Impellizzeri Daniela, Ahmad Akbar, Di Paola Rosanna, Campolo Michela, Navarra Michele, Esposito Emanuela, Cuzzocrea Salvatore
Department of Biological and Environmental Sciences, University of Messina, Viale Ferdinando Stagno D'Alcontres, Messina 31-98166, Italy.
Department of Drug Sciences and Health Products, University of Messina, SS. Annunziata, Messina 98168, Italy.
Immunobiology. 2015 Sep;220(9):1039-49. doi: 10.1016/j.imbio.2015.05.013. Epub 2015 May 11.
Toll-like receptors (TLRs) are signaling receptors in the innate immune system that is specific immunologic response to systemic bacterial infection and injury. TLRs contribute to the initial induction of neuroinflammation in the CNS. In spinal cord injury (SCI) intricate immune cell interactions are triggered, typically consisting of a staggered multiphasic immune cell response, which can become deregulated. The present study aims to evaluate the role of TLR4 signaling pathway in the development of secondary damage in a mouse model of SCI using TLR4-deficient (TLR4-KO) mice such as C57BL/10ScNJ and C3H/HeJ mice. We evaluated behavioral changes, histological, immunohistochemistry and molecular assessment in TLR4-KO after SCI. SCI was performed on TLR4-KO and wild-type (WT) mice by the application of vascular clips (force of 24g) to the dura via a four-level T5-T8 laminectomy. Mice were sacrificed at 24h after SCI to evaluate the various parameters. SCI TLR4 KO mice developed severer hind limb motor dysfunction and neuronal death by histological evaluation, myeloid differentiation primary response 88 (Myd88) expression as well as an increase in nuclear factor NF-κB activity, tumor necrosis factor (TNF)-α and interleukin (IL)-1β levels, glial fibrillary acidic protein (GFAP), microglia marker (CD11β), inducible nitric oxide synthases (iNOS), poly-ADP-ribose polymerase (PARP) and nitrotyrosine expression compared to WT mice. Moreover, the absence of TLR4 also caused a decrease in phosphorylated interferon regulatory transcription factor (p-IRF3) and interferon (IFN-β) release. In addition, SCI TLR4 KO mice showed in spinal cord tissues a more pronounced up-regulation of Bax and a down-regulation of Bcl-2 compared to SCI WT mice. Finally, we clearly demonstrated that TLR4 is important for coordinating post-injury sequel and in regulating inflammation after SCI.
Toll样受体（TLRs）是先天性免疫系统中的信号受体，对全身性细菌感染和损伤具有特异性免疫反应。TLRs有助于中枢神经系统中神经炎症的初始诱导。在脊髓损伤（SCI）中，会引发复杂的免疫细胞相互作用，通常包括多阶段交错的免疫细胞反应，这种反应可能会失调。本研究旨在使用C57BL/10ScNJ和C3H/HeJ等TLR4缺陷（TLR4-KO）小鼠，评估TLR4信号通路在SCI小鼠模型继发性损伤发展中的作用。我们评估了SCI后TLR4-KO小鼠的行为变化、组织学、免疫组织化学和分子评估。通过四级T5-T8椎板切除术，对TLR4-KO和野生型（WT）小鼠的硬脑膜施加血管夹（24g力）来制造SCI。在SCI后24小时处死小鼠以评估各项参数。通过组织学评估、髓样分化初级反应88（Myd88）表达以及核因子NF-κB活性、肿瘤坏死因子（TNF）-α和白细胞介素（IL）-1β水平、胶质纤维酸性蛋白（GFAP）、小胶质细胞标志物（CD11β）、诱导型一氧化氮合酶（iNOS）、聚ADP-核糖聚合酶（PARP）和硝基酪氨酸表达增加，SCI TLR4 KO小鼠出现了更严重的后肢运动功能障碍和神经元死亡，与WT小鼠相比。此外，TLR4的缺失还导致磷酸化干扰素调节转录因子（p-IRF3）和干扰素（IFN）-β释放减少。此外，与SCI WT小鼠相比，SCI TLR4 KO小鼠脊髓组织中Bax的上调更为明显，而Bcl-2的下调更为明显。最后，我们清楚地证明了TLR4对于协调损伤后后遗症和调节SCI后的炎症很重要。
