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Gas-liquid chromatographic analysis of sural nerves in peripheral neuropathies
593
Journal of the neurological Sciences Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
Gas-Liquid Chromatographic Analysis of Sural Nerves in Peripheral Neuropathies O. APPENZELLER* AND J. MACGEE Neurology Service and Medical Research Laboratories of the Veterans Administration Hospital and the Departments of Neurology, Experimental Medicine and Biological Chemistry, University of Cincinnati, College of Medicine, Cincinnati, Ohio (U.S.A.) (Received 7 May, 1968)
INTRODUCTION
The diagnosis of peripheral neuropathies has been greatly aided by electrophysiological studies and biopsy examination of peripheral nerves. The pathogenesis and classification of the neuropathies, however, remains difficult because such studies rarely distinguish between the various types of neuropathy. Moreover, clinically, peripheral nerve dysfunction may be severe without correlating well with pathological changes at necropsy examination. The technique of gas-liquid chromatography offers a number of advantages in the analysis of small quantities of tissues and it is particularly suited for detecting and separating substances present in microgram quantities. This technique should, therefore, be useful for the examination of tissues which are not obtainable in large quantities during life and in which subtle changes are expected. The analysis of tissues by gas-liquid chromatography requires usually initial fractionation and the fractionated materials only are measured. We used a technique which gave information about the entire sample without extensive fractionation and have applied it to human sural nerves obtained at autopsy and biopsy. This method gave elution curves which were "fingerprints" of the tissue analyzed (MACGEE 1968). We have attempted to relate changes in elution patterns of sural nerves from cases of peripheral neuropathy with the histological appearance of these nerves and describe the findings in this report. MATERIALS AND METHODS
Seventy-seven sural nerves were available for study. Twenty-four were obtained at autopsy and 53 by biopsy. The mean age and diagnoses of patients in this study are given in Table 1. The normal nerves obtained by biopsy were obtained from 7 patients * Present address: Division of Neurology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87106 (U.S.A.).
J. neurol. Sci. (1968) 7:593-603
594
o. APPENZELLER, J. MACGEE TABLE 1 CAUSES OF DEATH, C L I N I C A L
DIAGNOSES AND DIAGNOSTIC GROUPING OF SURAL NERVES
Number o f nerves
Mean age o f patients (years)
14
62
no rmal
3
61
miscellaneous
3 4
52 66
alcoholic neuropathy nutritional deficiencies
7
48
normal
13
58
miscellaneous
19 6 5
53 53 62
3
60
alcoholic neuropathy diabetic neuropathy respiratory peripheral neuropathy carcinomatous neuropathy
Diagnostic grouping ofsural nerves
Causes o f death
At autopsy
myocardial infarction (4)* cerebrovascular accident (2) pneumonia (6) cerebral trauma (2) Landry-Guillain-Barr6 syndrome (1) chronic obstructive bronchopulmonary disease (1) pulmonary infarction, peripheral neuropathy, ? cause (1) chronic pancreatitis and peritonitis (3) carcinoma with metastases (4)
By biopsy
amyotropic lateral sclerosis (2) proximal spinal muscular atrophy (1) myasthenia gravis (l) seizures (2) hyperlipemia (1) peripheral neuropathy ? cause (3) familial predominantly motor neuropathy (2) rheumatoid neuropathy (1) subacute combined degeneration (1) chronic lead and arsenic poisoning (1) tabes dorsalis (1) peroneal pressure palsy (1) Landry-Guillain-Barr6 syndrome (1) polyarteritis nodosa (1) furadantin neuropathy (1) chronic alcoholism diabetes mellitus chronic obstructive bronchopulmonary disease and wasting carcinoma
* Number of sural nerves from patients with this diagnosis.
who agreed to the p r o c e d u r e after full e x p l a n a t i o n a n d who suffered f r o m d i s o r d e r s n o t k n o w n to affect sensory p e r i p h e r a l nerves; none o f them showed any clinical evidence o f p e r i p h e r a l n e u r o p a t h y (Table 1). A t autopsy, 4 cm o f the right sural nerve were r e m o v e d at the ankle. A t biopsy, a similar length was r e m o v e d after local anesthesia o f the same region. Each specimen was placed on c a r d b o a r d a n d divided into 3 parts. One was fixed in 10% f o r m o l saline a n d e m b e d d e d in paraffin; sections were cut at 7 ff a n d stained with h e m a t o x y l i n a n d eosin. The second p a r t was fixed in l % o s m i u m tetroxide, e m b e d d e d in paraffin a n d sectioned at 4 #. L o n g i t u d i n a l a n d cross-sections o f these specimens were examined. The cross-sections o f whole nerves were p h o t o g r a p h e d and printed at 400 x magnification. Fibers were c o u n t e d a n d J. neurol. Sci. (1968) 7:593-603
GAS-LIQUID CHROMATOGRAPHICANALYSISOF SURAL NERVES
595
the diameter of the fibers of the entire cross-section was measured on the photographs. The third part of 16 sural nerves obtained at autopsy and of 43 biopsy specimens was immediately frozen and after cleaning subjected to gas-liquid chromatography. A Barber-Colman Series 5000 dual column temperature programmed gas chromatograph with Argon 00Sr ionization detectors and 6 ft. stainless steel columns packed with 4% SE-30 on Gas Chrome P, 80-100 mesh were used. The method for the preparation of tissues is described fully elsewhere (MACGEE 1968). Briefly, an aliquot of 100 mg of wet blotted tissue was heated with tetramethylammonium hydroxide reagent which dissolved it. The digested tissue was evaporated to dryness, cooled and then extracted with 0.5 ml of ethanol. When less than 100 mg of tissue was available a constant relation of tissue weight to the reagent and alcohol volumes was maintained. To gain a quantitative estimate of the composition of the nerves, the area of the 9 most prominent peaks of the chromatograms from 6 autopsy and 35 biopsy specimens was summated and the percentage area occupied by each peak calculated. RESULTS The examination of sections stained with hematoxylin and eosin showed occasional inflammatory perivascular cell infiltrates consisting mainly of mononuclear and occasional polymorphonuclear cells. They were situated predominantly in the perineurium and were not related to areas of myelin loss (Fig. 1). These were most
Fig. 1. Predominantly mononuclear perivascular cell infiltrate in perineurium of sural nerve from 72-years-old subject without clinical symptoms or signs of neuropathy. Hematoxylin and eosin; × 520. J. neurol. Sci.
(1968) 7:593-603
596
o. APPENZELLER~ J. MACGEE
often seen in specimens from elderly subjects and were found occasionally in sural nerves from patients who had no symptoms or signs of peripheral nerve disease. In one case with rheumatoid arthritis and severe peripheral neuropathy the infiltrates were a striking feature of the biopsy specimen (Fig. 2). In longitudinal sections of osmium-fixed sural nerves from normal elderly subjects occasional myelin ovoids could be seen (Fig. 3). Varying degrees of myelin loss and myelin ovoids arranged in rows suggestive of Wallerian degeneration were found in patients with neuropathies. These were not specific and did not correlate with the clinical severity of the disease or type of neuropathy (Figs. 4, 5, 6).
Fig. 2. Cross section of sural nerve biopsy from patient with rheumatoid arthritis and neuropathy showing mononuclear cell infiltrates in vessels of the epi- and perineurium and arteritis. Hematoxylin and eosin; x 100.
Fig. 3. Longitudinal section of sural nerve from 68-year-old man without clinical evidence of neuropathy showing a myelin ovoid. Osmium tetroxide; x 520. J. neurol. Sci. (1968) 7:593-603
GAS-LIQUID CHROMATOGRAPHIC ANALYSIS OF SURAL NERVES
597
Fig. 4. Longitudinal section of sural nerve biopsy from patient with chronic obstructive bronchopulmonary disease and neuropathy showing myelin ovoids arranged in rows suggestive of Wallerian degeneration. Osmium tetroxide; x 400.
Fig. 5. Longitudinal section from sural nerve biopsy from patient with rheumatoid arthritis and neuropathy showing extensive destruction of myelinated fibers. Osmium tetroxide; x 400. J. neurol. Sci. (1968) 7:593-603
598
o. APPENZELLER, J. MACGEE
The number of fibers and the fiber diameter spectra of sural nerves from patients with a variety of neuropathies were not uniform within any diagnostic grouping and did not correlate with the clinical severity of the neuropathy or histological appearance of the nerves (Fig. 7).
Fig. 6. Cross-section showing fascicle of sural nerve biopsy from patient with chronic alcoholism and neuropathy. Marked reduction in number of myelinated fibers. Osmium tetroxide; x 250.
TABLE 2 MEAN P E R C E N T A G E AREA O C C U P I E D BY PEAKS A -
I OF E L U T I O N C U R V E " F I N G E R P R I N T S "
FROM S U R A L
NERVES
Sural nerves
No o f nerves
A
B
C
D
E
F
G
H
:
Normal autopsy Normal biopsy Respiratory neuropathy Diabetic neuropathy Carcinomatous neuropathy Alcoholicneuropathy Miscellaneous
6 6 5 4 2 11 7
0.8 0.7 1.7" 1.2 1.0 0.8 1.0
1.5 1.3 2.3 1.6 1.2 1.4 1.5
12.3 11.8 13.6 9.3 8.6 15.0 11.8
16.1 16.6 15.1 14.2 12.6 16.8 17.5
1 s
58.3 56.3 54.7 59.5 62.4 59.0 55.4
5.1 5.8 4.2 4.5 5.2 3.2* 4.1
3.4 4.7 3.7 4.1 4.9 1.7" 3.6
1.1 1.1 0.6 0.9 1.5 0.9 0.9
1.0 1.3 3.2 4.3* 2.4 1.2 3.8
* Statistically significantly different from normal sural nerves obtained by biopsy, P < 0.01. .I. neurol. Sci. (1968) 7:593-603
599
GAS-LIQUID CHROMATOGRAPHIC ANALYSIS OF SURAL NERVES ALCOHOLIC NEUROPATHY
!!t ,.
n..35..5
2O
10
2 4 6 8
10
12
14
2 4 6 8
10
p
12
14
~
~1~.1--1 8
•
IJ
10
I 12
I 14
P
60-
50n -- 1486 a" 47
40-
~
n
=
n :
2301 o" 71
2 8 6 0 o* 49
3 0 -
20-
10--
I "2
4
I,.
8
10
12
14
1 II 2
4
Illl_l_l 8 10 12
6
|4
2
4
6
8
10
12
14
p
p
Fig. 7. Number of fibers (n) and fiber diameter spectra from 6 patients with chronic alcoholism and neuropathy. The ages of the patients are also shown. No consistent pattern in the number of remaining fibers or diameter spectra is seen.
AB
0
CII
D E
FG
H
I
I
30 40 20 MINUTES Fig. 8. Gas-liquid c h r o m a t o g r a m ~ o m n o r m a l s u r a l nerve biopsy. 10
J. neurol. Sci. (1968) 7:593-603
600
o . APPENZELLER, J. MACGEE
AB
CD
j
E
H
I
i
l
FG
~, IOK
I,
I
0
I
10
I
20 MINUTES
I
30
40
Fig. 9. Gas-liquid chromatogram from sural nerve biopsy of patient with diabetes and neuropathy. Note increase in peak I and decrease in peaks C and D. AB
I
0
I
10
C~D
E~FG
l:r
--
H
/
I
20 MINUTES
I
I
30
40
Fig. 10. Gas-liquid chromatogram from sural nerve biopsy of patient with carcinoma and neuropathy. Note decrease in peaks C and D. Gas-liquid chromatographic elution patterns ("fingerprints") were similar in normal nerves obtained at autopsy or at biopsy, but marked differences between these and nerves from patients with neuropathy were seen (Figs. 8, 9, 10). The mean percentage area occupied by peaks A---I of the "fingerprints" are given in Table 2. The composition of nerves in various major diagnostic groupings was clearly distinguishable from normal biopsy specimens. There was an increase in peaks A and I in respiratory and diabetic neuropathy, a decrease in peak C in diabetic and carcinomatous neuropathy and in peaks F and G in alcoholic neuropathy. The findings in cases of the miscellaneous group were not clearly distinguishable from those obtained from normal nerves except for peak I which was markedly increased. This group included 1 sural nerve each from patients with subacute combined degeneration of the cord, longstanding lead-arsenic poisoning, acute furadantin neuropathy, rheuJ. neurol. Sci.
(1968) 7:593-603
GAS-LIQUID
CHROMATOGRAPHIC
ANALYSIS OF SURAL NERVES
601
matoid neuropathy and 3 patients with chronic, predominantly motor neuropathy without other associated disease. It appears then that the neuropathy associated with diabetes, chronic respiratory disease and alcoholism may be recognizable by gas-liquid chromatography. A tentative identification of peaks A - - I of the "fingerprints" in this study is given in Table 3.
TABLE 3 "IENTATIVE IDENTIFICATION OF PEAKS A -- I IN ELUTION CURVE ~FINGERPRINTS ~
Peak
Possible fatty acid
A B C D E F G H I
? myristic ? palmitic oleic stearic ? ? cholesterol
OF SURAL NERVES
No. C: Double bonds
14:0 16:0 18:1 18:0
DISCUSSION
The sural nerve is usually selected for diagnostic biopsy. While the pathological changes in this nerve may not always reflect those found at necropsy in the rest of the peripheral nervous system the changes in this nerve usually give the nearest approximation to those in other nerves obtainable during life. The pathological reactions of peripheral nerves have been extensively studied but are limited. The best known of these are Wallerian degeneration and segmental demyelination. In addition, a third pathological response of peripheral nerves to injury is the formation of "onion bulbs" as seen in a variety of chronic neuropathies and recently subjected to extensive scrutiny (WEBSTERet al. 1967). These pathological changes occur in neuropathies of different etiology. Thus, segmental demyelination, characteristically seen in diphtheritic neuritis (FISHER AND ADAMS 1956) is also found in diabetic neuropathy (THOMASAND LASCELLES1966). Furthermore, proliferation of Schwann cells giving rise to "onion bulbs" is not only seen in hereditary hypertrophic neuropathies but also in Refsum's disease, a metabolic disorder in which consistent excess excretion of phytanic acid in the urine has been found (ELDJARN et al. 1966). This proliferation is also occasionally seen in diabetic and respiratory peripheral neuropathies (THOMAS AND LASCELLES1966; APPENZELLERAND PARKS 1967). It was, therefore, not surprising that in this study there was no relation between histological lesions on the one hand and the clinical severity or type of neuropathy on the other. On examination of sural nerves, areas of segmental myelin loss and features suggestive of Wallerian degeneration were found. In some nerves from elderly normal subjects occasional myelin ovoids were seen and this confirmed earlier observations of segmental myelin loss in apparently normal elderly subjects (VlZOSO 1950). J. neurol. Sci. (1968) 7:593-603
602
o . APPENZELLER, J. MACGEE
The assessment of loss of myelinated nerve fibers in sural nerves is difficult in cross sections because it has been shown that there is normally a great variability in the number of myelinated fibers in nerve biopsies (SWALLOW 1966). The observation that the number of fibers and the fiber diameter spectra of sural nerves from patients with peripheral neuropathies were not uniform in any one group was consistent with the great variation found with age in apparently normal nerves. Nevertheless, severe loss of myelinated nerve fibers was easily recognized in some sural nerves but could not be correlated with the clinical severity of the disease. An analysis of sciatic nerves by gas-liquid chromatography in animals with experimental allergic neuritis proved useful in detecting changes in the "fingerprints" of these nerves. In this initial study no relation between the clinical or histological and biochemical alterations was found, but the "fingerprints" of sciatic nerves were altered by the experimental procedures, whereas those obtained from other parts of the nervous system of these animals remained unchanged (APPENZELLER et al. 1967). Because of these findings we applied similar methods to human sural nerves. There were no quantitative differences in gas-liquid chromatogram peaks of control autopsy and biopsy material even though the biopsy specimens were obtained from younger patients, but changes in the quantities of certain peaks distinguished sural nerves obtained from patients with metabolic disturbances associated with neuropathy from normals and from those suffering from carcinomatous neuropathy or malnutrition. The apparent similarity in composition of normal sural nerves and those from patients with a variety of disorders affecting the nervous system was probably due to the heterogeneity of this group. It might be that if a large number of nerves from such patients were collected, differences in the composition of the various groups might be recognized. That gas-liquid chromatography differentiated between various groups of peripheral neuropathies was not surprising. It has been suggested that neuropathies occurring in association with diabetes, uremia or chronic respiratory disease are in some way related to an altered metabolism of peripheral nerves, perhaps affecting predominantly Schwann cells (THOMAS AND LASCELLES 1966; APPENZELLER AND PARKS 1967). The expression of metabolic abnormalities by changes in the composition of nerves is in keeping with this hypothesis. Another possible explanation for the different composition of sural nerves in various neuropathies is that it reflects a varying replacement of lost fibers by connective or other tissues which could be responsible for the changes in the elution patterns.
SUMMARY
Sural nerves obtained at autopsy and biopsy were examined histologically and subjected to gas-liquid chromatography. There was no correlation between the clinical severity or type of neuropathy and the histological appearance or the fiber numbers and diameter spectra of the nerves. Gas-liquid chromatography distinguished sural nerves obtained from patients with neuropathy occurring in association with diabetes, chronic respiratory disease and alcoholism from controls. It is suggested that this method of analysis of sural nerves may prove useful in diagnosis and in the understanding of the pathogenesis of peripheral neuropathies. J. neurol. Sci.
(1968) 7:593-603
GAS-LIQUID CHROMATOGRAPHIC ANALYSIS OF SURAL NERVES
603
REFERENCES APPENZELLER,O., J. MACGEE AND S. YINGVORAPANT(1967) Gas-liquid chromatographic analysis of sciatic nerves and spinal cords in experimental allergic neuritis, J. NeuroL Neurosurg. Psychiat., 30: 279. APPENZELLER, O. AND R. PARKS (1967) Respiratory neuropathy, Amer. Rev. resp. Dis., 96: 167. ELDJARN, L., K. TRY, O. STOKKE,A. W. MUNTHE-KAAS,S. REFSUM,D. STEINBERG,J. AVIGANAND C. MITZE (1966) Dietary effects on serum-phytanic acid levels and on clinical manifestations in heredopathia atactica polyneuritiformis, Lancet, i: 691. FISHER, C. M. AND R. n . ADAMS(1956) Diphtheritic polyneuritis. A pathological study, J. Neuropath. exp. Neurol., 15 : 243. MACGEE, J. 0968) Characterization of mammalian tissue and microorganisms by gas-liquid chromatography, J. Gas Chromat., 6: 48. SWALLOW, i . (1966) Fiber size and content of the anterior tibial nerve of the foot, J. Neurol. Neurosurg. Psychiat., 29: 205. THOMAS, P. K. AND R. G. LASCELLES(1966) The pathology of diabetic neuropathy, Quart. J. Med., 35: 489. Vmoso, A. D. (1950) Relationship between internodal lengths and growth in human nerves, J. Anat. (Lond.), 84: 342. WEBSTER, H. DE F., J. i . SCHRODER,A. K. ASBURYAND R. D. ADAMS(1967) The role of Schwann cells in the formation of "onion bulbs" found in chronic neuropathies, J. Neuropath. exp. Neurol., 26: 276.
J. neurol. Sci. (1968) 7:593-603
Journal of the neurological Sciences Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
Gas-Liquid Chromatographic Analysis of Sural Nerves in Peripheral Neuropathies O. APPENZELLER* AND J. MACGEE Neurology Service and Medical Research Laboratories of the Veterans Administration Hospital and the Departments of Neurology, Experimental Medicine and Biological Chemistry, University of Cincinnati, College of Medicine, Cincinnati, Ohio (U.S.A.) (Received 7 May, 1968)
INTRODUCTION
The diagnosis of peripheral neuropathies has been greatly aided by electrophysiological studies and biopsy examination of peripheral nerves. The pathogenesis and classification of the neuropathies, however, remains difficult because such studies rarely distinguish between the various types of neuropathy. Moreover, clinically, peripheral nerve dysfunction may be severe without correlating well with pathological changes at necropsy examination. The technique of gas-liquid chromatography offers a number of advantages in the analysis of small quantities of tissues and it is particularly suited for detecting and separating substances present in microgram quantities. This technique should, therefore, be useful for the examination of tissues which are not obtainable in large quantities during life and in which subtle changes are expected. The analysis of tissues by gas-liquid chromatography requires usually initial fractionation and the fractionated materials only are measured. We used a technique which gave information about the entire sample without extensive fractionation and have applied it to human sural nerves obtained at autopsy and biopsy. This method gave elution curves which were "fingerprints" of the tissue analyzed (MACGEE 1968). We have attempted to relate changes in elution patterns of sural nerves from cases of peripheral neuropathy with the histological appearance of these nerves and describe the findings in this report. MATERIALS AND METHODS
Seventy-seven sural nerves were available for study. Twenty-four were obtained at autopsy and 53 by biopsy. The mean age and diagnoses of patients in this study are given in Table 1. The normal nerves obtained by biopsy were obtained from 7 patients * Present address: Division of Neurology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87106 (U.S.A.).
J. neurol. Sci. (1968) 7:593-603
594
o. APPENZELLER, J. MACGEE TABLE 1 CAUSES OF DEATH, C L I N I C A L
DIAGNOSES AND DIAGNOSTIC GROUPING OF SURAL NERVES
Number o f nerves
Mean age o f patients (years)
14
62
no rmal
3
61
miscellaneous
3 4
52 66
alcoholic neuropathy nutritional deficiencies
7
48
normal
13
58
miscellaneous
19 6 5
53 53 62
3
60
alcoholic neuropathy diabetic neuropathy respiratory peripheral neuropathy carcinomatous neuropathy
Diagnostic grouping ofsural nerves
Causes o f death
At autopsy
myocardial infarction (4)* cerebrovascular accident (2) pneumonia (6) cerebral trauma (2) Landry-Guillain-Barr6 syndrome (1) chronic obstructive bronchopulmonary disease (1) pulmonary infarction, peripheral neuropathy, ? cause (1) chronic pancreatitis and peritonitis (3) carcinoma with metastases (4)
By biopsy
amyotropic lateral sclerosis (2) proximal spinal muscular atrophy (1) myasthenia gravis (l) seizures (2) hyperlipemia (1) peripheral neuropathy ? cause (3) familial predominantly motor neuropathy (2) rheumatoid neuropathy (1) subacute combined degeneration (1) chronic lead and arsenic poisoning (1) tabes dorsalis (1) peroneal pressure palsy (1) Landry-Guillain-Barr6 syndrome (1) polyarteritis nodosa (1) furadantin neuropathy (1) chronic alcoholism diabetes mellitus chronic obstructive bronchopulmonary disease and wasting carcinoma
* Number of sural nerves from patients with this diagnosis.
who agreed to the p r o c e d u r e after full e x p l a n a t i o n a n d who suffered f r o m d i s o r d e r s n o t k n o w n to affect sensory p e r i p h e r a l nerves; none o f them showed any clinical evidence o f p e r i p h e r a l n e u r o p a t h y (Table 1). A t autopsy, 4 cm o f the right sural nerve were r e m o v e d at the ankle. A t biopsy, a similar length was r e m o v e d after local anesthesia o f the same region. Each specimen was placed on c a r d b o a r d a n d divided into 3 parts. One was fixed in 10% f o r m o l saline a n d e m b e d d e d in paraffin; sections were cut at 7 ff a n d stained with h e m a t o x y l i n a n d eosin. The second p a r t was fixed in l % o s m i u m tetroxide, e m b e d d e d in paraffin a n d sectioned at 4 #. L o n g i t u d i n a l a n d cross-sections o f these specimens were examined. The cross-sections o f whole nerves were p h o t o g r a p h e d and printed at 400 x magnification. Fibers were c o u n t e d a n d J. neurol. Sci. (1968) 7:593-603
GAS-LIQUID CHROMATOGRAPHICANALYSISOF SURAL NERVES
595
the diameter of the fibers of the entire cross-section was measured on the photographs. The third part of 16 sural nerves obtained at autopsy and of 43 biopsy specimens was immediately frozen and after cleaning subjected to gas-liquid chromatography. A Barber-Colman Series 5000 dual column temperature programmed gas chromatograph with Argon 00Sr ionization detectors and 6 ft. stainless steel columns packed with 4% SE-30 on Gas Chrome P, 80-100 mesh were used. The method for the preparation of tissues is described fully elsewhere (MACGEE 1968). Briefly, an aliquot of 100 mg of wet blotted tissue was heated with tetramethylammonium hydroxide reagent which dissolved it. The digested tissue was evaporated to dryness, cooled and then extracted with 0.5 ml of ethanol. When less than 100 mg of tissue was available a constant relation of tissue weight to the reagent and alcohol volumes was maintained. To gain a quantitative estimate of the composition of the nerves, the area of the 9 most prominent peaks of the chromatograms from 6 autopsy and 35 biopsy specimens was summated and the percentage area occupied by each peak calculated. RESULTS The examination of sections stained with hematoxylin and eosin showed occasional inflammatory perivascular cell infiltrates consisting mainly of mononuclear and occasional polymorphonuclear cells. They were situated predominantly in the perineurium and were not related to areas of myelin loss (Fig. 1). These were most
Fig. 1. Predominantly mononuclear perivascular cell infiltrate in perineurium of sural nerve from 72-years-old subject without clinical symptoms or signs of neuropathy. Hematoxylin and eosin; × 520. J. neurol. Sci.
(1968) 7:593-603
596
o. APPENZELLER~ J. MACGEE
often seen in specimens from elderly subjects and were found occasionally in sural nerves from patients who had no symptoms or signs of peripheral nerve disease. In one case with rheumatoid arthritis and severe peripheral neuropathy the infiltrates were a striking feature of the biopsy specimen (Fig. 2). In longitudinal sections of osmium-fixed sural nerves from normal elderly subjects occasional myelin ovoids could be seen (Fig. 3). Varying degrees of myelin loss and myelin ovoids arranged in rows suggestive of Wallerian degeneration were found in patients with neuropathies. These were not specific and did not correlate with the clinical severity of the disease or type of neuropathy (Figs. 4, 5, 6).
Fig. 2. Cross section of sural nerve biopsy from patient with rheumatoid arthritis and neuropathy showing mononuclear cell infiltrates in vessels of the epi- and perineurium and arteritis. Hematoxylin and eosin; x 100.
Fig. 3. Longitudinal section of sural nerve from 68-year-old man without clinical evidence of neuropathy showing a myelin ovoid. Osmium tetroxide; x 520. J. neurol. Sci. (1968) 7:593-603
GAS-LIQUID CHROMATOGRAPHIC ANALYSIS OF SURAL NERVES
597
Fig. 4. Longitudinal section of sural nerve biopsy from patient with chronic obstructive bronchopulmonary disease and neuropathy showing myelin ovoids arranged in rows suggestive of Wallerian degeneration. Osmium tetroxide; x 400.
Fig. 5. Longitudinal section from sural nerve biopsy from patient with rheumatoid arthritis and neuropathy showing extensive destruction of myelinated fibers. Osmium tetroxide; x 400. J. neurol. Sci. (1968) 7:593-603
598
o. APPENZELLER, J. MACGEE
The number of fibers and the fiber diameter spectra of sural nerves from patients with a variety of neuropathies were not uniform within any diagnostic grouping and did not correlate with the clinical severity of the neuropathy or histological appearance of the nerves (Fig. 7).
Fig. 6. Cross-section showing fascicle of sural nerve biopsy from patient with chronic alcoholism and neuropathy. Marked reduction in number of myelinated fibers. Osmium tetroxide; x 250.
TABLE 2 MEAN P E R C E N T A G E AREA O C C U P I E D BY PEAKS A -
I OF E L U T I O N C U R V E " F I N G E R P R I N T S "
FROM S U R A L
NERVES
Sural nerves
No o f nerves
A
B
C
D
E
F
G
H
:
Normal autopsy Normal biopsy Respiratory neuropathy Diabetic neuropathy Carcinomatous neuropathy Alcoholicneuropathy Miscellaneous
6 6 5 4 2 11 7
0.8 0.7 1.7" 1.2 1.0 0.8 1.0
1.5 1.3 2.3 1.6 1.2 1.4 1.5
12.3 11.8 13.6 9.3 8.6 15.0 11.8
16.1 16.6 15.1 14.2 12.6 16.8 17.5
1 s
58.3 56.3 54.7 59.5 62.4 59.0 55.4
5.1 5.8 4.2 4.5 5.2 3.2* 4.1
3.4 4.7 3.7 4.1 4.9 1.7" 3.6
1.1 1.1 0.6 0.9 1.5 0.9 0.9
1.0 1.3 3.2 4.3* 2.4 1.2 3.8
* Statistically significantly different from normal sural nerves obtained by biopsy, P < 0.01. .I. neurol. Sci. (1968) 7:593-603
599
GAS-LIQUID CHROMATOGRAPHIC ANALYSIS OF SURAL NERVES ALCOHOLIC NEUROPATHY
!!t ,.
n..35..5
2O
10
2 4 6 8
10
12
14
2 4 6 8
10
p
12
14
~
~1~.1--1 8
•
IJ
10
I 12
I 14
P
60-
50n -- 1486 a" 47
40-
~
n
=
n :
2301 o" 71
2 8 6 0 o* 49
3 0 -
20-
10--
I "2
4
I,.
8
10
12
14
1 II 2
4
Illl_l_l 8 10 12
6
|4
2
4
6
8
10
12
14
p
p
Fig. 7. Number of fibers (n) and fiber diameter spectra from 6 patients with chronic alcoholism and neuropathy. The ages of the patients are also shown. No consistent pattern in the number of remaining fibers or diameter spectra is seen.
AB
0
CII
D E
FG
H
I
I
30 40 20 MINUTES Fig. 8. Gas-liquid c h r o m a t o g r a m ~ o m n o r m a l s u r a l nerve biopsy. 10
J. neurol. Sci. (1968) 7:593-603
600
o . APPENZELLER, J. MACGEE
AB
CD
j
E
H
I
i
l
FG
~, IOK
I,
I
0
I
10
I
20 MINUTES
I
30
40
Fig. 9. Gas-liquid chromatogram from sural nerve biopsy of patient with diabetes and neuropathy. Note increase in peak I and decrease in peaks C and D. AB
I
0
I
10
C~D
E~FG
l:r
--
H
/
I
20 MINUTES
I
I
30
40
Fig. 10. Gas-liquid chromatogram from sural nerve biopsy of patient with carcinoma and neuropathy. Note decrease in peaks C and D. Gas-liquid chromatographic elution patterns ("fingerprints") were similar in normal nerves obtained at autopsy or at biopsy, but marked differences between these and nerves from patients with neuropathy were seen (Figs. 8, 9, 10). The mean percentage area occupied by peaks A---I of the "fingerprints" are given in Table 2. The composition of nerves in various major diagnostic groupings was clearly distinguishable from normal biopsy specimens. There was an increase in peaks A and I in respiratory and diabetic neuropathy, a decrease in peak C in diabetic and carcinomatous neuropathy and in peaks F and G in alcoholic neuropathy. The findings in cases of the miscellaneous group were not clearly distinguishable from those obtained from normal nerves except for peak I which was markedly increased. This group included 1 sural nerve each from patients with subacute combined degeneration of the cord, longstanding lead-arsenic poisoning, acute furadantin neuropathy, rheuJ. neurol. Sci.
(1968) 7:593-603
GAS-LIQUID
CHROMATOGRAPHIC
ANALYSIS OF SURAL NERVES
601
matoid neuropathy and 3 patients with chronic, predominantly motor neuropathy without other associated disease. It appears then that the neuropathy associated with diabetes, chronic respiratory disease and alcoholism may be recognizable by gas-liquid chromatography. A tentative identification of peaks A - - I of the "fingerprints" in this study is given in Table 3.
TABLE 3 "IENTATIVE IDENTIFICATION OF PEAKS A -- I IN ELUTION CURVE ~FINGERPRINTS ~
Peak
Possible fatty acid
A B C D E F G H I
? myristic ? palmitic oleic stearic ? ? cholesterol
OF SURAL NERVES
No. C: Double bonds
14:0 16:0 18:1 18:0
DISCUSSION
The sural nerve is usually selected for diagnostic biopsy. While the pathological changes in this nerve may not always reflect those found at necropsy in the rest of the peripheral nervous system the changes in this nerve usually give the nearest approximation to those in other nerves obtainable during life. The pathological reactions of peripheral nerves have been extensively studied but are limited. The best known of these are Wallerian degeneration and segmental demyelination. In addition, a third pathological response of peripheral nerves to injury is the formation of "onion bulbs" as seen in a variety of chronic neuropathies and recently subjected to extensive scrutiny (WEBSTERet al. 1967). These pathological changes occur in neuropathies of different etiology. Thus, segmental demyelination, characteristically seen in diphtheritic neuritis (FISHER AND ADAMS 1956) is also found in diabetic neuropathy (THOMASAND LASCELLES1966). Furthermore, proliferation of Schwann cells giving rise to "onion bulbs" is not only seen in hereditary hypertrophic neuropathies but also in Refsum's disease, a metabolic disorder in which consistent excess excretion of phytanic acid in the urine has been found (ELDJARN et al. 1966). This proliferation is also occasionally seen in diabetic and respiratory peripheral neuropathies (THOMAS AND LASCELLES1966; APPENZELLERAND PARKS 1967). It was, therefore, not surprising that in this study there was no relation between histological lesions on the one hand and the clinical severity or type of neuropathy on the other. On examination of sural nerves, areas of segmental myelin loss and features suggestive of Wallerian degeneration were found. In some nerves from elderly normal subjects occasional myelin ovoids were seen and this confirmed earlier observations of segmental myelin loss in apparently normal elderly subjects (VlZOSO 1950). J. neurol. Sci. (1968) 7:593-603
602
o . APPENZELLER, J. MACGEE
The assessment of loss of myelinated nerve fibers in sural nerves is difficult in cross sections because it has been shown that there is normally a great variability in the number of myelinated fibers in nerve biopsies (SWALLOW 1966). The observation that the number of fibers and the fiber diameter spectra of sural nerves from patients with peripheral neuropathies were not uniform in any one group was consistent with the great variation found with age in apparently normal nerves. Nevertheless, severe loss of myelinated nerve fibers was easily recognized in some sural nerves but could not be correlated with the clinical severity of the disease. An analysis of sciatic nerves by gas-liquid chromatography in animals with experimental allergic neuritis proved useful in detecting changes in the "fingerprints" of these nerves. In this initial study no relation between the clinical or histological and biochemical alterations was found, but the "fingerprints" of sciatic nerves were altered by the experimental procedures, whereas those obtained from other parts of the nervous system of these animals remained unchanged (APPENZELLER et al. 1967). Because of these findings we applied similar methods to human sural nerves. There were no quantitative differences in gas-liquid chromatogram peaks of control autopsy and biopsy material even though the biopsy specimens were obtained from younger patients, but changes in the quantities of certain peaks distinguished sural nerves obtained from patients with metabolic disturbances associated with neuropathy from normals and from those suffering from carcinomatous neuropathy or malnutrition. The apparent similarity in composition of normal sural nerves and those from patients with a variety of disorders affecting the nervous system was probably due to the heterogeneity of this group. It might be that if a large number of nerves from such patients were collected, differences in the composition of the various groups might be recognized. That gas-liquid chromatography differentiated between various groups of peripheral neuropathies was not surprising. It has been suggested that neuropathies occurring in association with diabetes, uremia or chronic respiratory disease are in some way related to an altered metabolism of peripheral nerves, perhaps affecting predominantly Schwann cells (THOMAS AND LASCELLES 1966; APPENZELLER AND PARKS 1967). The expression of metabolic abnormalities by changes in the composition of nerves is in keeping with this hypothesis. Another possible explanation for the different composition of sural nerves in various neuropathies is that it reflects a varying replacement of lost fibers by connective or other tissues which could be responsible for the changes in the elution patterns.
SUMMARY
Sural nerves obtained at autopsy and biopsy were examined histologically and subjected to gas-liquid chromatography. There was no correlation between the clinical severity or type of neuropathy and the histological appearance or the fiber numbers and diameter spectra of the nerves. Gas-liquid chromatography distinguished sural nerves obtained from patients with neuropathy occurring in association with diabetes, chronic respiratory disease and alcoholism from controls. It is suggested that this method of analysis of sural nerves may prove useful in diagnosis and in the understanding of the pathogenesis of peripheral neuropathies. J. neurol. Sci.
(1968) 7:593-603
GAS-LIQUID CHROMATOGRAPHIC ANALYSIS OF SURAL NERVES
603
REFERENCES APPENZELLER,O., J. MACGEE AND S. YINGVORAPANT(1967) Gas-liquid chromatographic analysis of sciatic nerves and spinal cords in experimental allergic neuritis, J. NeuroL Neurosurg. Psychiat., 30: 279. APPENZELLER, O. AND R. PARKS (1967) Respiratory neuropathy, Amer. Rev. resp. Dis., 96: 167. ELDJARN, L., K. TRY, O. STOKKE,A. W. MUNTHE-KAAS,S. REFSUM,D. STEINBERG,J. AVIGANAND C. MITZE (1966) Dietary effects on serum-phytanic acid levels and on clinical manifestations in heredopathia atactica polyneuritiformis, Lancet, i: 691. FISHER, C. M. AND R. n . ADAMS(1956) Diphtheritic polyneuritis. A pathological study, J. Neuropath. exp. Neurol., 15 : 243. MACGEE, J. 0968) Characterization of mammalian tissue and microorganisms by gas-liquid chromatography, J. Gas Chromat., 6: 48. SWALLOW, i . (1966) Fiber size and content of the anterior tibial nerve of the foot, J. Neurol. Neurosurg. Psychiat., 29: 205. THOMAS, P. K. AND R. G. LASCELLES(1966) The pathology of diabetic neuropathy, Quart. J. Med., 35: 489. Vmoso, A. D. (1950) Relationship between internodal lengths and growth in human nerves, J. Anat. (Lond.), 84: 342. WEBSTER, H. DE F., J. i . SCHRODER,A. K. ASBURYAND R. D. ADAMS(1967) The role of Schwann cells in the formation of "onion bulbs" found in chronic neuropathies, J. Neuropath. exp. Neurol., 26: 276.
J. neurol. Sci. (1968) 7:593-603