The effect of propofol on α‐amino‐5‐methyl‐4‐isoxazole propionate(AMPA) induced neurotoxicity in rat mixed cortical cultures

BACKGROUND: Excessive extracellular glutamate accmulation due to cerebral ischemia is belived to induce excitotoxic cascade by overstimulation of glutamate receptors such as Nmethyl- D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainite. The pattern of AMPA mediated neurotoxicity (necrosis vs apoptosis) and the neuroprotective effect of propofol to AMPA mediated neurotoxicity still remain unclear. We applied 72 hr, 50 μM AMPA stimulation to cultured rat cortical neurons and have examined the pattern of AMPA neurotoxicity and the neuroprotective effects of propofol. Also, we have examined the effect of propofol on AMPA mediated intraneural calcium influx. METHODS: Sprague-Dawley rats (250-300 g) were used. Thirteen-day-old primary mixed cortical cultures were exposed to 50 μM AMPA stimulation (AMPA group). Propofol 0.1, 1, 25, 50, μM were co-administered with 50 μM AMPA (propofol-treated group) or propofol 50 μM alone. In control and Full-kill group, no drug or NMDA 100 μM were administered. 72 hr after, the survived cells were counted using trypan-blue staining. And, the cell death rate (CDR) were calculated (control group CDR set as 0, Full kill group CDR set as 100). After confirming the presence of neuronal cells by neuronal nuclei (NeuN) and Hoechst stain, we quantified apoptotic cells by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end-labeling (TUNEL) assay. The effect of propofol on AMPA mediated intracellular calcium increase was measured with calcium-sensitive fluorochrome calcium green-5N-acetoxymethylester with confocal laser scanning microscopy. Statistical analysis was done by Oneway-ANOVA test and Bonferroni test. P<0.05 was considered as statistically significant. RESULTS: AMPA 50 μM stimulation demonstrated 49.31% CDR and adding propofol 50 μM decreased CDR to 29.38% (P<0.05). In TUNEL assay, cells with no drug treatment demonstrated 12.33% apoptotic cell rate (ACR) and 50 μM AMPA incrcresed ACR to 28.01% (P<0.05). Adding 50 μM propofol to AMPA decreased the ACR to 20% (P<0.05). In calcium imaging, the maximum intracellular calcium influx was decreased in AMPA 50 μM + propofol 50 μM group compared to AMPA 50 μM group (3164.98 nA vs 2276.98 nA, respectively)(P<0.05). CONCLUSION: 50 μM propofol demonstrated neuroprotective effects against AMPA 50 μM stimulation and the underlying mechanism propofol’s effect may be caused partly by the reduction of the intracellulr calcium influx.

Author dc:creator

• 서, 명신

Contributors dc:contributor

• 이, 숙영
• 대학원 의학과
• 200424449

• α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
• cell death
• propofol

Language dc:language

ko

Identifier

http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000009274
000000009274

OAI identifier oai:identifier

oai:repository.ajou.ac.kr:201003/1820