Malignant Hyperthermia Susceptibility: Cardiac. Histomorphometry ofDogs and Young and Market-weight Swine. P.J. O'Brien, T.F. Fletcher, A.L. Metz, H.J. Kurtz,.
Malignant Hyperthermia Susceptibility: Cardiac Histomorphometry of Dogs and Young and Market-weight Swine P.J. O'Brien, T.F. Fletcher, A.L. Metz, H.J. Kurtz, B.K. Reed, W.E. Rempel, E.G. Clark and C.F. Louis*
ABSTRACT The defect causing malignant hyperthermia has been proposed to involve cardiac as well as skeletal muscle. We tested the hypothesis that histomorphometric parameters for ventricular wall from malignant hyperthermia-susceptible swine and dogs were abnormal. Hearts were obtained from: mature dogs, age- and weightmatched young swine (89 ± 15 days, 30 ± 3 kg); and market-weight swine (102± 10 kg). Using light microscopy, estimates were made for muscle nuclear dimensions and the volumefraction of nuclei, sarcoplasm, blood vessels, and interstitial space. Cardiac maturation in both MH and normal swine was accompanied by decreased myocyte volume-fraction due to decreased nuclear volume-fraction and increased interstitial space volumefraction. Sarcoplasm and vasculature volume-fraction were unchanged after maturation. Nuclear volume-fraction was slightly greater (p < 0.05) in the right ventricle than the left for malignant hyperthermia and normal swine. Myocyte nuclear dimensions were generally similar among animals. Dogs and the oldest group of swine were not significantly different. Myocytes of all swine contained multiple nuclei, closely spaced in rows of 2 to 12. In contrast, most myocytes of mature dogs apparently contained one or two nuclei. Histomorphometric values were not significantly different between normal and malignant hyperthermia young swine and dogs. However, within the market-weight swine,
volume-fraction for malignant hyperthermia myocytes and myocyte nuclei was decreased and interstitial space was increased compared to normal. These differences were attributed to the increased age of the malignant hyperthermia market-weight swine (177± 6 versus 249 ± 12 days). We concluded that cardiac histomorphometric parameters are normal in malignant hyperthermia-susceptible swine and dogs.
s'accompagna, tant chez les porcs susceptibles a l'hyperthermie maligne
que chez les normaux, d'une diminution de la proportion du volume des myocytes, attribuable a une diminution de la proportion du volume des noyaux et a une augmentation de la proportion du volume du tissu interstitiel. La proportion du volume du sarcoplasme et des vaisseaux sanguins s' avera semblable. La proportion du volume nucleaire se revela un peu plus elevee (p < 0,05) dans la paroi du Key words: Malignant hyperthermia, ventricule droit que dans celle du heart growth and development, histo- gauche, tant chez les porcs susceptibles logical techniques, swine diseases, dog a l'hyperthermie maligne que chez les diseases. normaux. Les dimensions des noyaux des myocytes s'averent generalement semblables, chez les sujets des deux en cause; elles n'accuserent pas especes RESUME de difference appreciable entre les Une hypothese veut que le defaut chiens et les porcs ayant atteint le responsable de l'hyperthermie maligne poids du marche. Les myocytes de implique le myocarde et les muscles tous les porcs contenaient plusieurs squelettiques. Les auteurs verifierent noyaux, entasses en rangees de deux a l'hypothese selon laquelle les para- 12. Par contre, la plupart des myocytes metres histomorphometriques de la des chiens adultes semblaient ne conparoi des ventricules cardiaques des tenir qu'un ou deux noyaux. Les porcs et des chiens susceptibles a valeurs histomorphometriques n'affil'hyperthermie maligne seraient anor- cherent pas de difference appreciable maux. Ils se procurerent a cette fin des entre les chiens et les jeunes porcs coeurs de chiens adultes et de jeunes normaux ou susceptibles a l'hyperporcs, d'age et de poids comparables thermie maligne. Chez les porcs ayant (89+ 15 jours et 30± 3 kg), ainsi que de atteint le poids du marche, la proporcs ayant atteint le poids du marche portion du volume des myocytes et (102±10 kg). Ils determinerent ensuite, de leurs noyaux, parmi les sujets a l'aide d'un microscope photonique, susceptibles a l'hyperthermie maligne, les dimensions des noyaux des fibres diminua toutefois en meme temps musculaires et le rapport entre le qu'augmentait l'espace interstitiel, convolume des noyaux, du sarcoplasme, trairement aux sujets normaux. Les des vaisseaux sanguins et du tissu auteurs attribuerent ces differences a interstitiel. La maturation cardiaque l'age plus avance des porcs susceptibles
*Department of Veterinary Biology (O'Brien, Fletcher, Reed, Louis), Department of Veterinary Pathobiology (Metz) and Department of Veterinary Diagnostic Investigation (Kurtz), College of Veterinary Medicine; Department of Animal Science (Rempel), College of Agriculture, University of Minnesota, St. Paul, Minnesota 55108 and Department of Veterinary Pathology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan S7N OWO (Clark). Present address of Dr. O'Brien: Department of Pathology, Ontario Veterinary College, University of Guelph, Guelph. Ontario N I G 2W 1. Supported in part by National Institutes of Health Grant GM-31382. Submitted November 25, 1985.
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Can J Vet Res 1987; 51: 50-55
rows. Preliminary observations (during an uncontrolled study) by one of us (H.K.) resulted in an impression of increased nuclei in hearts from rapidly-growing swine that had died due to M H. In addition, cardiac nuclei in these swine appeared abnormally large, pleomorphic, and closely spaced Mots cles: hyperthermie maligne, in rows. We therefore tested the croissance et developpement cardia- hypothesis that nuclear dimensions ques, techniques histologiques, mala- and the relative nuclear volume of hearts from MH-susceptible individdies porcines, maladies canines. uals were abnormal. Histomorphometric analysis was performed on both normal and MH-susceptible: dogs, age- and weight-matched young swine, and market-weight swine. INTRODUCTION MATERIALS AND METHODS Malignant hyperthermia (MH) is a Hearts were acquired from three hypermetabolic myopathy which is triggered in genetically-susceptible groups of animals: 1) eight mature individuals by potent, volatile anes- dogs, of which five were without thetics such as halothane, or by cardiovascular disease, and three depolarizing muscle relaxants such as had died from MH (12); 2) ten agesuccinylcholine (1). In swine, it is re- and weight-matched (89 ± 15 days, ferred to as porcine stress syndrome 30 ± 3kg) rapidly-growing swine because it may be triggered by exer- ("young" swine), of which five were tional, thermal, anoxic or mechanical normal Yorkshires, and five were Piestressors as well as anesthesia (2). trains which had been diagnosed as Although MH has a myogenic MH-susceptible using the halothane etiology, defects have been identified challenge test (4); and 3) eight marin MH erythrocytes (3,4) and have ket-weight (102± 10 kg) swine of which been postulated to occur in other MH five were normal Yorkshire x Duroc x tissues, including the heart. Cardiac Hampshire ("intermediate" age swine: dysfunction and degeneration are 177 ± 6 days), and three ("oldest" known to occur secondarily to the swine: 249 ± 12 days) were from a herd massive increase in blood catechola- of Pietrains of which approximately mines, as well as the hyperkalemia, 90% were homozygous for the MH acidosis, hypoxia and hyperthermia genetic defect (3). which occur during MH (5,6). Portions of left and right ventricuThat a primary defect occurs in M H lar free wall were fixed in 10% neutral hearts has been proposed based on the buffered formalin. Longitudinally- and similar nature of cardiac and skeletal transversely-oriented muscle was emmuscle, and on observations of non- bedded in paraffin, sectioned at a specific abnormalities in electrocar- thickness of 5 to 6 microns, and stained diograms (7), angiograms and thal- with hematoxylin and eosin. An array of sampling sites was lium -201 scans (8), and ventricular endomyocardial biopsy specimens (9) obtained by randomly placing each of MH people. In addition, swine with tissue slide onto graph paper and MH-susceptibility have been reported marking the slide with a felt-tip pen to have abnormally small heart at each point where line intersections weights, when they are expressed per were overlain with tissue. This procedure was used to generate a minimum unit muscle weight (10). Abnormalities of nuclei in skeletal of 24 sampling sites per animal. Under muscle of MH humans ( 11), swine low power (x 100), a tissue field, which (12), and dogs (13) have been reported: was adjacent to each marked point, increased total number of nuclei, was located. Then, under oil immerincreased number of internal nuclei, sion (x1000), the entire tissue field and occurrence of multiple internal was brought into focus. By this means nuclei arranged closely together in the tissue was optically thin-sectioned a I'hyperthermie maligne ayant atteint le poids du marche (177 ± 6 versus 249 ± 12 jours). Ils conclurent que les parametres histomorphometriques cardiaques sont normaux, chez les porcs et les chiens non susceptibles a I'hyperthermie maligne.
in order to minimize bias due to section thickness (Holmes effect, 14). An ocular containing a graticule with 12 right angles (points) was turned blindly. The tissue constituents which were both in focus and superimposed at points of the graticule were then tallied. Thus, for each tissue slide, a point-fraction was determined for myocyte nuclei and sarcoplasm, interstitial space (intermyofiber space was assumed to represent interstitial space, see Figure 1), and blood vessel lumen or wall. Assuming that tissue sections are representative samples of ventricular wall, the point-fraction is an unbiased estimate of volume-fraction (VV%, 14). Volume fraction is expressed as percent in this paper (VV%). The ventricular wall volume was defined as the sum of the volumes of: nuclei and sarcoplasm, the interstitial space, and the blood vessels. A similar sampling procedure was used to measure the major and minor axes of 20 nuclei per tissue slide. As additional sampling qualifications, muscle fibers had to be longitudinally oriented throughout the field of view and the nucleus had to be entirely within the thickness of the tissue section as determined by focusing above, through, and below the nucleus. The so-qualified nucleus, which was closest to the center of the field, was selected for measurement. Its largest profile was brought into focus, and major and minor axes were measured to the nearest 0.6 ,um, by means of an ocular scale graticule. In order to estimate nuclear volume, the nucleus was modeled as a prolate spheroid, a structure for which volume is generated by rotation of the elliptical profile about its major axis. Nuclear volume was computed by: V = (4/3) a x b2, where V = nuclear volume, a = major semiaxis and b = minor
semiaxis.
Histomorphometric data were analyzed statistically. For differences between right and left ventricles, a paired, two-tailed Student's t-test was performed using the 14 swine (9 young and 5 intermediate) for which these data were available. Data for right and left ventricles were combined for comparisons of estimates for MH versus normal swine or dogs, young versus intermediate swine, intermediate versus oldest swine, and oldest swine versus dogs, using two-tailed Student's 51
D ax ci;Ex r ; S0P^gfg Fig. 1. Ventricular myocardium from young normal swine (A), young malignant hyperthermia-susceptible swine (B), market-weight (oldest) malignant hyperthermia-susceptible swine (C) and mature dogs (D). H & E. X340.
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t-tests. Statistical computations were TABLE 1. Histomorphometric Parameters of Heart Ventricular Wall Estimated for Dogs and performed on a computer using SPSS Variably Aged Swine (Statistical Package for the Social ~Swine Mature Oldest Young Sciences, 15). Parameter Young Intermediate Dogs (n 3) (n10) (n 8) 5) ~~(n RESULTS Vessel Vv% 3.3 1.2 2.8 0.4 3.2 1.5 2.0 ± 1.1 Myocyte nuclei and sarcoplasm, Interstitial space Vv% 9.7 ±2.0' 12.8 ±2.6d 17.7 ±3.5 16.7 ± 8.3 interstitial space and vessels could be Myocyte Vv% 87.4 ±2.0c 84.4 ±3.1c 79.1 ±2.5 81.2 ±8.2 discerned with the hematoxylin and Sarcoplasm VV% 78.3 ±2.5 80.3 ±2.9 77.1 ±2.4 78.6 ±8.7 eosin stain. In all cases, heart tissue Muscle nucleus VV% 9.1 ± ISa 4.1 ± 1.4c 2.0 ±0.2 2.7 ± 1.1 appeared histologically normal. No Nuclear width (Mm) 4.9 ± 0.3a 4.2 ± 0.2 4.7 ± 0.6 4.5 ± 0.3 differences were apparent between Nuclear length (Aim) 10.8 ±0.5 11.3 ±0.6 11.3 ± 1.1 11.5 ±0.6 right and left ventricular walls. In the Nuclear volume (Am3) 138 18a 103 15 134 35 120 17 young swine, nuclei appeared more Data are expressed as x ± SD numerous and more variable in size Vv% = volume-fraction expressed as percent and shape, compared to the other Significant differences between adjacent means at ap < 0.002, bp < 0.02, cp < 0.03, dp < 0.06 hearts. In these young swine, myocyte nuclei were typically aligned closelyspaced in rows of 2 to 12. In intermediate and oldest swine, nuclear rows appeared to have fewer, more widelyspaced nuclei. Rows of nuclei were 100] not observed in mature dog hearts. -~90] Nuclear size, morphology, number, and arrangement appeared similar for MH versus normal age-matched Q)) 80] swine, or dogs (Fig. 1). C) 70
Porcine Heart Ventricular Wall Differences Due To Aae
HISTOMORPHOM FTRY
Right and left ventricular walls were compared histomorphometrically for four M H and five normal young swine, as well as five normal intermediate-age swine. Right versus left side differences were not statistically significant, except for nuclear VV% (p < 0.05), which was slightly greater in the right ventricle than the left (8.0± 3.4 versus 6.9 +
2.6%). Comparison of ventricular wall data in MH versus control animals revealed no significant differences for young swine or for dogs. However, comparison of the MH oldest swine with the normal intermediate age swine revealed several significant differences corresponding to age-related features. Age accounted for the principal morphometric differences found in ventricular wall (Table I). In both dogs and the oldest swine, the ventricular wall volume was composed of approx-
imately 80% myocytes, 17% interstitial space and 2-3% vessels. The myocyte nuclear VV% was about 2%. However, in the intermediate-age and young swine there was a progressive increase (as age decreased) in myocyte VV%, decrease in interstitial space VV%, and increase in myocyte nuclear VV%
Young
Medidte AGE
Oldest
Fig. 2. Porcine heart ventricular wall differences due to age.
(Table I). The significant differences among swine revealed distinct agerelated trends (Fig. 2). Cardiac maturation in swine was associated with a decreased VV% of myocytes due to decreased nuclear VV%, and an increased VV% of interstitial space. However, sarcoplasmic and vascular VV% remained constant during maturation. Myocyte nuclear size and shape were generally similar among the hearts studied. Nuclear length was not significantly different between groups, and averaged I 1.1 ± 0.63 ,IAm for the 26
animals. Nuclear diameter was not significantly different among dogs, young swine and oldest swine. However, nuclei of intermediate-age swine were significantly narrower than those of young swine in this study (Table I).
DISCUSSION Morphometric estimates of tissue compartments in heart ventricular wall were necessarily compromised in this study by distortion due to fixation and paraffin embedment, as well as the 53
limitations due to the staining method and magnification employed. However, valid comparison among animals can be made based on the assumption that tissue compartments are similarly altered among animals. H istomorphometric analysis indicated that nuclear dimensions were similar for animals, but young swine seemed to have rounder, more voluminous nuclei. The nuclear size similarity contrasts with the subjective impression of nuclear pleomorphism in market-weight swine and more so in young swine. This disparity may be due to hypernucleosis, that is, myocyte multinucleation and/ or decreased interstitium VV% (16), which is likely to increase the probability of observing fragments of nuclei in sectioned tissue. Our preliminary observation of apparently increased nuclear size in M H swine was not supported by histomorphometric analysis. A principal finding in this study was the histological manifestations of maturation in swine ventricular wall. The more mature larger ventricle has a higher VV% of interstitial space and, consequently a lower density of myocytes. The decrement of myocyte VV% was accounted for by a three to fourfold decreased nuclear VV%. This study is supportive of the report by Bishop and Hine (17) indicating that cardiac nuclear VV% decreased approximately tenfold during growth of dogs from birth to maturity and the observations of increased frequency of double nuclei or rows of nuclei, in rapidly-growing children (16) and dogs (17). The VV% of blood vessels, sarcoplasm, and presumably myofibrils, is apparently conserved during maturation. Moreover, these VV% are similar in mature individuals of disparate species, such as swine and dogs. Conservation of the VV% of blood vessels and myofibrils during growth, may be a reflection of their critical role in cardiac function. The VV% of interstitium (interstitial space plus vasculature), sarcoplasm, and nuclei for hearts of both mature dogs and oldest swine were 19-21%, 79-8 1%, and 2-3%, respectively. These compare favorably with average values reported for left ventricles of mature rats, 18%, 81%, and I %, respectively (18). 54
The left ventricular wall has a greater volume than the right, corresponding to the greater pressure demand on the left ventricle. By analogy with growth of the whole heart, the absolute volume difference between left and right ventricles may be considered to be a reflection of differential growth. Consistent with a differential growth effect, we found a significant decrease in nuclear volumedensity in the left ventricular wall and a similar sarcoplasmic density for both sides. Myocytes and interstitial space Vv% were not significantly different between sides. Thus, the differential growth of ventricle sides was subtle relative to that for different age swine, and it reflects a difference in ventricular size rather than age. A size-effect on cardiac nuclear VV% was reported by Astorri et al (20), who showed that increased weights of left ventricles of adult human hearts was correlated with lower nuclear VV%. However, in our study, maturation effects were correlated more with age than size. Histomorphometric data were similar for hearts from age- and weight-matched young swine, whereas data from weight-matched market-swine with different ages were dissimilar. The lower nuclear Vv% of the MH oldest swine compared to the normal intermediate-age swine is apparently due to age and not M H-susceptibility. Nuclear VV% were similar for MH compared to normal for both young swine and dogs. Furthermore, differences between M H and normal market swine are not due to differences in heart size, since heart weight per unit body weight is similar for MH and normal swine, although MH heart weights are less than normal when expressed per unit muscle weight (10). Also, smaller sized hearts would be expected to have an increase, rather than a decrease, in nuclear VV% (19). Thus, we conclude that differences between the MH and normal market swine are due to age and not differences in MH-susceptibility or heart size. Cardiac maturation involves cell hyperplasia, which is generally thought to cease during the early neonatal period, followed by hypertrophy (17). Initially in the hypertrophic growth phase, multi-nucleated myocytes are
present due to the occurrence of karyokinesis without cytokinesis. In hearts from dogs, rats and people, these nuclei occur in pairs or occasionally in rows of up to four (I 7,19). This hypernucleosis has been proposed ( 17) to occur during rapid hypertrophic growth in response to intense protein synthetic demands. Histomorphometric changes occurring during maturation (this study) are similar to those found during hypertrophy of adult rat hearts due to renal hypertension (18); viz. decreased nuclear VV%, compensatory increased interstitium VV%, and constant sarcoplasmic VV%. Furthermore, increased frequency of myocyte binucleation has been reported (16) in hypertrophied, adult human hearts. In addition, 100% increases in the number of myocytes and connective tissue cells may occur during hypertrophy of the adult human heart (19-21). However, the magnitude of the changes occurring in adult hearts due to hypertrophy are much less than those which occur during maturation. That swine have a greater rate of cardiac growth than dogs or people, is suggested by the greater length of rows of nuclei in swine (17,19). We found increased numbers of multinucleated cardiac myocytes in the oldest swine (versus mature dogs), despite their having nuclear VV% similar to those of mature dog hearts. That swine cardiac myocytes are multinucleated with up to 32 nuclei in a few has been well described in the German literature since 1900. This multinucleation was attributed to inhibition of cytokinesis (22). Greater cardiac growth demands are probably associated with the lower heart to body weight ratios of modern, domestic, market-weight swine (10,23,24) compared to adult people (16) or dogs (17),(0.27-0.32% versus 0.4-0.7% and 0.8-1%, respectively) and compared to mature wild pigs (0.6%, 24) or domestic, marketweight swine of the last quarter of the 19th century (0.5%, 24). Furthermore, swine hearts may be compromised by having a primitive coronary vascular system (25) and large myocyte diameters (26). A reasonable interpretation of the
morphometric changes during maturation is that growth demand for an enlarged ventricular wall is met by
hypertrophy: karyokinesis followed by elongation and increased girth of sarcoplasm. Thus, sarcoplasmic VV% is maintained, but nuclear VV% decreases, thereby causing decreases in myocyte VV% during maturation. This interpretation is supported by the report of similar changes occurring during hypertrophy of adult rat hearts (19). That myocyte elongation entrapped additional interstitial space, thereby compensating for decreased myocyte VV% due to loss of nuclear VV%, was suggested by the finding that the sum of interstitial space and nuclear VV% was approximately constant among animals (19 ± 4.1%). We concluded that cardiac histological features are not abnormal in MH-susceptible animals. No evidence was found for a primary defect in cardiac muscle of swine or dogs. The differences between MH and normal market-weight swine could be accounted for entirely by age. This dramatizes the importance of using age-matched controls when examining cardiac muscle for histological abnormalities. Swine with MH-susceptibility have a higher probability of premature death. Therefore, they are more likely to die with hypernucleotic hearts than normal swine. However, at market-weight, because MH-susceptible swine (26-29), especially Pietrains (26,29, see Materials and Methods of this report), have slower growth rates, they may have lower myocyte and nuclear VV%, and higher interstitial space VV%. In any event, cardiac histological features apparently have no potential as diagnostic indicators of M H-susceptibility status in either dogs or swine.
ACKNOWLEDGMENTS The helpful assistance generously provided by Drs. T.P. O'Leary, C.M. Czarnecki and W. Hartman, College of Veterinary Medicine, University of Minnesota is gratefully acknowledged. Dr. J.E. Edwards, United Hospitals, St. Paul and Dr. R.B. Estensen, Medical School, University of Minnesota are thanked for their valuable advice.
REFERENCES 1. ELLIS FR, HEFFRON JJA. Clinical and biochemical aspects of malignant hyperpyrexia. Recent Adv Anaesth Analg 1985; 15: 173-207. 2. CASSENS RG, MARPLE CN, EIKELENBOOM G. Animal physiology and meat quality. Adv Food Sci 1980; 24: 71-155. 3. O'BRIEN PJ, FORSYTH GW, OLEXSON DW, THATTE HS, ADDIS PB. Canine malignant hyperthermia susceptibility: erythrocytic defects - osmotic fragility, glucose-6-phosphate dehydrogenase deficiency and abnormal Ca2+ homeostasis. Can J Comp Med 1984; 48: 381-389. 4. O'BRIEN PJ, ROONEY MT, REIK TR, THATTE HS, REMPEL WE, ADDIS PB, LOUIS CF. Porcine malignant hyperthermia susceptibility: erythrocytic osmotic fragility. AmJ Vet Res 1985; 46: 1451-1456. 5. GRONERT GA. Malignant hyperthermia. Anesthesiology 1980; 53: 395-423. 6. JOHANSSON G, OLSSON K, HAGGENDAL J, JONSSON L, THORENTOLLING K. Effect of stress on myocardial cells and blood levels of catecholamines in normal and amygdalectomized pigs Can J Comp Med 1982; 46: 176-182. 7. HUCKELL VF, STANILOFF HM, BRITT BA, MORCH JE. Electrocardiographic abnormalities associated with malignant hyperthermia susceptibility. J Electrocardiol 1982; 15: 137-142. 8. HUCKELL VF, STANILOFF HM, BRITT BA, WAXMAN MB, MORCH JE. Cardiac manifestations of malignant hyperthermia susceptibility. Circulation 1978; 58: 916-925. 9. MAMBO NC, SILVER MD, McLAUGHLIN PR, HUCKELL VF, McEWAN PM, BRITT BA, MORCH JE. Maligant hyperthermia susceptibility. A light and electron microscopic study of endomyocardial biopsy specimens from nine patients. Human Pathol 1980; 11: 381-388. 10. ELIZONDO G, ADDIS PB, REMPEL WE, MADERO C, MARTIN FB, ANDERSON DB, MARPLE DN. Stress response and muscle properties in Pietrain (P). Minnesota No. I (M) and PXM pigs. J Anim Sci 1976; 43: 1004-1014. 11. ISAACS H, FRERE G, MITCHELL J. Histological, histochemical and ultramicroscopic findings in muscle biopsies form carriers of the trait for malignant hyperpyrexia. Br J Anaesth 1973; 45: 860-868. 12. O'BRIEN PJ, CRIBB PH, WHITE RJ, OLFERT ED, STEISS JE. Canine malignant hyperthermia: diagnosis of susceptibility in a breeding colony. Can Vet J 1983; 24: 172-177. 13. HWANG PT, McGRATH CJ, ADDIS PB, REMPEL WE, THOMPSON EW, ANTONIK A. Blood creatine kinase as a predictor of the porcine stress syndrome. J Anim Sci 1978; 47: 630-633. 14. WEIBEL ER. Steriological methods. Vol. 1. Practical methods for biological morphometry. London: Academic Press, 1979. 15. NIE NH, HULL CH, JENKINS JG STEINBRENNER K, BENT DH. Statistical package for the social sciences (SPSS). 2nd ed. Montreal: McGraw-Hill, 1975.
16. BAROLDI G, FALZI G, LAMPERTICO P. The nuclear patterns of the cardiac muscle fiber. Cardiologia 1967; 51: 109-123. 17. BISHOP SP, HINE P. Cardiac muscle cytoplasmic and nuclear development during canine neonatal growth. In: Roy P-E, Harris P, eds. Recent advances in studies on cardiac structure and metabolism. Vol. 8. The cardiac sarcoplasm. Baltimore, Maryland: University Park Press, 1975: 77-98. 18. ANVERSA P, LOUD AV, GIACOMELLI F, WIENER J. Absolute morphometric studv of myocardial hypertrophy in experimental hypertension. 11. Ultrastructure of myocytes and interstitium. Lab Invest 1978: 38: 597-607. 19. ASTORRI E, BOLOGNESI R, COLLA B, CHIZZOLA A, VISIOLI 0. Left ventricular hypertrophy: a cytometric study on 42 human hearts. J Mol Cell Cardiol 1977; 9: 763-775. 20. ADLER CP, FRIEDBURG H. Myocardial DNA content, ploidy level and cell number in geriatric hearts: postmortem examination of human myocardium in old age. J Mol Cell Cardiol 1986: 18: 39-53. 21. SANDRITTER W, ADLER CP. Numerical hyperplasia in human heart hypertrophy. Experientia 1971; 27: 1435-1437. 22. LANGES K, ARNOLD G, PFITZER P. Postnatal DNA-syntheses and mitoses in hearts of dwarf pigs. J Mol Cell Cardiol 1983; 15: 831-844. 23. UNSHELM J, HOHNS H, OLDIGS B, RUHL B. Physiological and morphological studies in pigs of different types bred and different body sizes. In: Hessel-de Heer JCM, Schmidt GR, Sybesma W, Van Der Wal PG, eds. Proceedings of the Second International Symposium on Condition and Meat Quality of Pigs. Wageningen. The Netherlands: Centre for Agriculture Publishing and Documentation, 1971: 171-179. 24. ENGELHARDT WV. Swine cardiovascular physiology - a review. In: Bustad LK, McLellan, eds. Swine in Biomedical Research 1966: 307-329. 25. JONSSON L, JOHANSSON G, LANNEK N, LINDBERG P. Intramural blood supply of porcine heart. A postmortem angiographic studv. Anat Rec 1974; 178: 647-656. 26. UNSHELM J. Review of papers Section 3: Transport. Physiological and morphological factors inducing stress-susceptibility in pigs. In: Hessel-de HeerJCM, Schmidt GR, Sybesma W, Van Der Wal PG, eds. Proceedings of the Second International Symposium on Condition and Meat Quality of Pigs. Wageningen. The Netherlands: Centre for Agriculture Publishing and Documentation, 1971: 171-179. 27. WEBB AJ. The halothane test: a practical method of eliminating porcine stress syndrome. Vet Rec 1980; 106: 410-412. 28. JENSEN P, BARTON-GADE PA. Performance and carcass characteristics of pigs with known genotypes for halothane susceptibility. In: Ludigsen JB, ed. Stress susceptibility and meat quality in pigs. EAAP Publication No. 33, 1985: 80-87. 29. METZ SHM, DEKKER RA. The contribution to the regulation of fat deposition in growing Large White and Pietrain pigs. Anim Prod 1981: 33: 149-157.
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