Name: murine aortic crush injury: an efficient in vivo model of smooth muscle cell proliferation and endothelial function
Uploaded: 2017-06-11T14:00:00
Description: Watch this Scientific Journal Video about Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function at JoVE.com

We thank Hsia Ru-ching PhD, from the Electron Microscopy Core Facility of University of Maryland School of Medicine, for her technical support in processing the scanning electronic microscopy samples.

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
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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...

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.

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murine aortic crush injury: an efficient in vivo model of smooth muscle cell proliferation and endothelial function

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 p27kip1 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.

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...

Watch this Scientific Journal Video about Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function at JoVE.com

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

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.

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

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell ...

The overall goal of this procedure is to create an animal model that allows the simultaneous assessment of vascular smooth muscle cell proliferation, and reendothelialization. This method can help answer key questions in the field of vascular biology such as what is the mechanism of restenosis after vascular interventions? This technique allows the simultaneous in vivo assessment of vascular smooth muscle cell proliferation and reendothelialization.Both key events that potentiate or attenuate restenosis after vascular interventions. Demonstrating the procedure will be Dr.Dan Yu, a post doctoral fellow from my laboratory. After confirming a lack of response to toe pinch, remove the hair on the ventral abdomen of an anesthetized, 10 to 12-week old male, C57BL/6 mouse from the sternum to the inguinal region.Next disinfect the shoulder area with 70%ethanol and use a 25-gauge needle or smaller to administer an appropriate analgesia. Then place the animal in the supine position on a surgical tray heated by an isothermal pad. Scrub the surgical sight with betadine or chlorhexidine and an alcohol rinse.Repeat both steps two additional times. Apply ointment to the animal's eyes. After making a median abdominal laparotomy incision, reflect the small bowel and duodenum laterally to the right.Using a new cotton-tipped applicator, expose the retroperitoneum and the abdominal aorta from the left renal vein to the aortic bifurcation. Then use another sterile cotton-tipped applicator to deliver 30 consecutive, five-second crushes to the aorta. After the last crush injury has been administered, remove the cotton roll and allow the viscera to return to their native positions.Then use a braided, absorbable suture to close the fascia followed by closure of the skin with a running 6-0, nonabsorbable, monofilament suture and place the animal in a recovery cage with clean bedding with monitoring until full recumbency. To analyze the endothelial integrity of the injured cardiac tissue, at the appropriate experimental time point insert a 21-gauge needle into the left ventricle of the exsanguinated heart. Inject PBS until the effluent from the right atrium is clear then perfuse the animal with five milliliters of 0.3%Evans blue dye at physiological pressure over five minutes followed by five milliliters of fresh PBS.At the end of the PBS infusion, open the abdomen with a midline incision and mobilize the small intestine to the right side of the abdomen, as just demonstrated, to expose the infrarenal aorta. Sharply dissect the aorta from the adjacent tissues and excise the vessel from the left renal vein to the aortic bifurcation for storage in 4%paraformaldehyde. At the end of the fixation, open the aorta longitudinally and pin the tissue to a wax sheet exposing the entirety of the luminal surface for the qualitative assessment of the endothelial integrity by the degree of Evans blue dye.Crush injury induces aortic wall thickening compared to the aortas of animals treated with a sham procedure with the greatest wall thickness observed three days after injury. The injury also induces a rounded appearance in the cells with a corresponding irregular contour of the lumenal surface. EdU incorporation indicates that the increased wall thickening of the injured aorta is at least in part mediated by the increased proliferation of medial vascular smooth muscle cells.Phospho-Erk1/2 expression is observed at over 200%greater than the baseline expression three days after the injury with a decreased expression of p27Kip1 measured at early time points. Both phospho-Erk1/2 and p27Kip1 expression return to baseline levels one month after injury suggesting that the observed thickening of the aortic wall is due also in part to increased medial proliferation. As assessed by scanning electron microscopy, crush injury of the luminal surface of the aorta results in the disruption of the endothelial cells and a partial denudation of the endothelium.Further, damage to the barrier function of the endothelium permits staining of the basement membrane with Evans blue dye while the endothelium of mice treated with a laparotomy alone maintains its barrier function and its white appearance. Once mastered, this technique can be completed in 20 minutes if it's properly performed. While attempting this procedure, it's important to remember to be consistent with the amount of force applied to the aorta and to blind the operating scientist to the experimental condition of the mouse to avoid bias.Don't forget that working with Evans blue dye and paraformaldehyde can be hazardous and that precautions such as wearing gloves should always be taken while using these materials.

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