| [Objective]The present study investigated the effect of sensory ganglia on the growth of cranial motor axons and its molecular mechanism by co-culturing a sensory ganglion and rhombomere8from hindbrain of chick embryo. It aims to demonstrate the factors of influencing growth of cranial motor axons more comprehensively and further to provide experimental evidence for nervous system development, repair and regeneration of motor neuron injury and other nervous system diseases.[Methods]The bilateral rhombomere8(r8) were dissected from the hindbrain of HH17-19chicken embryos and co-cultured in collagen Ⅰ gels with a sensory ganglion, including trigeminal, petrosal, nodose, geniculate, vestibuloacoustic and dorsal root ganglion which were isolated from different stages (HH18,25,30and36) chicken embryos. Neural explants were cultured in normal medium for4days. All cultures were immuno-stained with BEN antibodies, photographed and analyzed.The r8were cultured in collagen Ⅰ gels with medium which were added proteins of neurotrophic factors, BDNF (10ng/ml,25ng/ml,50ng/ml) or NGF (10ng/ml,25ng/ml,50ng/ml). All cultures were immuno-stained with3A10antibodies after cultured for4days and photographed. The number and length of cranial motor axons from r8were measured for statistical analysis.The r8were co-cultured with beads loaded with protein of BDNF (0.1μg/μl,0.5μg/μl,1.0μg/μl), NGF (0.1μg/μl,0.5μg/μl,1.0μg/μl) or BSA in collagen Ⅰ gels for4days. All cultures were immuno-stained with3A10antibodies, photographed and analyzed.Neutralizing antibodies against neurotrophic factors (20μg/ml BDNF and120μg/ml NGF) were added in medium for r8co-cultured with trigeminal ganglion, respectively. All cultures were immuno-stained with BEN antibodies after cultured for4days and photographed.The inhibitors of molecules which are associated with neurotrophins signaling pathway were added in medium for r8co-cultured with BDNF-beads, NGF-beads and trigeminal ganglion, respectively. The cultures were divided into Control group, DMSO group, DMSO+Tyrphostin A9group, DMSO+U73122group, DMSO+LY294002group and DMSO+U0126group according to the differences of medium. All cultures were immuno-stained with BEN antibodies after cultured for4days and photographed.When r8were co-cultured with sensory ganglion or beads, the side which was facing the sensory ganglion or beads was called ganglion or beads side, however, the side which was facing away the sensory ganglion or beads was called control side. Quantitative index of the cranial motor neuron axon growth is the number and length of new axons from r8. The number and length of cranial motor axons from r8were measured for statistical analysis.[Results](1) The effects of sensory ganglia on growth of cranial motor axons from r8. The axons from ganglion side of r8which was co-cultured with a trigeminal ganglion from HH25,30and36chicken embryos were significantly more and longer than from control side of r8(p<0.01, p<0.01). The axons from r8showed asymmetrical growth. However, when the ganglion was isolated from HH18chicken embryos, the axons from r8showed symmetrical growth. The axons from ganglion side of r8which was co-cultured with a petrosal, nodose or vestibuloacoustic ganglion from HH30and36chicken embryos were significantly more and longer than from control side of r8(p<0.01or p<0.05). The axons from r8showed asymmetrical growth. However, when these ganglia were isolated from HH18and25chicken embryos, the axons from r8showed symmetrical growth. The axons from ganglion side of r8which was co-cultured with a geniculate ganglion from HH36chicken embryos were significantly more and longer than from control side of r8(p<0.01, p<0.01). The axons from r8showed asymmetrical growth. However, when the ganglion was isolated from HH18,25and30chicken embryos, the axons from r8showed symmetrical growth. When r8was co-cultured with a dorsal root ganglion was isolated from HH18,25,30and36chicken embryos, the axons from ganglion side of r8were significantly more and longer than from control side (p<0.01or p<0.05).(2) The effects of BDNF and NGF protein added in medium on growth of cranial motor axons from r8:The axons from r8of the BDNF groups were significantly more and longer than from control group (p<0.01, p<0.01) and showed asymmetrical growth.. The axons from r8of the NGF groups were significantly more than from control group (p<0.01). The axons from r8of25ng/ml and50ng/ml NGF groups were significantly longer than from control group (p<0.05, p<0.05). However, the axons from r8of10ng/ml NGF groups were not significantly different with control group (p>0.05).(3) The effects of BDNF-beads and NGF-beads on growth of cranial motor axons from r8. In BDNF-beads groups, the axons from beads side of r8were significantly more and longer than from control side of r8(p<0.01, p<0.01), except for0.1μg/μl BDNF-beads group. In all NGF-beads groups, the axons from beads side of r8were significantly more and longer than from control side of r8(p<0.01or p<0.05). The axons from ganglion side of r8which was co-cultured with BSA-beads were not significantly different with from control side (p>0.05, p>0.05).(4) The effect of BDNF-Ab and NGF-Ab on cranial motor axon growth. The axons from ganglion side of r8which was co-cultured with a trigeminal ganglion from HH25chicken embryos were not significantly different with from control side in BDNF-Ab group and NGF-Ab group (p>0.05, p>0.05). But in Control group, the axons from ganglion side of r8were significantly more and longer than from control side of r8(p<0.01, p<0.01).(5) The effects of inhibitors on cranial motor axon growth.①The effect of inhibitors on growth of cranial motor axon from r8which was co-cultured with BDNF-beads:In DMSO group, the axons from r8showed symmetrical growth and were not significantly different with Control group. In DMSO+Tyrphostin A9group, the axons from r8had no growth. The axons from r8showed symmetrical growth in DMSO+U73122group, DMSO+LY294002group and DMSO+U0126group.②The effect of inhibitors on growth of cranial motor axon from r8which was co-cultured with NGF-beads. The result is same with①.③The effect of inhibitors on growth of cranial motor axon from r8which was co-cultured with a trigeminal ganglion:In DMSO group, the axons from r8showed asymmetrical growth and were not significantly different with control group. In DMSO+Tyrphostin A9group, the axons from r8had no growth. The axons from r8in DMSO+U73122group, DMSO+LY294002group and DMSO+U0126group showed asymmetrical growth and no significant difference with DMSO group.[Conclusion]These results demonstrate that trigeminal, petrosal, nodose, geniculate, vestibuloacoustic and dorsal root ganglion of chick embryo can promote the growth of cranial motor axons. The promotion of these sensory ganglia, except for dorsal root ganglion, on cranial motor axons growth is stage-dependent. Our results suggest that BDNF and NGF mediate the effect of sensory ganglia on growth of cranial motor axons. PLCyγ、PI-3K and MEK participated in the Trk-mediated BDNF and NGF signaling pathway of cranial motor axons growth. |
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