2.5. Evaluation of the effect of Tetrapleura tetraptera methanol fruit extract on astrocytes derived from Neural Stem Cells subjected to anoxia

Astrocytes derived from human induced pluripotent stem cell (hiPSC) were used for this experiment. Human fetal brain tissue was procured following the informed consent of the mother. The brain tissues were processed according to approval by the Institutional Human Ethics and Stem Cell and Research Committee of National Brain Research Centre, India, in compliance with the Indian Council of Medical Research (ICMR). The telencephalon of human aborted fetuses (10–15 weeks old) was used to isolate fetal Neural Stem Cells (fNSCs). Isolation and differentiation of fNSCs to astrocytes were done as described by Fatima et al. [47] and Bhagat et al. [48].

Briefly, isolated neural stem cells (NSCs) were cultured in poly-d-lysine (Sigma-Aldrich, St. Louis, MO, USA) coated culture dishes in neurobasal media (Invitrogen, San Diego, CA, USA). The media was supplemented with Neural Survival Factor-1, N2 supplement, bovine serum albumin, glutamine, 25 ng/mL basic fibroblast growth factor (bFGF), 20 ng/mL epidermal growth factor (EGF), penicillin and streptomycin solutions, and gentamycin. The expression of neural stem cell markers, SRY-Box Transcription factor 2 (SOX2) and Nestin, and lineage-specific markers, Glial fibrillary acidic protein (GFAP) and microtubule-associated protein 2 (MAP2) were assessed for NSCs characterization using western blotting and immunocytochemistry. Almost 99 % of fNSCs showed immunoreactivity towards SOX2 and Nestin, and were negative for both GFAP and MAP2. Differentiation of NSCs into astrocytes was achieved by withdrawing neurobasal media and growing NSCs in miminum essential medium (MEM) supplemented with 10 % fetal bovine serum (FBS). Cells were maintained in MEM with FBS for atleast 21 days before using them for the experiments. Following trypsinization at the confluency of 80–90 %, cells were seeded for experiments in 8 well chamber slides or 12 well plates.

The prepared astrocytes were counted to know the number of cells per volume of the media. Briefly, 10 μL of the cell was added onto a hemocytometer and viewed under the microscope for cell counting. The population of cells was then calculated:

No.ofcellsperml=No.ofcellscountedonthefourgrids×dilutionfactorNoofgridscounted×10-4

For the 12 well plates, 100,000 cells were seeded; and for the 8 well chamber slide, 20,000 cells were seeded.

Severe hypoxic condition (anoxia) was set by flushing the hypoxia chambers with a gas mixture of ∼0.2 % oxygen (replaced with 94.9 % nitrogen) and 5.1 % carbon dioxide [13], while normoxia chambers were maintained at 18.0 % oxygen and 5.1 % carbon dioxide and 76.9 % nitrogen, with a pressure of 1.75 bar for both conditions.

Viable but activated astrocytes were analyzed morphologically using the live/dead assay.

The live/dead assay is used for assessing cell viability/death and was carried out using a live/dead viability/cytotoxicity assay kit for mammalian cells (Invitrogen Detection Technologies, Thermofisher, UK) according to the manufacturer’s instructions. This method is used for assessing cell viability/death in live cell culture of cells. It uses two components, namely; Calcein AM (CaAM), which is component A and Ethidium homodimer-1 (EthD-1), which is component B. Dead cells are characterized by intense fluorescence at over 600 nm and little fluorescence around 530 nm.

Astrocytes seeded in an 8-well chamber slide were allowed to adhere and grow to confluence for 14 h at 37 °C in a CO₂ incubator. Thereafter, cells were placed in a normoxic or severe hypoxic condition for 3 and 6 h. After 3 and 6 h, 100 μL of EthD-1 (1 μL/mL) and CaAM (0.2 μL/mL) were separately prepared in cell media (MEM), added to the cells, and then, incubated at 37^ºC for 10 min. Fluorescent images of the cells were taken at excitation/emission(ex/em) wavelength of 495 nm/515 nm for the green filter (live cells) and ex/em wavelength of 495 nm/635 nm for the red filter (dead cells) using an Invitrogen Floid microscope (Fisher Scientific, Sweden). Percentage cell death was calculated to get the number of dead cells.

Astrocytes were seeded onto a 24 well-plate (Corning Flat Bottom Transparent Polystyrol well plate) at a density of 5 × 10⁴ cells/well and were allowed to stay for 14 h at 37^ºC in a CO₂ incubator. The cells (n = 3) were treated for 24 h with 0.5, 1 and 10 μg/mL of TT dissolved in 0.01 % DMSO in the culture medium. Treatment with DMSO (0.01 % v/v culture medium) alone serves as the control. Thereafter, MTT assay was carried out.

MTT is a colorimetry method used for assessing cell viability and cytotoxicity for drug screening [49]. The MTT assay is based on the reduction of MTT (yellow colored) due to the activity of NAD(P)H-dependent oxidoreductase [50].

After 24 h of astrocyte treatment with the extract, 10 μl of MTT working solution (5 mg/mL) was added and then incubated in a CO₂ incubator for 3 h at 37^ºC. The medium was removed, and the formazan crystals formed were dissolved by adding 200 μl of DMSO and incubated in the dark inside a CO₂ incubator for 1 h at 37^ºC. Finally, the purple color of the dissolved formazan crystals was quantified in a plate reader (Infinite 200Pro Tecan, Switzerland) at 650 nm.

Cells (astrocytes) were seeded in a 24 well-plate at a density of 5 * 10⁴ cells/well (n = 3) and allowed to stay for 14 h at 37^ºC in a CO₂ incubator. Treatment with TT was performed 4 h before exposure to either 3 h normoxic (normoxia) or anoxic (anoxia) conditions. Two concentrations of TT (1 μg/mL and 10 μg/mL) were used and the treatments were as follows: cells treated with 1 μg/mL TT prior to anoxia (anoxia + TT 1 μg/mL); cells treated with 10 μg/mL TT prior to anoxia (anoxia + TT 10 μg/mL); cells treated with 10 μg/mL TT prior to normoxia (normoxia + TT 10 μg/mL). DMSO was used as a vehicle for TT.

After treatment, MTT assay was carried out to determine the percentage cell viability as previously described. Each assay was repeated twice.

The mitochondrial membrane potential (MMP) was measured by staining astrocytes with JC-1 (5,59,6,69-tetrachloro-1,19,3,39-tetraethylbenzimidazolyl-carbocyanine iodide) according to the method of Korenic et al. [51].

JC-1 is a ljipophilic and cationic dye that exhibits a fluorescence emission shift upon aggregation from 530 nm (green monomer) to 590 nm (red “J-aggregates’’ monomer). In healthy cells with high MMP, JC-1enters the mitochondrial matrix in a potential-dependent manner and forms aggregates.

Cells were seeded into groups as previously described. After treatment, cells were stained with 2.5 mg/mL JC-1 at 37^ºC for 15 min. Thereafter, cells were rinsed three times with PBS and the dye was allowed to equilibrate at room temperature for another 10 min before imaging using an Invitrogen Floid microscope (Fisher Scientific, Sweden). Stained polarized mitochondria were detected with red fluorescence while the loss of mitochondrial integrity was detected with green fluorescence. The fluorescence was read on a plate reader (Infinite 200Pro Tecan, Switzerland) with an excitation wavelength of 485 nm and the absorbance at 530 nm and 590 nm. The JC-1 fluorescence values were first normalized separately to their respective controls for red and green signals before the relative MMP was calculated.