Peripheral Nerve Morphometry in Myotonic Dystrophy Martin Pollock, MD, Peter James
Dyck,
MD
\s=b\ Biopsy specimens of muscle and cutaneous branches of the common peroneal nerve from four patients with myotonic dystrophy (MD) were evaluated by
morphometric techniques and compared to age-matched control nerves. Myeli-
nated fiber densities were normal in the superficial and deep peroneal nerves of patients with MD. Internode length and frequency of abnormal fibers was not significantly different in MD and control nerves, nor were the number of myelin lamellae, number of neurofilaments per unit area, or number of microtubules per unit area to axis cylinder area. There was no evidence of morphologic abnormality of peripheral nerve in MD. (Arch Neurol 33:33-39, 1976)
dystrophy (MD) has long Myotonie been considered primary dis¬ of a
ease
muscle.
Recently, however,
the myopathie nature of MD and other muscular dystrophies has been chal¬ lenged by reports of an early wide¬ spread loss of functioning motor units,13 suggesting that the primary abnormality in muscular dystrophy lies in motoneurons.3 Although weakness, wasting, and
constitute the principal manifestations of MD, there are ab¬ normalities in other tissues, and the underlying defect in this multisystem disease may be a membrane abnor¬ mality. A defect in the muscle mem¬ brane seems likely in view of the continued firing of myotonic muscle fibers after nerve or neuromuscular blockade.46 A diffuse disorder of membranes is also suggested by im¬
the
and because of the rare association of MD with overt clinical evidence of neurop¬ athy.9·10 Four paid volunteers provided control nerve for electron microscopy. In¬ formed consent was obtained from pa¬ tients and volunteers for combined biopsy of muscle and cutaneous nerve. All agreed to undergo detailed neurologic examina¬ tion, quantitative sensory testing,1113 electromyographic and nerve conduction examination, and laboratory investigation. Biopsy specimens of the lateral fascicle of the deep peroneal nerve and the super¬ ficial peroneal nerve were taken in each patient and volunteer just proximal to the extensor retinaculum. Close attention was paid to fixation schedules, level of nerve biopsy, and age, because these variables may profoundly influence fiber density.14"16 Specimens were fixed in 2% glutaraldehyde in 0.1M cacodylate buffer (pH 7.40) with 0.025M calcium chloride at room tempera¬ ture, followed by 1% osmium tetroxide in the same buffer. The portion of nerve for teasing was fixed in glutaraldehyde for 10
membranes.8 We carried out a histologie eval¬ uation of muscle and cutaneous nerves in patients with MD to deter¬ mine whether morphologic correlates exist for either a primary abnormal¬ ity of motor nerve or a generalized disturbance of membranes. SUBJECTS AND METHODS Four patients with unequivocal evidence of MD who had normal results of nerve conduction studies were selected. The lat-
Table
publication
Feb 10, 1975. Accepted From the Mayo Clinic and Mayo Foundation, Rochester, Minn. Dr Pollock is now with the Department of Medicine, University of Otago, Dunedin, New Zealand. Reprint requests to Mayo Clinic and Mayo Foundation, Rochester, MN 55901 (Dr Dyck).
ter
paired protein phosphorylation in myotonic muscle7 and erythrocyte
1.—Pricking Pain Sensation Threshold (millicalories/sq cm/sec) Myotonic Dystrophy (N
for
requirement was necessary because of susceptibility of MD patients with se¬ vere wasting to entrapment neuropathies
myotonia
Mean
Hand 276
=
4)*
Foot 259
Control Hand 276
(N
=
69) Foot 278
SD_35_10_35_45
Range_242-325_249-273_207-389_181-354
*
No
significant difference from controls by Student t test for means of two samples.
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to 15 minutes and in osmium tetroxide for 1.5 hours. Portions to be embedded in epoxy were fixed for 1.5 to 2 hours in glutaralde¬
hyde and for 1.5 hours in osmium tetroxide. The method of preparing single teased fibers has been previously described.17·18
One hundred fibers from each nerve, with a minimum of four internodes, were graded descriptively as follows (Fig 1): A, normal appearance; B, marked irregularity of
myelin; C, segmental demyelination; D, segmental demyelination and remyelination as evidenced by at least a 50% differ¬ ence in myelin thickness; E, linear rows of myelin ovoids; F, segmental remyelination with internodes showing 50% or more dif¬ ference in myelin thickness; G, focal ir¬ regularity of myelin-forming globules or sausages; and H, myelin ovoids contiguous to two or more thinly myelinated inter¬ nodes. For comparison, teased fibers from age-matched control nerve were graded on the same basis. Slides containing MD and control teased fibers were presented for grading in a random manner (using tables of random numbers) without identifying marks. Teased fiber internode length was also measured by using an ocular microme¬ ter. For each teased fiber, mean internode length and coefficient of variation (of in¬ ternode length; SD x 100/mean) were cal¬ culated. From the mean internode length of each fiber, histograms were drawn for the 100 fibers of each nerve. For statistical analysis, a small fiber was defined as hav¬ ing a mean internode length of less than 625/i and a large fiber, as having a mean in¬ ternode length of 625µ or more.19
Fig 1 .—Descriptive grading of teased myelinated fibers. A, normal appearance; B, exces¬ sive irregularity of myelin not due to preparatory artifact; C, segmental demyelination and thickness of myelin of internode with thinnest myelin is 50% or more of that of internode with thickest myelin; D, segmentai demyelination and thickness of myelin of inter¬ node with thinnest myelin is less than 50% of that of Internode with thickest myelin; E, linear rows of myelin ovoids and balls at same stage of degeneration; F, 50% or more dif¬ ference in myelin thickness; G, excessive variability of myelin thickness forming "glob¬ ules" or "sausages"; H, thinly myelinated fiber with myelin ovoids or balls contiguous to two or more internodes. Table 2.—Cutaneous Thermal Discrimination* Myotonie Dystrophy (
Foot Mean SD
Range Hand Mean
SD
Range *
t
=
4)|
Copper and Polyvinyl
Copper
Copper
Chloride
and Glass
and Steel
96.5 4.1 92-100
81.0 6.0 74-86
94.5 4.4 90-100
81.0 7.7 72-90
Control
(
=
30)
Copper and Polyvinyl
Copper
Chloride
and Glass
54.0 7.3 50-66
93.3 6.3 78-100
81.7 8.7 82-98
61.3 10.2 40-88
58.5 2.5 56-62
94.3 5.4 82-100
82.8 8.8 66-94
60.5 10.6 34-90
Percent of correct responses in 50 trials. No significant difference from controls by Student t test for
means
of two
Copper and Steel
samples.
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Epoxy-embedded tissue, sectioned at 1.5/1, was photographed through a phasecontrast microscope, and the numbers and diameters of myelinated fibers were deter¬ mined, from photographic enlargements, with a particle-size analyzer, using the standard measuring range and settings to express results as an exponential fre¬ quency distribution curve. Up to eight pho¬ tographs per nerve were taken system¬ atically (every third field as encountered serially in an and y traverse of trans¬ verse fascicular area). Exact magnifica¬ tion was obtained from photographs of a reticle. Fibers not measured because of ar¬ tifact were used in the calculation of the total number of fibers per square millime¬ ter. Diameter histograms and numbers of myelinated fibers per square millimeter (density) were determined by programmed calculation and plotting. Measurements were made on electron micrographs of 100 deep peroneal myeli¬ nated fibers, as encountered serially in and y traverses. The micrographs were corrected for magnification and were read with their identification numbers covered.
Major dense lines of each myelinated fiber counted through a dissecting micro¬
were
scope. The numbers of microtubules and
neurofilaments were counted in a selected axonal area (A) 1 cm wide, which was drawn across the longest diameter of each fiber (Fig 2). In determining numbers of organelles, nodal and paranodal regions were excluded. The area of A and the area of the axis cylinder in square microns and the number of microtubules and neurofila¬ ments per square micron (density) were then determined by using programmed digitization and calculation. These data were stored on magnetic tape for subse¬
quent analysis.
RESULTS The diagnosis of MD was con¬ firmed in all cases. On quantitative sensory testing of the hands and feet, the patients with MD had normal
touch-pressure (Fig 3), pain (Table 1),
and temperature (Table 2) sensations. The degree of weakness in distal leg muscles varied. Two patients had complete foot drop, one had moderate weakness, and one had only minimal weakness. Motor and sensory nerve conduction velocities, latencies, and amplitudes of evoked responses were normal. No patient with MD had a diabetic glucose tolerance curve, al¬ though all had an excessive insulin response to a glucose load. None of the group with MD gave a history of long-
phenytoin (diphenylhydantoin) therapy. Comparison of mean myelinated fi¬ ber densities for both nerves, using a two-sample t test, indicated no evi¬ dence of a statistically significant dif¬ ference between groups (Table 3, Fig
Fig 2.—Transverse section of nerve fiber from patient with myotonic dystrophy, showing selected area (A) and total area (TA) in sq/i, number of myelin lamellae (L), and number of microtubules (Mt) and neurofilaments (Nf) per sqµ. Table
The grading of 100 teased myeli¬ nated fibers of deep peroneal and su¬ perficial peroneal nerves from myo¬ tonic and control subjects (Tables 4 and 5) revealed a low frequency of ab¬ normal findings. The most common abnormality was remyelinated inter¬ nodes, followed by linear rows of myelin ovoids, and segmental demye¬
as seen in Fig 5. The mean internode lengths of small and large fibers of superficial peroneal nerves from patients with MD did not differ significantly from the controls (Tables 6 and 7). By con¬ trast, the mean internode length of
lination,
Fibers in Peroneal Nerves
Density (No./sq mm) Age, yr
Case
Deep
Peroneal
Superficial Peroneal
Myotonic dystrophy 133-73 4-74 16-74 129-73 Mean SD
term
4).
3.—Density of Myelinated
48 48 33 36
Controls Mean ± SD
9,401 9,909
8,255 10,831 10,902 9,488 9,869 ±1,257
8,880 9,672 ±692
9,238 ±2,200*
8,152 ±1,942|
10,497
_
*
Mean from 11 control subjects. seven control subjects.
t Mean from
Table 4.—Condition of Teased Myelinated Fibers of Lateral Fascicles of Deep Peroneal Nerve Distribution of Types of Fibers, % Case
Myotonic dystrophy 4-74 16-74 129-73 133-73 Control 44-71 113-71 79-71 51-71
See text for
93 94 97 85
96 87 90 93
description
of fiber types.
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12
"
fibers of deep peroneal nerves from patients with MD was decreased 11% below the control mean value (P=.05). However, a comparison of mean internode length of small fibers and of combined small and large fi¬ bers of the deep peroneal nerves from patients and controls showed no sta¬ tistically significant difference. The mean coefficient of variation of inter¬ node length was smaller for large fi¬ bers in patients and controls. Comparison of control and MD elec¬ tron micrographs revealed no differ¬ ence in the incidence of peripheral nerve abnormalities. The regression equation (y a + bx) for the regres¬ sions of logarithm (to base e) of area of axis cylinder on number of myelin lamellae and of densities of micro¬ tubules and of neurofilaments on area of axis cylinder was determined for each nerve in the control and test groups. The mean intercept, slope, and standard deviation of the resid¬ uals were then computed (Table 8). Comparison of regression coefficients and the standard deviation of the re¬ siduals between myotonic and control groups, using a two-sample t test, in¬ dicated no significant difference be¬ tween groups. Mean regression lines for control and myotonic groups (Fig 6, 7, and 8) were obtained by comput¬ ing mean intercepts and slopes. The mean (± SD) area of axis cylinders of MD and control nerves was 5.4 sq/i ± 2.4 sq/i and 6.0 sqµ ± 1.6 sqµ; the mean number of myelin lamellae was 66 for both groups. The same mean number of myelin lamellae per fiber in patients and controls suggests that nerve fibers of similar size were com¬
large 100
+
-
-+
+
+
++ +
+ +
co
50
=
20
40
60
80
Age
10
+
+
:
* ©
E
7
+
+
+-H-
-
+
+
+
++
+
++
+ +
20
40
80
60
Age Fig 3.—Touch-pressure sensation thresholds of great toe of controls (plus sign) and of patients (plus sign within circle) with myotonic dystrophy. Upper graph, Percent of 25 grid points in which threshold level could be determined. Lower graph, Each point represents mean value of touch-pressure sensation threshold of sensitive grid points.
pared. COMMENT
Tableo.—Condition of Teased
Myelinated Fibers of Superficial Distribution of Types of
Peroneal Nerve
Fibers, %
*
Case
Myotonic dystrophy-f
*
4-74 129-73 133-73
95 96 95
Control 113-71 79-71 51-71
96 93 92 nerve
could not be
loss of either sensory or motoneu¬ Also, the incidence of abnormal teased fibers in patients with MD did not differ significantly from that seen in controls. Segmentai remyelination was the commonest abnormality, with a maximal incidence of 12%, and lina
rons.
See text for description of fiber types.
t One MD
These results provide evidence against a morphologic abnormality of peripheral nerve in myotonic dys¬ trophy. In particular, normal den¬ sities of myelinated fibers in cutane¬ ous and muscle nerves argue against
adequately teased
because of excessive fixation.
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Table 6.—Internode
Lengths of Deep Peroneal Nerve Small and Large Fiberst
Large Fibers
Small Fibers Case
Mean IL,'
CV,f
Mean
%
Myotonic dystrophy
Mean IL, µ
Mean CV, %
Adjusted IL, µ
CV of IL, %
Mean
Adjusted
133-73 129-73 16-74 4-74 Mean SD
359 381 383 311 359 ± 34
13.6 13.3 16.4 13.0 14.1 ±1.6
917 835 807 900 865 ± 52
10.6 10.5 10.4 12.0 10.9 ±7.5
537 574 510 428 512 ±62
12.6 12.1 14.6 13.0 13.1 ±1.1
Control (N = 7) Mean ± SD
333 ± 45
15.1 ±2.0
970 ± 81
11.5 ±2.2
608 ± 76
13.3±1.5
_
*
t
Internode length. Coefficient of variation. of small and large fibers based
^Adjustment
on
percent of small and large fibers in diameter histograms.
Table 7.—Internode
Lengths of Superficial Peroneal
Nerve Small and Large
Small Fibers Case
Large Fibers
Mean
Mean IL,
CV,t
%
Mean IL,* µ
Mean
Adjusted IL,* µ
CV,t %
Mean
Myotonic dystrophy 133-73 129-73 16-74 4-74 Mean ± SD Control (N = 6) Mean ± SD
Fibersij: Adjusted
CVt of IL,* %
334 325 438 372 367 ± 51
11.8 12.6 11.0 12.1 11.9 ±0.7
823 798 796 771 797 ± 0.9
8.3 8.4 10.3 9.3 9.1 ± 0.9
461 443 642 565 528 ± 93
10.9 11.5 10.6 10.8 10.9 ±0.4
322 ± 32
16.0 ±1.1
859 ± 68
13.2 ±1.7
538 ± 56
14.8 ±1.0
Internode length. t Coefficient of variation. £ Adjustment of small and large fibers based *
ear rows
of
myelin
ovoids
were
on
percent of small and large fibers in diameter histograms.
pres¬
Table
ent in only three of seven myotonic nerves, and at most, affected 1% of
MD peripheral nerve fibers. The mean internode length of large fibers from deep peroneal nerve was significantly decreased in the pa¬ tients with MD (P=.05), but all other internode length values in MD nerves did not differ significantly from con¬ trol values. Several deep peroneal nerve specimens from myotonic pa¬ tients were overfixed. Large myeli¬ nated fibers are most susceptible to excessive fixation and may become unusually brittle and adherent. It is therefore possible that the decreased
internode length of deep peroneal nerve in MD relates to a preferential selection of small fibers during teas¬ ing. The normal coefficient of vari¬ ation of internode length in myotonic nerves militates against an increased incidence of primary or secondary
segmental demyelination. Despite unremarkable light micro-
8.—Regression Analyses* Controls
Myotonie Dystrophy SD of
Intercept 0.3113 ±0.3415 68.0581 ±12.3961 226.9901 =164.8457
Values
are
Residuals Slope Intercept Slope Log, area of axis cylinder vs No. of myelin lamellae 0.0152 ±0.0040
0.0168 0.5761 0.2484 ±0.1477 ±0.1719 ±0.0022 Density of microtubules vs axis cylinder area —3.7714 36.8763 62.1793 -2.8400 ±0.5398 =12.3615 =2.1435 =16.3609 Density of neurofilaments vs axis cylinder area 207.7902 226.7051 -0.4030 -2.4635 =127.7004 =11.2683 =194.2126 =9.7940
expressed
as mean ±
SD of Residuals 0.5357 ±0.0752
25.4348 =21.3720
71.6758 =47.8905
SD.
scopic findings in MD nerve, elec¬ microscopy might reveal subtle changes indicative of "sick" motoneu¬ rons. However, quantitative electron microscopy revealed no noticeable tron
differences between MD and control
nerves.
McComas estimated that the exten¬ digitorum brevis muscle is sup¬ plied by a mean of only 199 motor units.1 Moreover, the lateral fascicle sor
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of the
deep peroneal nerve has a high
percentage of fibers supplying Golgi
tendon organs and joints and termi¬ nating as free endings. Our failure to detect histologie lesions in peripher¬ al nerve of patients with MD may therefore be a result of the small number of alpha motor fibers in the deep peroneal nerve. This point prob¬ ably could be resolved by a similar evaluation of a nerve, such as that to
Myotonic 4,000-
2,000-
B I
I
I
I
r——r-—r^-
_
o
a.
4,000-
Control
> _
2,000-
— -
10 Fiber diameters, µ of Fig 4.—Diameter histogram myelinated fibers per square millimeter of fascicular peroneal nerve of patient with myotonic dystrophy and age-matched control.
area
of
deep
Fig 5.—Teased fiber abnormalities in myotonic dystrophy and control nerves. From top to bottom, remyelination after previous segmental demyelination (F), regenerated fiber (H), and linear rows of myelin ovoids (E).
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7.—Mean regression lines for density of microtubules and of axis cylinder for patients with myotonic dystrophy (M) and controls (C). Relevant statistics in Table 8.
Fig
area
Fig 6.—Mean regression lines for area of axis cylinder (log to base e) and number of myelin lamellae for four patients with myotonic dystrophy (M) and four controls (C). Relevant statistics in Table 8.
Fig 8.—Mean regression lines for density of neurofilaments and of axis cylinder for patients with myotonic dystrophy (M) and controls (C). Relevant statistics in Table 8. area
the peroneus brevis, with a higher proportion of motor fibers than the
deep peroneal
nerve.
Alternatively,
a
disturbance of axolemma, as part of a generalized disorder of membranes, may not be associated with a recog¬ nizable structural abnormality. This investigation was supported in part by Muscular Dystrophy Center grant MDAA-4, Na¬ tional Institutes of Health, Public Health Ser¬ vice research grants NSO-5811, NSO-7541, and AM-2-22Ö0, and the Gallmeyer, Upton, and Hel¬ ler Funds. Peter O'Brien, PhD, provided assistance in the statistical
analysis
of data.
References 1. McComas AJ, Sica REP, Currie S: Muscular Evidence for a neural factor. Lancet 226:1263-1264, 1970. 2. McComas AJ, Campbell MJ, Sica RE: Electrophysiological study of dystrophia myotonica. J Neurol Neurosurg Psychiatry 34:132-139,1971. 3. McComas AJ, Sica RE, Upton AR: Multiple muscle analysis of motor units in muscular dystrophy. Arch Neurol 30:249-251, 1974. 4. Denny-Brown D, Nevin S: The phenomenon of myotonia. Brain 64:1-18, 1941.
dystrophy:
5. Landau WM: The essential mechanism in An electromyographic study. Neurology 2:369-388, 1952. 6. Floyd WF, Kent P, Page F: An electromyographic study of myotonia. Electroencephalogr Clin Neurophysiol 7:621-630, 1955. 7. Roses AD, Appel SH: Myotonic muscular dystrophy: A diffuse membrane disorder, abstracted. Neurology 24:365, 1974. 8. Roses AD, Appel SH: Protein kinase activity in erythrocyte ghosts of patients with myotonic muscular dystrophy (inborn error of metabolism/membrane). Proc Natl Acad Sci USA 70:1855-1859, 1973. 9. Kalyanaraman K, Smith BH, Chadha AL: Evidence for neuropathy in myotonic muscular dystrophy. Bull Los Angeles Neurol Soc 38:188\x=req-\ 196, 1973. 10. Pilz H, Prill A, Volles E: Kombination von Myotonischer Dystrophie mit "Idiopathischer" Neuropathie: Mitteilung klinischer Befunde bei 10 F\l=a"\llenmit gleichzeitiger Proteinvermehrung im Liquor. Z Neurol 206:253-265, 1974. 11. Hardy JD, Wolff HG, Goodell H: Pain Sensations and Reactions. Baltimore, Williams & Wilkins Co, 1952. 12. Dyck PJ, Lambert EH, Nichols PC: Quantitative measurement of sensation related to compound action potential and number and sizes of myelinated and unmyelinated fibers of sural nerve in health, Friedreich's ataxia, hereditary sensory neuropathy, and tabes dorsalis, in Handbook of Electroencephalography and Clinical
myotonia:
Downloaded From: http://archneur.jamanetwork.com/ by a Western University User on 06/07/2015
Neurophysiology. Amsterdam, Elsevier Publishing Co, 1971, vol 9, pp 83-118. 13. Dyck PJ, Curtis DJ, Bushek W, et al: Description of "Minnesota thermal disks" and nor-
mal values of cutaneous thermal discrimination in man. Neurology 24:325-330, 1974. 14. Rexed B: Contributions to the knowledge of the postnatal development of the peripheral nervous system in man: A study of the bases and scope of systematic investigations into the fibre size in peripheral nerves. Acta Psychiat Neurol,
suppl 33, pp 1-206, 1944. 15. Dyck PJ, Ellefson RD, Lais AC, et al: Histologic and lipid studies of sural nerves in inherited hypertrophic neuropathy: Preliminary report of a lipid abnormality in nerve and liver in Dejerine-Sottas disease. Mayo Clin Proc 45:286\x=req-\ 327, 1970.
16. Garven HS, Gairns FW, Smith G: The fibre populations of the nerves of the leg in chronic occlusive arterial disease in man. Scott Med J 7:250-265, 1962. 17. Dyck PJ, Lofgren EP: Method of fascicular biopsy of human peripheral nerve for electrophysiologic and histologic study. Mayo Clin Proc 41:778-784, 1966. 18. Dyck PJ, Lofgren EP: Nerve biopsy: Choice of nerve, method, symptoms, and usefulness. Med Clin North Am 52:885-893, 1968. 19. Stevens JC, Lofgren EP, Dyck PJ: Histometric evaluation of branches of peroneal nerve: Technique for combined biopsy of muscle nerve and cutaneous nerve. Brain Res 52:37-59, 1973. nerve
Dyck,
MD
\s=b\ Biopsy specimens of muscle and cutaneous branches of the common peroneal nerve from four patients with myotonic dystrophy (MD) were evaluated by
morphometric techniques and compared to age-matched control nerves. Myeli-
nated fiber densities were normal in the superficial and deep peroneal nerves of patients with MD. Internode length and frequency of abnormal fibers was not significantly different in MD and control nerves, nor were the number of myelin lamellae, number of neurofilaments per unit area, or number of microtubules per unit area to axis cylinder area. There was no evidence of morphologic abnormality of peripheral nerve in MD. (Arch Neurol 33:33-39, 1976)
dystrophy (MD) has long Myotonie been considered primary dis¬ of a
ease
muscle.
Recently, however,
the myopathie nature of MD and other muscular dystrophies has been chal¬ lenged by reports of an early wide¬ spread loss of functioning motor units,13 suggesting that the primary abnormality in muscular dystrophy lies in motoneurons.3 Although weakness, wasting, and
constitute the principal manifestations of MD, there are ab¬ normalities in other tissues, and the underlying defect in this multisystem disease may be a membrane abnor¬ mality. A defect in the muscle mem¬ brane seems likely in view of the continued firing of myotonic muscle fibers after nerve or neuromuscular blockade.46 A diffuse disorder of membranes is also suggested by im¬
the
and because of the rare association of MD with overt clinical evidence of neurop¬ athy.9·10 Four paid volunteers provided control nerve for electron microscopy. In¬ formed consent was obtained from pa¬ tients and volunteers for combined biopsy of muscle and cutaneous nerve. All agreed to undergo detailed neurologic examina¬ tion, quantitative sensory testing,1113 electromyographic and nerve conduction examination, and laboratory investigation. Biopsy specimens of the lateral fascicle of the deep peroneal nerve and the super¬ ficial peroneal nerve were taken in each patient and volunteer just proximal to the extensor retinaculum. Close attention was paid to fixation schedules, level of nerve biopsy, and age, because these variables may profoundly influence fiber density.14"16 Specimens were fixed in 2% glutaraldehyde in 0.1M cacodylate buffer (pH 7.40) with 0.025M calcium chloride at room tempera¬ ture, followed by 1% osmium tetroxide in the same buffer. The portion of nerve for teasing was fixed in glutaraldehyde for 10
membranes.8 We carried out a histologie eval¬ uation of muscle and cutaneous nerves in patients with MD to deter¬ mine whether morphologic correlates exist for either a primary abnormal¬ ity of motor nerve or a generalized disturbance of membranes. SUBJECTS AND METHODS Four patients with unequivocal evidence of MD who had normal results of nerve conduction studies were selected. The lat-
Table
publication
Feb 10, 1975. Accepted From the Mayo Clinic and Mayo Foundation, Rochester, Minn. Dr Pollock is now with the Department of Medicine, University of Otago, Dunedin, New Zealand. Reprint requests to Mayo Clinic and Mayo Foundation, Rochester, MN 55901 (Dr Dyck).
ter
paired protein phosphorylation in myotonic muscle7 and erythrocyte
1.—Pricking Pain Sensation Threshold (millicalories/sq cm/sec) Myotonic Dystrophy (N
for
requirement was necessary because of susceptibility of MD patients with se¬ vere wasting to entrapment neuropathies
myotonia
Mean
Hand 276
=
4)*
Foot 259
Control Hand 276
(N
=
69) Foot 278
SD_35_10_35_45
Range_242-325_249-273_207-389_181-354
*
No
significant difference from controls by Student t test for means of two samples.
Downloaded From: http://archneur.jamanetwork.com/ by a Western University User on 06/07/2015
to 15 minutes and in osmium tetroxide for 1.5 hours. Portions to be embedded in epoxy were fixed for 1.5 to 2 hours in glutaralde¬
hyde and for 1.5 hours in osmium tetroxide. The method of preparing single teased fibers has been previously described.17·18
One hundred fibers from each nerve, with a minimum of four internodes, were graded descriptively as follows (Fig 1): A, normal appearance; B, marked irregularity of
myelin; C, segmental demyelination; D, segmental demyelination and remyelination as evidenced by at least a 50% differ¬ ence in myelin thickness; E, linear rows of myelin ovoids; F, segmental remyelination with internodes showing 50% or more dif¬ ference in myelin thickness; G, focal ir¬ regularity of myelin-forming globules or sausages; and H, myelin ovoids contiguous to two or more thinly myelinated inter¬ nodes. For comparison, teased fibers from age-matched control nerve were graded on the same basis. Slides containing MD and control teased fibers were presented for grading in a random manner (using tables of random numbers) without identifying marks. Teased fiber internode length was also measured by using an ocular microme¬ ter. For each teased fiber, mean internode length and coefficient of variation (of in¬ ternode length; SD x 100/mean) were cal¬ culated. From the mean internode length of each fiber, histograms were drawn for the 100 fibers of each nerve. For statistical analysis, a small fiber was defined as hav¬ ing a mean internode length of less than 625/i and a large fiber, as having a mean in¬ ternode length of 625µ or more.19
Fig 1 .—Descriptive grading of teased myelinated fibers. A, normal appearance; B, exces¬ sive irregularity of myelin not due to preparatory artifact; C, segmental demyelination and thickness of myelin of internode with thinnest myelin is 50% or more of that of internode with thickest myelin; D, segmentai demyelination and thickness of myelin of inter¬ node with thinnest myelin is less than 50% of that of Internode with thickest myelin; E, linear rows of myelin ovoids and balls at same stage of degeneration; F, 50% or more dif¬ ference in myelin thickness; G, excessive variability of myelin thickness forming "glob¬ ules" or "sausages"; H, thinly myelinated fiber with myelin ovoids or balls contiguous to two or more internodes. Table 2.—Cutaneous Thermal Discrimination* Myotonie Dystrophy (
Foot Mean SD
Range Hand Mean
SD
Range *
t
=
4)|
Copper and Polyvinyl
Copper
Copper
Chloride
and Glass
and Steel
96.5 4.1 92-100
81.0 6.0 74-86
94.5 4.4 90-100
81.0 7.7 72-90
Control
(
=
30)
Copper and Polyvinyl
Copper
Chloride
and Glass
54.0 7.3 50-66
93.3 6.3 78-100
81.7 8.7 82-98
61.3 10.2 40-88
58.5 2.5 56-62
94.3 5.4 82-100
82.8 8.8 66-94
60.5 10.6 34-90
Percent of correct responses in 50 trials. No significant difference from controls by Student t test for
means
of two
Copper and Steel
samples.
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Epoxy-embedded tissue, sectioned at 1.5/1, was photographed through a phasecontrast microscope, and the numbers and diameters of myelinated fibers were deter¬ mined, from photographic enlargements, with a particle-size analyzer, using the standard measuring range and settings to express results as an exponential fre¬ quency distribution curve. Up to eight pho¬ tographs per nerve were taken system¬ atically (every third field as encountered serially in an and y traverse of trans¬ verse fascicular area). Exact magnifica¬ tion was obtained from photographs of a reticle. Fibers not measured because of ar¬ tifact were used in the calculation of the total number of fibers per square millime¬ ter. Diameter histograms and numbers of myelinated fibers per square millimeter (density) were determined by programmed calculation and plotting. Measurements were made on electron micrographs of 100 deep peroneal myeli¬ nated fibers, as encountered serially in and y traverses. The micrographs were corrected for magnification and were read with their identification numbers covered.
Major dense lines of each myelinated fiber counted through a dissecting micro¬
were
scope. The numbers of microtubules and
neurofilaments were counted in a selected axonal area (A) 1 cm wide, which was drawn across the longest diameter of each fiber (Fig 2). In determining numbers of organelles, nodal and paranodal regions were excluded. The area of A and the area of the axis cylinder in square microns and the number of microtubules and neurofila¬ ments per square micron (density) were then determined by using programmed digitization and calculation. These data were stored on magnetic tape for subse¬
quent analysis.
RESULTS The diagnosis of MD was con¬ firmed in all cases. On quantitative sensory testing of the hands and feet, the patients with MD had normal
touch-pressure (Fig 3), pain (Table 1),
and temperature (Table 2) sensations. The degree of weakness in distal leg muscles varied. Two patients had complete foot drop, one had moderate weakness, and one had only minimal weakness. Motor and sensory nerve conduction velocities, latencies, and amplitudes of evoked responses were normal. No patient with MD had a diabetic glucose tolerance curve, al¬ though all had an excessive insulin response to a glucose load. None of the group with MD gave a history of long-
phenytoin (diphenylhydantoin) therapy. Comparison of mean myelinated fi¬ ber densities for both nerves, using a two-sample t test, indicated no evi¬ dence of a statistically significant dif¬ ference between groups (Table 3, Fig
Fig 2.—Transverse section of nerve fiber from patient with myotonic dystrophy, showing selected area (A) and total area (TA) in sq/i, number of myelin lamellae (L), and number of microtubules (Mt) and neurofilaments (Nf) per sqµ. Table
The grading of 100 teased myeli¬ nated fibers of deep peroneal and su¬ perficial peroneal nerves from myo¬ tonic and control subjects (Tables 4 and 5) revealed a low frequency of ab¬ normal findings. The most common abnormality was remyelinated inter¬ nodes, followed by linear rows of myelin ovoids, and segmental demye¬
as seen in Fig 5. The mean internode lengths of small and large fibers of superficial peroneal nerves from patients with MD did not differ significantly from the controls (Tables 6 and 7). By con¬ trast, the mean internode length of
lination,
Fibers in Peroneal Nerves
Density (No./sq mm) Age, yr
Case
Deep
Peroneal
Superficial Peroneal
Myotonic dystrophy 133-73 4-74 16-74 129-73 Mean SD
term
4).
3.—Density of Myelinated
48 48 33 36
Controls Mean ± SD
9,401 9,909
8,255 10,831 10,902 9,488 9,869 ±1,257
8,880 9,672 ±692
9,238 ±2,200*
8,152 ±1,942|
10,497
_
*
Mean from 11 control subjects. seven control subjects.
t Mean from
Table 4.—Condition of Teased Myelinated Fibers of Lateral Fascicles of Deep Peroneal Nerve Distribution of Types of Fibers, % Case
Myotonic dystrophy 4-74 16-74 129-73 133-73 Control 44-71 113-71 79-71 51-71
See text for
93 94 97 85
96 87 90 93
description
of fiber types.
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12
"
fibers of deep peroneal nerves from patients with MD was decreased 11% below the control mean value (P=.05). However, a comparison of mean internode length of small fibers and of combined small and large fi¬ bers of the deep peroneal nerves from patients and controls showed no sta¬ tistically significant difference. The mean coefficient of variation of inter¬ node length was smaller for large fi¬ bers in patients and controls. Comparison of control and MD elec¬ tron micrographs revealed no differ¬ ence in the incidence of peripheral nerve abnormalities. The regression equation (y a + bx) for the regres¬ sions of logarithm (to base e) of area of axis cylinder on number of myelin lamellae and of densities of micro¬ tubules and of neurofilaments on area of axis cylinder was determined for each nerve in the control and test groups. The mean intercept, slope, and standard deviation of the resid¬ uals were then computed (Table 8). Comparison of regression coefficients and the standard deviation of the re¬ siduals between myotonic and control groups, using a two-sample t test, in¬ dicated no significant difference be¬ tween groups. Mean regression lines for control and myotonic groups (Fig 6, 7, and 8) were obtained by comput¬ ing mean intercepts and slopes. The mean (± SD) area of axis cylinders of MD and control nerves was 5.4 sq/i ± 2.4 sq/i and 6.0 sqµ ± 1.6 sqµ; the mean number of myelin lamellae was 66 for both groups. The same mean number of myelin lamellae per fiber in patients and controls suggests that nerve fibers of similar size were com¬
large 100
+
-
-+
+
+
++ +
+ +
co
50
=
20
40
60
80
Age
10
+
+
:
* ©
E
7
+
+
+-H-
-
+
+
+
++
+
++
+ +
20
40
80
60
Age Fig 3.—Touch-pressure sensation thresholds of great toe of controls (plus sign) and of patients (plus sign within circle) with myotonic dystrophy. Upper graph, Percent of 25 grid points in which threshold level could be determined. Lower graph, Each point represents mean value of touch-pressure sensation threshold of sensitive grid points.
pared. COMMENT
Tableo.—Condition of Teased
Myelinated Fibers of Superficial Distribution of Types of
Peroneal Nerve
Fibers, %
*
Case
Myotonic dystrophy-f
*
4-74 129-73 133-73
95 96 95
Control 113-71 79-71 51-71
96 93 92 nerve
could not be
loss of either sensory or motoneu¬ Also, the incidence of abnormal teased fibers in patients with MD did not differ significantly from that seen in controls. Segmentai remyelination was the commonest abnormality, with a maximal incidence of 12%, and lina
rons.
See text for description of fiber types.
t One MD
These results provide evidence against a morphologic abnormality of peripheral nerve in myotonic dys¬ trophy. In particular, normal den¬ sities of myelinated fibers in cutane¬ ous and muscle nerves argue against
adequately teased
because of excessive fixation.
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Table 6.—Internode
Lengths of Deep Peroneal Nerve Small and Large Fiberst
Large Fibers
Small Fibers Case
Mean IL,'
CV,f
Mean
%
Myotonic dystrophy
Mean IL, µ
Mean CV, %
Adjusted IL, µ
CV of IL, %
Mean
Adjusted
133-73 129-73 16-74 4-74 Mean SD
359 381 383 311 359 ± 34
13.6 13.3 16.4 13.0 14.1 ±1.6
917 835 807 900 865 ± 52
10.6 10.5 10.4 12.0 10.9 ±7.5
537 574 510 428 512 ±62
12.6 12.1 14.6 13.0 13.1 ±1.1
Control (N = 7) Mean ± SD
333 ± 45
15.1 ±2.0
970 ± 81
11.5 ±2.2
608 ± 76
13.3±1.5
_
*
t
Internode length. Coefficient of variation. of small and large fibers based
^Adjustment
on
percent of small and large fibers in diameter histograms.
Table 7.—Internode
Lengths of Superficial Peroneal
Nerve Small and Large
Small Fibers Case
Large Fibers
Mean
Mean IL,
CV,t
%
Mean IL,* µ
Mean
Adjusted IL,* µ
CV,t %
Mean
Myotonic dystrophy 133-73 129-73 16-74 4-74 Mean ± SD Control (N = 6) Mean ± SD
Fibersij: Adjusted
CVt of IL,* %
334 325 438 372 367 ± 51
11.8 12.6 11.0 12.1 11.9 ±0.7
823 798 796 771 797 ± 0.9
8.3 8.4 10.3 9.3 9.1 ± 0.9
461 443 642 565 528 ± 93
10.9 11.5 10.6 10.8 10.9 ±0.4
322 ± 32
16.0 ±1.1
859 ± 68
13.2 ±1.7
538 ± 56
14.8 ±1.0
Internode length. t Coefficient of variation. £ Adjustment of small and large fibers based *
ear rows
of
myelin
ovoids
were
on
percent of small and large fibers in diameter histograms.
pres¬
Table
ent in only three of seven myotonic nerves, and at most, affected 1% of
MD peripheral nerve fibers. The mean internode length of large fibers from deep peroneal nerve was significantly decreased in the pa¬ tients with MD (P=.05), but all other internode length values in MD nerves did not differ significantly from con¬ trol values. Several deep peroneal nerve specimens from myotonic pa¬ tients were overfixed. Large myeli¬ nated fibers are most susceptible to excessive fixation and may become unusually brittle and adherent. It is therefore possible that the decreased
internode length of deep peroneal nerve in MD relates to a preferential selection of small fibers during teas¬ ing. The normal coefficient of vari¬ ation of internode length in myotonic nerves militates against an increased incidence of primary or secondary
segmental demyelination. Despite unremarkable light micro-
8.—Regression Analyses* Controls
Myotonie Dystrophy SD of
Intercept 0.3113 ±0.3415 68.0581 ±12.3961 226.9901 =164.8457
Values
are
Residuals Slope Intercept Slope Log, area of axis cylinder vs No. of myelin lamellae 0.0152 ±0.0040
0.0168 0.5761 0.2484 ±0.1477 ±0.1719 ±0.0022 Density of microtubules vs axis cylinder area —3.7714 36.8763 62.1793 -2.8400 ±0.5398 =12.3615 =2.1435 =16.3609 Density of neurofilaments vs axis cylinder area 207.7902 226.7051 -0.4030 -2.4635 =127.7004 =11.2683 =194.2126 =9.7940
expressed
as mean ±
SD of Residuals 0.5357 ±0.0752
25.4348 =21.3720
71.6758 =47.8905
SD.
scopic findings in MD nerve, elec¬ microscopy might reveal subtle changes indicative of "sick" motoneu¬ rons. However, quantitative electron microscopy revealed no noticeable tron
differences between MD and control
nerves.
McComas estimated that the exten¬ digitorum brevis muscle is sup¬ plied by a mean of only 199 motor units.1 Moreover, the lateral fascicle sor
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of the
deep peroneal nerve has a high
percentage of fibers supplying Golgi
tendon organs and joints and termi¬ nating as free endings. Our failure to detect histologie lesions in peripher¬ al nerve of patients with MD may therefore be a result of the small number of alpha motor fibers in the deep peroneal nerve. This point prob¬ ably could be resolved by a similar evaluation of a nerve, such as that to
Myotonic 4,000-
2,000-
B I
I
I
I
r——r-—r^-
_
o
a.
4,000-
Control
> _
2,000-
— -
10 Fiber diameters, µ of Fig 4.—Diameter histogram myelinated fibers per square millimeter of fascicular peroneal nerve of patient with myotonic dystrophy and age-matched control.
area
of
deep
Fig 5.—Teased fiber abnormalities in myotonic dystrophy and control nerves. From top to bottom, remyelination after previous segmental demyelination (F), regenerated fiber (H), and linear rows of myelin ovoids (E).
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7.—Mean regression lines for density of microtubules and of axis cylinder for patients with myotonic dystrophy (M) and controls (C). Relevant statistics in Table 8.
Fig
area
Fig 6.—Mean regression lines for area of axis cylinder (log to base e) and number of myelin lamellae for four patients with myotonic dystrophy (M) and four controls (C). Relevant statistics in Table 8.
Fig 8.—Mean regression lines for density of neurofilaments and of axis cylinder for patients with myotonic dystrophy (M) and controls (C). Relevant statistics in Table 8. area
the peroneus brevis, with a higher proportion of motor fibers than the
deep peroneal
nerve.
Alternatively,
a
disturbance of axolemma, as part of a generalized disorder of membranes, may not be associated with a recog¬ nizable structural abnormality. This investigation was supported in part by Muscular Dystrophy Center grant MDAA-4, Na¬ tional Institutes of Health, Public Health Ser¬ vice research grants NSO-5811, NSO-7541, and AM-2-22Ö0, and the Gallmeyer, Upton, and Hel¬ ler Funds. Peter O'Brien, PhD, provided assistance in the statistical
analysis
of data.
References 1. McComas AJ, Sica REP, Currie S: Muscular Evidence for a neural factor. Lancet 226:1263-1264, 1970. 2. McComas AJ, Campbell MJ, Sica RE: Electrophysiological study of dystrophia myotonica. J Neurol Neurosurg Psychiatry 34:132-139,1971. 3. McComas AJ, Sica RE, Upton AR: Multiple muscle analysis of motor units in muscular dystrophy. Arch Neurol 30:249-251, 1974. 4. Denny-Brown D, Nevin S: The phenomenon of myotonia. Brain 64:1-18, 1941.
dystrophy:
5. Landau WM: The essential mechanism in An electromyographic study. Neurology 2:369-388, 1952. 6. Floyd WF, Kent P, Page F: An electromyographic study of myotonia. Electroencephalogr Clin Neurophysiol 7:621-630, 1955. 7. Roses AD, Appel SH: Myotonic muscular dystrophy: A diffuse membrane disorder, abstracted. Neurology 24:365, 1974. 8. Roses AD, Appel SH: Protein kinase activity in erythrocyte ghosts of patients with myotonic muscular dystrophy (inborn error of metabolism/membrane). Proc Natl Acad Sci USA 70:1855-1859, 1973. 9. Kalyanaraman K, Smith BH, Chadha AL: Evidence for neuropathy in myotonic muscular dystrophy. Bull Los Angeles Neurol Soc 38:188\x=req-\ 196, 1973. 10. Pilz H, Prill A, Volles E: Kombination von Myotonischer Dystrophie mit "Idiopathischer" Neuropathie: Mitteilung klinischer Befunde bei 10 F\l=a"\llenmit gleichzeitiger Proteinvermehrung im Liquor. Z Neurol 206:253-265, 1974. 11. Hardy JD, Wolff HG, Goodell H: Pain Sensations and Reactions. Baltimore, Williams & Wilkins Co, 1952. 12. Dyck PJ, Lambert EH, Nichols PC: Quantitative measurement of sensation related to compound action potential and number and sizes of myelinated and unmyelinated fibers of sural nerve in health, Friedreich's ataxia, hereditary sensory neuropathy, and tabes dorsalis, in Handbook of Electroencephalography and Clinical
myotonia:
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Neurophysiology. Amsterdam, Elsevier Publishing Co, 1971, vol 9, pp 83-118. 13. Dyck PJ, Curtis DJ, Bushek W, et al: Description of "Minnesota thermal disks" and nor-
mal values of cutaneous thermal discrimination in man. Neurology 24:325-330, 1974. 14. Rexed B: Contributions to the knowledge of the postnatal development of the peripheral nervous system in man: A study of the bases and scope of systematic investigations into the fibre size in peripheral nerves. Acta Psychiat Neurol,
suppl 33, pp 1-206, 1944. 15. Dyck PJ, Ellefson RD, Lais AC, et al: Histologic and lipid studies of sural nerves in inherited hypertrophic neuropathy: Preliminary report of a lipid abnormality in nerve and liver in Dejerine-Sottas disease. Mayo Clin Proc 45:286\x=req-\ 327, 1970.
16. Garven HS, Gairns FW, Smith G: The fibre populations of the nerves of the leg in chronic occlusive arterial disease in man. Scott Med J 7:250-265, 1962. 17. Dyck PJ, Lofgren EP: Method of fascicular biopsy of human peripheral nerve for electrophysiologic and histologic study. Mayo Clin Proc 41:778-784, 1966. 18. Dyck PJ, Lofgren EP: Nerve biopsy: Choice of nerve, method, symptoms, and usefulness. Med Clin North Am 52:885-893, 1968. 19. Stevens JC, Lofgren EP, Dyck PJ: Histometric evaluation of branches of peroneal nerve: Technique for combined biopsy of muscle nerve and cutaneous nerve. Brain Res 52:37-59, 1973. nerve
