A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

Summary

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.

Abstract

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified version of carotid partial ligation methods [1,2] to show that it acutely induces low and oscillatory flow conditions, two key characteristics of disturbed flow, in the mouse common carotid artery. Using this model, we have provided direct evidence that disturbed flow indeed leads to rapid and robust atherosclerosis development in Apolipoprotein E knockout mouse [3]. We also developed a method of endothelial RNA preparation with high purity from the mouse carotid intima [3]. Using this mouse model and method, we found that partial ligation causes endothelial dysfunction in a week, followed by robust and rapid atheroma formation in two weeks in a hyperlipidemic mouse model along with features of complex lesion formation such as intraplaque neovascularization by four weeks. This rapid in vivo model and the endothelial RNA preparation method could be used to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases. Also, it could be used to test various therapeutic interventions targeting endothelial dysfunction and atherosclerosis in considerably reduced study duration.

Protocol

1. Partial ligation of the left carotid artery

1. Mice are used at ~ 8 weeks of age. The weight of the mice is recommended to be at least 18 grams.
2. Sterilize the instruments for 30 second in bead instrument sterilizer and allow to cool down
3. Induce anesthesia with Ketamine (80mg/kg) and Xylazine (10mg/kg) intraperitoneal (i.p.) injection or alternative anesthesia as approved by IACUC. For i.p. injection, inject mouse in the left low quadrant of abdomen.
4. Place mouse in a warming chamber until completely anesthetized. Verify depth of anesthesia with response to toe pinch.
5. Place mouse on a surgical s....

Discussion

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 caro.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Normal Saline (0.9% sodium chloride)	Baxter Internationl Inc.	2B1323
Insulin syringe (3/10 ml 29g syringe)	Tyco Healthcare, Covidien	8881600145
HBSS	Cellgro	21-023
miRNeasy mini kit (including, QIAzol)	Qiagen	217004
Vevo 770 High-Resolution Micro-Imaging System	VisualSonics, inc.	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 Surgical Instruments Co.	RS-5675	or similar
Dissecting Forceps	Roboz Surgical Instruments Co.	RS-5005	or similar
Tissue Mend II	Webster Veterinary	07-856-7946