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Neuroprotective effects of mild hypoxia in organotypic hippocampal slice cultures.

Kim SH, Lee WS, Lee NM, Chae SA, Yun SW - Korean J Pediatr (2015)

Bottom Line: In the 7-DIV group compared to control tissue, hypoxia-exposed tissue showed decreased damage in two regions (CA1: 5.59%±2.99% vs. 4.80%±1.37%, P=0.900; DG: 33.88%±12.53% vs. 15.98%±2.37%, P=0.166), but this decrease was not statistically significant.In the 14-DIV group, hypoxia-exposed tissue showed decreased damage compared to control tissues; this decrease was not significant in the CA3 (24.51%±6.05% vs. 18.31%±3.28%, P=0.373) or DG (15.72%±3.47% vs. 9.91%±2.11%, P=0.134), but was significant in the CA1 (50.91%±5.90% vs. 32.30%±3.34%, P=0.004).Although only CA1 tissues cultured for 14 DIV showed significantly less damage after exposure to hypoxia, the other tissues examined in this study showed a tendency towards less damage after hypoxic exposure.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Chung-Ang University College of Medicine, Seoul, Korea.

ABSTRACT

Purpose: The aim of this study was to investigate the potential effects of mild hypoxia in the mature and immature brain.

Methods: We prepared organotypic slice cultures of the hippocampus and used hippocampal tissue cultures at 7 and 14 days in vitro (DIV) to represent the immature and mature brain, respectively. Tissue cultures were exposed to 10% oxygen for 60 minutes. Twenty-four hours after this hypoxic insult, propidium iodide fluorescence images were obtained, and the damaged areas in the cornu ammonis 1 (CA1), CA3, and dentate gyrus (DG) were measured using image analysis.

Results: In the 7-DIV group compared to control tissue, hypoxia-exposed tissue showed decreased damage in two regions (CA1: 5.59%±2.99% vs. 4.80%±1.37%, P=0.900; DG: 33.88%±12.53% vs. 15.98%±2.37%, P=0.166), but this decrease was not statistically significant. In the 14-DIV group, hypoxia-exposed tissue showed decreased damage compared to control tissues; this decrease was not significant in the CA3 (24.51%±6.05% vs. 18.31%±3.28%, P=0.373) or DG (15.72%±3.47% vs. 9.91%±2.11%, P=0.134), but was significant in the CA1 (50.91%±5.90% vs. 32.30%±3.34%, P=0.004).

Conclusion: Although only CA1 tissues cultured for 14 DIV showed significantly less damage after exposure to hypoxia, the other tissues examined in this study showed a tendency towards less damage after hypoxic exposure. Therefore, mild hypoxia might play a protective role in the brain.

No MeSH data available.


Related in: MedlinePlus

Propidium iodide fluorescence image (×40). A single researcher measured the individual areas of the cornu ammonis 1 (CA1), CA3, and dentate gyrus (DG). Scale bar=400 µm.
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Figure 1: Propidium iodide fluorescence image (×40). A single researcher measured the individual areas of the cornu ammonis 1 (CA1), CA3, and dentate gyrus (DG). Scale bar=400 µm.

Mentions: Fluorescent images of the slices were captured under an inverted microscope (IX 71, Olympus, Tokyo, Japan) 24 hours after hypoxic injury. To introduce total damage, slices were then exposed to 2mM N-methyl-D-aspartate (NMDA) in serum-free medium for 30 minutes at 37℃ with 5% CO2 in a humidified incubator. Slices were then cultured with 1 mL of serum-free media with 1-µg propidium iodide (Sigma Aldrich), and fluorescent images were obtained 24 hours after total damage. Images were analyzed with Image J (ver. 1.47c, National Institutes of Health, Bethesda, MD, USA), using a defined threshold as the amount of fluorescent area with a single researcher measuring individual areas above the threshold in the cornu ammonis 1 (CA1), CA3, and dentate gyrus (DG) (Fig. 1). After NMDA processing, images were assumed to include the total area of the hippocampal tissue. We then estimated the extent of damage 24 hours after hypoxic injury. Tissues in the 7 DIV and 14 DIV groups were compared, and hypoxia-exposed tissues were compared with the control group (Fig. 2). The ratio of damaged area at 24 hours to the total damaged area after NMDA treatment was also calculated.


Neuroprotective effects of mild hypoxia in organotypic hippocampal slice cultures.

Kim SH, Lee WS, Lee NM, Chae SA, Yun SW - Korean J Pediatr (2015)

Propidium iodide fluorescence image (×40). A single researcher measured the individual areas of the cornu ammonis 1 (CA1), CA3, and dentate gyrus (DG). Scale bar=400 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4414629&req=5

Figure 1: Propidium iodide fluorescence image (×40). A single researcher measured the individual areas of the cornu ammonis 1 (CA1), CA3, and dentate gyrus (DG). Scale bar=400 µm.
Mentions: Fluorescent images of the slices were captured under an inverted microscope (IX 71, Olympus, Tokyo, Japan) 24 hours after hypoxic injury. To introduce total damage, slices were then exposed to 2mM N-methyl-D-aspartate (NMDA) in serum-free medium for 30 minutes at 37℃ with 5% CO2 in a humidified incubator. Slices were then cultured with 1 mL of serum-free media with 1-µg propidium iodide (Sigma Aldrich), and fluorescent images were obtained 24 hours after total damage. Images were analyzed with Image J (ver. 1.47c, National Institutes of Health, Bethesda, MD, USA), using a defined threshold as the amount of fluorescent area with a single researcher measuring individual areas above the threshold in the cornu ammonis 1 (CA1), CA3, and dentate gyrus (DG) (Fig. 1). After NMDA processing, images were assumed to include the total area of the hippocampal tissue. We then estimated the extent of damage 24 hours after hypoxic injury. Tissues in the 7 DIV and 14 DIV groups were compared, and hypoxia-exposed tissues were compared with the control group (Fig. 2). The ratio of damaged area at 24 hours to the total damaged area after NMDA treatment was also calculated.

Bottom Line: In the 7-DIV group compared to control tissue, hypoxia-exposed tissue showed decreased damage in two regions (CA1: 5.59%±2.99% vs. 4.80%±1.37%, P=0.900; DG: 33.88%±12.53% vs. 15.98%±2.37%, P=0.166), but this decrease was not statistically significant.In the 14-DIV group, hypoxia-exposed tissue showed decreased damage compared to control tissues; this decrease was not significant in the CA3 (24.51%±6.05% vs. 18.31%±3.28%, P=0.373) or DG (15.72%±3.47% vs. 9.91%±2.11%, P=0.134), but was significant in the CA1 (50.91%±5.90% vs. 32.30%±3.34%, P=0.004).Although only CA1 tissues cultured for 14 DIV showed significantly less damage after exposure to hypoxia, the other tissues examined in this study showed a tendency towards less damage after hypoxic exposure.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Chung-Ang University College of Medicine, Seoul, Korea.

ABSTRACT

Purpose: The aim of this study was to investigate the potential effects of mild hypoxia in the mature and immature brain.

Methods: We prepared organotypic slice cultures of the hippocampus and used hippocampal tissue cultures at 7 and 14 days in vitro (DIV) to represent the immature and mature brain, respectively. Tissue cultures were exposed to 10% oxygen for 60 minutes. Twenty-four hours after this hypoxic insult, propidium iodide fluorescence images were obtained, and the damaged areas in the cornu ammonis 1 (CA1), CA3, and dentate gyrus (DG) were measured using image analysis.

Results: In the 7-DIV group compared to control tissue, hypoxia-exposed tissue showed decreased damage in two regions (CA1: 5.59%±2.99% vs. 4.80%±1.37%, P=0.900; DG: 33.88%±12.53% vs. 15.98%±2.37%, P=0.166), but this decrease was not statistically significant. In the 14-DIV group, hypoxia-exposed tissue showed decreased damage compared to control tissues; this decrease was not significant in the CA3 (24.51%±6.05% vs. 18.31%±3.28%, P=0.373) or DG (15.72%±3.47% vs. 9.91%±2.11%, P=0.134), but was significant in the CA1 (50.91%±5.90% vs. 32.30%±3.34%, P=0.004).

Conclusion: Although only CA1 tissues cultured for 14 DIV showed significantly less damage after exposure to hypoxia, the other tissues examined in this study showed a tendency towards less damage after hypoxic exposure. Therefore, mild hypoxia might play a protective role in the brain.

No MeSH data available.


Related in: MedlinePlus