Sharma Sourabh, Vidal Manuel, Paul Souvik, Han Arum, Menon Ramkumar, Richardson Lauren S
Division of Basic Science and Translational Research, OBGYN Department, University of Texas Medical Branch at Galveston, Galveston, TX, United States.
College of Medicine, San Beda University, Manila, Philippines.
Front Cell Dev Biol. 2025 Mar 12;13:1543710. doi: 10.3389/fcell.2025.1543710. eCollection 2025.
Glutamate dysregulation leading to neuronal excitotoxicity and neuroinflammation are associated with neurological disorders, specifically autism spectrum disorders (ASD) in preterm neonates. The lack of physiologically relevant models has limited mechanistic understanding of glutamate dysregulation and neuroinflammation during pregnancy. This study evaluated the effect of environmental pollutant and flame retardant, Polybrominated Diphenyl Ethers (PBDE) 99 and 47, on cell viability, glutamate dysregulation, and neuroinflammation using a microphysiologic system (MPS) of human fetal blood-brain-barrier organ on a chip (FB-OOC).
The FB-OOC is composed of 3-cell culture chambers, connected by microchannels, containing 1) human brain microvessel endothelial cells (HBMEC), 2) human vascular pericytes (HBVP), and 3) a triculture of neurons, astrocytes, and microglia in a 5:2:1 ratio, respectively. To assess the effect of toxicants on glutamate dysregulation and neuroinflammation, control (standard media) endothelial cells were exposed to PBDE 99 and 47 (150 ng/mL). To mimic the passage of PBDE through the placenta, endothelial cells in FB-OOC were exposed to conditioned PDBE media (1:1) derived from a placenta-OOC. In parallel, triculture cells were directly treated in a 96-well plate. Dextran propagation over 72 h confirmed FB barrier function. The activation status of microglia was determined using immunocytochemistry for CD11 b and Iba1, respectively. Cell morphology (microscopy), cell cytotoxicity (Lactate Dehydrogenase and glutamate assays), and cytokines (multiplex assay) were measured.
Physiologic FB-OOCs were characterized by 1) viable cell cultures expressing standard cell morphologies and cell-specific markers, 2) barrier formation confirmed by decreased dextran propagation over 72 h, and 3) baseline glutamate and pro-inflammatory cytokine production. On-chip PBDE and placenta-derived metabolites of PBDE treatment in the endothelial chamber induced cell cytotoxicity and significant upregulation of glutamate in the triculture but did not induce neuroinflammation nor microglia activation compared to the controls. Conversely, 2D triculture experiments showed direct PBDE treatment-induced significant neuroinflammation (TNF-α, GM-CSF, IL-8) compared to PBDE placenta-derived metabolites or controls.
This study established an FB model that recreated intercellular interactions. We report PBDE-induced glutamate dysregulation, often associated with the development of ASD, independent of neuroinflammation.
谷氨酸调节异常导致神经元兴奋性毒性和神经炎症，这与神经系统疾病相关，尤其是早产儿的自闭症谱系障碍（ASD）。缺乏生理相关模型限制了对孕期谷氨酸调节异常和神经炎症机制的理解。本研究使用人胎儿血脑屏障器官芯片微生理系统（MPS）评估环境污染物和阻燃剂多溴二苯醚（PBDE）99和47对细胞活力、谷氨酸调节异常和神经炎症的影响。
血脑屏障器官芯片由3个细胞培养室组成，通过微通道相连，分别包含1）人脑微血管内皮细胞（HBMEC）、2）人血管周细胞（HBVP）和3）神经元、星形胶质细胞和小胶质细胞以5:2:1比例组成的三培养体系。为评估毒物对谷氨酸调节异常和神经炎症的影响，将对照（标准培养基）内皮细胞暴露于PBDE 99和47（150 ng/mL）。为模拟PBDE通过胎盘的过程，将血脑屏障器官芯片中的内皮细胞暴露于来自胎盘器官芯片的条件性多溴二苯醚培养基（1:1）。同时，将三培养细胞直接在96孔板中处理。72小时内葡聚糖扩散情况证实了血脑屏障功能。分别使用针对CD11b和Iba1的免疫细胞化学法测定小胶质细胞的激活状态。测量细胞形态（显微镜观察）、细胞毒性（乳酸脱氢酶和谷氨酸测定）和细胞因子（多重测定）。
生理性血脑屏障器官芯片的特征为：1）活细胞培养物表达标准细胞形态和细胞特异性标志物；2）72小时内葡聚糖扩散减少证实了屏障形成；3）基线谷氨酸和促炎细胞因子产生。与对照组相比，在内皮细胞培养室中进行多溴二苯醚及胎盘来源的多溴二苯醚代谢物处理，诱导了细胞毒性以及三培养体系中谷氨酸的显著上调，但未诱导神经炎症或小胶质细胞激活。相反，二维三培养实验显示，与多溴二苯醚胎盘来源的代谢物或对照组相比，直接用多溴二苯醚处理诱导了显著的神经炎症（TNF-α、GM-CSF、IL-8）。
本研究建立了一个重现细胞间相互作用的血脑屏障模型。我们报告了多溴二苯醚诱导的谷氨酸调节异常，这通常与自闭症谱系障碍的发展相关，且与神经炎症无关。
