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Animal model of axonal Guillain-Barr syndrome induced by sensitization with GM1 ganglioside.

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Animal Model of Axonal Guillain-Barré
Syndrome Induced by Sensitization with
GM1 Ganglioside
Nobuhiro Yuki, MD, PhD,1 Mitsunori Yamada, MD, PhD,2 Michiaki Koga, MD, PhD,1
Masaaki Odaka, MD, PhD,1 Keiichiro Susuki, MD,1 Yumi Tagawa, MD, PhD,1 Shuichi Ueda, MD, PhD,3
Takeshi Kasama,4 Akio Ohnishi, MD, PhD,5 Shintaro Hayashi, MD, PhD,2 Hitoshi Takahashi, MD, PhD,2
Mikiko Kamijo, MD, PhD,6 and Koichi Hirata, MD, PhD1
Some humans develop the axonal form of Guillain-Barré syndrome after receiving bovine brain ganglioside. On sensitization with the ganglioside mixture, all of a group of rabbits injected developed high anti-GM1 IgG antibody titers,
flaccid limb weakness of acute onset, and a monophasic illness course. Pathological findings for the peripheral nerves
showed predominant Wallerian-like degeneration, with neither lymphocytic infiltration nor demyelination. IgG was
deposited on the axons of the anterior roots, and GM1 was proved to be present on the axons of peripheral nerves.
Sensitization with purified GM1 also induced axonal neuropathy, indicating that GM1 was the immunogen in the
mixture. A model of human axonal Guillain-Barré syndrome has been established that uses inoculation with a bovine
brain ganglioside mixture or isolated GM1. This model may help to clarify the molecular pathogenesis of the syndrome
and to develop new treatments for it.
Ann Neurol 2001;49:712–720
The presence of axonal Guillain-Barré syndrome (GBS)
has been established.1– 4 Cumulative evidence supports
the speculation that gangliosides, highly expressed molecules in nerve tissues, are the target antigens of the
autoantibodies in axonal GBS. First, patients frequently have IgG antibodies to several gangliosides
during the acute phase of axonal GBS.5–7 Second,
some patients develop axonal GBS subsequent to
Campylobacter jejuni enteritis, and the C. jejuni strains
isolated from these patients have lipopolysaccharides
bearing ganglioside-like structure.8,9 Third, gangliosides extracted from bovine brain have been used to
treat various neurological disorders, and reports of patients developing axonal GBS after ganglioside administration have been recorded.10,11
There are only two reports describing neurological
dysfunction in GM1-immunized animals. In the first
study, GM1-immunized rabbits developed a spastic paralysis, whereas GD1a-immunized rabbits had a flaccid
paralysis.12 Histologically, phagocytic cells containing
myelin debris could be observed. In the other study,
rabbits developed a subclinical neuropathy.13 There
was mild axonal degeneration in the sciatic nerve and
IgM deposits at the nodes of Ranvier. These findings
have failed confirmation in rodents. In contrast, the
sensitization of rabbits with GD1b did induce sensory
neuropathy associated with anti-GD1b antibody.14
These suggested that failure to induce neuropathy by
sensitization with gangliosides might depend on species
susceptibility and on the immunization procedure
used. In this study, therefore, rabbits were inoculated
with a bovine brain ganglioside (BBG) mixture according to the procedure of Kusunoki et al.14 The rabbits
developed acute motor axonal neuropathy associated
with anti-GM1 IgG antibody. Experimental motor axonal neuropathy also was induced by sensitization with
GM1.
From the Departments of 1Neurology, 3Histology and Neurobiology, Dokkyo University School of Medicine, Tochigi; 2Department
of Pathology, Brain Research Institute, Niigata University, Niigata;
4
Instrumental Analysis Research Center for Life Science, Tokyo
Medical and Dental University, Tokyo; 5Department of Neurology,
School of Medicine, University of Occupational and Environmental
Health, Fukuoka; and 6Department of Anatomy, Aichi Medical
University, Aichi, Japan.
Received Sep 19, 2000, and in revised form Jan 22, 2001. Accepted
for publication Jan 22, 2001.
712
© 2001 Wiley-Liss, Inc.
Materials and Methods
Immunization Procedure
Male Japanese white rabbits ( JW/CSK), weighing 2.0 –2.5
kg, were obtained from SLC (Hamamatsu Japan). A 2.5 mg
portion of a BBG mixture (GM1 21%, GD1a 40%, GD1b
16%, GT1b 19%; CronassialTM; Fidia, Padova, Italy) or 0.5
Published online 23 March 2001.
Address correspondence to Dr Yuki, Department of Neurology,
Dokkyo University School of Medicine, Kitakobayashi 880, Mibu,
Shimotsuga, Tochigi 321-0293, Japan.
E-mail: yuki@dokkyomed.ac.jp
mg of GM1 isolated from bovine brain (SygenTM; Fidia) was
dissolved in 0.5 ml of keyhole limpet hemocyanin (KLH; 2
mg/ml; Sigma, St. Louis, MO) in phosphate-buffered saline
(PBS). A 0.5 ml portion of Freund’s complete adjuvant
(FCA; Sigma) was added and the mixture emulsified. A 1 ml
sample of the emulsion of the BBG mixture or GM1 was
injected subcutaneously to the back and intraperitoneally at
3 week intervals until limb weakness developed or 6 months
had passed after the first inoculation. Control rabbits were
injected under the same protocol with the same inoculums
but without gangliosides. The rabbits were checked daily for
clinical signs and weighed twice per week. Weekly plasma
samples were taken by ear vein puncture. All the animal procedures conformed to the Dokkyo University School of
Medicine institutional guidelines.
Pathological Studies
Rabbits were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and the left sciatic nerves removed from some of them for teased fiber analysis. Nerve
specimens were immersed directly in 3% glutaraldehyde for
10 minutes and stained with 1% osmium tetroxide, then immersed in 60% glycerol, stored in 100% glycerol, and examined after teasing. At least 100 teased myelinated fibers were
prepared for each sciatic nerve. The teased fiber preparations
were blindly assessed by one of the authors (A.O.). Differences in frequency distribution of Wallerian-like degeneration were statistically examined with the Mann-Whitney U
test.
After nerve removal, the rabbits were perfused transcardially with 0.4 liters/kg of heparinized PBS (10 IU/ml) followed by 1.2 liters/kg of 3% glutaraldehyde-1% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. After this
perfusion, the right sciatic nerve, spinal cord with anterior
and posterior nerve roots, and brain were removed and immersed in the same fixative. Multiple blocks of tissue from
the brain and spinal cord were embedded in paraffin, after
which 4 ␮m thick sections were cut and stained with hematoxylin and eosin or Klüver-Barrera. The sciatic nerves and
lumbar segment of the spinal cord with anterior and posterior nerve roots were all postfixed with 1% osmium tetroxide, dehydrated in a graded ethanol series, then embedded in
Epon 812 (Polysciences, Warrington, PA). Sections 1 ␮m
thick were cut from each sample and stained with 0.5% toluidine blue and 1% safranine.
The image analysis program MacscopeTM (Mitani, Fukui,
Japan) was used for morphological examination of the sciatic
nerves. Digital photo images were obtained with a light microscope with a final magnification of ⫻400 and an attached
CCD camera interfaced with a computer. The computerassisted image analysis allowed for the determination of total
fascicular area and density of degenerative or surviving axons.
The number of these axons was counted manually from every three flame areas (single flame, 0.03/mm2) in the three
largest fascicles. Degenerative axon profiles and macrophages
were counted according to the criteria of Midroni and Bilbao.15 The profile of the surviving axons was characterized as
round myelin profiles that enclosed a transcular space. This
evaluation for the determination of surviving axons has been
done by other investigators.16 The morphometric analysis
was performed by one of the authors (M.Ka.), who was unaware of the clinical information. Data were analyzed by a
one-way analysis of variance (ANOVA) with appropriate
Bonferroni-corrected posthoc Student’s t-test comparisons.
Immunohistochemical Studies
The rabbits were deeply anesthetized, perfused transcardially
with 1 liter/kg of PBS, pH 7.4, and then with 1 liter/kg of
2% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4.
After perfusion, the sciatic nerves and spinal nerve roots were
removed. The samples were postfixed on ice for 30 minutes
with the same fixative, then cryoprotected in 20% sucrose
solution at 4°C overnight, after which they were frozen in
isopentane at –70°C and then sectioned by cryostat. Sections
20 ␮m thick were dried on gelatin-coated glass slides and
then incubated at 20°C for 2 hours with peroxidaseconjugated protein G (2 ␮g/ml; Sigma). Next, they were incubated for 5 minutes at 20°C in a solution of 50 mM Trishydrochloric acid buffer, pH 7.4, containing 0.02% 3,3⬘diaminobenzidine tetrahydrochloride (DAB) and 0.006%
hydrogen peroxide. The reaction was terminated by washing
the slides with PBS. Treatment with 0.05% osmium tetroxide in distilled water for 2 minutes was done to intensify the
DAB reaction products. As described elsewhere,17 the sciatic
nerves and spinal nerve roots of normal rabbits were incubated at 4°C overnight with peroxidase-conjugated cholera
toxin B-subunit (2 ␮g/ml; List Biological Laboratories,
Campbell, CA), then developed with DAB, followed by osmium tetroxide.
Sciatic nerves were removed from deeply anesthetized normal rabbits, quickly frozen in cold isopentane, and stored at
– 80°C. Cryostat sections (6 ␮m thick) from these nerves
were fixed with cold acetone at –20°C for 7 minutes. After a
wash with PBS, the sections were incubated successively at
20°C for 15 minutes with 10% normal goat serum, at 4°C
overnight with plasma from a BBG-immunized rabbit (1:100
dilutions), and at 20°C for 1 hour with peroxidaseconjugated goat anti-rabbit IgG (1:200; Southern Biotechnology, Birmingham, AL). After each step, the sections were
washed thoroughly with PBS. Finally, the immunoreaction
products were developed with DAB solution.
Thin-Layer Chromatography (TLC) Immunostaining
and TLC Blotting/Secondary Ion Mass Spectrometry
Total gangliosides were extracted from the peripheral nerves
of normal rabbits. The monosialosyl-, disialosyl-, and polysialosylganglioside fractions were prepared as described elsewhere.18 The BBG mixture; authentic GM1; or the
monosialosyl-, disialosyl-, and polysialosylgangliosides from
rabbit peripheral nerves were layered on TLC plates (Merk,
Darmstadt, Germany). The plates were developed with chloroform/methanol/12 mM magnesium chloride in water (5:
4:1, by volume), dipped in n-hexane-containing 0.4% polyisobutylmethacrylate for 1 minute, then dried under an air
stream. Each plate was overlaid with rabbit plasma (1:50 dilutions with PBS-0.5% casein) and kept at 4°C overnight.
Next, the plates were washed and overlaid with peroxidaseconjugated anti-rabbit ␮- or ␥-chain-specific antibodies (1:
500 dilutions; Nordic, Tilburg, The Netherlands), kept at
20°C for 2 hours, then washed. Binding activities were made
Yuki et al: Animal Model of Axonal GBS
713
visible with 4-chloro-1-naphtol. After marking the immunostainings with a 6B pencil, the TLC plates were dipped in
chloroform/methanol (1:1 by volume) to remove the visible
product and polymer. TLC blotting and negative secondary
ion mass spectrometry were done as described elsewhere.19
After being coated with the polymer, the plates were incubated at 20°C for 2 hours with peroxidase-conjugated cholera toxin B-subunit (0.2 ␮g/ml). Binding was made visible
with 4-chloro-1-naphtol.
Enzyme-Linked Immunosorbent Assay
The enzyme-linked immunosorbent assay (ELISA) was done
as reported elsewhere.20 Five picomoles of GM2, GM1,
GD1a, GD1b, GT1b, or GQ1b were placed in individual
wells of microtiter plates. Plasma samples were diluted serially starting at 1:500. The diluted plasma was added to each
well, and the plates were incubated at 4°C overnight.
Peroxidase-conjugated anti-rabbit ␮- or ␥-chain-specific antibody (1:2,000) was added, after which the plates were kept
at 20°C for 2 hours, then developed with o-phenylenediamine.
Results
Clinical and Pathological Findings for BBGImmunized Rabbits
None of the ten control rabbits inoculated with KLH
and FCA showed limb weakness until 6 months after
the first inoculation. In contrast, all of the 13 rabbits
immunized with BBG, KLH, and FCA developed flaccid paresis of the hind limbs, the onset ranging from
35 to 57 days (median 43 days) after the initial inoculation (Table 1, Fig 1a,b). Eleven of the rabbits began
to lose weight 3–20 days (median 6 days) before the
onset of limb weakness. Rabbits Cr-2, Cr-3, and Cr-5
had body and limb tremors several days before the onset of limb weakness. Tetraparesis developed rapidly in
rabbits Cr-3 and Cr-4, with respiration becoming labored and gasping. Limb weakness progressed for 4 –13
days (median 5 days) after onset in 9 rabbits (Cr-5 to
Cr-13) then reached a plateau. One rabbit (Cr-6)
could lift neither its head nor body but was able to
walk 2 weeks after the onset of limb weakness.
No significant changes were found in the brains or
spinal cords of any of the rabbits immunized with
BBG. In contrast, the sciatic nerves showed severe to
mild Wallerian-like degeneration, with rare demyelination or remyelination (Fig 1c). Macrophage invasion
was prominent in endoneurial-perivascular areas, but
no lymphocytic infiltration was found in any of the
sciatic nerve regions. Quantitative comparisons showed
that the mean degenerative axon density was significantly increased in 11 BBG-immunized rabbits [Table
1; 1,289 ⫾ 476/mm2 (mean ⫾ standard deviation)]
than in 4 adjuvant controls (498 ⫾ 152/mm2; p ⫽
Table 1. Rabbits Inoclulated with Bovine Brain Gangliosides or GM1
Quantitative Analysis of Sciatic Nerves
Rabbit
Immunogen
Inoculation
Times
Cr-1
Cr-2
Cr-3
Cr-4
Cr-5
Cr-6
Cr-7
Cr-8
Cr-9
Cr-10
Cr-11
Cr-12
Cr-13
Sy-1
Sy-2
Sy-3
Sy-4
Sy-5
Sy-6
Sy-7
Sy-8
Sy-9
Sy-10
Sy-11
BBG
BBG
BBG
BBG
BBG
BBG
BBG
BBG
BBG
BBG
BBG
BBG
BBG
GM1
GM1
GM1
GM1
GM1
GM1
GM1
GM1
GM1
GM1
GM1
2
3
3
2
2
2
3
3
3
3
3
3
3
2
9
3
9
2
5
5
9
4
4
4
Onset of Limb
Weakness (Day)
Days from
Onset to Peak
42
48
45
38
40
35
54
43
48
48
57
43
43
37
Not clarified
Not clarified
Undetermined
Undetermined
4
6
13
4
5
4
5
5
5
Not clarified
62
Not clarified
41
90
90
177
70
70
72
7
2
Not clarified
6
27
10
2
Poorest
Functional
Grade
Days from
Onset to
Sacrifice
1
1
4
4
4
4
2
4
4
3
4
4
4
1
0
1
0
2
2
1
2
3
5
5
52
53
2
4
29
31
32
9
8
11
13
20
16
29
11
9
119
81
43
35
Not done
Not done
Degenerative Axon
Density (n/mm2)
Surviving Axon
Density (n/mm2)
1,446
795
8,266
7,462
952
1,127
1,455
1,163
730
10,355
8,692
7,673
7,264
15,916
1,428
1,399
2,481
1,271
336
394
1,238
536
1,122
1,115
1,091
800
802
10,306
10,084
9,091
10,456
12,340
10,988
10,712
10,152
11,646
11,923
14,446
9,528
13,717
Functional grade: 0 ⫽ normal; 1 ⫽ showing weakness of the hind limbs; 2 ⫽ showing mild weakness of the four limbs but able to walk; 3 ⫽
showing moderate weakness of the four limbs and unable to walk; 4 ⫽ showing severe weakness of the four limbs, which were spread out; 5 ⫽
dead. Because rabbits Cr-1, Cr-2, Sy-1, Sy-3, and Sy-7 had only weakness of the hind limbs, the days from onset to peak were not clarified.
Sy-2 and Sy-4 were killed, respectively, 285 and 292 days after the first inoculation. Because rabbits Cr-3 and Cr-4 were killed, respectively,
2 and 4 days after the onset of limb weakness, the days from onset to peak could not be determined. Sciatic nerves of Cr-3 and Cr-9 were not
available for the morphometric examination.
714
Annals of Neurology
Vol 49
No 6
June 2001
Fig 1. (a) Rabbit Cr-6 (see Table 1) with limb weakness induced by sensitization with bovine brain ganglioside (BBG) mixture;
14 days after onset. It could not maintain a normal standing position nor lift its head or body. The muscles of the extremities and
trunk were weak and slack, offering less than the usual resistance to passive movement. (b) Rabbit Cr-4 with limb weakness induced by sensitization with the BBG mixture; 4 days after onset. It lay splayed out, all its extremities extended and head on the
floor, instead of sitting in the usual compact, hunched posture. This rabbit attempted to stand but could not. (c) Transverse section
of the sciatic nerve from BBG-immunized rabbit Cr-6. Many myelin ovoids produced by Wallerian-like degeneration of the myelinated fibers are present. Toluidine blue-safranine stain. (d) Transverse section of the anterior root from BBG-immunized rabbit
Cr-6. Clusters of small myelinated fibers indicative of regenerating sprouted fibers are present (arrowheads). Toluidine bluesafranine stain. (e) Myelin ovoids indicative of Wallerian-like degeneration of myelinated fibers from BBG-immunized rabbit Cr-11
are present in three sciatic nerve teased fiber preparations. (f ) Transverse section of the spinal anterior nerve root from a BBGimmunized rabbit with high anti-GM1 IgG antibody titer. Some axons are deeply stained by peroxidase-conjugated protein G (arrowheads). Scale bars ⫽ 10 ␮m in c,d,f, 20 ␮m in e.
0.0024). The surviving axon density was significantly
decreased in the BBG-immunized rabbits (9,597 ⫾
2,419/mm2) than in the adjuvant controls (14,971 ⫾
3,131/mm2; p ⫽ 0.0006). The anterior spinal nerve
roots from rabbit Cr-6 in the recovery phase of weakness had occasional clusters of small fibers showing various degrees of myelination, indicative of axonal sprout
regeneration (Fig 1d). No pathological changes were
seen in the posterior roots. Teased fiber studies showed
a significantly higher percentage of fibers that had undergone Wallerian-like degeneration in the BBGimmunized rabbits (12% in Cr-11, 21% in Cr-12, and
24% in Cr-13; Fig 1e) than in 3 adjuvant controls
(0%, 0%, and 3%; p ⫽ 0.046). In contrast, the paralyzed rabbits had low percentages of fibers that had undergone paranodal demyelination (1% in Cr-11, 0% in
Cr-12, and 0% in Cr-13) as did the controls (0%, 1%,
and 2%). Protein G positively stained some axons in
Yuki et al: Animal Model of Axonal GBS
715
spinal anterior roots obtained from BBG-immunized
rabbits (Fig 1f ), evidence of the presence of IgG in or
around the axons. Similar findings were obtained when
anterior roots were immunostained with anti-rabbit
IgG antibody. No pathological changes or immunoglobulin deposits were detected in the sciatic nerves or
anterior roots of the normal rabbits and adjuvant controls.
Induction of Antiganglioside Antibody
Plasma obtained within 1 week after the onset of limb
weakness was used. TLC immunostaining showed that
the rabbit IgGs strongly bound to GM1 (Fig 2a). An
ELISA confirmed that the immune response was directed predominantly against GM1 (Table 2). Neither
the IgMs nor the IgGs obtained from these rabbits before inoculation nor those from the control rabbits
bound to GM1. Anti-GM1 IgM antibodies were detected in the 13 paralyzed rabbits 2–3 weeks after their
first sensitization with BBG (Table 3). Titers increased
for 1–5 weeks (median 2 weeks) after detection (range
2,000 – 64,000; median 32,000). Anti-GM1 IgG antibodies were detected in these rabbits 3– 4 weeks after
the first inoculation. The titers increased for 1– 6 weeks
(median 3 weeks) after detection (range 4,000 –512,000;
median 32,000). Ten of the thirteen developed flaccid
paresis of the hind limbs within 1–3 weeks (median 1
week) after the peak anti-GM1 IgG titer was reached.
Axonal Neuropathy Induced by Sensitization
with GM1
Nine of eleven rabbits immunized with GM1, KLH,
and FCA developed flaccid paresis of the hind limbs
(Table 1). Quadriparesis and respiratory paresis rapidly
progressed in rabbits Sy-10 and Sy-11, which died, respectively, 11 and 3 days after the onset of limb weakness. Limb weakness progressed for 2, 6, 7, and 27
days, respectively, after onset in rabbits Sy-6, Sy-8,
Sy-5, and Sy-9, then reached a plateau. Weakness lessened in 2 rabbits (Sy-6 and Sy-7) 11 and 20 days after
onset and disappeared in 2 (Sy-7 and Sy-8) 5 and 29
days after onset. TLC immunostaining and the ELISA
showed that anti-GM1 IgM antibodies were induced
and that a switch from IgM to IgG occurred in all 11
rabbits with or without development of paralysis. IgG
from a paralyzed rabbit reacted with the GM1 of rabbit peripheral nerve (Fig 2b). Mild to moderate
Wallerian-like degeneration was present in Eponembedded sciatic nerve sections obtained from rabbits
with limb weakness (Table 1); the degenerative axon
density was increased (929 ⫾ 310/mm2). There was
mild to extensive fiber loss; the surviving axon density
was decreased (12,044 ⫾ 1,680/mm2). Neither lymphocytic infiltration nor demyelination was found.
Teased fiber preparations showed Wallerian-like degeneration in 23%, 21%, and 26% of the large myelinated
fibers in Sy-6, Sy-8, and Sy-9, respectively, whereas
paranodal demyelination was seen in 0%, 0%, and 2%
of these fibers.
Target Molecule for the IgG Autoantibody
Immunoblotting showed that none of the IgGs from
the paralyzed rabbits bound to proteins from rabbit peripheral nerves (data not shown). In contrast, TLC immunostaining showed that the IgG strongly reacted
with a band in the monosialosylganglioside fraction
from rabbit peripheral nerve (Fig 2b). The mobility of
the band was similar to that of authentic GM1. Figure
2c shows the TLC blotting/negative secondary ion
mass spectrum of the IgG-reactive band. On the TLC
plates, cholera toxin B-subunit, which specifically recognizes GM1, reacted with a band in the monosialosylganglioside fraction (Fig 2b), which had mobility
similar to that of the IgG-reactive band. These findings
suggest that GM1 is present in rabbit peripheral
nerves. The cholera toxin B-subunit bound to the axons of normal rabbit sciatic nerves (Fig 2d) and anterior and posterior roots, evidence that GM1 is expressed on those axons. Immunohistochemical
experiments with plasma from a BBG-immunized rabbit that had high anti-GM1 activity clearly showed that
its plasma IgG reacted positively with the axon surfaces
of sciatic nerves from a normal rabbit (Fig 2e,f ).
Discussion
The clinical features of paresis with an acute,
monophasic course in BBG-immunized rabbits were
very similar to those of patients with GBS. Axonal
GBS has predominant axonal involvement, characterized by Wallerian-like degeneration of nerve fibers,
with only minimal demyelination and minimal lymphocytic infiltration.1– 4 Our paralyzed rabbits showed
similar pathological changes with predominant axonal
degeneration of the peripheral nerves. Results of teased
fiber studies confirmed a higher percentage of
Wallerian-like degeneration of the large myelinated fibers of the sciatic nerves. In addition, there was regenerative sprouting of anterior root fibers taken from a
rabbit during the recovery phase. Anti-GM1 IgG antibody specifically is present in GBS during the acute
phase and is significantly associated with axonal
GBS.5,6 The paralyzed rabbits also had the anti-GM1
IgG antibody. As in rodents and humans,17 GM1 is
expressed on the axons of rabbit peripheral nerves, as
our study indicates. We showed that the IgG with antiGM1 activity from a paralyzed rabbit strongly bound
to the axon. Moreover, as in Chinese patients with axonal GBS,21 IgG was deposited on the motor nerve
axons in the paralyzed rabbits. The cholera toxin
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Vol 49
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Yuki et al: Animal Model of Axonal GBS
717
Table 2. Antiganglioside Antibodies in Plasma Obtained within 1 Week after the Onset of Limb Weakness
Rabbits Sensitized with Bovine Brain Ganglioside
Cr-1
Cr-2
Cr-3
Cr-4
Cr-5
Cr-6
Cr-7
Cr-8
Cr-9
Cr-10
Cr-11 Cr-12
Cr-13
IgM antibody to
GM2
0
0
0
0
0
0
0
0
0
0
0
0
0
GM1
1,000 8,000 8,000
8,000 32,000 32,000 8,000 16,000 64,000 64,000 1,000 4,000 16,000
GD1a
0 500
500
2,000 2,000 16,000
500
0 4,000 4,000
0 500
500
GD1b
0 2,000 2,000
4,000 16,000 16,000 4,000 8,000 16,000 16,000
0 1,000 2,000
GT1b
0
0
0
0
0
0
0
0
0
0
0
0
0
GQ1b
0
0
0
0
0
0
0
0
0
0
0
0
0
IgG antibody to
GM2
0
0
0
0
0
0
0
0
0
0
0
0
0
GM1
4,000 4,000 32,000 128,000 32,000 128,000 32,000 16,000 64,000 32,000 16,000 4,000 4,000
GD1a
0
0 1,000
0 1,000
0
500
0 8,000
500
0
0
0
GD1b
0
0 4,000
4,000 4,000 32,000 1,000
500 32,000 1,000
500 500
500
GT1b
0
0
0
0
0
0
0
0
0
0
0
0
0
GQ1b
0
0
0
0
0
0
0
0
0
0
0
0
0
bound with both anterior and posterior roots, indicative of the presence of GM1 on sensory fibers as well as
motor fibers. Why neither the IgG deposition nor the
Wallerian-like degeneration was seen in the posterior
roots is unknown. Motor nerve terminals are proposed
to be the target site of anti-GM1 antibody in acute
motor axonal neuropathy.10,22 The pathology of the
terminals should be investigated in this animal model.
The pathophysiological role of anti-GM1 antibodies
is still a matter of controversy. In some studies antiGM1 sera caused conduction block of the motor
Š
nerves,23,24 whereas in others it did not.25,26 Immunohistochemical findings in our study, however, provide
evidence that IgG antibodies directed at GM1 on motor nerve axons could cause motor axonal neuropathy.
We showed that sensitization with GM1 highly induced acute motor axonal neuropathy similar to that
induced by the BBG mixture injection, strong support
that GM1 is the immunogen in the BBG mixture and
the target molecule for the autoantibodies. GM1 and
galactocerebroside are expressed in both the peripheral
nervous system (PNS) and central nervous system
Fig 2. (a) Antiganglioside antibody from rabbits that developed limb weakness after sensitization with a bovine brain ganglioside
(BBG) mixture. A: Thin-layer chromatogram stained with the orcinol reagent for hexose. B: Immunostained chromatogram that
had been overlaid first with plasma from the rabbits, then with peroxidase-conjugated anti-rabbit ␥-chain specific antibodies. Lanes
1–10 show plasma from rabbits Cr-1 to Cr-10 in Table 1. Orcinol reagent stains GM1, GD1a, GD1b, and GT1b. Plasma IgGs
from the rabbits strongly bind to GM1, and some weakly react with GD1b. (b) Target molecule for IgG autoantibodies among
peripheral nerve gangliosides. A: Thin-layer chromatogram stained with the orcinol reagent. B: Immunostained chromatogram incubated first with plasma from rabbit Cr-6 inoculated with BBG then with peroxidase-conjugated anti-rabbit ␥-chain specific antibodies. C: Binding of the peroxidase-conjugated cholera toxin B-subunit. D: Immunostained chromatogram incubated first with
plasma from rabbit Sy-5 inoculated with GM1, then with peroxidase-conjugated anti-rabbit ␥-chain specific antibodies. Lane 1:
BBG mixture (CronassialTM). Lane 2: Authentic GM1 (SygenTM) from bovine brain. Lanes 3–5: Monosialosyl-, disialosyl-, and
polysialosylganglioside fractions, respectively, from rabbit peripheral nerves. The mobility of the monosialosylganglioside, with which
the cholera toxin B-subunit and IgGs from rabbits Cr-6 and Sy-5 react, is similar to that of authentic GM1. (c) Negative secondary ion mass spectrum of the monosialosylganglioside that reacted with IgG from rabbit Cr-6. Cer ⫽ ceramide; Hex ⫽ hexose;
HexNAc ⫽ N-acetylhexosamine; NeuAc ⫽ N-acetylneuraminic acid. The ion at m/z 1,544 consisted of stearic acid (C18:0) and
sphingenine (d18:1), and that at m/z 1,572 of C18:0 and icosasphingenine (d20:1). The 7 major fragment ions of the sugar sequence ions in the spectrum were representative ceramides (m/z 564, 592; out of spectrum), glucosylceramides (a; m/z 726, 754),
lactosylceramides (b; m/z 888, 916), gangliotriaosylceramides (c; m/z 1,091, 1,119), gangliotetraosylceramides (d; m/z 1,253,
1,281), II 3-N-acetylneuraminosyllactosylceramides (e; m/z 1,179, 1,207), and II 3-N-acetylneuraminosylgangliotriaosylceramides (f;
m/z 1,382, 1,410). The two fragment ions that corresponded to the nonreducing terminal side of the carbohydrate chain were m/z
833 (g; [(Hex-HexNAc-[NeuAc]Hex-OH)-H2-H] –) and m/z 995 (h; [(Hex-Hex-HexNAc-[NeuAc]Hex-OH)-H2-H] –). The m/z
290 and 308 ions, which corresponded to N-acetylneuraminic acid, also were present. Its spectrum and its mobility on the thinlayer chromatogram plate similar to that of authentic GM1 and its binding with the cholera toxin B-subunit suggest that the structure of this ganglioside is Gal ␤1–3 GalNAc ␤1– 4 (NeuAc ␣2–3) Gal ␤1– 4 Glc ␤1–1⬘ Cer. (d)Cross section of normal rabbit
sciatic nerve stained with the peroxidase-conjugated cholera toxin B-subunit. Axons are stained. (e,f ) Cross (e) and longitudinal (f )
sections of normal rabbit sciatic nerve immunostained with plasma IgG that has anti-GM1 activity from the BBG-immunized rabbit Cr-4. Axon surfaces are positively stained. Scale bars ⫽ 10 ␮m.
718
Annals of Neurology
Vol 49
No 6
June 2001
Table 3. Anti-GM1 Antibody Titers in the Rabbits Sensitized with Bovine Brain Ganglioside
Rabbit
Isotype 1
Cr-1
IgM
IgG
IgM
IgG
IgM
IgG
IgM
IgG
IgM
IgG
IgM
IgG
IgM
IgG
IgM
IgG
IgM
IgG
IgM
IgG
IgM
IgG
IgM
IgG
IgM
IgG
Cr-2
Cr-3
Cr-4
Cr-5
Cr-6
Cr-7
Cr-8
Cr-9
Cr-10
Cr-11
Cr-12
Cr-13
Week(s) After the First Inoculation
2
3
4
5
6
7
8
9
10
11
12
13
14
0
0 1,000 2,000
2,000
2,000
1,000
500
500
500 1,000 1,000 1,000
0
0 1,000 4,000
4,000
4,000
4,000
4,000
4,000 4,000 4,000 4,000 4,000
0
0 8,000 8,000
8,000
8,000
8,000 16,000 16,000 16,000 16,000 16,000 16,000 16,000
0
0
0 4,000
4,000
4,000
4,000
8,000 16,000 32,000 32,000 32,000 32,000 32,000
0
0 1,000 8,000
8,000
8,000
8,000
0
0 1,000 8,000 16,000 32,000 16,000
0 2,000 32,000 32,000 16,000
8,000
0
0 1,000 32,000 128,000 128,000
0
0 16,000 32,000 64,000 32,000 32,000 64,000 64,000 32,000
0
0 8,000 32,000 64,000 32,000 32,000 64,000 64,000 64,000
0 500 16,000 16,000 32,000 32,000 16,000 16,000
8,000
0
0 8,000 32,000 64,000 128,000 256,000 512,000 256,000
0
0
500 4,000 16,000 16,000 16,000
8,000
4,000 2,000
0
0
0 4,000 32,000 32,000 32,000 32,000 32,000 32,000
0
0 1,000 8,000 16,000 32,000 16,000
0
0
0
500
4,000 16,000 16,000
0 500 8,000 32,000 32,000 64,000 64,000
0
0 1,000 8,000 32,000 32,000 64,000
0 2,000 32,000 64,000 64,000 64,000 64,000 32,000
0
0
500 8,000 16,000 32,000 32,000 32,000
0
0
500 2,000
2,000
2,000
2,000
2,000
1,000 1,000
0
0
0
500
2,000
2,000
4,000 16,000 16,000 16,000
0
0 4,000 16,000 16,000 16,000
4,000
1,000
1,000
0
0
0 4,000
4,000
8,000
4,000
8,000
8,000
0
0 4,000 16,000 32,000 16,000 16,000
4,000
2,000
0
0
0
500
1,000
2,000
4,000
4,000
1,000
(CNS), but sensitization with these molecules produces
only peripheral neuropathy.27 The blood–nerve barrier
that guards the PNS is not as tight as the blood–brain
barrier; therefore, small amounts of circulating IgG,
which cannot enter the CNS, can penetrate the endoneurial space. This relative leakiness may make the
PNS, especially the roots and nerve terminals, more
vulnerable than constituents of the CNS to IgG
antibody-mediated disorders. We assume that inoculation with the BBG mixture or the isolated GM1 induced high anti-GM1 IgG antibody titers, after which
the autoantibodies bound to the GM1 expressed on
the axons in the rabbit PNS, producing dysfunction of
the motor nerves followed by Wallerian-like degeneration.
Studies on BBG or purified GM1 as the agent for
treating a variety of neurological disorders were initially
reported with enthusiasm to be successful, but later
double-blind controlled studies have failed to confirm
these findings.28 CronassialTM (BBG mixture) and SygenTM (isolated GM1), which had been available on
the Italian market, were used in our study. Although
whether there is an epidemiological relationship between exogenous gangliosides and GBS is still not
clear,29,30 our results indicate that in some cases a BBG
or GM1 injection may trigger axonal GBS. As in the
patients with axonal GBS after BBG therapy,10,31 in-
terestingly, the immune response in the BBGinoculated rabbits predominantly was directed against
GM1. The BBG mixture contains more GD1a than
GM1, but it is unknown why the immune response
against GD1a was poor in the rabbits. All the antiGD1b antibody titers were lower than the anti-GM1
antibody titers (Table 2). The anti-GD1b antibodies in
the rabbits were absorbed with GM1 (data not shown),
indicating that the antibodies cross-react with GM1.
In conclusion, experimental inoculation of rabbits
with BBG or GM1 provides a new and useful model
for determination of the molecular pathogenesis of axonal GBS. This model should be useful for developing
new treatments for patients with GBS.
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