Auðra Burkauskienë Institute of Anatomy, Kaunas University of Medicine, Lithuania

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1 145 Age-related changes in the structure of myocardial collagen network of auricle of the right atrium in healthy persons and ischemic heart disease patients Institute of Anatomy, Kaunas University of Medicine, Lithuania Key words: heart, myocardial collagen network, ischemic heart disease, age, video morphometric analysis. Summary. The objective of the study was to examine and evaluate morphometrically agerelated changes in the structure of myocardium collagen network of auricle of the right atrium in control persons, who were not diagnosed with cardiac pathology leading to heart lesion or overload, and in ischemic heart disease patients. Material of 56 persons of both genders aged years was used for study purposes. Biopsy material of 17 healthy persons (control, average age 60.53±9.89 years) and autopsy material of 39 ischemic heart disease patients (average age 63.83±15.67 years) taken from the basis of auricle of the right atrium (specimen size 2 mm 2 mm) were examined. Morphometric analysis of collagen network was performed using histologic and video morphometric methods. After this investigation we evaluated quantitative parameters of the bundles of collagen net namely area, number, perimeter. The percentile occupied area of bundles in control was 17.6±2.5%; ischemic heart disease patients 26.8±2.9%; number of bundles was 4179±1073 and 2523±867; perimeter 24163±3308 μm and 23426±409 μm, respectively. After investigation of age-related changes of collagen network in control and ischemic heart disease patients, which did not statistically significantly differed by age, we determined that collagen network area in auricle of the right atrium increased with age in both s, however, spatial distribution of collagen network was different. Collagen network area enlarged with lengthening of its fibers along cardiomyocytes in control. In ischemic heart disease, it enlarged both in parallel to cardiomyocytes and by separate collagen fibers merging into bigger bundles. Fibrillar collagen network area and its total perimeter of healthy persons increased with age, and number of fibers did not change. Consequently, collagen fiber area of one location increased with age; its shape, judging by in parallel increasing total perimeter, became branchier, i.e. proliferated in endomysium in parallel with cardiomyocytes. In ischemic heart disease fibrillar collagen network percentage area increased with age, however, total perimeter and number of separate fibers in visual field decreased. Consequently, in ischemic heart disease separate collagen fibers merged, their locations enlarged, taking an integral structure, which allowed assuming development of interstitial fibrosis. Introduction Recently more attention is being paid to prevention and treatment of cardiovascular diseases; scientists work intensively, performing research in remodeling of cardiomyocytes and collagen. Collagen is one of the most important structures of the connective tissue. It is the main structural protein in the interstitium. Currently functional significance of myocardial collagen is emphasized due to its importance for contraction and nutrition of cardiomyocytes (1 4). Five molecular isoforms of collagen are found in the heart: type I, III, IV, V and VI collagens. Fibrillar types of I and III collagens are the most abundant and constitute 90% of total amount in the myocardium. Type I collagen consists of thick and tensile fibers. Type III collagen forms thin reticular, more elastic network than type I collagen, and usually is found together with type I collagen, forming collagen fibers in myocardium between myocytes and muscle fibers (5). Many publications could be found on remodeling Correspondence to A. Burkauskienë, Institute of Anatomy, Kaunas University of Medicine, A. Mickevièiaus 9, Kaunas, Lithuania

2 146 mechanisms, changes in collagen network in different cases of heart pathology, and on experimental research using different preparations, which influence remodeling of collagen network (1, 6 11). Currently research studies of changes occurring during the remodeling process of cardiomyocytes and collagen in cases of arterial hypertension, myocarditis and dilated cardiomyopathy predominate (3, 5, 8, 12, 13). In more detail remodeling processes are examined in aspect of changes in cardiomyocytes and contractile collagen network in patients suffering from ischemic heart disease and after myocardial infarction (14 16). However, very few clinical trials of this type are dedicated for analysis of age-related remodeling processes. There are few extensive morphometric studies, which evaluate fibrillar collagen network of human atrial myocardium. Even though many studies were performed investigating structural, functional and biochemical changes of collagen, the main attention was paid to changes of collagen in ventricles but not in atria. No studies were performed with the aim to evaluate age-related changes of contractile collagen network of human atrial myocardium (17). The objective of the study was to examine and evaluate morphometrically age-related changes in the structure of myocardium collagen network of auricle of the right atrium in control persons, who were not diagnosed cardiac pathology leading to heart lesion or overload, and in ischemic heart disease patients. Material and methods Study material consisted of 56 persons (control ) and ischemic heart disease (IHD) patients of both genders aged years. 17 healthy people aged years (average age 60.53±9.89 years) and 39 IHD patients, aged years (average age 63.83±15.67 years) were investigated. Study material was taken from the basis of auricle of the right atrium. It was always taken from the same location. Either autopsy or biopsy material was examined. For control, autopsy material was taken no later than 3 4 hours after death at the Clinic of Pathologic Anatomy of Kaunas University of Medicine. For IHD biopsy material was taken during coronary artery bypass grafting operation at the Clinic of Cardiac Surgery of Kaunas University of Medicine. Morphometric analysis of collagen network was performed using histologic and video morphometric methods (18). Study material (specimen size 2 mm 2 mm) was fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (ph 7.4) for 1 2 days, embedded in paraffin and then sectioned into 6 μm slices using microtome. Collagen was stained with Picrosirius red. Structure of collagen fibers was enhanced using polarizing light microscopy (Fig. 1). Preparations were analyzed using optic microscope Micros (Austria), magnifying 20x. Ten images of each person were registered for examination of myocardium collagen network. Images were selected visually based on optimal brightness of examined structures. Area of one visual field was 0.25 mm 2. Totally 560 atrial myocardium fields were examined using semi-automated image analysis system. These images were recorded onto personal computer using color digital video camera Pixera and analyzed using special image processing program Image-Pro Plus v. 4. Using this program color image is transformed to binary image, i. e. black and white image. For enhancement of structures of collagen network we used 255 halftone image levels (255=white, 0=black). Detailed quantitative analysis of myocardium collagen network was performed: bundle area, their perimeter and number were calculated. Area was expressed in percent as comparative value between examined structures and the whole visual field. Data was processed and statistically analyzed using Microsoft Excel program. Distribution of morphologic examination data of collagen network in both s was analyzed using Kolmogorov-Smirnov criterion. It was determined that null hypothesis on normal distribution of analyzed data should not be rejected, therefore sample average approximation method and dispersion analysis were used. Sample average and dispersion confidence intervals of morphometric parameters were evaluated. Established short interval length as well as small ratio between standard deviation and parameter average (for all parameters less than 5%) show sufficient sample size and that it well reflects general population. For comparison of averages of control and IHD, two-factor variance analysis (ANOVA) was performed, evaluating parameter dispersions of and case (nested design). Correlation between age of study participants and percentage area, number and perimeter of collagen structures was determined using regressive analysis. Results Video morphometric analysis revealed that area of collagen bundles in control was 17.6±2.5%

3 Age-related changes in the structure of myocardial collagen network in health and ischemic heart disease 147 Fig. 1. Evaluation of quantitative changes of myocardium collagen network preparations stained with Picrosirius red using standard light microscopy (left) and polarized light microscopy (right) and in IHD 26.8±2.9%; number of bundles was 4179±1073 and 2523±867, respectively; perimeter was μm and 23426±409 μm, respectively. Area of collagen bundles was 51.7% bigger in IHD than in control one (p<0.001); number of bundles was 39.6% smaller (p<0.001), and perimeter of collagen network was only 3.1% smaller in IHD than in control (p<0.05) (Fig. 2). 32 Area, % Number of bundles, n Perimeter, μm 28 * Control IHD Control IHD Control IHD Fig. 2. Comparison of collagen network area, number of bundles and perimeter in control and IHD (average and standard deviation, *p<0.001, **p<0.05)

4 148 Linear correlation between fibrillar collagen network area of myocardium of auricle of the right atrium and number of its separate bundles was weak both in control and in IHD (Fig. 3, 4). Correlation between collagen network area and perimeter was close to the second-degree polynomial, however, in control with an increase of area, perimeter also increased (r=0.74, p<0.001). In IHD it was opposite: with an increase of area, perimeter decreased, however, this correlation was weak (r=0.23, p<0.001). Correlation between collagen parameters and age in control is presented in Figure 5. Even though collagen bundle area, expressed in percent, increased linearly with age (r=0.95, p<0.001), change in number of bundles was not observed (p>0.05). Collagen network perimeter increased linearly with age (r=0.73, p<0.001). In IHD (40 80 years) collagen bundle area increased linearly with age (r=0.85, p<0.001), number of bundles (r=0.53, p<0.001) and perimeter decreased Number, n Perimeter, μm Number of bundles (left axis) y= x 34.2x 2 ; r=0.74; p< Area, % Perimeter (right axis) y= x; r=0.15; p<0.05. Fig. 3. Correlation between auricle of the right atrium myocardium collagen network area, number of bundles and perimeter in control Number, n Perimeter, μm Area, % Number of bundles (left axis) Perimeter (right axis) y= x 0.35x x 3 ; y= x; r=0.15; p< r=0.23; p< Fig. 4. Correlation between auricle of the right atrium myocardium collagen network area, number of bundles and perimeter in IHD

5 Age-related changes in the structure of myocardial collagen network in health and ischemic heart disease 149 Area, % y=0.16*x+7.58; r=0.95; p= Number of bundles, n y=2.69*x ; p=0.8. Perimeter, μm y=130.2*x ; r=0.73; p= Fig. 5. Relationship of collagen network area, number of bundles and perimeter with age in control

6 Area, % 150 (r=0.44, p<0.01) (Fig. 6). After investigation of agerelated changes in collagen network in control and IHD s, which did not statistically significantly differed by age, we determined that in both s fibrillar collagen network of auricle of the right atrium Area, % increased with age (Fig. 7, 8), however spatial distribution of collagen network differed. In control collagen area increased via lengthening of its fibers along cardiomyocytes; in IHD it increased both in parallel to cardiomyocytes, and by merging of col y=0.25*x+11.66; r=0.85; p= Number of bundles, n Perimeter, μm y= 46.47*x ; r=0.53; p= y= 152.7*x+32669; r=0.44; p= Fig. 6. Relationship of collagen network area, number of bundles and perimeter with age in IHD

7 Age-related changes in the structure of myocardial collagen network in health and ischemic heart disease 151 A B C Fig. 7. Age-related quantitative changes in myocardium collagen network of auricle of the right atrium in control patients A normal (40 year old person), B slightly increased (65 year old person), C moderately increased (81 year old person). A B C Fig. 8. Age-related quantitative changes in myocardium collagen network of auricle of the right atrium in IHD patients A slightly increased (40 year old person), B moderately increased (65 year old person), C markedly increased (81 year old person). lagen fibers into bigger conglomerates. In control fibrillar collagen network area and its total perimeter increased with age, however, number of fibers did not change. Consequently, location area of collagen fibers increased with age, its shape, judging by in parallel increasing total perimeter, became branchier, i.e. proliferated in endomysium along cardiomyocytes. In IHD fibrillar collagen network percentage area increased with age, however total perimeter and number of fibers in the visual field decreased. Consequently in IHD separate collagen fibers merged, their location areas increased taking integral structure, which allowed assuming development of interstitial fibrosis. This confirms that fibrillar collagen network of myocardium of auricle of the right atrium changes with age. Both in control and IHD fi-

8 152 brillar collagen network percentage area equally increases starting with the fifth decade of life. Discussion Collagen is the only human protein, physicochemical characteristics of which change with age, its amount in myocardium decreases, even though its area increases. Similar age-related development of fibrillar collagen network is determined in the studies performed by other authors (10). Literature data show that decrease of average optical density with age is related to increased amount of binding tissue (proteoglycans) among collagen fibrils (main structural unit, which forms collagen fiber), even though diameter of fibers also increases in advanced age (17). With an increase of amount of proteoglycans, amount of collagen and elasticity of myocardium decrease. Increased total fibrillar collagen network percentage capacity and bigger separate location areas increase resilience of myocardium, change direction of force vectors and disturb its contractile function (7, 10, 18, 19). Even though for IHD patients hemodynamic overload falls on the right atrium in the last place of all heart parts, we determined that its myocardium collagen network percentage area was bigger, perimeter of collagen fibers did not differ, and perimeter of separate bundles was smaller. Consequently, in IHD amount of collagen in auricle of the right atrium myocardium not only increased, but also its fibers merged into bigger bundles. This reactive interstitial fibrosis develops when synthesis of type I and III collagens increases, and when their degradation is unchanged or decreased. Together increased collagen concentration is defined as fibrosis, which can start both in case when cardiomyocyte hypertrophy is present or not. Collagen accumulation is determined by volume and pressure overload (12, 20, 21). There is very little data on remodeling of atria and age-related changes of collagen network. We could not find similar studies, which would analyze agerelated changes of collagen network of the atrium. There are more studies analyzing structural and functional changes in case of heart diseases: hypertension, valve pathology and ischemic heart disease (3, 9, 12, 16, 20, 22). It is determined that origin of atrial fibrosis is influenced by extracellular signal-regulated protein kinase and angiotensin receptors when atrial fibrillation is present. Renin-angiotensin-aldosterone activation determines cell growth, fibroblast proliferation and atrial fibrosis (8, 11, 14, 23). Due to increased atrial strain, natriuretic peptides are excreted. In experiments with rats, analyzing model of heart fibrosis, it was determined that when type B natriuretic peptide was not excreted, ventricular fibrosis developed (24 26). In IHD patients, we did not observe atrial fibrillation, however area of collagen network was 50% bigger compared with healthy patients; i.e. myocardial ischemia is related not only to changes in collagen network of ventricles but also of atria. Supposedly, in cases of short-term ischemia episodes, reserve, flow and contractile function changes of compensative character occur in atrium. Increased flow function at first compensates disturbances of atrial systole; later changes in these functions become more severe and insufficiency develops (21). In case of ischemia reverse correlation exists between activity of ventricle and atrium; when ventricular stroke volume index decreases, atrial stroke volume index increases. With decrease in ventricular ejection fraction, atrial pump or contractile function increases (3, 21, 26, 27). Strengthening of contractile function might be a response to an increased preload and post load due to dilated ventricular tightness. In addition that collagen network area was determined, perimeter of its fibers and number of bundles were also determined, which not only widens possibilities for quantitative assessment, but also provides objective information on qualitative characteristics of collagen network. Similar collagen network perimeter of IHD patients and healthy persons and in case of IHD smaller number of collagen bundles indicates the presence of bigger and more rounded collagen fiber bundles. D. Pangonytë et al performed analogous measurements investigating changes in myocardial collagen network of the left ventricle (14, 15). However, in myocardium of left ventricle in IHD patients they found bigger perimeter of collagen network as well as number of bundles, i.e. forming of collagen fibers in parallel to cardiomyocytes. Conclusions Quantitative parameters of collagen network bundles: area, number and perimeter, evaluated using video microscopic system, provide additional information on structural changes occurring in myocardium of auricle of right atrium and their correlation with ischemic heart disease. Remodeling of collagen network of auricle of right atrium occurs both due to age and myocardial ischemia. In ischemic heart disease percentage area (51.7%) of collagen network bundles was bigger; number (39.6%) and perimeter (3.1%) were smaller compared with data of control. Collagen network bundle percentage area increases

9 Age-related changes in the structure of myocardial collagen network in health and ischemic heart disease 153 equally both in healthy and ischemic heart disease patients, starting with the fifth decade of life. In control number of collagen bundles does not change with age, however, their perimeter increases; in ischemic heart disease patients both number of collagen bundles and perimeter decrease. Sveikø þmoniø ir serganèiøjø iðemine ðirdies liga deðiniojo prieðirdþio ausytës miokardo kolageno tinklo sandaros pokyèiai, susijæ su amþiumi Kauno medicinos universiteto Anatomijos institutas Raktaþodþiai: ðirdis, miokardo kolageno tinklas, iðeminë ðirdies liga, amþius, vaizdo morfometrinis tyrimas. Santrauka. Darbo tikslas. Iðtirti ir morfometriðkai ávertinti deðiniojo prieðirdþio ausytës miokardo kolageno tinklo sandaros pokyèius, atsirandanèius su amþiumi, þmonëms, kuriems nenustatyta ðirdies patologija, galëjusi sukelti ðirdies paþeidimà arba perkrovà sveikiems asmenims ir sergantiesiems iðemine ðirdies liga. Iðtirta 56 abiejø lyèiø metø þmoniø biopsinë ir autopsinë medþiaga, kurios buvo paimta ið deðiniojo prieðirdþio ausytës pagrindo (mëginio dydis 2 mm 2 mm). Biopsinës medþiagos paimta ið 17 sveikø þmoniø (kontrolinë grupë), kuriø amþiaus vidurkis 60,83±9,89 metø; autopsinës ið 39 serganèiøjø IÐL, kuriø amþiaus vidurkis 63,83±15,67 metø. Kolageno tinklo morfometriniam tyrimui panaudoti histologinis ir vaizdo mikroskopinis metodai. Atlikæ ðá tyrimà, ávertinome kolageno tinklo pluoðtø kiekybinius parametrus: plotà, skaièiø, perimetrà. Pluoðtø uþimamas plotas sveikø kontrolinës grupës tiriamøjø buvo 17,6±2,5 proc.; IÐL serganèiøjø grupës 26,8±2,9 proc.; pluoðtø skaièius atitinkamai 4179±1073 ir 2523±867; perimetras 24163±3308 μm ir 23426±409 μm. Iðtyrus sveikø þmoniø grupæ ir serganèiøjø IÐL, kuriø amþius reikðmingai nesiskyrë, kolageno tinklo pokyèius, susijusius su amþiumi, nustatëme, kad tiek sveikø tiriamøjø, tiek serganèiøjø IÐL deðiniojo prieðirdþio ausytës kolageno tinklo plotas su amþiumi didëjo, taèiau ðiø dviejø grupiø tiriamøjø kolageno tinklo erdvinis iðsidëstymas skyrësi. Sveikø tiriamøjø kolageno plotas didëjo ilgëjant jo skaiduloms iðilgai kardiomiocitø, o serganèiøjø IÐL didëjo tiek paraleliai kardiomiocitams, tiek ir susiliejant atskiroms kolageno skaiduloms á didesnius pluoðtus. Sveikø asmenø fibrilinio kolageno tinklo plotas ir jo suminis perimetras su amþiumi didëjo, o atskirø skaidulø skaièius nekito. Taigi kolageno skaidulø vieno þidinio uþiimamas plotas didëjo su amþiumi, jo forma, sprendþiant pagal lygiagreèiai didëjantá suminá perimetrà, darësi labiau ðakota, tai yra plito endomizyje paraleliai kardiomiocitø. Serganèiøjø IÐL fibrilinio kolageno tinklo uþimamas procentinis plotas didëjo su amþiumi, taèiau maþëjo suminis perimetras ir atskirø skaidulø skaièius regëjimo lauke. Taigi serganèiøjø IÐL atskiros kolageno skaidulos susiliejo, jø þidiniai didëjo, ágaudami vientisà struktûrà, todël galima daryti prielaidà, kad ðiuo atveju formavosi intersticinë fibrozë. Adresas susiraðinëti: A. Burkauskienë, KMU Anatomijos institutas, A. Mickevièiaus 9, Kaunas References 1. Bishop JE, Lindahl G. Regulation of cardiovascular collagen synthesis by mechanical load. Cardiovasc Res 1999;42(1): Burgess ML, McCrea JC, Hedrick HL. Age-associated changes in cardiac matrix and integrins. Mech Ageing Dev 2001;122(15): Frohlich ED. Ischemia and fibrosis: the risk mechanisms of hypertensive heart disease. Braz J Med Biol Res 2000;33(6): Rossi MA, Abreu MA, Santoro LB. Images in cardiovascular medicine. Connective tissue skeleton of the human heart: a demonstration by cell-maceration scanning electron microscope method. Circulation 1998;97(9): Stalioraitytë E, Pangonytë D. Kardialinë patologija. (Cardiac pathology.) Vilnius; Anker SD, Rauchhaus M. Heart failure as a metabolic problem. Eur J Heart Fail 1999;1(2): Bolognese L, Cerisano G, Buonamici P, Santini A, et al. Influence of infarct-zone viability on left ventricular remodeling after acute myocardial infarction. Circulation 1997; 96(10): Brilla CG, Maisch B, Weber KT. Renin-angiotensin system and myocardial collagen matrix: remodeling in hypertensive heart disease: in vivo and in vitro studies on collagen matrix: regulation. Clin Invest 1993;71 Suppl 5: Cohn JN, Ferrari R, Sharpe N. Cardiac remodeling concepts and clinical implications: A consensus paper from an International Forum on Cardiac Remodeling. J Am Coll Cardiol 2000;35(3): Debessa CRG, Maifrino LB, de Souza RR. Age related

10 154 changes of the collagen network of the human heart. Mech Ageing Dev 2001;122(10): Paradis P, Dali-Youcef N, Paradis FW, Thibault G, Nemer M. Overexpression of angiotensin II type I receptor in cardiomyocytes induces cardiac hypertrophy and remodeling. Proc Natl Acad Sci USA 2000;97(2): Goldfine SM, Pena M, Magid NM, Liu SK, Borer JS. Myocardial collagen in cardiac hypertrophy from chronic aortic regurgitation. Am J Ther 1998;5(3): Rossi M. Pathologic fibrosis and connective tissue matrix in left ventricular hypertrophy due to chronic arterial hypertension in humans. J Hypertens 1998;16(7): Pangonytë D. Ischeminio miokardo kolageno matrikso pokyèiai. (Changes in matrix of collagen of ischemic myocardium.) Medicina (Kaunas) 1996;32 Suppl 3: Pangonytë D. Ikiinfarktinis kairiojo skilvelio miokardo fibrilinio kolageno remodeliavimasis. (Remodeling of fibrillar collagenous matrix of ischemic myocardium.) Lithuanian Journal of Cardiology 2001;8(2): Grimm D, Kromer EP, Bocker W, Bruckschledgel G, Holmer SR, Riegger GA, Schunkert H. Regulation of extracellular matrix proteins in pressure overload cardiac hypertrophy: effect of angiotensin converting enzyme inhibition. J Hypertens 1998;16(9): Burkauskienë A, Stropus R, Ðurna R, Pangonytë D, Vaièekauskas V. Deðiniojo prieðirdþio ausytës fibrilinio kolageno amþinë dinamika. (Age dynamics of fibrillar collagen of auricle of right atrium.) Lithuanian Journal of Cardiology 2002, 9(4): Burkauskienë A, Ðurna R. Video mikroskopiniai ðirdies morfologiniai tyrimai. (Videomicroscopic heart morphologic research.) In: Biomedicininë inþinerija. Kaunas; p Eghbali ME, Eghbali M, Robinson TF. Collagen accumulation in the heart ventricles as a function of growth and aging. Cardiovasc Res 1989;23(8): Bishop JE. Regulation of cardiovascular collagen deposition by mechanical forces. Mol Med Today 1998;4(2): Lijnen P, Petrov V. Renin angiotensin system, hypertrophy and gene expression in cardiac myocytes. J Mol Cell Cardiol 1999;31(5): Kahan T. The importance of left ventricular hypertrophy in human hypertension. J Hypertens. 1998;16 Suppl 7: Stalioraitytë E, Ptasekas R, Pangonytë D, et al. Patologinë anatomija (Pathologic anatomy.) Kaunas; Di Bello V, Pedrinelli R, Bianchi M, Giorgi D, Bertini A, Valenti G, et al. Ultrasonic myocardial texture in hypertensive mild-to-moderate left ventricular hypertrophy: a videodensitometric study. Amer J Hypertens 1998;11(2): Brilla CG, Scheer C, Rupp H. Renin-angiotensin system and myocardial collagen matrix: modulation of cardiac fibroblast function by angiotensin II type 1 receptor antagonism. J Hypertens 1997;15 Suppl 6: Burlew BS, Weber KT. Connective tissue and the heart. Functional significance and regulatory mechanisms. Cardiol Clin 2000;18(3): Ju H, Dixon IM. Extracellular matrix and cardiovascular diseases. Can J Cardiol 1996;12(12): Yoshihara F, Nishikimi T, Sasako Y, Hino J, et al. Plasma atrial natriuretic peptide concentration inversely correlates with left atrial collagen volume fraction in patients with atrial fibrillation: plasma ANP as a possible biochemical marker to predict the outcome of the maze. J Am Coll Cardiol 2002; 39(2): Van Den Berg MP, Crijns HJ, Van Veldhuisen DJ, Van Gelder IC, De Kam PJ, Lie KI. Atrial natriuretic peptide in patients with heart failure and chronic atrial fibrillation: role of duration of atrial fibrillation. Am Heart J 1998;135(2 Pt 1): Received 23 April 2004, accepted 28 June 2004 Straipsnis gautas , priimtas