experimental Animals and Feed SourcesRandomly selected 12 male, 8 month old Sprague Dawley (SD) rats weighted about 270 to 300 g. These animals were fed at the animal house of Sun Yat-Sen University with the temperature of (25±1) centigrade. The relative humidity of (50±5)%, 12h light cycle on a day and related feed were purchased from the the experimental animal center of Sun Yat-Sen University.

Grouping and the Route of AdministrationAccorded to the the random number table, all SD rats were randomly divided into three groups as the blank group, 4wk for acrolein and 8wk for acrolein. The amount of 2.5 mg/kg acrolein dissolved into 200μL fresh water which was daily gavaged in SD rats within 2h as the treatment group. At the same time, the control group was given the same volume of fresh water.

Hematoxylin-Eosin Staining for Paraffin SectionEyes, collected from these experimental rats, were removed into the fixed radium of 4% paraformaldehyde for the whole night. Then these organizations were dehydration by gradient alcohol, embedded in the paraffin and sliced with HE staining. Observed and photoed them by optical microscope.

Statistical MethodsUsing the SPSS 13.0 statistical software to make a statistical analysis. Experiment data were expressed as mean± standard deviation by single factor analysis of variance in the complete random block design. The data was statistically significant whenP<0.05.

RESULTS

HE staining of optic nerve and choroida control group: the structure of the retina was regular with the complete RPE-Bruch film continuity by optical microscope (Figure 1A).

Acrolein 4wk group: the structure of the retina was regular with the complete RPE-Bruch film continuity by optical microscope (Figure 1B).

Acrolein 8wk group: the RPE-Bruch film continuity interrupted with the invasion of choroidal neovascularization into the neuroepithelial layer as a typical uplift (Figure 1C, 1D).

DISCUSSION

More than 300 kinds of substances, as formaldehyde, acetaldehyde, acrolein, crotonaldehyde and so on, have been found in the daily food or raw materials like bread, fish, alcohol and etc. In addition, the contact of oil, sugar and fatty substance can make the production of aldehydes by peroxidation during the cooking. But these aldehydes which contained of aldehyde (CHO) and hydroxyl (-OH) or hydrogen (H) atoms have a strong reduction and oxidation. The maximum amount of average adult daily contact is about 7mg/kg in the epidemiological studyings, included of 5mg/kg[7-11]acrolein, 2 mg/kg formaldehyde and acetaldehyde exposure. Otherwise, foreign scholars constructed the acute myocardial toxicity model with the concentration of 5 mg/kg/day acrolein in the treatment for 30min, the chronic vascular tissue and nerve tissue toxicity model with the concentration of 2.5 mg/kg/day acrolein in the treatment for 8wk to make a great contribution in the animal toxicity model. Therefore, we take 2.5 mg/kg/day acrolein as a 50% total unsaturated aldehydes to investigate the chronic injury of rat retina and choroid.

We found the interruption of RPE-Bruch membrane and invasion of CNV from the choroidal tissue which might be possible to form as follows[12-15]. The production of vascular endothelial growth factor (VEGF) not only promotes the formation of new blood vessels, but also can stimulate endothelial cells division, increasing of microvascualr permeability and the formation of some proteases. Nicotine in cigarettes can increase the ratio of VEGF and pigment epithelium-derived factor(PEDF) which could cause the formation of CNV in RPE cells. The levels of VEGF and TGFβ2 were double than that of the control group on the acute toxicity of RPE cells model[16]. Another, the level of VEGF was 1.417 times to that of the TGF-β/SMAD3. We believe the induction of TGF-β which was an important factor in CNV activity and fibrosis could cause the formation of VEGF and CNV. On the other hand, acrolein can cause the production of cyclooxigenase-2 (COX-2), nitric oxide (NO), and the oxidative damage of nitric, proteins and other biological molecules. It was found the deficiency in COX-2 which made a decline on the expression of VEGF and formation of CNV[17]. NO can also promote the release of proteases to stimulate the proliferation, migration of endothelial cells and increasing permeability of capillaries as an important signal molecule transduction. Therefore, ACinvitroandvivocan cause the chronic effects on the formation of CNV which expand the development of CNV disease in rats.

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DOI:10.3980/j.issn.1672-5123.2016.4.01

通訊作者：鄒秀蘭，畢業于德國柏林Charité醫學院，博士研究生，碩士研究生導師，研究方向：眼底病. 149642427@qq.com

作者簡介：王觀峰，畢業于暨南大學，碩士研究生，住院醫師，研究方向：眼底病。

基金項目：廣州醫科大學附屬第三醫院院內課題(No.2013Y06)；廣東省科技廳課題(No.2012B031800419)
(作者單位：510150中國廣東省廣州市,廣州醫科大學附屬第三醫院眼科)

目的：探討丙烯醛(AC)誘導大鼠脈絡膜新生血管(CNV)的形成。

方法：選取遠交群大鼠(SD)12只, 隨機將大鼠分為三組, 即空白組，AC誘導4wk組和AC誘導8wk組, 其中空白對照組每日200μL新鮮自來水灌胃, 實驗組為200μL AC溶液(2.5 mg/kg/d)每日灌胃,分為誘導4wk組和8wk組，取材后視網膜組織石蠟包埋,切片及HE染色。

結果：空白對照組及AC誘導4wk組均見RPE-Bruch膜連續性完整, 未見明顯異常；AC誘導8wk組發現RPE-Bruch膜連續性缺失, 脈絡膜新生血管長入視網膜神經上皮層內。

結論：長期使用AC可以誘導大鼠脈絡膜新生血管形成。

引用：王觀峰，鄒秀蘭，李東豪，王琛，李文立，皮榮標. 丙烯醛誘導大鼠脈絡膜新生血管的形成.國際眼科雜志2016;16(4):591-593