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Direct immunofluorescence findings in peripheral nerve from patients with diabetic neuropathy.

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Direct Immunofluorescence Findings
in Peripheral Nerve from Patients
with Diabetic Neuropathy
Anna R. Graham, MD," and Peter C . Johnson, MDt
Direct immunofluorescence examination was performed on peripheral nerve from 16 patients with diabetes mellitus
and 53 additional patients with peripheral neuropathy of diverse cause. Six nerves from patients with diabetes mellitus
yielded positive findings: 4 had granular and lamellar deposition of IgM within the perineurium (of which 2 also had
fibrinogen, IgA, C3, and albumin and 1 also had IgG); 1 had IgM, C3, and C4, and perineurial fibrinogen; the sixth
contained linear perineurial C3 and fibrinogen. These 6 nerves contained axonal degeneration (3),axonal degeneration
with chronic demyelination (l), microvasculitis with wallerian degeneration (l), and no pathological change (1).
Sixteen of 53 nerves from nondiabetics yielded positive findings with immunofluorescence, possibly as a result of
vascular leakage or as a manifestation of impaired removal of plasma proteins. T h e deposition of immunoreactants, as
well as other plasma proteins, in peripheral nerve from patients with diabetes mellitus probably represents a "trapping" phenomenon reflecting altered basement membrane permeability. Alternatively, the changes could reflect a
defect in the blood-nerve barrier in diabetic microangiopathy.
Graham AR, Johnson PC: Direct immunoffuorescence findings in peripheral nerve from patients with
diabetic neuropathy Ann Neurol 17:450-454, 1985
Immunoglobulin and complement deposition in peripheral nerve has been reported chiefly in cases of
neuropathy associated with paraproteinemias [ 1, 3, 7,
10, 13, 15, 16, 181. These findings of immunoreactants
have been variously ascribed to nonspecific plasma
protein leakage [20) and to antibody-complement
components directed against peripheral nerve constituents I1 11. Immunoreactants have also been reported
in peripheral nerve arterioles and perineurium in conjunction with rheumatoid arthritis 12 11, within intraneural blood vesseis, and on the Schwann cell plasmalemma of nerve fibers in patients with chronic
relapsing polyneuropathy [4], and in a variety of hereditary neurological disorders, collagen vascular diseases, and toxic neuropathies 1221. We report the
findings of immunoreactants in peripheral nerve from
patients with diabetic peripheral neuropathy, and present a review of our experiences with a variety of other
disorders.
Clinical Material and Methods
Patients
Peripheral nerves were derived from biopsy and postmortem
material obtained in 1980 through 1983 from the University
of Arizona Health Sciences Center or received there in con-
Irom the *Department of Pathology, University of Arizona Health
'Sciences Center, Tucson, A 2 85724 arid tSt: Joseph's Hospital,
Barrow Neurological Institute, 350 W Thomas Rd, Phoenix, A 2
85013.
450
sultation. Seventy-one specimens from 69 patients were
available for direct immunoff uorcscence study. All had tissue
for light microscopy or plastic-embedded 1 p sectioning.
Biopsy specimens were obtained from the sural nerve in 67
of the 69 patients. Two of the specimens were identified only
as originating from the leg. Two patients had more than one
nerve subjected to biopsy. Electron microscopy, nerve teasing, or both were also performed in selected cases.
Patients, 46 males and 23 females, ranged in age from 16
to 83 years. Sixteen had diabetes mellitus (DM) (3 early or
mild, 2 diet controlled, the remainder insulin dependent).
Four had clinical evidence of hereditary motor and sensory
neuropathy type I (HMSN type I; Charcot-Marie-Tooth disease). Eight patients had vasculitis ( 1 case consistent with the
pattern of polyarteritis nodosa, 1 case associated with cryoglobulinemia, and 6 cases not otherwise characterized), and 1
patient had circulating immune complexes without vasculitis.
Two patients had alcoholic neuropathy, and a third alcoholic
patient developed a Guillain-BarrC-like syndrome following
a toxic interaction of alcohol and disulfiram (previously reported l17)). Two patients had neuropathy complicating
rheumatoid arthritis. Two patients had multiple myeloma
and another a nonmyeloma monoclonal gammopathy. In addition, there were patients with a clinical peripheral nerve
disorder associated with myotonic dystrophy, porphyria,
metastatic tumor involving the sacral plexus, heat stroke, and
prior encephalitis. Twenty-eight patients had no identified
cause for their peripheral neuropathy.
Recelved Aug 9. 1984. and in revlsed form Oct 26. Accepted for
publication 0;t 27, 1984.
Address reprint requests to Dr Graham.
Light and Electron Microscopy
Tissue for light microscopy was fixed in Karnovsky’s solution
and processed routinely. Sections were cut at 5 p and stained
with H&E. Additional tissue was processed in Epon for
semithin and thin sectioning, and in glycerin for nerve teasing, using methods previously described [27.
~mmunojuorescence
Tissue for immunofluorescence was immersed in OCT
mounting medium (Lab-Tek Products, Naperville, IN),
snap-frozen in isopentane chilled in liquid nitrogen, and cut
at 6 to 8 p in a cryostat. The sections were fixed in acetone
for 5 minutes, rinsed in phosphate-buffered saline for 5 minutes, and treated with antihuman IgA, IgG, IgM, C3, C4,
fibrinogen, and albumin (Cappell Laboratories, Cochranville,
PA) for 30 minutes. They were then rinsed three times in
phosphate-buffered saline over a total time period of 6 minutes and covered with coverslips with Gelvatol as a mounting
medium (Monsanto, Indian Orchard, MA). They were examined using a Zeiss epi-illumination ultraviolet microscope
employing BP 450 to 490 and KP 560 filters.
Results
Light and Electron Microscopy and Teased
Newe Fiber Preparations
There were several major patterns of abnormality revealed by these morphological techniques. These are
summarized in Table 1, together with the clinical
profiles of the patients. The first pattern was axonal
degenerative neuropathy, which was seen in specimens
from 6 diabetic and 15 nondiabetic patients. The second pattern, that of demyelinating neuropathy, was
observed in nerves of 2 diabetic and 8 nondiabetic
patients. Abnormally thin myelin andor hypertrophic
onion bulb formation was seen in nerves of the 4 patients with clinical HMSN type I. Four patients, l a
diabetic, had vasculitis with wallerian degeneration; 5
additional patients had vasculitis without evidence of
wallerian degeneration. S i x biopsy specimens showed
nonspecific myelinated fiber loss (1 in conjunction
with the vasculitis consistent with polyarteritis nodosa
and 5 of unknown cause). Four patients’ nerves
showed wallerian degeneration. Fifteen nerve biopsy
specimens, 7 from diabetics and 8 from nondiabetics,
demonstrated no pathological change by light microsCOPY.
Immunojuorescence
Twenty-two biopsies revealed the presence of immunoglobulin andor complement in varying patterns,
6 in patients with DM and 16 in nondiabetic patients
(Table 2). The DM patterns included granular and
lamellar perineurial (Fig IA), axonal (Fig 2 ) , and linear
perineurial (Fig 3). The 1 p section correlate to one of
these immunofluorescence patterns was the finding of
focal, and likely ischemically mediated, destruction of
the perineurial barrier, resulting in perineurial and endoneurial edema with varying degrees of axonal at-
Table I . Findings b = ~Light and Electron Microscopy
and Teased Nerve Fiber Preparations
~
~~
~
~
~~
Finding
~~
No. of Patients
DIABETICS
Axonal degenerative neuropathy
Demyelinating neuropathy
Vasculitis with wallerian degeneration
No pathological change evident on light
microscopy
6
2
1
7
NONDIABETICS
Axonal degenerative neuropathy
Metastatic carcinoma
Circulating immune complex disease
without vasculitis
Idiopathic
Demyelinating neuropathy
Postencephalitis
Monoclonal gammopathy
(nonmyeloma)
Idiopathic
Chronic demyelination with or without
hypertrophy (clinical hereditary
motor and sensory neuropathy
type 1)
Vasculitis with wallerian degeneration
Vasculitis without wallerian degeneration
Rheumatoid arthritis
Polyarteritis nodosa
Nonspecific vasculitis
Walletian degeneration
Alcohol related
Myeloma
Post-heat stroke
Nonspecific myelinated fiber loss
No pathological change evident on light
microscopy
15
1
1
13
8
1
1
6
4
3
5
2
1
2
4
2
1
1
6
8
rophy and myelinated fiber loss (Fig lB, C). Three of
the nerves from patients with DM that yielded positive
findings on immunofluorescence had axonal degeneration evident on light microscopy, 1 had a microvasculitis with wallerian degeneration, and another had
no pathological changes evident. The sixth D M nerve
biopsy demonstrated axond degeneration and chronic
dernyelination.
The nerves from 4 patients with clinically defined
HMSN type I had granular and lamellar perineurial
deposition of immunoreactants. Two of these biopsies
revealed abnormally thin myelin and hypertrophic
fibers, and 2 specimens had decreased numbers of myelinated fibers and hypertrophic onion bulb formation.,
The nerve from 1 of the 2 patients with IgG myeloma had severe wallerian degeneration and perineu-
Graham and Johnson: Immunofluorescence and Neuropathy 45 1
Table 2. Findings by Immunofuorescence
Finding
No. of Patients
DIABETICS
~~
~~
Perineurial granular and lamellar
deposition
IgA
IgG
IgM
c3
Fibrinogen
Albumin
Axonal deposition with IgM, C 3 , C4,
and perineurial fibrinogen
Perineurial linear deposition with C 3 and
fibrinogen
4
2
1
4
2
2
2
1
1
~~
NONDIABETICS
Perineurial granular and lamellar
deposition
Hereditary motor and sensory
neuropathy type I
IgA
IgG
IgM
Myeloma-IgG and C 3
Postencephalitis-IgM and fibrinogen
Metastatic carcinoma-IgM and
fibrinogen"
Idiopathic
Ig-4
IgG
IgM
11
4
2
1
3
1
1
1
4
2
1
4
3
c3
Fibrinogen
Albumin
Endoneurial granular deposition, alcohol
related
C3 and C4
IgA, IgG, IgM, C3, C4, and
fibrinogen
Myelin deposition
Myeloma-IgG
Monoclonal gammopathy-IgM
Vascular deposition: rheumatoid
arthritis-IgM, C3, C4, and
fibrinogen
2
2
2
1
1
2
1
1
1
"Granular C3 and C4 deposition along rnyelin was also present.
452 Annals of Neurology Vol 17 N o 5 May 1985
C
Fig 1. (A) Granular and lamellar perineurial pattern. (FITCconjugated goat antihuman IgM; X 350 before 33% reduction.)
(B) Edema of the perineurium and right half of the fascicle,
with loss of myelinated fibers in the edematous area. (Paraphenylenediamine; x 160 before 30% reduction.) (C) Higher
mugntjication showing detail of upper right of B , showing
marked perineurial and endoneurial edema, and a granulation
tissue response with neovascularization.Early perineurial edema
involving only the innermost lamellae of the fascicle is evident on
the right, demonstrating that this focal lesion is probably ischemically mediated. (Paragon; x 400 before 30% reduction.)
F i g 2. Axonal pattern. (FITC-conjugatedgoat antihuman
IgM; x 550 before 35% reduction.)
Fig 3. Linear perineurial pattern. (FITC-conjugatedgoat antihuman C3; x 350 before 35% reduction.)
rial immunofluorescence positivity. The nerve from
the other patient (whose myeloma was IgG kappa
type) demonstrated striking localization of IgG and
kappa light chains selectively to damaged myelin in a
biopsy specimen obtained from his clinically affected
side; there was no immunoreactant deposition in nerve
obtained from his contralateral, clinically unaffected
side. The biopsy specimen of the patient with an IgM
kappa monoclonal gammopathy, without hematological confirmation of myeloma, showed 3 + intensity
IgM deposition along myelin and had a demyelinating
lesion evident on light microscopy.
The remaining 9 patients with positive findings on
immunofluorescence showed a variety of patterns. The
dominant pattern was a perineurial granular and/or
lamellar distribution, seen in six specimens, with endoneurial positivity in four. Two of the nerves had
decreased numbers of myelinated fibers (both clinically
idiopathic neuropathies), two had segmental demyelination (one clinically idiopathic and one postencephalitic), and two had an axonal degeneration neuropathy
(one clinically idiopathic and one with metastatic carcinoma involving the sacral plexus, the latter being the
biopsy specimen with myelin positivity as well). The
second immunofluorescence pattern was a granular endoneurial localization, seen in 1 nerve biopsy specimen
demonstrating wallerian degeneration by light microscopy (the clinical setting being a Guillain-BarrBlike
syndrome following disulfiram-alcohol interaction toxicity [17}) and in a nerve specimen from a patient
having a history of alcoholism and severe ascending
sensorimotor loss but with no abnormality seen by
light microscopy. The ninth patient’s nerve had immunoreactants and fibrinogen within vessel walls, in
conjunction with histological findings of an acute vasculitis, perivasculitis, and severe active wallerian degeneration. Clinically, the patient had a rheumatoid
vasculitis.
Discussion
Our results support the well-established finding of deposition of immunoglobulins in nerves of patients with
paraproteinemias [I, 3, 7, 10, 13, 15, 16, IS]. We also
identified vascular immunoglobulins and complement
in a nerve biopsy specimen from a patient with
rheumatoid vasculitis, as previously described by Van
Lis and Jennekens [21}, and within perineurium and
endoneurium of specimens with chronic axonal degeneration and hypertrophic neuropathy, similar to the
findings of Van Lis and Jennekens [22}. The uniform
finding of a variety of immunoreactants in the nerves
of these patients with HMSN type I remains unexplained. The diversity of plasma proteins found
strongly suggests increased vascular permeability, a
possibility also entertained by Van Lis and Jennekens
[22]. Such increased vascular permeability may be expected in very active neuropathies, such as that found
in the case of disulfiram-alcohol overdose, but HMSN
type I is a very slowly progressive neuropathy. It is
possible that HMSN type I has increased vascular permeability as a feature of its pathogenesis. Alternately,
some patients with HMSN may have a superimposed chronic inflammatory-demyelinating polyradiculopathy, such as that found in the prednisoneresponsive group of patients described by Dyck and
colleagues [ 5 1. In such patients immunoreactants
within peripheral nerve may represent part of an active
inflammatory process potentially amenable to steroid
therapy. We identified albumin less frequently in these
latter biopsy specimens than has been described [22},a
finding possibly related to poorer fixation of lowmolecular-weight proteins [6].
We observed immunoreactants in 6 of 16 nerves
from patients with diabetic neuropathy. The distribution was principally perineurial ( 5 of the 6 cases), displaying granular andor lamellar to linear patterns, with
IgM the most frequent finding. In I patient the pattern
Graham and Johnson: Imrnunofluorescence and Neuropathy
453
was axonal. The deposition of immunoglobulins and
complement in diabetics’ nerves probably represents a
“trapping” phenomenon reflecting altered basement
membrane permeability, and, in the perineurium,
basement membrane thickening (as noted by Johnson
and colleagues 191) similar to that observed in glomerular and renal tubular basement membrane 1141,
rather than a primary immunologically mediated abnormality. Alternately, abnormal amounts of immunoreactants may gain access to the perineurium by
means of a damaged blood-nerve barrier in diabetic
microangiopathy [9]. The apparent lack of concordance between histological and immunofluorescence
patterns, and the fact that only some of the nerves
from diabetics were abnormal, may reflect the focal
nature of lesions in diabetic neuropathy noted by Johnson {S] and Sugimura and Dyck [19J A comparable
disparity between morphological changes and abnormal immunoglobulin and complement localization is
observed in early glomerular mesangial changes in the
diabetic mouse, as reported by Lee and Graham 112).
Presented in part at the Spring Meeting of the International
Academy of Pathology, Feb 28-Mar 4, 1983.
Supported in part by Grant NS-15204 from the National Institutes
of Health.
The authors thank Dr Jack M. Layton for his continued support and
leadership, Ms Judy M. Guillot and Ms Susan Doll for their technical
assistance, and Ms Nancy Suttle for her secretarial support.
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