J. Vet. Med. A 39,321-327 (1992) 0 1992 Paul Parey Scientific Publishers, Berlin and Hamburg ISSN 0931- 184X
Departments of Veterinary Diagnosis, Pathology and Anatomy and Physiology, College of Veterinary Medicine, and Department of Statistics, Kansas State University, Manhattan, KS 66506
Histochemical and Morphometric Studies of Peripheral Muscle in Bovine Progressive Degenerative Myeloencephalopathy of Brown Swiss Cattle R. OYSTER, H. W. LEIPOLD,D. TROYER, W. CASHand D. JOHNSON Corresponding author: H. W. LEIPOLD, Department of Pathology, VCS Building, Kansas State University, Manhattan, KS 66506
With 2 figures and 2 tables (Received for publicatwn August 21, 1991)
Summary Histochemical and morphometric analysis of selected skeletal muscles was performed on 14 pure bred, Brown Swiss cattle. Nine cattle were clinically affected with bovine progressive degenerative myeloencephalopathy (BPDME) while five served as controls. Statistically significant trend differences were not observed for the parameters of mean cross sectional area, and mean fiber type percentages for types, I, IIA, and IIB fibers between affected and control test groups. In general, patterns of hypertrophy or atrophy, fiber type grouping, fiber type predominance, or fiber cross sectional profile alteration were not observed in the muscles examined from affected cattle. The findings suggest that BPDME, or weaver syndrome, ist not a muscular dystrophy and that muscle pathology is not a primary part of the syndrome nor would muscle pathology be expected to contribute significantly to the clinical signs of the disease.
Introduction Bovine progressive degenerative myeloencephalopathy (BPDME) (or *weaver syndrome’’) is a disease of pure bred Brown Swiss cattle characterized by progressive hind et al., limb ataxia and weakness that eventually leads to permanent recumbency (OYSTER 1991a, b, c; STUART and LEIPOLD,1983,1985). Reported here are the results of histochemical and morphometric studies of selected skeletal muscles from a group of nine affected and five control cattle used in a concentrated neuromuscular investigation of this syndrome (OYSTER et al., 1991a, b, c). The main purpose of this study was to identify and tabulate the fiber types and morphometric parameters in selected skeletal muscles from cattle affected with BPDME, compare them to parameters derived from a group of normal control cattle, and report any significant alterations from normal that would correlate with a primary myopathic or neurogenic disorder of skeletal muscle. Material and Methods Animals Fourteenpure bredBrownSwisscattleranginginagefrom6 to 78monthswereutilizedinthisstudy. US.Copyright Clearance Center Code Smtmmt:
0931-184X/92/3905 -0321$02.50/0
322
OYSTER, LEIPOLD, TROYER, CASHand JOHNSON
The criteria necessary for a diagnosis of bovine progressive degenerative myeloencephalopathy et al., 1991a, b, c; STUARTand LEIPOLD,1983, 1985). These have been well documented (OYSTER criteria, along with data collected from extensive clinical, laboratory, and electrophysiological testing, as well as histopathological and electron microscopic examinations were utilized in the placement of cattle into test groups. This resulted in nine affected and five control cattle. Histochemistry and Fiber Typing At necropsy, muscle samples from the midbelly region were taken from triceps brachii, longissimus dorsi, iliopsoas, rectus femoris, vastus lateralis, gluteobiceps, semitendinosus, semimembranosus, peroneus tertius, and the lateral head of the gastrocnemius. The fresh skeletal muscle samples were covered with gauze moistened with physiological saline and held at 20 degrees Celsius until processed. Cooled samples were transported from the necropsy facility to the histochemistry laboratory (Vertebrate Morphology Laboratory, Dept. of Anatomy and Physiology, College of Veterinary Medicine, KSU), where they were trimmed with razor blades into 1 cm blocks, oriented transversely, and mounted on cork squares utilizing frozen tissue embedding media (Histo PrepTM, Frozen Tissue Embedding Media, Fisher Diagnostics, Fischer-Scientific, Orangeburg, New York) as an adhesive. These samples were then snap frozen by direct immersion for approximately 10 seconds into isopentane (2-methyl-butane, Fisher-Scientific Company, Chemical Manufacturing Division, Fairlawn, New Jersey) cooled to roughly minus 120 degrees Celsius in liquid nitrogen. Frozen samples were placed on dry ice to allow the iso-pentane to evaporate. They were then placed into heat-sealed, air-tight plastic containers, labelled, and stored in an ultra-low freezer at minus 70 degrees Celsius until sectioning was performed. In preparation for sectioning, muscle blocks were placed in a cryostat chamber maintained at minus 20 degrees Celsius for temperature equilibration. Sections were cut at 12 micron thickness with a Slee Cryostat (Slee Manufacturing Company, London, England). A combination histochemical stain using the nicotinic acid dehydrogenase-tetrazolium reductase technique (NADH/TR) and the acid stable adenosinetriphosphatase procedure (ATPase) was utilized to allow for the simultaneous determination of type I, IIA, and IIB muscle fibers on one slide preparation (TROYERet al., 1990). Morphometry Morphometric analysis and counting of individual fiber types was accomplished with the use of an integrated image analysis system (Micro Comp, Southern Micro Instruments, Inc., Atlanta, Georgia). It utilized the attributes of light microscopy, video camera images, and computer technology to produce a video monitor color image of cross sections of histochemically stained muscle fibers, which could be counted and measured with the help of planar morphometry computer software. Between 200 and 300 individual fibers were evaluated from each muscle. Cross-sectional areas of individual muscle fibers and percentages of each histochemical fiber type were recorded for each muscle sampled. Numbers of fiber type were expressed as a percentage of the total number of fibers counted. Statistics Statistical analysis of the data was initiated in order to be able to make inferences concerning parameter differences between control and principal groups. Mean cross-sectional area values and their standard errors ( S . E.) for each fiber type from each muscle sampled were computed for control animals and affected cattle (Table 1). A weighted analysis of variance comparing the means for the affected and control groups was performed utilizing a general linear models procedure (KSU Computing Center, General Linear Models Procedure Software from S.A.S. Institute, Inc., Cary, North Carolina) and least square mean values were computed. The arcsin of muscle fiber type percentages for each fiber type within each muscle sampled was computed (Table 2). Utilizing these arcsin values, composite means for each muscle fiber type within each muscle sampled for control and affected groups were compared utilizing the same general linear models procedure described above. P-values generated from this procedure were utilized to make inferences concerning numerical differences in data between control and affected groups.
Results Histochemistry The combined histochemical staining technique utilized in this study resulted in delineation of the three major fiber types on a single slide. Type I fibers stained black. Type
Histochemical and Morphometric Studies of Peripheral Muscle
323
Fig. 1. Vastus lateralis muscle from affected calf R-88-484 stained with a combination histochemical technique to sequentially delineate type I (dark or black stained), type IIA (intermediate stained), and type IIB (unstained) fibers. NADH-TR/Acid ATP-ase stain, x 88
IIB fibers were relatively unreactive to the oxidative and acid-stable ATPase stains utilized in this procedure. They were either clear and unstained or took on the light blue color of the hematoxylin counterstain. Type IIA fibers were identified by their dark blue color, which was especially concentrated in the mitochondria-rich periphery of the fiber. Cross sections of the various muscles stained with this combination procedure revealed the presence of a normal mosaic of fiber types, with uniform distribution of the fiber types within the muscle fasciculus. Fiber type grouping or predominance was not observed in samples collected from any of the muscles studied. Qualitative differences between control and affected groups were not observed in the cross-sectional fiber type profiles of any of the muscles sampled (Figs. 1-2).
Fig. 2. Vastus lateralis muscle from control calf R-88-489 stained with a combination histochemical technique to sequentially delineate type I (dark or black stained), type IIA (intermediate stained), and type IIB (unstained) fibers. Vastus lateralis muscle, NADH-TR/Acid A n - a s e stain, x 88
324
OYSTER, LEIPOLD,TROYER, CASHand JOHNSON Table 1. Muscle Fiber Cross Sectional Area Statistics
'Muscle
Type
ZTrt
'Mean
S.E.
n
p-value
TRI
I
A
1058.69 1465.32 1492.05 1825.56 2080.22 2508.14 1026.67 1312.39 1570.36 2058.44 2713.27 3426.76 1337.29 1727.65 1591.62 2074.03 2817.54 3781.03 1265.27 1345.93 2 123.09 2296.49 3366.19 3762.24 1130.75 1280.40 1600.25 221 1.34 2136.61 3206.45 804.83 1503.24 1462.73 2006.26 2461.56 3172.48 1046.98 1326.68 1690.85 1892.16 2716.83 3400.85 1264.31 1485.93 1405.23 1797.34 2770.64 2934.04 1433.12 1694.79 2076.36 1892.45 3351.03 3159.63 1181.50 1560.06 1679.72 1736.05 2887.13 3119.42
139.09 251.84 144.79 195.16 224.44 316.19 145.04 211.17 151.79 239.37 217.15 326.22 80.67 114.25 109.09 205.46 212.28 416.23 190.32 263.53 204.91 348.06 379.64 509.97 106.27 168.90 124.52 247.55 196.53 308.36 146.37 404.24 124.64 174.55 300.69 406.66 124.00 355.24 150.06 430.53 188.88 455.50 141.62 259.06 130.35 227.81 282.52 377.34 126.53 193.12 170.81 278.19 295.80 405.13 117.16 224.25 65.46 100.07 230.01 257.83
7 5 7 5 7 5 8 5 8 5 8 5 9 5 9 5 9 5 7 3 7 3 7 3 8 5 8 5 8 5 7 5 7 5 7 5 8 2 8 2 8 2 8 5 8 5 8 5 9 4 9 4 9 4 6 4 6 4 6 4
0.1879
TRI
IIA
TRI
IIB
LD
I
LD
IIA
LD
IIB
IP
I
IP
IIA
IP
IIB
RF
I
RF
IIA
RF
IIB
VL
I
VL
IIA
VL
IIB
GB
I
GB
IIA
GB
IIB
ST
I
ST
IIA
ST
IIB
SM
I
SM
IIA
SM
IIB
PT
I
PT
IIA
PT
IIB
GA
I
GA
IIA
GA
IIB
C A C A C A C A C A C
A C A C A C A C A C A
C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A
C A C
0.1999 0.2956 0.2885 0.1130 0.0822 0.0163 0.0603 0.0615 0.8103 0.6790 0.5507 0.4690 0.0496 0.0138 0.1353 0.0297 0.1901 0.4785 0.6705 0.2028 0.4686 0.1633 0.7354 0.2812 0.5845 0.7101 0.1730 0.6502 0.5203
'Muscle Abbreviations: TRI - triceps brachii, L D - longissimus dorsi, IP - iliopsoas, RF - rectus femoris, VL - vastus lateralis, GB - gluteobiceps, SM - semimembranosus, ST - semitendinosus, PT - peroneus tertius, GA - gastrocnemius; ZTrt: C - control, A - affected; 3Mean: values are expressed in square microns.
325
Histochemical and Morphometric Studies of Peripheral Muscle Table 2. Muscle Fiber Type Percentage Statistics ~~
‘Muscle
Type
ZTn
3 Mean
4Arcsinmean
n
p-value
TRI
I IIA
TRI
IIB
LD
I
LD
IIA
LD
IIB
IP
I
IP
IIA
IP
IIB
RF
I
RF
IIA
36.12 33.97 24.23 25.22 40.17 40.89 33.51 35.80 23.36 18.02 43.59 46.30 43.84 50.74 25.67 20.94 31.21 28.88 23.03 21.35 30.48 26.30 46.75 52.59 30.35 22.39 32.30 24.62 37.89 53.14 31.10 24.71 33.09 32.23 36.08 43.53 28.73 20.44 22.40 18.53 50.04 61.18 22.73 17.93 37.38 34.52 40.15 49.26 34.21 34.16 23.21 19.47 43.07 46.58 28.15 26.60 32.54 20.42 39.46 54.03
0.36955 0.34660 0.24469 0.25498 0.41341 0.42130 0.34176 0.36612 0.23578 0.18114 0.45102 0.48143 0.45386 0.53214 0.25965 0.21092 0.3 174 1 0.29300 0.23237 0.21512 0.30977 0.26608 0.48649 0.55381 0.30834 0.22586 0.32886 0.24873 0.38858 0.56020 0.31628 0.24964 0.33727 0.32817 0.36910 0.45036 0.29137 0.20580 0.22593 0.18634 0.52410 0.65830 0.22935 0.1 8032 0.38307 0.35249 0.41314 0.5 1505 0.34917 0.34865 0.23423 0.19599 0.44524 0.48449 0.2 8540 0.26925 0.33139 0.20565 0.40565 0.57075
7 5 7 5 7 5 8 5 8 5 8 5 9 5 9 5 9 5 7 3 7
0.6332
TRI
A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C
RF
IIB
VL
I
VL
IIA
VL
IIB
GB
I
GB
IIA
GB
IIB
ST
I
ST
IIA
ST
IIB
SM
I
SM
IIA
SM
IIB
PT
I
PT
IIA
PT
IIB
GA
I
GA
IIA
GA
IIB
0.8597 0.8868 0.6879 0.1678 0.5125 0.3665 0.4165 0.5918 0.7977 0.4839
3 7 3 8 5 8
0.1815 0.0567 0.1960
5 8 5 7 5 7 5 7 5 8 2 8 2 8 2 8 5 8 5 8 5 9 4 9 4 9 4 6 4 6 4 6 4
0.0109 0.1340 0.8781 0.1757 0.4072 0.6461 0.3969 0.3205 0.6490 0.2737 0.9920 0.3544 0.5934 0.6825 0.0634 0.1088 ~
‘Muscle Abbreviations: TRI - triceps brachii, L D - longissimus dorsi, IP - iliopsoas, RF - R C N S femoris, VL - vastus lateralis, GB - gluteobiceps, SM - semimembranosus, ST - semitendinosus, PT - peroneus tenius, GA - gastrocnemius; ZTn: C - control, A - affected; 3Mean: values are expressed in square microns; ‘Arcsinmean: mathematical arcsin of the mean percentage.
326
OYSTER, LEIPOLD,TROYER, CASHand JOHNSON
Fiber Typing In general, fiber type counts for each muscle, expressed as a percentage of the total number of fibers counted from each muscle, did not vary significantly between control and affected groups as a whole at the 0.05 level of significance (Tables 1 and 2). The percentage of type IIB fibers within the vastus lateralis muscles of affected animals was significantly less than the percentage for this muscle in the control group at the 0.05 level of significance. The percentage of type I fibers in the vastus lateralis muscle in affected cattle was greater than the percentage observed in that muscle in control animals at the 0.05 level of significance. This would imply a possible correlated shift in muscle fibers within the vastus lateralis muscle from type IIB to type I fibers in affected cattle. This was considered an isolated finding and was not representative of the findings in the other muscles compared between test groups. Morphomety In general, no significant differences in cross-sectional areas of each fiber type from each muscle tested were detected between affected and control groups. However, significantly smaller size was noted at the 0.05 level of significance for the type IIA fibers of the gluteobiceps, type I fibers of the iliopsoas, and type IIA and type IIB fibers in the vastus lateralis muscles of affected animals when statistically compared to mean fiber type sizes of the same muscles in control animals. These differences were isolated in nature and did not represent a trend of atrophy of any one specific fiber type in muscles sampled from affected animals. Discussion Histochemical processing of muscle samples is a useful tool allowing for the differentiation of primary muscle lesions (myopathic) from muscle lesions occurring secondary to nerve damage (neuropathic or neurogenic) (MCGAVIN,1983). Skeletal muscle samples processed histochemically are quite well suited for morphometric studies which in turn allow for the comparison of the size and number of each muscle fiber type within each muscle sampled from a single individual and between different individuals (BENNINGTON and KRUPP,1984; SWASHand SCHWARTZ, 1985). Definitive evidence of myopathic or neurogenic lesions was not observed in the skeletal muscles of test cattle in this study. Statistical analysis of the morphometrical and histochemical data in this study did not suggest the presence of generalized trends supportive of selective atrophy or hypertrophy of a particular fiber type or alteration of fiber type percentage profiles that would support muscle pathology as a primary part of this disease. The significant differences in cross sectional area and fiber type percentage between affected and control animals noted in Tables 1 and 2 were isolated, incidental, and were not considered significant in the overall interpretation of the data. Microscopic examination did not provide evidence consistent with generalized fiber type grouping, fiber type predominance, or myopathic lesions of muscular dystrophy. In addition, muscle lesions reported in earlier studies (LEIPOLD et al., 1973; STUART and LEIPOLD,1985) of this disease were not observed in skeletal muscle examined from affected test cattle in this study. The absence of skeletal muscle pathology in this histochemical and morphological investigation suggests that BPDME or weaver syndrome is not a muscular dystrophy. Electromyograms did not support myopathic disease in these cattle (OYSTER et al., 1991c). Muscle pathology is not a primary part of the syndrome nor would muscle pathology be expected to contribute significantly to the clinical signs of this disease. Acknowledgement This research was supported by the National Association of Animal Breeders, Columbia, MO, and was part of the Regional Project NC-2. Contribution no. 92-100-5 from the Kansas Agricultural Experiment Station.
Histochemical and Morphometric Studies of Peripheral Muscle
327
References BENNINGTON, J. L., and M. KRUPP, 1984: Morphometric analysis of muscle. In: R. R. Heffner, Jr. (Ed.), Muscle Pathology. Churchill Livingstone, New York. LEIPOLD,H. W., B. BLAUGH,K. HUSTON, C. G. EDGERLY, and C. M. HIBBS, 1973: Weaver syndrome in Brown Swiss cattle: Clinical signs and pathology. Vet. Med. Small A i m . Clin. 68,645-647. MCGAVIN,M. D., 1983: Muscle biopsy in veterinary practice. In: J. W. Alexander, and R. E. Roberts (Eds.), Veterinary Clincs of North America: Small Animal Practice, Symposium on Orthopedic Diseases. W. B. Saunders, Philadelphia 135-144. OYSTER, R., H. W. LEIPOLD,D. TROYER, and W. CASH,1991 a: Clinical studies of bovine progressive degenerative myeloencephalopathy of Brown Swiss cattle. Progress Vet. Neurol. 2, 159- 165. OYSTER, R., H.W. LEIPOLD,D.TROYER, and W.CASH, D.JOHNSON,and H.D. STOWE,1991b: Laboratory Studies of bovine progressive degenerative myeloencephalopathy in Brown Swiss cattle. Bovine Pract. 26, 77-83. OYSTER, R., W. CASH, D.TROYER, J. E. VESTWEBER, H. W. LEIPOLD,and D. JOHNSON,1991 c: Electrophysiological studies in bovine progressive degenerative rnyeloencephalopathy of Brown Swiss cattle. Progress in Vet. Neurol. 2, 243-251. STUART,L. D.,, and H. W. LEIPOLD,1983: Bovine progressive degenerative myeloencephalopathy rweaver”) of Brown Swiss cattle 11: Clinical and laboratory findings. Bovine Pract. 18, 133- 146. STUART,L. D.,, and H. W. LEIPOLD,1985: Lesions in bovine progressive degenerative myeloencephalopathy (-weaver”) of Brown Swiss cattle. Vet. Pathol. 22, 13-23. 1984: Biopsy Pathology of Muscle. Chapman and Hall, London, SWASH,M., and M. S. SCHWARTZ, England. TROYER, D. L., R. A. OYSTER, and H. W. LEIPOLD,1990: A rapid and reliable sequential staining method for bovine muscle. Anat. Histol. Embryol. 19, 154-157.
Departments of Veterinary Diagnosis, Pathology and Anatomy and Physiology, College of Veterinary Medicine, and Department of Statistics, Kansas State University, Manhattan, KS 66506
Histochemical and Morphometric Studies of Peripheral Muscle in Bovine Progressive Degenerative Myeloencephalopathy of Brown Swiss Cattle R. OYSTER, H. W. LEIPOLD,D. TROYER, W. CASHand D. JOHNSON Corresponding author: H. W. LEIPOLD, Department of Pathology, VCS Building, Kansas State University, Manhattan, KS 66506
With 2 figures and 2 tables (Received for publicatwn August 21, 1991)
Summary Histochemical and morphometric analysis of selected skeletal muscles was performed on 14 pure bred, Brown Swiss cattle. Nine cattle were clinically affected with bovine progressive degenerative myeloencephalopathy (BPDME) while five served as controls. Statistically significant trend differences were not observed for the parameters of mean cross sectional area, and mean fiber type percentages for types, I, IIA, and IIB fibers between affected and control test groups. In general, patterns of hypertrophy or atrophy, fiber type grouping, fiber type predominance, or fiber cross sectional profile alteration were not observed in the muscles examined from affected cattle. The findings suggest that BPDME, or weaver syndrome, ist not a muscular dystrophy and that muscle pathology is not a primary part of the syndrome nor would muscle pathology be expected to contribute significantly to the clinical signs of the disease.
Introduction Bovine progressive degenerative myeloencephalopathy (BPDME) (or *weaver syndrome’’) is a disease of pure bred Brown Swiss cattle characterized by progressive hind et al., limb ataxia and weakness that eventually leads to permanent recumbency (OYSTER 1991a, b, c; STUART and LEIPOLD,1983,1985). Reported here are the results of histochemical and morphometric studies of selected skeletal muscles from a group of nine affected and five control cattle used in a concentrated neuromuscular investigation of this syndrome (OYSTER et al., 1991a, b, c). The main purpose of this study was to identify and tabulate the fiber types and morphometric parameters in selected skeletal muscles from cattle affected with BPDME, compare them to parameters derived from a group of normal control cattle, and report any significant alterations from normal that would correlate with a primary myopathic or neurogenic disorder of skeletal muscle. Material and Methods Animals Fourteenpure bredBrownSwisscattleranginginagefrom6 to 78monthswereutilizedinthisstudy. US.Copyright Clearance Center Code Smtmmt:
0931-184X/92/3905 -0321$02.50/0
322
OYSTER, LEIPOLD, TROYER, CASHand JOHNSON
The criteria necessary for a diagnosis of bovine progressive degenerative myeloencephalopathy et al., 1991a, b, c; STUARTand LEIPOLD,1983, 1985). These have been well documented (OYSTER criteria, along with data collected from extensive clinical, laboratory, and electrophysiological testing, as well as histopathological and electron microscopic examinations were utilized in the placement of cattle into test groups. This resulted in nine affected and five control cattle. Histochemistry and Fiber Typing At necropsy, muscle samples from the midbelly region were taken from triceps brachii, longissimus dorsi, iliopsoas, rectus femoris, vastus lateralis, gluteobiceps, semitendinosus, semimembranosus, peroneus tertius, and the lateral head of the gastrocnemius. The fresh skeletal muscle samples were covered with gauze moistened with physiological saline and held at 20 degrees Celsius until processed. Cooled samples were transported from the necropsy facility to the histochemistry laboratory (Vertebrate Morphology Laboratory, Dept. of Anatomy and Physiology, College of Veterinary Medicine, KSU), where they were trimmed with razor blades into 1 cm blocks, oriented transversely, and mounted on cork squares utilizing frozen tissue embedding media (Histo PrepTM, Frozen Tissue Embedding Media, Fisher Diagnostics, Fischer-Scientific, Orangeburg, New York) as an adhesive. These samples were then snap frozen by direct immersion for approximately 10 seconds into isopentane (2-methyl-butane, Fisher-Scientific Company, Chemical Manufacturing Division, Fairlawn, New Jersey) cooled to roughly minus 120 degrees Celsius in liquid nitrogen. Frozen samples were placed on dry ice to allow the iso-pentane to evaporate. They were then placed into heat-sealed, air-tight plastic containers, labelled, and stored in an ultra-low freezer at minus 70 degrees Celsius until sectioning was performed. In preparation for sectioning, muscle blocks were placed in a cryostat chamber maintained at minus 20 degrees Celsius for temperature equilibration. Sections were cut at 12 micron thickness with a Slee Cryostat (Slee Manufacturing Company, London, England). A combination histochemical stain using the nicotinic acid dehydrogenase-tetrazolium reductase technique (NADH/TR) and the acid stable adenosinetriphosphatase procedure (ATPase) was utilized to allow for the simultaneous determination of type I, IIA, and IIB muscle fibers on one slide preparation (TROYERet al., 1990). Morphometry Morphometric analysis and counting of individual fiber types was accomplished with the use of an integrated image analysis system (Micro Comp, Southern Micro Instruments, Inc., Atlanta, Georgia). It utilized the attributes of light microscopy, video camera images, and computer technology to produce a video monitor color image of cross sections of histochemically stained muscle fibers, which could be counted and measured with the help of planar morphometry computer software. Between 200 and 300 individual fibers were evaluated from each muscle. Cross-sectional areas of individual muscle fibers and percentages of each histochemical fiber type were recorded for each muscle sampled. Numbers of fiber type were expressed as a percentage of the total number of fibers counted. Statistics Statistical analysis of the data was initiated in order to be able to make inferences concerning parameter differences between control and principal groups. Mean cross-sectional area values and their standard errors ( S . E.) for each fiber type from each muscle sampled were computed for control animals and affected cattle (Table 1). A weighted analysis of variance comparing the means for the affected and control groups was performed utilizing a general linear models procedure (KSU Computing Center, General Linear Models Procedure Software from S.A.S. Institute, Inc., Cary, North Carolina) and least square mean values were computed. The arcsin of muscle fiber type percentages for each fiber type within each muscle sampled was computed (Table 2). Utilizing these arcsin values, composite means for each muscle fiber type within each muscle sampled for control and affected groups were compared utilizing the same general linear models procedure described above. P-values generated from this procedure were utilized to make inferences concerning numerical differences in data between control and affected groups.
Results Histochemistry The combined histochemical staining technique utilized in this study resulted in delineation of the three major fiber types on a single slide. Type I fibers stained black. Type
Histochemical and Morphometric Studies of Peripheral Muscle
323
Fig. 1. Vastus lateralis muscle from affected calf R-88-484 stained with a combination histochemical technique to sequentially delineate type I (dark or black stained), type IIA (intermediate stained), and type IIB (unstained) fibers. NADH-TR/Acid ATP-ase stain, x 88
IIB fibers were relatively unreactive to the oxidative and acid-stable ATPase stains utilized in this procedure. They were either clear and unstained or took on the light blue color of the hematoxylin counterstain. Type IIA fibers were identified by their dark blue color, which was especially concentrated in the mitochondria-rich periphery of the fiber. Cross sections of the various muscles stained with this combination procedure revealed the presence of a normal mosaic of fiber types, with uniform distribution of the fiber types within the muscle fasciculus. Fiber type grouping or predominance was not observed in samples collected from any of the muscles studied. Qualitative differences between control and affected groups were not observed in the cross-sectional fiber type profiles of any of the muscles sampled (Figs. 1-2).
Fig. 2. Vastus lateralis muscle from control calf R-88-489 stained with a combination histochemical technique to sequentially delineate type I (dark or black stained), type IIA (intermediate stained), and type IIB (unstained) fibers. Vastus lateralis muscle, NADH-TR/Acid A n - a s e stain, x 88
324
OYSTER, LEIPOLD,TROYER, CASHand JOHNSON Table 1. Muscle Fiber Cross Sectional Area Statistics
'Muscle
Type
ZTrt
'Mean
S.E.
n
p-value
TRI
I
A
1058.69 1465.32 1492.05 1825.56 2080.22 2508.14 1026.67 1312.39 1570.36 2058.44 2713.27 3426.76 1337.29 1727.65 1591.62 2074.03 2817.54 3781.03 1265.27 1345.93 2 123.09 2296.49 3366.19 3762.24 1130.75 1280.40 1600.25 221 1.34 2136.61 3206.45 804.83 1503.24 1462.73 2006.26 2461.56 3172.48 1046.98 1326.68 1690.85 1892.16 2716.83 3400.85 1264.31 1485.93 1405.23 1797.34 2770.64 2934.04 1433.12 1694.79 2076.36 1892.45 3351.03 3159.63 1181.50 1560.06 1679.72 1736.05 2887.13 3119.42
139.09 251.84 144.79 195.16 224.44 316.19 145.04 211.17 151.79 239.37 217.15 326.22 80.67 114.25 109.09 205.46 212.28 416.23 190.32 263.53 204.91 348.06 379.64 509.97 106.27 168.90 124.52 247.55 196.53 308.36 146.37 404.24 124.64 174.55 300.69 406.66 124.00 355.24 150.06 430.53 188.88 455.50 141.62 259.06 130.35 227.81 282.52 377.34 126.53 193.12 170.81 278.19 295.80 405.13 117.16 224.25 65.46 100.07 230.01 257.83
7 5 7 5 7 5 8 5 8 5 8 5 9 5 9 5 9 5 7 3 7 3 7 3 8 5 8 5 8 5 7 5 7 5 7 5 8 2 8 2 8 2 8 5 8 5 8 5 9 4 9 4 9 4 6 4 6 4 6 4
0.1879
TRI
IIA
TRI
IIB
LD
I
LD
IIA
LD
IIB
IP
I
IP
IIA
IP
IIB
RF
I
RF
IIA
RF
IIB
VL
I
VL
IIA
VL
IIB
GB
I
GB
IIA
GB
IIB
ST
I
ST
IIA
ST
IIB
SM
I
SM
IIA
SM
IIB
PT
I
PT
IIA
PT
IIB
GA
I
GA
IIA
GA
IIB
C A C A C A C A C A C
A C A C A C A C A C A
C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A
C A C
0.1999 0.2956 0.2885 0.1130 0.0822 0.0163 0.0603 0.0615 0.8103 0.6790 0.5507 0.4690 0.0496 0.0138 0.1353 0.0297 0.1901 0.4785 0.6705 0.2028 0.4686 0.1633 0.7354 0.2812 0.5845 0.7101 0.1730 0.6502 0.5203
'Muscle Abbreviations: TRI - triceps brachii, L D - longissimus dorsi, IP - iliopsoas, RF - rectus femoris, VL - vastus lateralis, GB - gluteobiceps, SM - semimembranosus, ST - semitendinosus, PT - peroneus tertius, GA - gastrocnemius; ZTrt: C - control, A - affected; 3Mean: values are expressed in square microns.
325
Histochemical and Morphometric Studies of Peripheral Muscle Table 2. Muscle Fiber Type Percentage Statistics ~~
‘Muscle
Type
ZTn
3 Mean
4Arcsinmean
n
p-value
TRI
I IIA
TRI
IIB
LD
I
LD
IIA
LD
IIB
IP
I
IP
IIA
IP
IIB
RF
I
RF
IIA
36.12 33.97 24.23 25.22 40.17 40.89 33.51 35.80 23.36 18.02 43.59 46.30 43.84 50.74 25.67 20.94 31.21 28.88 23.03 21.35 30.48 26.30 46.75 52.59 30.35 22.39 32.30 24.62 37.89 53.14 31.10 24.71 33.09 32.23 36.08 43.53 28.73 20.44 22.40 18.53 50.04 61.18 22.73 17.93 37.38 34.52 40.15 49.26 34.21 34.16 23.21 19.47 43.07 46.58 28.15 26.60 32.54 20.42 39.46 54.03
0.36955 0.34660 0.24469 0.25498 0.41341 0.42130 0.34176 0.36612 0.23578 0.18114 0.45102 0.48143 0.45386 0.53214 0.25965 0.21092 0.3 174 1 0.29300 0.23237 0.21512 0.30977 0.26608 0.48649 0.55381 0.30834 0.22586 0.32886 0.24873 0.38858 0.56020 0.31628 0.24964 0.33727 0.32817 0.36910 0.45036 0.29137 0.20580 0.22593 0.18634 0.52410 0.65830 0.22935 0.1 8032 0.38307 0.35249 0.41314 0.5 1505 0.34917 0.34865 0.23423 0.19599 0.44524 0.48449 0.2 8540 0.26925 0.33139 0.20565 0.40565 0.57075
7 5 7 5 7 5 8 5 8 5 8 5 9 5 9 5 9 5 7 3 7
0.6332
TRI
A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C A C
RF
IIB
VL
I
VL
IIA
VL
IIB
GB
I
GB
IIA
GB
IIB
ST
I
ST
IIA
ST
IIB
SM
I
SM
IIA
SM
IIB
PT
I
PT
IIA
PT
IIB
GA
I
GA
IIA
GA
IIB
0.8597 0.8868 0.6879 0.1678 0.5125 0.3665 0.4165 0.5918 0.7977 0.4839
3 7 3 8 5 8
0.1815 0.0567 0.1960
5 8 5 7 5 7 5 7 5 8 2 8 2 8 2 8 5 8 5 8 5 9 4 9 4 9 4 6 4 6 4 6 4
0.0109 0.1340 0.8781 0.1757 0.4072 0.6461 0.3969 0.3205 0.6490 0.2737 0.9920 0.3544 0.5934 0.6825 0.0634 0.1088 ~
‘Muscle Abbreviations: TRI - triceps brachii, L D - longissimus dorsi, IP - iliopsoas, RF - R C N S femoris, VL - vastus lateralis, GB - gluteobiceps, SM - semimembranosus, ST - semitendinosus, PT - peroneus tenius, GA - gastrocnemius; ZTn: C - control, A - affected; 3Mean: values are expressed in square microns; ‘Arcsinmean: mathematical arcsin of the mean percentage.
326
OYSTER, LEIPOLD,TROYER, CASHand JOHNSON
Fiber Typing In general, fiber type counts for each muscle, expressed as a percentage of the total number of fibers counted from each muscle, did not vary significantly between control and affected groups as a whole at the 0.05 level of significance (Tables 1 and 2). The percentage of type IIB fibers within the vastus lateralis muscles of affected animals was significantly less than the percentage for this muscle in the control group at the 0.05 level of significance. The percentage of type I fibers in the vastus lateralis muscle in affected cattle was greater than the percentage observed in that muscle in control animals at the 0.05 level of significance. This would imply a possible correlated shift in muscle fibers within the vastus lateralis muscle from type IIB to type I fibers in affected cattle. This was considered an isolated finding and was not representative of the findings in the other muscles compared between test groups. Morphomety In general, no significant differences in cross-sectional areas of each fiber type from each muscle tested were detected between affected and control groups. However, significantly smaller size was noted at the 0.05 level of significance for the type IIA fibers of the gluteobiceps, type I fibers of the iliopsoas, and type IIA and type IIB fibers in the vastus lateralis muscles of affected animals when statistically compared to mean fiber type sizes of the same muscles in control animals. These differences were isolated in nature and did not represent a trend of atrophy of any one specific fiber type in muscles sampled from affected animals. Discussion Histochemical processing of muscle samples is a useful tool allowing for the differentiation of primary muscle lesions (myopathic) from muscle lesions occurring secondary to nerve damage (neuropathic or neurogenic) (MCGAVIN,1983). Skeletal muscle samples processed histochemically are quite well suited for morphometric studies which in turn allow for the comparison of the size and number of each muscle fiber type within each muscle sampled from a single individual and between different individuals (BENNINGTON and KRUPP,1984; SWASHand SCHWARTZ, 1985). Definitive evidence of myopathic or neurogenic lesions was not observed in the skeletal muscles of test cattle in this study. Statistical analysis of the morphometrical and histochemical data in this study did not suggest the presence of generalized trends supportive of selective atrophy or hypertrophy of a particular fiber type or alteration of fiber type percentage profiles that would support muscle pathology as a primary part of this disease. The significant differences in cross sectional area and fiber type percentage between affected and control animals noted in Tables 1 and 2 were isolated, incidental, and were not considered significant in the overall interpretation of the data. Microscopic examination did not provide evidence consistent with generalized fiber type grouping, fiber type predominance, or myopathic lesions of muscular dystrophy. In addition, muscle lesions reported in earlier studies (LEIPOLD et al., 1973; STUART and LEIPOLD,1985) of this disease were not observed in skeletal muscle examined from affected test cattle in this study. The absence of skeletal muscle pathology in this histochemical and morphological investigation suggests that BPDME or weaver syndrome is not a muscular dystrophy. Electromyograms did not support myopathic disease in these cattle (OYSTER et al., 1991c). Muscle pathology is not a primary part of the syndrome nor would muscle pathology be expected to contribute significantly to the clinical signs of this disease. Acknowledgement This research was supported by the National Association of Animal Breeders, Columbia, MO, and was part of the Regional Project NC-2. Contribution no. 92-100-5 from the Kansas Agricultural Experiment Station.
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327
References BENNINGTON, J. L., and M. KRUPP, 1984: Morphometric analysis of muscle. In: R. R. Heffner, Jr. (Ed.), Muscle Pathology. Churchill Livingstone, New York. LEIPOLD,H. W., B. BLAUGH,K. HUSTON, C. G. EDGERLY, and C. M. HIBBS, 1973: Weaver syndrome in Brown Swiss cattle: Clinical signs and pathology. Vet. Med. Small A i m . Clin. 68,645-647. MCGAVIN,M. D., 1983: Muscle biopsy in veterinary practice. In: J. W. Alexander, and R. E. Roberts (Eds.), Veterinary Clincs of North America: Small Animal Practice, Symposium on Orthopedic Diseases. W. B. Saunders, Philadelphia 135-144. OYSTER, R., H. W. LEIPOLD,D. TROYER, and W. CASH,1991 a: Clinical studies of bovine progressive degenerative myeloencephalopathy of Brown Swiss cattle. Progress Vet. Neurol. 2, 159- 165. OYSTER, R., H.W. LEIPOLD,D.TROYER, and W.CASH, D.JOHNSON,and H.D. STOWE,1991b: Laboratory Studies of bovine progressive degenerative myeloencephalopathy in Brown Swiss cattle. Bovine Pract. 26, 77-83. OYSTER, R., W. CASH, D.TROYER, J. E. VESTWEBER, H. W. LEIPOLD,and D. JOHNSON,1991 c: Electrophysiological studies in bovine progressive degenerative rnyeloencephalopathy of Brown Swiss cattle. Progress in Vet. Neurol. 2, 243-251. STUART,L. D.,, and H. W. LEIPOLD,1983: Bovine progressive degenerative myeloencephalopathy rweaver”) of Brown Swiss cattle 11: Clinical and laboratory findings. Bovine Pract. 18, 133- 146. STUART,L. D.,, and H. W. LEIPOLD,1985: Lesions in bovine progressive degenerative myeloencephalopathy (-weaver”) of Brown Swiss cattle. Vet. Pathol. 22, 13-23. 1984: Biopsy Pathology of Muscle. Chapman and Hall, London, SWASH,M., and M. S. SCHWARTZ, England. TROYER, D. L., R. A. OYSTER, and H. W. LEIPOLD,1990: A rapid and reliable sequential staining method for bovine muscle. Anat. Histol. Embryol. 19, 154-157.
