Faculty of Medicine & Dentistry, Department of Pediatrics, University of Alberta, Edmonton, Alberta, Canada.
PLoS One. 2021 Mar 18;16(3):e0248777. doi: 10.1371/journal.pone.0248777. eCollection 2021.
Perinatal brain injury results in neurodevelopmental disabilities (neuroDDs) that include cerebral palsy, autism, attention deficit disorder, epilepsy, learning disabilities and others. Commonly, injury occurs when placental circulation, that is responsible for transporting nutrients and oxygen to the fetus, is compromised. Placental insufficiency (PI) is a reduced supply of blood and oxygen to the fetus and results in a hypoxic-ischemic (HI) environment. A significant HI state in-utero leads to perinatal compromise, characterized by fetal growth restriction and brain injury. Given that over 80% of perinatal brain injuries that result in neuroDDs occur during gestation, prior to birth, preventive approaches are needed to reduce or eliminate the potential for injury and subsequent neuroDDs. Sulforaphane (SFA) derived from cruciferous vegetables such as broccoli sprouts (BrSps) is a phase-II enzyme inducer that acts via cytoplasmic Nrf2 to enhance the production of anti-oxidants in the brain through the glutathione pathway. We have previously shown a profound in vivo neuro-protective effect of BrSps/SFA as a dietary supplement in pregnant rat models of both PI and fetal inflammation. Strong evidence also points to a role for SFA as treatment for various cancers. Paradoxically, then SFA has the ability to enhance cell survival, and with conditions of cancer, enhance cell death. Given our findings of the benefit of SFA/Broccoli Sprouts as a dietary supplement during pregnancy, with improvement to the fetus, it is important to determine the beneficial and toxic dosing range of SFA. We therefore explored, in vitro, the dosing range of SFA for neuronal and glial protection and toxicity in normal and oxygen/glucose deprived (OGD) cell cultures.
OGD simulates, in vitro, the condition experienced by the fetal brain due to PI. We developed a cell culture model of primary cortical neuronal, astrocyte and combined brain cell co-cultures from newborn rodent brains. The cultures were exposed to an OGD environment for various durations of time to determine the LD50 (duration of OGD required for 50% cell death). Using the LD50 as the time point, we evaluated the efficacy of varying doses of SFA for neuroprotective and neurotoxicity effects. Control cultures were exposed to normal media without OGD, and cytotoxicity of varying doses of SFA was also evaluated. Immunofluorescence (IF) and Western blot analysis of cell specific markers were used for culture characterization, and quantification of LD50. Efficacy and toxicity effect of SFA was assessed by IF/high content microscopy and by AlamarBlue viability assay, respectively.
We determined the LD50 to be 2 hours for neurons, 8 hours for astrocytes, and 10 hours for co-cultures. The protective effect of SFA was noticeable at 2.5 μM and 5 μM for neurons, although it was not significant. There was a significant protective effect of SFA at 2.5 μM (p<0.05) for astrocytes and co-cultures. Significant toxicity ranges were also confirmed in OGD cultures as ≥ 100 μM (p<0.05) for astrocytes, ≥ 50 μM (p<0.01) for co-cultures, but not toxic in neurons; and toxic in control cultures as ≥ 100 μM (p<0.01) for neurons, and ≥ 50 μM (p<0.01) for astrocytes and co-cultures. One Way ANOVA and Dunnett's Multiple Comparison Test were used for statistical analysis.
Our results indicate that cell death shows a trend to reduction in neuronal and astrocyte cultures, and is significantly reduced in co-cultures treated with low doses of SFA exposed to OGD. Doses of SFA that were 10 times higher were toxic, not only under conditions of OGD, but in normal control cultures as well. The findings suggest that: 1. SFA shows promise as a preventative agent for fetal ischemic brain injury, and 2. Because the fetus is a rapidly growing organism with profound cell multiplication, dosing parameters must be established to insure safety within efficacious ranges. This study will influence the development of innovative therapies for the prevention of childhood neuroDD.
围产期脑损伤可导致神经发育障碍（neuroDDs），包括脑瘫、自闭症、注意力缺陷障碍、癫痫、学习障碍等。通常情况下，当胎盘循环受损，无法向胎儿输送营养和氧气时，就会发生损伤。胎盘功能不全（PI）是指胎儿血液和氧气供应减少，导致缺氧缺血（HI）环境。宫内严重 HI 状态会导致围产期损伤，表现为胎儿生长受限和脑损伤。由于导致 neuroDDs 的 80%以上的围产期脑损伤发生在妊娠期间，即分娩前，因此需要采取预防措施来减少或消除潜在的损伤和随后的 neuroDDs。来自西兰花芽（BrSps）等十字花科蔬菜的萝卜硫素（SFA）是一种 II 期酶诱导剂，通过细胞质 Nrf2 作用增强大脑中抗氧化剂的产生，途径是谷胱甘肽途径。我们之前的研究表明，BrSps/SFA 作为一种膳食补充剂，在 PI 和胎儿炎症的妊娠大鼠模型中具有显著的体内神经保护作用。强有力的证据还表明 SFA 可作为各种癌症的治疗方法。矛盾的是，SFA 具有增强细胞存活的能力，并且在癌症情况下，增强细胞死亡。鉴于我们发现 SFA/西兰花芽作为妊娠期间膳食补充剂的益处，可改善胎儿健康，因此确定 SFA 的有益和毒性剂量范围非常重要。因此，我们在体外研究了 SFA 对正常和氧/葡萄糖剥夺（OGD）细胞培养中的神经元和神经胶质保护和毒性的剂量范围。
OGD 模拟了 PI 导致胎儿大脑经历的情况。我们从新生啮齿动物大脑中开发了原代皮质神经元、星形胶质细胞和混合脑细胞共培养物的细胞培养模型。将培养物暴露于 OGD 环境中不同时间，以确定 LD50（导致 50%细胞死亡所需的 OGD 持续时间）。使用 LD50 作为时间点，我们评估了不同剂量的 SFA 对神经保护和神经毒性作用的疗效。对照培养物暴露于不含 OGD 的正常培养基中，并且还评估了不同剂量 SFA 的细胞毒性。使用免疫荧光（IF）和 Western blot 分析细胞特异性标志物来进行培养物特征描述和 LD50 的定量。通过 IF/高内涵显微镜和 AlamarBlue 活力测定分别评估 SFA 的疗效和毒性作用。
我们确定神经元的 LD50 为 2 小时，星形胶质细胞为 8 小时，共培养物为 10 小时。SFA 的保护作用在神经元中在 2.5 μM 和 5 μM 时是明显的，尽管它并不显著。SFA 在 2.5 μM 时对星形胶质细胞和共培养物具有显著的保护作用（p<0.05）。在 OGD 培养物中也确认了明显的毒性范围，即≥100 μM（p<0.05）用于星形胶质细胞，≥50 μM（p<0.01）用于共培养物，但神经元无毒；在对照培养物中，神经元≥100 μM（p<0.01），星形胶质细胞和共培养物≥50 μM（p<0.01）有毒。我们使用单因素方差分析和 Dunnett 多重比较检验进行统计分析。
我们的结果表明，神经元和星形胶质细胞培养物中的细胞死亡显示出减少的趋势，并且在接受低剂量 SFA 处理并暴露于 OGD 的共培养物中显著减少。在正常对照培养物中，即使在 OGD 条件下，SFA 的剂量也高达 10 倍，具有毒性。这表明：1. SFA 有望成为预防胎儿缺血性脑损伤的一种预防性药物，2. 由于胎儿是一个快速生长的生物体，具有深刻的细胞增殖，必须建立剂量参数，以确保在有效范围内的安全性。这项研究将影响预防儿童神经发育障碍的创新疗法的发展。
