Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Summary

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo.

Abstract

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27^kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.

Introduction

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. All revascularization procedures are plagued by restenosis. Present strategies to prevent restenosis (drug-eluting stents and drug-coated balloons) inhibit both vascular smooth muscle cell (VSMC) and endothelial cell proliferation (EC). Consequently, these interventions prevent VSMC mediated restenosis, but also prevent the regeneration of the endothelium. Without an intact endothelium, patients are required to be on potent antiplatelet agents to decrease the risk of in situ thrombosis at the risk of bleeding complications. An ideal therapy would inhibit VSMC proliferation while promoting regeneration of the endothelium. Thus, there is a need to simultaneously study VSMC proliferation and endothelial barrier function in vivo.

Presently, there are several mouse models of restenosis ¹. These models include carotid ligation and femoral artery wire injury ². Aortic models include stent placement ³, balloon injury ⁴, and aortic allograft ⁵. All of the present models are limited. Carotid ligation generates a flow-mediated neointimal lesion and does not have endothelial injury. Additionally, both carotid and femoral arteries have many fold fewer cell layers than human vessels, limiting their translational value. The mouse aorta which is approximately 1.3 mm in diameter, is the only vessel that approximates a clinically relevant (coronary) human artery (3).

Despite the translational potential of murine aortic models of disease, current models have limitations. These models require advanced microsurgical skills and specialized equipment such as angioplasty balloons and stents. Herein, we present a novel, reproducible technique to simultaneously induce VSMC proliferation and disrupt endothelial barrier function.

Protocol

Ethics Statement: The protocols for animal handling were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Maryland (protocol number 0416009) and conducted according to AAALAC-International standards.

1. Surgical Procedure

1. Anesthetic Technique
   1. Sterilize all instruments used in survival surgery with steam sterilization at 121 °C for 30 min.
   2. Induce anesthesia via an induction tank with 100% O₂ and 2.5% isoflurane delivered via precision vaporizer. Post-induction, discontinue the isoflurane and flush the chamber with O₂. Maintain anesthesia with 1.5-2% isoflurane via face mask and 1 L/min O₂ by inhalation.
   3. Attach both the induction chamber and the face mask to a charcoal scavenger for waste gas adsorption to protect personnel. Ensure an adequate anesthetic plane by demonstrating that there is no response to noxious stimuli (toe pinch).
   4. Create an operative field consisting of a surgical tray with isothermal pad to provide thermal support during surgery. An additional isothermal pad will provide thermal support to animals in their recovery cage.
2. Animal Preparation
   1. Carry out the following investigation on 10-12 weeks old male C57BL/6 mice.
   2. Remove the hair on the ventral abdominal surface of the animal from the sternum to the inguinal region with a depilatory agent or an electrical clipper with a number 40 blade.
      1. In case of depilatory agent, apply this compound to the surgical area for 2-3 min and then remove it with cotton. We use a commercially available compound of calcium hydroxide and sodium hydroxide.
   3. Prep the area over the shoulder with 70% alcohol and subcutaneously inject carprofen (5 mg/kg) with a 25 gauge or smaller bore needle. This treatment will provide postoperative analgesia for the animal.
   4. Transfer the animal to the surgical field and position in dorsal recumbency.
   5. Prepare the surgical site by scrubbing 8-12% providone-iodine with a clean cotton applicator or cotton gauze. Then rinse the skin twice with 70% alcohol.
   6. Place ocular lubricant in both eyes to reduce the incidence of corneal desiccation. Cover the surgical site with a sterile drape.
3. Operative Technique
   1. Make a median abdominal laparotomy incision approximately 2-2.5 cm in length with a scalpel beginning immediately caudal the xiphoid process and extending towards the pelvis.
   2. Mobilize the small bowel and duodenum and reflect laterally to the right. Roll up a strip of sterile cotton gauzed and soaked with sterile saline for injection to allow packing of the viscera to improve exposure.
   3. With the small intestine mobilized to the right side of the abdomen, expose the retroperitoneum and expose the abdominal aorta from the left renal vein to the aortic bifurcation (Figure 1).
   4. With a sterile cotton-tipped applicator, deliver 30 consecutive crushes, each five seconds in duration.
   5. Remove the packing and allow the viscera to return to their native position.
   6. Close fascia with a running 4-0 absorbable monofilament suture (polydioxanone). Skin is closed with a running 6-0 non-absorbable monofilament suture (nylon).
4. Recovery and Post-Procedure Care
   1. After the procedure, place the animal in a recovery cage with clean bedding on an isothermal pad to continue thermal support until the animal is able to ambulate normally. Do not leave the animal unattended until it demonstrates the ability to maintain sternal recumbency.
   2. Monitor the animal every hour for the first 4 h after surgery. Once the animal is ambulating normally return it to the assigned husbandry room. The animal will not be housed in the company of another animal until it has fully recovered.
   3. Monitor the animal twice daily for the first 72 h after surgery and at least 3 times a week thereafter. Monitoring includes weighing the animal three times a week.
   4. Administer carpofen (5 mg/kg) subcutaneously twice daily for the first 72 h after surgery.

2. Procurement of Tissue

1. Method of Euthanasia
   1. Euthanize the animals at predetermined time points.
   2. Induce anesthesia via an induction tank with 100% O₂ and 2.5% isoflurane delivered via precision vaporizer.
      1. To study the integrity of the endothelium, Administer Evans Blue dye to the animal.
         NOTE: Evans Blue is a negatively charged azo dye with a high binding affinity for albumin and can only stain blood vessels in the absence of an intact endothelium⁶.
      2. For endothelial integrity studies, at the time of euthanasia, perfuse the animals with 5 mL of 0.3 % Evans Blue dye followed by 5 mL PBS at physiological pressure for 5 min. The animal is maintained in a surgical plane of anesthesia during the perfusion procedure.
   3. After achieving a deep anesthetic plane, open the chest with a sternotomy. Make a laceration in the right atrium to allow drainage of blood from the animal and access the left ventricle is accessed with a 21 G needle and inject phosphate buffered saline (PBS) until the effluent from the right atrium is clear.
   4. After perfusion with PBS, enter the abdomen through a midline incision.
   5. Once again, mobilize the small intestine to the right side of the abdomen exposing the infrarenal aorta, sharply dissect the aorta from adjacent tissues and excise it from the left renal vein to the aortic bifurcation.
   6. Store the excised aorta in a 4% paraformaldehyde solution until tissue processing occurs.
      1. For endothelial integrity studies, open the aorta longitudinally and pin it to a wax sheet exposing the entirety of the luminal surface⁶. Carry out a qualitative assessment of endothelial integrity by the degree of staining with Evans Blue dye⁶.
      2. For other histologic assays, section the aorta transversely and embed it in optimal cutting temperature (OCT) compounds as dictated by the histological method to be used ⁷.

Results

Transverse sections aorta embedded in OCT were sectioned, and stained with hematoxylin and eosin then counter stained with Verhoeff-Van Gieson (VVG) stain to identify the internal and external elastic lamina ⁷. Crush injury induced aortic wall thickening compared to the aortas of animals treated with a sham procedure (laparotomy and small bowel mobilization alone). Wall thickness, as assessed by the distance from adventitia to the lumen, was greatest three days aft...

Discussion

We have characterized the effects of a murine aortic injury model that results in medial hyperplasia and endothelial barrier dysfunction. Partial EC detachment along the aorta intima accompanied the loss of cell-cell contact and enhancement of cell protrusions. Correspondingly, endothelial barrier function was significantly impaired, which stimulated the mitogen-sensitive signaling pathways, leading to proliferation of VSMCs and thickening of the vessel wall. The strengths of this model is that it is technically easier t...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ocular lubricant	Dechra	17033-211-38	Pharmaceutical agents
Isoflurane	VetOne	502017	Pharmaceutical agents
Carprofen	Zoetis	26357	Pharmaceutical agents
Precision vaporizer	Summit Medical	10675	Surgical supplies
Charcoal scavenger	Bickford Inc.	80120	Surgical supplies
Isothermal pad	Harvard Apparatus	50-7053-R	Surgical supplies
Sterile cotton-tipped applicator	Fisher Scientific	23-400-124	Surgical supplies
4-0 absorbable monofilament suture	Ethicon, Inc	J310	Surgical supplies
5-0 non-absorbable monofilament suture	Ethicon,Inc	1666	Surgical supplies
21-gauge x 1 inch needle	BD Biosciences	305165	Surgical supplies
25-gauge x 1 inch  needle	BD Biosciences	305125	Surgical supplies
Dry sterilizer	Cellpoint	7770	Surgical supplies
Fine scissors	Fine Science Tools	14058-09	Surgical instruments
Adson forceps	Fine Science Tools	11006-12	Surgical instruments
Dumont #5 fine forceps	Fine Science Tools	11254-20	Surgical instruments
Vannas Spring Scissors 3 mm cutting edge	Fine Science Tools	15000-00	Surgical instruments
Needle driver	Fine Science Tools	91201-13	Surgical instruments
Scalpel handle #4	Fine Science Tools	10004-13	Surgical instruments
Scalpel blades #10	Fine Science Tools	10010-00	Surgical instruments
PBS	Lonza	17-516F	Reagents for tissue processing
Evans Blue	Sigma-Aldrich	E2129	Reagents for tissue processing
Paraformaldehyde	Sigma-Aldrich	P6148	Reagents for tissue processing
Modeling wax	Bego	40001	Reagents for tissue processing
OCT compound	Tissue-Tek Sakura	4583	Reagents for tissue processing
Mayer's hematoxylin solution	Sigma-Aldrich	MHS16	Reagents for immunohistological analysis
Eosin Y solution alcoholic	Sigma-Aldrich	HT110316	Reagents for immunohistological analysis
Elastin stain kit	Sigma-Aldrich	HT25A	Reagents for immunohistological analysis
Click-it Edu Alexa-488 Imaging Kit	Invitrogen	C10337	Reagents for immunohistological analysis
Anti-Erk1/2 antibody	Cell Signaling Technology	4695	Reagents for immunohistological analysis
Anti-phospho-Erk1/2 antibody	Cell Signaling Technology	4370	Reagents for immunohistological analysis
Anti-p27kip1 antibody	Cell Signaling Technology	3698	Reagents for immunohistological analysis
Trichloroacetic acid	Sigma-Aldrich	T9159	Reagents for immunohistological analysis