Characterisation of Primary Human Hippocampal Astrocyte Cell Culture Following Exposure to Hypoxia

Nurul Atikah Nor Nazli; Sangu Muthuraju; Farizan Ahmad; Abdul Aziz Mohamed Yusoff; Hasnan Jaafar; Shaharum Shamsuddin; Jafri Malin Abdullah

doi:10.21315/mjms2023.30.1.8

Characterisation of Primary Human Hippocampal Astrocyte Cell Culture Following Exposure to Hypoxia

Malays J Med Sci. 2023 Feb;30(1):92-106. doi: 10.21315/mjms2023.30.1.8. Epub 2023 Feb 28.

Authors

Nurul Atikah Nor Nazli^{1

2}, Sangu Muthuraju^{1

2}, Farizan Ahmad^{1

2}, Abdul Aziz Mohamed Yusoff^{1

2}, Hasnan Jaafar³, Shaharum Shamsuddin⁴, Jafri Malin Abdullah^{1

2}

Affiliations

¹ Department of Neurosciences, School of Medical Sciences, Universiti Sains Malaysia, Kelantan, Malaysia.
² Brain and Behaviour Cluster, School of Medical Sciences, Universiti Sains Malaysia, Kelantan, Malaysia.
³ Department of Pathology, School of Medical Sciences, Universiti Sains Malaysia, Kelantan, Malaysia.
⁴ Department of Biomedicine, School of Health Sciences, Universiti Sains Malaysia, Kelantan, Malaysia.

Abstract

Background: The present study aimed to understand the characterisation of human hippocampal astrocyte following hypoxia exposure. Based on the preliminary screening, 15 min was chosen as the time point and the cells were exposed to different oxygen percentages.

Methods: The Trypan blue viability assay used to examine cell death. Immunofluorescence assay, glial fibrillary acidic protein (GFAP) was used to portray the morphology of astrocytes. The hypoxia-inducible factor 1 (HIF-1) staining was performed to confirm hypoxia induced cell death and there was a dramatic expression of HIF-1α displayed in exposed astrocyte cells compared to the control. In molecular level, genes were chosen, such as glyceraldehyde 3-phosphate dehydrogenase (GAPDH), GFAP, HIF-1α and B-cell lymphoma 2 (Bcl-2) and ran the reverse transcription-polymerase chain reaction (RT-PCR).

Results: Microscope revealed a filamentous and clear nucleus appearance in a control whereas the rupture nuclei with no rigid structure of the cell were found in the 3% oxygen. The control and hypoxia cells were also stained with the annexin V-fluorescein isothiocyanate (annexin V-FITC). Fluorescence microscope reveals astrocyte cells after hypoxia showed higher expression of nuclei but not in control. Merging PI and FITC showed the differences of nuclei expression between the control and hypoxia. In the molecular analysis, there were significant changes of GFAP, HIF-1α and Bcl-2 in hypoxia exposed cells when compared to the control group.

Conclusion: Cells that were exposed to hypoxia (3% oxygen for 15 min) clearly showed damage. General view of human hippocampal astrocyte genomic response to hypoxia was obtained.

Keywords: B-cell lymphoma 2; HIF-1α; annexin V-fluorescein isothiocyanate staining; cell viability; glial fibrillary acidic protein marker; glyceraldehyde 3-phosphate dehydrogenase; human hippocampal astrocytes; hypoxia; morphological changes; oxygen percentage.