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Validation of a rabbit model of neuropathy induced by immunization with gangliosides
Journal of the Neurological Sciences 272 (2008) 110 – 114 www.elsevier.com/locate/jns
Validation of a rabbit model of neuropathy induced by immunization with gangliosides A.L. Moyano a,1 , R. Comín a,1 , R.D. Lardone a , M.E. Alaniz a , R. Theaux b , F.J. Irazoqui a , G.A. Nores a,⁎ a
Departamento de Química Biológica “Dr. Ranwel Caputto”, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba and CIQUIBIC-CONICET, Ciudad Universitaria, 5000 Córdoba, Argentina b División de Neuropatología, Departamento de Patología, Facultad de Medicina, Universidad Católica de Córdoba, Córdoba, Argentina Received 18 January 2008; received in revised form 16 April 2008; accepted 15 May 2008 Available online 24 June 2008
Abstract The induction of neurological signs by immunization of rabbits with gangliosides has been a controversial topic for many years. Recently, Yuki et al. [N. Yuki, M. Yamada, M. Koga, M. Odaka, K. Susuki, Y. Tagawa, et al. Animal model of axonal Guillain–Barré syndrome induced by sensitization with GM1 ganglioside. Ann Neurol 2001;49:712–720.] described an immunization protocol, including keyhole lympet hemocyanin in addition to ganglioside that induced a neurological disease resembling human Guillain–Barré syndrome. We employed this protocol in our laboratory and succeeded in reproducing the disease. Five different experiments were performed during a period of two years by different operators, using different batches of drugs, in a total of 26 rabbits. Despite minor variations in onset time and severity of the induced disease, the model proved to be reproducible. Both gangliosides and keyhole limpet hemocyanin are required for induction of disease. © 2008 Elsevier B.V. All rights reserved. Keywords: Ganglioside; Anti-GM1 antibodies; Guillain–Barré; syndrome; Experimental neuropathy
1. Introduction Guillain–Barré syndrome (GBS) is an antibody-mediated acute neuropathy, considered a prototype of autoimmune disease [1]. The ganglioside GM1, a self-glycan present in neural tissues, has been proposed as one of the main antigenic targets [2]. Although not a common disease, GBS is a disabling process with a high rate of mortality [3]. Plasma exchange and intravenous immunoglobulins are useful therapeutic tools, but there is strong interest in the development of new therapies based on immune intervention at early stages of the disease. Research along this line is focused on the immune mechanisms involved in pathogenesis and pathophysiology, and several ⁎ Corresponding author. Facultad de Ciencias Químicas (UNC), Ciudad Universitaria, 5000 Córdoba, Argentina. Tel.: +54 351 433 4168. E-mail address: [email protected] (G.A. Nores). 1 Both authors contributed equally to this work. 0022-510X/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2008.05.006
laboratories have been searching for an animal model of the disease. In the mid-70's, Nagai et al. [4] described “ganglioside syndrome”, a motor neuropathy induced by immunization with GM1. This concept was long considered controversial, but is receiving renewed attention in view of recent evidence for the key role of anti-ganglioside antibodies in neuropathy. One reason for the controversy is that preparation of rabbit anti-GM1 antibodies is a common procedure in several laboratories where ganglioside function is studied, including ours [5], and these rabbits do not exhibit motor neuropathy. Inoculation of rabbits with GM1 in Freund's adjuvant results in high titer of anti-GM1 IgG antibodies, but causes only minor electrophysiological and pathological changes [6] and no clinical signs [7]. In 1996, using a slightly different immunization protocol and GD1b as immunogen, Kusunoki et al. [8] succeeded in inducing an experimental sensory ataxic neuropathy in rabbits. Based on this new protocol, Yuki et al. [9] developed an experimental model of GBS: rabbits immunized with bovine brain gangliosides or
A.L. Moyano et al. / Journal of the Neurological Sciences 272 (2008) 110–114
GM1 produced high titers of anti-GM1 IgG antibodies, but after 5 to 10 weeks they also showed clinical and pathological changes similar to those found in GBS patients. A significant modification in the new protocol was the use of keyhole limpet hemocyanin (KLH) as carrier for the immunogen; this protein was not used in previous protocols. However, Dasgupta et al. [10] reported that even using KLH, repeated immunizations with GM1 produced high titer of anti-GM1 IgG antibodies but did not induce neurological abnormalities. This report and the absence of reports from other labs describing successful induction of motor neuropathy by immunization with gangliosides illustrate the difficulty in the reproducibility of the model. We also failed to produce “ganglioside syndrome” in a 2002 experiment [7]. We now report a successful induction of a disease similar to that described by Yuki et al. [9], following their immunization protocol. 2. Materials and methods 2.1. Glycolipids Glycolipids GM1, GD1a, GD1b, GT1b, galactocerebroside and sulfatide were purified from human brain [11] and used as antigens. Asialo-GM1 (GA1) was prepared by acid hydrolysis of bovine brain gangliosides [12]. Total ganglioside fraction from bovine brain (Nx) was prepared from bovine brain by Folch extraction [13], DEAE-Sephadex [14], alkaline methanolysis, and reversed phase chromatography [15]. The ganglioside composition of this preparation was similar to that of commercial preparation Cronassial®.
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immunostaining as previously described [7]. Antibody affinity for GM1 was estimated by inhibition of antibody binding by soluble antigen [11]. Antibody binding to GM1 was quantified by enzyme-linked immunosorbent assay (ELISA) [7]. Titer values were calculated as the reciprocal of the serum dilution needed to obtain half-maximal antibody binding [16]. Rabbit sera were purified by GM1 affinity columns as described [7]. 2.4. Pathological studies Rabbits were deeply anesthetized by intramuscular injection of ketamine hydrochloride (35 mg/kg) and acepromazine (0.75 mg/kg), sciatic nerves were removed, and the animals were sacrificed. Nerve specimens were fixed by immersion in 4% (v/v) glutaraldehyde and 4% (w/v) formaldehyde in 0.1 M cacodylate buffer. Samples were treated with 1% osmium tetroxide, dehydrated, and embedded in Araldite. Sections (1 µm thick) were stained with toluidine blue and examined by an Axioplan light microscope (Carl Zeiss, Oberkochem, Germany). Digital images were obtained with a CCD camera (MICROMax, Princeton Instruments, Downingtown, PA, USA). 3. Results The protocol for our “experiment 1” was as described by Yuki et al. [9]. Rabbits were inoculated subcutaneously on the back and then intraperitoneally at 3 week intervals. GM1 and KLH emulsified in CFA were used as immunogens.
2.2. Immunization of rabbits New Zealand male white rabbits, weighing 2–3 kg, were immunized with GM1 or bovine brain gangliosides (BBG). For each immunization, 0.5 mg GM1 (Sygen®, GramonBagó, Uruguay), 2.5 mg commercial BBG (Cr, Cronassial®, Fidia, Padova, Italy), or 2.5 mg BBG prepared in our laboratory (Nx) were dissolved in 500 μl PBS containing 1 mg KLH (Sigma, St. Louis, MO, USA), and emulsified in 500 μl complete Freund's adjuvant (CFA). The animals were injected subcutaneously on the back, and intraperitoneally at 3week intervals until neurological signs appeared, or until 4 months after the first inoculation. Control animals were injected with the same inoculum without KLH or ganglioside, under the same protocol. Rabbits were weighed weekly. Blood samples were taken by ear vein puncture; sera were separated from blood clots and frozen at −70 °C until use. Five experiments with a total of 26 rabbits were performed. All experiments were performed in accordance with international and institutional guidelines for animal care. 2.3. Antibody binding assays Binding of rabbit antibody to GA1, GM1, GD1a, GD1b, GT1b, sulfatide and galactocerebroside was studied by TLC-
Fig. 1. Time course of disease. Body weight variation determined as the percentage of difference relative to week 0 of rabbits immunized with GM1/ KLH (● ○); KLH (■ □) or GM1 (▲ Δ). Animals were immunized at the indicated times (arrows) as described in Materials and methods. Horizontal bar indicates the appearance of neurological signs in rabbits immunized with GM1/KLH.
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Table 1 Disease induction in rabbits immunized with gangliosides Experiment
1
2
3
4
5
Rabbit
1.1 1.2 1.3 1.4 1.5 1.6 2.1 2.2 2.3 2.4 2.5 2.6 3.1 3.2 3.3 3.4 4.1 4.2 4.3 4.4 5.1 5.2 5.3 5.4 5.5 5.6
Immunogen KLH
Ganglioside
− − + + + + − − + + + + + + + + + + − − + + + + + +
GM1 GM1 − − GM1 GM1 GM1 GM1 − − GM1 GM1 GM1 GM1 BBG-Cr BBG-Cr BBG-Cr BBG-Cr BBG-Cr BBG-Cr GM1 GM1 BBG-Cr BBG-Cr BBG-Nx BBG-Nx
Disease onset (week)
Maximal clinical grade
− − − − 8 8 − − − − − 6 12 15 3 5 6 10 − − 7 8 6 5 5 7
0 0 0 0 2 2 0 0 0 0 0 4 4 2 3 4 4 4 0 0 2 2 4 4 3 4
Anti-GM1 IgG antibody Titer 0 0 0 0 810 1750 0 0 0 0 2370 2780 7180 4390 7410 9050 1030 3240 0 0 2070 1110 1500 1360 1300 3180
High affinity
ND ND
Absent Present Present Present ND ND ND ND
Present Present ND ND ND ND
Clinical grade: 0 = normal; 1 = weight lost; 2 = reversible weakness of the hind limbs, difficulty to walk; 3 = reversible weakness of the four limbs; 4 = nonreversible, severe weakness of the four limbs, death. Weight loss was considered the onset of the disease. Anti-GM1 IgG-antibody binding was determined at the time of appearance of neurological signs. Titer values were calculated as the reciprocal of the serum dilution needed to obtain half-maximal antibody binding [16]. Antibody affinity for GM1 was estimated by inhibition of antibody binding by soluble antigen [11]. Presence of high affinity antibodies refers to the presence in a serum of antibody binding inhibitable by soluble GM1 at 10− 8 M. Titer value 0 = binding not detectable at 1/100 dilution. BBG-Cr: commercial preparation of bovine brain gangliosides (Cronassial®); BBG-Nx: bovine brain gangliosides prepared in our laboratory.
Eight to 9 weeks after the initial immunization, both animals began to lose weight (Fig. 1). A few days later, they exhibited neurological signs such as tremor and hind limb weakness. These signs disappeared after a few more days,
and body weight was recovered. Control animals inoculated with either GM1 or KLH alone did not lose weight nor exhibit neurological signs. With some protocol variants, experiment 1 was repeated 4 times during a period of 2 years.
Fig. 2. Anti-GM1 IgG antibodies in ganglioside/KLH immunized rabbits. Serum was taken at the time of appearance of neurological signs and passed through a GM1 affinity column. Dilutions of whole serum (A, D, G) and the fractions retained (B, E, H) and not retained by GM1-column (C, F, I) of rabbits immunized with GM1 (# 1.6), BBG-Cr (# 5.3), and BBG-Nx (# 5.6) were assayed for IgG-immunoreactivity by TLC-immunostaining. GM1 and related glycolipids were used as antigen (orcinol plate). Serum dilution: 1/5000.
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Fig. 3. Light microscopy of toluidine-blue stained transverse sections of the sciatic nerve. A) Normal rabbit. B) Rabbit # 5.1(immunized with GM1/KLH). C) Rabbit # 3.4 (immunized with BBG-Cr/KLH). Note fibers with axonal degeneration (arrows). Scale bars = 20 µm.
Results are shown in Table 1. In this new set of experiments, control rabbits (immunized without KLH or gangliosides) did not show clinical signs. Rabbits immunized with GM1 and KLH either showed similar response as in experiment 1 (i.e. rabbit 5.2), developed more severe disease (rabbit 2.6), or showed no clinical signs (rabbit 2.5). In another experiment, rabbits were immunized with Cronassial®, a commercial preparation of BBG. Consistent with the findings of Yuki et al. [9], BBG were more efficient in inducing disease. Signs appeared early and most of the animals developed irreversible severe weakness of the four limbs. Similar results were obtained when animals were immunized with BBG prepared in our laboratory (Nx, experiment 5, Table 1). Considering the five experiments performed, 15 of 16 rabbits immunized with gangliosides plus KLH lose weight and develop neurological signs. Rabbit serum samples were screened for immunoreactivity against GM1 and structurally related glycolipids by TLCimmunostaining. As in our previous study [7], pre-immune sera displayed IgM-reactivity against GM1, GA1, and GD1b, but not against GD1a or GT1b (results not shown). In addition to this reactivity, rabbits immunized with GM1 or BBG together with KLH also showed IgG-reactivity against the above glycolipids (Fig. 2A, D, G). Antibody binding to all the glycolipids was totally abrogated by pre-absorption of serum with GM1 affinity column (Fig. 2), indicating that all IgG antibodies induced by GM1, as well as by BBG, are directed to GM1. Serum from control animals had nondetectable IgG-immunoreactivity. Anti-GM1 IgG antibody titer was measured by ELISA (Table 1). As expected, rabbits immunized with ganglioside and KLH had high titer values. High affinity of anti-GM1 antibodies have has been associated to disease onset in Yuki's rabbits [11]. Most of our rabbits immunized with GM1 and KLH showed the presence of high affinity antibodies (Table 1). Interestingly, the rabbit that developed a relatively high titer of antibodies but lacked those with high affinity did not get sick (Table 1, rabbit 2.5). Serum was also checked for immunoreactivity against galactocerebroside and sulfatide, two glycolipids that have been described as targets for antibodies associated to
neuropathy [17,18]. At the appearance of neurological signs, while anti-GM1 IgG-immunoreactivity towards GM1 was high, antibody binding to these glycolipids was not detected (results not shown). Transverse sections of sciatic nerves were screened for lesions. Consistent with the findings of Yuki et al. [9], rabbits immunized with GM1/KLH or BBG/KLH showed variable degrees of axonal degeneration (Fig. 3). Demyelination and remyelination were not observed. 4. Discussion An animal model developed by biomedical researchers can be one of their best tools for the management of human diseases. To be useful, a model must have “reproducibility”, i.e., it should be easily produced in laboratories other than the one that described it. Until recently, an animal model of GBS was not available. An early report by Nagai et al. [4], of a motor neuropathy induced by immunization with GM1, was controversial because of lack of reproducibility. The more recent model described by Yuki et al. [9] appeared to fulfill the requirements for a model of GBS. However, despite the fact that Yuki's group successfully reproduced the experimental disease [19], even using a different rabbit breed [20], the model was questioned by other labs [10,21]. We attempted for several years to induce motor neuropathy by immunization with GM1 [7], but did not succeed until we used Yuki's immunization protocol. The disease induced in our rabbits is similar to that in Yuki's rabbits, i.e., both are flaccid, mainly axonal, motor neuropathies with similar onset time. The disease is associated with the presence of anti-GM1 IgG antibodies, which were detected not only in rabbits immunized with GM1 but also in those immunized with a mixture of BBG. It should be noted that, although the BBG mixture used as immunogen contained gangliosides other than GM1 (such as GD1a and GT1b), only immunoreactivity against GM1 was produced. This finding is consistent with the hypothesis that “disease-associated” anti-GM1 antibodies originated from “normal occurring” counterparts (“binding site drift” hypothesis; Lopez et al.
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[22]), and could explain why both immunogens induce a similar disease since, in both cases, only anti-GM1 antibodies are generated. This is the first study in which the GBS model induced by ganglioside immunization was successfully reproduced outside Japan. The disease was induced in 5 different experiments in our laboratory, conducted by different operators over a period of more than 2 years, during which time we also used different lots of drugs (adjuvant, gangliosides, KLH). The reproducibility of the experiments increases our confidence in the model as a useful tool for study of GBS and related diseases. In both our experiments and those of Yuki et al. [9], onset time and severity of the induced disease were highly variable. This variability, which is more evident in rabbits immunized with GM1, reflects a random component in the origin of the disease. This random component has at least two possible explanations: i. It is a characteristic intrinsically associated with the disease. For example, we described a “random component” in the origin of disease-associated antiGM1 IgM-antibodies in neuropathy patients [23]. ii. On the other hand, the variability may have a methodological origin; i.e., uncontrolled technical details in the immunization process may be important in overcoming self-tolerance to GM1. This explanation could reconcile the early observation of Nagai et al. [4], and later studies in which GM1 immunization produced variable disease ([9]; present work), subclinical disease [6] or even no disease[7,10]. Acknowledgements This work was supported by grants from SeCyT (UNC), CONICET, and ANPCyT, Argentina. A.L.M. had fellowship assistance from ANPCyT. R.C., M.E.A., and R.D.L. had fellowship assistance from CONICET. F.J.I. and G.A.N. are Career Investigators of CONICET. The authors thank Dr. Steve Anderson for editing and Dr. Carlos Mas for his help in images preparation.
References [1] Willison HJ, Yuki N. Peripheral neuropathies and anti-glycolipid antibodies. Brain 2002;125:2591–625. [2] Sadiq SA, Thomas FP, Kilidireas K, Protopsaltis S, Hays AP, Lee KW, et al. The spectrum of neurologic disease associated with anti-GM1 antibodies. Neurology 1990;40:1067–72. [3] Kuwabara S. Guillain–Barré syndrome: epidemiology, pathophysiology and management. Drugs 2004;64:597–610. [4] Nagai Y, Momoi T, Saito M, Mitsuzawa E, Ohtani S. Ganglioside syndrome, a new autoimmune neurologic disorder, experimentally induced with brain gangliosides. Neurosci Lett 1976;2:107–11.
[5] Panzetta P, Gravotta D, Maccioni HJ. Biosynthesis and expression of gangliosides during differentiation of chick embryo retina cells in vitro. J Neurochem 1987;49:1763–71. [6] Thomas FP, Trojaborg W, Nagy C, Santoro M, Sadiq SA, Latov N, et al. Experimental autoimmune neuropathy with anti-GM1 antibodies and immunoglobulin deposits at the nodes of Ranvier. Acta Neuropathol (Berl) 1991;82:378–83. [7] Lopez PH, Villa AM, Sica RE, Nores GA. High affinity as a disease determinant factor in anti-GM(1) antibodies: comparative characterization of experimentally induced vs. disease-associated antibodies. J Neuroimmunol 2002;128:69–76. [8] Kusunoki S, Shimizu J, Chiba A, Ugawa Y, Hitoshi S, Kanazawa I. Experimental sensory neuropathy induced by sensitization with ganglioside GD1b. Ann Neurol 1996;39:424–31. [9] Yuki N, Yamada M, Koga M, Odaka M, Susuki K, Tagawa Y, et al. Animal model of axonal Guillain–Barré syndrome induced by sensitization with GM1 ganglioside. Ann Neurol 2001;49:712–20. [10] Dasgupta S, Li D, Yu RK. Lack of apparent neurological abnormalities in rabbits sensitized by gangliosides. Neurochem Res 2004;29:2147–52. [11] Comín R, Lopez PHH, Yuki N, Nores GA. High affinity of anti-GM1 antibodies is associated with disease onset in experimental neuropathy. J Neurosci Res 2006;84:1085–90. [12] Cumar FA, Barra HS, Maccioni HJ, Caputto R. Sulfation of glycosphingolipids and related carbohydrates by brain preparations from young rats. J Biol Chem 1968;243:3807–16. [13] Folch-Pi J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 1957;226:497–509. [14] Yu RK, Ledeen RW. Gangliosides of human, bovine, and rabbit plasma. J Lipid Res 1972;13:680–6. [15] Williams MA, McCluer RH. The use of Sep-Pak C18 cartridges during the isolation of gangliosides. J Neurochem 1980;35:266–9. [16] Mizutamari RK, Wiegandt H, Nores GA. Characterization of antiganglioside antibodies present in normal human plasma. J Neuroimmunol 1994;50:215–20. [17] Kusunoki S, Shiina M, Kanazawa I. Anti-Gal-C antibodies in GBS subsequent to mycoplasma infection: evidence of molecular mimicry. Neurology 2001;57:736–8. [18] Dabby R, Weimer LH, Hays AP, Olarte M, Latov N. Antisulfatide antibodies in neuropathy: clinical and electrophysiologic correlates. Neurology 2000;54:1448–52. [19] Susuki K, Nishimoto Y, Yamada M, Baba M, Ueda S, Hirata K, et al. Acute motor axonal neuropathy rabbit model: immune attack on nerve root axons. Ann Neurol 2003;54:383–8. [20] Susuki K, Nishimoto Y, Koga M, Nagashima T, Mori I, Hirata K, Yuki N. Various immunization protocols for an acute motor axonal neuropathy rabbit model compared. Neurosci Lett 2004;368:63–7. [21] Nachamkin I. Rabbit model of Guillain–Barre syndrome. Ann Neurol 2002;52:127–8. [22] Lopez PH, Lardone RD, Irazoqui FJ, Maccioni M, Nores GA. The origin of anti-GM1 antibodies in neuropathies: the “binding site drift” hypothesis. Neurochem Res 2002;27:687–95. [23] Lopez PH, Lardone RD, Irazo qui FJ, Villa AM, Di EM, Saizar RD, et al. Variable patterns of anti-GM(1) IgM-antibody populations defined by affinity and fine specificity in patients with motor syndromes: evidence for their random origin. J Neuroimmunol 2001;119:131–6.
Validation of a rabbit model of neuropathy induced by immunization with gangliosides A.L. Moyano a,1 , R. Comín a,1 , R.D. Lardone a , M.E. Alaniz a , R. Theaux b , F.J. Irazoqui a , G.A. Nores a,⁎ a
Departamento de Química Biológica “Dr. Ranwel Caputto”, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba and CIQUIBIC-CONICET, Ciudad Universitaria, 5000 Córdoba, Argentina b División de Neuropatología, Departamento de Patología, Facultad de Medicina, Universidad Católica de Córdoba, Córdoba, Argentina Received 18 January 2008; received in revised form 16 April 2008; accepted 15 May 2008 Available online 24 June 2008
Abstract The induction of neurological signs by immunization of rabbits with gangliosides has been a controversial topic for many years. Recently, Yuki et al. [N. Yuki, M. Yamada, M. Koga, M. Odaka, K. Susuki, Y. Tagawa, et al. Animal model of axonal Guillain–Barré syndrome induced by sensitization with GM1 ganglioside. Ann Neurol 2001;49:712–720.] described an immunization protocol, including keyhole lympet hemocyanin in addition to ganglioside that induced a neurological disease resembling human Guillain–Barré syndrome. We employed this protocol in our laboratory and succeeded in reproducing the disease. Five different experiments were performed during a period of two years by different operators, using different batches of drugs, in a total of 26 rabbits. Despite minor variations in onset time and severity of the induced disease, the model proved to be reproducible. Both gangliosides and keyhole limpet hemocyanin are required for induction of disease. © 2008 Elsevier B.V. All rights reserved. Keywords: Ganglioside; Anti-GM1 antibodies; Guillain–Barré; syndrome; Experimental neuropathy
1. Introduction Guillain–Barré syndrome (GBS) is an antibody-mediated acute neuropathy, considered a prototype of autoimmune disease [1]. The ganglioside GM1, a self-glycan present in neural tissues, has been proposed as one of the main antigenic targets [2]. Although not a common disease, GBS is a disabling process with a high rate of mortality [3]. Plasma exchange and intravenous immunoglobulins are useful therapeutic tools, but there is strong interest in the development of new therapies based on immune intervention at early stages of the disease. Research along this line is focused on the immune mechanisms involved in pathogenesis and pathophysiology, and several ⁎ Corresponding author. Facultad de Ciencias Químicas (UNC), Ciudad Universitaria, 5000 Córdoba, Argentina. Tel.: +54 351 433 4168. E-mail address: [email protected] (G.A. Nores). 1 Both authors contributed equally to this work. 0022-510X/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2008.05.006
laboratories have been searching for an animal model of the disease. In the mid-70's, Nagai et al. [4] described “ganglioside syndrome”, a motor neuropathy induced by immunization with GM1. This concept was long considered controversial, but is receiving renewed attention in view of recent evidence for the key role of anti-ganglioside antibodies in neuropathy. One reason for the controversy is that preparation of rabbit anti-GM1 antibodies is a common procedure in several laboratories where ganglioside function is studied, including ours [5], and these rabbits do not exhibit motor neuropathy. Inoculation of rabbits with GM1 in Freund's adjuvant results in high titer of anti-GM1 IgG antibodies, but causes only minor electrophysiological and pathological changes [6] and no clinical signs [7]. In 1996, using a slightly different immunization protocol and GD1b as immunogen, Kusunoki et al. [8] succeeded in inducing an experimental sensory ataxic neuropathy in rabbits. Based on this new protocol, Yuki et al. [9] developed an experimental model of GBS: rabbits immunized with bovine brain gangliosides or
A.L. Moyano et al. / Journal of the Neurological Sciences 272 (2008) 110–114
GM1 produced high titers of anti-GM1 IgG antibodies, but after 5 to 10 weeks they also showed clinical and pathological changes similar to those found in GBS patients. A significant modification in the new protocol was the use of keyhole limpet hemocyanin (KLH) as carrier for the immunogen; this protein was not used in previous protocols. However, Dasgupta et al. [10] reported that even using KLH, repeated immunizations with GM1 produced high titer of anti-GM1 IgG antibodies but did not induce neurological abnormalities. This report and the absence of reports from other labs describing successful induction of motor neuropathy by immunization with gangliosides illustrate the difficulty in the reproducibility of the model. We also failed to produce “ganglioside syndrome” in a 2002 experiment [7]. We now report a successful induction of a disease similar to that described by Yuki et al. [9], following their immunization protocol. 2. Materials and methods 2.1. Glycolipids Glycolipids GM1, GD1a, GD1b, GT1b, galactocerebroside and sulfatide were purified from human brain [11] and used as antigens. Asialo-GM1 (GA1) was prepared by acid hydrolysis of bovine brain gangliosides [12]. Total ganglioside fraction from bovine brain (Nx) was prepared from bovine brain by Folch extraction [13], DEAE-Sephadex [14], alkaline methanolysis, and reversed phase chromatography [15]. The ganglioside composition of this preparation was similar to that of commercial preparation Cronassial®.
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immunostaining as previously described [7]. Antibody affinity for GM1 was estimated by inhibition of antibody binding by soluble antigen [11]. Antibody binding to GM1 was quantified by enzyme-linked immunosorbent assay (ELISA) [7]. Titer values were calculated as the reciprocal of the serum dilution needed to obtain half-maximal antibody binding [16]. Rabbit sera were purified by GM1 affinity columns as described [7]. 2.4. Pathological studies Rabbits were deeply anesthetized by intramuscular injection of ketamine hydrochloride (35 mg/kg) and acepromazine (0.75 mg/kg), sciatic nerves were removed, and the animals were sacrificed. Nerve specimens were fixed by immersion in 4% (v/v) glutaraldehyde and 4% (w/v) formaldehyde in 0.1 M cacodylate buffer. Samples were treated with 1% osmium tetroxide, dehydrated, and embedded in Araldite. Sections (1 µm thick) were stained with toluidine blue and examined by an Axioplan light microscope (Carl Zeiss, Oberkochem, Germany). Digital images were obtained with a CCD camera (MICROMax, Princeton Instruments, Downingtown, PA, USA). 3. Results The protocol for our “experiment 1” was as described by Yuki et al. [9]. Rabbits were inoculated subcutaneously on the back and then intraperitoneally at 3 week intervals. GM1 and KLH emulsified in CFA were used as immunogens.
2.2. Immunization of rabbits New Zealand male white rabbits, weighing 2–3 kg, were immunized with GM1 or bovine brain gangliosides (BBG). For each immunization, 0.5 mg GM1 (Sygen®, GramonBagó, Uruguay), 2.5 mg commercial BBG (Cr, Cronassial®, Fidia, Padova, Italy), or 2.5 mg BBG prepared in our laboratory (Nx) were dissolved in 500 μl PBS containing 1 mg KLH (Sigma, St. Louis, MO, USA), and emulsified in 500 μl complete Freund's adjuvant (CFA). The animals were injected subcutaneously on the back, and intraperitoneally at 3week intervals until neurological signs appeared, or until 4 months after the first inoculation. Control animals were injected with the same inoculum without KLH or ganglioside, under the same protocol. Rabbits were weighed weekly. Blood samples were taken by ear vein puncture; sera were separated from blood clots and frozen at −70 °C until use. Five experiments with a total of 26 rabbits were performed. All experiments were performed in accordance with international and institutional guidelines for animal care. 2.3. Antibody binding assays Binding of rabbit antibody to GA1, GM1, GD1a, GD1b, GT1b, sulfatide and galactocerebroside was studied by TLC-
Fig. 1. Time course of disease. Body weight variation determined as the percentage of difference relative to week 0 of rabbits immunized with GM1/ KLH (● ○); KLH (■ □) or GM1 (▲ Δ). Animals were immunized at the indicated times (arrows) as described in Materials and methods. Horizontal bar indicates the appearance of neurological signs in rabbits immunized with GM1/KLH.
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Table 1 Disease induction in rabbits immunized with gangliosides Experiment
1
2
3
4
5
Rabbit
1.1 1.2 1.3 1.4 1.5 1.6 2.1 2.2 2.3 2.4 2.5 2.6 3.1 3.2 3.3 3.4 4.1 4.2 4.3 4.4 5.1 5.2 5.3 5.4 5.5 5.6
Immunogen KLH
Ganglioside
− − + + + + − − + + + + + + + + + + − − + + + + + +
GM1 GM1 − − GM1 GM1 GM1 GM1 − − GM1 GM1 GM1 GM1 BBG-Cr BBG-Cr BBG-Cr BBG-Cr BBG-Cr BBG-Cr GM1 GM1 BBG-Cr BBG-Cr BBG-Nx BBG-Nx
Disease onset (week)
Maximal clinical grade
− − − − 8 8 − − − − − 6 12 15 3 5 6 10 − − 7 8 6 5 5 7
0 0 0 0 2 2 0 0 0 0 0 4 4 2 3 4 4 4 0 0 2 2 4 4 3 4
Anti-GM1 IgG antibody Titer 0 0 0 0 810 1750 0 0 0 0 2370 2780 7180 4390 7410 9050 1030 3240 0 0 2070 1110 1500 1360 1300 3180
High affinity
ND ND
Absent Present Present Present ND ND ND ND
Present Present ND ND ND ND
Clinical grade: 0 = normal; 1 = weight lost; 2 = reversible weakness of the hind limbs, difficulty to walk; 3 = reversible weakness of the four limbs; 4 = nonreversible, severe weakness of the four limbs, death. Weight loss was considered the onset of the disease. Anti-GM1 IgG-antibody binding was determined at the time of appearance of neurological signs. Titer values were calculated as the reciprocal of the serum dilution needed to obtain half-maximal antibody binding [16]. Antibody affinity for GM1 was estimated by inhibition of antibody binding by soluble antigen [11]. Presence of high affinity antibodies refers to the presence in a serum of antibody binding inhibitable by soluble GM1 at 10− 8 M. Titer value 0 = binding not detectable at 1/100 dilution. BBG-Cr: commercial preparation of bovine brain gangliosides (Cronassial®); BBG-Nx: bovine brain gangliosides prepared in our laboratory.
Eight to 9 weeks after the initial immunization, both animals began to lose weight (Fig. 1). A few days later, they exhibited neurological signs such as tremor and hind limb weakness. These signs disappeared after a few more days,
and body weight was recovered. Control animals inoculated with either GM1 or KLH alone did not lose weight nor exhibit neurological signs. With some protocol variants, experiment 1 was repeated 4 times during a period of 2 years.
Fig. 2. Anti-GM1 IgG antibodies in ganglioside/KLH immunized rabbits. Serum was taken at the time of appearance of neurological signs and passed through a GM1 affinity column. Dilutions of whole serum (A, D, G) and the fractions retained (B, E, H) and not retained by GM1-column (C, F, I) of rabbits immunized with GM1 (# 1.6), BBG-Cr (# 5.3), and BBG-Nx (# 5.6) were assayed for IgG-immunoreactivity by TLC-immunostaining. GM1 and related glycolipids were used as antigen (orcinol plate). Serum dilution: 1/5000.
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Fig. 3. Light microscopy of toluidine-blue stained transverse sections of the sciatic nerve. A) Normal rabbit. B) Rabbit # 5.1(immunized with GM1/KLH). C) Rabbit # 3.4 (immunized with BBG-Cr/KLH). Note fibers with axonal degeneration (arrows). Scale bars = 20 µm.
Results are shown in Table 1. In this new set of experiments, control rabbits (immunized without KLH or gangliosides) did not show clinical signs. Rabbits immunized with GM1 and KLH either showed similar response as in experiment 1 (i.e. rabbit 5.2), developed more severe disease (rabbit 2.6), or showed no clinical signs (rabbit 2.5). In another experiment, rabbits were immunized with Cronassial®, a commercial preparation of BBG. Consistent with the findings of Yuki et al. [9], BBG were more efficient in inducing disease. Signs appeared early and most of the animals developed irreversible severe weakness of the four limbs. Similar results were obtained when animals were immunized with BBG prepared in our laboratory (Nx, experiment 5, Table 1). Considering the five experiments performed, 15 of 16 rabbits immunized with gangliosides plus KLH lose weight and develop neurological signs. Rabbit serum samples were screened for immunoreactivity against GM1 and structurally related glycolipids by TLCimmunostaining. As in our previous study [7], pre-immune sera displayed IgM-reactivity against GM1, GA1, and GD1b, but not against GD1a or GT1b (results not shown). In addition to this reactivity, rabbits immunized with GM1 or BBG together with KLH also showed IgG-reactivity against the above glycolipids (Fig. 2A, D, G). Antibody binding to all the glycolipids was totally abrogated by pre-absorption of serum with GM1 affinity column (Fig. 2), indicating that all IgG antibodies induced by GM1, as well as by BBG, are directed to GM1. Serum from control animals had nondetectable IgG-immunoreactivity. Anti-GM1 IgG antibody titer was measured by ELISA (Table 1). As expected, rabbits immunized with ganglioside and KLH had high titer values. High affinity of anti-GM1 antibodies have has been associated to disease onset in Yuki's rabbits [11]. Most of our rabbits immunized with GM1 and KLH showed the presence of high affinity antibodies (Table 1). Interestingly, the rabbit that developed a relatively high titer of antibodies but lacked those with high affinity did not get sick (Table 1, rabbit 2.5). Serum was also checked for immunoreactivity against galactocerebroside and sulfatide, two glycolipids that have been described as targets for antibodies associated to
neuropathy [17,18]. At the appearance of neurological signs, while anti-GM1 IgG-immunoreactivity towards GM1 was high, antibody binding to these glycolipids was not detected (results not shown). Transverse sections of sciatic nerves were screened for lesions. Consistent with the findings of Yuki et al. [9], rabbits immunized with GM1/KLH or BBG/KLH showed variable degrees of axonal degeneration (Fig. 3). Demyelination and remyelination were not observed. 4. Discussion An animal model developed by biomedical researchers can be one of their best tools for the management of human diseases. To be useful, a model must have “reproducibility”, i.e., it should be easily produced in laboratories other than the one that described it. Until recently, an animal model of GBS was not available. An early report by Nagai et al. [4], of a motor neuropathy induced by immunization with GM1, was controversial because of lack of reproducibility. The more recent model described by Yuki et al. [9] appeared to fulfill the requirements for a model of GBS. However, despite the fact that Yuki's group successfully reproduced the experimental disease [19], even using a different rabbit breed [20], the model was questioned by other labs [10,21]. We attempted for several years to induce motor neuropathy by immunization with GM1 [7], but did not succeed until we used Yuki's immunization protocol. The disease induced in our rabbits is similar to that in Yuki's rabbits, i.e., both are flaccid, mainly axonal, motor neuropathies with similar onset time. The disease is associated with the presence of anti-GM1 IgG antibodies, which were detected not only in rabbits immunized with GM1 but also in those immunized with a mixture of BBG. It should be noted that, although the BBG mixture used as immunogen contained gangliosides other than GM1 (such as GD1a and GT1b), only immunoreactivity against GM1 was produced. This finding is consistent with the hypothesis that “disease-associated” anti-GM1 antibodies originated from “normal occurring” counterparts (“binding site drift” hypothesis; Lopez et al.
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[22]), and could explain why both immunogens induce a similar disease since, in both cases, only anti-GM1 antibodies are generated. This is the first study in which the GBS model induced by ganglioside immunization was successfully reproduced outside Japan. The disease was induced in 5 different experiments in our laboratory, conducted by different operators over a period of more than 2 years, during which time we also used different lots of drugs (adjuvant, gangliosides, KLH). The reproducibility of the experiments increases our confidence in the model as a useful tool for study of GBS and related diseases. In both our experiments and those of Yuki et al. [9], onset time and severity of the induced disease were highly variable. This variability, which is more evident in rabbits immunized with GM1, reflects a random component in the origin of the disease. This random component has at least two possible explanations: i. It is a characteristic intrinsically associated with the disease. For example, we described a “random component” in the origin of disease-associated antiGM1 IgM-antibodies in neuropathy patients [23]. ii. On the other hand, the variability may have a methodological origin; i.e., uncontrolled technical details in the immunization process may be important in overcoming self-tolerance to GM1. This explanation could reconcile the early observation of Nagai et al. [4], and later studies in which GM1 immunization produced variable disease ([9]; present work), subclinical disease [6] or even no disease[7,10]. Acknowledgements This work was supported by grants from SeCyT (UNC), CONICET, and ANPCyT, Argentina. A.L.M. had fellowship assistance from ANPCyT. R.C., M.E.A., and R.D.L. had fellowship assistance from CONICET. F.J.I. and G.A.N. are Career Investigators of CONICET. The authors thank Dr. Steve Anderson for editing and Dr. Carlos Mas for his help in images preparation.
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