Received on February 1, 1999; revised on April 20, 1999; accepted on April 20, 1999
We have examined the time course of the neuronal death and regeneration of rat axotomized hypoglossal nerve with various conditions of the nerve resection, and established a useful system to measure neurotrophic activities of bioactive substances. In this system, neuronal death can be evaluated by counting surviving neurons in the nucleus of hypoglossal neuron at the brain stem, and the degree of the regeneration can be measured by counting horseradish peroxidase-positive cells at the same region after injection of horseradish peroxidase into tongue. Using this system, the effects of brain gangliosides on rat hypoglossal nerve regeneration following 5 mm transection were examined. The addition of a ganglioside mixture from bovine brain as well as the autograft strongly prevented the death of neurons and promoted the regeneration of the lesioned nerve at 10 weeks after the operation. Further analyses on the dose effects and injection sites of gangliosides were performed. Although the mechanisms of the neurotrophic effects of the gangliosides are unknown, the therapeutic application of gangliosides for neuronal degeneration is a promising approach.
Key words: ganglioside/hypoglossal nerve/nerve regeneration/neurotrophic factor/neuronal death
A current treatment for neural paralysis following trauma or surgical procedures is an attempt to stimulate nerve axonal regeneration by providing a tract with an autograft of relatively less important nerves such as cutaneous nerves of a lower extremity (Millesi et al., 1972). If some artificial materials can substitute for the autografts, additional surgery and resulting paralysis of the donor site could be avoided and the quality of life may be much improved.
There have been many studies suggesting a neurotrophic activity of gangliosides in cultured neuronal cells and in experimental animals (Schengrund, 1990). Many of these studies were performed by adding gangliosides to the culture medium of neuronal cell lines or primary neuron cultures (Ferrari et al., 1983, 1995; Favaron et al., 1988), and observing the subsequent changes of phenotypes such as morphology and the mode of proliferation. Gangliosides have been directly administered to experimental animals after generating artificial neurological damage or disorders by mechanical or chemical manipulation (Karpiak, 1984; Karpiak and Mahadik, 1984), the injection of toxic reagents (Schneider et al., 1992) or ischemic treatment (Karpiak et al., 1986), for example. Despite of many reports on the effects of gangliosides as a neurotrophic factor, there have been no studies to investigate the mechanisms of their actions.
Recently, the monosialoganglioside GM1 has been reported to enhance the activity of nerve growth factor (NGF) in NGF-responsive cells and to stimulate neuronal sprouting both in vitro and in vivo(Cuello et al., 1989).GM1 binds to the high-affinity NGF receptor trkA (Mutoh et al., 1995; Rabin and Mocchetti, 1995)and activates its function. However, detailed studies of the effects of gangliosides on long-term nerve regeneration in vivo have not been reported.
In the present study, we have examined the neuronal death and regeneration of rat axotomized hypoglossal nerve with various conditions of the nerve resection, and established a useful system to measure neurotrophic activities of bioactive substances. Then, the effects of brain gangliosides on rat hypoglossal nerve regeneration following the nerve transection were examined. The addition of a ganglioside mixture from bovine brain strongly prevented the death of neurons and promoted the regeneration of the lesioned nerve, as evaluated by the number of surviving neuronal cells and that of horseradish peroxidase (HRP)-positive cells in the nucleus of the hypoglossal nerve. Further analyses to investigate the dose effects and preference of injection sites of gangliosides were performed. The mechanisms of the neurotrophic effects of the gangliosides and the possible application of gangliosides for the therapy of neuronal degeneration are discussed.
Establishment of an effective system
We chose the rat hypoglossal nerve as an experimental system since it contains almost exclusively motor axons. The corresponding cell bodies are comprised of a homogenous, well-delineated nucleus in the ventral-medial region of the brain stem (Figure 1). Moreover, the retrograde and degenerative changes occurring after hypoglossal nerve lesion have been investigated in great detail (Snider and Thanedar, 1989).
Fig. 1. A scheme for the assay system of nerve regeneration with rat hypoglossal nerve. Usually 5 mm of axon was resected. HRP was injected into tongue and hypoglossal nerve nuclei was examined 10 weeks after the operation.
When the numbers of surviving neurons were examined based on the cresyl violet staining, significant differences were revealed in the number of surviving neurons between simply-cut group and 2.5 mm resected group, and other groups with longer sizes resected (Figure 2a). In the former two groups, more than 90% of neurons were surviving at 20 weeks after operation. In contrast, rats resected more than 5 mm showed about 20% of living neurons compared to the untreated side. As for the regeneration of axotomized hypoglossal nerve as counted by HRP-stained neurons at the nucleus, no significant living neurons were detected in the groups underwent the resection of more than 5 mm, while the simply-cut group and 2.5 mm resected group showed about 100% or 85% HRP-positive cells (RHN/LHN), respectively (Figure 2b). According to these results, we decided to further examine the neurotrophic effects of gangliosides by resection of 5 mm in length at 10 weeks after operation (Figure 3).
Fig. 2. Time course of neuron death and regeneration after resection of hypoglossal nerve with various sizes. Axons were resected as indicated at the bottom in the figure. Percent ratio of surviving neuron number and HRP positive neurons were counted as described in Materials and methods, and presented as RHN/LHN.
Fig. 3. Resection of rat hypoglossal nerve and recovery after 10 weeks. (a) RHN before resection (3×). (b) RHN just after 5 mm resection showing the cut edges (arrowheads) (3×). (c) LHN (arrow) and RHN (arrowhead) treated with 2 µg/10 µl of G-mix at 10 weeks after the operation (1.5×).
Protection of neuronal death and regeneration of axotomized hypoglossal nerve by G-mix
Pathological examination of resection sites. Using this newly established system, we analyzed the neurotrophic effects of bovine brain ganglioside mixture (G-mix). As shown in Figure 3c, right hypoglossal nerve (RHN) treated by G-mix after 5 mm resection showed complete regeneration macroscopically, and became rather thick at 10 weeks after operation. The operation sites were examined under microscope and showed contrastive effects of G-mix on the recovery of the resected hypoglossal nerve axon. As shown in Figure 4b, 5 mm resected samples showed marked degeneration of axons. On the other hand, ganglioside-treated samples exhibited well regenerated axons with even larger diameter (Figure 4c).
Analyses of the hypoglossal nerve nuclei. Then, hypoglossal nerve nuclei was examined by injecting HRP into the tongue at 10 weeks after the resection. As shown in Figure 5, significant differences were revealed in the number of surviving neurons between the 5 mm-resected group (a) and the autografted group (b) or ganglioside-injected group (c) (p < 0.001). The majority of the cell bodies on the right side with the simple lesion were lost after 10 weeks (Figure 5a). The percent of the number of neurons in the RHN over the number in the untreated side (LHN) was 14.4 ± 5.3% (mean ± SD, n = 4) in the 5 mm resected group. In contrast, the administration of bovine brain gangliosides (2 µg) to the proximal stump of the nerve in which the lesion had been made resulted in the survival of almost all neurons (Figure 5c). The ganglioside-injected group and the autografted group showed almost equivalent results (Figure 6a, lanes 1, 3, 4).
The number of HRP-positive neurons were compared after the injection of HRP. In the unoperated rats injected with HRP into the tongue, the number of HRP-positive neurons over that of cresyl violet-stained neurons in the RHN was 81.5 ± 4.9% (mean ± SD, n = 30) and that in the LHN was same as in the RHN. In the LHN of operated rats, the percent of the number of HRP-positive neurons over that of cresyl violet-stained motor neurons was 80.6 ± 4.5% (mean ± SD, n = 13), and was not significantly different from that of the intact animals. There were consistent numbers of HRP-positive neurons in the LHN in all groups (Figure 5a-c). The percent of the number of HRP-positive motor neurons in the RHN over that in the LHN was 0% (n = 4) in the 5 mm-resected group, 80.1 ± 10.3% (mean ± SD, n = 4) in the autografted group, and 85.2 ± 4.5% (mean ± SD, n = 5) in the ganglioside-injected group (Figure 5b, lanes 1, 3, 4). The percentage of HRP-positive neurons in the ganglioside-injected group was almost equivalent to that of the autografted group. There were significant differences between the 5 mm resected group and the autografted group or ganglioside-injected group (p < 0.001, respectively).Influence of injection sites of G-mix
The site of injection of G-mix was compared between the tongue and the lesion sites. Both ways of injection showed fairly well results in the survival of neuron (Figure 6a) and also in the regeneration (Figure 6b) compared to the autograft. Therefore, injected G-mix did not need to directly reach to the injection sites.
Fig. 4. Microscopic examination of the resection sites. (a) Untreated LHN (50×). (b) RHN 10 weeks after 5 mm resection (50×). (c) RHN treated with 2 µg/10 µl of G-mix at 10 weeks after the operation (50×).
Fig. 5. Section of the brain stem of rats at 10 weeks after the nerve resection. The hypoglossal nucleus of the intact side is located on the left-hand side. The hypoglossal nucleus containing the cell bodies of the neurons in which the resections and treatments were performed are located on the right-hand side. (a) A control animal after resection of 5 mm nerve. Almost all cell bodies on the side with the lesion were completely lost. (b) Autografted animal. Almost all cell bodies are seen, but about 85% of the HRP-positive cell bodies on the right-hand side nucleus are present compared to the unoperated side. (c) ganglioside-injected animal. The number of HRP-positive motor neurons on the ganglioside-injected side was almost equivalent to that of the unoperated side. Scale bar, 100 µm.
Fig. 6. Effects of the injection routs of G-mix on the number of survival of neurons and HRP-positive neurons after resection of the hypoglossal nerve. (a) Number of surviving neurons as a ratio of RHN/LHN. (b) Number of HRP-positive neurons as a ratio of RHN/LHN. The hypoglossal nerve was resected and either way of G-mix injection was performed as described in Materials and methods. The results are mean ± SE. *, p < 0.005.
Dose effects of G-mix
We then examined the effects of the doses of added gangliosides on the regeneration of the resected hypoglossal nerves. As shown in Figure 7a, percent ratios of surviving neurons of G-mix-added side to the unoperated side (RHN/LHN) were more than 80% in all conditions tried including those with 0.002 µg. However, samples added more amounts of G-mix showed higher survival rates. For the effects on the regeneration, dose effects were more striking. HRP-positive neurons were about 50% in the group with 0.002 µg G-mix, and the percent ratio increased with increasing amounts of G-mix up to 90% in the group of 2 µg (Figure 7b).
Fig. 7. Dose effects of added ganglioside mixture on the number of surviving neurons (a) and the HRP-positive neurons (b) after resection of the hypoglossal nerve. a, number of the surviving neurons as a ratio of RHN/LHN. The hypoglossal nerve was resected as described in Materials and methods, and treated with indicated amounts of G-mix. The results are mean ± SE. (b) Number of the HRP-positive neurons as a percent ratio of RHN/LHN. *, p < 0.05; **, p < 0.01; ***, p < 0.005
In many therapeutic trials of gangliosides for brain damages or neuronal degeneration with various causes, ganglioside treatment often showed positive effects presumably due to neurotrophic activity. However, it has been difficult to objectively evaluate the effects of gangliosides on neuronal functions in vivo with definite and quantitative criteria. Our system of hypoglossal nerve resection was found to be very useful to evaluate the resulting effects, and was relatively easy to perform. The degree of regeneration of the axotomized nerves was very clearly demonstrated by the numbers of HRP-stained cells at the nuclei, and actual recovery of the cleaved axon was confirmed in the histological analysis of the lesion sites. The results of our present experiments show that the local application of gangliosides prevented the lesion-induced death of motor neurons in the hypoglossal brain stem nucleus, and promoted the regeneration of the axotomized hypoglossal nerve in rats.
There have been many reports on the effects of the polypeptide neurotrophin family on the prevention of neuron death caused by degeneration after axotomy. For example, ciliary neurotrophic factor (CNTF) (Sendtner et al., 1990), brain-derived neurotrophic factor (BDNF) and NT3 (Oppenheim et al., 1992; Sendtner et al., 1992; Yan et al., 1992) were somewhat effective in the regeneration of motor neurons, such as those in the sciatic nerve (Yan et al., 1992) and facial nerve (Sendtner et al., 1992; Yan et al., 1992). In these systems, neonatal rats (Sendtner et al., 1990, 1992; Yan et al., 1992) or chick embryos (Oppenheim et al., 1992) were used, and the nerve fibers were simply transected. However, the recovery rate was around 50%. In contrast, gangliosides used in our study showed much higher recovery rates in the neurons which were resected at a 5 mm length using adult rats. Therefore, the neurotrophic effects of gangliosides demonstrated here seem to be equivalent to or even better than those of the NGF family members.
The mechanisms by which the gangliosides prevented the death of hypoglossal neurons and promoted their regeneration are not known at this time. However, it was reported that neuronal injury induced the alteration of ganglioside profiles (Bahr and Schlosshauer, 1989). The mRNA expression of various neurotrophins and their Trk receptors was also differentially regulated after a peripheral nerve axotomy (Funakoshi et al., 1993). For example, TrkB and p75NTR were demonstrated to be upregulated after nerve injury (Frisen et al., 1993; Funakoshi et al., 1993). Furthermore, gangliosides have been reported to potentiate the in vivo and in vitro effects of NGF on central cholinergic neurons (Cuello et al., 1989). Recently, Mutoh et al. reported that the function of a high-affinity NGF receptor (trkA) was markedly enhanced by the ganglioside GM1 (Mutoh et al., 1995). On the other hand, injury to hypoglossal motor neurons resulted in an increase in the mRNA levels of extracellular regulated kinase (ERK, or MAP kinase) and ERK kinase (MEK, or MAP kinase) genes (Kiryu et al., 1995).
Taking these findings into account, it is likely that the administration of gangliosides to injured hypoglossal neurons modulated the neurotrophin family/neurotrophin receptor interactions. Namely, gangliosides administered at the injury sites might promote the increased expression of these neurotrophic factors and receptors and/or directly enhance the functions of those receptors resulting in the upregulation of the mRNAs of those kinase genes, and in the enhanced regeneration of neurons in an autocrine manner.
The observations that the neuron death can be prevented and the regeneration of an axotomized hypoglossal nerve can be promoted by the injection of gangliosides may provide the basis for a novel therapeutic approach to the treatment of motor neuron injury and degeneration, and also to the enhancement of the amelioration of other central and peripheral neuronal disorders (Svennerholm, 1994). Further analyses of the neurotrophic activity of gangliosides including the comparison among carbohydrate structures and signaling pathway which mediates the stimulation of gangliosides in the cells remain to be performed.
Gangliosides
Gangliosides were purchased from Seikagaku Kogyo (Tokyo, Japan). They were applied for thin layer chromatography before use to confirm the composition and concentration.
Resection of rat hypoglossal nerve
Adult Wistar rats were anesthetized by an intraperitoneal injection of 20-30 mg/kg sodium pentobarbital, and various sizes of segments of the right hypoglossal nerve (RHN) were removed, i.e., 2.5 mm, 5 mm, 7.5 mm, and fully removed. In the ganglioside-injected group, various amount of ganglioside mixture (G-mix) (2 µg, 0.2 µg, 0.02 µg and 0.002 µg) dissolved in phosphate-buffered saline (PBS) was injected into the nerve stump site. In the autografted group, the 5 mm cut hypoglossal nerve was placed back between the excised nerve stumps and sutured to the stump. To compare the influence of injection sites of G-mix, G-mix was injected into tongue or the lesion site.
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 various parts of the tongue of the rat as described previously (Streit and Reubi, 1977). After 24 h, the animals were anesthetized deeply and perfused intracardially with 0.9% saline containing heparin-Na, and fixed 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[prime]-diaminobenzidine and hydrogen peroxide at room temperature for 40 min (Svennerholm, 1994), mounted on 3-aminopropyl-trienthoxysilane (Aldrich, Milwaukee, Wl)-coated glass slides, and counterstained with 1% cresyl violet (Chroma, Kongen, Germany). Only cells containing a clearly visible HRP vesicle in the cytoplasm were counted in every fifth section, as described previously (Taniuchi et al., 1986). Same counting procedure was performed for the untreated left hypoglossal nerve (LHN) and its result was used to obtain the percent ratio (RHN/LHN). To follow the time course of neuronal death and nerve regeneration, mice were sacrificed at 2, 4, 6, 8, 10 and 20 weeks after the operation, and served for the histological analyses as described above.
All these experimental protocols were approved by the Review Committee of Nagasaki University School of Dentistry, and meet the guidelines of 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.
Statistical analysis
The results obtained were analyzed for significance based on Tukey's method (Stuart and Ord, 1991).
We thank Professor A.Rokutanda, Department of Oral Anatomy, Nagasaki University School of Dentistry for valuable discussion; the staff of Laboratory Animal Research in the Animal Center for Biomedical Research, Nagasaki University School of Medicine for care of chronic experimental animal; and Mr. Sumihisa Honda, Scientific Data Center for the Atomic Bomb Disaster, Nagasaki University School of medicine for his advice on the statistical analyses. This work was supported by a Grant-in-Aid for Scientific Research of Priority Areas (10178104), and that of Core of Excellence from the Ministry of Education, Science, Sports and Culture of Japan.
NGF, nerve growth factor; HRP, horseradish peroxidase; RHN, right hypoglossal nerve; LHN, left hypoglossal nerve; G-mix; ganglioside mixture; PBS, phosphate-buffered saline; ERK, extracellular regulated kinase (or MAP kinase).
1To whom correspondence should be addressed