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Open Access Study protocol

Detecting DNA synthesis of neointimal formation after catheter balloon injury in GK and in Wistar rats: using 5-ethynyl-2'-deoxyuridine

Jingsheng Guo1, Dongye Li1*, Shiru Bai1, Tongda Xu1, Zhongmin Zhou2 and Yanbin Zhang1*

Author Affiliations

1 Institute of Cardiovascular Disease Research, Xuzhou Medical College, 84 West Huaihai Road, Xuzhou, Jiangsu, Peoples Republic of China

2 Department of Internal Medicine, Aultman Hospital & Canton Medical Education Foundation, Northeast Ohio Medical University, Canton, OH, USA

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Cardiovascular Diabetology 2012, 11:150  doi:10.1186/1475-2840-11-150

Published: 13 December 2012

Abstract

Background

Neointimal formation plays an important role in the pathogenesis of coronary restenosis after percutaneous coronary intervention (PCI), especially in patients with diabetes mellitus. Recently, some studies have shown that 5-ethynyl-2'-deoxyuridine (EdU) incorporation can serve as a novel alternative to the 5-bromo-2'-deoxyuridine (BrdU) antibody detection method for detection of DNA synthesis in regenerating avian cochlea, chick embryo and the adult nervous system. However, few studies have been performed to assess the suitability of EdU for detecting DNA synthesis in vascular neointima.

Methods

The carotid artery balloon injury model was established in Goto-Kakizaki (GK) and Wistar rats. A Cell-LightTM EdU Kit was used to detect EdU-labeled cell nuclei of common carotid arteries at day 7 after catheter balloon injury. Different methods of injecting EdU were tested. The protein levels of proliferating cell nuclear antigen (PCNA) and p-Akt (Ser473), as well as the mRNA levels of PCNA were evaluated by Western blotting and quantitative real-time PCR (qRT-PCR), respectively. Immunohistochemical staining was also employed to visualize PCNA-positive cells.

Results

At day 7 after catheter balloon injury, far more EdU-positive and PCNA-positive cells were observed in GK rats. When comparing groups that received different EdU doses, it was found that the percentage of EdU-positive cells at a dose of 100 mg/kg body weight was than at doses of 25 mg/kg and 50 mg/kg. The number of positive cells was significantly higher in the repeated injection group compared to the single injection group. Further, after balloon injury DNA synthesis in GK rats was more notable than in Wistar rats. Neointimal formation in GK rats was more obvious than in Wistar rats. The protein levels of PCNA and p-Akt (Ser473) and the mRNA levels of PCNA were increased in injured rats as compared to uninjured rats, and were significantly higher in GK rats than in Wistar rats.

Conclusion

By intraperitoneal injections of EdU at a dose of 100 mg/kg three times, EdU incorporation can detect carotid arterial DNA synthesis caused by neointimal formation in GK rats and Wistar rats at day 7 after balloon injury by the EdU click reaction quickly and effectively. Moreover, more obvious DNA synthesis in the vascular neointima could be observed in GK rats than in Wistar rats.

Keywords:
Neointimal formation; DNA synthesis; Diabetes mellitus; EdU; Catheter balloon injury; PCNA