Abstract
BACKGROUND:Researches on establishing restenosis models in rats emphasize the importance of 2.0 Forgarty balloon catheter import, but little has been mentioned about domestic-made balloon catheter.
OBJECTIVE: To explore the characteristics of vessel restenosis model by denuding arterial endothelium with domestic-made 2.0 Forgarty balloon catheter in rats.
DESIGN: A randomized controlled animal experiment.
SETTING: Laboratory of Pathophysiology, Hunan University of Traditional Chinese Medicine.
MATERIALS: This study was performed at the Laboratory of Pathophysiology, Hunan University of Traditional Chinese Medicine between September 2006 and January 2007. Healthy male SD rats, weighing 300-350 g, were selected. The protocol was performed in accordance with ethical guidelines for the use and care of animals. Domestic-made 2.0 Forgarty balloon catheters were made of balloons and catheters. Proliferating cell nuclear antigen (PCNA), collagen I, and SMα-actin immunohistochemical staining kits were provided by Wuhan Boster Bioengineering Co., Ltd, China.
METHODS: The rats were randomly divided into 4 groups: sham-operated group (n = 5), 7th day model group (n = 4), 14th day model group (n = 5), and 21st day model group (n = 4). The vessel restenosis models were established in the latter 3 groups by denuding arterial endothelium with domestic-made balloon catheter in rats. In the sham-operated group, rats were without insertion of balloon catheter. Injured thoracic aortas were taken out for HE staining to observe histological changes of vascular intima. At the meantime, expressions of SMα-actin, PCNA and collagen I were determined by immunohistochemical method in each group.
MAIN OUTCOME MEASURES: Histological changes of intima at the thoracic aorta as well as expressions of SMα-actin, PCNA and collagen I.
RESULTS: Eighteen rats were included in the final analysis. The aortal vascular walls remained integrity without narrowness of the lumen areas and proliferation of the endothelium, vascular smooth muscle cells (VSMCs) ranged regularly in the mesolamella in the sham-operated group. The endothelium had little proliferation in the 7th day model group. The intima thickened and the area of lumen became narrow in the 14th day model group. The intimal hyperplasia was diffusive and progressive which mainly included VSMCs, the area of lumen narrowed markedly and cells ranged disorderly in mesolamella in the 21st day model group. In the 14th day model group, PCNA level was higher compared to the remaining 3 groups (P < 0.01). SMα-actin level were significantly higher in the 14th and 21st day model groups than in the sham-operated group and 7th day model group (both P < 0.01). There were no significant differences in collagen I among the groups (P > 0.05).
CONCLUSION: Vascular intima hyperplasia appeared markedly 14-21 days after domestic-made balloon catheter-induced rat aorta injury. The results suggested that intima hyperplasia is mainly induced by proliferation of VSMCs.

INTRODUCTION

Percutaneous transluminal coronary angioplasty (PTCA) can alleviate clinical symptoms of cardiovascular disease, while restenosis incidence rate after PTCA remains high, which affected the long-term curative effects. Restenosis mechanisms after balloon inflation include the elastic retraction of blood vessels, the thrombosis at injury site, the proliferation and migration of vascular smooth muscle cells (VSMCs) and the accumulation of extracellular matrix [1]. In order to find a reliable and cheap method, and replace the imports, we tried to establish the vessel restenosis models by denuding arterial endothelium with domestic-made balloon catheters in rats, and investigated the model characteristics.

MATERIALS AND METHODS

Materials
This study was performed at the Laboratory of Pathophysiology (grade 3 laboratory of State Administration of Traditional Chinese Medicine), Hunan University of Traditional Chinese Medicine between September 2006 and January 2007. Eighteen healthy male SD rats, weighing 300-350 g, were provided by the Laboratory Animal Center for Disease Control and Prevention of Hunan Province, and used in this study (certification No. 20-010). The protocol was performed in accordance with ethical guidelines for the use and care of animals. Balloons were provided by Tianjin Zhongtuo Latex Science and Technology Co., Ltd., China. Catheters (refitted by 5? pinhead) were gifted by professor Han Mei, who came from Laboratory of Biochemistry, Institute of Basic Medical Sciences, Hebei Medical University. A domestic-made 2.0F balloon catheter was assembled with a balloon and a catheter: balloon about 2 cm in length was covered on one end of the catheter, two wedge-shaped grooves around water hole of the catheter which were designed up and down were tightened by the thread respectively. The other end of the catheter was connected with the No.16 needle and fixed. After balloon catheter was ready, physiological

balloon (just like soybean size), and the balloon could retract easily (Figure 1).
Reagents: PCNA, collagen I, SMα-actin immunohisto-chemical staining kits, and DAB kits were provided by Wuhan Boster Bioengineering Co., Ltd, China. SP-9001 immunohis-tochemical staining kits and poly-L-lysine were provided by Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd, China. The other chemicals and solvents were of analytical grade.

Methods
Animal models and experimental groups: The rats were ran-domly divided into 4 groups: sham-operated group (n = 5), 7th day model group (n = 4), 14th day model group (n = 5), and 21st day model group (n = 4). In the latter 3 groups, the rats were anesthetized with 10% chloral hydrate and a median incision was made on each rat neck. Left common carotid artery 1-1.5 cm was dissociated, and distal end of the artery was ligated. After the proximal end of left common carotid artery was oc-cluded with an artery clamp, a V-shaped incision was made at the distal end, and a domestic 2.0F balloon catheter was inserted. The catheter was carefully manipulated to get through aortic arch down to abdominal aorta, and the depth was about 6-7 cm. Physiological saline 0.4-0.6 mL was injected into the catheter to fill the balloon like soybean size. Balloon was inflated until we had an obvious sense of resistance when it was pulled back. The same resistance was kept to pull the catheter to the aortic arch for 3 times, and the catheter was rotated 180°, so that the bal-loon was transferred to the other side of the blood vessel. The procedure was repeated 3 times again with the same way. En-dothelium was completely stripped. After the catheter was withdrawn, the proximal end of left common carotid artery was ligated for hemostasis, and then incisions were sutured. Left common carotid artery was dissociated, but balloon catheter was not inserted in the sham-operated group. After the operation, penicilin 200 000 units were intraperitoneally injected for 3 days.
Experimental sampling: At the different time points, a vessel was taken from the thoracic aorta injury site for a morpho-logical observation and determination of the SM α- actin, collagen I and PCNA expressions. The rats in the sham-operated group were euthanized after intervention and all observation indices equaled to those in the model groups.
Determination of vascular morphology: The rats were bled to death and their chests were opened after anesthesia, then 1cm vascular segments at the same position where the endo-thelium was denuded were picked up. After the vessels were fixed with 4% paraformaldehyde, gradient dehydration and embedment were performed, and sections were stained rou-tinely with hematoxylin & eosin. Then 3 sections were ran-domly selected from each rat specimen for optical observation, photography and image analysis. According to the histology, the part within the internal elastic membrane was intima, the part between the internal elastic membrane and the external was tunica media. The intramembranous area of the internal elastic tunica, the external elastic tunica and the cross-sections of the lumen were measured by using a MIAS medical image analysis system, and the perimeters of the internal elastic tu-nica, the external elastic tunica and the lumen were measured too. On the basis of the results, following indices were calcu-lated : the area of the intima = (the intramembranous area of the internal elastic tunica - the area of the lumen), the thick-ness of the intima= [(the perimeter of the internal elastic tu-nica -the perimeter of the lumen)/2∏],the hyperplasia ratio of the intimal area= [(the area of the intima)/(the area of the in-tima +the area of the tunica media)×100%], the hyperplasia ratio of the intimal thickness= [(the thickness of the in-tima/(the thickness of the intima + the thickness of the meso-lamella) ×100%).
Immunohistochemistry: Immunohistochemical staining was performed for SMα-actin, PCNA and collagen I expressions. Brown yellow particles that appeared in the cells were re-garded as positive signals. PBS were used to replace the first antibodies as the negative controls.
Statistical analysis: SPSS 11.5 statistical software was adopted by the third author for analysis. All data were ex-pressed as Mean±SD. One-way analysis of variance (ANOVA) was used for multi-group comparison. Among every two groups, LSD test was used for analysis of homogeneity of variance, and Dounnett’ T3 test was used for heterogeneity of variance.

RESULTS

Vascular morphological changes after the aortal endothe-lium denuded with domestic-made balloon catheter in rats
The aortal vascular walls remained integrity without narrow-ness of the lumen areas and proliferation of the endothelium, VSMCs ranged regularly in the mesolamella in the sham-operated group. The endothelium had little proliferation in the 7th day model group. The intima thickened and the area of lumen became narrow in the 14th day model group. The intimal hyperplasia was diffusive progressively which mainly included VSMCs, the area of lumen narrowed markedly and cells ranged disorderly in mesolamella in the 21st day model group. The results suggested that the animal model of vascular restenosis had formed on the 21st day after the intimal denuda-tion (Figure 2).

Comparisons of vascular morphological indices (Table 1)
In the 21st day model group, intima presented a marked prolif-eration, and the area and thickness of intima, the hyperplasia ratio of intimal area and intimal thickness were significantly higher compared to other groups (P < 0.01).

Comparison of PCNA expression among the groups
PCNA-positive cells could not be observed in the sham-operated group. There were very few positive cells in the 7th day model group. A large number of positive cells were observed, PCNA expression in the neotenic intima and tunica media increased statistically in the 14th day model group. Many positive cells in the 21st day model group could be still observed, but were less than in the 14th day model group (Fig-ure 3). Quantitative comparison of PCNA expression in each group can be seen in Figure 4. There were significant dif-ferences in the PCNA expression between the 14th day model group and the others (P < 0.01).

Comparison of SMα-actin expression among the groups
SMα-actin positive cells were not observed in the sham-operated group. There were very few positive cells in the 7th day model group. Lots of positive cells were observed, and SMα-actin expression increased markedly in the 14th and 21st day model groups(Figure 5). SMα-actin expression was significantly higher in the 14th and the 21st day model groups than in the sham-operated and the 7th day model group (P < 0.01) (Figure 4).

Comparison of collagen Ⅰ expression
There were no significant differences among the 4 groups (P>0.05). Quantitative Comparison could be seen in Figure 4.

DISCUSSION

Rat model has become the preferable experiment model be-cause of its simple reproduction, high success rate, short ex-perimental cycle, very low price, the moderate lumen size of thoracic aortic and the thicker muscular layer of meso-lamella[2]. The imported balloon catheters were very expensive and difficult to match and apply in small animals particularly, so we used domestic-made balloon catheters to establish a reliable and inexpensive method. We made the vessel resteno-sis model by denuding the arterial endothelium in rats and investigated the model characteristics.
The morphometric results in this experiment showed that in-timal hyperplasia were obvious in the 21st day model group. So it was a good time point for observation on the 21st day after operation. PCNA is a kind of auxiliary protein for DNA poly-merase, which participates in the synthesis of DNA. It is a spe-cific indicator to reflect cell proliferation [3]. A large number of PCNA positive cells could be found on the 14th day after opera-tion, which indicated that PCNA expression increased markedly at this time point. SMα-actin is a specific symbol for VSMCs. VSMCs can obtain proliferative and synthetic capabilities only by changing differentiated state into dedifferentiated state, so in the initial recovery stages of the vascular injuries, VSMC pro-liferated vigorously and the content of SMα-actin reduced. When lesions gradually recovered, VSMC stopped proliferating and the content of SMα-actin increased. A small number of positive cells could be found on the 7th day, but a lot of positive cells were observed on the 14th and 21st days after operation. The morphological and immunohistochemical results suggested that vascular smooth muscle cells were close to or had recov-ered the differentiated state on the 14th days or the 21st day after operation. Collagen is a primary ingredient of normal vascular wall and neotenic intimal extracellular matrix. In the 19 known species of collagen, type Ⅰ and Ⅲ collagen account for 60% and 30% of the total vascular collagen, respectively. Collagen Ⅰ can spur VSMCs from the contractile state to the synthetic state, so collagen Ⅰ can effectively reflect the synthesis state of the damaged vascular extracellular matrix. However, colla-gens always change obviously in different atherosclerotic plaques, even in the same one. For example, fibrous plaque can contain little type Ⅰ and Ⅲ collagen but be rich in type Ⅳ and V [4]. Therefore, it maybe a reason why collagen Ⅰ ex-pression has no change in the experiment. At present, the com-mon model all over the world is the arterial injury model in-duced by a 2.0F Forgaty balloon catheter, which leads to the intimal hyperplasia and vascular restenosis [5-8]. Compared to it, a domestic-made balloon catheter is simple and practical, but probably results in slighter injury in modeling, unevenness of endothelial denudation and inconsistency of intimal hyperplasia thickness. If we keep the same balloon filling volume and the same insert-pull speed, maintain consistent balloon friction feeling in the whole experiment, and increase the times of injury, we can acquire the ideal results.

REFERENCES

1 Schwartz RS. Pathophysiology of restenosis, interaction of thrombosis hyperplasia and/or remodeling.Am J Cardiol 1998; 81(7A):14E-17E
2 Mcewan J. Therapeutic approaches to the control of fibrocellular intimal hyperplasia after angioplasty.Br Heart J 1993;70(1):1-3
3 Takada M, Tanaka H, Yamada T, et al. Antibody to thrombin receptor inhibits neointimal smooth muscle cell accumulation without causing inhibition of platelet aggregation or altering hemostatic parameters after angioplasty in rat. Circ Res 1998;82(9):980-987
4 Wen Jk, Han M. Vascullar Smooth Muscle Cell. Beijing: Science Publishing House 2005:166-166
5 Chen J, Han Y, Lin C, et al. PDGF-D contributes to neointimal hyperplasia in rat model of vessel injury. Biochem Biophys Res Commun 2005;329(3):976-983
6 Fischer JW, Hawkins S, Clowes AW. Pharmacologic inhibition of nitric oxide synthases and cyclooxygenases enhances intimal hyperplasia in balloon-injured rat carotid arteries. J Vasc Surg 2004; 40(1): 115-122
7 Miyahara T, Koyama H, Miyata T，et al. Inflammatory responses involving tumor necrosis factor receptor-associated factor 6 contribute to in-stent lesion formation in a stent implantation model of rabbit carotid artery. J Vasc Surg 2006;43(3):592-600
8 Nuthakki VK, Fleser PS, Malinzak LE, et al.Lysyl oxidase expression in a rat model of arterial balloon injury. J Vasc Surg 2004;40(1): 123-129

国产球囊导管制作大鼠主动脉损伤血管
再狭窄模型的特性及可行性*★

吴 露1，张 伟1，邓常青2
1湖南中医药大学2005级研究生班，湖南省长沙市 410007; 2湖南中医药大学病理生理学实验室，湖南省长沙市 410007
吴 露★，女，1981年生，陕西省安康市人，汉族，湖南中医药大学在读硕士，助教，主要从事心脑血管疾病防治机制的研究。
通讯作者：邓常青，博士后，教授，湖南中医药大学病理生理学实验室，湖南省长沙市 410007
国家自然科学基金资助项目(30572301)*
摘要
背景：研究应用国产球囊导管可否替代2.0F进口球囊导管制作大鼠主动脉损伤血管再狭窄模型。
目的：验证国产2.0F球囊导管致大鼠主动脉损伤血管再狭窄模型的特点及可行性。
设计：随机对照动物实验。
单位：湖南中医药大学病理生理学实验室。
材料：实验于2006-09/2007-01在湖南中医药大学病理生理学实验室完成。选用健康雄性SD大鼠，体质量300~350 g。实验过程中对动物的处置符合动物伦理学标准。国产2.0F球囊导管由球囊和导管组装而成。增殖细胞核抗原、Ⅰ型胶原、SMα-actin免疫组化染色试剂盒由武汉博士德生物工程有限公司提供。
方法：①随机数字表法法将大鼠分为假手术组(n = 5)、模型7 d组(n = 4)、模型14 d组(n = 5)和模型21 d组(n = 4)，后3组大鼠经麻醉后采用国产2.0F球囊导管建立大鼠主动脉内皮剥脱模型，假手术组只进行颈总动脉近心端切口，但不插入球囊损伤。②模型7，14，21 d组大鼠于术后7，14，21 d取损伤部位胸主动脉段进行切片及HE染色，观察大鼠血管内膜组织学变化，同时采用免疫组织化学法检测血管平滑肌细胞表型标志物SM-肌动蛋白、增殖细胞核抗原及Ⅰ型胶原表达情况。假手术组于干预后直接取材观察，检测项目同各模型组。
主要观察指标：大鼠胸主动脉段内膜组织学变化及SM-肌动蛋白、增殖细胞核抗原和Ⅰ型胶原表达。
结果：纳入大鼠18只均进入结果分析。①假手术组各层结构完整，管腔面积无缩小，内膜光滑无增生，中膜血管平滑肌细胞排列整齐。模型7 d组增生不明显，模型14 d组内膜有增厚，管腔面积有缩小，模型21 d组内膜呈进行性弥漫性增厚，管腔面积明显缩小，增厚的内膜中以平滑肌细胞为主，中膜细胞排列紊乱。②模型14 d组增殖细胞核抗原水平高于其他3组(P < 0.01)， 样型14 d组21 d组SM-肌动蛋白含量高于；模型21 d组SM-肌动蛋白含量低于假手术组及模型7 d组，差异均有显著性意义（P < 0.01）；各组Ⅰ型胶原表达水平差异均无显著性意义(P > 0.05)。
结论：国产2.0F球囊导管致大鼠主动脉损伤后14~21 d可出现明显血管内膜增生，该增生主要是由血管平滑肌细胞增殖所致。
关键词： 球囊导管；血管平滑肌细胞； SM-肌动蛋白；增殖细胞核抗原；Ⅰ型胶原
中图分类号: R318 文献标识码: B 文章编号: 1673-8225(2008)17-03372-04
吴露，张伟，邓常青.国产球囊导管制作大鼠主动脉损伤血管再狭窄模型的特性及可行性[J].中国组织工程研究与临床康复，2008，12(17):3372-3375
[www.zglckf.com/zglckf/ejournal/upfiles/08-17/17k-3372(ps).pdf]
（Edited by Ding C/Cui X/Song LP/Wang L）