Sulforaphane (SFA) protects neuronal cells from oxygen & glucose deprivation (OGD)

Abstract

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.

Fig 1. Immunofluorescence analysis to confirm the characterization of control cortical brain cell cultures.

DAPI is a staining control and represents the nuclei of all cells. Cell specific markers used were: NSE for neurons, GFAP for astrocytes, and CD68 for microglia. A) Neuron culture B) Astrocyte culture, C) Co-culture. Scale shown is 20 μm.

Fig 2. LD50 for neuronal cell culture was determined using a live dead assay, and further analyzed by western blot and densitometry.

A) Neuronal cell cultures achieved an equal to or above 50% cell death at 2 hours OGD, data was normalized to control (0h OGD). B, C) Actin represents the loading control. Cell specific markers used were: NSE for neurons, GFAP for astrocytes, and CD68 for microglia. Data represented as mean±SEM, n≥3. One way ANOVA, and Dunnett’s Multiple Comparison Test was completed; *p<0.05, **p<0.01, ***p<0.001, ****p<0.001, compared to 0 hour OGD control.

Fig 3. LD50 for astrocyte cell culture was determined using a live dead assay, and further analyzed by western blot and densitometry.

A) Astrocyte cell cultures achieved an equal to or above 50% cell death at 8 hours OGD, data was normalized to control (0h OGD). B, C) Actin represents the loading control. Cell specific markers used were: NSE for neurons, GFAP for astrocytes, and CD68 for microglia. Data represented as mean±SEM, n≥3. One way ANOVA, and Dunnett’s Multiple Comparison Test was completed; *p<0.05, **p<0.01, ***p<0.001, ****p<0.001, compared to 0 hour OGD control.

Fig 4. LD50 for co-culture was determined using a live dead assay, and further analyzed by western blot and densitometry.

A) Co-cultures achieved an equal to or above 50% cell death at 10 hours OGD, data was normalized to control (0h OGD). B, C) Actin represents the loading control. Cell specific markers used were: NSE for neurons, GFAP for astrocytes, and CD68 for microglia. Data represented as mean±SEM, n≥3. One way ANOVA, and Dunnett’s Multiple Comparison Test was completed; *p<0.05, **p<0.01, ***p<0.001, ****p<0.001, compared to 0 hour OGD control.

Fig 5. SFA dose response for each brain cell-type culture in an OGD environment was determined using a live dead assay at previously determined LD50.

A) Neuronal cultures at LD50 of 2 hours OGD, no significant protection or toxicity, B) Astrocyte cultures at LD50 of 8 hours OGD, protection of SFA at 2.5 μM, significant toxicity of SFA ≥ 100 μM, C) Co-cultures at LD50 of 10 hours OGD, protection of SFA at 2.5 μM and toxicity of SFA ≥ 50 μM. Data represented as Mean±SEM, n≥3, One way ANOVA, and Dunnett’s Multiple Comparison Test was completed for all cultures; *p<0.05, **p<0.01, ***p<0.001, ****p<0.001, compared to respective controls (0 μM SFA).

Fig 6. SFA dose response for each brain cell-type control culture was determined using an AlamarBlue assay.

A) Neuronal cultures showed toxicity of SFA ≥ 100 μM, B) Astrocyte cultures showed toxicity of SFA ≥ 50 μM, C) Co-cultures showed toxicity of SFA ≥ 50 μM. Data represented as Mean±SEM, n≥3, One way ANOVA, and Dunnette’s Multiple Comparison Test was completed for all cultures; *p<0.05, **p<0.01, ***p<0.001, ****p<0.001, compared to respective controls (0 μM SFA).