Painful Diabetic A
Neuropathy
Morphometric Study
Mark J.
Brown, MD; John R. Martin, MD; Arthur K. Asbury,
\s=b\ We investigated three diabetic patients whose neuropathy was characterized by pain, hypesthesia, and autonomic dysfunction, with preservation of epicritic sensation and muscle-stretch reflexes. Two sural nerves were studied qualitatively and quantitatively, using teased fiber, light, and electron microscopical techniques. The most striking alterations were encountered in unmyelinated and
myelinated fibers. Unmyelinated fiber sprouting was evident. The clinical features, which suggested smallfiber involvement, correlated with the pathological findings in biopsied cutaneous nerve. The balance of evidence indicates that the painful small-fiber neuropathy of diabetes is an axonal disorder. (Arch Neurol 33:164-171, 1976) small
nerve
Accepted
for publication March 5, 1975. Neurology Research Laboratory, San Francisco Veterans Administration Hospital, and the Department of Neurology, University of California, San Francisco. Drs Brown and Asbury are now with the Department of Neurology, Hospital of the University of Pennsylvania, Philadelphia. Dr Martin is presently with the Department of Pathology, College of Physicians and Surgeons, Columbia University, New York. Read in part before the annual meeting of the American Association of Neuropathologists, BosFrom the
ton, June 9, 1974. Reprint requests
to Department of Neurology, Hospital of the University of Pennsylvania, Philadelphia, PA 19104 (Dr Brown).
MD
symmetric Distal, neuropathy
sensory-motor
frequent and well-recognized complication of dia¬ is
a
betes mellitus. Manifestations vary1-9; a patient may be free of symptoms and signs, but have subtle electrophysiologic abnormalities. With the most common clinical picture, vibra¬ tion and position perception are impaired, ankle jerks are lost, and motor and sensory nerve conduction velocities are slowed. Sometimes dys¬ esthesia, distal cutaneous hypesthesia, and autonomie symptoms are promi¬ nent. At a presumably later stage, defective proprioception and dimin¬ ished muscular strength may result in unsteadiness of gait. Pathological studies of this form of diabetic neurop¬ athy from biopsy or postmortem material have documented both pri¬ mary segmental demyelination and axonal loss with secondary involve¬ ment of myelin sheaths.816 There is conflicting pathological evidence for a vascular abnormality in the distal,
symmetric sensory-motor neuropathy
of diabetes.1214~18 This contrasts with diabetic cranial mononeuropathy and mononeuropathy multiplex in which vasa nervorum alterations and nerve infarction have been documented.19'20
Using clinical and electrophysiologic methods, we have recently surveyed 40 adult male patients with diabetes and varying degrees of neuropathy. Clinical patterns of neu¬ ropathy encountered presented a con-
tinuum. At
patients
end of the spectrum, identified with painless,
one
were
distal sensory loss, primarily proprioception and position sensibility, and loss of tendon jerks. At the other end, three patients were found with severe
burning pain, cutaneous hypesthesia, autonomie disturbance, and relative preservation of tendon jerks and large-fiber sensory modalities. This article presents the quantitative mor¬ phologic data from biopsied sural nerves of two of the three patients. Pathological evidence suggests that unmyelinated fibers and, to a lesser extent, small myelinate fibers were disproportionately involved. REPORT OF CASES Case 1.—A 45-year-old diabetic man was admitted for neurologic evaluation after a year of increasingly severe burning pain and deep aching in his feet. He had been fired from his factory job because of clum¬ siness in handling small metal parts. He described impotence for nine months, constipation, occasional dizziness on stand¬ ing, and mild dependent ankle edema. He did not sweat below the knees. An insulindependent diabetic for 12 years, he had only mild hypoglycémie reactions, and degree of control was considered adequate. He ate well, did not use alcohol, and had no known exposure to neurotoxins. There was a family history of diabetes, but not of neurologic disorder. He seemed in good health. His legs were scarred from previous injuries, and he had necrobiosis lipoidica diabeticorum. Blood pressure was 122/80 mm Hg supine and
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Table 1.—Nerve Conduction
Velocity Studies Sensory
Motor Median
CV,*
m/sec Case 1 59 Case 2 48 Case 3 47 Control >49
Ulnar
CV,
m/sec 51 45 45 >47
Peroneal
CV, m/sec 40 32
NO§ >40
Median
Sural
Ulnar
DL,t
SAP,*
DL,
SAP,
DL,
msec
;¡v
msec
¡,y
msec
4.8
4.6 5.8 9
4.0 3.9 NO 7
SAP,
5.3 NO 10
Conduction velocity. Distal latency. t Sensory action potential amplitude. § No potential obtained. *
t
Table
of Sural Nerve Fibers and Cell Nuclei Expressed Number per Square Millimeter of Nerve Cross Section
2.—Density
Fibers and Nuclei
Myelinated fibers* Myelinated fibersf Unmyelinated fibers* Schwann cell nuclei associated with myelinated fibersf Schwann cell nuclei associated with unmyelinated fibersf Other Schwann cell nucleif Total Schwann cell nucleif Fibroblast nucleif
Case 1 2,890
Case 2
Control
32,200
3,130 3,155 19,700
9,630 9,500 33,400
165
330
675
2^640
980
1,675
2,405
1,445 2,755
3,165
150
265
320
1,180 1,060
From thick sections, final magnification X 1000 f From ultrathin sections, final magnification 1,600 From ultrathin sections, final magnification X 20,000 *
as
815
122/80 mm Hg standing, without increase of pulse rate. Pedal pulses were full. Fundi were normal. There was a right Horner syndrome. Strength was normal, and all stretch reflexes were present. Hypesthesia to pinprick and light touch was found in a patchy distribution, but generally it con¬ formed to a glove and stocking distribu¬ tion. Histamine injection (0.1ml of 1:10,000, intracutaneously) produced a normal wheal, but a markedly reduced flare over the dorsum of the foot. Response at the anterior thigh was normal. Pallesthesia and proprioception were intact. Fasting hyperglycemia was verified. Spinal fluid protein was 66 mg/100 ml. Electrodiagnostic studies showed normal motor conduction velocities, but low-ampli¬ tude sensory action potentials with pro¬ longed latencies (Table 1). The following test results were normal: complete blood cell (CBC) count, erythrocyte sedimenta¬ tion rate (ESR), urinalysis (UA), blood urea nitrogen (BUN), serum creatinine (Cr), cholesterol, serum glutamic oxaloacetic transaminase (SGOT), serum protein elec¬ trophoresis, serum thyroxine (T,), antinu¬ clear factor (ANF), VDRL, and chest roentgenogram. Sural nerve biopsy was
performed. Eighteen
pain had then were unchanged, except that vibration and posi¬ tion senses were impaired in the feet. Case 2.-A 44-year-old diabetic man was disabled by continuous deep limb pains for two years. They reminded him of a ciga¬ rette burn. In addition, he had shooting pains and intermittent cramps in both calves and feet. These occurred either spon¬ taneously or after light tactile stimulation. Finally, he had aerai "pins and needles" paresthesias and was aware of numbness in his fingertips and below the knees, where he had suffered painless injuries. He had had no erections for at least one year and reported constipation and urinary hesitancy. He did not sweat below the waist. Nine years earlier when diabetes mellitus was first diagnosed, he had a sudden, unexplained attack of blindness without residual visual defect. Symptoms of muscle weakness in the proximal part of the leg and tender thighs led to the diag¬ nosis of femoral neuropathy at age 42. This, too, cleared completely. The degree of diabetic control with hypoglycémie agents administered orally was "fair"; he never decreased.
Fig
months later his
Physical findings
1.—Cross section from sural
nerves.
Left, Control. Upper right, Case 1. Lower
Case 2. Myelinated fiber density is reduced in diabetic material (phase con¬ trast optics, original magnification
right,
X675).
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had ketoacidosis. As a business represent¬ ative for an industrial firm, he had had slight exposure to metal salts and organic solvents but not for eight years. He rarely used alcohol. His family was free of diabetes or known neurologic disease. The general physical examination was unremarkable, and he looked well. Blood pressure was 125/80 mm Hg supine and standing. Muscle strength was excellent. All tendon jerks were present, but the left Achilles reflex was obtained only after reinforcement. Pinprick, temperature, and light touch thresholds were elevated below the wrist and knee. He would not tolerate light stroking of his toes, as this produced pain. Pallesthesia was mildly impaired at the ankle. Position sense was normal. Histamine given intradermally produced a normal wheal, but reduced flare, on the dorsum of his foot. Fasting blood glucose level was repeat¬ edly elevated. The thickness of the base¬ ment membrane of the capillaries in the quadriceps muscle was 3,011 ± 276 Ang-
Fig 3—Left, Relationship and diabetic sural diabetic material.
nerves.
2500 Case I
2000-
1500-
2,890 MF/
500 Control
9,630 MF/sq.
mm.
10
16
Case 2 500
3,130 MF/sq.
500-
8
10
T
12
16
14
Myelinated
2
12
10
6
4
mm.
~1
14
Fiber Diameter (ju)
Fig 2.—Myelinated fiber densities in control and diabetic sural nerves. Loss of smaller disproportionately greater than loss of larger ones in diabetic patients. Proportion of small myelinated fibers (^5µ in diameter) in control was 59.1%; case 1,
fibers is
44.5%; and
case
2, 50.7%.
between length and diameter of internodes from teased fibers. Calculated regression lines similar for control Right, Internodes on single fiber plotted against fiber's widest diameter. Variation may be slightly greater in
CONTROL
¡E
0.8
O
o
UJ -
i í
0.6
UJ
Q
O
O
0
3
4
m
0.4
ft.
.1 5
6
7
8
9
10
11
12
3456789
INTERNODE DIAMETER («)
10
WIDEST INTERNODE DIAMETER
1.2
(li)
1.2 CASE 2
1.0
CASE 2
1.0 E
0.8-
£
0.6
O Z
14
12
1000-
CONTROL
O
sq.mm.
5
0.6
Q
0.4
O 0.2-
0
3
4
5
6
7
8
9
10
INTERNODE DIAMETER |u)
11
12
13
0
4^^-1-1-1-1-,-1-1-1-1-1 0
3
4
5
6
7
8
9
10
WIDEST INTERNODE DIAMETER (u|
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11
12
13
Fig 4.—Electron photomicrographs of control sural nerve. Left, Well-preserved membranes including sheaths of myelinated fibers (M). Right, Most unmyelinated fibers (U) invested by cytoplasm of only one Schwann cell, with well-defined mesaxon. Occasional unmyelinated fibers (star) surrounded by processes from two or more Schwann cells (x 14,600). (normal, 1,325 A; diabetic, above 1,600 A). Motor conduction velocities were
Stroms
mildly slowed, sensory action potential amplitudes were reduced, and terminal latencies were prolonged (Table 1). Labora¬ tory tests with normal findings included CBC count, ESR, UA, BUN, Cr, cholesterol, SGOT, serum protein electrophoresis, T„ B,_, VDRL, chest roentgenogram, and barium meal and enema. Sural nerve
serum
biopsy was performed. Phenytoin therapy reduced the extent and frequency of muscle cramps and shooting leg pains. When the patient was seen three months after biopsy, his neuro¬ logic status had not changed. Case 3.—A 42-year-old employed diabetic machinist complained of "wooden" feet and impotence. For ten years he had been aware of insidiously progressive numbness that ascended to his midthighs and fingers. He had numerous unrecognized injuries. Dysesthesias, foot cramps, and stabbing leg pains were superimposed on a constant
aching, tearing sensation in his limbs. In the past, light touch had triggered parox¬ ysms of foot pain. He had not had a penile erection in more than five years; a trial with a prosthetic penile implant was unsuc¬ cessful. He had dependent ankle edema at the end of the workday. A known diabetic and insulin-dependent for 14 years, he had always been in good control. He was treated for retinopathy with photocoagulation and had proteinuria for at least one year. There was no exposure to neurotoxins, including alcohol. Family history was negative for diabetes and neurologic disor¬ ders. He appeared healthy, but with a depressed affect. Blood pressure was 160/90 mm Hg supine and standing. He had bilateral retinal microaneurisms, hyperpigmented scars on his lower legs, and mild ankle edema. Dorsalis pedis pulses were strong. Toe dorsiflexion was modera¬ tely weak, and the extensor digitorium brevis muscles were not palpable. Ankle
jerks were present, but diminished. He was hypesthetic to pinprick, light touch, and temperature change below the elbow and midthigh. Pallesthesia was moderately reduced in the lower extremities and mildly depressed in the upper ones. Recog¬ nition of position change was impaired in the toes. Histamine flare was decreased on the dorsum of the foot, but not on the anterior thigh. Studies with abnormal results included elevated blood glucose level, ESR of 42 mm/hr, BUN level of 44 mg/100 ml, Cr level of 2.2 mg/100 ml, creatinine clearance of 37 ml/min, and a urine protein content of 10.5 gm/24 hr. Bladder capacity was enlarged to 1,000 ml, with weak detrusor function. Motor nerve conduction velocities were somewhat slowed (Table 1). The median nerve sensory action potential was of reduced amplitude and prolonged laten¬ cy. The following studies yielded normal results: CBC count, cholesterol, SGOT, serum protein electrophoresis, antinuclear
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Fig 5.—Electron photomicrograph from sural nerve of patient 1, a diabetic with painful neuropathy. Left, Normal, unmyelinated fiber (U) ultrastructure. Right, Small, unmyelinated fibers cluster in Schwann cell cytoplasm (arrows) (original magnification 14,600).
antibody,
serum B,2, VDRL, and chest roentgenogram. Sural nerve biopsy was
performed. Phenytoin therapy did not alter the pain, and at follow-up six months later, the neurologic symptoms were unchanged. Control.—A 48-year-old man died sud¬ denly of cardiac arrhythmia. At the time, he was undergoing investigations for congestive heart failure. He did not abuse alcohol. Neurologic history and examina¬ not
tion had been negative. Laboratory studies with normal results included: CBC count, UA, BUN, Cr, blood glucose, and chest
roentgenogram. Autopsy findings were atherosclerosis, left ventricular aneurysm, and pulmonary scarring from old tubercu¬ losis.
METHODS Sural nerve biopsy was performed at the ankle under local anesthesia after the risks of the procedure were fully explained to the patient-volunteers. A portion of each
nerve was fixed in 10% formaldehyde, embedded in paraffin, and stained. An¬ other segment was fixed in 3.6% glutaral¬ dehyde in phosphate buffer and postfixed in buffered 2% osmium tetroxide. After dehydration, it was embedded in epoxy resin. Longitudinal and full cross sections, 1.5fi-thick, were mounted on glass slides. Endoneurial area was calculated from lowpower photomicrographs of each fascicle. Representative fields covering about 60% of the endoneurial area were photographed and printed at a final magnification of x 1,000. Myelinated fiber diameters were determined from these photographs. Sin¬ gle osmicated myelinated fibers were teased from their fascicles. Internodal lengths and diameters were measured along approximately 40 such fibers from each biopsy specimen. Ultrathin sections were stained with uranyl acetate and lead citrate. Unmyeli¬ nated fibers were studied, measured, and counted from 200 electron photomicro-
graphs from each biopsy specimen, which were printed at a final magnification of 20,000. Each fascicle was represented according to its contribution to the total endoneurial was
area.
About 2.5% of that
area
sampled. The density of both Schwann
cell and fibroblast nuclei, and a second estimate of the myelinated fiber popula¬ tion, were calculated from low-power elec¬ tron micrographs of the same grids (Table
2). RESULTS
appeared normal surgical removal. Micro¬ scopically, the perineurium and ves¬ The sural
nerves
at the time of
sels
were
unremarkable, and there
amyloid deposition or inflam¬ matory infiltrate. Degenerating fi¬ bers were rarely seen in cross or longi¬ tudinal sections. Total myelinated fiber density was reduced to about one was no
third of the normal value in all fasci-
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best
in the
of myelinated fibers had not oc¬ curred. The ultrastructural membrane de¬ tail of both diabetic and autopsy material was well-preserved (Fig 4 and 5). In the control, swollen mito¬
phase microscopical preparations (Pig 1). Though both large and small myelinated fiber populations were affected, there was a relatively greater loss of smaller myelinated fibers (Fig 2). The lengths of all inter¬ nodes on a teased, single myelinated fiber were plotted against the fiber's widest diameter according to the method of Fullerton et al.-'1 By inspec¬ tion, the degree of variability was only slightly greater in the diabetic material (Fig 3, right), indicating that demyelination-remyelination was not prominent in the pathological process. Myelinated fibers undergoing active axonal degeneration were occasionally cíes; this
was
seen
contrast
chondria and loss of neurotubules were recognized as artifacts. In the diabetic nerves, we found no myelin sheath disorganization, neurofilament dissolution, or membranous organelle proliferation in the axons themselves. Occasional Schwann cells had in¬ creased amounts of endoplasmic retic¬ ulum and mitochondria, but this was inconsistent. Endoneurial capillaries were normal in appearance; we did not survey their basement membrane thickness. Myelinated fiber density, as determined in the electron micro¬ graphs, was the same as that found
seen, three of 49 fibers in case 1 and of 35 in case 2 vs one of 47 in the
one
control
When
nerve.
an
internode's
length was plotted against its own width, disregarding the rest of the fiber, and the regression line then calculated, the diabetic fibers were not different from the control (Fig 3,
using lower-magnification phase con¬ trast photomicrographs. Schwann cell nuclei associated with myelinated
fibers were reduced to a degree appro¬ priate for the amount of myelinated fiber loss (Table 2). Those Schwann cells not clearly associated with any
left). This observation suggests that
regeneration
of
significant
numbers
Fig 6.—Unmyelinated fiber densities in control and diabetic sural nerves. Type 1 axon-Schwann cell (Ax-ScC) configuration is axon surrounded by single Schwann cell. Type 2 is axon surrounded by two or more Schwann cell processes, juxtaposed against another axon, or partially uncovered. Number of large unmyelinated fibers
ScC
fiber type ("other Schwann cell nuclei" in Table 2) are considered to.be unmyelinated fiber-related. This group of Schwann nuclei, taken to¬ gether with those that are clearly associated with unmyelinated fibers, number approximately the same as in control nerve. Total numbers of Schwann cell nuclei were not in¬ creased, suggesting that Schwann cells had not proliferated in response to the pathological process. The densi¬ ty of fibroblast nuclei was similar to that of the control. The total number of unmyelinated fibers per unit area was normal in case 1 and mildly reduced in case 2 (Fig 6), when compared to either our control values or those of others.---3 Analysis of the unmyelinated fibers by size indicated a striking absence of the large fibers more than 1µ in diam¬ eter and an accompanying increase in the small ones (Fig 6). Only 4% of all unmyelinated fibers were larger than lju in the diabetic patients (control, 33%). Looking at the small end of the fiber spectrum, approximately 75% of
more than 1µ reduced in diabetic tissue; small unmyelinated fibers increased in number. Frequency of type 2 configurations abnormally high in case 1 and 2, indicating regenerative sprout¬
ing.
16,000
Configuration
—
0- Type D- Type
14,000
1 2
Case 1
Case 2
32,200 UF/sq.
19,700 UF/sq.
mi
12,000 10,000
10,000
8000
8000
8000
6000
6000
6000
4000-
4000
2000
2000-
Control
33,400 UF/sq. a>
"o
0.2
0.6
1.0
1.4
1.8
2.2
0.2
Unmyelinated
0.6
1.0
1.4
1.8
2.2
Fiber Diameter (a»)
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0.2
0.6
1.0
1.4
1.8
the total unmyelinated fibers were smaller than 0.6µ in the diabetics (con¬ trol, 25%). In addition to these quanti¬ tative changes, there were qualitative alterations in the axon-Schwann cell relationships in the diabetic material (Fig 6). Whereas 88% of control unmyelinated fibers were fully in¬ vested by the cytoplasm of a single Schwann cell, usually with a readily identifiable mesaxon (Fig 4), only 52% and 65% of the diabetic unmyelinated fibers were in this simple "type 1" relationship (Fig 5). The remainder of the diabetic unmyelinated fibers were in a "type 2" axon-Schwann cell rela¬ tionship, that is, either incompletely surrounded by a Schwann cell or, less
commonly, wrapped by
two
or more
separate tongues of Schwann cell cytoplasm. While it was unusual to find more than three unmyelinated fibers associated with a single Schwann cell in the control, three or more unmyelinated fibers per Schwann cell were frequently ob¬ served in the diabetic material. Fur¬ ther, only the diabetic nerves con¬ tained clusters of small, naked unmye¬ linated fibers that abutted one anoth¬ er, forming complexes that were surrounded by Schwann cell cyto¬
plasm (Fig 5).
COMMENT
One might predict on clinical grounds that three diabetic patients with painful neuropathy would show disproportionate pathological involve¬ ment of unmyelinated and small myelinated fibers. Quantitative fiber analysis of two biopsied sural nerves bore out this impression. The diagnosis of diabetic neurop¬ athy was made by the temporal asso¬ ciation with diabetes mellitus and by
the absence of another basis for neuropathy after the history, physical examination, and laboratory studies, including biopsy, were completed. Pa¬ tient 3, in whom biopsy was not performed, had renal impairment to a degree not thought to affect periph¬ eral nerve.24 Functions attributable to large my¬ elinated fibers were little altered in our patients. Muscle stretch reflexes, which depend on the integrity of the largest myelinated fibers, were al-
ways
present. Vibration and position not lost. The
electrophysiologic studies, which similarly test the largest myelinated fiber popula¬ tion, indicated only mild conduction velocity slowing, consistent with the paucity of segmental demyelination in teased fiber preparations. Reduc¬ tion of amplitude of sensory action potentials paralleled roughly the loss of largest myelinated fibers in the biopsy specimens. Unmyelinated fiber dysfunction In cutaneous was more striking. senses were
nerves, these C fibers carry out both
afferent and autonomie efferent func¬ tions.27' Clinically, both functions were affected in our patients. Elevated pain, temperature, and touch thresh¬ olds suggested involvement of affer¬ ent C fibers, perhaps along with small myelinated A delta fibers, while impo¬ tence and decreased peripheral sweat¬ ing pointed to involvement of sympa¬ thetic fibers. Anatomically, there was a near absence of larger unmyelinated fibers, and small myelinated fibers were reduced to approximately one quarter of control. Concurrent unmye¬ linated fiber loss and altered proto¬ pathic sensation has been observed in several other neuropathies2"28 with an absent C wave when in vitro nerve conduction velocity was determined. Very small unmyelinated fibers, less than 0.6µ in diameter, were strik¬ ingly increased in number, both abso¬ lutely and relative to the overall fiber spectrum. Their relationships with Schwann cells were often complex, and frequently several tiny fibers shared the same Schwann cell invagi¬ nation. This pattern of multiple, small, unsegregated fibers is charac¬ teristic of developing,2" regenerat¬ ing,30 or aging31 nerve and represents the process of sprouting. Ochoa32 first described the electron microscopical features of nerve sprouting in a human polyneuropathy, that of isoniazid intoxication. Our study confirms ultrastructurally that sprouting also can occur in the neuropathy of
diabetes mellitus.
Pathologically, segmental demyeli¬
nation is prominent in some cases of diabetic neuropathy.13141"33 Because myelin sheath is specialized Schwann cell surface membrane, segmental loss
of myelin is indicative of a Schwann cell disorder, either primary or sec¬ ondary. Metabolic alterations have, in fact, been imputed to Schwann cells in experimental diabetic neuropathy.8·34·35 Recently, however, research¬ ers have questioned the pathological importance of segmental demyelina¬ tion in human diabetic neuropathy and instead reemphasized axonal in¬ volvement.15 In our patients the major neuropathologic feature was loss of axons. This involved primarily large unmyelinated fibers, but also included small myelinated and, to a lesser extent, large myelinated fibers. Near absence of active degeneration of axons or myelin attests to the chronicity of the process. Teased nerve fiber preparations showed little in the way of demyelination and remyelinations. On the basis of these observa¬ tions, we favor axonal degeneration as the most likely cause for neuropathy in these patients. The fact that nerve conduction velocities were only mini¬ mally slowed supports this formula¬ tion. In most polyneuropathies with axonal degeneration the largest mye¬ linated fibers are affected earliest and most severely.23-31-36-37 It is of interest that unmyelinated and small myelinated fibers seem to be rela¬ tively more vulnerable in our pa¬ tients. Unmyelinated fiber abnormalities have been described qualitatively in diabetic neuropathy.1015-16·33-3* How¬ ever, without quantitative analysis, the patterns of degeneration (or regeneration) could not be assessed. We found no qualitative changes in axons or Schwann cells, aside from complex axon-Schwann cell relation¬ ships. Quantitatively, our patients had a virtual absence of large unmyeli¬ nated fibers. In addition, we consider the evidence for sprouting without early myelination to be a sign of unmyelinated fiber alteration. Our qualitative findings are at variance with some of those described by others,1516·33-35 but may only represent differences in patient selection. The pathophysiology of pain is always of interest, and patients such as ours are natural subjects in its study. There is a poor correlation between myelinated fiber loss and the
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of pain.38 It is tempting to relate pain of peripheral nerve origin to alterations of the unmyelinated fiber population. A possibility that intrigues us is that regenerating sprouts may themselves produce pain. This is consonant with the clinical observations that advancing pain and paresthesias accompany regrowth of a transected nerve and recovery from
occurence
some
polyneuropathies. Further
port for this mechanism
comes
sup¬
from
the recent demonstration that in rats regenerating nerve fibers fire sponta¬ neously, and after stimulation they
discharge abnormally.40 The painful small-fiber neuropathy
of diabetes is a characteristic syn¬ drome at one pole of the diabetic distal sensory-motor neuropathy spec-
Clinical features of the disorder consistent with the observed path¬ ological alterations. trum. are
Austin Sumner, MD, assisted with the electro-
physiologic studies. Marvin Siperstein, MD, PhD, measured the thickness of the basement mem¬ brane of the capillaries in the quadriceps muscle of patient 2. Basil Rapoport, MB, referred patient 3 for evaluation.
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study of
published.
10. Martin MM: Involvement of autonomic fibres in diabetic neuropathy. Lancet 1:561\x=req-\ 565, 1953. 11. Dolman CL: The morbid anatomy of diabetic neuropathy. Neurology 13:135-142, 1963. 12. Greenbaum D, Richardson PC, Salmon MV, et al: Pathological observations on six cases of diabetic neuropathy. Brain 87:201-214, 1964. 13. Thomas PK, Lascelles RG: The pathology of diabetic neuropathy. Q J Med 35:489-509, 1966. 14. Chopra JS, Hurwitz LJ, Montgomery DAD: The pathogenesis of sural nerve changes in diabetes mellitus. Brain 92:391-418, 1969. 15. Bischoff A: Ultrastructural pathology of peripheral nervous system in early diabetes, in Camerini-Davalos RA, Cole HS (eds): Vascular and Neurologic Changes in Early Diabetes. New York, Academic Press Inc, 1973, pp 441-449. 16. Vital CI, Vallat JM, LeBlanc M, et al: Les nerve
neuropathies periph\l=e'\riquesdu
diab\l=e'\tesucr\l=e'\: Etude ultrastructurale de 12-cas biopsies. J Neurol Sci 18:381-398, 1973. 17. Fagerberg S: Diabetic neuropathy. Acta Med Scand 345(suppl): 5-60, 1959. 18. Chopra JS, Hurwitz LJ: A comparative study of peripheral nerve conduction in diabetes and non-diabetic chronic occlusive peripheral
vascular disease. Brain 92:83-96, 1969. 19. Raff MC, Asbury AK: Ischemic mononeuropathy and mononeuropathy multiplex in diabetes mellitus. N Engl J Med 279:17-22, 1968. 20. Asbury AK, Aldredge H, Hershberg R, et al: Oculomotor palsy in diabetes mellitus: A clinico-pathologic study. Brain 93:555-566, 1970. 21. Fullerton PM, Gilliatt RW, Lascelles RG, et al: The relation between fiber diameter and internodal length in chronic neuropathy, abstracted. J Physiol 178:26-28P, 1965. 22. Ochoa J, Mair WGP: The normal sural nerve in man: I. Ultrastructure and numbers of fibers and cells. Acta Neuropathol 13:197-216, 1969. 23. Dyck PJ, Lambert EH, Nichols PC: Quantitative measurement of sensation related to compound action potential and number and sizes of myelinated and unmyelinated fibers of sural nerve in health, Friedreich's ataxia, herditary sensory neuropathy, and tabes dorsalis, in Reymond A (ed): Handbook of Electroencephalography and Clinical Neurophysiology. Amsterdam, Elsevier, 1971, vol 9, pp 83-118. 24. Nielsen VK, Winkel P: The peripheral nerve function in chronic renal failure: III. A multivariate statistical analysis of factors presumed to affect the development of clinical neuropathy. Acta Med Scand 190:119-125, 1971. 25. Mountcastle VB: Sensory receptors and neural encoding: Introduction to sensory processes, in Mountcastle VB (ed): Medical Physiology. St Louis, CV Mosby Co, 1974, p 296. 26. Dyck PJ, Lambert EH: Dissociated sensation in amyloidosis. Arch Neurol 20:490-507, 1969. 27. Aguayo AJ, Nair CPV, Bray G: Peripheral nerve abnormalities in the Riley-Day syndrome. Arch Neurol 24:106-116, 1971. 28. Bischoff A: Ultrastructural pathology of
the
peripheral nervous system. Z Neurol
274, 1973.
205:257\x=req-\
29. Davison AN, Duckett S, Oxberry JM: Correlative morphological and biochemical studies of the human fetal sciatic nerve. Brain Res 58:327-342, 1973. 30. Aguayo AJ, Peyronnard JM, Bray GM: A quantitative ultrastructural study of regeneration from isolated proximal stumps of transected unmyelinated nerves. J Neuropathol Exp Neurol 32:256-270, 1973. 31. Ochoa J, Mair WGP: The normal sural nerve in man: II. Changes in the axons and Schwann cells due to aging. Acta Neuropathol 13:217-239, 1969. 32. Ochoa J: Isoniazid neuropathy in man: Quantitative electron microscope study. Brain 93:831-850, 1970. 33. Luse SA, Moon TR: An ultrastructural study of peripheral nerves in human diabetes mellitus and in steroid-induced hyperglycemia in the rabbit. Anat Rec 154:380, 1966. 34. Gabbay KH, O'Sullivan JB: The sorbitol pathway: Enzyme localization and content in normal and diabetic nerve and cord. Diabetes 17:239-243, 1968. 35. Ward JD, Baker RWR, Davis BH: Effect of blood sugar control on the accumulation of sorbitol and fructose in nervous tissues. Diabetes 21:1173-1178, 1972. 36. Thomas P: The quantitation of nerve biopsy findings. J Neurol Sci 11:285-295, 1970. 37. Dyck PJ, Johnson WJ, Lambert EH, et al: Segmental demyelination secondary to axonal degeneration in uremic neuropathy. Mayo Clin Proc 46:400-430, 1971. 38. Thomas PK: The ultrastructural pathology of unmyelinated nerve fibers, in Desmedt JE (ed): New Developments in Electromyography and Clinical Neurophysiology. Basel, Switzerland, Karger, 1973, pp 227-239. 39. Thomas PK: The anatomical substratum of pain: Evidence derived from morphometric studies on peripheral nerve. Arch Neurol 30:418, 1974. 40. Wall PD, Gutnick M: Properties of afferent nerve impulses originating from a neuroma. Nature 248:740-743, 1974.
Downloaded From: http://archneur.jamanetwork.com/ by a Monash University Library User on 06/18/2015
Neuropathy
Morphometric Study
Mark J.
Brown, MD; John R. Martin, MD; Arthur K. Asbury,
\s=b\ We investigated three diabetic patients whose neuropathy was characterized by pain, hypesthesia, and autonomic dysfunction, with preservation of epicritic sensation and muscle-stretch reflexes. Two sural nerves were studied qualitatively and quantitatively, using teased fiber, light, and electron microscopical techniques. The most striking alterations were encountered in unmyelinated and
myelinated fibers. Unmyelinated fiber sprouting was evident. The clinical features, which suggested smallfiber involvement, correlated with the pathological findings in biopsied cutaneous nerve. The balance of evidence indicates that the painful small-fiber neuropathy of diabetes is an axonal disorder. (Arch Neurol 33:164-171, 1976) small
nerve
Accepted
for publication March 5, 1975. Neurology Research Laboratory, San Francisco Veterans Administration Hospital, and the Department of Neurology, University of California, San Francisco. Drs Brown and Asbury are now with the Department of Neurology, Hospital of the University of Pennsylvania, Philadelphia. Dr Martin is presently with the Department of Pathology, College of Physicians and Surgeons, Columbia University, New York. Read in part before the annual meeting of the American Association of Neuropathologists, BosFrom the
ton, June 9, 1974. Reprint requests
to Department of Neurology, Hospital of the University of Pennsylvania, Philadelphia, PA 19104 (Dr Brown).
MD
symmetric Distal, neuropathy
sensory-motor
frequent and well-recognized complication of dia¬ is
a
betes mellitus. Manifestations vary1-9; a patient may be free of symptoms and signs, but have subtle electrophysiologic abnormalities. With the most common clinical picture, vibra¬ tion and position perception are impaired, ankle jerks are lost, and motor and sensory nerve conduction velocities are slowed. Sometimes dys¬ esthesia, distal cutaneous hypesthesia, and autonomie symptoms are promi¬ nent. At a presumably later stage, defective proprioception and dimin¬ ished muscular strength may result in unsteadiness of gait. Pathological studies of this form of diabetic neurop¬ athy from biopsy or postmortem material have documented both pri¬ mary segmental demyelination and axonal loss with secondary involve¬ ment of myelin sheaths.816 There is conflicting pathological evidence for a vascular abnormality in the distal,
symmetric sensory-motor neuropathy
of diabetes.1214~18 This contrasts with diabetic cranial mononeuropathy and mononeuropathy multiplex in which vasa nervorum alterations and nerve infarction have been documented.19'20
Using clinical and electrophysiologic methods, we have recently surveyed 40 adult male patients with diabetes and varying degrees of neuropathy. Clinical patterns of neu¬ ropathy encountered presented a con-
tinuum. At
patients
end of the spectrum, identified with painless,
one
were
distal sensory loss, primarily proprioception and position sensibility, and loss of tendon jerks. At the other end, three patients were found with severe
burning pain, cutaneous hypesthesia, autonomie disturbance, and relative preservation of tendon jerks and large-fiber sensory modalities. This article presents the quantitative mor¬ phologic data from biopsied sural nerves of two of the three patients. Pathological evidence suggests that unmyelinated fibers and, to a lesser extent, small myelinate fibers were disproportionately involved. REPORT OF CASES Case 1.—A 45-year-old diabetic man was admitted for neurologic evaluation after a year of increasingly severe burning pain and deep aching in his feet. He had been fired from his factory job because of clum¬ siness in handling small metal parts. He described impotence for nine months, constipation, occasional dizziness on stand¬ ing, and mild dependent ankle edema. He did not sweat below the knees. An insulindependent diabetic for 12 years, he had only mild hypoglycémie reactions, and degree of control was considered adequate. He ate well, did not use alcohol, and had no known exposure to neurotoxins. There was a family history of diabetes, but not of neurologic disorder. He seemed in good health. His legs were scarred from previous injuries, and he had necrobiosis lipoidica diabeticorum. Blood pressure was 122/80 mm Hg supine and
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Table 1.—Nerve Conduction
Velocity Studies Sensory
Motor Median
CV,*
m/sec Case 1 59 Case 2 48 Case 3 47 Control >49
Ulnar
CV,
m/sec 51 45 45 >47
Peroneal
CV, m/sec 40 32
NO§ >40
Median
Sural
Ulnar
DL,t
SAP,*
DL,
SAP,
DL,
msec
;¡v
msec
¡,y
msec
4.8
4.6 5.8 9
4.0 3.9 NO 7
SAP,
5.3 NO 10
Conduction velocity. Distal latency. t Sensory action potential amplitude. § No potential obtained. *
t
Table
of Sural Nerve Fibers and Cell Nuclei Expressed Number per Square Millimeter of Nerve Cross Section
2.—Density
Fibers and Nuclei
Myelinated fibers* Myelinated fibersf Unmyelinated fibers* Schwann cell nuclei associated with myelinated fibersf Schwann cell nuclei associated with unmyelinated fibersf Other Schwann cell nucleif Total Schwann cell nucleif Fibroblast nucleif
Case 1 2,890
Case 2
Control
32,200
3,130 3,155 19,700
9,630 9,500 33,400
165
330
675
2^640
980
1,675
2,405
1,445 2,755
3,165
150
265
320
1,180 1,060
From thick sections, final magnification X 1000 f From ultrathin sections, final magnification 1,600 From ultrathin sections, final magnification X 20,000 *
as
815
122/80 mm Hg standing, without increase of pulse rate. Pedal pulses were full. Fundi were normal. There was a right Horner syndrome. Strength was normal, and all stretch reflexes were present. Hypesthesia to pinprick and light touch was found in a patchy distribution, but generally it con¬ formed to a glove and stocking distribu¬ tion. Histamine injection (0.1ml of 1:10,000, intracutaneously) produced a normal wheal, but a markedly reduced flare over the dorsum of the foot. Response at the anterior thigh was normal. Pallesthesia and proprioception were intact. Fasting hyperglycemia was verified. Spinal fluid protein was 66 mg/100 ml. Electrodiagnostic studies showed normal motor conduction velocities, but low-ampli¬ tude sensory action potentials with pro¬ longed latencies (Table 1). The following test results were normal: complete blood cell (CBC) count, erythrocyte sedimenta¬ tion rate (ESR), urinalysis (UA), blood urea nitrogen (BUN), serum creatinine (Cr), cholesterol, serum glutamic oxaloacetic transaminase (SGOT), serum protein elec¬ trophoresis, serum thyroxine (T,), antinu¬ clear factor (ANF), VDRL, and chest roentgenogram. Sural nerve biopsy was
performed. Eighteen
pain had then were unchanged, except that vibration and posi¬ tion senses were impaired in the feet. Case 2.-A 44-year-old diabetic man was disabled by continuous deep limb pains for two years. They reminded him of a ciga¬ rette burn. In addition, he had shooting pains and intermittent cramps in both calves and feet. These occurred either spon¬ taneously or after light tactile stimulation. Finally, he had aerai "pins and needles" paresthesias and was aware of numbness in his fingertips and below the knees, where he had suffered painless injuries. He had had no erections for at least one year and reported constipation and urinary hesitancy. He did not sweat below the waist. Nine years earlier when diabetes mellitus was first diagnosed, he had a sudden, unexplained attack of blindness without residual visual defect. Symptoms of muscle weakness in the proximal part of the leg and tender thighs led to the diag¬ nosis of femoral neuropathy at age 42. This, too, cleared completely. The degree of diabetic control with hypoglycémie agents administered orally was "fair"; he never decreased.
Fig
months later his
Physical findings
1.—Cross section from sural
nerves.
Left, Control. Upper right, Case 1. Lower
Case 2. Myelinated fiber density is reduced in diabetic material (phase con¬ trast optics, original magnification
right,
X675).
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had ketoacidosis. As a business represent¬ ative for an industrial firm, he had had slight exposure to metal salts and organic solvents but not for eight years. He rarely used alcohol. His family was free of diabetes or known neurologic disease. The general physical examination was unremarkable, and he looked well. Blood pressure was 125/80 mm Hg supine and standing. Muscle strength was excellent. All tendon jerks were present, but the left Achilles reflex was obtained only after reinforcement. Pinprick, temperature, and light touch thresholds were elevated below the wrist and knee. He would not tolerate light stroking of his toes, as this produced pain. Pallesthesia was mildly impaired at the ankle. Position sense was normal. Histamine given intradermally produced a normal wheal, but reduced flare, on the dorsum of his foot. Fasting blood glucose level was repeat¬ edly elevated. The thickness of the base¬ ment membrane of the capillaries in the quadriceps muscle was 3,011 ± 276 Ang-
Fig 3—Left, Relationship and diabetic sural diabetic material.
nerves.
2500 Case I
2000-
1500-
2,890 MF/
500 Control
9,630 MF/sq.
mm.
10
16
Case 2 500
3,130 MF/sq.
500-
8
10
T
12
16
14
Myelinated
2
12
10
6
4
mm.
~1
14
Fiber Diameter (ju)
Fig 2.—Myelinated fiber densities in control and diabetic sural nerves. Loss of smaller disproportionately greater than loss of larger ones in diabetic patients. Proportion of small myelinated fibers (^5µ in diameter) in control was 59.1%; case 1,
fibers is
44.5%; and
case
2, 50.7%.
between length and diameter of internodes from teased fibers. Calculated regression lines similar for control Right, Internodes on single fiber plotted against fiber's widest diameter. Variation may be slightly greater in
CONTROL
¡E
0.8
O
o
UJ -
i í
0.6
UJ
Q
O
O
0
3
4
m
0.4
ft.
.1 5
6
7
8
9
10
11
12
3456789
INTERNODE DIAMETER («)
10
WIDEST INTERNODE DIAMETER
1.2
(li)
1.2 CASE 2
1.0
CASE 2
1.0 E
0.8-
£
0.6
O Z
14
12
1000-
CONTROL
O
sq.mm.
5
0.6
Q
0.4
O 0.2-
0
3
4
5
6
7
8
9
10
INTERNODE DIAMETER |u)
11
12
13
0
4^^-1-1-1-1-,-1-1-1-1-1 0
3
4
5
6
7
8
9
10
WIDEST INTERNODE DIAMETER (u|
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11
12
13
Fig 4.—Electron photomicrographs of control sural nerve. Left, Well-preserved membranes including sheaths of myelinated fibers (M). Right, Most unmyelinated fibers (U) invested by cytoplasm of only one Schwann cell, with well-defined mesaxon. Occasional unmyelinated fibers (star) surrounded by processes from two or more Schwann cells (x 14,600). (normal, 1,325 A; diabetic, above 1,600 A). Motor conduction velocities were
Stroms
mildly slowed, sensory action potential amplitudes were reduced, and terminal latencies were prolonged (Table 1). Labora¬ tory tests with normal findings included CBC count, ESR, UA, BUN, Cr, cholesterol, SGOT, serum protein electrophoresis, T„ B,_, VDRL, chest roentgenogram, and barium meal and enema. Sural nerve
serum
biopsy was performed. Phenytoin therapy reduced the extent and frequency of muscle cramps and shooting leg pains. When the patient was seen three months after biopsy, his neuro¬ logic status had not changed. Case 3.—A 42-year-old employed diabetic machinist complained of "wooden" feet and impotence. For ten years he had been aware of insidiously progressive numbness that ascended to his midthighs and fingers. He had numerous unrecognized injuries. Dysesthesias, foot cramps, and stabbing leg pains were superimposed on a constant
aching, tearing sensation in his limbs. In the past, light touch had triggered parox¬ ysms of foot pain. He had not had a penile erection in more than five years; a trial with a prosthetic penile implant was unsuc¬ cessful. He had dependent ankle edema at the end of the workday. A known diabetic and insulin-dependent for 14 years, he had always been in good control. He was treated for retinopathy with photocoagulation and had proteinuria for at least one year. There was no exposure to neurotoxins, including alcohol. Family history was negative for diabetes and neurologic disor¬ ders. He appeared healthy, but with a depressed affect. Blood pressure was 160/90 mm Hg supine and standing. He had bilateral retinal microaneurisms, hyperpigmented scars on his lower legs, and mild ankle edema. Dorsalis pedis pulses were strong. Toe dorsiflexion was modera¬ tely weak, and the extensor digitorium brevis muscles were not palpable. Ankle
jerks were present, but diminished. He was hypesthetic to pinprick, light touch, and temperature change below the elbow and midthigh. Pallesthesia was moderately reduced in the lower extremities and mildly depressed in the upper ones. Recog¬ nition of position change was impaired in the toes. Histamine flare was decreased on the dorsum of the foot, but not on the anterior thigh. Studies with abnormal results included elevated blood glucose level, ESR of 42 mm/hr, BUN level of 44 mg/100 ml, Cr level of 2.2 mg/100 ml, creatinine clearance of 37 ml/min, and a urine protein content of 10.5 gm/24 hr. Bladder capacity was enlarged to 1,000 ml, with weak detrusor function. Motor nerve conduction velocities were somewhat slowed (Table 1). The median nerve sensory action potential was of reduced amplitude and prolonged laten¬ cy. The following studies yielded normal results: CBC count, cholesterol, SGOT, serum protein electrophoresis, antinuclear
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Fig 5.—Electron photomicrograph from sural nerve of patient 1, a diabetic with painful neuropathy. Left, Normal, unmyelinated fiber (U) ultrastructure. Right, Small, unmyelinated fibers cluster in Schwann cell cytoplasm (arrows) (original magnification 14,600).
antibody,
serum B,2, VDRL, and chest roentgenogram. Sural nerve biopsy was
performed. Phenytoin therapy did not alter the pain, and at follow-up six months later, the neurologic symptoms were unchanged. Control.—A 48-year-old man died sud¬ denly of cardiac arrhythmia. At the time, he was undergoing investigations for congestive heart failure. He did not abuse alcohol. Neurologic history and examina¬ not
tion had been negative. Laboratory studies with normal results included: CBC count, UA, BUN, Cr, blood glucose, and chest
roentgenogram. Autopsy findings were atherosclerosis, left ventricular aneurysm, and pulmonary scarring from old tubercu¬ losis.
METHODS Sural nerve biopsy was performed at the ankle under local anesthesia after the risks of the procedure were fully explained to the patient-volunteers. A portion of each
nerve was fixed in 10% formaldehyde, embedded in paraffin, and stained. An¬ other segment was fixed in 3.6% glutaral¬ dehyde in phosphate buffer and postfixed in buffered 2% osmium tetroxide. After dehydration, it was embedded in epoxy resin. Longitudinal and full cross sections, 1.5fi-thick, were mounted on glass slides. Endoneurial area was calculated from lowpower photomicrographs of each fascicle. Representative fields covering about 60% of the endoneurial area were photographed and printed at a final magnification of x 1,000. Myelinated fiber diameters were determined from these photographs. Sin¬ gle osmicated myelinated fibers were teased from their fascicles. Internodal lengths and diameters were measured along approximately 40 such fibers from each biopsy specimen. Ultrathin sections were stained with uranyl acetate and lead citrate. Unmyeli¬ nated fibers were studied, measured, and counted from 200 electron photomicro-
graphs from each biopsy specimen, which were printed at a final magnification of 20,000. Each fascicle was represented according to its contribution to the total endoneurial was
area.
About 2.5% of that
area
sampled. The density of both Schwann
cell and fibroblast nuclei, and a second estimate of the myelinated fiber popula¬ tion, were calculated from low-power elec¬ tron micrographs of the same grids (Table
2). RESULTS
appeared normal surgical removal. Micro¬ scopically, the perineurium and ves¬ The sural
nerves
at the time of
sels
were
unremarkable, and there
amyloid deposition or inflam¬ matory infiltrate. Degenerating fi¬ bers were rarely seen in cross or longi¬ tudinal sections. Total myelinated fiber density was reduced to about one was no
third of the normal value in all fasci-
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best
in the
of myelinated fibers had not oc¬ curred. The ultrastructural membrane de¬ tail of both diabetic and autopsy material was well-preserved (Fig 4 and 5). In the control, swollen mito¬
phase microscopical preparations (Pig 1). Though both large and small myelinated fiber populations were affected, there was a relatively greater loss of smaller myelinated fibers (Fig 2). The lengths of all inter¬ nodes on a teased, single myelinated fiber were plotted against the fiber's widest diameter according to the method of Fullerton et al.-'1 By inspec¬ tion, the degree of variability was only slightly greater in the diabetic material (Fig 3, right), indicating that demyelination-remyelination was not prominent in the pathological process. Myelinated fibers undergoing active axonal degeneration were occasionally cíes; this
was
seen
contrast
chondria and loss of neurotubules were recognized as artifacts. In the diabetic nerves, we found no myelin sheath disorganization, neurofilament dissolution, or membranous organelle proliferation in the axons themselves. Occasional Schwann cells had in¬ creased amounts of endoplasmic retic¬ ulum and mitochondria, but this was inconsistent. Endoneurial capillaries were normal in appearance; we did not survey their basement membrane thickness. Myelinated fiber density, as determined in the electron micro¬ graphs, was the same as that found
seen, three of 49 fibers in case 1 and of 35 in case 2 vs one of 47 in the
one
control
When
nerve.
an
internode's
length was plotted against its own width, disregarding the rest of the fiber, and the regression line then calculated, the diabetic fibers were not different from the control (Fig 3,
using lower-magnification phase con¬ trast photomicrographs. Schwann cell nuclei associated with myelinated
fibers were reduced to a degree appro¬ priate for the amount of myelinated fiber loss (Table 2). Those Schwann cells not clearly associated with any
left). This observation suggests that
regeneration
of
significant
numbers
Fig 6.—Unmyelinated fiber densities in control and diabetic sural nerves. Type 1 axon-Schwann cell (Ax-ScC) configuration is axon surrounded by single Schwann cell. Type 2 is axon surrounded by two or more Schwann cell processes, juxtaposed against another axon, or partially uncovered. Number of large unmyelinated fibers
ScC
fiber type ("other Schwann cell nuclei" in Table 2) are considered to.be unmyelinated fiber-related. This group of Schwann nuclei, taken to¬ gether with those that are clearly associated with unmyelinated fibers, number approximately the same as in control nerve. Total numbers of Schwann cell nuclei were not in¬ creased, suggesting that Schwann cells had not proliferated in response to the pathological process. The densi¬ ty of fibroblast nuclei was similar to that of the control. The total number of unmyelinated fibers per unit area was normal in case 1 and mildly reduced in case 2 (Fig 6), when compared to either our control values or those of others.---3 Analysis of the unmyelinated fibers by size indicated a striking absence of the large fibers more than 1µ in diam¬ eter and an accompanying increase in the small ones (Fig 6). Only 4% of all unmyelinated fibers were larger than lju in the diabetic patients (control, 33%). Looking at the small end of the fiber spectrum, approximately 75% of
more than 1µ reduced in diabetic tissue; small unmyelinated fibers increased in number. Frequency of type 2 configurations abnormally high in case 1 and 2, indicating regenerative sprout¬
ing.
16,000
Configuration
—
0- Type D- Type
14,000
1 2
Case 1
Case 2
32,200 UF/sq.
19,700 UF/sq.
mi
12,000 10,000
10,000
8000
8000
8000
6000
6000
6000
4000-
4000
2000
2000-
Control
33,400 UF/sq. a>
"o
0.2
0.6
1.0
1.4
1.8
2.2
0.2
Unmyelinated
0.6
1.0
1.4
1.8
2.2
Fiber Diameter (a»)
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0.2
0.6
1.0
1.4
1.8
the total unmyelinated fibers were smaller than 0.6µ in the diabetics (con¬ trol, 25%). In addition to these quanti¬ tative changes, there were qualitative alterations in the axon-Schwann cell relationships in the diabetic material (Fig 6). Whereas 88% of control unmyelinated fibers were fully in¬ vested by the cytoplasm of a single Schwann cell, usually with a readily identifiable mesaxon (Fig 4), only 52% and 65% of the diabetic unmyelinated fibers were in this simple "type 1" relationship (Fig 5). The remainder of the diabetic unmyelinated fibers were in a "type 2" axon-Schwann cell rela¬ tionship, that is, either incompletely surrounded by a Schwann cell or, less
commonly, wrapped by
two
or more
separate tongues of Schwann cell cytoplasm. While it was unusual to find more than three unmyelinated fibers associated with a single Schwann cell in the control, three or more unmyelinated fibers per Schwann cell were frequently ob¬ served in the diabetic material. Fur¬ ther, only the diabetic nerves con¬ tained clusters of small, naked unmye¬ linated fibers that abutted one anoth¬ er, forming complexes that were surrounded by Schwann cell cyto¬
plasm (Fig 5).
COMMENT
One might predict on clinical grounds that three diabetic patients with painful neuropathy would show disproportionate pathological involve¬ ment of unmyelinated and small myelinated fibers. Quantitative fiber analysis of two biopsied sural nerves bore out this impression. The diagnosis of diabetic neurop¬ athy was made by the temporal asso¬ ciation with diabetes mellitus and by
the absence of another basis for neuropathy after the history, physical examination, and laboratory studies, including biopsy, were completed. Pa¬ tient 3, in whom biopsy was not performed, had renal impairment to a degree not thought to affect periph¬ eral nerve.24 Functions attributable to large my¬ elinated fibers were little altered in our patients. Muscle stretch reflexes, which depend on the integrity of the largest myelinated fibers, were al-
ways
present. Vibration and position not lost. The
electrophysiologic studies, which similarly test the largest myelinated fiber popula¬ tion, indicated only mild conduction velocity slowing, consistent with the paucity of segmental demyelination in teased fiber preparations. Reduc¬ tion of amplitude of sensory action potentials paralleled roughly the loss of largest myelinated fibers in the biopsy specimens. Unmyelinated fiber dysfunction In cutaneous was more striking. senses were
nerves, these C fibers carry out both
afferent and autonomie efferent func¬ tions.27' Clinically, both functions were affected in our patients. Elevated pain, temperature, and touch thresh¬ olds suggested involvement of affer¬ ent C fibers, perhaps along with small myelinated A delta fibers, while impo¬ tence and decreased peripheral sweat¬ ing pointed to involvement of sympa¬ thetic fibers. Anatomically, there was a near absence of larger unmyelinated fibers, and small myelinated fibers were reduced to approximately one quarter of control. Concurrent unmye¬ linated fiber loss and altered proto¬ pathic sensation has been observed in several other neuropathies2"28 with an absent C wave when in vitro nerve conduction velocity was determined. Very small unmyelinated fibers, less than 0.6µ in diameter, were strik¬ ingly increased in number, both abso¬ lutely and relative to the overall fiber spectrum. Their relationships with Schwann cells were often complex, and frequently several tiny fibers shared the same Schwann cell invagi¬ nation. This pattern of multiple, small, unsegregated fibers is charac¬ teristic of developing,2" regenerat¬ ing,30 or aging31 nerve and represents the process of sprouting. Ochoa32 first described the electron microscopical features of nerve sprouting in a human polyneuropathy, that of isoniazid intoxication. Our study confirms ultrastructurally that sprouting also can occur in the neuropathy of
diabetes mellitus.
Pathologically, segmental demyeli¬
nation is prominent in some cases of diabetic neuropathy.13141"33 Because myelin sheath is specialized Schwann cell surface membrane, segmental loss
of myelin is indicative of a Schwann cell disorder, either primary or sec¬ ondary. Metabolic alterations have, in fact, been imputed to Schwann cells in experimental diabetic neuropathy.8·34·35 Recently, however, research¬ ers have questioned the pathological importance of segmental demyelina¬ tion in human diabetic neuropathy and instead reemphasized axonal in¬ volvement.15 In our patients the major neuropathologic feature was loss of axons. This involved primarily large unmyelinated fibers, but also included small myelinated and, to a lesser extent, large myelinated fibers. Near absence of active degeneration of axons or myelin attests to the chronicity of the process. Teased nerve fiber preparations showed little in the way of demyelination and remyelinations. On the basis of these observa¬ tions, we favor axonal degeneration as the most likely cause for neuropathy in these patients. The fact that nerve conduction velocities were only mini¬ mally slowed supports this formula¬ tion. In most polyneuropathies with axonal degeneration the largest mye¬ linated fibers are affected earliest and most severely.23-31-36-37 It is of interest that unmyelinated and small myelinated fibers seem to be rela¬ tively more vulnerable in our pa¬ tients. Unmyelinated fiber abnormalities have been described qualitatively in diabetic neuropathy.1015-16·33-3* How¬ ever, without quantitative analysis, the patterns of degeneration (or regeneration) could not be assessed. We found no qualitative changes in axons or Schwann cells, aside from complex axon-Schwann cell relation¬ ships. Quantitatively, our patients had a virtual absence of large unmyeli¬ nated fibers. In addition, we consider the evidence for sprouting without early myelination to be a sign of unmyelinated fiber alteration. Our qualitative findings are at variance with some of those described by others,1516·33-35 but may only represent differences in patient selection. The pathophysiology of pain is always of interest, and patients such as ours are natural subjects in its study. There is a poor correlation between myelinated fiber loss and the
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of pain.38 It is tempting to relate pain of peripheral nerve origin to alterations of the unmyelinated fiber population. A possibility that intrigues us is that regenerating sprouts may themselves produce pain. This is consonant with the clinical observations that advancing pain and paresthesias accompany regrowth of a transected nerve and recovery from
occurence
some
polyneuropathies. Further
port for this mechanism
comes
sup¬
from
the recent demonstration that in rats regenerating nerve fibers fire sponta¬ neously, and after stimulation they
discharge abnormally.40 The painful small-fiber neuropathy
of diabetes is a characteristic syn¬ drome at one pole of the diabetic distal sensory-motor neuropathy spec-
Clinical features of the disorder consistent with the observed path¬ ological alterations. trum. are
Austin Sumner, MD, assisted with the electro-
physiologic studies. Marvin Siperstein, MD, PhD, measured the thickness of the basement mem¬ brane of the capillaries in the quadriceps muscle of patient 2. Basil Rapoport, MB, referred patient 3 for evaluation.
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