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Peripheral neuropathy in chronic disease of the respiratory tract
Peripheral
Neuropathy
in Chronic
of the Respiratory O~ITO
.\PPENZELLER,
M.D.,
PH.D.,~ RALPH
Disease
Tract*
D. PARKS, M.D. and JOSEPH MACGEE,
Cincinnati,
I’H.D
Ohio
Eight patients with chronic obstructive bronchopulmonary disease were studied. In seven who had wasting associated with chronic respiratory tract disease, evidence of peripheral neuropathy was found on clinical and electromyographic examinations. Histologic changes were also present in muscle and sural nerve biopsy specimens from these patients. Biochemical analysis of these sural nerve specimens showed them to differ from those in normal control subjects and patients with alcoholic neuropathy. It is suggested that the neuropathy in chronic obstructive bronchopulmonary disease might be due to abnormalities in the metabolism of Schwann cells.
I
muscles, other than the gastrocnemius muscle, as well as conduction velocities in the fastest conducting motor fibers in the median, ulnar and peroneal nerves,
N SOMEpatients with chronic obstructive bronchopulrnonary disease, wasting occurs in the terminal stages of the disorder. In such patients mortality is high, with death occurring soon after the onset of weight loss. The cause of this weight loss is not clear but it does not seem to be related to a sudden deterioration in pulmonary function or to a decrease in caloric intake [I 1. The appearance of symptoms and signs suggestive of peripheral neuropathy in patients with chronic obstructive bronchopulmonary disease and wasting may lead to the suspicion of an underlying carcinoma. We have examined eight patients with chronic obstructive bronchopulmonary disease, in seven of whom clinical, electrornyographic or histologic evidence of peripheral neuropathy was found, but none showed evidence of malignancy. In addition, biochemical analyses of the peripheral nerves of these patients showed them to differ in composition from the ner\res of normal control subjects and patients with other neuropathies.
were measured in all patients. Sural nerve and gastrocnemius muscle biopsy specimens were obtained from six patients with Masting associated with chronic respiratory tract disease, five of whom had clinical evidence of neuropathy. The sural nerve specimens were divided into three parts. One was fixed in 10 per cent form01 saline solution and stained with hematoxylin and eosin. The second part was fixed in 1 per cent osmium tetroxide and cut at 4 p. Longitudinal sections were examined for areas of myelin loss. The cross sections of whole nerves were photographed and printed at X400 magnification. The diameter of the fibers was measured on the photographs and all fibers of the entire cross section were counted. The third part of the specimen was subjected to gas-liquid chromatography. The chromatographic unit was a Barber-Colman Series 5000 dual-column temperature programmed gas chromatograph, with Argon SF ionization detectors Six foot stainless steel columns packed with 4 per cent SE-30 on Gas-Chrom P, 80-100 mesh were used. The method for the preparation of the tissue is fully described elsewhere [J]. Briefly, an aliquot of 100 mg. of wet blotted tissue was heated with tetramethylammonium hydroxide reagent, which completely solubilized it. The digested sample was then evaporated to dryness and, after cooling, extracted with 0.5 ml. of ethanol. The relation
MATERIALS AND METHODS All available patients with chronic obstructive bronchopulmonary disease and weight loss were examined clinically and subjected to pulmonary function tests [2,3]. Electromyographic sampling with concentric needle electrcdes of upper and lower limb
* From the Neurology, Pulmonary Diseases and Metabolism Sections, Veterans Administration Hospital, and the Departments of Medicine and Biological Chemistry, University of Cincinnati College of Medicine, Cincinnati, Ohio 45220. Requests for reprints should be addressed to Otto Appenzeller, M.D. Manuscript received June 5, 1967. t Present address: Division of Neurology, The University of New Mexico School of Medicine, 2211 Lomas Blvd., N.E., Albuquerque, New Mexico 87106. VOI.
44,
JUNE
1968
873
2 > r
G.W., 71, M
43, M
E.M.,
2 U z
P
G.F., 74, M
;I >
P
0
50
22
15
0
Ankle
Ankle
Ankle
0
Dorsiflexion of ankle
Foot muscles
Dorsiflexion of ankle
Mid-calf
28
W.B., 55, M
68, M
Foot muscles
Mid-calf
A.P., a’ m
Foot muscles Foot muscles
Ankle
25
28
C.W., 49, M
E.P., 45, M
Foot muscles
Mid-calf
11
H.S., 62, M
Weakness
Findings
Sensory Loss
Weight Loss (lb.)
Patient, Age (yr.) and Sex
Clinical
SUMMARYOF CLINICALFINDINGS,
Normal
Denervation
Denervation
Denervation
Normal
Normal
Denervation
Denervation
Electromyography
46
20
29
29
ovoids
ovoids
Normal
Loss of myelin; degeneration
Myelin
Myelin
20
?Wallerian
myelin
Swelling ovoids
28 of fibers,
... Myelin ovoids; Schwann cell proliferation
29
...
FINDINGS
34
HISTOLOGIC
TABLE I AND
Sural Nerve Biopsy
STUDIES
Conduction Velocity, Peroneal Nerve (M./set.)
ELECTRODIAGNOSTIC
WITH
. *.
variation
in
Normal
Small fibers, fiber size
variation
in
Myophagia, central nucleation, variation in fiber size
Small fibers, fiber size
variation
atrophy Small fibers, fiber size
Grouped
...
in
OBSTRUCTIVE
Gastrocnemius Muscle Biopsy
OF PATIENTS
Remarks
DISEASE
Gained 15 lb. in weight, mo. later, conduction velocity 40 M./set.
5
Gained 20 lb. in weight, 9 mo. later, conduction velocity 50 M./set.
. . ..
After 9 mo. no weight change; conduction velocity 22 M./set.
LUNG
Peripheral
TABLE PULMONARY
FUNCTION
TESTS
RV Patients
vc
(cc.)
4,220 3,854 2,750 3,886 3,603 2,781 2,239 2,755
I-IS. C.W. E.P. W.R. A.P. G.F. E.M. G.\Y.
TLC
(cc.)
7,477 5,824 7,217 7,450 6,811 6,477 7,356 5,510
OF
PATIENTS
II WITH
oBS’I‘R”CTI”E
: TLC (%) 50 45 66 71 64 56 62 49
X75
et al.
Neuropathy-&pen,-eller
LUNG DISEASE
FEVI .o FRC
(cc.)
(70)
MVV (L./mm)
PF (L./‘min.
J
__.___-..-. 4,892 4,280 5,798 5,980 5,139 5,261 5,274 3,913
54 45 27 29 33 45 33 59
70 48 32 31 27 47 31 74
220 15U 14(1 90 170 3 40 140 165
NOTE : VC = vital capacity; TLC = total lung capacity; RV:TLC = ratio residual volume to total lung capacity ; FRC = functional residual capacity; FEV r.,, = forced expiratory volume at one second; MVV = maximal voluntary ventilation; and PF = peak flow.
of sample weight to reagent volume and to the volume of alcohol was kept constant. Adjustments in the volume of reagents and of alcohol were made if less than 100 mg. of tissue was available. The gas-liquid chromatograms obtained by this method gave “fingerprints” of the whole tissue. The muscle was fixed in a 10 per cent form01 saline solution and longitudinal and cross sections stained with hematoxylin and eosin were examined. RESULTS
Clinical Examination. Complaints directly attributable to peripheral neuropathy were found in one patient. On examination, evidence of weight loss and of mild reduction of joint position, vibration, touch and temperature sensation in a stocking distribution were found in seven of the patients with chronic obstructive bronchopulmonary disease. These patients also had slight weakness of the ankles and small muscles of the feet, and contracture of the shoulder joint was noted in one. Deep tendon reflexes were preserved in all (Table I). Results of pulmonary function tests in these patients appear in Table II. Electromyograply. Denervation changes were foulld in the extensor digitorum brevis of the foot in five of seven patients with clinical evidence of neuropathy. The other muscles were normal. The conduction velocities to the thenar and hypothenar muscles were normal. The fastest motor nerve fibers to the extensor digitorum brevis had conduction velocities ranging from 20 to 34 (M.) per second in seven patients with clinical evidence of neuropathy (normal, 40 to 60 11. per second). Three patients were reexamined five to nine months later; in two there was a marked improvement in conduction velocities (Table I).
VOL.
44,
JUNE
1968
Sural Nerve Biopsies. In one patient with clinical evidence of neuropathy the sural nerve specimen showed an increase in the number of Schwann cell nuclei and widening of the endoneural spaces (Fig. 1). Osmium-fixed longitudinal sections disclosed myelin in macrophages and occasional fibers undergoing Wallerian degeneration evidenced by myelin ovoids arranged in rows (Fig. 2). On inspection of transverse sections, some fascicles contained numerous swollen fibers and the density of myelinated fibers seemed to be reduced in others. The number of nerve fibers and the percentage of fibers of various sizes in each of the sural nerve specimens obtained from six patients with chronic ohstructive bronchopulmonary disease is shown in Figure 3. Normally there is a considerable \-ariation in the number and frequency distribution of various-sized fibers but a decrease in total fiber count and in the proportion of large fibers with advancing age has been found in the anterior tibia1 nerve [5]. Nevertheless, all the nerve specimens, except one from a patient (G.W.) who had no clinical or electromyographic evidence of neuropathy, were thought to show a loss of myelin sheath. The percentage of fibers of various sizes did not seem to conform to a single pattern in these sural nerves. The gas-liquid chromatograms (“fingerprints”) of the sural nerve biopsy specimens from five patients with chronic obstructive bronchopulmonary disease and neuropathy, from eleven control subjects (five specimens obtained by biopsy and six at autopsy) and from eleven patients with alcoholic neuropathy were compared (Fig. 4 and 5). In addition, the area occupied by the peaks of the elution patterns from each
876
Peripheral
Neuropathy-Appenzeller
et al.
FIG. 1. Cross sections of sural nerve specimens from normal control subject (left) and patient with chronic respiratory tract disease and neuropathy (rip-ht), showing an increase in the number of Schwann cell nuclei and widening of endoneural spaces. Hematoxylin and eosin stain, original magnification X 900. FIG. 2. Longitudinal section of osmium fixed nerve from patient with chronic respiratory tract disease and neuropathy, showing myelin avoids. Original magnification X 250.
nerve was summated and the percentage of t1.e total area occupied by individual peaks calculated. In Table III are shown the mean percentage of the total area occupied by each peak in the various groups. Statistically significant differences, when compared with normal biopsy specimens, were found in peak A of nerve specimens from patients with chronic obstructive bronchopulmonary disease and neuropathy and in peak F of nerve specimens from patients
with alcoholic neuropathy. There was no statistically significant difference in the percentage area occupied by the various peaks of the normal biopsy and autopsy specimens. A tentative identification of some peaks of the “fingerprints” is given in Table IV. Muscle Biopsies. The gastrocnemius muscle biopsy specimens showed denervation atrophy in one, and fiber necrosis with myophagia, central nucleation and marked variation in fiber AMERICAN
JOURNAL
OF
MEDICINE
Peripheral
Neuropat lly-
L-Ipj~enzdlrrr! al.
H7?
E.P.
A.P.
W.B. n = 2388 0’ 55
u
E.M.
1
u
u
FIG. 3. Fiber number and diameter spectra of sural nerve specimens from patients with chronic obstructive bronchopulmonary disease in this study. The letters refer to the initials of the patients and correspond to those in Table I.
Gas-liquid chromatograms of sural nerve specimens from normal control subject (above) and patient with FIG. 4. chronic respiratory tract disease (below). Note increase in peaks A, B and I in the lower chromatogram. The three fold decrease in the sensitivity of the upper tracing (3K and 30K) was necessary to illustrate these differences. The integrations and figures given in Table III were obtained at a uniform sensitivity of the recording apparatus used in this study. chromatograms of sural nerves from normal FIG. 5. Gas-liquid neuropathy. Note decrease in height of peaks F and G in the lower VOL.
44,
JUNE
1968
control subject chromatogram.
(above) and patient
with
alcoholic
Peripheral
878
Neuropathy-A@enzeller TABLE
MEAN
PERCENTAGE
AREA
OCCUPIED
BY
PEAKS
A THROUGH
SPECIMENS(n =
NUMBEROF
et al.
III
I OF
THE NERVES
GAS-LIQUID
CHROMATOGRAMS
FROM
SEVERAL
NERVE
GROUP)
IN EACH
Sural Nerves Source
n
A
B
C
D
E
F
G
H
I
Normal subjects autopsy specimen Biopsy specimen Subjects with alcoholic neuropathy Subjects with chronic pulmonary disease
6 5 11 5
0.8 0.9 0.8 1.7*
1.5 1.5 1.4 2.3
12.3 11.4 16.0 13.6
16.1 15.1 16.8 15.1
58.3 58.5 59.0 54.7
5.1 5.0 3.2* 4.2
3.4 3.9 1.7 3.7
1.1 0.9 0.9 0.6
1.0 2.1 1.2 3.2
* Statistically significantly different P < 0.01. in another. The remaining three biopsy specimens showed generally small fibers and some random variation in fiber size. size
COMMENTS
The etiology of wasting associated with chronic respiratory tract disease remains obscure but it is known to be indicative of a poor prognosis. To elucidate the cause of wasting associated with chronic respiratory tract disease, the theoretic caloric requirements in such patients were compared with actual caloric intake and this was found adequate. Moreover, no relation between the onset of wasting and deterioration in pulmonary function tests was lound. It was suggested, nevertheless, that the wasting may be associated with a deficient protein intake in such patients [I]. Some patients with chronic obstructive bronchopulmonary disease and wasting have an associated peripheral neuropathy on clinical and electromyographic examinations. In addition, in this study evidence for neuropathy was obtained on histologic examination of specimens TABLE TENTATIVE
IDENTIFICATION
IV OF PEAKS A-I IN GAS-
LIQUID CHROMATOGRAMS OF SURAL NERVES
Peak
Identification
A B C D E F G H I
? Myristic ? Palmitic Oleic Stearic ? ? Cholesterol
of sural nerves and muscles, and alterations in the composition of these nerves were found also. The clinical features of the neuropathy were not uniform and some patients showed a marked improvement in nutrition and nerve conduction velocities some months after their initial examination. The most common cause of marked slowing of conduction velocity is segmental myelin loss in motor nerves. In this study it was impossible, however, to confirm this histologically, since motor nerves are not easily amenable to biopsy; the sural nerve is composed of sensory fibers only. Nevertheless, evidence for loss of myelin and Wallerian degeneration of some fibers was found in biopsy specimens and this was consistent with the clinical findings in these patients. After treatment, recovery from acute neuropathy in uncontrolled diabetes is not uncommon. In this disorder, as in the neuropathy described here, the mode of presentation of patients may vary, but segmental and Wallerian degeneration occurs and the temporary conduction block in diabetic neuropathy is attributed to segmental myelin loss rather than Wallerian degeneration. This type of myelin loss can be shown to occur also in experimentally induced disorders of the peripheral nervous system [6,7] in which rapid recovery is associated with remyelination. It is likely, therefore, that the recovery of nerve conduction velocities in our patients was related to their clinical improvement, particularly as further deterioration in conduction velocity was found in one patient who remained otherwise unchanged. Dyck and Lambert [8] examined conduction velocities in excised sural nerves from patients with a variety of polyneuropathies and found them lower than expected from the diameter of myelinated fibers measured on cross sections of these nerves. Attempts to reconstruct the comAMERICAN
JOURNAL
OF
MEDICINE
Peripheral
Neuropathy-_,4pbenzc/ler
pound action potentials from the spectra of nerve fiber diameters of these nerves were unsuccessful. In one nerve, myelinated fibers 6 M in diameter, which normally conduct at 25 to 35 M. per seco~~tl, were found, whereas the fastest conduction ill this salne sural nerve was only 4 M. per second. It is not surprising, therefore, that in patients with chronic disease of the respiratory tract there was marked \-ariation in the number of rnyelinated fibers and no uniformity in fiber diameter spectra of the sural nerves. B.iopsy specimens from patients who had poliomyelitis in the past were obtained, in a study designed to examine the histologic effects of long-standing denervation of skeletal muscle. All features hitherto considered typical of were found in these chronically “myopathy” denervated muscles. These included random variation in fiber size, central nucleation and rnyophagia [!I]. S imilar features found in the muscle biopsy specimens of patients in this study are therefore not inconsistent with the assumption that they represented denervation rather than myopathic changes. Gas-liquid chromatographic analysis of sciatic nerves in experimental allergic neuritis in the guinea pig was found to be a sensitive method for the recognition of changes in the composition of these nerves [70]. By the use of this technic, biochemical alterations in the nerves were found without clinical or histologic evidence of disease in these animals. These changes were confined to the sciatic nerves and reverted to normal after two months. In patients with chronic disease of the respiratory tract and neuropathy the clinical, electromyographic and histologic findings were indistinguishable from other peripheral neuropathies. In an attempt to separate this neuropathy from other polyneuropathies, we subjected part of the sural nerve biopsy specimens to gas-liquid chromatography. The elution patterns obtained enabled us to differentiate biochemically the neuropathy of patients with chronic respiratory tract disease from control subjects and patients with alcoholic neuropathy. The etiology of neuropathy occurring in association with chronic respiratory tract disease is not clear, but one possible factor in the appearance of neuropathy in patients with wasting is malnutrition. All patients with neuropathy were malnourished and it is tempting, therefore, to assume that this was the cause of their neuropathy. The neuropathies associated with chronic VOL.
44,
JUNE
1968
et ol.
S?!)
alcoholism have been attributed to ~nalrl~~trition. But it is still not clear whether alcohol itself, apart from malnutrition, exerts a direct influence on the peripheral nervous s)-strln [ I I]. The different rlution patterns obtaincxd in sllral ner\:e specimens from patients with alcoholic neuropathp do not, therefore, necessarily / ndicate that neuropathy in patients with wasting associated with chronic respiratory tract disease is unrelated to malnutrition. Peripheral neuropathy sometimes appears as a complication of drug therapy. All patients in this study had been on a standard regimen of bronchodilators, digitalis and, intermittently, diuretics and a variety of antibiotics. None had received steroids. To our knowledge, the drugs administered to these patients have not been incriminated in the causation of neuropathies. A number of metabolic disorders have been associated with peripheral neuropathy but the etiology of the neuropathy in these diseases is disputed. Thus, diabetic neuropath), was thought to be ischemic due to occlusion of \.asa nervorum [ 721. Fagerberg [ 7~1 considered diabetic neuropathy to be due to a specific diabetic angiopathy, associated with the accumulation of periodic acid-Schiff-positive material in intraneural arterioles, but more recent studies failed to substantiate these findings and diabetic neuropathy has been attribllted to metabolic disturbances [ 741. Uremic ne\lropathy also is thought to be due to a nletabolic disorder dependent on severe renal failure, although the neuropathy occurs in malnourished patients who often have abnormal glucose tolerance curves as well [ 7.51. In patients with chronic respiratory tract disease and neuropathy, no abnormal glucose tolerance was ftlund and it is unlikely that they suffered from specific deficiencies of B vitamins, such as occllr in beriberi and alcoholic neuropathy. An alternative to the hypothesis that the neuropathy is due to malnutrition is that it is the result of a metabolic disturbance. The recovery of conduction velocity after appropriate treatment is best explained on this basis. The myelin sheath of peripheral nerves is derived from Schwann cells [7ti] so that an interference with the metabolism of Schwann cells may readily account for the myelin breakdown. It has been suggested that in diphtheritic neuritis the protein metabolism of Schwann cells is abnormal and results in the segmental deobserved in this disease 1771. myelination
880
Peripheral
Neuropathy-Appenzeller
Moreover, the resting respiration of the normal nerve is predominantly due to Schwann cell activity [ 78,791. Majno and Karnovsky [20] studied the lipogenic activity of Schwann cells and found that this decreased progressively toward the periphery, suggesting that peripheral Schwann cells might have an increased susceptibility in demyelinating neuropathies. This could explain the clinical and pathologic findings in metabolic neuropathies which appear first and are more severe in the periphery of the limbs. Schwann cell proliferation has been noted in diabetic neuropathy [74,27] and has been a feature in one of our patients. This may be accounted for by repeated injury to Schwann cells and suggests that it is not specific for the hereditary hypertrophic neuropathies in which it is characteristically seen, but may be a feature of metabolic neuropathies as well. Peripheral neuropathic sequelae of occlusive vascular disease of the limbs in the absence of metabolic disorders are minor [22]. In such patients anoxia secondary to the ischemia is usually of acute onset and is different from the oft-rehypoxemia, mild hypercapnia and curring cardiac failure of patients with obstructive bronchopulmonary disease. It is possible, therefore, that this long-lasting disturbance alters the metabolism of Schwann cells in some way and leads to the breakdown of peripheral myelin in patients with chronic respiratory tract disease and neuropathy. We wish to acknowledge Acknowledgment: the excellent technical assistance of Mr. Kenneth G. Allen and Mrs. Louise P. Tobias.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17. REFERENCES
E., VAN DE WOESTIJNE,K. P. and GYSELEN,A. Weight changes in the terminal stages of chronic obstructive pulmonary disease. Relation to respiratory function and prognosis. Am. Rev. Resp. Dis., 95: 556, 1967. 2. BALDWIN, E. DE F., COWRNARD,A. and RICHARDS, D. W., JR. Pulmonary insufficiency. I. Physiological classification, clinical methods of analysis, standard values in normal subjects. Medicine, 27: 243, 1948. 3. MENEELY, G. R., BALL, C. O., KORY, R. C., CALLAWAY, J. J., MERRILL, J. M., MABE, R. E., ROEHM, D. C. and KALTREIDER,N. L. A simplified closed circuit helium dilution method for the determination of the residual volume of the lungs. Am. J. Med., 28: 824, 1960. 4. MACGEE, J. Characterization of mammalian tissues 1. VANDENBERGH,
18.
19.
20.
21.
22.
et al.
and microorganisms by gas-liquid chromatography. J. Gas. Chromatog., 6: 48, 1968. SWALLOW, M. Fibre size and content of the anterior tibia1 nerve of the foot. J. Neural., Neurosurg. LT Psychiat., 29: 205,1966. MCDONALD, W. I. The effects of experimental demyelination on conduction in peripheral nerve: a histological and electrophysiological study. I. Clinical and histological observations. Brain, 86: 481,1963. CRAGG, B. G. and THOMAS, P. K. Changes in nerve conduction in experimental allergic neuritis. J. Neural., Neurosurg. & Psych&., 27: 106, 1964. DYCK, P. J. and LAMBERT, E. H. Compound action potentials and fiber diameter spectra of excised sural nerves in human neuropathies. Neurology, 17: 286,1967. DRACHMAN,D. B., MURPHY, S. R., NIGAM, M. P. and HILLS, J. R. “Myopathic” changes in chronically denervated muscle. Arch. Newof., 16: 14, 1967. APPENZELLER,O., MACGEE, J. and YINGVORAPANT, S. Gas-liquid chromatographic analysis of sciatic nerves and spinal cords in experimental allergic neuritis. J. Neural., Neurosurg. & Psych&., 30: 279, 1967. VICTOR, M. and DREYFUS P. M. Nutritional diseases of the nervous system. A statement of some current problems and suggestions for further investigation. World Neural., 2: 862, 1961. WOLTMAN, H. W. and WILDER, R. M. Diabetes mellitus; pathologic changes in spinal cord and peripheral nerves. Arch. Int. Med., 44: 576, 1929. FAGERBERG,S. E. Diabetic neuropathy: A clinical and histological study on the significance of vascular affections. Acta med. scandinau., supp. 345,1959. THOMAS, P. K. and LASCELLE, R. G. The pathology of diabetic neuropathy. Quart. J. Med. n.s., 35: 489, 1966. ASBURY, A. K., VICTOR, M. and ADAMS, R. D. Uremic polyneuropathy. Arch. Neural., 8: 413, 1963. GEREN, BEN B. The formation from the Schwann cell surface of myelin in the peripheral nerves of chick embryos. Exper. Cell. Res., 7: 558,1954. CAVANAGH,J. B. and JACOBS,J. M. Some quantitative aspects of diphtheritic neuropathy. Brit. J. Exper. Path., 45: 309, 1964. HELLER, I. H. and HESSE, S. Action of insulin on the respiration of rat sciatic nerve. Lancet, 2: 406, 1960. FIELD, R. A. and ADAMS, L. C. Insulin response of peripheral nerve. I. Effects on glucose metabolism and permeability. Medicine, 43 : 275, 1964. MAJNO, G. and KARNOVSKY, M. L. A biochemical and morphologic study of myelination and demyelination. I. Lipide biosynthesis in vitro by normal nervous tissue. J. Exper. Med., 107: 475, 1958. GREENBAUM,D. Observations on the homogeneous nature and pathogenesis of diabetic neuropathy. Brain, 87: 215,1964. HUTCHINSON,E. C. and LIVERSEDGE,L. A. Neuropathy in peripheral vascular disease; its bearing on diabetic neuropathy. Quart. J. Med. n.s., 25: 267 1956.
AMERICAN
JOURNAL
OF
MEDICINE
Neuropathy
in Chronic
of the Respiratory O~ITO
.\PPENZELLER,
M.D.,
PH.D.,~ RALPH
Disease
Tract*
D. PARKS, M.D. and JOSEPH MACGEE,
Cincinnati,
I’H.D
Ohio
Eight patients with chronic obstructive bronchopulmonary disease were studied. In seven who had wasting associated with chronic respiratory tract disease, evidence of peripheral neuropathy was found on clinical and electromyographic examinations. Histologic changes were also present in muscle and sural nerve biopsy specimens from these patients. Biochemical analysis of these sural nerve specimens showed them to differ from those in normal control subjects and patients with alcoholic neuropathy. It is suggested that the neuropathy in chronic obstructive bronchopulmonary disease might be due to abnormalities in the metabolism of Schwann cells.
I
muscles, other than the gastrocnemius muscle, as well as conduction velocities in the fastest conducting motor fibers in the median, ulnar and peroneal nerves,
N SOMEpatients with chronic obstructive bronchopulrnonary disease, wasting occurs in the terminal stages of the disorder. In such patients mortality is high, with death occurring soon after the onset of weight loss. The cause of this weight loss is not clear but it does not seem to be related to a sudden deterioration in pulmonary function or to a decrease in caloric intake [I 1. The appearance of symptoms and signs suggestive of peripheral neuropathy in patients with chronic obstructive bronchopulmonary disease and wasting may lead to the suspicion of an underlying carcinoma. We have examined eight patients with chronic obstructive bronchopulmonary disease, in seven of whom clinical, electrornyographic or histologic evidence of peripheral neuropathy was found, but none showed evidence of malignancy. In addition, biochemical analyses of the peripheral nerves of these patients showed them to differ in composition from the ner\res of normal control subjects and patients with other neuropathies.
were measured in all patients. Sural nerve and gastrocnemius muscle biopsy specimens were obtained from six patients with Masting associated with chronic respiratory tract disease, five of whom had clinical evidence of neuropathy. The sural nerve specimens were divided into three parts. One was fixed in 10 per cent form01 saline solution and stained with hematoxylin and eosin. The second part was fixed in 1 per cent osmium tetroxide and cut at 4 p. Longitudinal sections were examined for areas of myelin loss. The cross sections of whole nerves were photographed and printed at X400 magnification. The diameter of the fibers was measured on the photographs and all fibers of the entire cross section were counted. The third part of the specimen was subjected to gas-liquid chromatography. The chromatographic unit was a Barber-Colman Series 5000 dual-column temperature programmed gas chromatograph, with Argon SF ionization detectors Six foot stainless steel columns packed with 4 per cent SE-30 on Gas-Chrom P, 80-100 mesh were used. The method for the preparation of the tissue is fully described elsewhere [J]. Briefly, an aliquot of 100 mg. of wet blotted tissue was heated with tetramethylammonium hydroxide reagent, which completely solubilized it. The digested sample was then evaporated to dryness and, after cooling, extracted with 0.5 ml. of ethanol. The relation
MATERIALS AND METHODS All available patients with chronic obstructive bronchopulmonary disease and weight loss were examined clinically and subjected to pulmonary function tests [2,3]. Electromyographic sampling with concentric needle electrcdes of upper and lower limb
* From the Neurology, Pulmonary Diseases and Metabolism Sections, Veterans Administration Hospital, and the Departments of Medicine and Biological Chemistry, University of Cincinnati College of Medicine, Cincinnati, Ohio 45220. Requests for reprints should be addressed to Otto Appenzeller, M.D. Manuscript received June 5, 1967. t Present address: Division of Neurology, The University of New Mexico School of Medicine, 2211 Lomas Blvd., N.E., Albuquerque, New Mexico 87106. VOI.
44,
JUNE
1968
873
2 > r
G.W., 71, M
43, M
E.M.,
2 U z
P
G.F., 74, M
;I >
P
0
50
22
15
0
Ankle
Ankle
Ankle
0
Dorsiflexion of ankle
Foot muscles
Dorsiflexion of ankle
Mid-calf
28
W.B., 55, M
68, M
Foot muscles
Mid-calf
A.P., a’ m
Foot muscles Foot muscles
Ankle
25
28
C.W., 49, M
E.P., 45, M
Foot muscles
Mid-calf
11
H.S., 62, M
Weakness
Findings
Sensory Loss
Weight Loss (lb.)
Patient, Age (yr.) and Sex
Clinical
SUMMARYOF CLINICALFINDINGS,
Normal
Denervation
Denervation
Denervation
Normal
Normal
Denervation
Denervation
Electromyography
46
20
29
29
ovoids
ovoids
Normal
Loss of myelin; degeneration
Myelin
Myelin
20
?Wallerian
myelin
Swelling ovoids
28 of fibers,
... Myelin ovoids; Schwann cell proliferation
29
...
FINDINGS
34
HISTOLOGIC
TABLE I AND
Sural Nerve Biopsy
STUDIES
Conduction Velocity, Peroneal Nerve (M./set.)
ELECTRODIAGNOSTIC
WITH
. *.
variation
in
Normal
Small fibers, fiber size
variation
in
Myophagia, central nucleation, variation in fiber size
Small fibers, fiber size
variation
atrophy Small fibers, fiber size
Grouped
...
in
OBSTRUCTIVE
Gastrocnemius Muscle Biopsy
OF PATIENTS
Remarks
DISEASE
Gained 15 lb. in weight, mo. later, conduction velocity 40 M./set.
5
Gained 20 lb. in weight, 9 mo. later, conduction velocity 50 M./set.
. . ..
After 9 mo. no weight change; conduction velocity 22 M./set.
LUNG
Peripheral
TABLE PULMONARY
FUNCTION
TESTS
RV Patients
vc
(cc.)
4,220 3,854 2,750 3,886 3,603 2,781 2,239 2,755
I-IS. C.W. E.P. W.R. A.P. G.F. E.M. G.\Y.
TLC
(cc.)
7,477 5,824 7,217 7,450 6,811 6,477 7,356 5,510
OF
PATIENTS
II WITH
oBS’I‘R”CTI”E
: TLC (%) 50 45 66 71 64 56 62 49
X75
et al.
Neuropathy-&pen,-eller
LUNG DISEASE
FEVI .o FRC
(cc.)
(70)
MVV (L./mm)
PF (L./‘min.
J
__.___-..-. 4,892 4,280 5,798 5,980 5,139 5,261 5,274 3,913
54 45 27 29 33 45 33 59
70 48 32 31 27 47 31 74
220 15U 14(1 90 170 3 40 140 165
NOTE : VC = vital capacity; TLC = total lung capacity; RV:TLC = ratio residual volume to total lung capacity ; FRC = functional residual capacity; FEV r.,, = forced expiratory volume at one second; MVV = maximal voluntary ventilation; and PF = peak flow.
of sample weight to reagent volume and to the volume of alcohol was kept constant. Adjustments in the volume of reagents and of alcohol were made if less than 100 mg. of tissue was available. The gas-liquid chromatograms obtained by this method gave “fingerprints” of the whole tissue. The muscle was fixed in a 10 per cent form01 saline solution and longitudinal and cross sections stained with hematoxylin and eosin were examined. RESULTS
Clinical Examination. Complaints directly attributable to peripheral neuropathy were found in one patient. On examination, evidence of weight loss and of mild reduction of joint position, vibration, touch and temperature sensation in a stocking distribution were found in seven of the patients with chronic obstructive bronchopulmonary disease. These patients also had slight weakness of the ankles and small muscles of the feet, and contracture of the shoulder joint was noted in one. Deep tendon reflexes were preserved in all (Table I). Results of pulmonary function tests in these patients appear in Table II. Electromyograply. Denervation changes were foulld in the extensor digitorum brevis of the foot in five of seven patients with clinical evidence of neuropathy. The other muscles were normal. The conduction velocities to the thenar and hypothenar muscles were normal. The fastest motor nerve fibers to the extensor digitorum brevis had conduction velocities ranging from 20 to 34 (M.) per second in seven patients with clinical evidence of neuropathy (normal, 40 to 60 11. per second). Three patients were reexamined five to nine months later; in two there was a marked improvement in conduction velocities (Table I).
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Sural Nerve Biopsies. In one patient with clinical evidence of neuropathy the sural nerve specimen showed an increase in the number of Schwann cell nuclei and widening of the endoneural spaces (Fig. 1). Osmium-fixed longitudinal sections disclosed myelin in macrophages and occasional fibers undergoing Wallerian degeneration evidenced by myelin ovoids arranged in rows (Fig. 2). On inspection of transverse sections, some fascicles contained numerous swollen fibers and the density of myelinated fibers seemed to be reduced in others. The number of nerve fibers and the percentage of fibers of various sizes in each of the sural nerve specimens obtained from six patients with chronic ohstructive bronchopulmonary disease is shown in Figure 3. Normally there is a considerable \-ariation in the number and frequency distribution of various-sized fibers but a decrease in total fiber count and in the proportion of large fibers with advancing age has been found in the anterior tibia1 nerve [5]. Nevertheless, all the nerve specimens, except one from a patient (G.W.) who had no clinical or electromyographic evidence of neuropathy, were thought to show a loss of myelin sheath. The percentage of fibers of various sizes did not seem to conform to a single pattern in these sural nerves. The gas-liquid chromatograms (“fingerprints”) of the sural nerve biopsy specimens from five patients with chronic obstructive bronchopulmonary disease and neuropathy, from eleven control subjects (five specimens obtained by biopsy and six at autopsy) and from eleven patients with alcoholic neuropathy were compared (Fig. 4 and 5). In addition, the area occupied by the peaks of the elution patterns from each
876
Peripheral
Neuropathy-Appenzeller
et al.
FIG. 1. Cross sections of sural nerve specimens from normal control subject (left) and patient with chronic respiratory tract disease and neuropathy (rip-ht), showing an increase in the number of Schwann cell nuclei and widening of endoneural spaces. Hematoxylin and eosin stain, original magnification X 900. FIG. 2. Longitudinal section of osmium fixed nerve from patient with chronic respiratory tract disease and neuropathy, showing myelin avoids. Original magnification X 250.
nerve was summated and the percentage of t1.e total area occupied by individual peaks calculated. In Table III are shown the mean percentage of the total area occupied by each peak in the various groups. Statistically significant differences, when compared with normal biopsy specimens, were found in peak A of nerve specimens from patients with chronic obstructive bronchopulmonary disease and neuropathy and in peak F of nerve specimens from patients
with alcoholic neuropathy. There was no statistically significant difference in the percentage area occupied by the various peaks of the normal biopsy and autopsy specimens. A tentative identification of some peaks of the “fingerprints” is given in Table IV. Muscle Biopsies. The gastrocnemius muscle biopsy specimens showed denervation atrophy in one, and fiber necrosis with myophagia, central nucleation and marked variation in fiber AMERICAN
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Neuropat lly-
L-Ipj~enzdlrrr! al.
H7?
E.P.
A.P.
W.B. n = 2388 0’ 55
u
E.M.
1
u
u
FIG. 3. Fiber number and diameter spectra of sural nerve specimens from patients with chronic obstructive bronchopulmonary disease in this study. The letters refer to the initials of the patients and correspond to those in Table I.
Gas-liquid chromatograms of sural nerve specimens from normal control subject (above) and patient with FIG. 4. chronic respiratory tract disease (below). Note increase in peaks A, B and I in the lower chromatogram. The three fold decrease in the sensitivity of the upper tracing (3K and 30K) was necessary to illustrate these differences. The integrations and figures given in Table III were obtained at a uniform sensitivity of the recording apparatus used in this study. chromatograms of sural nerves from normal FIG. 5. Gas-liquid neuropathy. Note decrease in height of peaks F and G in the lower VOL.
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1968
control subject chromatogram.
(above) and patient
with
alcoholic
Peripheral
878
Neuropathy-A@enzeller TABLE
MEAN
PERCENTAGE
AREA
OCCUPIED
BY
PEAKS
A THROUGH
SPECIMENS(n =
NUMBEROF
et al.
III
I OF
THE NERVES
GAS-LIQUID
CHROMATOGRAMS
FROM
SEVERAL
NERVE
GROUP)
IN EACH
Sural Nerves Source
n
A
B
C
D
E
F
G
H
I
Normal subjects autopsy specimen Biopsy specimen Subjects with alcoholic neuropathy Subjects with chronic pulmonary disease
6 5 11 5
0.8 0.9 0.8 1.7*
1.5 1.5 1.4 2.3
12.3 11.4 16.0 13.6
16.1 15.1 16.8 15.1
58.3 58.5 59.0 54.7
5.1 5.0 3.2* 4.2
3.4 3.9 1.7 3.7
1.1 0.9 0.9 0.6
1.0 2.1 1.2 3.2
* Statistically significantly different P < 0.01. in another. The remaining three biopsy specimens showed generally small fibers and some random variation in fiber size. size
COMMENTS
The etiology of wasting associated with chronic respiratory tract disease remains obscure but it is known to be indicative of a poor prognosis. To elucidate the cause of wasting associated with chronic respiratory tract disease, the theoretic caloric requirements in such patients were compared with actual caloric intake and this was found adequate. Moreover, no relation between the onset of wasting and deterioration in pulmonary function tests was lound. It was suggested, nevertheless, that the wasting may be associated with a deficient protein intake in such patients [I]. Some patients with chronic obstructive bronchopulmonary disease and wasting have an associated peripheral neuropathy on clinical and electromyographic examinations. In addition, in this study evidence for neuropathy was obtained on histologic examination of specimens TABLE TENTATIVE
IDENTIFICATION
IV OF PEAKS A-I IN GAS-
LIQUID CHROMATOGRAMS OF SURAL NERVES
Peak
Identification
A B C D E F G H I
? Myristic ? Palmitic Oleic Stearic ? ? Cholesterol
of sural nerves and muscles, and alterations in the composition of these nerves were found also. The clinical features of the neuropathy were not uniform and some patients showed a marked improvement in nutrition and nerve conduction velocities some months after their initial examination. The most common cause of marked slowing of conduction velocity is segmental myelin loss in motor nerves. In this study it was impossible, however, to confirm this histologically, since motor nerves are not easily amenable to biopsy; the sural nerve is composed of sensory fibers only. Nevertheless, evidence for loss of myelin and Wallerian degeneration of some fibers was found in biopsy specimens and this was consistent with the clinical findings in these patients. After treatment, recovery from acute neuropathy in uncontrolled diabetes is not uncommon. In this disorder, as in the neuropathy described here, the mode of presentation of patients may vary, but segmental and Wallerian degeneration occurs and the temporary conduction block in diabetic neuropathy is attributed to segmental myelin loss rather than Wallerian degeneration. This type of myelin loss can be shown to occur also in experimentally induced disorders of the peripheral nervous system [6,7] in which rapid recovery is associated with remyelination. It is likely, therefore, that the recovery of nerve conduction velocities in our patients was related to their clinical improvement, particularly as further deterioration in conduction velocity was found in one patient who remained otherwise unchanged. Dyck and Lambert [8] examined conduction velocities in excised sural nerves from patients with a variety of polyneuropathies and found them lower than expected from the diameter of myelinated fibers measured on cross sections of these nerves. Attempts to reconstruct the comAMERICAN
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pound action potentials from the spectra of nerve fiber diameters of these nerves were unsuccessful. In one nerve, myelinated fibers 6 M in diameter, which normally conduct at 25 to 35 M. per seco~~tl, were found, whereas the fastest conduction ill this salne sural nerve was only 4 M. per second. It is not surprising, therefore, that in patients with chronic disease of the respiratory tract there was marked \-ariation in the number of rnyelinated fibers and no uniformity in fiber diameter spectra of the sural nerves. B.iopsy specimens from patients who had poliomyelitis in the past were obtained, in a study designed to examine the histologic effects of long-standing denervation of skeletal muscle. All features hitherto considered typical of were found in these chronically “myopathy” denervated muscles. These included random variation in fiber size, central nucleation and rnyophagia [!I]. S imilar features found in the muscle biopsy specimens of patients in this study are therefore not inconsistent with the assumption that they represented denervation rather than myopathic changes. Gas-liquid chromatographic analysis of sciatic nerves in experimental allergic neuritis in the guinea pig was found to be a sensitive method for the recognition of changes in the composition of these nerves [70]. By the use of this technic, biochemical alterations in the nerves were found without clinical or histologic evidence of disease in these animals. These changes were confined to the sciatic nerves and reverted to normal after two months. In patients with chronic disease of the respiratory tract and neuropathy the clinical, electromyographic and histologic findings were indistinguishable from other peripheral neuropathies. In an attempt to separate this neuropathy from other polyneuropathies, we subjected part of the sural nerve biopsy specimens to gas-liquid chromatography. The elution patterns obtained enabled us to differentiate biochemically the neuropathy of patients with chronic respiratory tract disease from control subjects and patients with alcoholic neuropathy. The etiology of neuropathy occurring in association with chronic respiratory tract disease is not clear, but one possible factor in the appearance of neuropathy in patients with wasting is malnutrition. All patients with neuropathy were malnourished and it is tempting, therefore, to assume that this was the cause of their neuropathy. The neuropathies associated with chronic VOL.
44,
JUNE
1968
et ol.
S?!)
alcoholism have been attributed to ~nalrl~~trition. But it is still not clear whether alcohol itself, apart from malnutrition, exerts a direct influence on the peripheral nervous s)-strln [ I I]. The different rlution patterns obtaincxd in sllral ner\:e specimens from patients with alcoholic neuropathp do not, therefore, necessarily / ndicate that neuropathy in patients with wasting associated with chronic respiratory tract disease is unrelated to malnutrition. Peripheral neuropathy sometimes appears as a complication of drug therapy. All patients in this study had been on a standard regimen of bronchodilators, digitalis and, intermittently, diuretics and a variety of antibiotics. None had received steroids. To our knowledge, the drugs administered to these patients have not been incriminated in the causation of neuropathies. A number of metabolic disorders have been associated with peripheral neuropathy but the etiology of the neuropathy in these diseases is disputed. Thus, diabetic neuropath), was thought to be ischemic due to occlusion of \.asa nervorum [ 721. Fagerberg [ 7~1 considered diabetic neuropathy to be due to a specific diabetic angiopathy, associated with the accumulation of periodic acid-Schiff-positive material in intraneural arterioles, but more recent studies failed to substantiate these findings and diabetic neuropathy has been attribllted to metabolic disturbances [ 741. Uremic ne\lropathy also is thought to be due to a nletabolic disorder dependent on severe renal failure, although the neuropathy occurs in malnourished patients who often have abnormal glucose tolerance curves as well [ 7.51. In patients with chronic respiratory tract disease and neuropathy, no abnormal glucose tolerance was ftlund and it is unlikely that they suffered from specific deficiencies of B vitamins, such as occllr in beriberi and alcoholic neuropathy. An alternative to the hypothesis that the neuropathy is due to malnutrition is that it is the result of a metabolic disturbance. The recovery of conduction velocity after appropriate treatment is best explained on this basis. The myelin sheath of peripheral nerves is derived from Schwann cells [7ti] so that an interference with the metabolism of Schwann cells may readily account for the myelin breakdown. It has been suggested that in diphtheritic neuritis the protein metabolism of Schwann cells is abnormal and results in the segmental deobserved in this disease 1771. myelination
880
Peripheral
Neuropathy-Appenzeller
Moreover, the resting respiration of the normal nerve is predominantly due to Schwann cell activity [ 78,791. Majno and Karnovsky [20] studied the lipogenic activity of Schwann cells and found that this decreased progressively toward the periphery, suggesting that peripheral Schwann cells might have an increased susceptibility in demyelinating neuropathies. This could explain the clinical and pathologic findings in metabolic neuropathies which appear first and are more severe in the periphery of the limbs. Schwann cell proliferation has been noted in diabetic neuropathy [74,27] and has been a feature in one of our patients. This may be accounted for by repeated injury to Schwann cells and suggests that it is not specific for the hereditary hypertrophic neuropathies in which it is characteristically seen, but may be a feature of metabolic neuropathies as well. Peripheral neuropathic sequelae of occlusive vascular disease of the limbs in the absence of metabolic disorders are minor [22]. In such patients anoxia secondary to the ischemia is usually of acute onset and is different from the oft-rehypoxemia, mild hypercapnia and curring cardiac failure of patients with obstructive bronchopulmonary disease. It is possible, therefore, that this long-lasting disturbance alters the metabolism of Schwann cells in some way and leads to the breakdown of peripheral myelin in patients with chronic respiratory tract disease and neuropathy. We wish to acknowledge Acknowledgment: the excellent technical assistance of Mr. Kenneth G. Allen and Mrs. Louise P. Tobias.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17. REFERENCES
E., VAN DE WOESTIJNE,K. P. and GYSELEN,A. Weight changes in the terminal stages of chronic obstructive pulmonary disease. Relation to respiratory function and prognosis. Am. Rev. Resp. Dis., 95: 556, 1967. 2. BALDWIN, E. DE F., COWRNARD,A. and RICHARDS, D. W., JR. Pulmonary insufficiency. I. Physiological classification, clinical methods of analysis, standard values in normal subjects. Medicine, 27: 243, 1948. 3. MENEELY, G. R., BALL, C. O., KORY, R. C., CALLAWAY, J. J., MERRILL, J. M., MABE, R. E., ROEHM, D. C. and KALTREIDER,N. L. A simplified closed circuit helium dilution method for the determination of the residual volume of the lungs. Am. J. Med., 28: 824, 1960. 4. MACGEE, J. Characterization of mammalian tissues 1. VANDENBERGH,
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and microorganisms by gas-liquid chromatography. J. Gas. Chromatog., 6: 48, 1968. SWALLOW, M. Fibre size and content of the anterior tibia1 nerve of the foot. J. Neural., Neurosurg. LT Psychiat., 29: 205,1966. MCDONALD, W. I. The effects of experimental demyelination on conduction in peripheral nerve: a histological and electrophysiological study. I. Clinical and histological observations. Brain, 86: 481,1963. CRAGG, B. G. and THOMAS, P. K. Changes in nerve conduction in experimental allergic neuritis. J. Neural., Neurosurg. & Psych&., 27: 106, 1964. DYCK, P. J. and LAMBERT, E. H. Compound action potentials and fiber diameter spectra of excised sural nerves in human neuropathies. Neurology, 17: 286,1967. DRACHMAN,D. B., MURPHY, S. R., NIGAM, M. P. and HILLS, J. R. “Myopathic” changes in chronically denervated muscle. Arch. Newof., 16: 14, 1967. APPENZELLER,O., MACGEE, J. and YINGVORAPANT, S. Gas-liquid chromatographic analysis of sciatic nerves and spinal cords in experimental allergic neuritis. J. Neural., Neurosurg. & Psych&., 30: 279, 1967. VICTOR, M. and DREYFUS P. M. Nutritional diseases of the nervous system. A statement of some current problems and suggestions for further investigation. World Neural., 2: 862, 1961. WOLTMAN, H. W. and WILDER, R. M. Diabetes mellitus; pathologic changes in spinal cord and peripheral nerves. Arch. Int. Med., 44: 576, 1929. FAGERBERG,S. E. Diabetic neuropathy: A clinical and histological study on the significance of vascular affections. Acta med. scandinau., supp. 345,1959. THOMAS, P. K. and LASCELLE, R. G. The pathology of diabetic neuropathy. Quart. J. Med. n.s., 35: 489, 1966. ASBURY, A. K., VICTOR, M. and ADAMS, R. D. Uremic polyneuropathy. Arch. Neural., 8: 413, 1963. GEREN, BEN B. The formation from the Schwann cell surface of myelin in the peripheral nerves of chick embryos. Exper. Cell. Res., 7: 558,1954. CAVANAGH,J. B. and JACOBS,J. M. Some quantitative aspects of diphtheritic neuropathy. Brit. J. Exper. Path., 45: 309, 1964. HELLER, I. H. and HESSE, S. Action of insulin on the respiration of rat sciatic nerve. Lancet, 2: 406, 1960. FIELD, R. A. and ADAMS, L. C. Insulin response of peripheral nerve. I. Effects on glucose metabolism and permeability. Medicine, 43 : 275, 1964. MAJNO, G. and KARNOVSKY, M. L. A biochemical and morphologic study of myelination and demyelination. I. Lipide biosynthesis in vitro by normal nervous tissue. J. Exper. Med., 107: 475, 1958. GREENBAUM,D. Observations on the homogeneous nature and pathogenesis of diabetic neuropathy. Brain, 87: 215,1964. HUTCHINSON,E. C. and LIVERSEDGE,L. A. Neuropathy in peripheral vascular disease; its bearing on diabetic neuropathy. Quart. J. Med. n.s., 25: 267 1956.
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