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Y.-N. Chen, Z. Yu, W. Mao, S. Uchida, M. Aihara, M. Araie; Effects of Hyperbaric Pressure Loading on Glutamate Neurotoxicity in Purified Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4361.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the effect of hyperbaric pressure on purified retinal ganglion cells (RGCs), and also its additive effect on glutamate-induced neurotoxicity in RGCs using a high pressure incubation chamber.
RGC cultures were obtained from retina of 6- to 8-day-old rats utilizing the two-step immunopanning procedure. The RGCs were cultured in serum-free medium. After 3-day incubation, the culture medium was changed to B27 neurobasal medium with or without glutamate (5µM or 25µM) and the culture was moved to a special incubator in which constant high pressures can be maintained. The RGCs were divided into the following groups: (1) normobaric group, 1.0 ATA (atmosphere absolute), (2) hyperbaric group, 15mmHg + 1.0 ATA, (3) hyperbaric group, 30mmHg +1.0 ATA. After 72 hours, using the calcein-AM assay, the percentage of surviving RGCs was determined by comparison with the control group (normobaric group without glutamate). In addition, we also added MK801, an NMDA antagonist, or DNQX, an AMPA/kainite antagonist to the RGCs to evaluate its neuroprotective effect on glutamate-induced neurotoxicity in RGCs under pressure.
Without glutamate addition, the viability percentage was 100.4% in 15mmHg group and 101.1% in 30mmHg group. There was no meaningful difference of cell viabilities between the normobaric group and the hyperbaric groups. With 5µM glutamate addition, the viability was 100.4% in normobaric group, 97.2% in 15mmHg group and 73.3% in 30mmHg group. With 25µM glutamate addition, the viability was 69.1% in normobaric group, 55.7% in 15mmHg group and 54.5% in 30mmHg group. 5µM glutamate showed no neurotoxicity under normobaric or 15mmHg conditions but could induce RGC deaths in 30mmHg conditions. With addition of 25µM glutamate, the viabilities of RGCs in both 15mmHg and 30mmHg pressure loading groups are lower than that in normobaric group. DNQX showed stronger protective effect than MK801 in each pressure group (n=10)
In purified RGCs, hyperbaric pressure loading alone could not induce cell death but exacerbated glutamate-induced neurotoxicity. AMPA/kainite receptor may be related to the mechanism of glutamate-induced neurotoxicity in purified RGCs.
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