TY - JOUR
T1 - Characterisation of Primary Human Hippocampal Astrocyte Cell Culture Following Exposure to Hypoxia
AU - Nazli, Nurul Atikah Nor
AU - Muthuraju, Sangu
AU - Ahmad, Farizan
AU - Yusoff, Abdul Aziz Mohamed
AU - Jaafar, Hasnan
AU - Shamsuddin, Shaharum
AU - Abdullah, Jafri Malin
N1 - Funding Information:
We acknowledge the Fundamental Research Grant Scheme (FRGS) from the Ministry of Education (MOE) Malaysia for their financial support to perform this work.
Publisher Copyright:
© Penerbit Universiti Sains Malaysia, 2023.
PY - 2023/2
Y1 - 2023/2
N2 - 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.
AB - 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.
KW - annexin V-fluorescein isothiocyanate staining
KW - B-cell lymphoma 2
KW - cell viability
KW - glial fibrillary acidic protein marker
KW - glyceraldehyde 3-phosphate dehydrogenase
KW - HIF-1α
KW - human hippocampal astrocytes
KW - hypoxia
KW - morphological changes
KW - oxygen percentage
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U2 - 10.21315/mjms2023.30.1.8
DO - 10.21315/mjms2023.30.1.8
M3 - Article
AN - SCOPUS:85149458763
SN - 1394-195X
VL - 30
SP - 92
EP - 106
JO - Malaysian Journal of Medical Sciences
JF - Malaysian Journal of Medical Sciences
IS - 1
ER -