Wowro Sylvia J, Tong Giang, Krech Jana, Rolfs Nele, Berger Felix, Schmitt Katharina R L
Department of Congenital Heart Disease/Pediatric Cardiology, Universitäres Herzzentrum Berlin - Medical Heart Center of Charité and German Heart Institute Berlin, Berlin, Germany.
Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany.
Front Cell Neurosci. 2019 Jun 25;13:273. doi: 10.3389/fncel.2019.00273. eCollection 2019.
Hypothermia attenuates cerebral ischemia-induced neuronal cell death associated with neuroinflammation. The calcineurin inhibitor cyclosporin A (CsA) has been shown to be neuroprotective by minimizing activation of inflammatory pathways. Therefore, we investigated whether the combination of hypothermia and treatment with CsA has neuroprotective effects in an oxygen-glucose deprivation/reperfusion (OGD/R) injury model in neuronal and BV-2 microglia monocultures, as well as in an organotypic hippocampal slice culture (OHSC).
Murine primary neurons, BV-2 microglia, and OHSC were pretreated with CsA and exposed to 1 h OGD (0.2% O) followed by reperfusion at normothermia (37°C) or hypothermia (33.5°C). Cytotoxicity was measured by lactate dehydrogenase and glutamate releases. Damage-associated molecular patterns (DAMPs) high mobility group box 1 (HMGB1), heat shock protein 70 (Hsp70), and cold-inducible RNA-binding protein (CIRBP) were detected in cultured supernatant by western blot analysis. Interleukin-6 (IL-6), Interleukin-1α and -1β (IL-1α/IL1-β), tumor necrosis factor-α (TNF-α), monocyte chemotactic protein 1 (MCP1), inducible nitric oxide synthase (iNOS), glia activation factors ionized calcium-binding adapter molecule 1 (Iba1), and transforming growth factor β1 (TGF-β1) gene expressions were analyzed by RT-qPCR.
Exposure to OGD plus 10 μM CsA was sufficient to induce necrotic cell death and subsequent release of DAMPs in neurons but not BV-2 microglia. Moreover, OGD/R-induced secondary injury was also observed only in the neurons, which was not attenuated by cooling and no increased toxicity by CsA was observed. BV-2 microglia were not sensitive to OGD/R-induced injury but were susceptible to CsA-induced toxicity in a dose dependent manner, which was minimized by hypothermia. CsA attenuated IL-1β and Iba1 expressions in BV-2 microglia exposed to OGD/R. Hypothermia reduced IL-1β and iNOS expressions but induced TNF-α and Iba1 expressions in the microglia. However, these observations did not translate to the OHCS model, as general high expressions of most cytokines investigated were observed.
Treatment with CsA has neurotoxic effects on primary neurons exposed to OGD but could inhibit BV-2 microglia activation. However, CsA and hypothermia treatment after ischemia/reperfusion injury results in cytotoxic neuroinflammation in the complex OHSC.
低温可减轻与神经炎症相关的脑缺血诱导的神经元细胞死亡。钙调神经磷酸酶抑制剂环孢素A（CsA）已被证明通过最小化炎症途径的激活而具有神经保护作用。因此，我们研究了低温与CsA联合治疗在神经元和BV-2小胶质细胞单培养以及器官型海马脑片培养（OHSC）的氧-葡萄糖剥夺/再灌注（OGD/R）损伤模型中是否具有神经保护作用。
将小鼠原代神经元、BV-2小胶质细胞和OHSC用CsA预处理，然后暴露于1小时的OGD（0.2%氧气），随后在正常体温（37°C）或低温（33.5°C）下进行再灌注。通过乳酸脱氢酶和谷氨酸释放来测量细胞毒性。通过蛋白质印迹分析在培养上清液中检测损伤相关分子模式（DAMPs）高迁移率族蛋白B1（HMGB1）、热休克蛋白70（Hsp70）和冷诱导RNA结合蛋白（CIRBP）。通过RT-qPCR分析白细胞介素-6（IL-6）、白细胞介素-1α和-1β（IL-1α/IL1-β）、肿瘤坏死因子-α（TNF-α）、单核细胞趋化蛋白1（MCP1）、诱导型一氧化氮合酶（iNOS）、胶质细胞激活因子离子钙结合衔接分子1（Iba1）和转化生长因子β1（TGF-β1）的基因表达。
暴露于OGD加10μM CsA足以诱导神经元坏死性细胞死亡和随后DAMPs的释放，但对BV-2小胶质细胞无此作用。此外，仅在神经元中观察到OGD/R诱导的继发性损伤，冷却并未减轻该损伤，且未观察到CsA增加毒性。BV-2小胶质细胞对OGD/R诱导的损伤不敏感，但对CsA诱导的毒性呈剂量依赖性敏感，低温可将其最小化。CsA减弱了暴露于OGD/R的BV-2小胶质细胞中IL-1β和Iba1的表达。低温降低了小胶质细胞中IL-1β和iNOS的表达，但诱导了TNF-α和Iba1的表达。然而，这些观察结果并未转化到OHCS模型中，因为观察到所研究的大多数细胞因子普遍高表达。
CsA治疗对暴露于OGD的原代神经元具有神经毒性作用，但可抑制BV-2小胶质细胞的激活。然而，缺血/再灌注损伤后CsA和低温治疗在复杂的OHSC中导致细胞毒性神经炎症。
