Failure to Confirm a Vascular Cause of Muscular Dystrophy Walter G.
Bradley, DM; Michael
Margaret Johnson, PhD;
D. O'Brien, MD; Dennis N. David J. Newell, PhD
Walder, MD; Dorothy Murchison, BSc;
The vascular hypothesis of the cause of muscular dystrophy suggests that ischemia is responsible for the muscle fiber necrosis. A xenon 133 clearance study of muscle blood flow in Duchenne and other muscular dystrophies showed no obvious difference between the response to exercise and arterial occlusion compared with control subjects. Radioautographic study of distribution of 4-125l-antipyrine in skeletal muscle of mice with muscular dystrophy showed no abnormal areas of ischemia.
A statistical examination was also made of the grouping of damaged fibers, one of the observations on which the vascular hypothesis was based. Only 0.9% of fibers undergoing phagocytosis occurred in groups of four or more fibers in greater frequency than would have been expected by chance, and 70% of such fibers were isolated. These studies argue strongly against the vascular hypothesis of the cause of muscular dystrophy.
Demos12 advanced a vascu¬ lar of the cause of muscular dystrophy, suggesting that the muscle degeneration was due to an abnormality of skeletal muscle blood flow. He studied the circulation times in a group of normal individuals and a group of patients with various myopa¬ thies. The latter were found to have a greater than normal range of circula¬ tion times, some being longer and some shorter than normal. A subse¬ quent double-blind trial of a vasodilating agent, bamethan sulfate (phydroxyphenylbutyl amino ethanol; Butylsympatol), in a group of pa¬ tients with various myopathies was reported to show a substantial im¬ provement in the clinical state of the patients receiving the drug and a fall in the serum creatine kinase activity.3
Further experience has reinforced his enthusiasm for the procedure.1 Demos has also recently reported finding abnormalities in an enzyme, diphenoloxidase, in patients with myopa¬ thies.:>,i He suggested that these abnormalities in an enzyme that may play a part in the inactivation of cate¬ cholamines lend further support to a vascular hypothesis of the cause of muscular dystrophy. The vascular hypothesis of the cause of muscular dystrophy has recently been propounded more strongly by Hathaway and et al.7 They stated that one pathological feature of the early stages of Duchenne muscular dystro¬ phy is the tendency for grouping of small numbers of fibers at the same stage of breakdown in an area of muscle. If the underlying mechanism of the disease were a biochemical abnormality in the individual muscle fibers, then a random distribution of breakdown of single muscle fibers would be expected. They produced a similar pathological picture of groups of damaged fibers by microembolization of rabbit muscle with dextran
In 1961,hypothesis
-
-
for publication Sept 3, 1974. From the Muscular Dystrophy Research Laboratories, Newcastle General Hospital, Newcastle upon Tyne, England. Reprint requests to Muscular Dystrophy Research Laboratories, Newcastle General Hospital, Newcastle upon Tyne, NE4 6BE, England
Accepted
(Prof. Bradley).
(Arch
Neurol
32:466-473, 1975)
Downloaded From: http://archneur.jamanetwork.com/ by a Western University User on 06/07/2015
it was recognized that structural abnormalities of the intramuscular blood vessels were not a feature of Duchenne muscular dystro¬ phy.8 A further experimental model was, therefore, advanced to overcome this problem. A combination of aortic ligation and intraperitoneal injection of the vasoactive compounds 5hydroxytryptamine and norepinephrine produced similar grouping of damaged fibers.8 They, therefore, suggested that a decrease in muscle blood flow might underlie the degen¬ eration of the muscle in Duchenne muscular dystrophy. In order to provide direct evidence on this hypothesis, we have examined the intramuscular blood flow in patients with Duchenne and other forms of muscular dystrophy and in mice with inherited muscular dystro¬ phy (Bar Harbor 129 Re dy/dy). The pathological picture in the skeletal muscle of these animals is similar to that of human Duchenne muscular dystrophy, with both isolated and small groups of damaged muscle fibers.9 The xenon 133 (133Xe) clear¬ ance technique was used to study muscle blood flow in the patients. 4125I-antipyrine was used as a marker of plasma water in the mice. The diffusion of this marker from the blood vessels into the muscle and back into the blood vessels was studied at intervals after its intravascular injec¬ tion by the technique of radioautography of water-soluble substances.10 Muscle blood flow was studied under conditions of maximal vasodila¬ tation produced either by exercise or arterial occlusion, because in resting
particles. However,
muscle, only a relatively small propor¬ tion of the muscle capillaries are patent at any one time.11 Under the condition of maximal vasodilatation, vascular insufficiency of muscle is seen either as a decreased blood flow or as a prolonged period of reactive hyperemia. A vascular abnormality in the radioautographic study is shown by decreased inflow and clearance of radioactivity from the muscle and by ischemie areas that remain free of
radioactivity. SUBJECTS AND METHODS Human Studies
patients with muscular dystrophy studied, two with Duchenne, one with limb-girdle, and one with facioscapulohumeral muscular dystrophy. There was no family history in either patient with Duchenne muscular dystrophy, though the Four
were
clinical course and biochemical and electrophysiological studies confirmed the diag¬ nosis. Patient 1 (Duchenne) was age 9 years and was still just able to walk. Patient 2 (Duchenne) was age 19 years and was in an advanced state; he died the
following year. Patient 3, age 47 years, had typical limb-girdle muscular dystrophy that had begun about the age of 20 years. Biochemical, electrophysiological, and pathological studies confirmed the diagno¬ sis. He was still able to walk at the time of the study. Patient 4, a 43-year-old man, had facioscapulohumeral muscular dystrophy with no family history. Facial weakness began in early childhood and limb-girdle weakness at about the age of 17 years. The muscles studied in the patients varied in their degree of weakness from normal to severely weak. Two normal males, ages 31 and 56, were studied as controls.
Methods One hundred microcuries of 133Xe in 0.1 ml of physiological saline solution was injected into the muscle and the radioac¬ tivity recorded by a portable apparatus12 using a 1.27-cm thallium-activated sodium iodide crystal (modified EKCO N632) and a photomultiplier tube (EMI type 9650B) in conjunction with a portable telometer pack with a ratemeter (Nuclear Enterprises). The experimental procedure is shown in Fig 1. The radioactive count rate over the depot falls as 133Xe is cleared by the muscle blood flow. The value of the resting clea¬ rance was measured as the slope of the line relating counts per second to time. There are two phases of increased clearance during exercise, the fast initial rate and
the slower maintained rate. The slope of both clearances was similarly measured. After arterial occlusion, the clearance is in¬ creased to repay to metabolic debt incurred due to ischemia. The slope of this increased clearance was also measured. The blood flow was recorded during a five-minute rest, followed by two minutes of exercise (contraction of the muscle against a 2.73-kg (6-lb) weight once a second in time with a metronome). The changes in blood flow after exercise were followed until the resting rate was reestablished. This test was followed by a two-minute period of total arterial occlusion with a pneumatic cuff placed on the limb proximal to the muscle under investigation and inflated to a pressure of 180 to 200 mm Hg. Arterial occlusion was verified by the cessation of 133Xe clearance. Muscle blood flow is expressed as the half-time in minutes of the 133Xe clearance and as the ratio between 133Xe clearance half-times at rest and during the experi¬ mental maneuvers to increase the blood flow. Values for the initial resting level, initial slope with exercise, the flow during exercise, the second resting levels, the initial slope after occlusion, and the third resting levels were obtained.
Animal
Experiments mice Dystrophie (dy/dy) of
the Bar Harbor 129 Re strain were used and compared with their littermates of the same sex. Animals of 1 to 3 months of age were studied. The dystrophic animals showed moderately severe clinical and pathological signs of the muscle disease.
Operative Technique Mice
toneally
anesthetised with intraperiadministered pentobarbital (pen-
were
0.12 mg/10 gm body weight, and anesthesia was maintained with a mixture of halothane and oxygen delivered through a face mask. The skin of both forelegs and hind legs, was carefully removed, and the denuded legs were care¬ fully wrapped in gauze swabs soaked in warm physiological saline solution. Body temperature, monitored by subcutaneous thermocouple, was maintained at 38 C by a heat lamp. Fine-needle stimulating cath¬ odes were inserted into the region of the sciatic notch on both sides, the anode being a similar fine needle in the subcutaneous tissue of the dorsal region. The animal was then allowed to rest for ten minutes before supramaximal stimuli at one each second were applied for five minutes to the electrodes, using square wave pulses of 0.1 msec delivered by an isolated stimulator. The intensity of the
tobarbitone),
Downloaded From: http://archneur.jamanetwork.com/ by a Western University User on 06/07/2015
stimulus was adjusted to produce a maximal twitch of the hind limbs. With the intensity and electrode position used, the upper limbs also contracted with each stimulus.
Injection
Procedure
Two groups of animals, three dystrophic mice and three littermate controls, were studied. In the first group, 200/J (70 microcuries) of 4-125I-antipyrine (0.35 microcuries/ml) were injected into the jugular bulb at the end of the five-minute stimulation period. The right foreleg was rapidly cut from the body with bone forceps at three seconds, the right hind leg at six seconds, the left foreleg at ten seconds, and the left hind leg at 20 seconds after injection. Immediately after removal, the individual legs were rapidly frozen in isopentane cooled in liquid nitrogen. The second group of animals was treated in an identical fashion, except that 200 µ (130 microcuries) of 4-125I-antipyrine (0.65 microcuries/ml) was injected, and the legs were removed in the following order: right foreleg, 40 seconds; right hind leg, 80 seconds; left foreleg, 120 seconds; left hind leg, 160 seconds after injection.
Radioautographic Techniques Great care was taken to ensure that the muscle remained frozen at below —20 C from the time of rapid initial freezing of the legs immediately after separation from the body to the development of the final radioautograms. Muscle was cut off the bones of the individual legs at —20 C, mounted in a compound embedding me¬ dium for frozen tissue (Tissue-Tek O.C.T.) and cut in total darkness at 8µ thickness on a cryostat. Microscope slides were predipped in undiluted emulsion (Ilford K2), dried in air at room temperature, and stored at —20 C in lightproof boxes in the
cryostat.
Cryostat
were
sections of the
cut in the
dark,
were
muscle, which picked up on
these emulsion-coated slides. The slides with sections were stored in lightproof boxes at —70 C for six weeks. They were then allowed to warm to room tempera¬ ture, and the radioautogram was developed by the technique previously reported.13 The sections were stained with hematoxylineosin. Serial sections of those sections studied by radioautography were stained with hematoxylin-eosin and by histo¬ chemical techniques for myosin ATPase and nicotinamide adenine nucleotide dehy¬
drogenase. Radioautograms were studied qualita¬ tively under the light microscope, and quantitative silver grain counting was
undertaken time points
on
material from the shorter
(first group of animals) under 1,000 magnification (oil immersion ob¬ jective). For the latter, attention was directed to areas of lowest grain density over the muscle section. Major arteries and veins were avoided, and the areas counted lay entirely within the body of the muscle. The total number of silver grains in a standard area of 1.6/1,600 sq firn was count¬ ed. This total included grains lying over both muscle fibers and endomysial connec¬ tive tissue. In the dystrophic animals, areas showing marked degeneration or regener¬ ation of fibers were avoided, counting being restricted to areas containing rela¬ tively normal-appearing muscle fibers. In the normal animals, this area included part or all of two or three muscle fibers and in the dystrophic animals part or all of three to six fibers. Three standard areas of each of three sections were counted for each animal. The background radioactivity was measured by counting three standard areas without muscle, immediately adjacent to the mus¬
cle sections studied. The background grain density was subtracted from the measured tissue grain density to give the corrected tissue grain density.
Statistical Analysis of Distribution of Necrotic Fibers A statistical analysis of the frequency of single fibers and of groups of fibers undergoing phagocytosis was made in muscle biopsy specimens from 42 patients with Duchenne muscular dystrophy. The diagnosis was based on a typical history, clinical picture, serum creatine kinase activity, and muscle pathological condition. In about half, there was a family history of the disease and typical electrophysiological
changes. In almost all cases, the biopsy specimen had been taken in the early clin¬ ical or preclinical phase of the disease.
Fibers were counted when obviously necrotic and invaded by phagocytes. All fibers within two fiber diameters of the periphery of each fasciculus were disre¬ garded. Two or more fibers undergoing
phagocytosis
were accepted as a group, they were directly contiguous separated by normal fibers. In the
either when or
not
few instances when the connective tissue space separating them was narrowed to less than the diameter of the smaller of the necrotic fibers by intervening normal fibers, they were classed as separate fibers. Only five such adjacent-but-not-contiguous
pairs
of fibers undergoing phagocytosis rejected from consideration in groups of fibers by virtue of this criterion. were
Exercise Maintained
Slope
Exercise Initial
Slope
N
Postocclusion Initial Slope \
Counts per Second
t
Time
Exercise
Occlusion
(2 min)
(2 min)
Fig 1.—Diagram of clearance of ,33Xe from muscle plotted on semilogarithmic paper. Parts of clearance that were measured were successively: initial resting slope, initial and maintained slope with exercise, postexercise reestablished resting rate, and postocclusion initial rate. Clearance ceases during arterial occlusion. RESULTS Human Experiments The clearance rates expressed as half-times in minutes of 133Xe clear¬ ance for the initial resting level, initial and maintained level during exercise, the postexercise resting lev¬ el, and the initial postarterial occlu¬ sion level are shown in Table 1. As de¬ scribed later (Comment), these indi¬ vidual values are greatly dependent on the proportion of fat in the muscle being studied. The values in dys¬ trophic muscles were, however, of the same order as those in the two con¬ trols with the exception of patient 2. Dystrophic muscle blood flow in¬ creased during exercise and following arterial occlusion. Similarly, though it was not possible to obtain an accurate measurement of the duration of reac¬ tive hyperemia, there was no obvious difference between the patients and controls; in all cases, the resting flow was restored to normal in less than 15 seconds. In order to show the degree to which blood flow may be increased by exer¬ cise or ischemia, the values of in¬ creased clearance are expressed as a ratio of the preceding resting clear-
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in Table 2. All the dystrophic muscles showed a large increase in blood flow (as indicated by the ratio) in response to exercise and ischemia, and the extent of this increase did not appear substantially different from that in the two controls. ance
Animal Studies
The skeletal muscle in the mice with muscular dystrophy showed a similar pattern of pathological change to Duchenne muscular dystrophy, with both scattered isolated fibers and other damaged fibers occurring in small groups (Fig 2). Qualitative
Radioautographic Data Three Seconds After Injection of 4125I-antipyrine.— In both normal and dystrophic muscle, the distribution of silver grains was patchy, the highest activity being over the blood vessels and connective tissue septae (Fig 3). In one of the three dystrophic speci¬ mens, the grain count was higher over areas showing the most severe active
dystrophic change, that is, fiber necrosis, phagocytosis, generation.
muscle and re¬
Table 1.—,33Xe Clearance Half-Times Exercise
Degree of
Patient
Age (yr), Sex 31,M
Diagnosis Normal,
Muscle Gastrocnemius
56, M
Normal,
Gastrocnemius
48, 43, 9, 19, 48,
Limb-girdle FSH* Duchenne Duchenne
Gastrocnemius Gastrocnemius Gastrocnemius Gastrocnemius
Limb-girdle
Biceps
Weakness of Muscle Tested None
Maintained
Postexercise
Slope
Slope
Resting
13.8
1.5
2.7
19.9
None
39.8
2.3
7.7
22.4
3.1
None
32.9 78.1 15.1 291.5 30.0
1.1 1.1 1.5 30.1 2.1
6.9 5.1 7.2 44.9 8.9
26.7 96.5 33.7 188.5 96.3
9.7 1.2 5.4
control
control
M M M M M
Initial
Postocclusion Initial Slope 3.1
Resting
Slight Moderate Moderate Severe
18.7
FSH, Facioscapulohumeral.
Degree of Weakness of Muscle
Subject
Age, yr 31 56 48
43 19 48
Maintained
Slope
Slope
Postocclusion Initial Slope
Slight
9.2 17.3 29.9 71.0
5.1 5.2 4.8 15.3
6.4 7.2 2.7 80.4
Moderate Moderate Severe
10.0 9.7 14.3
2.1 6.5 3.4
6.2
Tested None None None
Gastrocnemius Gastrocnemius Gastrocnemius
Biceps
Six Seconds After Injection.- The distribution of silver grains over the normal muscle was much more even than at three seconds after injection (Fig 3), the density over blood vessels and muscle fibers being approximate¬ ly equal. Over the dystrophic muscle, there was considerable variation of grain density (Fig 3). In one of the three dystrophic specimens, the high¬ est
density
was over areas
showing dys¬
the greatest amount of active
trophic change.
Ten Seconds After Injection.— The exact time at which this leg was removed varied from 9 to 12 seconds
in different
animals, though this did
not appear to affect
substantially the distribution of radioactivity. Over the normal muscle, there was slightly more variation between areas than at six seconds (Fig 3). However, there was considerably more variation be¬ tween different areas over the dys¬ trophic muscle, though this was less than over the dystrophic muscle at six seconds after injection and not related to the activity of the dystrophic change (Fig 3).
Twenty Seconds After Injection.- The
grain density
over
Exercise Initial
Muscle Gastrocnemius Gastrocnemius Gastrocnemius
both the normal
count over individual muscle fibers and their histochemical type. The density of grains over normal muscle was highest at three to six seconds after injection and fell pro¬ gressively thereafter. The same pat¬ tern was seen over the dystrophic muscle, though the distribution re¬ mained more patchy than normal for a
grain
Table 2.—133Xe Clearance Half-Time Ratios
5.1
and
dystrophic muscle was relatively and in the dystrophic material showed no relationship to the degree of active dystrophic change (Fig 3). even
40 to 160 Seconds After Injection.— The distribution of grains over the normal and dystrophic muscle was even at all these later time points. Only at three seconds after injec¬ tion were areas found in the dys¬ trophic muscle with a density of silver grains equivalent to background lev¬ els, and similar areas were also found in the normal muscle at this time. No evidence was, therefore, found of totally ischemie areas in the muscle of dystrophic mice. The grain density over the dys¬ trophic muscle tended to be slightly higher than over the normal muscle, but this was probably due to the slightly lower body weight of the
dystrophic mice, which, therefore, a slightly higher dose of radioactivity per gram of body weight received
than the normal littermate controls. In both normal and dystrophic muscle, the radioactivity appeared eventually to distribute itself evenly throughout the muscle. No relationship was detected at any time between the
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longer period.
Quantitative
Radioautographic Data The minimum grain count per stan¬ dard area of 1,600 sq µ of each
animal is shown in Fig 4. In Table 3 is shown the comparison of the mini¬ mum grain counts between the groups of normal and dystrophic animals at each time point. At three and six seconds after injection, there was no statistical difference between normal and dystrophic muscle, while at 10 and 20 seconds after injection, the mini¬ mum grain density was higher than normal. This difference was probably due to the slightly higher dose of 4-12'I-antipyrine per gram of body weight that the dystrophic animals received. The difference disappeared when a correction was applied for the
body weights.
Quantitative
Histological
Data
The distribution of fibers undergo¬ ing necrosis and phagocytosis in Duchenne muscle biopsy specimens is shown in Table 4. Seven hundred fifty-two such fibers were counted, and the number occurring singly was 529 (70%). These figures do not corre¬ spond to a Poisson distribution, there being a slight excess of necrotic fibers
in clumps of four and more (P.10
Bradley, DM; Michael
Margaret Johnson, PhD;
D. O'Brien, MD; Dennis N. David J. Newell, PhD
Walder, MD; Dorothy Murchison, BSc;
The vascular hypothesis of the cause of muscular dystrophy suggests that ischemia is responsible for the muscle fiber necrosis. A xenon 133 clearance study of muscle blood flow in Duchenne and other muscular dystrophies showed no obvious difference between the response to exercise and arterial occlusion compared with control subjects. Radioautographic study of distribution of 4-125l-antipyrine in skeletal muscle of mice with muscular dystrophy showed no abnormal areas of ischemia.
A statistical examination was also made of the grouping of damaged fibers, one of the observations on which the vascular hypothesis was based. Only 0.9% of fibers undergoing phagocytosis occurred in groups of four or more fibers in greater frequency than would have been expected by chance, and 70% of such fibers were isolated. These studies argue strongly against the vascular hypothesis of the cause of muscular dystrophy.
Demos12 advanced a vascu¬ lar of the cause of muscular dystrophy, suggesting that the muscle degeneration was due to an abnormality of skeletal muscle blood flow. He studied the circulation times in a group of normal individuals and a group of patients with various myopa¬ thies. The latter were found to have a greater than normal range of circula¬ tion times, some being longer and some shorter than normal. A subse¬ quent double-blind trial of a vasodilating agent, bamethan sulfate (phydroxyphenylbutyl amino ethanol; Butylsympatol), in a group of pa¬ tients with various myopathies was reported to show a substantial im¬ provement in the clinical state of the patients receiving the drug and a fall in the serum creatine kinase activity.3
Further experience has reinforced his enthusiasm for the procedure.1 Demos has also recently reported finding abnormalities in an enzyme, diphenoloxidase, in patients with myopa¬ thies.:>,i He suggested that these abnormalities in an enzyme that may play a part in the inactivation of cate¬ cholamines lend further support to a vascular hypothesis of the cause of muscular dystrophy. The vascular hypothesis of the cause of muscular dystrophy has recently been propounded more strongly by Hathaway and et al.7 They stated that one pathological feature of the early stages of Duchenne muscular dystro¬ phy is the tendency for grouping of small numbers of fibers at the same stage of breakdown in an area of muscle. If the underlying mechanism of the disease were a biochemical abnormality in the individual muscle fibers, then a random distribution of breakdown of single muscle fibers would be expected. They produced a similar pathological picture of groups of damaged fibers by microembolization of rabbit muscle with dextran
In 1961,hypothesis
-
-
for publication Sept 3, 1974. From the Muscular Dystrophy Research Laboratories, Newcastle General Hospital, Newcastle upon Tyne, England. Reprint requests to Muscular Dystrophy Research Laboratories, Newcastle General Hospital, Newcastle upon Tyne, NE4 6BE, England
Accepted
(Prof. Bradley).
(Arch
Neurol
32:466-473, 1975)
Downloaded From: http://archneur.jamanetwork.com/ by a Western University User on 06/07/2015
it was recognized that structural abnormalities of the intramuscular blood vessels were not a feature of Duchenne muscular dystro¬ phy.8 A further experimental model was, therefore, advanced to overcome this problem. A combination of aortic ligation and intraperitoneal injection of the vasoactive compounds 5hydroxytryptamine and norepinephrine produced similar grouping of damaged fibers.8 They, therefore, suggested that a decrease in muscle blood flow might underlie the degen¬ eration of the muscle in Duchenne muscular dystrophy. In order to provide direct evidence on this hypothesis, we have examined the intramuscular blood flow in patients with Duchenne and other forms of muscular dystrophy and in mice with inherited muscular dystro¬ phy (Bar Harbor 129 Re dy/dy). The pathological picture in the skeletal muscle of these animals is similar to that of human Duchenne muscular dystrophy, with both isolated and small groups of damaged muscle fibers.9 The xenon 133 (133Xe) clear¬ ance technique was used to study muscle blood flow in the patients. 4125I-antipyrine was used as a marker of plasma water in the mice. The diffusion of this marker from the blood vessels into the muscle and back into the blood vessels was studied at intervals after its intravascular injec¬ tion by the technique of radioautography of water-soluble substances.10 Muscle blood flow was studied under conditions of maximal vasodila¬ tation produced either by exercise or arterial occlusion, because in resting
particles. However,
muscle, only a relatively small propor¬ tion of the muscle capillaries are patent at any one time.11 Under the condition of maximal vasodilatation, vascular insufficiency of muscle is seen either as a decreased blood flow or as a prolonged period of reactive hyperemia. A vascular abnormality in the radioautographic study is shown by decreased inflow and clearance of radioactivity from the muscle and by ischemie areas that remain free of
radioactivity. SUBJECTS AND METHODS Human Studies
patients with muscular dystrophy studied, two with Duchenne, one with limb-girdle, and one with facioscapulohumeral muscular dystrophy. There was no family history in either patient with Duchenne muscular dystrophy, though the Four
were
clinical course and biochemical and electrophysiological studies confirmed the diag¬ nosis. Patient 1 (Duchenne) was age 9 years and was still just able to walk. Patient 2 (Duchenne) was age 19 years and was in an advanced state; he died the
following year. Patient 3, age 47 years, had typical limb-girdle muscular dystrophy that had begun about the age of 20 years. Biochemical, electrophysiological, and pathological studies confirmed the diagno¬ sis. He was still able to walk at the time of the study. Patient 4, a 43-year-old man, had facioscapulohumeral muscular dystrophy with no family history. Facial weakness began in early childhood and limb-girdle weakness at about the age of 17 years. The muscles studied in the patients varied in their degree of weakness from normal to severely weak. Two normal males, ages 31 and 56, were studied as controls.
Methods One hundred microcuries of 133Xe in 0.1 ml of physiological saline solution was injected into the muscle and the radioac¬ tivity recorded by a portable apparatus12 using a 1.27-cm thallium-activated sodium iodide crystal (modified EKCO N632) and a photomultiplier tube (EMI type 9650B) in conjunction with a portable telometer pack with a ratemeter (Nuclear Enterprises). The experimental procedure is shown in Fig 1. The radioactive count rate over the depot falls as 133Xe is cleared by the muscle blood flow. The value of the resting clea¬ rance was measured as the slope of the line relating counts per second to time. There are two phases of increased clearance during exercise, the fast initial rate and
the slower maintained rate. The slope of both clearances was similarly measured. After arterial occlusion, the clearance is in¬ creased to repay to metabolic debt incurred due to ischemia. The slope of this increased clearance was also measured. The blood flow was recorded during a five-minute rest, followed by two minutes of exercise (contraction of the muscle against a 2.73-kg (6-lb) weight once a second in time with a metronome). The changes in blood flow after exercise were followed until the resting rate was reestablished. This test was followed by a two-minute period of total arterial occlusion with a pneumatic cuff placed on the limb proximal to the muscle under investigation and inflated to a pressure of 180 to 200 mm Hg. Arterial occlusion was verified by the cessation of 133Xe clearance. Muscle blood flow is expressed as the half-time in minutes of the 133Xe clearance and as the ratio between 133Xe clearance half-times at rest and during the experi¬ mental maneuvers to increase the blood flow. Values for the initial resting level, initial slope with exercise, the flow during exercise, the second resting levels, the initial slope after occlusion, and the third resting levels were obtained.
Animal
Experiments mice Dystrophie (dy/dy) of
the Bar Harbor 129 Re strain were used and compared with their littermates of the same sex. Animals of 1 to 3 months of age were studied. The dystrophic animals showed moderately severe clinical and pathological signs of the muscle disease.
Operative Technique Mice
toneally
anesthetised with intraperiadministered pentobarbital (pen-
were
0.12 mg/10 gm body weight, and anesthesia was maintained with a mixture of halothane and oxygen delivered through a face mask. The skin of both forelegs and hind legs, was carefully removed, and the denuded legs were care¬ fully wrapped in gauze swabs soaked in warm physiological saline solution. Body temperature, monitored by subcutaneous thermocouple, was maintained at 38 C by a heat lamp. Fine-needle stimulating cath¬ odes were inserted into the region of the sciatic notch on both sides, the anode being a similar fine needle in the subcutaneous tissue of the dorsal region. The animal was then allowed to rest for ten minutes before supramaximal stimuli at one each second were applied for five minutes to the electrodes, using square wave pulses of 0.1 msec delivered by an isolated stimulator. The intensity of the
tobarbitone),
Downloaded From: http://archneur.jamanetwork.com/ by a Western University User on 06/07/2015
stimulus was adjusted to produce a maximal twitch of the hind limbs. With the intensity and electrode position used, the upper limbs also contracted with each stimulus.
Injection
Procedure
Two groups of animals, three dystrophic mice and three littermate controls, were studied. In the first group, 200/J (70 microcuries) of 4-125I-antipyrine (0.35 microcuries/ml) were injected into the jugular bulb at the end of the five-minute stimulation period. The right foreleg was rapidly cut from the body with bone forceps at three seconds, the right hind leg at six seconds, the left foreleg at ten seconds, and the left hind leg at 20 seconds after injection. Immediately after removal, the individual legs were rapidly frozen in isopentane cooled in liquid nitrogen. The second group of animals was treated in an identical fashion, except that 200 µ (130 microcuries) of 4-125I-antipyrine (0.65 microcuries/ml) was injected, and the legs were removed in the following order: right foreleg, 40 seconds; right hind leg, 80 seconds; left foreleg, 120 seconds; left hind leg, 160 seconds after injection.
Radioautographic Techniques Great care was taken to ensure that the muscle remained frozen at below —20 C from the time of rapid initial freezing of the legs immediately after separation from the body to the development of the final radioautograms. Muscle was cut off the bones of the individual legs at —20 C, mounted in a compound embedding me¬ dium for frozen tissue (Tissue-Tek O.C.T.) and cut in total darkness at 8µ thickness on a cryostat. Microscope slides were predipped in undiluted emulsion (Ilford K2), dried in air at room temperature, and stored at —20 C in lightproof boxes in the
cryostat.
Cryostat
were
sections of the
cut in the
dark,
were
muscle, which picked up on
these emulsion-coated slides. The slides with sections were stored in lightproof boxes at —70 C for six weeks. They were then allowed to warm to room tempera¬ ture, and the radioautogram was developed by the technique previously reported.13 The sections were stained with hematoxylineosin. Serial sections of those sections studied by radioautography were stained with hematoxylin-eosin and by histo¬ chemical techniques for myosin ATPase and nicotinamide adenine nucleotide dehy¬
drogenase. Radioautograms were studied qualita¬ tively under the light microscope, and quantitative silver grain counting was
undertaken time points
on
material from the shorter
(first group of animals) under 1,000 magnification (oil immersion ob¬ jective). For the latter, attention was directed to areas of lowest grain density over the muscle section. Major arteries and veins were avoided, and the areas counted lay entirely within the body of the muscle. The total number of silver grains in a standard area of 1.6/1,600 sq firn was count¬ ed. This total included grains lying over both muscle fibers and endomysial connec¬ tive tissue. In the dystrophic animals, areas showing marked degeneration or regener¬ ation of fibers were avoided, counting being restricted to areas containing rela¬ tively normal-appearing muscle fibers. In the normal animals, this area included part or all of two or three muscle fibers and in the dystrophic animals part or all of three to six fibers. Three standard areas of each of three sections were counted for each animal. The background radioactivity was measured by counting three standard areas without muscle, immediately adjacent to the mus¬
cle sections studied. The background grain density was subtracted from the measured tissue grain density to give the corrected tissue grain density.
Statistical Analysis of Distribution of Necrotic Fibers A statistical analysis of the frequency of single fibers and of groups of fibers undergoing phagocytosis was made in muscle biopsy specimens from 42 patients with Duchenne muscular dystrophy. The diagnosis was based on a typical history, clinical picture, serum creatine kinase activity, and muscle pathological condition. In about half, there was a family history of the disease and typical electrophysiological
changes. In almost all cases, the biopsy specimen had been taken in the early clin¬ ical or preclinical phase of the disease.
Fibers were counted when obviously necrotic and invaded by phagocytes. All fibers within two fiber diameters of the periphery of each fasciculus were disre¬ garded. Two or more fibers undergoing
phagocytosis
were accepted as a group, they were directly contiguous separated by normal fibers. In the
either when or
not
few instances when the connective tissue space separating them was narrowed to less than the diameter of the smaller of the necrotic fibers by intervening normal fibers, they were classed as separate fibers. Only five such adjacent-but-not-contiguous
pairs
of fibers undergoing phagocytosis rejected from consideration in groups of fibers by virtue of this criterion. were
Exercise Maintained
Slope
Exercise Initial
Slope
N
Postocclusion Initial Slope \
Counts per Second
t
Time
Exercise
Occlusion
(2 min)
(2 min)
Fig 1.—Diagram of clearance of ,33Xe from muscle plotted on semilogarithmic paper. Parts of clearance that were measured were successively: initial resting slope, initial and maintained slope with exercise, postexercise reestablished resting rate, and postocclusion initial rate. Clearance ceases during arterial occlusion. RESULTS Human Experiments The clearance rates expressed as half-times in minutes of 133Xe clear¬ ance for the initial resting level, initial and maintained level during exercise, the postexercise resting lev¬ el, and the initial postarterial occlu¬ sion level are shown in Table 1. As de¬ scribed later (Comment), these indi¬ vidual values are greatly dependent on the proportion of fat in the muscle being studied. The values in dys¬ trophic muscles were, however, of the same order as those in the two con¬ trols with the exception of patient 2. Dystrophic muscle blood flow in¬ creased during exercise and following arterial occlusion. Similarly, though it was not possible to obtain an accurate measurement of the duration of reac¬ tive hyperemia, there was no obvious difference between the patients and controls; in all cases, the resting flow was restored to normal in less than 15 seconds. In order to show the degree to which blood flow may be increased by exer¬ cise or ischemia, the values of in¬ creased clearance are expressed as a ratio of the preceding resting clear-
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in Table 2. All the dystrophic muscles showed a large increase in blood flow (as indicated by the ratio) in response to exercise and ischemia, and the extent of this increase did not appear substantially different from that in the two controls. ance
Animal Studies
The skeletal muscle in the mice with muscular dystrophy showed a similar pattern of pathological change to Duchenne muscular dystrophy, with both scattered isolated fibers and other damaged fibers occurring in small groups (Fig 2). Qualitative
Radioautographic Data Three Seconds After Injection of 4125I-antipyrine.— In both normal and dystrophic muscle, the distribution of silver grains was patchy, the highest activity being over the blood vessels and connective tissue septae (Fig 3). In one of the three dystrophic speci¬ mens, the grain count was higher over areas showing the most severe active
dystrophic change, that is, fiber necrosis, phagocytosis, generation.
muscle and re¬
Table 1.—,33Xe Clearance Half-Times Exercise
Degree of
Patient
Age (yr), Sex 31,M
Diagnosis Normal,
Muscle Gastrocnemius
56, M
Normal,
Gastrocnemius
48, 43, 9, 19, 48,
Limb-girdle FSH* Duchenne Duchenne
Gastrocnemius Gastrocnemius Gastrocnemius Gastrocnemius
Limb-girdle
Biceps
Weakness of Muscle Tested None
Maintained
Postexercise
Slope
Slope
Resting
13.8
1.5
2.7
19.9
None
39.8
2.3
7.7
22.4
3.1
None
32.9 78.1 15.1 291.5 30.0
1.1 1.1 1.5 30.1 2.1
6.9 5.1 7.2 44.9 8.9
26.7 96.5 33.7 188.5 96.3
9.7 1.2 5.4
control
control
M M M M M
Initial
Postocclusion Initial Slope 3.1
Resting
Slight Moderate Moderate Severe
18.7
FSH, Facioscapulohumeral.
Degree of Weakness of Muscle
Subject
Age, yr 31 56 48
43 19 48
Maintained
Slope
Slope
Postocclusion Initial Slope
Slight
9.2 17.3 29.9 71.0
5.1 5.2 4.8 15.3
6.4 7.2 2.7 80.4
Moderate Moderate Severe
10.0 9.7 14.3
2.1 6.5 3.4
6.2
Tested None None None
Gastrocnemius Gastrocnemius Gastrocnemius
Biceps
Six Seconds After Injection.- The distribution of silver grains over the normal muscle was much more even than at three seconds after injection (Fig 3), the density over blood vessels and muscle fibers being approximate¬ ly equal. Over the dystrophic muscle, there was considerable variation of grain density (Fig 3). In one of the three dystrophic specimens, the high¬ est
density
was over areas
showing dys¬
the greatest amount of active
trophic change.
Ten Seconds After Injection.— The exact time at which this leg was removed varied from 9 to 12 seconds
in different
animals, though this did
not appear to affect
substantially the distribution of radioactivity. Over the normal muscle, there was slightly more variation between areas than at six seconds (Fig 3). However, there was considerably more variation be¬ tween different areas over the dys¬ trophic muscle, though this was less than over the dystrophic muscle at six seconds after injection and not related to the activity of the dystrophic change (Fig 3).
Twenty Seconds After Injection.- The
grain density
over
Exercise Initial
Muscle Gastrocnemius Gastrocnemius Gastrocnemius
both the normal
count over individual muscle fibers and their histochemical type. The density of grains over normal muscle was highest at three to six seconds after injection and fell pro¬ gressively thereafter. The same pat¬ tern was seen over the dystrophic muscle, though the distribution re¬ mained more patchy than normal for a
grain
Table 2.—133Xe Clearance Half-Time Ratios
5.1
and
dystrophic muscle was relatively and in the dystrophic material showed no relationship to the degree of active dystrophic change (Fig 3). even
40 to 160 Seconds After Injection.— The distribution of grains over the normal and dystrophic muscle was even at all these later time points. Only at three seconds after injec¬ tion were areas found in the dys¬ trophic muscle with a density of silver grains equivalent to background lev¬ els, and similar areas were also found in the normal muscle at this time. No evidence was, therefore, found of totally ischemie areas in the muscle of dystrophic mice. The grain density over the dys¬ trophic muscle tended to be slightly higher than over the normal muscle, but this was probably due to the slightly lower body weight of the
dystrophic mice, which, therefore, a slightly higher dose of radioactivity per gram of body weight received
than the normal littermate controls. In both normal and dystrophic muscle, the radioactivity appeared eventually to distribute itself evenly throughout the muscle. No relationship was detected at any time between the
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longer period.
Quantitative
Radioautographic Data The minimum grain count per stan¬ dard area of 1,600 sq µ of each
animal is shown in Fig 4. In Table 3 is shown the comparison of the mini¬ mum grain counts between the groups of normal and dystrophic animals at each time point. At three and six seconds after injection, there was no statistical difference between normal and dystrophic muscle, while at 10 and 20 seconds after injection, the mini¬ mum grain density was higher than normal. This difference was probably due to the slightly higher dose of 4-12'I-antipyrine per gram of body weight that the dystrophic animals received. The difference disappeared when a correction was applied for the
body weights.
Quantitative
Histological
Data
The distribution of fibers undergo¬ ing necrosis and phagocytosis in Duchenne muscle biopsy specimens is shown in Table 4. Seven hundred fifty-two such fibers were counted, and the number occurring singly was 529 (70%). These figures do not corre¬ spond to a Poisson distribution, there being a slight excess of necrotic fibers
in clumps of four and more (P.10
