Lithium enhances axonal regeneration in peripheral nerve by inhibiting glycogen synthase kinase 3β activation

Huanxing Su, Qiuju Yuan, Dajiang Qin, Xiaoying Yang, Wai-Man Wong, Kwok-Fai So, Wutian Wu, Huanxing Su, Qiuju Yuan, Dajiang Qin, Xiaoying Yang, Wai-Man Wong, Kwok-Fai So, Wutian Wu

Abstract

Brachial plexus injury often involves traumatic root avulsion resulting in permanent paralysis of the innervated muscles. The lack of sufficient regeneration from spinal motoneurons to the peripheral nerve (PN) is considered to be one of the major causes of the unsatisfactory outcome of various surgical interventions for repair of the devastating injury. The present study was undertaken to investigate potential inhibitory signals which influence axonal regeneration after root avulsion injury. The results of the study showed that root avulsion triggered GSK-3β activation in the injured motoneurons and remaining axons in the ventral funiculus. Systemic application of a clinical dose of lithium suppressed activated GSK-3β in the lesioned spinal cord to the normal level and induced extensive axonal regeneration into replanted ventral roots. Our study suggests that GSK-3β activity is involved in negative regulation for axonal elongation and regeneration and lithium, the specific GSK-3β inhibitor, enhances motoneuron regeneration from CNS to PNS.

Figures

Figure 1
Figure 1
Lithium treatment suppressed GSK-3β activation in the avulsion-injured ventral horn. Immunostaining with p-GSK-3βTyr216 on cross-sections of the spinal cord of animals: (A) normal animals, (B) animals which received root avulsion, and (C) animals which received lithium treatment after root avulsion. ((a), (b), (c)) The arrow-pointed areas under higher magnifications. (D) The number of p-GSK-3βTyr216-positive neurons in the ventral horn at 24 h after root avulsion was significantly increased compared with that in the normal ventral horn and treatment with lithium markedly reduced avulsion-induced GSK-3β activation to the normal level (*P < 0.001; scale bar: 180 μm in (A), (B), and (C); 40 μm in (a), (b), and (c)).
Figure 2
Figure 2
Lithium treatment increased axonal regeneration and dendritic emanation of motoneurons after replantation of avulsed ventral roots. (A) A representative micrograph of spinal cross-sections showing FG-positive neurons (arrows) present in the ventral horn of the animals with ventral root reimplantation (VRI) plus saline treatment as controls. (a1) Micrographs made under higher magnification of the rectangular area in A showing that FG-labeled neurons extended their axons into the replanted ventral roots; (a2) micrographs made under higher magnification of the arrow-pointed area in A showing dendritic emanation of FG-labeled neurons in the ventral horn. (B) A representative micrograph of spinal cross-sections showing FG-positive neurons (arrows) present in the ventral horn of the animals with ventral root reimplantation (VRI) plus lithium treatment. (b1) Micrographs made under higher magnification of the rectangular area in B showing that FG-labeled neurons extended their axons into the replanted ventral roots; (b2) micrographs made under higher magnification of the arrow-pointed area in B showing dendritic emanation of FG-labeled neurons in the ventral horn. (C) A representative micrograph of spinal cross-sections showing FG-positive neurons (arrows) present in the ventral horn of the animals with ventral root reimplantation (VRI) plus SB injection. (c1) Micrographs made under higher magnification of the rectangular area in C showing that FG-labeled neurons extended their axons into the replanted ventral roots; (c2) micrographs made under higher magnification of the arrow-pointed area in C showing dendritic emanation of FG-labeled neurons in the ventral horn. (D) The number of regenerating motoneurons that extended axons into replanted ventral roots in the lithium-treated animals was significantly higher than that in the saline control animals (*P < 0.001). (E) The number of dendritic emanation from regenerating motoneurons in the lithium-treated animals was significantly higher than that in the saline control animals (#P < 0.05). Scale bar: 180 μm in A, B, and C; 75 μm in (a1), (a2), (b1), (b2), (c1), and (c2).
Figure 3
Figure 3
Effects of lithium treatment and ventral root reimplantation (VRI) on the survival of host motoneurons as revealed by neutral red staining 6 weeks after root avulsion. (A) Normal animals. (B) Animals receiving root avulsion only. (C) Animals receiving ventral root reimplantation (VRI) plus saline injection. (D) Animals receiving ventral root reimplantation (VRI) plus lithium treatment. (E) VRI significantly increased the survival rate of motoneurons compared to controls (*P < 0.001 compared to root avulsion only). Lithium treatment did not further increase the motoneuron survival after VRI. There were no statistically significant differences in the survival rate of motoneurons between saline- and lithium-treated animals. Scale bar: 150 μm.

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