Shunt Surgery, Right Heart Catheterization, and Vascular Morphometry in a Rat Model for Flow-induced Pulmonary Arterial Hypertension

Summary

This protocol describes a surgical procedure to create a model for flow-induced pulmonary arterial hypertension (PAH) in rats and the procedures to analyze the principle hemodynamic and histological end-points in this model.

Abstract

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt (MCT+Flow). The shunt is created by inserting an 18-G needle from the abdominal aorta into the adjacent caval vein. Increased pulmonary flow has been demonstrated as an essential trigger for a severe form of PAH with distinct phases of disease progression, characterized by early medial hypertrophy followed by neointimal lesions and the progressive occlusion of the small pulmonary vessels. To measure the right heart and pulmonary hemodynamics in this model, right heart catheterization is performed by inserting a rigid cannula containing a flexible ball-tip catheter via the right jugular vein into the right ventricle. The catheter is then advanced into the main and the more distal pulmonary arteries. The histopathology of the pulmonary vasculature is assessed qualitatively, by scoring the pre- and intra-acinar vessels on the degree of muscularization and the presence of a neointima, and quantitatively, by measuring the wall thickness, the wall-lumen ratios, and the occlusion score.

Introduction

The goal of this method is to create a reproducible model for severe, flow-induced pulmonary arterial hypertension in rats and to measure its principle hemodynamic and histopathological end points.

Pulmonary arterial hypertension (PAH) is a clinical syndrome that encompasses a progressive increase in pulmonary vascular resistance leading to right ventricular failure and death. Within the superordinate disease spectrum of pulmonary hypertensive diseases (PH), PAH is the most severe form and one that remains without a cure¹. The underlying arteriopathy in PAH is characterized by a typical form of vascular remodeling that occludes the vessel lumen. Muscularization of normal non-muscularized vessels and hypertrophy of the medial vessel layer are regarded as early disease phenomena in PAH, are also seen in other forms of PH², and are thought to be reversible³. As PAH advances, the intimal layer begins to remodel, eventually forming characteristic neointimal lesions². Neointimal-type pulmonary vascular remodeling is exclusive to PAH and is currently regarded to be irreversible⁴.

As PAH is a rare disease, advances in its pathobiological comprehension and development of novel therapies have relied heavily on animal models. The monocrotalin (MCT) model in rats is a simple single hit model that has been, and still is, used frequently. MCT is a toxin that causes injury to the pulmonary arterioles and regional inflammation⁵. 60 mg/kg MCT leads to an increase in the mean pulmonary artery pressure (mPAP), pulmonary vascular resistance (PVR), and right ventricular hypertrophy (RVH) after 3 – 4 weeks⁶. The histomorphology is characterized by isolated medial hypertrophy without neointimal lesions⁵. The MCT rat model thus represents a moderate form of PH, and not PAH, although it is commonly presented as the latter.

In children with PAH associated with a congenital left-to-right shunt (PAH-CHD), increased pulmonary blood flow is regarded as the essential trigger for the development of neointimal lesions^7,8,9. In rats, increased pulmonary blood flow can be induced by the creation of a shunt between the abdominal aorta and the vena cava, a technique first described in 1990¹⁰. Alternatives to create increased pulmonary flow are by unilateral pneumonectomy or by subclavian to pulmonary artery anastomosis¹¹. Conceptual disadvantages of these models consist of potential compensatory growth of the remaining lung and adaptive pathway activation induced by the pneumonectomy, or of iatrogenic injury of the pulmonary vasculature due to pulmonary artery anastomosis, both confounding the effects of increased pulmonary blood flow.

When an aorto-caval shunt is created and increased pulmonary blood flow is induced as a second hit in MCT-treated rats, characteristic neointimal lesions occur, and a severe form of PAH and associated right ventricular failure (RVF) develop 3 weeks after the increased flow¹². The hemodynamic progression of PAH in this model can be assessed in vivo by echocardiography and right heart catheterization. The vascular histomorphology, vessel wall thickness, degree of arteriolar occlusion, and parameters for right ventricular failure form the pillars of the ex vivo characterization of PAH.

This method describes detailed protocols for the aorto-caval shunt (AC-shunt) surgery, right heart catheterization, and qualitative and quantitative assessment of vascular histomorphology.

Protocol

Procedures involving animal subjects have been approved by the Dutch Central Committee for Animal Experiments and the Animal Care Committee at University Medical Center Groningen (NL). Both Wistar and Lewis rats with weights between 180 and 300 g were used. 1. Housing and Acclimatization After arrival at the central animal facility, house rats in groups of 5 per cage. During a 7-day acclimatization period, accustom the rats to human handling, but do not perform any experimental pro…

Representative Results

Representative results are presented in Figure 4. The presented results show characteristics of MCT+FLOW in Lewis rats in the following groups: Control (n = 3), MF8 (n = 5), MF14 (n = 5), MF28 (n = 5), and MF-RVF (n = 10). Statistical analyses were performed using the one-way ANOVA with Bonferroni correction. 60 mg/kg MCT and increased pulmonary blood flow lead to a mean rise in systolic right ventricular pressu…

Discussion

This method describes the surgical procedure of an aorto-caval shunt in rats pre-treated with MCT to create flow-induced PAH and the techniques to assess the principle hemodynamic and histopathological end points that characterize PAH and this model.

Critical Steps within the Protocol and Troubleshooting

Surgery and post-surgery. During the aorto-caval shunt surgery, the most critical step is the dissection of the aorta and vena cava. The membranes that enclose the aor…

Materials

Shunt Surgery
Sterile surgical gloves
Duratears Eye ointment	Alcon	10380
Chloride-Hexidine
Cotton swabs
Histoacryllic tissue glue	B. Braun Medical	1050052
Silkam 5-0 sutures black non-resorbable	B. Braun Medical	F1134027
Safil 4-0 sutures violet resorbable	B. Braun Medical
18 G needle	Luer	NN1838R BD	tip bent in 45 degrees orifice to the outside
Gauzes 10×10 cm	Paul Hartmann	407825
Temgesic Buprenorphine	RB Pharmaceuticals	5429	subcutaneous injection
Sodium Chloride 0.9 %
Ventilation mask Rat
Scalple blade
Biemer clamp 18 mm, 5 mm opening	AgnTho	64-562
Heat mat
Kocher Clamp
Shaving machine
Microscope	Leica
Right Heart Catheterization
Name	Company	Catalog Number	Comments
Sterile surgical gloves
Eye ointment	Duratears
Chloride-Hexidine
Cotton swabs
Gauzes 10×10 cm	Paul Hartmann	407825
Silkam 5-0 sutures black non-resorbable	B. Braun Medical	F1134027
Needle 20 G	Luer		Tip slightly bent to the inside
Cannula 20 G	Luer		to introduce catheter, tip pre-formed in 20 degrees
Silastic Catheter 15 cm long			0.5 mm ball 2 mm from tip
Pressure transducer	Ailtech
Bedside monitor Cardiocap/5	Datex-Ohmeda
Shaving machine
10mL Syringe
Sodium Chloride 0.9 %			for flushing
Vascular Morphology
Name	Company	Catalog Number	Comments
50ml Syringe
4 % Formaldehyde
18 G cannula with tube
Verhoef staining kit	Sigma-Aldrich	HT254	http://www.sigmaaldrich.com/catalog/product/sigma/ht254?lang=en&region=US
Digital slide scanner	Hamamatsu	C9600
Image-J
Elastic (Connective Tissue Stain)	Abcam	ab150667	http://www.abcam.com/elastic-connective-tissue-stain-ab150667.html
			http://www.abcam.com/ps/products/150/ab150667/documents/ab150667-Elastic%20Stain%20Kit%20(website).pdf