MORPHOMETRlC STUDY OF SKELETAL MUSCLE ULTRASTRUCTURE ANDREW G. ENGEL, MD, T. SANTA, MD, H. H. STONNINGTON, MD, F. JERUSALEM, MD, M. TSUJIHATA, MD, A. K. W. BROWNELL, MD, H. SAKAKIBARA, MD, B. Q. BANKER, MD, K. SAHASHI, MD, and E. H. LAMBERT, MD
One of the features of the IVth International Congress on Neuromuscmlar Diseases, heId in Montreal September 17-2 1, 1978, \$.as a series of invited posters. In order to achieve a wider audience for this valuable material, we have made arrangements to publish a number of these posters in ~ M U S L l3 ~ P i V m ~ ?To ~ . preserve the original effect of t h e posters, they arc being reproduced almost exactly as they appeared at the Congress, without editing o r other alterations. We hope you will find this feature a useful addition to your Journal. Editor’s Note:
From the Department of Neurology Mayo Clinic Rochester, Minnesota Address reprint requests to Or Engel at the Department of Neurology, Mayo Clqnic, Rochester MN 55901
0148-639)60203/0229$00 OO/O 1979 Mayo Clinic
MUSCLE & NERVE
ELECTRON MICROGRAPHS OF SKELETAL MUSCLE REPRESENTING MUSCLE FIBERS, MOTOR ENDPLATES, INTRAMUSCULAR BLOOD VESSELS AND NERVES CAN BE QUANTITATIVELY ANALYZED ACCORDING TO STEREOLOGICAL PRINCIPLES. DIFFERENT STRUCTURES AND PROBLEMS CALL FOR DIFFERENT TEST SYSTEMS, BUT ALL TEST SYSTEMS ARE BASED ON SIMILAR GENERAL PRINCIPLES.
GENERAL PRINCIPLES OF STEREOLOGY
Section through an n-dimensional object yields an n-1 dimensional figure: are seen as areas, surfaces as lines and lines as points.
Area fractions in micrographs correspond to volume fractions of the cell. Membrane profile lengths per unit fiber area are related to membrane surface areas per unit fiber volume. The conversion factor is 4 / ~ .
Morphometric studies provide estimates of tissue or cell parameters. The reliability of the estimates i s increased by systematic, random and adequate sampling of the specimen, enlarging the sample size and standard methods of tissue preparation.
MEASUREMENTS OF MICROGRAPHS Areas can be measured by point sampling, line sampling, planimetry or with a digitizer attached to programmable calculator. Linear profiles can be measured by line sampling or with a map reader or digitizer. Point sampling: The test grid consists of dots arrayed in a square lattice. Area = n x dZ,where n is the number of dots falling on the area and d the spacing of the grid. The fractional volume of a cell component is n/N, where n is the number of dots falling on the Cell component and N, the number of dots falling on the cell. Line sampling:
The test grid consists of regularly spaced parallel lines.
The fractional volume of cell component is proportionate to the fraction of the scanning line which overlies that component. The length of randomly orientated linear membrane profiles is equal to N P K , where I is the number of intersections with the scanning line and K the length of the scanning line per unit grid area. Preferentially orientated membrane profiles require randomly orientated scanning lines. This condition can be approximated by using a grid made up of zig-zag lines placed at 25 to 35 degrees relative to the direction of preferential orientation.
MUSCLE & NERVE
Estimation of mitochondria1 fraction 01 fiber volume by point sampling. Mitochodrial profiles are outlined. Points falling on mitochondria and half of those hitting outlines of profiles are counted. Mitochondria1 dols account 4.4 per cent of all dots on the fiber. Corresponding estimate obtained with digitizer was 4.5 per cent.
Estimation of sarcotubular surface density by line sampling. Zig-zag scanning grid is applied on a 28.91 p m Z fiber region enlarged 38.555 times. The grid constant i s 12 cm/cmZ. There are 307 intersections between scanning line and sarcotubular profiles. The sarcotubular density is estimated to be 3.89 pm21pm3.
Estimation of sarcoplasmic reticulum (SR)and transverse tubular (T) surface densities. A l l T and rare SR profiles are rendered electron dense by osmium-ferrocyanide method. There are 468 SR and 68 T intersections with the scanning line. Estimates of SR and T surface densitites (~mZ/pmJ) are 5.94 and 0.86, respectively.
GENERAL REFERENCES ~~
Loud AV: J Cell Biol 15:181-187. 1962
Weibel ER et al: J Cell Biol 3023-28, 1966 Loud AV: J Cell Biol 37:27-46, 1968 Weibel ER et al: J Cell Biol 42:68-91, 1969
Underwood EE: Quantitative Slereology. AddisonWesley Publishing Co., Reading, Mass., 1970 Elias HE at al: Physiol Rev 51:158-200, 1971
Eirenberg BR e l al: J Cell Biol 60:732-754, 1974 Eisenberg BR, Mobley BA: Tissue & Cell 7: 383-387, 1975
MUSCLE & NERVE
MORPHOMETRIC ANALYSIS OF INTRAMUSCULAR CAPILLARIES LIGHT MICROSCOPY The mean muscle fiber area per capillary can be determined in phase micrographs of semi-thin sections of randomly selected, transversely orientated blocks. The fiber areas are measured with a digitizer and every capillary is counted directly. The ratios found in normal children are independent of age and the mean muscle fiber diameter.
ELECTRON MICROSCOPY For each biopsy, a predetermined number of transversely orientated blocks are chosen at random for thin sectioning. The first two or three capillaries encountered during electron microscopic examination of a given block are photographed. For each capillary, the following parameters can be analyzed: Luminal, endothelial, basement membrane and pericyte areas (digitizer) Mitochondria1 and endoplasmic reticulum fractions of endothelial cells and pericytes (point and line sampling) Numerical density of endothelial vesicles Minimum basement membrane thickness
The muscle fiber area per capillary and capillary fine structure were analyzed in Duchenne dystrophy and in normal children (Brain 97:115122, 1974). The mean muscle fiber area per capillary was not affected, but the mean capillary dimensions were significantly increased in Duchenne dystrophy. Diagram shows schematized control and Duchenne capillary drawn to the scale 01 the mean figures. 30 control capillaries of 5 children and 71 capillaries of 9 Duchenne patients were analyzed. L, lumen; E. endothelium; EL. basal lamina; P,pericyte.
D u c HE “ E
Capillary area f u m y b
Endolhelial lraciion of capillary area (%)
Basemeni membrane area as lracfion 01 capillary area I%)
Pericyie area as lraciion ol capillary area ( 9 0 )
Control capillaries (“41)
12 50+1 31