National Research Council of Italy-Cell Biology and Neurobiology Institute/Istituto Di Ricovero e Cura a Carattere Scientifico Fondazione Santa Lucia, Rome, Italy.
PLoS One. 2012;7(10):e47977. doi: 10.1371/journal.pone.0047977. Epub 2012 Oct 24.
In recent years a growing debate is about whether botulinum neurotoxins are retrogradely transported from the site of injection. Immunodetection of cleaved SNAP-25 (cl-SNAP-25), the protein of the SNARE complex targeted by botulinum neurotoxin serotype A (BoNT/A), could represent an excellent approach to investigate the mechanism of action on the nociceptive pathways at peripheral and/or central level. After peripheral administration of BoNT/A, we analyzed the expression of cl-SNAP-25, from the hindpaw's nerve endings to the spinal cord, together with the behavioral effects on neuropathic pain. We used the chronic constriction injury of the sciatic nerve in CD1 mice as animal model of neuropathic pain. We evaluated immunostaining of cl-SNAP-25 in the peripheral nerve endings, along the sciatic nerve, in dorsal root ganglia and in spinal dorsal horns after intraplantar injection of saline or BoNT/A, alone or colocalized with either glial fibrillar acidic protein, GFAP, or complement receptor 3/cluster of differentiation 11b, CD11b, or neuronal nuclei, NeuN, depending on the area investigated. Immunofluorescence analysis shows the presence of the cl-SNAP-25 in all tissues examined, from the peripheral endings to the spinal cord, suggesting a retrograde transport of BoNT/A. Moreover, we performed in vitro experiments to ascertain if BoNT/A was able to interact with the proliferative state of Schwann cells (SC). We found that BoNT/A modulates the proliferation of SC and inhibits the acetylcholine release from SC, evidencing a new biological effect of the toxin and further supporting the retrograde transport of the toxin along the nerve and its ability to influence regenerative processes. The present results strongly sustain a combinatorial action at peripheral and central neural levels and encourage the use of BoNT/A for the pathological pain conditions difficult to treat in clinical practice and dramatically impairing patients' quality of life.
近年来，人们对于肉毒神经毒素是否会从注射部位逆行运输存在越来越多的争议。免疫检测被肉毒神经毒素 A 型（BoNT/A）靶向的 SNARE 复合物的蛋白 SNAP-25 （cl-SNAP-25），可以代表一种极好的方法来研究外周和/或中枢水平伤害性通路的作用机制。在 BoNT/A 外周给药后，我们分析了 cl-SNAP-25 的表达，从足底神经末梢到脊髓，以及对神经病理性疼痛的行为影响。我们使用慢性坐骨神经缩窄损伤（CCI）作为神经病理性疼痛的 CD1 小鼠动物模型。我们评估了 cl-SNAP-25 在足底注射盐水或 BoNT/A 后，在外周神经末梢、坐骨神经、背根神经节和脊髓背角中的免疫染色，单独或与神经胶质纤维酸性蛋白（GFAP）、补体受体 3/分化簇 11b（CD11b）或神经元核（NeuN）共定位，取决于所研究的区域。免疫荧光分析显示 cl-SNAP-25 存在于从外周末梢到脊髓的所有检查组织中，提示 BoNT/A 的逆行运输。此外，我们进行了体外实验以确定 BoNT/A 是否能够与雪旺细胞（SC）的增殖状态相互作用。我们发现 BoNT/A 调节 SC 的增殖并抑制 SC 中的乙酰胆碱释放，证明了毒素的新生物学效应，并进一步支持毒素沿神经逆行运输及其影响再生过程的能力。这些结果强烈支持外周和中枢神经水平的组合作用，并鼓励使用 BoNT/A 治疗临床上难以治疗的病理性疼痛状况，这些状况严重影响患者的生活质量。
