Neural Tissue/Electrode Interface and Neural Tissue Engineering lab, Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15260, United States.
Neural Tissue/Electrode Interface and Neural Tissue Engineering lab, Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15260, United States; Center for Neural Basis of Cognition, Pittsburgh, PA 15213, United States.
Acta Biomater. 2022 Sep 1;149:273-286. doi: 10.1016/j.actbio.2022.06.038. Epub 2022 Jun 25.
Microelectrode arrays for neural recording suffer from low yield and stability partly due to the inflammatory host responses. A neuronal cell adhesion molecule L1 coating has been shown to promote electrode-neuron integration, reduce microglia activation and improve recording. Coupling L1 to surface via a nanoparticle (NP) base layer further increased the protein surface density and stability. However, the exact L1-microglia interaction in these coatings has not been studied. Here we cultured primary microglia on L1 modified surfaces (with and without NP) and characterized microglia activation upon phorbol myristate acetate (PMA) and lipopolysaccharide (LPS) stimulation. Results showed L1 coatings reduced microglia's superoxide production in response to PMA and presented intrinsic antioxidant properties. Meanwhile, L1 decreased iNOS, NO, and pro-inflammatory cytokines (TNF alpha, IL-6, IL-1 beta), while increased anti-inflammatory cytokines (TGF beta 1, IL-10) in LPS stimulated microglia. Furthermore, L1 increased Arg-1 expression and phagocytosis upon LPS stimulation. Rougher NP surface showed lower number of microglia attached per area than their smooth counterpart, lower IL-6 release and superoxide production, and higher intrinsic reducing potential. Finally, we examined the effect of L1 and nanoparticle modifications on microglia response in vivo over 8 weeks with 2-photon imaging. Microglial coverage on the implant surface was found to be lower on the L1 modified substrates relative to unmodified, consistent with the in vitro observation. Our results indicate L1 significantly reduces superoxide production and inflammatory response of microglia and promotes wound healing, while L1 immobilization via a nanoparticle base layer brings added benefit without adverse effects. STATEMENT OF SIGNIFICANCE: Surface modification of microelectrode arrays with L1 has been shown to reduce microglia coverage on neural probe surface in vivo and improves neural recording, but the specific mechanism of action is not fully understood. The results in this study show that surface bound L1 reduces superoxide production from cultured microglia via direct reduction reaction and signaling pathways, increases anti-inflammatory cytokine release and phagocytosis in response to PMA or LPS stimulation. Additionally, roughening the surface with nanoparticles prior to L1 immobilization further increased the benefit of L1 in reducing microglia activation and oxidative stress. Together, our findings shed light on the mechanisms of action of nanotextured and neuroadhesive neural implant coatings and guide future development of seamless tissue interface.
用于神经记录的微电极阵列由于炎症宿主反应，其产量和稳定性较低。已经表明神经元细胞粘附分子 L1 涂层可以促进电极-神经元整合，减少小胶质细胞激活并改善记录。通过纳米颗粒 (NP) 基底层将 L1 偶联到表面进一步增加了蛋白质表面密度和稳定性。然而，这些涂层中 L1-小胶质细胞的确切相互作用尚未研究。在这里，我们在 L1 改性表面（有和没有 NP）上培养原代小胶质细胞，并在佛波醇肉豆蔻酸酯 (PMA) 和脂多糖 (LPS) 刺激下对小胶质细胞的激活进行了表征。结果表明，L1 涂层减少了小胶质细胞对 PMA 的超氧化物产生，并表现出内在的抗氧化特性。同时，L1 降低了 LPS 刺激的小胶质细胞中的诱导型一氧化氮合酶 (iNOS)、NO 和促炎细胞因子 (TNF alpha、IL-6、IL-1 beta)，同时增加了抗炎细胞因子 (TGF beta 1、IL-10)。此外，L1 增加了 LPS 刺激后的 Arg-1 表达和吞噬作用。与光滑的相比，粗糙的 NP 表面显示出附着的小胶质细胞数量较少、IL-6 释放和超氧化物产生较低，以及较高的内在还原电位。最后，我们通过双光子成像检查了 L1 和纳米颗粒修饰在体内 8 周内对小胶质细胞反应的影响。与未修饰的相比，发现植入物表面上 L1 修饰基底的小胶质细胞覆盖率较低，这与体外观察一致。我们的结果表明，L1 显著降低了小胶质细胞的超氧化物产生和炎症反应，并促进了伤口愈合，而通过纳米颗粒基底层固定 L1 则带来了额外的好处，没有不良影响。
L1 已被证明可以减少体内神经探针表面的小胶质细胞覆盖，从而改善神经记录，但具体作用机制尚不完全清楚。本研究结果表明，表面结合的 L1 通过直接还原反应和信号通路减少培养中小胶质细胞的超氧化物产生，增加 PMA 或 LPS 刺激后的抗炎细胞因子释放和吞噬作用。此外，在固定 L1 之前用纳米颗粒粗糙化表面进一步增加了 L1 减少小胶质细胞激活和氧化应激的益处。总之，我们的研究结果阐明了纳米纹理和神经粘附神经植入物涂层的作用机制，并为无缝组织界面的未来发展提供了指导。
