2First Department of Oral and Maxillofacial Surgery, 3Department of Pediatric Dentistry, 4Department of Oral Anatomy, Nagasaki University School of Dentistry, 171, Sakamoto, Nagasaki, 8528102, 5Department of Applied Bio-organic Chemistry, Faculty of Agriculture, Gifu University, Gifu, 5011193, and 6Department of Biochemistry II, Nagoya University School of Medicine, Tsurumai, Nagoya, 4660065 Japan
Received on July 3, 2000; revised on September 22, 2000; accepted on September 22, 2000.
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Abstract |
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Key words: gangliosides/hypoglossal nerve/nerve regeneration/ceramide/carbohydrate
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Introduction |
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In many studies, ganglioside GM1 has been used, for example, in experimental rat Parkinsonism (Schneider et al., 1992). This is because GM1 is one of the major gangliosides in vertebrate brain tissues (Suzuki, 1965
). However, there have been no systematic studies on the neurotrophic effects of various glycosphingolipids, and no definite evidence to indicate that GM1 is a particularly excellent structure among ganglioside species.
In the present study, we compared the neurotrophic effects of various glycosphingolipids including neutral glycolipids using the rat hypoglossal nerve regeneration system. Then, we analyzed the effects of chemically synthesized gangliosides to eliminate the possibility that the effects were due to contaminants during purification. Furthermore, we compared the neurotrophic effects between the carbohydrate moiety and the lipid moiety to confirm the importance of the carbohydrate structures of gangliosides in their biological functions.
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Results |
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When the numbers of surviving neurons were examined, the majority of glycolipids examined showed fairly good effects as shown in Figure 1. Even LacCer treatment resulted in a neuron survival better than 60%, whereas the non-treated group showed about 15% survival. Significant differences were revealed in the number of surviving neurons between the untreated (5 mm resected group) and the autografted group or glycolipid-injected group (P < 0.005). As shown in Figure 3d, the majority of cell bodies on the right side (with the simple lesion) were lost after 10 weeks. The percent of the number of motor neurons in the right hypoglossal nerve (RHN) over the number in the left hypoglossal nerve (LHN) was 14.4 ± 5.3% (mean ± SD, n = 4). In contrast, the administration of any of the glycolipids to the proximal stump of the nerve in which the lesion had been made resulted in the survival of 6090% of motor neurons (Figure 1). Among glycolipids, GT1b showed an effect almost equivalent to those of the ganglioside mixture and autograft, and it was significantly better than those of LacCer (P < 0.005), GD1a (P < 0.005), GQ1b (P < 0.005), and GD1b (P < 0.05).
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Discussion |
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The present findings clearly showed that the carbohydrate structures of gangliosides are critical for the prevention of the lesion-induced death of motor neurons in the hypoglossal nerve nucleus, and in the promotion of the regeneration of the axotomized hypoglossal nerve. In particular, regeneration based on HRP-stained neurons in brain stem nuclei markedly varied depending on the glycolipid species. GT1b and GD1b showed better effects than GM1 or GD1a (representative structures of the a-series) in the regeneration of neurons (the number of HRP-positive neurons). GT1b, in particular, showed the best results among structures examined. GQ1b did not show so excellent effects as expected from a previous report (Tsuji et al., 1983). The relative importance of the carbohydrate structures in these compounds was also supported by the finding that oligosaccharides of GT1b or GD1b showed fairly marked effects, while ceramide did not. However, the presence of the 2-(trimethysilyl)-ethyl structure in these oligo-saccharides may have some significant roles to give a similar physico-chemical property as ceramide in native glycosphingolipids.
In addition, the finding that the effects on the neuron survival rate and those on the frequency of HRP-positive cells were not necessarily identical among the purified single gangliosides suggested that the prevention of neuronal cell death and the enhancement of neuronal regeneration were achieved by different mechanisms, that is, the former effect was relatively universal to glycolipids and the latter was relatively specific to the restricted carbohydrate structures in gangliosides. The protective effects of polypeptide neurotrophins on the motoneuronal death were examined only by surviving neuron numbers (Sendtner et al., 1990, 1992; Oppenheim et al., 1992
; Yan et al., 1992
). Therefore, the enhancement of neuronal regeneration might be unique for gangliosides.
The mechanisms by which gangliosides prevented the death of hypoglossal neurons and promoted their regeneration are presently not known. However, gangliosides have been reported to potentiate the in vivo and in vitro effects of NGF on central cholinergic neurons (Cuello et al., 1989; Panni et al., 1998
). Mutoh et al. and others reported that the function of a high-affinity NGF receptor (trkA) was markedly enhanced by the ganglioside GM1 (Mutoh et al, 1995
; Rabin and Mocchetti, 1995
). Recently, we demonstrated that introduction of the GD3 synthase gene into PC12 cells resulted in the continuous activation of NGF receptor TrkA and ERK1/2, leading to accelerated proliferation (Fukumoto et al., 2000
). These findings indicated that both exogenous and endogenous gangliosides can modulate the neurotrophin family/neurotrophin receptor interactions regardless of their mechanisms. Moreover, the mRNA expression of neurotrophins and their receptors was differentially regulated after peripheral nerve axotomy (Frisen et al., 1993
; Funakoshi et al., 1993
). Thus, gangliosides administered at injury sites might alter the expression of these neurotrophic factors and receptors and/or directly modulate the functions of those receptors resulting in the activation of downstream kinases or up-regulation of protein kinase genes such as mitogen-activated protein kinases (Kiryu et al., 1995
).
To clarify whether injected gangliosides were transported via axon to the brain stem, we tried immunodetection of GT1b after injection at the resection sites in GM2/GD2 synthase gene knock-out mice. We failed to detect GT1b (data not shown), which suggested that GT1b remained and exerted its role at the resection site, not at the brain stem.
The observations that motor neuron death can be prevented and the regeneration of an axotomized hypoglossal nerve can be promoted by exogenously added gangliosides may provide the basis for a novel therapeutic approach in the treatment of motor neuron injury, and also to the enhancement of the amelioration of other central and peripheral neuronal disorders (Svennerholm, 1994). These findings prompted us to analyze the influence of genetic disruption of complex gangliosides on the regeneration of cleaved hypoglossal nerve (Takamiya et al., 1996
). These analyses should reveal the real significance of endogenously-synthesized gangliosides in the nervous system, and provide experimental systems for the analysis of molecular mechanisms of ganglioside actions.
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Materials and methods |
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Resection of rat hypoglossal nerve
Experiments of rat hypoglossal nerve regeneration were performed as previously described (Itoh et al., 1999). Briefly, adult Wistar rats were anesthetized by an intraperitoneal injection of sodium pentobarbital, and a 5 mm segment of the right hypoglossal nerve (RHN) was removed (5 mm resected group). In the ganglioside-injected group, 2 µg (or 0.2 µg) ganglioside dissolved in phosphate-buffered saline (PBS) was injected into the nerve stump site. In the autografted group, the 5 mm sectioned hypoglossal nerve was placed back between the excised nerve stumps and sutured to the stump. Injection of synthetic gangliosides, neutral glycolipids, oligosaccharides and ceramides were also administered in a similar manner.
HRP injection and counting of neurons at the hypoglossal nerve nucleus
Ten weeks after these treatments, 20 µl of 30% horseradish peroxidase (HRP) (Toyobo, Osaka, Japan) in sterile saline was injected into the whole parts of the tongue of the rat as described previously (Streit and Reubi , 1977). After 24 h, the animals were anesthetized deeply and fixed by intracardial perfusion with 0.9% saline containing heparin-Na, then with 10% formalin in 0.1 M phosphate buffer. The lower brain stem was dissected, and 50 µm serial cross-sections were prepared on a freezing microtome. The sections were then incubated with a mixture of 3,3'-diaminobenzidine and hydrogen peroxide at room temperature for 40 min (Svennerholm, 1994
), mounted on 3-aminopropyl-trienthoxysilane (Aldrich, Milwaukee, WI)-coated glass slides, and counterstained with 1% cresyl violet (Chroma, Kongen, Germany). Only cells containing a clearly visible HRP vesicle in the cytoplasm were quantitated in every fifth section, as described previously (Taniuchi et al., 1986
).
All these experimental protocols were approved by the Review Committee of Nagasaki University School of Dentistry, and met the guide-lines of the Japanese Governmental Agency. All efforts were made to minimize animal suffering to reduce the number of animals used, and to utilize alternatives to in vivo techniques, if available.
Immunohistochemistry
Expression of gangliosides in peripheral hypoglossal nerves and hypoglossal nerve nuclei in the brain stem was examined using monoclonal antibodies specific for individual ganglioside structures with frozen sections. Briefly, frozen sections were fixed in cold acetone at 20°C for 10 min, then washed in PBS 3 times at room temperature. After blocking with 1% bovine serum albumin in PBS for 1 h at room temperature, anti-ganglioside antibodies were added and incubated for 1 h at room temperature. After washing with PBS, antibody binding was detected with biotin-labeled second antibody and ABC methods (Vectastain). After incubation with DAB for 10 min, samples were washed and counter stained with 2% methyl green for 10 min. The antibodies used were as follows; R24 (GD3, 1:2000 of ascites), 3F8 (GD2, 1 µg/ml), 2590 (GM3 0.5 µg/ml), 1011 (GM2, 1:200 of ascites), 370 (GD1b, 1:500 of ascites), 549 (GT1b, 1:200 of ascites). For GM1, biotin-conjugated choleratoxin B (List Biological Laboratories, Campbell, CA) and avidin-FITC (EY Laboratories, San Mateo, CA) were used.
Statistical analysis
The results obtained were analyzed for significance based on Tukeys method (Stuart and Ord , 1991).
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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