The effect of PEEP on the right ventricle. A transeosophageal echocardiography and tissue Doppler study

Authors: Margaritis A.¹ MD, PhD, Patsouras D.² MD,  Tsigaridas N.² MD, Rammos A.² MD, Bolosi M.³ MD,  Ygropoulou O.¹ MD, Tzimas P.³ MD, Papadopoulos G.³ MD, PhD.

 

¹ Department of Intensive Care, General Hospital of  Ioannina “G. Hatzikosta”, Greece
² Cardiology Department, General Hospital of Ioannina “G. Hatzikosta”, Greece
³ Department of Anesthesiology and Postoperative Intensive Care, University    Hospital of Ioannina, Greece

 

ABSTRACT

The aim of this study was to determine how low and moderate levels of PEEP affect right ventricular structure and function.

The study involved 14 patients under mechanical ventilation (pressure controlled). To perform the study, we used transesophageal echocardiography (M-mode, two-dimensional,tissue Doppler) and 10 parameters were recorded. Measurements were performed initially with PEEP 0 cmΗ₂Ο, then 10 minutes after applying PEEP 5cmΗ₂Ο and 10 minutes after applying PEEP 10cmΗ₂Ο.

After applying PEEP 5 cmH₂O, there was no statistically significant change observed in any parameter. In contrast, after the application of PEEP 10cmH₂O, significant changes in most parameters were recorded: St changed from 10,86±3,8 cm/sec (mean±SD) at ΡΕΕΡ 0 cmH₂O to 8,7±2,9 at PEEP 10 cmH₂O, TAPSE changed from 2,876±0,643cm to 2,385±0,556cm, IVA from 2,2692±0,93m/sec² to 2,8536±1,628m/sec², ΕΤ from 259,27±17,47 msec to 229,34±31,22msec,EDA from 21,372±5,01cm² to 18,87±4,15cm², left ventricular eccentricity index at end-diastole from 0,9±0,07 to 0,99±0,08. After applying PEEP 10cmH₂O, a trend towards an increase in MPI index of right ventricle and a decline in ESA of the right ventricle were recorded. With respect to other parameters no statistically significant differences were recorded.

After applying ΡΕΕΡ=5cmH₂O there is no significant change noted in the overall structure and function of right heart. After applying ΡΕΕΡ=10cmH₂O changes were recorded in the structure and function of right ventricle caused mainly by the reduction of preload  and less by the  augmentation of  afterload.

INTRODUCTION

End-expiratory alveolar collapse is a common event in patients under mechanical ventilation, causing atelectasis which impair gas exchange and oxygenation. Positive end-expiratory pressure (PEEP) is an established method for mitigating this trend towards alveolar collapse in mechanically ventilated patients. Yet, in patients with respiratory disease, high values of PEEP, necessary for retaining oxygenation, may have negative effect on cardiac function^1,2. RV function is the primary determinant of cardiac output in critically ill patients³ and its dysfunction is associated with poor outcomes and can be induced by mechanical ventilation and PEEP therapy⁴.

In 1948, Cournand et al. studied the intermittent positive-pressure breathing, with or without continuous positive airway pressure (CPAP) and concluded that ventilation with positive pressure reduces venous return and right ventricular filling, with a result of cardiac output decrease⁵.

Later on, other researchers studied PEEP effects and presumed that the decrease in cardiac output during mechanical ventilation is partially attributed to the myocardial systolic impairement^6-8, a theory not confirmed on subsequent studies^9,10. On the contrary, some studies revealed that PEEP application in patients with congestive heart failure could increase cardiac output^11,12. Moreover, it seems that PEEP application could reinforce right ventricle (RV) ejection either through increasing the functional residual capacity¹³ or through reducing the pulmonary hypoxic vasoconstriction, after the opening of atelectatic alveoli^14-16.

Therefore, the effects of PEEP on cardiac function vary and they are often unpredictable, either with a predominantly positive effect, or a negative one, depending on a patient’s condition. The ideal value of PEEP is not easily defined and remains atopic of controversy.

The aim of this study was to perform a step-wise PEEP escalation in anesthetized, fully mechanically ventilated patients and to assess their RV function with tissue Doppler and conventional echocardiography.

Material and methods

The study (observational) was conducted after the approval of the scientific committee of bioethics and is part of the main writer’s (of this article) PhD Thesis¹⁷.

The inclusion criteria were: intubated patients of intensive care unit, haemodynamic stability, sinus rhythm, without oxygenation and ventilation problems during the study.

All patients were sedated with midazolam and cis-atracurium, under pressure control mechanical ventilation. Blood pressure was measured invasively through radial artery catheterization.

Exclusion criteria were: patients with atrial fibrillation or conduction disorders (inability to obtain tissue Doppler imaging at tricuspid annulus), patients with stomach or oesophagus pathologies (contraindication for transesophageal echocardiography) and patients with hypovolemic shock, pneumothorax and bronchial asthma (absolute and relative contraindications for PEEP insertion).

In many critically ill patients, low quality images are obtained with transthoracic echo, because the acoustic windows are suboptimal¹⁸. So, transensophageal echocardiography was selected as the method of choice.

All echo measurements were conducted at three different PEEP values: at first at 0 PEEP, ten minutes after insertion of 5cmH₂O PEEP and ten minutes after insertion of 10cmH₂O PEEP. Previous studies have demonstrated that all cardiorespiratory effects take place just a few seconds after PEEP insertion¹⁹.

The GE Vivid 7 cardiac ultrasound machine with a high frequency (6MHz) transesophageal transducer for adults (6T) was used for acquisition of images. These images were stored in an external hard disk and all the measurements were done at second time using the manufacturing software (EchoPac GE Vingmed, Horten Norway).

The echocardiographic study was performed according to ASE/EAE guidelines for transesophageal echocardiography and for evaluation of right ventricle function²⁰. The indexes that are measured during the study were the following:

-St (Tricuspid annular Systolic velocity): Peak systolic velocity of the tricuspid annulus (tissue doppler).

-TAPSE (Tricuspid Annular Peak Systolic Excursion): Distance of systolic excursion of the RV annular plane towards the apex (M-Mode).

-MPI of RV (Myocardial Performance Index): The ratio of total isovolumic time (isovolumic contraction time and isovolumic relaxation time) divided by RV ejection time.

-IVA (Myocardial Isovolumic Acceleration Time): The ratio of peak isovolumic RV myocardial velocity divided by time to peak velocity, measured at the level of the lateral tricuspid annulus during the isovolumic contraction.

-ET (Ejection Time): Time of ejection to the pulmonary artery, measured in the right ventricle outflow tract.

-Eccentricity index of left ventricle in end-systole (EccIndS) and end-diastole (EccIndD): The ration of anterior-inferior and septal-posterolateral cavity dimensions, measured at end-systole and end-diastole.

-End diastolic (EDA) and end-systolic(ESA) right ventricle area.

-FAC (Fractional Area Change): The percentage change in right ventricle area between end-diastole and end-systole.

All the Doppler indexes mentioned above were measured for each patient and for every PEEP value several times (3 times) per respiratory circle, using eventually the mean value for statistical analysis. In that way each value was representative of the overall conditions during the respiration and could be reliably compared to each other, in the different PEEP values inserted.

Statistics

A non-parametric statistical test (Friedman ANOVA) was used for the comparison between the three different results of each index. Non-paramentric multiple comparisons using the Schaich- Hamerle test, were conducted for the cases there was statistical significance between the three different values of the indexes. The level of statistical significance was defined as p<0.05. The data analysis was completed using the statistical package SPSS v. 19.0.

Results

Patient’s demographic data are presented in table 1.

The results of all the indexes measured are presented in table 2.

Application of 5cmH₂O PEEP did not affect significantly any of the parameters measured.

On the other hand, application of 10cmH₂O PEEP induced significant changes in the following indexes: St was reduced from 10,86±3,8 cm/sec to 8,7±2,9cm/sec (Image 1), TAPSE was reduced from 2,876±0,643cm to 2,385±0,556cm, IVA of the RV increased from 2,2692±0,93m/sec² to 2,8536±1,628m/sec², ΕΤ in right ventricular outflow tract was reduced from 259,27±17,47msec to 229,34±31,22msec, Left ventricle eccentricity index in end-diastole increased from  0,9±0,07 to 0,99±0,08 (Image 2, 3), EDA of the RV decreased from 21,372±5,01cm² to 18,87±4,15cm².

Image 1: Results of peak systolic velocity of the tricuspid annulus (St) on Box & Whisker Plot diagram.

After applying PEEP the St decreases and the change becomes statistical significant between 0 and 10cmH₂O PEEP.

 

Image 2: Results of Eccentricity index of LV in diastole on Box & Whisker Plot diagram.

 

Image 3: An echo image of Eccentricity index of left ventricle in end-diastole  without PEEP (A) and after applying 10cmH₂O PEEP (B).

ΜΡΙ of the RV, did not reveal a statistically significant change, though a trend towards an increase was recorded when 10cmH₂O was used.

ESA of the RV displayed a trend towards reduction when 10cmH₂O was used (borderline non statistical significant, p=0,06).

FAC of the RV and eccentricity index of the left ventricle in end-systole do not change after PEEP application.

No changes of blood pressure and cardiac rhythm were noted after PEEP application.

Discussion

One of the main results of this study is that application of a 5cmH₂O PEEP in patients under mechanical ventilation does not affect neither the structure nor the function of the right ventricle. On the other hand, the effect of application of a 10cmH₂O PEEP is complex and provokes changes in the preload and the afterload²¹.

Previous research has proven that PEEP causes dilation of lungs and increase in intrathoracic pressure, inducing restriction of the diastolic filling of the right ventricle²² and an increase in pulmonary vascular resistance²³. The elevated intrathoracic pressure impedes the venous flow into the thorax, and compresses cardiac chambers, resulting in a reduction of the diastolic filling and the preload of the right ventricle^1,24. The increase in PEEP values causes increase in elasticity of the right ventricle^25,26. The increase of pulmonary vascular resistance is a result of lung dilatation, which impairs perfusion in some small pulmonary vessels and changes the diameter of pulmonary vessels²⁷. The above mentioned mechanisms result in a reduction of the preload of the right ventricle (due to the restriction of the diastolic filling of the right ventricle) and in an increase in the afterload of the right ventricle (due to the increase of pulmonary vascular resistance).

In our study, a significant reduction of St and of TAPSE was found, indicating right ventricle systolic dysfunction. St values <11.5cm/sec are related with systolic dysfunction of the right ventricle (RV EF<50%), with a sensitivity of 90% and a specificity of 85% respectively^28,29. However, recent guidelines for right ventricle assessment correlate St values<10cm/sec with right ventricle’s systolic dysfunction²⁰. Moreover, TAPSE (with normal values>15mm) is well correlated with ejection fraction (EF) of the right ventricle and reflects also the function of the free wall of the right ventricle along the longitudinal axis^30,31; though it is preload dependent¹⁹. In our study, there was a significant reduction of St below the normal cutoff (when PEEP of 10cmH₂Owas applied) and a significant reduction of TAPSE, though without falling below the lower normal cutoff.

On the other hand, FAC of the right ventricle did not change after PEEP increase. This index is well correlated with RV EF³⁰, though it is also preload and afterload dependent. Moreover, a borderline statistically significant increase of time of IVA(p=0,046) was found (normalvalues  1,4-3m/sec²)²⁰. It seems that IVA reflects the systolic function of the right ventricle and is less load dependent^28,32; IVA values of >1,1m/sec² arewell correlated with RV EF>45% measured with MRI, with a 90% sensitivity and specificity³⁰. IVA is proportionally correlated with RV systolic function^31-33. The results of FAC and IVA in our study indicate that application of 10cmH₂O PEEP does not affect the systolic function of the right ventricle. It is widely accepted that RV EF and the parameters that are related with it, can be influenced by preload and therefore do not illustrate accurately the systolic function of the right ventricle²⁰. Therefore, St and TAPSE reduction may reflect a reduction in the systolic function of the right ventricle, not because of a dysfunction of its contractility, but because of a distention inability of myocardial fiber of the right ventricle and a consequent reduction of its contraction, according to the Frank-Starling law.

In our study, it was found a reduction of ET in the right ventricular outflow tract and a trend towards an increase of MPI (normal values with tissue Doppler <0.55)²⁰. It is known from previous studies that the reduction in ET is conversely related with pulmonary artery pressure³⁵; while MPI index is usually increased in patients with systolic or diastolic dysfunction of the right ventricle^35,36, and in cases with high pulmonary artery pressures³⁵. MPI values have prognostic significance in patients with pulmonary hypertension^35,36. Therefore, the reduction of ET and the trend for an increase of MPI, which were found in this study, show an increase in pulmonary vascular pressures after application of 10cmH₂O PEEP.

A reduction of end-diastolic surface area and a trend towards a reduction of end-systolic surface area of the right ventricle is recorded in this study, as reflected in FAC of the right ventricle, which did not change after PEEP application. A recent study showed that the FAC changes significantly after applying 20cmH₂O PEEP⁴.

Application of 10 cmH₂O of PEEP did not change  Eccentricity Index of the left ventricle in end-systole, while in end-diastole there was a statistically significant  increase of this index, but values did not exceed 1.The major impact of RV function is usually considered to be a decrease in LV diastolic compliance if RV overdistention occurs, through the process of ventricular interdependence (changes of the right ventricle affect the structure of the left ventricle through interventricular septum)³⁷. This index is based on ventricular interdependence, meaning that. Normal values of Eccentricity index are equal to 1 both in systole and in diastole. Values>1 in end-diastole indicate volume overload of the right ventricle, while values >1 both in systole and in diastole indicate pressure overload of the right ventricle^30,38 (Image 3).

Values<1 are not taken into account. In our study, the increase of Eccentricity index in end-diastole after applying 10cmH₂O does not exceed 1 and so it cannot be taken into account. This finding is not opposed to the recorded reduction of end-diastolic surface area of the right ventricle. We could hypothesize that in PEEP values>10 cmH₂O the Eccentricity index would increase>1, with a consequent leftward movement of the interventricular septum.

Since the application of PEEP may have opposing effects, we think that our results are not against to the results of other studies^4,25,39-42.  The increase in pulmonary vascular resistance (increase of afterload), tend to lower the ejection of the right ventricle and increase its end-diastolic volume. On the other hand, the increase in elasticity of right ventricle tends to reduce its end-diastolic volume (reduction of preload). Hence, depending on which effect (on preload or on afterload) predominates after the increase of airway pressures, there will be a relative effect on end-diastolic volume of the right ventricle which is partially reflected through its end-diastolic surface area.

In conclusion, in our study it seems that in PEEP values of 10cmH₂O there is predominantly a reduction in the right ventricle preload, while an increase in the right ventricle afterload is also recorded. The gradual increase of Eccentricity Index in end diastole and the reduction of ET after PEEP increase, may indicate a possible significant increase in pulmonary vascular resistance after application of PEEP>10cmH₂O, having as a predominant consequence an increase of the right ventricle afterload, an increase in right ventricle diameter and a leftward shift of the interventricular septum.

In the study most of the indexes were derived through tissue Doppler. This method, as any doppler method, depends on the angle between ultrasound beam and the tissue. The movement of free wall of the right ventricle is not always parallel to the ultrasound beam, having as a consequence the underestimation of St and IVA absolute values. However, in each patient the alignment was the same for all three values of PEEP, making the results comparable and reliable. Measurements of MPI do not have these limitations, as MPI is calculated using specific time and is independent of velocities absolute values.

Tricuspid annular peak systolic velocity is a validated method for the estimation of right ventricle’s systolic function. However, it is dependent on the movement of the basis of the heart to the apex. New echocardiographic methods for the evaluation of systolic function of the right ventricle, like strain (myocardial deformation) could be used more accurately and reliably for the evaluation of biventricular systolic function on different PEEP values, as it is angle independent method.

The last limitation of the study is the small number of patients included. Consequently, the results cannot be definite and cannot be generated. However, they create a hypothesis which can be tested with larger studies.

Conclusion

In conclusion, from above, it appears that applying PEEP 5 cm H₂O has no effect in the structure and the function of the right heart. In contrast, applying PEEP 10 cmH₂O effects the structure and the function of the right ventricle; this is mainly attributed to reduced filling of the heart (reduced preload) and to a lesser extent to increased pulmonary vascular resistance (increased afterload). Further studies in the ICU population, using TEE imaging and tissue Doppler are warranted.

References

1. Jardin F., Vieillard-Baron Α. Right ventricular function and positive pressure ventilation in clinical practice: from hemodynamic subsets to respirator settings. Intens Car Med 2003; 29:1426-34.
2. Luecke and P. Pelosi. Positive end-expiratory pressure and cardiac output. Crit. Care 2005; 9: 607-21.
3. Michael R. Pinsky. My paper 20 years later: Effect of positive end-expiratory pressure on right ventricular function in humans. Intensive Care Med 2014; 40: 935–41.
4. Sam RO, Atta B, Paul GS, et al. Effect of positive end-expiratory pressure on porcine right ventricle function assessed by speckle tracking echocardiography. Anesthesiology 2015; 15:49.
5. Cournand A, Motley HL, Werko L, et al. Physiological studies of the effects of intermittent positive pressure breathing on cardiac output in man. Am J Physiol 1948; 152:162-74.
6. Liebman PR, Patten MT, Manny J, et al. The mechanism of depressed cardiac output on positive end-expiratory pressure(PEEP). Surgery 1978; 83(5):594-8.
7. Patten MT, Liebman PR, Manny J, et al. Humorally mediated alterations in cardiac performance as a consequence of positive end-expiratory pressure. Surgery 1978; 84(2):201-5.
8. Grindlinger GA, Manner J, Justice R, et al. Presence of negative inotropic agents in canine plasma duting positive end-epiratory pressure. Circ Res 1979; 45(4):460-7.
9. Berglund JE, Halden E, Jakobson S, et al. Echocardiographic analysis of cardiac function during high PEEP ventilation. Intensive Care Med 1994; 20:174-180.
10. Berglund JE, Halden E, Jakobson S. Maintained cardiac output during PEEP ventilation in open chest pigs.Acta Anaesth Scand 1997;41(5):618-23.
11. Kaneko Y, Floras JS, Usui K, et al.: Cardiovascular effects of continuous positive airway pressure in patients with heart failure and obstructive sleep apnea. N Engl J Med 2003; 348:1233-41.
12. Nadar S, Prasad N, Taylor RS, et al. Positive pressure ventilation in the management of acute and cronic cardiac failure: a systematic review and meta-analysis. Int J Cardiol 2005; 99:171-85.
13. Canada E, Benumoff JL, Tousdale FR. Pulmonary vascular resistance correlates in intact normal and abnormal canine lungs. Crit Care Med 1982; 10:719-23.
14. Bouferrache K, Vieillard-Baron A. Acute respiratory distress syndrome, mechanical ventilation and right ventricular function. Cur OpinCrit Car 2011; 17:30-35.
15. Miranda DR, Klompe L, Cademartiri F, et al. The effect of open lung ventilation on right ventricular and left ventricular function in lung-lavaged pigs. Crit Car 2006; 10:R86.
16. Gernoth C, Wagner G, Pelosi P, et al. Respiratory and hemodynamic changes during decremental open lung positive end expiratory pressure titration in patients with acute respiratory distress syndrome.Critical Care 2009; 13:R59.
17. Margaritis A. The effect of PEEP on structure and function of right and left ventricle. Doctoral Thesis No 5636/2014. Repository “Olympia” of University of Ioannina. Available from: http://olympias.lib.uoi.gr/jsp/handle/123456789/5636.
18. Beaulieu Y, Marik PE. Bedside ultrasonography in the ICU. Chest 2005; 128:881-95.
19. Schuster S, Erbel R, Weilemann L S, et al. Hemodynamics during PEEP ventilation in patients with severe left ventricular failure studied by transesophageal echocardiography. Chest 1990; 97:1181-9.
20. Rudski L, Lai W, Afilalo J, et al. Guidelines for the Echocardiographic Assessment of the Right Heart in Adults: A Report from the American Society of Echocardiography Endorsed by the European Association of Echocardiography, a registered branch of the European Society of Cardiology, and the Canadian Society of Echocardiography. J Am SocEchocardiogr 2010; 23:685-713.
21. Pinsky M: Cardiovascular Issues in Respiratory Care. Chest 2005; 128:592S-597S.
22. Dambrosio M, Cinnella G, Brienza N, et al. Effects of positive end-expiratory pressure on right ventricular function in COPD patients during ventilator failure. Intens Car Med 1996; 22:923-32.
23. Jardin F, Vieillard-Baron A. Is there a safe plateau pressure in ARDS? The right heart only knows. Intens Car Med 2007; 33:444-7.
24. Morgan B, Guntheroth W, Dillard D. Relationship of pericardial pressure to pleural pressure during quiet respiration and cardiac tamponade. Circ Res 1965; 26:493-8.
25. Jardin F, Brun-Ney D, Hardy A, et al. Combined thermodilution and two dimensional echocardiographic evaluation of right ventricular function duringrespiratory support with PEEP. Chest 1991; 99:162-8.
26. Vieillard-Baron A, Jardin F: Why protect the right ventricle in patients with acute respiratory distress syndrome? Cur OpinCrit Car 2003; 9:15-21.
27. Fougeres E, Teboul JL, Richard C, et al. Hemodynamic impact of a positive end-expiratory pressure setting in acute respiratory distress syndrome: Importance of the volume status. Crit Care Med 2010; 38:802-7.
28. Haddad F, Hunt S, Rosental D, et al. Right ventricular function in cardiovascular disease, Part I: Anatomy, Physiology, Aging and Functional assessment of right ventricle. Circulation 2008; 117:1436-48.
29. Meluzin J, Spinarova L, Bakala J, et al. Pulsed Doppler tissue imaging on the velocity of tricuspid annular systolic motion: a new, rapid, and non-invasive method of evaluating right ventricular systolic function. Eur Heart J 2001; 22:340-8.
30. Jurcut R, Giusca S, La gerche A, et al. The echocardiographic assessment of the right ventricle: what to do in 2010? Eur J of Echocardiography 2010; 11:81-96.
31. Kaul S, Tei C, Hopkins JM, et al. Assessment of right ventricular function using two-dimensional echocardiography. Am Heart J 1984; 107:526-31.
32. Vogel M, Derrick G, White PA, Cullen S, et al. Systemic ventricular function in patients with transposition of the great arteries after atrial repair: a tissue Doppler and conductance catheter study. J Am Coll Cardiol 2004; 43:100-6.
33. Bayram NA, Ciftci B, Bayram H, et al. Effects of continuous positive airway pressure therapy on right ventricular function assessment by tissue Doppler imaging in patients with obstructive sleep apnea syndrome. Echocardiography 2008;25:1071-8.
34. Schattke S, Knebel F, Grohmann A, et al. Early right ventricular systolic dysfunction in patients with systemic sclerosis without pulmonary hypertension: a Doppler Tissue and Speckle Tracking echocardiography. Cardiovasc Ultrasound 2010; 8:3.
35. Tei C, Dujardin K, Hodge D, et al. Doppler Echocardiographic Index for Assessment of Global Right Ventricular Function. J Am SocEchocardiogr 1996; 9:838-47.
36. Michaux I, Seeberger M, Schuman R, et al. Feasibility of measuring Myocardial Performance Index of the Right Ventricle in Anesthetized Patients. J CardiothVascAnesth 2010; 24:270-4.
37. Pinsky R. Michael: The right ventricle: interaction with the pulmonary circulation. Crit Care 2016; 20:266.
38. Ryan T, Petrovic O, Dillon JC, et al. An Echocardiographic index for separation of right ventricular volume and pressure overload. J Am CollCardiol 1985; 5:918-27.
39. Vieillard-Baron A, Loubieres Y, Schmitt JM, et al. Cyclic changes in right ventricular output impedance during mechanical ventilation. J ApplPhyiol. 1999; 87:1644–50.
40. Pinsky MR, DeSmet JM, Vincent JL. Effect of positive end-expiratory pressure on right ventricular function in humans. 1992; 146:681–687.
41. Jardin F, Delorme G, Hardy A, Auvert B, Beauchet A, Bourdarias JP: Reevaluation of hemodynamic consequences of positive pressure ventilation: emphasis on cyclic right ventricular afterloading by mechanical lung inAnesthesiology 1990; 72:966-70.
42. Cherpanath TG, Lagrand WK, Binnekade JM, et al. Impact of Positive End-Expiratory Pressure on thermodilution-derived right ventricular parameters in mechanically ventilated critically ill patients. J CardiothoracVascAnesth 2016; 30:632-8.

 

Acknowledgments

We thank Mr. George Dimakopoulos for the statistical analysis

 

Author Disclosures:

Authors Margaritis A., Patsouras D., Tsigaridas N., Rammos A., Bolosi M., Ygropoulou O., Tzimas P., Papadopoulos G. have no conflicts of interest or financial ties to disclose.

 

Corresponding Author:

Athanasios Margaritis

Address: Anilio Metsovo 44200, Greece
tel. (+30) 6976335002
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