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.
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.
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 lesions7,8,9. 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.
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
2. Preparation and Injection of Sterile Monocrotalin
3. Aorta-caval Shunt Surgery
4. Development of PAH
NOTE: In this protocol, the animal is euthanized by the extraction of the circulating blood volume while under anesthesia.
5. Right Heart Catheterization
6. Morphology Assessment and Morphometry
NOTE: In this protocol, the animal is euthanized by the extraction of the circulating blood volume while under anesthesia. Rats in any phase of PAH progression and control can be used in this protocol.
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 pressure (sRVP) (23 ± 6 to 56 ± 11 mmHg), systolic pulmonary artery pressure (sPAP) (20 ± 4 to 54.0 ± 10 mmHg), and mean pulmonary artery pressure (mPAP) (16 ± 3 to 36 ± 4 mmHg) at 28 days (MF28). They remain equally high up to the stage when right ventricular failure develops (MF-RVF) (Figure 4). At the early PAH stages (MF8 and MF14), no rise in sRVP, sPAP, and mPAP is observed. Diastolic PAP and right atrial pressure increase in the late phases, but not significantly. Wedge pressures do not change significantly during disease progression.
The right ventricular-to-left ventricular and septal weight ratio increases significantly from MF14 to MF-RVF, indicating right ventricular hypertrophy. The liver's wet-to-dry weight ratio is significantly increased at the MF-RVF stage, indicating liver edema and congestive right ventricular failure.
Muscularization of intra-acinar vessels < 50 µm increases progressively during PAH progression. Vessels of this size normally do not have a muscular medial layer in control rats. At MF14, almost half of these vessels (43 ± 17%) has a total muscular media (as in Figure 3B). At MF28 and MF-RVF, nearly every arteriole is muscularized (98.7 ± 2.5% and 100 ± 0%). Neointimal lesions first occur at MF21, while at MF28 and MF-RVF, around 65% of all arterioles have a neointimal layer (as in Figure 3C). The arteriolar wall-to-lumen ratio and occlusion scores both increase significantly from MF14 to MF28 (respectively, 10.4 ± 3.9 to 71.5 ± 30 (con: 7.1 ± 0.2) and 20.0 ± 2.8 to 54.7 ± 10.6 (con: 12.2 ± 0.3)). The hemodynamic and histomorphological characteristics of PAH progression in MCT+FLOW in Wistar rats are similar14.
Figure 1. Schematic Representation of the Aorto-caval Shunt Surgery. A) The aorta is tensioned and clamped superior to the insertion site. The vena cava is compressed inferior to the insertion site. The needle, bent at 45° and with the orifice to the outside, is inserted into the aorta at a 90° angle. B) The needle is positioned in the aorta with the tip inserted into the vena cava. Please click here to view a larger version of this figure.
Figure 2. Right Heart Catheterization Procedure and Representative Pressure Curves. A) The right jugular vein is tensioned with a ligature and taped onto the ventilation mask. The catheter is placed into the jugular vein. B) A bedside monitor displaying a right ventricular pressure wave. C) The catheter within the cannula placed in the right atrium after introduction into the right jugular vein. Below: a typical right atrial pressure wave. D) The catheter within the cannula placed in the right ventricle. Below: a typical right ventricular pressure wave in end-stage PAH. E) The catheter is advanced in the cannula to enter the main pulmonary artery. Below: a typical pulmonary arterial pressure wave. F) The catheter is advanced into the pulmonary arteries until a wedge pressure wave is displayed on the monitor. Below: a typical pulmonary wedge pressure wave. Please click here to view a larger version of this figure.
Figure 3. Vascular Morphology and Morphometry in Control and PAH Rats. A) A normal, non-muscularized vessel with an occlusion score of 3.7%. B) A totally muscularized arteriole with an occlusion score of 24.3%. C). A neointimal lesion with an occlusion score of 54.1%. D) An excluded vessel (longest/shortest diameter ratio of > 2 and an incomplete circular shape). E) The measurement of the total vessel and luminal area (in a vessel with a schematic representation of a neointimal lesion), including calculations. The bars represent 50 µm. Please click here to view a larger version of this figure.
Figure 4. Representative Results of Pulmonary Hemodynamics and Vascular Morphology/Morphometry. The statistical analyses were performed using the one-way ANOVA with Bonferroni corrections. Values are represented as the mean ± SEM. con: control; MF (monocrotalin + flow); RVF: right ventricular failure; s: systolic; d: diastolic; m: mean; RVP: right ventricular pressure; PAP: pulmonary arterial pressure; RAP: right atrial pressure. RV: right ventricle; LV: left ventricle; IVS: interventricular septum; BW: body weight. Please click here to view a larger version of this figure.
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 aorta and vena cava should be dissected enough to create 1) good visibility of the aortic area, where the needle will be inserted, and of the position of the needle in the vena cava after insertion and 2) sufficient space to clamp the aorta above the insertion site. The same membranes, however, are also used to conduct the aortic blood through the puncture site between both vessels (Figure 1). Dissecting the membranes too much will cause the shunt to leak. Tissue glue may solve the leakage, but it may then also seep into the shunt, compromising its size. When the glue has restricted flow through the shunt or either of the vessels, the glue can be removed gently, but rupture of the vena cava or of the membranes that conduct the shunt may occur. The size or adequacy of the shunt can be estimated by comparing the color difference and the degree of turbulence of the blood in the vena cava during compression and decompression of the proximal aorta with a cotton swab.
An 18-G needle has been shown to create an adequate shunt that results in a consistent and reproducible form of PAH progression in Lewis (this article) and Wistar (see Reference 14) rats and of right ventricular volume overload15. An 18-G needle created the most well-balanced shunt, with significantly increased pulmonary flow on the one hand and a low post-operative complication rate on the other hand.
The most common post-surgical problem is weight loss. Weight loss up to 10% in 1 week occurs in all rats after surgery, presumably due to lower intake the first few days after surgery. Rats are euthanized when the weight loss exceeds 15% in 1 week, as this is considered a sign of being unwell. Liquid chow can improve feeding in the first week after surgery. Rare postoperative complications are hind leg paralysis and bowel ischemia, which also result in euthanasia. In total, less than 5% of the rats had to be euthanized postoperatively.
Catheterization. Critical steps during the catheterization protocol start with the regulation of anesthesia. The depth of the anesthesia should be as minimal as possible (1.5 – 2% isoflurane in this protocol), as an increase in anesthetic depth appears to decrease right ventricular and pulmonary artery pressures, especially in rats with right ventricular failure. Measurements have a tendency to become unreliable when the protocol exceeds 20 min in duration.
The next critical step is the manipulation of the catheter in the RV and in the main pulmonary artery. This can be challenging. Flushing the catheter can help to curve the catheter in the outflow tract when the tip is stuck in the RV's trabeculae. The manipulation itself can cause RV dyskinesia, which shows irregular pressure curves on the bedside monitor. The introduction of the catheter into the right ventricle and the pulmonary artery should run smoothly. When the tip gets stuck at the pulmonary valve, a resistance is felt. Pressing through this resistance may cause the pulmonary valve to rupture, which limits the reliability of subsequent measurements.
In the present protocol, rats are sacrificed after the catheterization procedure. In theory, however, the jugular vein and surgical wound can be closed after the catheter is pulled out, as animals can live with only the remaining left jugular vein.
Morphometry. In the assessment of vascular wall thickness and occlusion scores, the most critical step is to identify the elastic laminae. From experience, the likelihood of success to this end is the greatest with a well-differentiated Verhoeff or Elastica-van Gieson staining. While the lumen can usually be discerned easily from the intima (to measure internal vascular area), distinction of the media from the adventitia may require a closer look (to measure the external vascular area). Some protocols measure intimal and medial thickness separately, defining the intima as the layer between the lumen and the internal elastic lamina, and the media as the layer between the internal and outer elastic lamina. This is usually possible in early-stage MCT+FLOW PAH. However, arterioles in advanced disease, particularly neointimal lesions, may display multiple elastic laminae and often lose the integrity of the elastic laminae (Figure 3C). In advanced lesions, the internal lamina elastica is therefore often difficult to identify, hindering the distinction between the media and the (neo-) intima. This reticulation of the intimal and medial layer prohibits separate layer thickness assessments. Therefore, the use of the total vessel wall thickness (to lumen ratio) and occlusion scores are preferred as an end point, instead of separate intimal and medial thicknesses.
Advantages and Limitations of Adding the Flow as a Trigger
The use of increased pulmonary blood flow to create PAH in rats has several advantages, the most prominent being that it is a known (patho-)physiological trigger for the disease, which favors translation to human PAH-CHD (Eisenmenger physiology), but also to other forms of PAH9. The model allows for the regulation of the flow by varying the size of the needle when creating the AC-shunt.
In human PAH-CHD, closure of the shunt will lead to the reversal of PAH in the early phase of disease, but to progression of PAH in advanced disease stages. Closure of the shunt in vivo would allow one to investigate the effect of removal of the trigger at different time points of disease progression and thus to investigate the mechanisms of (non-)reversal of PAH. Unfortunately, at present, shunt closure is not feasible in the current model. The effects of hemodynamic normalization (e.g., the removal of excess flow and the normalization of pulmonary artery pressure) in rats with flow-associated PAH can be investigated by transplantation of the affected left lung into a recipient rat with normal circulation. It has been shown previously that hemodynamic normalization, in rats by lung transplantation and in human PAH-CHD by closure of a cardiac shunt, leads to the regression of medial hypertrophy in early-stage PAH21. The effects of hemodynamic normalization in the advanced stages of experimental flow-PAH are currently unknown.
Significance with Respect to Alternative Models
The single-hit MCT model. A subcutaneous injection of 60 mg/kg MCT is a simple and effective way to create a model for pulmonary hypertension in rats. MCT induces pulmonary arterial endothelial cell injury, followed by hypertrophy of the muscular layer of the pulmonary arteries5. Although the exact mechanisms remain unclear, various pathways and growth factors have been identified that participate in medial hypertrophy following MCT. Pharmaceutical intervention upon these pathways has often successfully reduced medial hypertrophy and mPAP in MCT-rats. However, since medial hypertrophy is known to have a natural tendency to reverse in humans3 and has also been described to reverse spontaneously in MCT-rats16, the effect of these treatments should be appraised critically.
The double-hit MCT+FLOW model. The addition of increased pulmonary blood flow 7 days after MCT injection critically alters the (vascular) phenotype in a characteristic time-dependent fashion. At MF14 (7 days after the induction of increased flow), normally non-muscularized vessels start to develop a muscular medial layer. At MF21, the medial thickness increases and the first neointimal lesions occur. At MF28, a neointimal layer has developed in the majority of vessels. Between MF28 and MF35, most rats develop right heart failure and die of its sequellae. Previous studies in MCT+Flow rats have shown that the addition of flow to MCT leads to the activation of specific clusters of genes. In some clusters, flow opposed the effects induced by MCT; in others, flow enhanced these effects, and one cluster contained genes that were specifically up-regulated after flow17. One of those flow-specific genes is the early growth response-1 gene14 (Egr-1). Early inhibition of Egr-1 resulted in the attenuation of PAH and neointimal formation in MCT+Flow rats18. Egr-1 was also associated with neointimal remodeling in human PAH (PAH-CHD and idiopathic PAH)19. These observations add to the evidence that increased or disturbed pulmonary blood flow is an essential trigger for neointimal formation.
The single-hit flow-only model. In rats with an aorto-caval shunt without MCT-injection, pulmonary hypertension (mPAP > 25 mmHg) develops between 10 and 20 weeks after shunt induction20. At 20 weeks, the pulmonary vascular histology is dominated by medial hypertrophy of the pre-acinar arteries and neo-muscularization of the intra-acinar arterioles. Although some neointimal lesions have also been described in this model20, the development of these lesions needs to be confirmed and quantified.
The Sugen-Hypoxia model. Another common model for PAH with neointimal lesions is the Sugen5416-Hypoxia (SuHx) rat. Sugen5416 blocks the vascular endothelial growth factor (VEGF) receptor. This induces endothelial cell damage and a signaling cascade that, in combination with hypoxia, evokes endothelial apoptosis and proliferation22. After Sugen5416 injection, the rat is placed in a hypoxic chamber for 4 weeks, upon which PAH develops. The rat is then re-exposed to normoxia for 4 weeks. Pharmacological compounds that target endothelial apoptosis-resistance or the signaling cascades of TGF-B and BMP have shown the potential to reverse the neointimal lesions in this model23,24,25. A new variant of the SuHx model is the Sugen-pneumectomy model, which also results in severe PAH with neointimal lesions26. However, this model has not been fully characterized yet. A novel genetic method to induce PH in rats involves a mutation in the BMP-receptor-2 gene, which results in significant muscularization (PH) but no neointimal formation (PAH)27.
Comparable results have been reported regarding the number of neointimal lesions and the degree of luminal occlusion in the end-stage of untreated SuHx and MCT+Flow rats28. The main differences between both models are 1) that the mPAP in MCT+Flow progressively increases, whereas in SuHx, the mPAP has been shown to decrease gradually after re-exposure to normoxia28; 2) that the MCT+Flow model knows an early disease stage, characterized by medial hypertrophy and endothelial dysfunction; 3) that the time it takes both models to reach an end-stage where right ventricular failure begins to develop (4 weeks in MCT+Flow, 8 weeks in SuHx)28 is different; and 4) that Sugen5416 interferes in a molecular pathway (VEGF) whose role in the pathogenesis of PAH is still unclear. This may hinder the translation to human PAH.
Future Applications or Directions
The distinct disease phases of the MCT+Flow model allow one 1) to test the mechanisms of disease progression (human tissue in general is only available from post-mortem or explant procedures) and 2) to test interventions in different strategies. A preventive strategy could be initiated at the construction of the shunt (MF7). An early intervention can be initiated at MF14. This may be relevant as a treatment strategy prior to shunt closure in children with a congenital cardiac shunt and associated PAH that has progressed into the grey zone between reversible and irreversible disease. Reversal strategies can be initiated at MF21 or MF28. Later stages both show neointimal lesions, a manifestation of end-stage PAH.
In conclusion, the addition of increased pulmonary flow to MCT in rats creates a model of progressive and severe PAH that mimics human disease development. Right heart catheterization and the qualitative and quantitative assessment of the vascular histopathology form the cornerstones of the disease characterization in this and other models for PAH.
The authors have nothing to disclose.
This study was supported by the Netherlands Cardiovascular Research Initiative, the Dutch Heart Foundation, the Dutch Federation of University Medical Centers, the Netherlands Organization for Health Research and Development, and the Royal Netherlands Academy of Sciences (CVON nr. 2012-08, PHAEDRA, The Sebald fund, Stichting Hartekind).
Shunt Surgery |
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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 |
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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 |
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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®ion=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 |