Effect of Hypoxia on Adult Müller Glia Cultures

Abstract

Background: The retina, a light-sensitive tissue of the central nervous system that is located at the posterior part of the eye, is particularly vulnerable to alterations in oxygen levels. In various retinal diseases, such as central retinal vein occlusion, glaucoma, and diabetic retinopathy, hypoxia (a condition of low oxygen levels) is commonly observed. Müller glia, the principal glial cells in the retina, play a crucial role in supporting the metabolic needs of retinal neurons. They are also responsible for sensing oxygen levels and, in response to hypoxia, express Hypoxia-Inducible Factor 1 (HIF-1), a transcription factor that activates signaling pathways related to hypoxia. Methods: In this study, primary rat Müller glial cells were cultured and exposed to a 1% oxygen for 72 h. Following this, immunohistochemical assays were conducted to assess the effects of hypoxia on various parameters, including HIF-1α expression, cell survival, Müller glia-specific markers (CRALBP and GS), gliosis (GFAP expression), apoptosis (caspase-3 expression), cell proliferation (Ki-67 expression), and metabolic stress (indicated by the number of mitochondria per cell). Results: Under hypoxic conditions, a decrease in Müller glial survival and proliferation was observed. Conversely, there was an increase in HIF-1α expression, GFAP expression, caspase-3-positive cells, and the number of mitochondria per cell. However, no significant changes were noted in the expression of the Müller glial markers GS and CRALBP. Conclusions: In conclusion, hypoxia resulted in reduced proliferation and survival of Müller glial cells, primarily due to increased apoptosis and heightened metabolic stress.

Keywords: Müller glia; hypoxia; metabolic stress; retina.

Figure 1

Methodology used in this experiment. Created with www.BioRender.com (accessed on 2 June 2025).

Figure 2

Analysis of survival and expression of CRALBP and GS in MG cultures under control and hypoxia conditions. Images of MG cultures taken under control (A,C,E) and hypoxia (B,D,F) conditions, in which the expressions of CRALBP (red) (A,B,E,F), GS (red) (C,D), and Vimentin (E,F) are observed. The immunostaining intensities and patterns of CRALBP and GS appeared unchanged when comparing the two conditions. In the analysis of cell survival, a significant decrease in the total number of cells in hypoxia is observed compared to the control (G). Changes in vimentin organization as observed by confocal microscopy at 63×. Nuclei were labeled with DAPI (blue). * p-value ≤ 0.05. Scale bar = 50 µm.

Figure 3

Effect of hypoxia on HIF-1α expression. Images of MG cultures taken under control (A,B) and hypoxia (C,D) conditions ((B,D) images are zoomed-in images of (A,C), respectively). It can be observed that under hypoxic conditions, there is a greater expression of HIF-1α marker. Cells were labeled with anti-HIF-1α (red), and the cell nuclei were labeled with DAPI (blue). Scale bar = 50 µm.

Figure 4

Analysis of apoptosis by expressing caspase 3. Images of MG cultures were taken under control (A) and hypoxia (B) conditions, in which the expressions of caspase 3 (red) and vimentin (green) are observed. A significant increase in the number of caspase 3-positive cells can be observed under hypoxic conditions compared to the control (C). Nuclei were labeled with DAPI (blue). * p-value ≤ 0.05. Scale bar = 50 µm.

Figure 5

Effect of hypoxia on the expression of the gliosis marker GFAP under control and hypoxia conditions. Images of MG cultures taken under control (A) and hypoxia (B) conditions, in which the expression of GFAP (green) is observed. A greater expression of GFAP can be observed under hypoxic conditions compared to the control (C). Nuclei were labeled with DAPI (blue). * p-value ≤ 0.05. ** p-value ≤ 0.01. Scale bar = 50 µm.

Figure 6

Analysis of MG proliferation through Ki-67 expression. Images of MG cultures taken under control (A) and hypoxia (B) conditions, in which the expression of Ki-67 (red) and vimentin (green) is observed. A significant decrease in the percentage of cells expressing Ki-67 can be observed under hypoxic conditions compared to the control (C). Nuclei were labeled with DAPI (blue). * p-value ≤ 0.05. Scale bar = 50 µm.

Figure 7

Analysis of mitochondria from MG by VDAC1 labeling. Images of MG cultures taken under control (A) and hypoxia (B) conditions, in which mitochondria labeled with the anti-VDAC1 antibody (red) and vimentin (green) can be observed. A significant increase in the number of mitochondria can be observed under hypoxic conditions (C). A significant increase in the number of mitochondria per µm² cell can also be observed for the same cell area (D,E). Therefore, it can be observed that the number of mitochondria labeled with the anti-VDAC1 antibody increases significantly under hypoxic conditions compared to the control. Nuclei were labeled with DAPI (blue). * p-value ≤ 0.05. Scale bar = 50 µm.