Hlavac Nora, VandeVord Pamela J
Department of Biomedical Engineering and Mechanics, Virginia Polytechnic Institute, Blacksburg, VA, United States.
Department of Research, Salem Veterans Affairs Medical Center, Salem, VA, United States.
Front Neurol. 2019 Feb 22;10:99. doi: 10.3389/fneur.2019.00099. eCollection 2019.
Primary blast neurotrauma represents a unique injury paradigm characterized by high-rate overpressure effects on brain tissue. One major hallmark of blast neurotrauma is glial reactivity, notably prolonged astrocyte activation. This cellular response has been mainly defined in primary blast neurotrauma by increased intermediate filament expression. Because the intermediate filament networks physically interface with transmembrane proteins for junctional support, it was hypothesized that cell junction regulation is altered in the reactive phenotype as well. This would have implications for downstream transcriptional regulation via signal transduction pathways like nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Therefore, a custom high-rate overpressure simulator was built for testing using mechanical conditions based on intracranial pressure measurements in a rat model of blast neurotrauma. Primary rat astrocytes were exposed to isolated high-rate mechanical stimulation to study cell junction dynamics in relation to their mechano-activation. First, a time course for "classical" features of reactivity was devised by evaluation of glial fibrillary acidic protein (GFAP) and proliferating cell nuclear antigen (PCNA) expression. This was followed by gene and protein expression for both gap junction (connexins) and anchoring junction proteins (integrins and cadherins). Signal transduction analysis was carried out by nuclear localization of two molecules, NF-κB p65 and mitogen-activated protein kinase (MAPK) p38. Results indicated significant increases in connexin-43 expression and PCNA first at 24 h post-overpressure ( < 0.05), followed by structural reactivity (via increased GFAP, < 0.05) corresponding to increased anchoring junction dynamics at 48 h post-overpressure ( < 0.05). Moreover, increased phosphorylation of focal adhesion kinase (FAK) was observed in addition to increased nuclear localization of both p65 and p38 ( < 0.05) during the period of structural reactivity. To evaluate the transcriptional activity of p65 in the nucleus, electrophoretic mobility shift assay was conducted for a binding site on the promoter region for intracellular adhesion molecule-1 (ICAM-1), an antagonist of tight junctions. A significant increase in the interaction of nuclear proteins with the NF-κB site on the ICAM-1 corresponded to increased gene and protein expression of ICAM-1 ( < 0.05). Altogether, these results indicate multiple targets and corresponding signaling pathways which involve cell junction dynamics in the mechano-activation of astrocytes following high-rate overpressure.
原发性爆炸性神经创伤是一种独特的损伤模式，其特征是对脑组织产生高速超压效应。爆炸性神经创伤的一个主要标志是神经胶质反应，尤其是星形胶质细胞的长期激活。在原发性爆炸性神经创伤中，这种细胞反应主要通过中间丝表达的增加来定义。由于中间丝网络与跨膜蛋白在物理上相互作用以提供连接支持，因此推测在反应性表型中细胞连接调节也发生了改变。这将通过诸如活化B细胞核因子κ-轻链增强子（NF-κB）等信号转导途径对下游转录调节产生影响。因此，基于爆炸性神经创伤大鼠模型中的颅内压测量结果，构建了一种定制的高速超压模拟器，用于在机械条件下进行测试。将原代大鼠星形胶质细胞暴露于孤立的高速机械刺激下，以研究细胞连接动力学与其机械激活的关系。首先，通过评估胶质纤维酸性蛋白（GFAP）和增殖细胞核抗原（PCNA）的表达，设计了反应性“经典”特征的时间进程。随后检测了间隙连接（连接蛋白）和锚定连接蛋白（整合素和钙黏蛋白）的基因和蛋白表达。通过核定位两种分子NF-κB p65和丝裂原活化蛋白激酶（MAPK）p38进行信号转导分析。结果表明，超压后24小时，连接蛋白43表达和PCNA首先显著增加（P<0.05），随后是结构反应性（通过GFAP增加，P<0.05），对应于超压后48小时锚定连接动力学增加（P<0.05）。此外，在结构反应期，除了p65和p38的核定位增加外，还观察到粘着斑激酶（FAK）的磷酸化增加（P<0.05）。为了评估p65在细胞核中的转录活性，对细胞间粘附分子-1（ICAM-1，紧密连接的拮抗剂）启动子区域的结合位点进行了电泳迁移率变动分析。核蛋白与ICAM-1上NF-κB位点的相互作用显著增加，对应于ICAM-1的基因和蛋白表达增加（P<0.05）。总之，这些结果表明，在高速超压后星形胶质细胞的机械激活过程中，涉及细胞连接动力学的多个靶点和相应的信号通路。
