Asian Cardiovasc Thorac Ann 2006;14:183-188
© 2006 Asia Publishing EXchange Ltd
Experimental Study of Effect of Fontan Circuit on Pulmonary Microcirculation
Zongtao Yin, MD,
Zengwei Wang, MD,
Hongyu Zhu, MD,
Renfu Zhang, MD,
Huishan Wang, MD,
Xinmin Li, MD
Department of Cardiovascular Surgery, Shenyang Northern Hospital, Shenyang, China
For reprint information contact: Zongtao Yin, MD Tel: 86 133 0988 1423 Fax: 86 24 2391 2376 Email: yzt1210{at}hotmail.com, Department of Cardiovascular Surgery, Shenyang Northern Hospital, 83 Wenhua Road, Shenhe District, Shenyang 110016, China.
 |
ABSTRACT
|
|---|
Pulsatile pulmonary blood flow plays an important role in regulating shear-stress-mediated release of endothelium-derived nitric oxide and endothelin-1, and it reduces pulmonary vascular resistance by passive capillary recruitment. The aim of this study was to demonstrate changes in pulmonary capillary structure and endothelial function induced by the chronic nonpulsatile flow of the Fontan circulation. A canine model with nonpulsatile flow in the right lung was established, and sacrificed 3 months later. Compared to the left lung, wall thickness of the pulmonary arterioles was thinner, endothelin-1 expression was weaker, endothelial nitric oxide synthase activity was stronger, and there was a good correlation between the histomorphometric and immunohistochemical findings. These data indicate that long-term nonpulsatile flow can lead to endothelial dysfunction, which may be involved in distention and vascular structure remodeling due to the increase in pulmonary vascular resistance; but it also can lead to increased patency of the arteriovenous shunt, which might be at least partly involved in pulmonary arteriovenous fistula development and exercise limitation after the Fontan operation.
|
INTRODUCTION
|
|---|
The Fontan operation benefits patients with a single functioning ventricle and low pulmonary resistance. However, even perfect Fontan operations cannot completely avoid complications related to elevated systemic venous pressure, such as hypoxia, exercise intolerance, and refractory hydrothorax, which are common during the long-term follow-up.1 Several disadvantages of nonpulsatile blood flow after a bidirectional cavopulmonary shunt have been reported, and experimental studies have shown that it can markedly elevate pulmonary vascular resistance.2,3 Vascular endothelial cells play a crucial role in local regulation of pulmonary vascular tone and smooth muscle cell function by releasing a variety of bioactive substances. Among these, nitric oxide is a potent endothelium-derived vasodilator that prevents the proliferation of smooth muscle cells, and endothelin-1 (ET-1) is a potent endothelium-derived peptide with vasoconstrictive and mitogenic properties. In patients with pulmonary hypertension, the expression of endothelial nitric oxide synthase (eNOS) has been shown to decrease, but that of ET-1 increases.4,5 Thus, we postulated that disordered pulmonary microcirculation and endothelial dysfunction might be involved in the outcome of Fontan operations. This study on dogs was undertaken to test this hypothesis.
|
MATERIALS AND METHODS
|
|---|
The animals were quarantined for 2 weeks before the experiment. All received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Science and published by the National Institutes of Health (NIH Publication 86–23, revised 1985). Twenty-two adult mongrel dogs weighing 15–20 kg were anesthetized with intravenous pentobarbital sodium (30 mg·kg–1), intubated with a cuffed endotracheal tube, and mechanically ventilated with a volume-cycled ventilator on room air (tidal volume 10 mL·kg–1, respiration rate 15–20 breaths·min–1). The fraction of inspired oxygen was kept at 0.4 with 5 cm H2O positive end-expiratory pressure. An adequate level of anesthesia was maintained by intermittent infusion of 1 to 2 mg pentobarbital sodium. A thoracotomy was performed in the right 4th intercostal space. The superior vena cava (SVC), azygous vein, and the right pulmonary artery were widely dissected, and the azygous vein was ligated. After systemic heparinization (1 mg·kg–1), venous cannulation was performed with 28F venous cannulas between the SVC and the right atrial appendage. The venous cannulas were secured with ligatures to ensure diversion of blood through the conduit. After the proximal SVC was transected and the proximal part of the right atrial incision was closed, 2 crossclamps were placed on the proximal and distal parts of the right pulmonary artery. The SVC was anastomosed end-to-side to the right pulmonary artery, followed by release of the clamp and ligation of the proximal right pulmonary artery. Protamine was used to neutralize heparin. The right thoracotomy was closed with a drainage tube. The endotracheal tubes were removed 3–5 hr after the operation, and the drainage tubes were withdrawn 24–48 hr later. Intramuscular injection of pethidine (30 mg) was administered intermittently in the early postoperative period to relieve pain. Thus, a novel animal model was established without the use of cardiopulmonary bypass. In this model, blood from the SVC directly entered the right lung in a nonpulsatile fashion, and blood from the inferior vena cava perfused the left lung with pulsatile flow via the right ventricle.
Three months after the operation, the animals were anesthetized and after a median sternotomy, pressures were measured in the right and left pulmonary arteries and veins; blood samples were obtained from the SVC, inferior vena cava, right and left pulmonary veins; and biopsy specimens were taken from the left and right lungs. Serial paraffin-embedded sections (4 µm thick) were processed with hematoxylin and eosin stain, Perls stain for iron, and modified orcein stain for elastic fibers. In each biopsy specimen, the pulmonary vascular structure was analyzed by quantitative morphometric techniques with UTHSCSA Image Tool 3.0 (University of Texas Health Science Center at San Antonio, TX, USA). The muscularity of the pulmonary arteriole (accompanying alveolar ducts) was assessed by determining the wall thickness (calculated as a percentage of arteriolar external diameter) and wall area (calculated as a percentage of arteriolar area) of 20 distal arteries.
Serial paraffin-embedded sections of lung tissue were stained with human antibody to ET-1 and eNOS. All slides were stained and developed at the same time to avoid variations. Immunohistochemical staining was performed strictly according to the manufacturer’s instructions. Tissue sections were deparaffinized in dimethyl benzene, rehydrated through gradient concentrations of ethanol and water, and heated for 40 min in citrate buffer at pH 6. Slides were incubated in hydrogen peroxide to block endogenous peroxidase activity, rinsed in Tris-buffered saline solution, and incubated for 1 hr with a primary antibody: anti-ET-1 (Alexis) and anti-eNOS (NeoMarkers). Sections were incubated with a biotinylated secondary antibody for 15 min and stained with peroxidase-labeled streptavidin. Finally, slides were counterstained with Harris hematoxylin. A positive result was defined as brown cytoplasmic staining. Staining intensity was measured as the integrated total optical density and average optical density with a MetaMorph DP10/BX41 image analysis system (Universal Imaging Corp., Downingtown, PA, USA).
All data are presented as mean ± standard deviation. Statistical analyses were performed using Student’s t test for two-group comparisons and linear regression analysis to determine the association of histomorphometric and immunohistochemical data. Statistical significance was defined as p < 0.05. SPSS version 10.0 software (SPSS, Inc., Chicago, IL, USA) was used for statistical analyses.
|
RESULTS
|
|---|
There were 22 dogs used in the study, one died of incision infection and another died of anastomotic stoma obstruction. The mortality rate was 9%. Results were obtained from the other 20 animals. As shown in Table 1
and Figure 1, the left lung exhibited normal structure of the pulmonary arterioles (accompanying alveolar ducts); wall thickness was 14.96% ± 13.1% and wall area was 47.8% ± 12.2%. In contrast, wall thickness of the pulmonary arterioles in the right lung was 13.64% ± 12.8% (t = 2.12; p < 0.05); and wall area was 46.4% ± 11.7% (t = 2.14; p < 0.05). No significant tissue edema (endothelium and stroma) was present in either lung. ET-1 activity in the pulmonary distal arteries of the right lung was weaker than that of the left lung (t = 2.32; p < 0.05; Table 2, Figure 2). Endothelin-1 was mainly expressed in endothelial cells and smooth muscle cells of the muscular pulmonary arteries. There was a good correlation between the histomorphometric and immunohistochemical result (r = 0.87; p < 0.05). In contrast to the left lung, eNOS activity in the pulmonary distal arteries of the right lung increased markedly (t = 2.24; p < 0.05). The eNOS was mainly expressed in endothelial cells of the distal muscular pulmonary arteries and capillaries. There was a good correlation between the histomorphometric and immunohistochemical results (r = –0.86; p < 0.05; Table 3, Figure 3). The pressure gradient from the pulmonary artery to the pulmonary vein in the right lung was higher than that of the left lung (t = 2.18; p < 0.05), and the PO2 gradient in the right lung was lower than that of the left lung (t = 2.24; p < 0.0; Table 4).
View larger version (67K):
[in this window]
[in a new window]
|
Figure 1. Elastic fiber staining of lung biopsy specimen, original magnification for all panels x 400, (A) Pulmonary arteriole in the right lung, (B) Pulmonary arteriole in the left lung.
|
|
View larger version (72K):
[in this window]
[in a new window]
|
Figure 3. Endothelial nitric oxide synthase expression in a pulmonary arteriole: (A) right lung, (B) left lung.
|
|
|
DISCUSSION
|
|---|
The Fontan operation was proposed to separate the systemic venous and arterial circulation by direct anastomosis between the vena cava and the pulmonary arteries. It has been applied for correction of complex cardiac lesions for which biventricular repair is impossible. The Fontan circulation is unique in exposing the pulmonary vascular bed to nonpulsatile blood flow. In the absence of force from the ventricle, the pulmonary circulation is driven by the elevated vena caval pressure. In contrast to patients with biventricular physiology, those with univentricular physiology have less hemodynamic reserve. Small increases in pulmonary vascular resistance can result in decreased cardiac output. Therefore, it is critical to lower pulmonary vascular resistance for optimum functioning of the Fontan circulation.
It is well known that endothelial cells play an important role in the regulation of pulmonary vascular tone. The endothelium is not only a single cell layer of mechanical barrier between the blood and pulmonary vessel wall but it also regulates various important vascular functions such as vasomotion and blood flow. Endothelial cells can release a variety of substances such as ET-1 and NO to control vascular tone. Endogenous ET-1 of the smooth muscle cell induces vascular constriction via endothelin-A receptors.6 Nitric oxide produced by eNOS diffuses to adjacent smooth muscle to induce vascular relaxation. Normally these factors act in a coordinated fashion so that the vasodilator and vasoconstrictor are locally balanced to regulate the resistance of the vascular bed for steady pulmonary perfusion.7 Several disadvantages of nonpulsatile or decreased pulsation of pulmonary flow after the bidirectional cavopulmonary shunt have been reported, as well as the importance of pulsatile flow in both global and microvascular pulmonary circulation.2,8 However, there are few reports on the sequelae of pulmonary microcirculation changes caused by chronic nonpulsatile pulmonary flow after the Fontan operation.
It has been demonstrated that capillary recruitment varies directly with pressure, but not with flow. Capillary recruitment increases during pulsatile flow, and most of the capillaries recruited by the systolic pulse are perfused throughout the pulsatile cycle. Nonpulsatile flow can increase pulmonary vascular resistance by 30%, which is closely related to the disordered microcirculation. Capillary recruitment caused by nonpulsatile flow reduces the cross-sectional area of the pulmonary vascular bed, which is responsible for the increased pulmonary vascular resistance and decreased gas-exchange surface area.8–10 In our study, the pressure and the PO2 gradient between the pulmonary artery and the pulmonary vein in the right lung decreased more than that of the left lung, confirming that chronic nonpulsatile flow can increase pulmonary resistance.
Recent in vitro studies indicate that the different flow patterns result in alterations of fluid shear stress and subsequently modulate expression of some vasoactive factors.11 The pulsatility of blood flow may significantly influence both endothelial morphology and function through the molecular responses to shear stress.12 We postulated that endothelial dysfunction might be at least partly involved in the sequelae of nonpulsatile flow after the Fontan operation. Our results show that the expression of endothelial vasoactive factors changed greatly in lungs with severely attenuated pulsatile flow, and that these changes were quite different to those previously reported in pulmonary hypertension.13,14 Immunoreactivity was weaker for ET-1 but stronger for eNOS in the right lung than the left. We speculate that the decrease in shear stress due to nonpulsatile pulmonary flow might lead to endothelial dysfunction of the capillary vascular bed, and furthermore, it might be related to the up-regulation of the NO pathway and down-regulation of the ET-1 pathway, which are responsible for dilating pulmonary capillaries and lowering pulmonary resistance to obtain more pulmonary blood perfusion.
Endothelial dysfunction plays an integral role in mediating structural changes in the pulmonary vasculature. Disordered endothelial cell proliferation along with concurrent neoangiogenesis, when exuberant, result in the formation of a glomeruloid structure known as the plexiform lesion, which is a common pathological feature of pulmonary arterial hypertension.4,5 Endothelin-1 and eNOS can affect the growth of smooth muscle cells; the alteration in their production may be involved in the pathogenesis of pulmonary vascular atrophy and structural remodeling. In this study, we observed that the pulmonary arteriolar wall became thinner in the right lung, confirming that endothelial dysfunction with chronic nonpulsatile flow can lead to pulmonary vascular structural remodeling.
The typical pulmonary microcirculation consists of arterioles, metarterioles, precapillary sphincters, capillaries, thoroughfare channels, arteriovenous shunts, and venules. The arteriole, metarteriole and precapillary sphincter, which are enclosed by smooth muscle cells, control the pulmonary vascular tone. We believe that decreased ET-1 expression and increased eNOS expression may contribute to the dilation of pre-resistant capillaries of the pulmonary microcirculation. This helps to decrease the pulmonary vascular resistance, enhance blood flow, and increase blood volume returning to the left heart. On the other hand, besides the nonpulsatile-flow-induced capillary recruitment of pulmonary alveoli, dilation of pre-resistant vessels can result in more blood transiting from the arteriovenous shunt and the thoroughfare channel, which can increase intrapulmonary right-to-left shunting and lead to hypoxemia. It has also been confirmed in our study that the PO2 decreased significantly in the right lung compared to the left. The long-term patency of the arteriovenous shunt and microvascular structure remodeling may lead to a pulmonary arteriovenous fistula. It should be noted that the results of our study revealed decreases of 1.2% in wall thickness and 1.3% in wall area, which contradicts the pressure gradient increase of 1.3 mm Hg and PO2 gradient decrease of 3 mm Hg in the right pulmonary artery. Our explanation for this is that capillary dilation cannot compensate totally for the recruitment of capillaries in a Fontan circuit, even though it can help to increase pulmonary perfusion.
|
REFERENCES
|
|---|
- Fontan F, Kirklin JW, Fernandez G, Costa F, Naftel DC, Tritto F, et al. Outcome after a "perfect" Fontan operation. Circulation 1990;81:1520–36.[Abstract/Free Full Text]
- Brandes H, Albes JM, Conzelmann A, Wehrmann M, Ziemer G. Comparison of pulsatile and nonpulsatile perfusion of the lung in an extracorporeal large animal model. Eur Surg Res 2002;34:321–9.[Medline]
- Kurotobi S, Sano T, Kogaki S, Matsushita T, Miwatani T, Takeuchi M, et al. Bidirectional cavopulmonary shunt with right ventricular outflow patency: the impact of pulsatility on pulmonary endothelial function. J Thorac Cardiovasc Surg 2001;121:1161–8.[Abstract/Free Full Text]
- Moncada S, Palmer RM, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 1991;43:109–42.[Medline]
- Haynes WG, Webb DJ. Contribution of endogenous generation of endothelin-1 to basal vascular tone. Lancet 1994;344:852–4.[Medline]
- Yang Z, Krasnici N, Luscher TF. Endothelin-1 potentiates human smooth muscle cell growth to PDGF: effects of ETA and ETB receptor blockade. Circulation 1999;100:5–8.[Abstract/Free Full Text]
- Mombouli JV, Vanhoutte PM. Endothelial dysfunction: from physiology to therapy. J Mol Cell Cardiol 1999;31:61–74.[Medline]
- Presson RG Jr, Baumgartner WA Jr, Peterson AJ, Glenny RW, Wagner WW. Pulmonary capillaries are recruited during pulsatile flow. J Appl Physiol 2002;92:1183–90.[Abstract/Free Full Text]
- Johnson EH, Bennett SH, Goetzman BW. The influence of pulsatile perfusion on the vascular properties of the newborn lamb lung. Pediatr Res 1992;31(4 Pt 1):349–53.[Medline]
- Raj JU, Kaapa P, Anderson J. Effect of pulsatile flow on microvascular resistance in adult rabbit lungs. J Appl Physiol 1992;72:73–81.[Abstract/Free Full Text]
- Noris M, Morigi M, Donadelli R, Aiello S, Foppolo M, Todeschini M, et al. Nitric oxide synthesis by cultured endothelial cells is modulated by flow conditions. Circ Res 1995;76:536–43.[Abstract/Free Full Text]
- Rubanyi GM, Romero JC, Vanhoutte PM. Flow-induced release of endothelium-derived relaxing factor. Am J Physiol 1986;250(6 Pt 2):H1145–9.
- Giaid A, Yanagisawa M, Langleben D, Michel RP, Levy R, Shennib H, et al. Expression of endothelin-1 in the lungs of patients with pulmonary hypertension. N Engl J Med 1993;328:1732–9.[Abstract/Free Full Text]
- Giaid A, Saleh D. Reduced expression of endothelial nitric oxide synthase in the lungs of patients with pulmonary hypertension. N Engl J Med 1995;333:214–21.[Abstract/Free Full Text]
This article has been cited by other articles:
|
|
|
|
 
Z. Yin, C. Wang, H. Zhu, R. Zhang, H. Wang, and X. Li
Exercise Tolerance in Extracardiac Total Cavopulmonary Connection
Asian Cardiovasc Thorac Ann,
January 1, 2009;
17(1):
39 - 44.
[Abstract]
[Full Text]
[PDF]
|
|
|
TOP
ABSTRACT
INTRODUCTION
