The effect of Hypothermia and Progesterone (P4) against Glutamate challenged primary cortical astrocytes on S100β, Glutamate Uptake, GLT-1 and p62 / Fatin Nur Asyiqin Abd Talib

Glutamate excitotoxicity is a concept that underpinned the massive release of glutamate to extracellular space, thereby inducing neuronal and glial injuries. Hypothermia has been proposed to offer neuroprotection even though the mechanism underlying it is poorly understood. Furthermore, prolonged time is required for hypothermia to exert its effects as well as inconsistencies and variation in the outcomes patients. An adjuvant therapy with hypothermia may be an alternative to reduce exposure time and obtain consistent outcomes. Progesterone (P4) is a neurosteroid which has been shown to elicit neuroprotection in neuronal cells with ischemic injury. This study investigates the neuroprotective effects of hypothermia and P4 on astrocytes following glutamateinduced toxicity. The cultured primary cortical astrocyte cells were exposed to 50 µM of glutamate for 15 minutes followed by incubation under hypothermia conditions with and without P4 for 24 hours. After 24 hours, the viability of cells was assessed by 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The intracellular concentration of glutamate in astrocyte cells was estimated by the glutamate uptake assay. The levels of p62 and S100β were measured using ELISA. The membrane protein was extracted and estimated for GLT-1 by Western Blot. There were significant increases in the percentage of viable cells as well as the concentration of glutamate uptake by the astrocyte cells in mild hypothermia with P4 (88.27 ± 3.96%) and moderate hypothermia with P4 (80.94 ± 4.12%) as compared to normothermia after glutamate induced toxicity (51.05 ± 4.10%). Further analysis revealed that there was a significant effect of moderate hypothermia with P4 (142.78 ± 13.85 pg/ml) in increasing the S100β level in comparison to normothermia across the glutamate-induced toxicity groups (74.34 ± 4.42 pg/ml) and significantly increasing in glutamate uptake (p ≤ 0.01) after treated with both mild and moderate hypothermia and P4. There was a significant increase of membrane GLT-1 in both mild hypothermia (1.07 ± 0.01 FD) and moderate hypothermia (0.87 ± 0.08 FD) group when compared to the normothermia (0.53 ± 0.05 FD) group. The p62 level was shown significant reduced in both mild and moderate hypothermia and P4 (p ≤ 0.01) in comparison to normothermia across the glutamateinduced toxicity group. In conclusion, hypothermia and P4 reduced the glutamateinduced toxicity in the astrocyte cells by increasing glutamate uptake via GLT-1.