Murine Surgical Model of Topical Elastase Induced Descending Thoracic Aortic Aneurysm

Summary

We describe a surgical protocol to consistently induce robust descending thoracic aortic aneurysms in mice. The procedure involves left thoracotomy, thoracic aorta exposure, and placement of a sponge soaked in porcine pancreatic elastase on the aortic wall.

Abstract

According to the Center for Disease Control, aortic aneurysms (AAs) were considered a leading cause of death in all races and both sexes from 1999-2016. An aneurysm forms as a result of progressive weakening and eventual dilation of the aorta, which can rupture or tear once it reaches a critical diameter. Aneurysms of the descending aorta in the chest, called descending thoracic aortic aneurysms (dTAA), make up a large proportion of aneurysm cases in the United States. Uncontained dTAA rupture is almost universally lethal, and elective repair has a high rate of morbidity and mortality. The purpose of our model is to study dTAA specifically, to elucidate the pathophysiology of dTAA and to search for molecular targets to halt the growth or reduce the size of dTAA. By having a murine model to study thoracic pathology precisely, targeted therapies can be developed to specifically test dTAA. The method is based on the placement of porcine pancreatic elastase (PPE) directly on the outer murine aortic wall after surgical exposure. This creates a destructive and inflammatory reaction, which weakens the aortic wall and allows for aneurysm formation over weeks to months. Though murine models possess limitations, our dTAA model produces robust aneurysms of predictable size. Furthermore, this model can be used to test genetic and pharmaceutical targets which may arrest dTAA growth or prevent rupture. In human patients, interventions such as these could help avoid aneurysm rupture, and difficult surgical intervention.

Introduction

The purpose of this method is to study the development, pathophysiology, and structural changes in the murine descending thoracic aorta during aortic aneurysm formation. Our model offers a reproducible and consistent method to induce thoracic aortic aneurysms (dTAA) in mice thereby allowing for the testing of various genetic and pharmacologic inhibitors. This work can help identify drugs and gene-therapies which could be translated to a viable treatment strategy for humans with dTAA disease.

dTAAs form when the wall of the thoracic aorta becomes weakened and dilates over time until reaching a critical diameter when tearing or rupture can th....

Protocol

Animal protocols were approved by the University of Virginia Institutional Animal Care and Use Committee (No. 3634).

1. Induction of anesthesia and intubation

1. Place an 8-10-week-old male C57BL/6 mouse in a closed chamber with continuous flow of 5% isoflurane and oxygen mixture for 5 min, until respirations are visibly slowed.
   NOTE: Different strains, genders and ages of mice can be used depending on the experimental protocol. Female mice may be more difficult to intubate because of smaller size and thus smaller airway.
2. Intubate the mouse as described by Vandivort et al.¹³.
   NOTE: Th....

Results

The application of our protocol results in robust dTAA in mice compared to saline controls. The TAAs developed are fusiform in shape and occur only in the treated portion of the aorta (Figure 1 and Figure 2)¹¹. Figure 2 shows an example of a video micrometry measurement at tissue harvest. Using Equation 1, the aortic dilation is 130% in this example.

The original study by Johnston .......

Discussion

The thoracic and abdominal aorta are cellularly and embryologically distinct, which is relevant to aneurysmal disease^14,15,16. Therefore, a specific animal model to study TAA is needed. Though other murine dTAA models have been published⁸, ours is the only model to create descending thoracic aortic dilatation which can be considered truly aneurysmal (over 50% dilation). Furthermore, our model is relativel.......

Materials

Name	Company	Catalog Number	Comments
Angiocatheter (22G)			Used for ET Tube
Dumont Tweezers; Pattern #7 x2	Roboz	RS-4982
Graefe Tissue Forceps	Roboz	RS-5158
Harms Forceps x2	Roboz	RS-5097
Intracardiac Needle Holder; Extra Delicate; Carbide Jaws; 7" Length	Roboz	RS-7800
KL 1500 LED Light Source	Leica	150-400
M205A Dissction Microscope	Leica	CH 94-35
Iris Scissors, 11cm, Tungsten Carbide	World Precision Instruments	500216-G
Metal Clip board			Use with the Mouse Retractor Set
Mouse Retractor Set	Kent	SURGI-5001	Need 2 short and 1 tall fixators
Mouse Ventilator MiniVent Type 845, 115 V, Power Supply with US Connector	Harvard Apparatus	73-0043	MiniVent Ventilator for Mice (Model 845), Single Animal, Volume Controlled
Sigma Aldrich	Elastase from porcine pancreas	E0258-50MG	Can be purchased in various size bottles
Small Vessel Cauterizer Kit	Fine Science Tools	18000-00	Recommend using rechargable AA batteries
Spring Scissors, 10.5cm	World Precision Instruments	14127
Steril Swabs (Sponges)	Sugi	31603	Can be cut to size
Surgi Suite Surgical Platform	Kent		Attach to clip board
Tech IV Isoflurane Vap	Jorgensen Laboratories	J0561A	Anesthesia vaporizer