Sulforaphane Protects Brain Cells from Oxygen & Glucose Deprivation

• Author / Creator

  Ladak, Zeenat

• Perinatal brain injury results in neurodevelopmental disabilities (NDDs) that are inclusive of motor, cognitive and mental health disease. Some of these include cerebral palsy, intellectual disability, autism, attention deficit disorder and even schizophrenia. One of the most prominent etiologies is placental insufficiency which results in a hypoxic-ischemic (HI) environment in-utero. This leads to perinatal compromise, characterized by fetal growth restriction and brain injury. Since 80% of perinatal brain injuries occur during gestation, preventative approaches are needed to reduce or eliminate the potential for injury to the fetus and subsequent NDDs. Sulforaphane (SFA) derived from cruciferous vegetables such as broccoli sprouts (BrSps) is a phase-II enzyme inducer that enhances the production of antioxidants in the brain through the glutathione pathway. We have previously shown profound in-vivo neuro-protective effects of BrSp/SFA as a dietary supplement in pregnant rat models of both placental insufficiency and fetal inflammation. Strong evidence also points to a role for SFA as treatment for various cancers. Paradoxically, then SFA could enhance cell survival as a neuroprotectant, but with cancer, works to increase cell death. It is therefore important to determine the dosing parameters around which SFA is safe for the fetus, beneficial for brain protection, and which dosing range is toxic to cancer cells, and therefore may be very detrimental for use in the pregnant mother. 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 placental insufficiency. We developed a cell culture model of primary cortical neuronal, astrocytes 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 duration of OGD required to reach 50% cell death (LD50). We then evaluated the efficacy of varying doses of SFA for neuroprotective and neurotoxicity effects at LD50. 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 effect of SFA was assessed by Live/Dead assay (IF/high content microscopy), and toxicity effect of SFA was assessed by both AlamarBlue viability and Live/Dead assay.

  We determined LD50 to be 2 hours for neurons, 8 hours for astrocytes, and 10 hours for co-cultures, p<0.0001 compared to 0 hour OGD. The protective effect of SFA was noticeable at 2.5 µM for neurons, although not significant. There was a significant protective effect of SFA at 2.5 µM for astrocytes and co-cultures, p<0.05 compared to 0 µM SFA. Significant toxicity ranges were also confirmed in OGD cultures as ≥ 100 µM for astrocytes, ≥ 50 µM for co-cultures, p<0.05 compared to 0 µM SFA, but not toxic in neurons; and toxic in control cultures as ≥ 100 µM for neurons, and ≥ 50 µM for astrocytes and co-cultures, p<0.01 compared to 0 µM SFA. One Way ANOVA and Dunnett’s Multiple Comparison Test were used for statistical analysis.

  Our results indicate that cell death 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 SFA shows promise as a preventative agent for fetal ischemic brain injury that could be developed as a safe innovative therapy for the prevention of childhood NDD.

• Subjects / Keywords

  • sulforaphane
  • perinatal brain injury
  • neurodevelopmental disabilities
  • glutathione
  • anti-oxidants

• Graduation date

  Spring 2021

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-cs6z-y581

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.