ABSTRACT
Objective To investigate the effect of recombinant botulinum neurotoxin serotype A heavy chain (BoNT/A heavy chain)on local proteins which are related to nerve growth after spinal cord injury in rats,and to get some experimental evidence to explain the mechanism of BoNT/A heavy chain in stimulating neuritogenesis. Methods Recombinant botulinum neurotoxin serotype A heavy chain was applied locally or intrathecally to rats with ipsilateral semi-dissociated lumbar spinal injury. Local spinal tissue was extracted for general protein expression by two dimension electrophoresis plus nitrate silver staining after different time period of injury. Based on the results of 2-D gel electrophoresis,growth-associated protein 43(GAP-43)and of superior cervical ganglion 10(SCG 10)were selected to examine the changes of their expression and distribution features under BoNT/A heavy chain administration using SDS-PAGE,western blot and immunofluorescence. Results (1)The model of spinal cord injury(SCI)in this study was an ipsilateral semi-dissociated lumbar SCI in rat. The rats showed obvious motor and sensory dysfunction in the ipsilateral hind limb.(2)The results from 2-D gel electrophoresis plus nitrate silver staining showed that the administration of BoNT/A heavy chain based on SCI altered the local protein expression pattern. The decrease or increase in the expression of some protein dots /dots group was clearly seen after single SCI. However, these changes were transformed by BoNT/A heavy chain treatment,which appeared as a reversed pattern turning toward that in control group or further increased expression upon SCI,such as the dots located respectively at 35-45 kDa and 18-25 kDa level,pI between 5-7. In addition,the expression of the two dots located at the level as above increased after SCI only, and showed further increase in their expression with BoNT/A heavy chain intervention.(3)The changes of selective GAP-43 and SCG 10 expression and distribution by western blot and immunofluorescence indicated that the administration of BONT/A heavy chain based on SCI amplified the expression of GAP-43 and SCG 10(P < 0.05). Meanwhile,the positive immuonfluorescent staining for both GAP-43 and SCG 10 mainly distributed nearby the proximal area of injury, both cytoplasm and neuronal processes were positively stained. Conclusions Intrathecal delivery of BoNT/A heavy chain increases the expression of growth-associated proteins GAP 43 and SCG 10 after SCI in rats.
ABSTRACT
AIM:To observe the neuritogenic actions of botulinum neurotoxin serotype A heavy chain ( BoNT/A HC) on cultured Neuro-2a cells and to investigate the related signaling mechanisms for the effect of BoNT /A HC. METHODS:Neuro-2a cells were treated with different doses of BoNT/A HC (0.01, 0.1, 1 and 10 nmol/L), and then the cells were harvested at 24 h, 48 h and 72 h of BoNT/A HC exposure for detecting the neurite length and the percen-tage of the cells with neuronal processes by immunofluorescence staining .The most efficient dose of BoNT/A HC was cho-sen for exposure to Neuro-2a cells as the above.Whole cell protein was harvested at different time points for detecting the protein levels of phosphorylated ERK 1/2 ( p-ERK1/2 ) and phosphorylated Akt ( p-Akt ) by Western blot .RESULTS:Low doses of BoNT/A HC stimulated the neurite outgrowth , and increased the percentage of the cells with neurites com-pared with the negative controls (P<0.05), especially in the group with 1 nmol/L of BoNT/A HC treatment.Meanwhile, the phosphorylation of ERK 1/2 and Akt was increased after treated with BoNT/A HC.There was an increasing tendency for the phosphorylation of ERK1/2 after the exposure of the cells to BoNT/A HC.The obvious increase in p-ERK1/2 was seen from 60 min to 5 h with 1 nmol/L of BoNT/A HC treatment ( P<0.05 ) , and the increased protein level of p-Akt was mainly observed at 15 min and 60 min ( P<0.05 ) .CONCLUSION: BoNT/A HC stimulates the neuritogenesis .The neuritogenic mechanism of BoNT/A HC on Neuro-2a cells might be realized by activation of the phosphorylation of ERK 1/2 and Akt.