Sulforaphane (SFA) protects neuronal cells from oxygen & glucose deprivation (OGD)
Authors: Ladak Zeenat , Garcia Elizabeth , Yoon Jenny , Landry Takaaki , Armstrong Edward A. , Yager Jerome Y. , Persad Sujata 
Published: March 2021

Background
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.

Methods
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.

Results
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.

Conclusions
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.