Plasma nitric oxide and left ventricular function in rabbits after cardiac lymphatic obstruction

Th is study was designed to investigate the eff ect of cardiac lymphatic obstruction on plasma nitric oxide (NO) and left ventricular function. Th e plasma NO was measured in study group (n=) and control group rabbits (n=) before, and , , ,  and  days after the obstruction of cardiac lymphatic vessels. Left ventricular ejection fraction was measured with echocardiography. Th ere was a signifi cant reduction in the left ventricular ejection fraction following the lymphatic obstruction (.±. vs. .±., p<.). Plasma NO in the control group remained unchanged during the observation period (.±. vs. .±. μmol/L, p>.). In the study group, there was a small but signifi cant increase in the plasma NO on day ,  and  following the lymphatic obstruction (.±. vs. .±. μmol/L, p<.). Th e plasma NO returned to the baseline levels on day  but reduced to .±. μmol/L on  days after the lymphatic obstruction (p<.). In conclusion, cardiac lymphatic obstruction was associated with a signifi cant reduction in left ventricular function. It was also associated with an increase in the plasma NO in the fi rst  weeks but there was a signifi cant reduction in the NO levels three months after the lymphatic obstruction. ©  Association of Basic Medical Sciences of FBIH. All rights reserved


Introduction
Cardiac transplantation is a life-saving treatment for patients with end-stage heart failure.However, the outcomes of this treatment are limited by acute allograft rejection and chronic allograft coronary artery disease or allograft vasculopathy [, ].Endothelium-derived nitric oxide (NO) is a potent endogenous vasodilator, inducing vasodilation by stimulating soluble guanylate cyclase to produce cGMP [].Nitric oxide has been found to play a critical role in the acute allograft rejection, as well as in the development of allograft vasculopathy.Th e expression of the inducible isoform of nitric oxide synthase (NOS-) is upregulated during cardiac allograft rejection in endothelial cells, in vascular smooth muscle cells, and in cardiac myocytes [, ].Increased synthesis of nitric oxide by inducible nitric oxide synthase soon after the heart transplantation may contribute acute allograft rejection by inducing myocyte necrosis and ventricular failure [, ].After heart transplantation, the impairment of nitric oxide synthesis from the endothelium and myocytes is considered a major contributing factor for allograft vasculopathy [].
Lymphatic vessels are an important source of nitric oxide biosynthesis [-].Nitric oxide is released from lymphatic smooth muscle cells and the vascular endothelium after interacting with various vasoactive substances [, ].Cardiac lymphatic flow plays a pivotal role in maintaining the homeostasis by draining the myocardial interstitial fl uid and proteins back to the circulation.However, the lymphatic fl ow is often interrupted after heart transplantation, because the lymphatic vessels of the donor heart are not routinely connected with the recipient's during the surgical procedure.To date, there is little knowledge about the impact of the lymphatic fl ow interruption on the biosynthesis of nitric oxide, which may have signifi cant eff ect on the prevalence or severity of acute or chronic allograft rejection following heart transplantation.Th e primary purpose of this study was to investigate the plasma level of nitric oxide in a rabbit model before and after the lymphatic fl ow obstruction.

Surgical preparation of the animal model
Th e study was approved by the Institutional Review Board.Th irty-three New Zealand white rabbits of both sexes (body weight .-.kg, aged - months) were assigned to the study (lymphatic obstruction) group (n=,  males) and control group (n=,  males).Animals in the control group underwent the same open-chest surgery as the lymphatic obstruction group, but without the ligation of the lymphatic vessels.Under general anesthesia, left thoractomy was performed in the fourth intercostal space and the heart was suspended in a pericardial cradle.In the study group, . ml of  methylene blue was injected into the wall of the left and right ventricular apex, clearly marking the lymphatic vessels in the epicardium and the adjacent lymph nodes.Animals in the control group underwent the same open-chest surgery as the lymphatic obstruction group, but without the ligation of the lymphatic vessels.The main epicardial lymphatic vessels, as well as the large lymph nodes between the aortic root and the pulmonary artery, and between the posterior aorta and the right superior pulmonary artery were destroyed [].Th e chest was then closed by layers and the animals were returned to the animal house for standard care by the investigators and the curators.Penicillin (average . million units) was administered intramuscularly daily for a week after the surgery to prevent wound infection.

Measurement of plasma nitric oxide
Venous blood was obtained before, and at , , ,  and  days following the surgery.The plasma was collected from each sample by centrifuge at  rpm for min at ° C and stored at −°C until detection.Th awed samples ( μL), were diluted fourfold with deionized water and deproteinated by zinc sulfate.They were centrifuged at  rpm for  min at room temperature and  μL supernatant was injected into a chemiluminescence machine (Sievers  NO Analyzer, Sievers Instruments, Inc., Boulder, CO), for the measurement of plasma nitric oxide.

Assessment of left ventricular function
Left ventricular ejection fraction (EF) was measured by echocardiography (HP SONOC-, Agilent Technologies, MA, USA) before and after operation.

Statistical analysis
Data was expressed as mean ± standard deviation.Numerical data were analyzed by one-way ANOVA.Categorical data were examined by Chi-square test.p <. was considered statistically signifi cant.

Results
All animals survived the initial operation and completed the -day study.Pericardial fluid of - ml (between the left ventricular posterior wall and the pericardium) was detected by echocardiography in . (/) of the study and . (/) of the control group animals three days following the surgery (p <.).There was a gradual reduction in preva-lence of pericardial fl uid and at the end of week , none of the control or study group animals had pericardial fluid.Table  shows the average values of the left ventricular ejection fraction before and after operation.Th ere was no significant difference in the ejection fraction between the study and control groups before the surgery (p >.).In the control group, average ejection fraction remained unchanged following operation.Following operation, there was a significant reduction in the left ventricular ejection fraction in the study group between day  and .
As shown in Table , the average value of nitric oxide was similar between the control and the study group before the surgery.In the control group the average nitric oxide values remained unchanged following the sham surgery (p >.).
In the study group, the average plasma nitric oxide on day ,  and  were higher than the baseline value (p<.).The average nitric oxide at these time points were also higher than in the control group (p <.).However, in the study group, the average nitric oxide on day  was similar to the baseline value, or the average value of the control group (p >.).However, the plasma nitric oxide on day  was lower than the baseline value (p<.) and the nitric oxide in the control group (p<.).

Discussion
Previous studies have found that ligation of large cardiac lymphatic vessels, together with the destruction of the large cardiac lymph nodes, caused significant reduction or oc-   Data are expressed as mean ± SD *p <0.01 compared with the baseline value in the same group clusion in the cardiac lymph fl ow [, ].Th e lymph fl ow obstruction impairs the drainage of interstitial fluid and proteins in the myocardium, resulting in significant myocardial edema and fibrosis within the first four weeks of operation [].Cardiac lymphatic obstruction is also associated with a reduction in the left ventricular ejection fraction, compromising ventricular muscle contractility [].
It has been proposed that the interruption of the cardiac lymphatic fl ow may play an important role in the pathogenesis of the heart failure following heart transplantation [].
In the present study, pericardial eff usions were detected in approximately  of the animals  days after lymphatic obstruction.Pericardial eff usions were likely caused by myocardial edema, as our previous tissue examination on the same animal model showed a signifi cant degree of edema in the ventricular myocardium [].Th e present study also demonstrated that cardiac lymphatic fl ow obstruction was associated with a signifi cant elevation in plasma nitric oxide levels in the fi rst  weeks following the obstruction.Th ese results are consistent with the previous studies where elevated nitric oxide levels were identifi ed following acute allograft rejection [, ].
The pathways by which cardiac lymphatic obstruction increased the plasma nitric oxide are not entirely clear.Th e obstruction of lymphatic vessels and the adjacent lymph nodes may cause accumulation of lymph and increase in intravascular pressure in the blocked vessels.Th e rises of the intravascular pressure may stimulate biosynthesis, or the release of nitric oxide from the lymphatic vessels cells or the endothelium.In addition, acute obstruction of lymphatic fl ow leads to hypoxia and ischemia-like changes in the coronary arteries due to the reduced drainage of interstitial fl uid [].Hypoxia and ischemia are potent stimulators for the production of nitric oxide by the coronary endothelium [].Nitric oxide is an important factor in the regulation of blood fl ow and fl uid homeostasis [].After heart transplantation, the expression and activity of endothelial nitric oxide synthase in the donor heart can be impaired [].Th e reduced nitric oxide activity plays a key role in the development of allograft vasculopathy [].The proposed factors causing the endothelial dysfunction include preexisting arteriosclerotic disease in the graft, graft ischemia before transplantation, immunosuppressive agents such as cyclosporin A, and other classic risk factors such as hyperlipidemia, hypertension, diabetes, or hyperhomocysteinemia [].In the present study,  weeks after the lymphatic obstruction, the plasma nitric oxide levels declined to a level that is below the baseline value, suggesting that chronic lymphatic obstruction may be detrimental to nitric oxide production which in turn, participating the pathogenesis of graft vasculopathy.

Conclusion
In conclusion, cardiac lymphatic obstruction was associated with a reduction in left ventricular function.Th e plasma levels of nitric oxide were increased in the fi rst  weeks of lymphatic obstruction.After  weeks, the plasma nitric oxide levels tend to decrease to below the baseline value.Further studies are required to clarify the clinical signifi cance of the nitric oxide changes following the lymphatic obstruction.

Declaration of Interest
None to declare.

TABLE 2 .
Changes in plasma concentration of nitric oxide following cardiac lymphatic obstruction.