Histometric evaluation of branches of peroneal nerve: Technique for combined biopsy of muscle nerve and cutaneous nerve

Brain Research, 52 (1973) 37-59

37

© Elsevier Scientific Publishing Company, Amsterdam- Printed in The Netherlands

HISTOMETRIC EVALUATION OF BRANCHES OF PERONEAL NERVE: TECHNIQUE FOR COMBINED BIOPSY OF MUSCLE NERVE AND CUTANEOUS NERVE

JOHN C. STEVENS, ERIC P. LOFGREN ANUPETER JAMES DYCK

Mayo Clinic and Mayo Foundation, Rochester, Minn. 55901 (U.S.A.} (Accepted September 25th, 1972)

SUMMARY

A technique for combined biopsy of muscular and cutaneous branches of the common peroneal nerve is described. Nerves studied were the nerve to the peroneus brevis, the lateral fascicles of the deep peroneal (to the extensor digitorum brevis) nerve and the cutaneous branches of the superficial peroneal nerve. These nerves from normal subjects of various ages were evaluated by histometric techniques. Degenerative changes affecting teased myelinated fibers were infrequent, but their incidence increased significantly with age. Teased-fiber measurements were analyzed to provide the average internode length (IL) and internode diameter (ID) of the large and small fiber populations of the nerve. Estimates of the IL of the whole nerve were adjusted for sampling error inherent in fiber teasing. Values for lateral fascicles of the deep peroneal nerve from persons 18 years and older (IL) and 10 years and older (ID) are: mean IL, small fibers, all nerves, 338/am; mean IL, large fibers, all nerves, 962/am; mean IL, all nerves, 602/am; mean ID, small fibers, all nerves, 5.1/am; mean ID, large fibers, all nerves, 10.8/am; mean ID, all nerves, 7.7/am. Values for the superficial peroneal nerve from persons 18 years and older are: mean IL, small fibers, all nerves, 328/am; mean IL, large fibers, all nerves, 873/am; mean IL, all nerves, 554/am. Th~ adjusted mean IL of the lateral fascicles of the deep peroneal nerve and the superficial peroneal nerve decreased with increasing age. Variation in IL increased significantly with age and was greater in small fibers than in large fibers. Although histograms made from teased-fiber measurements of the nerve to the peroneus brevis were bimodal, accurate separation of fibers into large and small groups was not possible. The fiber density (number of fibers/sq, ram) in the lateral fascicles and in the superficial peroneal nerve decreased significantly with age, the averages for adults were 6,838 and 8,648/ sq. mm, respectively. Fiber-diameter histograms revealed that the superficial peroneal nerve contained the largest number of small fibers, the nerve to the peroneus brevis

38

J . C . STEVENS cl a].

contained the least, and the lateral fascicles of the deep peroneal nerve contained an intermediate number.

INTRODUCTION

In recent years, nerve biopsy, particularly of the sural nerve, has become a useful procedure for the study of peripheral neuropathy. Histologic and teased-fiber measurements of the normal sural nerve have been publishedlZ,a~,zl,2s, 29. Histologic measurements of numbers and sizes of myelinated fibers on transverse sections have been adequately evaluated in many previous studies, but teased-fiber measurements have seldom been based on a large enough sample of randomly selected fibers to be representative of the nerve. In addition, normative data for a muscle nerve are not available. Muscle nerve biopsy would be valuable in investigations, and occasionally also in diagnosis, of disorders which affect lower motor neurons. Ideally, biopsy would include fascicles of cutaneous and muscle nerves and muscle obtained through one incision. In this study, we explored the suitability of biopsy of nerve to the peroneus brevis (muscle nerve) and fascicles of the superficial peroneal nerve (cutaneous nerve) through one incision and of the lateral fascicles of the deep peroneal nerve (muscle nerve) and fascicles of the superficial peroneal nerve (cutaneous nerve) through another incision. We report normative histologic and teased-fiber measurements of these nerves as a function of age. MATERIALS AND METHODS

Six nerves to the peroneus brevis (N-PB) were taken from the upper to middle third of the leg, where the nerve lies between the peroneus longus and peroneus brevis. Cutaneous fascicles of 5 superficial peroneal nerves (SP-N) were taken at the same level. The lateral fascicles of 18 deep peroneal nerves and cutaneous fascicles from 12 superficial peroneal nerves were obtained at a level just proximal to the extensor retinaculum. Dissections of amputated legs showed that the lateral fascicles of the deep peroneal nerve (LFDP) above the ankle formed the lateral terminal branch of the deep peroneal nerve to the extensor digitorum brevis (EDB) in front of the ankle. The L F D P were taken just above the superior extensor retinaculum, via an incision of the deep fascia medial to the tendon of the extensor hallucis longus. The skin incision was made somewhat lateral to the tendon to make it easier to locate the cutaneous branches of SP-N. In living subjects, the pulse of the anterior tibial artery was used as a guide for the location of the LFDP. At biopsy, the deep peroneal nerve was split longitudinally and the lateral fascicles to be taken were electronically stimulated to be sure that motor fibers to the extensor digitorum brevis were present. A section of the remaining medial fascicles of the deep peroneal nerve (MFDP) was also taken in most cases. Nerves were obtained at leg amputations, at post mortem examinations, and

COMBINED MUSCLE-CUTANEOUS NERVE BIOPSY

39

from one paid volunteer by methods la and criteria for normality previously described 1°. Nerves were removed less than 4 h after death or amputation. Fixation was in 2 70 glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, at 0-4 °C, followed by 1 70 osmium tetraoxide in the same buffer. The portion of nerve for teasing was fixed in glutaraldehyde for 30-60 min, depending on the size of the nerve, and in osmium tetraoxide for 3 h. Portions to be embedded in epoxy were fixed for 2-2.5 h in glutaraldehyde and for 2 h in osmium tetraoxide. Our method of preparing single teased fibers has been described ls,14. Small bundles of fibers from each of the available fascicles were teased consecutively without regard to size. Fibers with a minimum of 4 nodes of Ranvier were typed according to the following criteria: type A, normal appearance; type B, irregularity of myelin sheath, normal variability of internode length and diameter, and no retraction at nodes of Ranvier; type C, irregularity of myelin sheath, normal variability of internode length and diameter, and retraction of myelin sheaths of a few microns at one or more nodes of Ranvier; type D, one or more demyelinated regions longer than a few microns, usually more variability in internode length and diameter than in normal fibers, usually irregularities of myelin sheaths of some internodes, and occasionally remyelinated internodes; type E, various stages of segmentation into myelin ovoids in linear rows, all internodes of fibers showing abnormalities at the same state of degeneration; and type F, more than one very recently remyelinated internode. Careful attention to the duration of glutaraldehyde fixation resulted in preparation of long, artifact-free, teased fibers (Fig. 1). Teased-fiber internode length and diameter were measured by using an ocular micrometer. The diameter was measured, at a point one-fourth of the distance along the length of the internode, with a 'high-dry' objective ( × 500). Diameters of teased fibers from SP-N were not measured. In most cases, 4-7 internodes per fiber were measured, and in all cases this was done on 100 fibers per nerve. For each teased fiber, the mean internode length (IL) and, if available, internode diameter (ID) and the coefficient of variation (CV) of IL and ID (SD × 100/mean) were calculated. By using the mean IL of a fiber, histograms were drawn for the 100 fibers of each nerve. It was apparent that, except in the very young, there were two populations of fibers, one with long internodes (large fibers) and one with short internodes (small fibers). From histograms of mean IL of teased fibers the approximate internode length at the point of separation of the two populations, determined by eye, of the nerves of subjects 18 years old and older was similar for both L F D P and SP-N and averaged 625 #m. For statistical analysis, a small fiber was considered to have a mean IL less than 625/~m and a large fiber, an IL of 625/zm or more. The point of separation for a nerve of persons less than 18 years old was determined from its IL histogram. From histograms based on ID, fibers of L F D P were divided into large and small, but with some difficulty. The point of separation used for adults was 7.6/~m. Fibers of the N-PB were not divided because the point of separation between groups could not be determined precisely enough from either the IL or ID histograms. The average values of IL and of CV of IL for the large-fiber and small-fiber groups will be designated

40

J. ( . S[EVENS ~)! ell.

Fig. 1. Upper, Consecutive strips, from top to bottom, of normal teased myelinated fiber from lateral fascicles of deep peroneal nerve (LFDP) of 10-year-old person. Lower, Teased myelinated fiber fi'om nerve to peroneus brevis (N-PB) of 4-year-old child, showing branching.

COMBINED MUSCLE-CUTANEOUS NERVE BIOPSY

41

'mean IL, large fibers' or 'mean IL, small fibers' and 'mean CV of IL, large fibers' or ' m e a n CV of IL, small fibers'. Since, in teasing, the correct proportion of small and large fibers, as it is found in the nerve, was not always taken, an adjustment (see below) was necessary to correct for this sampling error. The adjusted mean IL and the adjusted CV of IL of all 100 fibers of each nerve will be designated 'adjusted mean IL, nerve' and 'adjusted CV of IL, nerve'. The average of the 'adjusted' values for different specimens of the same nerve will be designated 'mean IL, all nerves' and 'mean CV of IL, all nerves'. The average values for the large-fiber and small-fiber groups of different specimens of the same nerve will be designated 'mean IL, small fibers, all nerves' and so forth. Epoxy-embedded tissue sectioned at 1-1.5 # m was photographed through the phase-contrast microscope, and the numbers and diameters of myelinated fibers (diameter histogram) were d6termined from photographic enlargements ( × 1,500) with a Zeiss particle size analyzer (TGZ3) using the standard measuring range and settings to express the results as an exponential frequency distribution curve. Photographs of a reticle were used to check final magnification. Up to 6 photographs per nerve were taken at random. Fascicles smaller than a frame of 135 m m film were not photographed except in the very young when all the fascicles were small. Fibers which were not measured because of artifact or a flattened profile were used in the calculation of the number of fibers per square millimeter of transverse fascicular area. Programs were written for a 9810A Hewlett-Packard calculator and plotter which was used to obtain various statistical results and to make the bar graphs shown in this paper. Differences in preparation and in method of measurement do not allow direct comparison between fiber diameter peaks in histograms plotted from diameter measurement and those in histograms plotted from teased-fiber measurements. The diameter histogram based on measurements of 400-2,000 fibers is a more accurate representation of a nerve's fiber-size distribution than is the IL histogram based on only I00 fibers. In addition there is the likelihood that the correct proportion of large and small fibers will not be teased. In order to obtain a more accurate average of a nerve's mean internode length, we made an adjustment for incorrect sampling of large and small teased fibers: adjusted mean IL, nerve = 700small fibers x mean S ÷ 70 large fibers x mean L in which: 70 small fibers -- 70 small fibers obtained from diameter histograms; 700 large fibers = 70 large fibers obtained from diameter histograms; mean S ----mean IL, small fibers; and mean L = mean IL, large fibers. Because small fibers have a larger CV of IL and CV of ID, another adjustment as necessary: adjusted CV of IL, nerve ---- ~ small fibers × CVs + ~ large fibers × CVL in which: CVs = mean CV of IL, small fibers; and CVt, ----mean CV of IL, large fibers.

42

J. C . S T E V E N S

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TABLE I CONDITION OF TEASED MYELINATED FIBERS OF NERVE TO PERONEUS BREVIS

Nerve

8-71 7-71 9-71 15-71 21-71

Age

6 days 5months 4 years 8 years 43 years

(N-PB)

No. fibers graded

Type of myelinatedfiber (%) *

250 277 244 204 260

98.0 100.0 98.8 98.5 96.9

A

B

C

0.4

D

E

F

0.8

0.8

0.4

0.4

0.8 1.5 2.3

0.4

* See text for description of fiber types.

RESULTS The conditions of teased myelinated fibers of N-PB, L F D P , a n d SP-N are summarized in Tables I - I l i . T w e n t y - n i n e of 35 nerves had 97 % or more type A ( n o r m a l appearance) fibers; the percentage of type A fibers decreased with age. The most comm o n a b n o r m a l i t y seen was linear rows of myelin ovoids. P a r a n o d a l or i n t e r n o d a l d e m y e l i n a t i o n was u n c o m m o n . The specimen of L F D P with the highest percentage of a b n o r m a l fibers came from a 30-year-old healthy physician. His neurologic examination gave n o r m a l findings a n d his fasting blood glucose value, c o n d u c t i o n velocities of nerves, a n d electromyogram were normal. The incidence of abnormalities increased with age; the slope of the regression line on age was significantly greater than 0

TABLE II CONDITION OF TEASED MYELINATED FIBERS OF LATERAL FASCICLES OF DEEP PERONEAL NERVE

Nerve

71-71 96-71 75-71 78-71 115-71 54-71 70-71 44-71 79-71 113-71 51-71 108-71 1-72 74-71

Age(years) No.fibers graded

Type of myelinated fiber (%) *

2 10 18 18 21 30 31 33 37 46 58 59 67 70

100.0 99.5 100.0 97.4 97.2 95.6 98.4 99.3 97.6 97.0 95.7 98.2 99.2 95.7

201 216 143 152 177 363 243 144 164 167 139 220 126 281

* See text for description of fiber types.

A

B

C

D

(LFDP)

E

F

2.0 0.6 3.0 1.2 0.7 1.8 3.0 4.3

0.6

1.4

0.4

0.5 2.2 0.4 0.6

0.8 4.3

1.4

43

COMBINED MUSCLE-CUTANEOUSNERVEBIOPSY TABLE III CONDITION OF TEASED MYELINATED FIBERS OF SUPERFICIAL PERONEAL NERVE ( S P - N )

Nerve

8-71 7-71 9-71 15-71 62-71 75-71 78-71 115-71 70-71 79-71 21-71 113-71 51-71 108-71 74-71 41-71

Age (years)

No. fibers graded

Type o f myelinated fiber (%) *

6 days 5months 4 8 15 18 18 21 31 37 43 46 58 59 70 71

127 113 130 127 161 144 177 113 180 173 126 206 175 235 254 113

100.0 100.0 100.0 100.0 99.4 100.0 99.4 99.1 99.4 98.8 97.6 98.1 97.7 99.1 95.3 93.8

A

B

C

D

E

F

0.6

1.6

0.9

0.9

0.6 0.9 0.6 1.2 0.8 1.9 2.3 0.9 4.7 4.4

* See text for description of fiber types.

(SP-N, P =< 0.0005; L F D P , P _--<0.05). Branching o f myelinated teased fibers was not seen in most o f the nerves studied. N-PB o f a 4-year-old child was exceptional, containing 12 sites o f branching a m o n g 244 fibers typed (Fig. 1). The branches were always smaller in size than the parent fiber. The reliability of our teased-fiber measurements was determined by the following analysis. To evaluate the effect of sample size, the average I L o f 25 fibers and o f 50 fibers of one nerve were c o m p a r e d with the average for 100 fibers and were f o u n d to differ by 9.6 and 3.1%, respectively. To set confidence intervals, we considered the L F D P with the largest SD of 100 mean I L (440 #m). With this SD one can be 95 ~o certain that x based on 100 fibers is within 88 ~ m o f the real value for the nerve. F o r m o s t nerves, confidence intervals were m u c h less. Measurement o f the first 4, o f the first 7, and o f all (mean, 15.8) internodes of 100 teased fibers gave the following mean I L and CV: 4 internodes, I L : 524/zm, CV -- 17.3; 7 internodes, I L ---- 516/zm, CV -- 19.5; 15.8 internodes, I L -- 514/~m, CV : 20.5. In most cases, we measured 4-7 internodes per fiber, which means that our measurements are probably in g o o d agreement with the real I L o f the nerve but that the CV is underestimated. To check observer error, 50 fibers of one nerve were measured twice by the same observer; the difference in the two mean I L was less than 1/zm. In Table IV, the average o f the mean I L and 1D for each o f 5 N-PB are given. It is evident that I L and I D increase to age 8 years. A l t h o u g h the values for the 8-yearold nerve are larger than those of the 43-year-old, this m a y be due to sampling error. Regression lines for the relationship of I D to I L are shown in Fig. 2. The regression

44

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TABLE

S T E V E N S t)l (2/.

IV

INTERNODE MEASUREMENTS* OF TEASED MYELINATED FIBERS OF HEALTHY NERVE TO PERONEUS BREVIS

(N-PB) Nerve

8-71 7-71 9-71 15-71 21-71

Age

Number**

6 days 5 months 4 years 8 years 43 years

10.9 11.5 10.4 9.2 15.8

Average (#m J***

SD (Itm)

CV§

IL

ID

IL

ID

1L

ID

242 284 476 691 514

6.6 7.3 8.7 9.7 7.7

46 68 194 180 368

1.7 2.4 3.5 2.6 2.5

14.0 10.1 10.6 9.5 20.5

15.4 16.6 16.0 19.2 18.6

* IL, internode length; ID, internode diameter. ** Mean number ofinternodes per teased fiber. *** Average of the mean of 100 fibers. § Mean of CV of 100 teased fibers.

line of the nerve of the 8-year-old is not as steep as that of the nerve of the 43-year-old. The CV of IL of nerve 8-71 from a 6-day-old infant is high. This resulted from the presence of occasional short internodes, of normal diameter and myelin thickness, in many fibers with otherwise uniformly long internodes. The measurements of IL made on L F D P are listed in Table V. The mean IL, small fibers and mean IL, large fibers of adult nerves do not change significantly with age. For nerves 18 years and older, the mean IL, small fibers, all nerves is 338/zm (SD, 44/zm); the mean IL, large fibers, all nerves is 962 #m (SD, 117 #m); and the

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Fig. 3. Calculated regression lines (y = a + bx) of internode diameter (ID) on internode length (IL) of teased myelinatedfibers of lateral fasciclesof deep peroneal nerve (LFDP) at various ages. mean IL, all nerves is 602/~m (SD, 88 #m). The adjusted mean IL, nerve decreases with age (P --< 0.0005). This may be due to loss of large fibers and repair of degenerative changes affecting large fibers (see below). At all ages, the mean CV of IL, small fibers is greater than the mean CV of IL, large fibers. The adjusted CV of IL, nerve increases with age (P =< 0.005). The ID measurements of LFDP are shown in Table VI. Considering the 11 nerves from persons of ages 10-70 years, the mean ID, small fibers, all nerves is 5.1 /~m (SD, 0.36/zm); the mean ID, large fibers, all nerves is 10.8 #m (SD, 0.64 #m); and the mean ID, all nerves is 7.7 #m (SD, 0.95/~m). The adjusted mean ID, nerve decreases with age (P =< 0.01). The adjusted CV of ID, nerve values are significantly higher than those of IL. This result is probably due to greater actual variation but in part results from imprecision of measuring the diameter. As has been shown for other nerves, there is a linear relationship between fiber diameter and internode length; large fibers have long internodes. Regression lines of IL on ID for L F D P were drawn for each of the nerves (Fig. 3). The slope of the line for the 2-year-old is significantly less than that for each of the other nerves (in each case, P _< 0.001). The slope of the line for the 10-year-old is less than that of the 18-, 21-, 31-, 46-, and 59-year-old at a highly significant level (in each case P __< 0.001), is less than that for the 58-year-old (P ~ 0.02), and is not different from that for the 30-, 33-, and 37-year-old. The slope of the line for the 70-year-old is less than that foi the 18-, 21-, 31-, and 46-year-old (P _< 0.001) and for the 58-year-old (P _< 0.005), and is not different from that for the 10-, 30-, 33-, and 37-year-old. Measurements of IL of teased fibers of SP-N are listed in Table VII. The frequency distributions of IL of teased fibers of nerves from the 6-day-old and 5-month-

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Fig. 4. Representative histograms of mean internode length (IL) of teased myelinated fibers of nerve to peroneus brevis (N-PB), of superficial peroneal nerve (SP-N), and of lateral fascicles of the deep peroneal nerve (LFDP).

old infants are still unimodal, which explains why no values are listed separately for small-fiber and large-fiber groups and why adjusted values were not obtained. The adjusted mean IL, nerve remains fairly constant after age 8 years. I f the nerves of persons 18 years and older are compared, there is a slight decrease of adjusted mean IL, nerve with age (P :~ 0.05). For the 11 adults, mean IL, small fibers, all nerves is 328 # m (SD, 25/~m); mean 1L, large fibers, all nerves is 873 # m (SD, 69 #m); and mean IL, all nerves is 554 # m (SD, 48/~m). The mean IL, ]arge fibers, all nerves is slightly smaller than the corresponding value for the LFDP, perhaps because of the greater number of large motor and proprioceptive fibers present in LFDP. The adTABLE VIII DENSITY OF MYELINATED FIBERS OF HEALTHY NERVE TO PERONEUS BREVIS

Nerve

Age

No./sq. mm

8-71 7-71 9-71 15-71 32-71 21-71

6 days 5 months 4 years 8 years 10 years 43 years

22,913 20,572 8,438 6,538 6,861 4,942

(N-PB)

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Fig. 5. Histograms of diameters of myelinated fibers of nerve to peroneus brevis (N-PB) at various ages.

justed CV of IL, nerve gradually increases with age (P ~ 0.0005), ranging from 9.1 in a 4-year-old to 19.9 in a 71-year-old. Representative histograms of internode measurements of teased fibers drawn for all nerves are shown in Fig. 4. The distribution of the length of internodes of teased fibers was bimodal except in the very young. The fiber density, expressed as number of fibers/sq, mm of transverse fascicular area, and the corresponding diameter histograms of N-PB are shown in Table VIII and Fig. 5. The fibers of the tiny nerves of infants 6 days and 5 months old were very tightly packed together, resulting in a high density. The bimodal fiber spectrums of N-PB are easily recognized because they lack the large small-fiber peak found in the mixed and sensory nerves studied. Table IX lists the fiber densities found in the L F D P and M F D P at the same level• The density is slightly less with increasing age (LFDP, P = 0.05; MFDP, P <= 0.05). The presumed loss involves large fibers predominantly since the percentage of the nerve made up of small fibers increases with age (LFDP, P ~ 0.0025; MFDP, P 0.005). The average fiber densities of the 18 L F D P and 14 M F D P are about the same although some pairs differ by 2,000-3,000 fibers/sq.mm. Additional counts of 3 nerves

51

COMBINED M U S C L E - C U T A N E O U S NERVE BIOPSY

TABLE IX DENSITY OF MYELINATED FIBERS OF HEALTHY DEEP PERONEAL NERVE

Nerve

71-71 32-71 96-71 46-71 62-71 48-71 35-71 75-71 78-71 54-71 70-71 44-71 79-71 113-71 51-71 108-71 1-72 74-71

Age (years)

Lateral fascicles No./sq. mm

Small fibers (%)

2 10 10 14 15 16 18 18 18 30 31 33 37 46 58 59 67 70

10,592 11,414 9,183 8,199 10,348 11,069 8,837 10,587 10,616 8,909 7,418 9,337 5,141 7,326 12,026 7,989 9,194 6,391

44 50 28 53 49 52 39 59 50 56 40 53 49 78 57 72 68 65

(LFDP AND MFDP) Medial fascicles No./sq. mm

Small fibers (%)

9,856

39

10,089 10,076 8,901

58 39 52

8,637 11,468

44 59

8,545 10,098 8,131 7,041 10,463 8,305 9,001 6,329

40 59 63 72 52 76 71 64

taken 5-7.5 cm proximally did not show higher densities, probably because of the short distance involved. The diameter histograms of selected L F D P from persons of various ages are shown in Fig. 6. For some nerves, the spectrum looks similar to that of the N-PB; for others, it resembles that of the SP-N. The diameter histograms of the M F D P are not shown because they were similar to those of the L F D P with certain exceptions (Fig. 7). The fiber densities for SP-N specimens are detailed in Table X. Nerves labeled ' P ' were taken from the middle third of the leg; the remainder were taken subcutaneously from just above the ankle. The difference in location is reflected by the high density of nerve fibers in the proximal specimens. The distal specimens show a decrease in density of fibers with increasing age (P --< 0.025), but the percentage of the total fiber population made up of small fibers does not decrease with age in this nerve. Small fibers comprise an average of 61 ~o of this nerve at ages 2-43 years, compared with 48 ~o for L F D P at ages 2-37 years. Adult SP-N fiber density is slightly less (P 0.05), averaging 6,838/sq. mm, than that of L F D P from adult persons, which averaged 8,648/sq. mm. Representative diameter histograms of SP-N from persons of various ages are shown in Fig. 8. Some diameter histograms of the 3 different nerves studied showed a suggestion o f a third fiber peak between the small-fiber and large-fiber peaks.

52

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The nerve to the peroneus brevis (N-PB) is of the correct size and length for nerve biopsy studies and is conveniently located next to the superficial peroneal nerve

53

COMBINED MUSCLE-CUTANEOUS NERVE BIOPSY fl:

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Fig. 7. Histograms of the diameters of myelinatext fibers of lateral and medial fascicles of the deep peroneal nerve of a 37-year-old person. Note the lesser number of small fibers in the lateral fascicles.

(SP-N). It is easily obtained at autopsy but should not be removed from patients who do not have a permanent foot drop because the peroneus brevis is a main evertor of the foot and is an important antagonist of the tibialis posteriorT, a6. Occasionally, NPB may be difficult to locate because the fascial plane between the peroneus brevis and the peroneus longus is hard to identify. In some patients the nerve may lie deep in the leg, making the procedure painful under local anesthesia. The lateral terminal branch of the deep peroneal nerve to the extensor digitorum brevis (EDB) can be sacrificed with little functional impairment; however, exposure is TABLE X DENSITY OF MYELINATED FIBERS OF HEALTHY SUPERFICIAL PERONEAL NERVE

Nerve* 8-71 7-71 71-71 9-71 15-71 32-71 62-71 75-71 70-71 79-71 21-71 113-71 51-71 108-71 1-72 74-71 41-71

P P P P P

Age (years)

No./sq. mm

Small fibers (%)

6 days] 4 5 months 2 4 8 10 15 18 31 39 43 46 58 59 67 70 71

32,837 27,483 9,877 18,153 16,921 13,531 9,968 7,516 8,270 5,395 9,938 5,979 9,995 5,139 7,278 4,052 4,820

Unimodal Unimodal 59 65 77 71 61 55 52 50 62 59 59 77 68 64 54

*P, proximal; see Methods.

(SP-N)

J . C . STEVENS eta/.

54

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240004

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8000

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-i

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MICRONS

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MYELINATED FIBER DIAMETERS IN Fig. 8. Histograms of diameters of myelinated fibers of superficial peroneal nerve (SP-N) at various ages. difficult because of overlying tendons and the extensor retinaculum, and there is risk of damage to these structures. The lateral fascicles of the deep peroneal nerve (LFDP) above the ankle form the lateral terminal branch to EDB, and the nerve is easily found in this location. Fascicular biopsy of the subcutaneous SP-N, which lies lateral to the incision, and peroneus brevis muscle biopsy can be carried out at the same time. A nerve stimulator is used to evoke a response in the EDB before the lateral fascicles are transected. This has been helpful when it was difficult to be sure which fascicles

COMBINED MUSCLE---CUTANEOUS NERVE BIOPSY

55

were destined to form the lateral terminal branch. Electrical stimulation of the remaining medial fascicles has always evoked a response in the EDB muscle, indicating that the motor fibers of the lateral terminal branch are not all collected in the LFDP, even inches above the branch point. In autopsy specimens, decussation between the lateral and medial fascicles has been seen in front of the ankle just before the terminal branching of the deep peroneal nerve. In 22 ~ of limbs, the lateral portion of the EDB also receives additional innervation from the accessory deep peroneal nerve 23. Biopsy of the LFDP, therefore, does not completely denervate the muscle. In normal nerve, histologic and teased-fiber preparations provide no indication of the number of motor fibers in the lateral terminal branch of the deep peroneal nerve. McComas et aL 27 studied the deep peroneal nerve of 10 normal boys, 4-16 years old, electrophysiologically and estimated that the EDB was supplied by a mean of 199 motor units. Although all nerves to muscle have a large proportion of afferent fibers 3a, the L F D P contain additional afferents from the joints and capsules of the dorsum of the foot 18,~5,a7. Certain fascicles also contain fibers of the medial terminal branch which supplies cutaneous sensation to the skin between the great and second toes. The use of deep peroneal nerve for biopsy can be criticized because degeneration and regeneration of nerve fibers in L F D P may result from trauma to the common peroneal nerve at the neck of the fibula and from retrograde change due to nerve injury at the ankle 1. Particularly in disorders of lower motor neurons, in which the histometric evaluation of SP-N gives normal results, evaluation of the type and incidence of pathologic change in fibers of L F D P will probably reflect the abnormalities of motor fibers. Degenerative changes in nerves are known to occur in 'normal' persons, and the incidence increases with age8,~8,41. Quantitation of these abnormalities is required in order to know what amount of degeneration is outside the normal range. Abnormal teased fibers in this study were classified into 5 categories and were seen infrequently. Segmental demyelination and remyelination (types D and F changes)were uncommon in all age groups and were absent in 24 of 35 nerves. In the 11 nerves in which it was present, it was found in more than 1 Yo of fibers in only two nerves. The incidence of linear rows of myelin ovoids increased with age. Under 18 years, ovoids were absent in 7 of 11 nerves and at most affected 1.5 ~ of fibers. Between ages 18 and 43 years, the maximum was 2.3~o; between ages 46 and 71 years, all nerves had one or more fibers affected, the maximum being 4.7 ~ . Using the same grading system in studying the sural nerve at ages 10--70 years, Dyck et al. 15 found axonal degeneration and paranodal or internodal myelin loss to be absent in about one-half of the nerves, with highest incidence of these abnormalities being 4 ~o. Duncan 8 concluded that the number of degenerating fibers present in any peripheral nerve is always less than 1 ~ but he did not examine distal limb nerves. Arnold and Harriman 2 studied 37 superficial peroneal nerves of various ages, typing 24 teased fibers per nerve. Segmental demyelination and remyelination was found in 12 of 37 specimens, usually affecting only 1 of the 24 fibers. This incidence is much higher than ours for the same nerve, considering that we examined many more fibers

56

J.c. STEVENSel a[.

per nerve. The explanation may be that different definitions of remyelination or 'pale internodes' were used. For inclusion in our type F, a fiber had to have more than one recently remyelinated segment, 'recently remyelinated' meaning that the new myelin sheath was just visible. Arnold and Harriman found only 3 nerves containing fibers undergoing Wallerian degeneration. Our incidence was much higher, but again the disparity may be due to a different sample size per nerve. The fiber diameter spectrums of sensory and motor nerves are bimoda116,1v,3°. In this study, the histograms of teased-fiber internode lengths (IL) were also bimodal. This was expected because in normal nerve there is a direct relationship between internode diameter (ID) and IL4,22,al, 46. The 1L between the two peaks on the histogram was used to determine an average IL for the separation of the two populations. The mean IL of small and large fibers is a more precise measurement of I L of biopsied nerve than has been available. The low variability of these measurements between the nerves in this study, and of the sural nerve, suggests that these indices are reproducible, In our opinion they reflect the actual distribution of internode lengths of nerves. It may be possible to determine these indices in pathologic nerves in order to evaluate abnormalities of small and large fibers separately. The adjusted IL of nerve was utilized to correct for an unequal sampling of large and small fibers, and it provides the best measure available of the IL of the whole nerve. Although 1D measurements of N-PB and L F D P were made, accurate measurement of teased-fiber diameter is difficult and time-consuming. Myelinated fiber diameter reaches adult size early in the second half of the first decade6, 21. However, l L is said to continue to increase probably for the duration of growth of the part in which the nerve lies4Z,45. Our measurements do not support this view because adult values of IL were attained prior to cessation of growth. Three sural nerves from patients 10, 11, and 14 years old, in the study by Dyck et al. ~5, also had adjusted mean IL of nerve values which were within the adult range. The variability of IL of SP-N and L F D P was found to increase with age. This probably results from segmental demyelination and remyelination. Increase in variability of IL may also result from axonal degeneration with secondary segmental demyelination and remyelination 11. Increased variability of IL with age also has been found by others 15,24,~6,45. The adjusted IL and ID, nerve values of L F D P and the adjusted IL, nerve of SP-N tended to decrease with the onset of maturity, partly because of the above changes and because of loss of fibers. Atherosclerotic changes in nutrient vessels to nerves are known to occur in old age6, 32 and are said to correlate with loss of nerve fibers and absence of deep tendon reflexes in the lower limbs 4°. Upper limb nerves are apparently less liable to damage. Other nonvascular factors, such as senile degeneration, affecting the cell bodies of the primary motor and sensory neurons could account for part of the loss of nerve fibers with increasing age a. The effects of the above changes may account for the progressive slowing of conduction in the nerves of the leg, which begins in young adults a. The fiber density and the size distribution of myelinated fiber diameters are influenced by the type of fixation a2. Glutaraldehyde was used in this study and is known to cause more shrinkage than does fixation in osmium tetraoxide alonO °. The

COMBINED MUSCLE--CUTANEOUS NERVE BIOPSY

57

level of nerve biopsy also affects fiber density because much higher values were found in proximal specimens of SP-N. Greater compaction of fibers proximally than distally has been found previously in the sural nerve a. It is also important to note that the proximal SP-N specimens came from children and infants, who have a higher fiber density than adults 19. Another variable affecting the range of our results is the variation in numbers and sizes of fibers in different fascicles of a nerve and the sometimes considerable difference in total fiber counts on the same nerve from the right and left sides of the body 38. We have measured fiber density by making counts from photographs of different fascicles and different areas of large fascicles. It is possible that diseased, shrunken nerves could still have a normal fiber density when the total fiber count is considerably decreased. Even in this eventuality, however, abnormalities would be evident in teased-fiber typing and measurements and in the fiber spectrum. The mean fascicular area of normal nerve does not decrease with ageZa, 8a, so that counts of the number of fibers per unit area still reflect the changes in the total number of fibers present. Quantitative histologic measurements of the muscle nerves and cutaneous nerves described here have not been published before. Greenfield and Carmichael z0 studied the terminal branches of the anterior tibial nerve between the first and second metatarsals and found 5,000-10,000 myelin sheaths/sq, m m in 5 normal controls. Swallow 39 studied the same nerve in the foot and found a mean density of 3,293-7,565 fibers/sq, m m in subjects under 10 years of age; the range of variation was too great for this author to recommend this nerve and site for biopsy. Quantitative histologic measurements of the normal distal sural nerve of subjects of various ages have been published12,21,28,29, 38 and will not be detailed. These studies suggest that the fiber density and spectrum of the distal sural nerve is similar to that of the distal superficial peroneal nerve (SP-N), which we studied, taking into account the variables discussed. The fiber density of the SP-N was slightly less than that of the deep peroneal nerve, while the N-PB appears to have a lesser density than both of the other nerves. Loss of fibers occurred in older L F D P and SP-N. In the case of L F D P , the loss mainly affected fibers of large diameter. Muscle nerves contain a smaller proportion of small myelinated fibers than do cutaneous nerves, and the diameters of the largest fibers of muscle nerve are greater than those of cutaneous nerveZ3-35,43, 44. This difference was obvious when the histograms of N-PB and the superficial nerves are compared. L F D P , a mixed nerve, had a spectrum which was similar in some cases to that of the N-PB but more often resembled that of a cutaneous nerve. Some of the fiber spectrums of all 3 nerves examined appeared to have an illdefined third peak between the large-fiber and small-fiber peaks. The fact that a third peak occurred in some of the SP-N suggests that the elevation was spurious. Study of motor nerves after section of the dorsal and ventral roots indicates that the m o t o r fibers are bimodally distributed but the sensory component has 3 fiber peaks a2. It is possible that the suggestion of a third peak seen in N-PB and L F D P is due to the sensory components.

J . c . STEVENS et al.

58 ACKNOWLEDGEMENTS

T h i s i n v e s t i g a t i o n was s u p p o r t e d in p a r t by R e s e a r c h G r a n t s NS-5811 a n d N S 7541 f r o m the N a t i o n a l I n s t i t u t e s o f H e a l t h , P u b l i c H e a l t h Service, a g r a n t f r o m the M a y o F o u n d a t i o n , a n d by t h e G a l l m e y e r , U p t o n , a n d M i l l e r F u n d s .

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