Pretreatment with mGluR2 or mGluR3 Agonists Reduces Apoptosis Induced by Hypoxia-Ischemia in Neonatal Rat Brains

Abstract

Hypoxia-ischemia (HI) in an immature brain results in energy depletion and excessive glutamate release resulting in excitotoxicity and oxidative stress. An increase in reactive oxygen species (ROS) production induces apoptotic processes resulting in neuronal death. Activation of group II mGluR was shown to prevent neuronal damage after HI. The application of agonists of mGluR3 (N-acetylaspartylglutamate; NAAG) or mGluR2 (LY379268) inhibits the release of glutamate and reduces neurodegeneration in a neonatal rat model of HI, although the exact mechanism is not fully recognized. In the present study, the effects of NAAG (5 mg/kg) and LY379268 (5 mg/kg) application (24 h or 1 h before experimental birth asphyxia) on apoptotic processes as the potential mechanism of neuroprotection in 7-day-old rats were investigated. Intraperitoneal application of NAAG or LY379268 at either time point before HI significantly reduced the number of TUNEL-positive cells in the CA1 region of the ischemic brain hemisphere. Both agonists reduced expression of the proapoptotic Bax protein and increased expression of Bcl-2. Decreases in HI-induced caspase-9 and caspase-3 activity were also observed. Application of NAAG or LY379268 24 h or 1 h before HI reduced HIF-1α formation likely by reducing ROS levels. It was shown that LY379268 concentration remains at a level that is required for activation of mGluR2 for up to 24 h; however, NAAG is quickly metabolized by glutamate carboxypeptidase II (GCPII) into glutamate and N-acetyl-aspartate. The observed effect of LY379268 application 24 h or 1 h before HI is connected with direct activation of mGluR2 and inhibition of glutamate release. Based on the data presented in this study and on our previous findings, we conclude that the neuroprotective effect of NAAG applied 1 h before HI is most likely the result of a combination of mGluR3 and NMDA receptor activation, whereas the beneficial effects of NAAG pretreatment 24 h before HI can be explained by the activation of NMDA receptors and induction of the antioxidative/antiapoptotic defense system triggered by mild excitotoxicity in neurons. This response to NAAG pretreatment is consistent with the commonly accepted mechanism of preconditioning.

Figure 1

TUNEL-positive cells in the CA1 region of the ipsilateral hemisphere in mGluR2/3 agonist-preconditioned animals. Brain tissue was examined 7 days after ischemia. (a) TUNEL-stained cells were calculated from the central CA1 area of 0.5 mm length; (b) representative pictures of TUNEL-stained cells, scale bar: 50 μm; (c) results expressed as the number of TUNEL-positive cells. The number of analyzed animals per group n = 3-5. The results on the graph are presented as the mean values ± SEM, statistically significant differences: ^∗∗∗p < 0.001 compared to HI; ^#p < 0.01 compared to the sham-operated group.

Figure 2

The effect of mGluR2/3 agonist application on HI induced changes in Bcl-2 (a) and Bax (b) expression in the brain. Results are presented as the mean ± SEM, n = 6–8; statistically significant differences: ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 compared to HI; ^#p < 0.01 compared to the sham-operated group.

Figure 3

The effect of LY379268 or NAAG application 24 h or 1 h before HI on caspase-9 (a) and caspase-3 (b) activity. Results are presented as the mean ± SEM, n = 6–8; statistically significant differences: ^#p < 0.001 compared to the sham-operated group. ^∗p < 0.05, ^∗∗p < 0.005, and ^∗∗∗p < 0.001 compared to HI; ^$p < 0.05 and ^$$p < 0.001 compared to NAAG group.

Figure 4

The effect of LY379268 or NAAG application 24 h or 1 h before HI on HIF-1α concentration. Results are presented as the mean ± SEM, n = 5; statistically significant differences: ^∗p < 0.01 and ^∗∗p < 0.001 compared to HI; ^#p < 0.001 compared to the sham-operated group.