This describes a partial carotid ligation surgery, which causes disturbed flow conditions and subsequent atherosclerosis development (in two weeks) with intraplaque neo-vascularization (in four weeks) in the mouse common carotid artery. We also describe a novel method of RNA isolation from the carotid intima, providing high purity endothelial RNA.
1. Partial ligation of the left carotid artery
2. Carotid ultrasound examination
3. Intimal RNA isolation from carotid
4. Representative Results:
We partially ligated mouse left carotid arteries and ultrasound examination was carried out one day following the procedure. As shown in Figure 1, successful partial ligation reduces blood flow velocity and reverses flow (disturbed flow) in the left common carotid artery during diastole. Partial ligation of ApoE knockout (KO) mice accompanied by a high fat diet induces robust atherosclerosis in the left common carotid artery(LCA), but not in the contralateral right carotid, by two weeks (Figure 2). RNA collected from the carotid intima contains endothelial marker genes such as PECAM-1, while free of medial smooth muscle cell marker genes such as a-SMA (Figure 3).
Figure 1: Schematic of carotid anatomy and ligations: Three branches of the left common carotid artery (LCA) [external carotid artery (ECA), internal carotid artery (ICA), and occipital artery (OA)] were ligated, while leaving the superior thyroid artery (STA) open.
Figure 2: The ultrasound images show flow velocity profiles and reveal that partial ligation induces flow reversal (indicated by arrows) in LCA during diastole. Flow in RCA remains unchanged after ligation.
Figure 3: Partial ligation and high-fat diet rapidly induces atherosclerosis in LCA of ApoE KO mice. ApoE KO mice were partially ligated and fed the high-fat diet for 1 to 3 weeks. Frozen sections from LCA were stained with Oil-Red-O.
Figure 4. Method of intimal RNA preparation. Intimal RNA and medial+adventitial (m+a) RNA were obtained from sham-operated RCA and LCA in C57BL/6 mice. RNAs were analyzed by semi-quantitative RT-PCR (A) and qPCR (B, C) for PECAM-1 and α-SMA using 18s as an internal control. Bar graphs are mean ± SEM, n=3.
In this procedure, proper identification of all the branches of the common carotid including the external carotid, internal carotid, occipital artery and superior thyroid artery is essential. It is also important to verify the flow pattern using ultrasound as demonstrated. In our experienced hands, our success rate of partial ligation surgeries based on the ultrasound studies of more than 500 mice is better than 90%. For the carotid intimal RNA preparations, it is important to pay attention to the appearance of the carotid after the first perfusion step. If there is evidence of flow blockage in the left common carotid at this point, we recommend not using these samples for further analysis.
This work was supported by funding from NIH grants HL87012 (HJ), HL75209 (HJ), UO1HL80711 (DH, DG, HJ) and a World Class University Project (HJ) from the Ministry of Science, Technology and Education of S. Korea.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Normal Saline (0.9% sodium chloride) | Baxter | 2B1323 | ||
Insulin syringe (3/10 ml 29g syringe) | Tyco Health care | 8881600145 | ||
HBSS | Cellgro, Mediatech, Inc. | 21-023 | ||
miRNeasy mini kit (including, QIAzol) | QIAGEN | 217004 | ||
Vevo 770 High-Resolution Micro-Imaging System | Visualsonics | a.Visualsonics 770 system b.Imaging stage c.Vevo integrated rail system d.RMV scan head for mice |
or similar system | |
6-0 Silk Suture, sterile | Covidien | s-1172 c2 | ||
Small scissors | Roboz | RS-5675 | or similar | |
Dissecting Forceps | Roboz | RS-5005 | or similar | |
Tissue Mend II | Webster Vet Supply | 07-856-7946 |