Effects Of Wnt-3a Signal Protein On Proliferation And Differentiation Of Endogenous Neural Stem Cells Into Injured Spinal Cord In Rats

Objective:To evaluate the functional recovery of acute spinal cord injuried rats treated with Wnt-3a signal protein administration and to study the effects of Wnt signal pathway which was actived by the recombinant Wnt-3a signal protein on proliferation and differentiation of endogenous neural stem cells in adult rat spinal cord injury .Methods:①Forty Female Sprague Dawley adult rats (200-250g; 6-8 weeks of age) were randomly divided into two groups: laminectomy followed by spinal cord contusion (Group 1, n=20), contusion followed by treatment with the recombinant Wnt-3a signal protein (Group 2, n=20).②Moderate spinal cord injury(SCI) model was built by revised Allen method in forty Sprague Dawley adult rats at T10. The recombinant Wnt-3a signal protein was injected into the spinal cord through micropipette at the lesion site three days after injury. Control rats underwent reinjections with identical doses of normal saline.③Basso-Beattie-Bresnahan (BBB) scores was performed within 24 hours of injury and again at 7-day intervals up to 28 days to show the motor function recovery.④Four rats in each group were transcardially perfused with 4% paraformaldehyde either 14 or 28 days after SCI. Some dissected spinal cords was fixed in 4% paraformaldehyde and embeded in paraffin wax in routine and made coronal section continuously. A section was randomly taken from the sections of each rat for hematoxylin-eosin (HE) staining, light microscope observation. And the other dissected spinal cords was fixed in 2.5% glutaraldehyde. Electron microscope showed ultrastructural finding of the injuried spinal cord between two groups.⑤Another four rats were randomly chosen from each group either 14 or 28 days after SCI . BrdU (5-Bromodeoxyuridines) was administered beginning 48 hours prior to perfusion intraperitoneally every 12 hours. The dissected spinal cords was frozen and cut in serial transverse sections and submitted to immunohistochemistry of BrdU (Bromodeoxyuridines),microtubule-associated protein II(MAP-2) and glial fibrillary acidic protein (GFAP) , markers for endogenous neural stem cells,neuron and astrocytes. The number of BrdU+/MAP-2+ newborn neurons and BrdU+/GFAP+ newborn astrocytes were counted and estimated by counting eight different fields under×400 magnification.Results:①Rats that received Wnt3a protein 3 days after SCI exhibited higher BBB locomotor function scores compared with normal saline injected control animals (Groups 2=10.32±1.12, Group 1=8.64±1.00; p<0.05) at 14 days after injury. By 28 days , the variability of BBB scores between two groups was more obvious (Groups2 =16.94±1.18, Group 1 =9.89±1.29;p<0.01) .②At 14 days after injury, the HE staining of histology showed a little better in the group 2 than that in the group 1. And the morphology in the group 2 was close to the normal spinal cord tissue at 28 days after injury. Electron microscope observation showed axonal degenerations in the white matter in the group 2 14 days post-injury were more better than that in the group 1. At 4 weeks post-injury, only less axonal degenerations could be found in the white matter and the thin myelin sheath was mainly observed in the group 2.③The number of BrdU+/MAP-2+ newborn neurons and BrdU+/GFAP+ newborn astrocytes showed much difference between in group 1 and group 2 at 14 days after injury(P<0.05). Immunohistochemical analysis showed significant increases the number of the inducing differentiated newborn neurons from endogenous neural stem cells in Wnt-3a-treated rats compared with control rats at 14 days after injury. But the number of BrdU+/MAP-2+ newborn neurons and BrdU+/GFAP+ newborn astrocytes showed little difference between in group 1 and group 2 at 28 days post- injury(P<0.05).Conclusion:①Exogenous Wnt3a signal protein administration on the contused adult rat spinal cord actived the Wnt signal pathway, promoted the differentiation of endogenous neural stem cells into neurons and remyelination and inhibited the differentiation of endogenous neural stem cells into astrocytes.②The BBB locomotor function scores is obvious higher in rats that received Wnt3a protein than that in Group 1 at 28 days after injury. The results showed Wnt3a can promote the functional recovery after spinal cord injury.