Bovine Morphometry

J. Comp. Path. 2004, Vol. 130, 235–245 Morphometry of Bovine Dilated Cardiomyopathy P. Nart, A. Williams, H. Thompson and G. T. Innocent* Department of Veterinary Pathology, *Comparative Epidemiology and Informatics Group, Institute of Comparative Medicine, Glasgow University Veterinary School, Bearsden Road, Glasgow G61 1QH, UK Summary Bovine dilated cardiomyopathy (BDCM) is a primary disease of the myocardium that has been described in Holstein-Friesian cattle w
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  Morphometry of Bovine Dilated Cardiomyopathy  P. Nart, A. Williams, H. Thompson and G. T. Innocent  *  Department of Veterinary Pathology, * Comparative Epidemiology and Informatics Group, Institute of Comparative Medicine, Glasgow University Veterinary School, Bearsden Road, Glasgow G61 1QH, UK  Summary  Bovine dilated cardiomyopathy (BDCM) is a primary disease of the myocardium that has been describedin Holstein-Friesian cattle worldwide in the last 20 years. The principal morphological changes in themyocardium are interstitial fibrosis and increased variability in cardiomyocyte size. Sections of heart muscle from nine cases of BDCM and nine unaffected controls matched for age, sex and breed werestudied by means of a computer-assisted image analyser to measure the degree of fibrosis, and thecardiomyocyte cellular and nuclear cross-sectional area and length. The amount of connective tissue inthe hearts of BDCM cases was increased by 6.7 times, the nuclear transverse cross-sectional area by 1.9times, and the cardiomyocyte length and cross-sectional area by 1.7 and 1.6 times, respectively. Thisresulted in an estimated 2.5-fold increase in mean cardiomyocyte volume. Animals with clinical signs of BDCM showed a mean loss of 51% of the total number of cardiomyocytes as compared with controls. Of the five parameters studied, the percentage of fibrosis was found to be the most consistent discriminatorfor BDCM. It is possible that the degree offibrosis could be used to distinguish BDCM from other cardiacdiseases of cattle. q 2003 Elsevier Ltd. All rights reserved. Keywords:  cardiomyocyte morphometry; cardiomyopathy; cattle; myocardium Introduction The first published report of bovine dilatedcardiomyopathy (BDCM) in Holstein-Friesiancattle came from Japan (Sonoda et al  ., 1982). It  was also reported in Switzerland (Martig et al  .,1982), Canada (where cases had been recordedsince 1971;Baird et al  ., 1986), Sweden (Olsson,1987), Australia (McLennan and Kelly, 1990), the United Kingdom (Bradley  et al  ., 1991) and Den-mark (Leifsson et al  ., 1994). The main clinical signsof BDCM are those associated with severe con-gestive heart failure, and the gross pathologicalfindings include a dilated heart, congested liver, ventral subcutaneous oedema and ascites or hydro-thorax. Histologically, the heart shows diffusefibrosis,andthecardiomyocytesfocaldegeneration,extensive vacuolation and variability in calibre(Bradley  et al  ., 1991). The aetiology of BDCM inthe Holstein-Friesian breed is still unknown, but Dolf  et al  . (1998)proposed that an autosomalrecessive gene might play a role. Dilated cardio-myopathy (DCM) in man may also follow a familialpattern (Towbin and Bowles, 2002) and BDCM hasbeen proposed as a model of human DCM becauseof similarities in clinical features and pattern of protein expression (Eschenhagen et al  ., 1995; Weekes et al  ., 1999).Morphometry is the process of producing, fromimages of two-dimensional histological sections,measurements from which three-dimensional volume, surface area, and number and length of tissue components can be determined. The tissue’sfunctional capacity can then be estimated by quantifying the volume fraction of the parenchymaand stroma and their proportional relationships(Loud and Anversa, 1984). Morphometric studies  J. Comp. Path. 2004, Vol. 130, 235–245–9975/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.doi: 10.1016/j.jcpa.2003.11.002Correspondence to: A. Williams, Department of Pathology andInfectious Diseases, Royal Veterinary College, Hawkshead Campus,Hawkshead Lane, North Mymms, Hertfordshire AL9 7TA, UK.  can be undertaken with a light or an electronmicroscope and the data obtained either by computer-assisted image analysis or by the moretraditional point-counting method with a squaregrid. In the latter, intersections of the grid(‘points’) falling on features of interest arecounted and expressed either as a percentage oras a proportion of total points. However, computer-assisted image analysis measures larger areas in ashorter time (Beltrami et al  ., 1996) and the resultsagree closely with those obtained by the classic‘point counting’ method (Porzio et al  ., 1995); it istherefore often the method of choice.Morphometric analysis of human DCM hasdemonstrated increased fibrosis, cardiomyocytehypertrophy and fibre elongation. Estimates of the degreeoffibrosisinDCMinmanrangefrom 14to 20%, as compared with a baseline of 3% incontrol hearts (Dick et al  ., 1982; Unverferth et al  .,1986; Beltrami et al  ., 1995; Ohtani et al  ., 1995).Studies of cardiomyocyte length in DCM showedincreases of 40% (Gerdes, 1992) and 59% (Bel- trami et al  ., 1995) in man and of 35% (Spinale et al  .,1991) and 50% (Kajstura et al  ., 1995) in exper-imental models of pacemaker-induced DCM indogs. Cardiomyocyte hypertrophy, which may bemeasured as increases in width, diameter or cross-sectional area, has been reported in human DCMas 50% (Rowan et al  ., 1988), 30% (Dick et al  ., 1982;Unverferth et al  ., 1986) and 20% (Beltrami et al  .,1995). Doubling of the nuclear cross-sectional area was reported by Rowan et al  . (1988), and anincrease in cardiomyocyte nuclear size was relatedby Figulla et al  . (1985) and Pelliccia et al  . (1994)todecreased functional status and poor prognosis.Left ventricular failure was associated with anestimated 39% loss of ventricular cardiomyocytes(Kajstura et al  ., 1995), and with a positive corre-lation between the extent of cardiomyocyte lossand cellular hypertrophy in samples taken at necropsy. However, there appeared to be little if any association between histological changes seenon cardiac biopsy in human DCM and clinical signsand prognosis (Baandrup et al  ., 1981); this may reflect the fact that biopsies are usually taken fromthe right ventricle, whereas cardiac function ismore dependentontheleft ventricle.Nevertheless,the degree offibrosis is correlated with a reductionin the ejection fraction or contractibility of theheart (Schwarz et al  ., 1983; Ohtani et al  ., 1995).Cardiomyocyte hypertrophy and interstitialfibrosis are the main qualitative morphologicalfindings in both human and bovine DCM (Robin-son and Ferrans, 1975; Furuoka et al  ., 2001).However, no previous studies have quantifiedcardiomyocyte hypertrophy and interstitial fibrosisin BDCM. The purpose of the present study,therefore, was to determine the main quantitativemorphological changes in BDCM by objectivestatistical analysis and to identify the most consist-entandrelevanthistologicalfindingsofthiscardiacdisorder. The total numbers of cardiomyocytes inthe hearts of animals with BDCM and controlanimals were estimated from the measurements inan attempt to understand the pathological mech-anisms of this disease. Materials and Methods Tissues Examined  Samples from the outer (free) wall of the left  ventricle of the heart were collected from nineHolstein-Friesian cattle (nos 1–9) in which BDCMhad been diagnosed at the Glasgow University  Veterinary School Pathology Department over aperiod of 9 years. The diagnosis was made on thebasis of clinical findings and gross pathology,followed by histopathological confirmation. Con-trol material was obtained from apparently normalhearts of nine unaffected animals (nos 10–18) –matched for age, sex and breed with the diseasedanimals – slaughtered at an abattoir.To estimate the degree of fibrosis in control andaffected hearts, longitudinal sections of myocar-dium stained with Sirius red and picric acid( Junqueira et al  ., 1979) were examined by light microscopy, and a ‘bit map’ image was developedfrom the srcinal image by means of the KS 300 V.3software package (Image Associates/Zeiss; Ober-kochen, Germany). The percentage of fibrosis wascalculated by comparing the proportion of fibroustissue (Sirius red positive,Fig. 1a; recognized as white in the bit map image) with the total of alltissue (black and white) (Fig. 1b).Estimations of cardiomyocyte area, nuclear areaand nuclear length were made from sectionsstained by haematoxylin and eosin (HE) (Bancroft and Stevens, 1996). Areas with transverse sectionsof myofibres were selected. The contour of thefibres was then drawn manually (as shown inFig.1c). The same method was used for nuclear areaand nuclear length. Estimates of cardiomyocytelength were made on sections stained with phos-photungstic acid haematoxylin (PTAH), whichclearly demonstrates intercalated discs (Bancroft and Stevens, 1996) (Fig. 1d). Quantitative measure- ments of area and length were taken automatically by a multipurpose colour image processor with theKS 300 V.3 software package. Two recorded P. Nart  et al. 236  command sequences (macros) were created foranalysing data. Numbers of Measurements Made and Statistical Analysis   A pilot study was first undertaken to determine thenumber of observations required to distinguishbetween affected and control animals in respect of the various cardiomyocyte parameters. Six samplestaken from each of four healthy animals and allnine cases of BDCM were measured for degree of fibrosis and cardiomyocyte cross-sectional area. Application of Bartlett’s test (Paradine and Rivett,1960) indicated that although the values forunaffected animals were homoscedastic (equal variance), the values for the animals with BDCMhad variances that were different from the healthy animals and from each other. Standard tests fordifferences between groups require that all groupshave the same variance. Due to the difficulties incalculating power when the variances between and within groups vary, the variances in the pilot study  were used to calculate minimum significant differ-ences in a t  -test with Welch’s correction forheteroscedasticity. The numbers of observations were chosen with a significance level of  P  , 0 : 05 inmind. Thus, 30 measurements of cardiomyocytelength, nuclear length and nuclear area and fivemeasurementsoffibrosisper animalwerejudgedtobe sufficient to distinguish between groups. Cross-sectional areas of 60 cardiomyocytes per animal Fig. 1a–d. (a) Sirius red staining of myocardium of case 1 showing fibrous tissue stained red. (b) Computer-generated bit map of Fig. 1arendered black and white to enable the computer to calculate the percentage of fibrosis (now white). (c) HE-stained section of the myofibres of case 1 in transverse section, and the hand drawn contours to delineate cross-sectionalarea. (d) PTAH staining of cardiomyocytes in longitudinal section with hand-drawn line between intercalated discs. Bar,200 m m (a,b); 20 m m (c); 100 m m (d). Bovine Dilated Cardiomyopathy  237   were measured, however, because of the greater variability of this parameter.Histological sections from all 18 animals werethen sampled systematically, since systematicsampling yields smaller errors than randomsampling in histometrics (Ebbeson and Tang,1967). In the present study, constraints arisingfromtherandomorientationofthecardiomyocytes within the section were overcome by carefulselection of appropriate fields (i.e., those in which the myocytes were aligned transversely orlongitudinally to the section plane), thereby ensur-ing accurate systematic measurements. Since thepilot study had identified the heteroscedasticnature of the data, and since repeated measures were taken for each animal, a method of analysis was required that was suitable for such data. Onesuch method is that of iterative generalized least squares, a generalization of the least squaresmethod for normally distributed data, whichprovides maximum likelihood estimators for multi-level mixed-effects models (Goldstein, 1986, 1989;Ihaka and Gentleman, 1996). This is implementedin the ‘gls’ function in the ‘R’ statistical packagethat was used to analyse the data arising from themorphometrical analyses in this study (Ihaka andGentleman, 1996). In the statistical tests, signifi-cance was set at the 5% level.Theanalysesofmeasurementsofeachparameterfor each animal were then converted into a‘boxplot’ representation (seeFigs 2–6). In theseboxplots, the extremes of the box indicate the 25thand75thquartiles.Valuesthatweremorethantwice(vertical lines, ending with an open horizontal bar)the interquartile range away from the median wereconsideredoutliersandmarkedwithanopencircle.The‘whiskers’ateachendoftheplotsextendtotheextremes of the non-outlier data. The traverse lineacross the boxplot represents the median value.Statistical analysis was used to determine if there were significant differences between diseased andcontrol animals for the five parameters studied. Animals with BDCM that appeared to differ lessmarkedly from the controls than the rest of theaffected group were then tested to determine if they were indeed significantly different from thecontrols. The sub-groups derived from comparingthese animals against the control groupare marked with a filled triangle or filled circle that indicatesthe value of the mean for that particular sub-group.The heart weight was recorded in four BDCMcases and four controls. The heart weights were Fig. 2. Percentage offibrosis. Vertical axis:fibrosis as percentageof thetotal area examined inhistological sections. Horizontal axis:bovine dilated cardiomyopathy (BDCM) cases (animals 1–9); controls (animals 10–18). A subgroup composed of animals 6and 9 (triangles) was also compared with the control group. The horizontal dotted lines represent the weighted mean of theanimals with BDCM and of control animals. P. Nart  et al. 238
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