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Abstract

Hypoxia-ischemia (H-I) at the time of birth may cause neonatal death or lead to persistent brain damage. The search for an effective treatment of asphyxiated infants has not resulted in an effective therapy, and hypothermia remains the only available therapeutic strategy. Among possible experimental therapies, the induction of ischemic tolerance is promising. Recent investigations have shown that activation of group II metabotropic glutamate receptors (mGluR2/3) can provide neuroprotection against H-I, but the mechanism of this effect is not clear. The aim of this study was to investigate whether an mGluR2/3 agonist applied before H-I reduces brain damage in an experimental model of birth asphyxia and whether a decrease in oxidative stress plays a role in neuroprotection. Neonatal H-I on seven-day-old rats was used as an experimental model of birth asphyxia. Rats were injected intraperitoneally with the mGluR2/3 agonist 24 or 1 h before H-I (5 mg/kg). reduced the infarct area in the ischemic hemisphere. Application of the agonist at both times also reduced the elevated levels of reactive oxygen species (ROS) in the ipsilateral hemisphere observed after H-I and prevented the increase in antioxidant enzyme activity in the injured hemisphere. The decrease in glutathione (GSH) level was also restored after agonist application. The results suggest that the neuroprotective mechanisms triggered by the activation of mGluR2/3 before H-I act through the decrease of glutamate release and its extracellular concentration resulting in the inhibition of ROS production and reduction of oxidative stress. This, rather than induction of ischemic tolerance, is probably the main mechanism involved in the observed neuroprotection.

2.1. Induction of Cerebral Hypoxia–Ischemia

Neonatal cerebral H-I was induced according to Rice et al. (1981). Briefly, seven-day-old Wistar rat pups (weight 12–18 g) were anaesthetized with isoflurane (4% for induction, and 1.5–2.0% for maintenance) in a mixture of nitrous oxide and oxygen (0.6:1). The left common carotid artery was exposed and cut between double ligatures of silk sutures or only exposed (sham control). Animals were placed in home cages, allowed to recover for 60 min, placed in a humidified chamber (35 °C), and exposed to hypoxic conditions (8% oxygen in nitrogen) for 75 min. This duration of hypoxia-ischemia is typically associated with infarction predominantly of the cerebral hemisphere ipsilateral to the carotid artery occlusion [,]. After hypoxic treatment the animals were returned to the cages and housed at room temperature (22 °C) with a 12:12 h light–dark cycle and with ample food and water.

2.2. Drug Application

Animals were injected intraperitoneally (i.p.) with , a highly selective agonist of group II mGluRs, for which its affinity to mGluR2 is two times greater than that to mGluR3. The animals received a single injection made 24 or 1 h before H-I in a dose of 5 mg/kg of body weight. The dose was based on previous findings [,]. Sham-operated and H-I control rats were injected with saline.

3.1. LY379268 Reduces H-I Evoked Brain Damage

H-I resulted in brain tissue damage in the ipsilateral brain hemisphere. In the present study the evaluation of the infarct area using TTC staining showed that H-I produced 42% infarction of the ipsilateral hemisphere (). applied 24 or 1 h before H-I significantly decreased the area of infarction to 18.8% and 8.5%, respectively (p < 0.001). However, the infarct area measured after application of 1 h before H-I was significantly smaller than when the agonist was applied 24 h before the insult (p < 0.001).

3.2. LY379268 Reduces the H-I Evoked Increase in ROS Level

H-I resulted in an increase in the ROS level in the left ischemic hemisphere by as much as a factor of three (p < 0.001; ). The application of at both times significantly reduced the ROS level (p < 0.005). applied 24 h before H-I decreased the ROS level by 30% (p < 0.001), whereas when applied 1 h before H-I, it decreased the ROS level by 45%. However, the difference between the ROS level after agonist application at 24 or 1 h before H-I was not statistically significant. Neither of the treatments changed the ROS level in the right hemisphere.

3.3. LY379268 Pretreatment Reduces the H-I Increased Activity of SOD and Catalase

SOD activity in the brain tissue measured 3 h after H-I increased four times compared to the SOD activity in the control sham-operated animals (). Application of 24 h before H-I decreased SOD activity by 25% compared to the H-I group and by 42% when application of the agonist was performed 1 h before H-I.