N-Acetylaspartylglutamate (NAAG) Pretreatment Reduces Hypoxic-Ischemic Brain Damage and Oxidative Stress in Neonatal Rats.

N-acetylaspartylglutamate (NAAG), the most abundant peptide transmitter in the mammalian nervous system, activates mGluR3 at presynaptic sites, inhibiting the release of glutamate, and acts on mGluR3 on astrocytes, stimulating the release of neuroprotective growth factors (TGF-ß). NAAG can also affect N-methyl-d-aspartate (NMDA) receptors in both synaptic and extrasynaptic regions. NAAG reduces neurodegeneration in a neonatal rat model of hypoxia-ischemia (HI), although the exact mechanism is not fully recognized. In the present study, the effect of NAAG application 24 or 1 h before experimental birth asphyxia on oxidative stress markers and the potential mechanisms of neuroprotection on 7-day old rats was investigated. The intraperitoneal application of NAAG at either time point before HI significantly reduced the weight deficit of the ischemic brain hemisphere, radical oxygen species (ROS) content and activity of antioxidant enzymes, and increased the concentration of reduced glutathione (GSH). No additional increase in the TGF-ß concentration was observed after NAAG application. The fast metabolism of NAAG and the decrease in TGF-ß concentration that resulted from NAAG pretreatment, performed up to 24 h before HI, excluded the involvement mGluR3 in neuroprotection. The observed effect may be explained by the activation of NMDA receptors induced by NAAG pretreatment 24 h before HI. Inhibition of the NAAG effect by memantine supports this conclusion. NAAG preconditioning 1 h before HI results in a mixture of mGluR3 and NMDA receptor activation. Preconditioning with NAAG induces the antioxidative defense system triggered by mild excitotoxicity in neurons. Moreover, this response to NAAG pretreatment is consistent with the commonly accepted mechanism of preconditioning. However, this theory requires further investigation.

Changes in the Expression of SNAP-25 Protein in the Brain of Juvenile Rats in Two Models of Autism.

The results of genetic studies suggest a possible role for SNAP-25 polymorphism in the development of autism spectrum disorders (ASDs); however, there are no data available on whether changes in SNAP-25 expression also affect animals in rodent models of ASD. The aim of the present study was to explore this issue. The studies included 1-month-old rats representing valproic acid (VPA)- and thalidomide (THAL)-induced models of autism. Their mothers received single doses of VPA (800 mg/kg) or THAL (500 mg/kg) per os on the 11th day of gestation. SNAP-25 protein content in the cerebellum, hippocampus, and frontal lobe was determined using Western blotting, while changes of mRNA levels of Snap25 gene were determined using real-time polymerase chain reaction. Compared to controls, SNAP-25 content was decreased by approximately 35% in all brain structures tested, in both males and females, exclusively in the VPA group. In contrast to this, Snap25 expression, studied in males, was increased in the hippocampus and cerebellum in both, VPA- and THAL-treated rats. We discuss the compliance of these results with the hypothesized role of SNAP-25 in the pathophysiology of ASD and the adequacy of the experimental models used.

Simultaneous Inhibition of BCR-ABL1 Tyrosine Kinase and PAK1/2 Serine/Threonine Kinase Exerts Synergistic Effect against Chronic Myeloid Leukemia Cells.

Tyrosine kinase inhibitors (TKIs) revolutionized the treatment of chronic myeloid leukemia in the chronic phase (CML-CP). However, it is unlikely that they can completely "cure" the disease. This might be because some subpopulations of CML-CP cells such as stem and progenitor cells are resistant to chemotherapy, even to the new generation of TKIs. Therefore, it is important to look for new methods of treatment to improve therapeutic outcomes. Previously, we have shown that class I p21-activated serine/threonine kinases (PAKs) remained active in TKI-naive and TKI-treated CML-CP leukemia stem and early progenitor cells. In this study, we aimed to determine if simultaneous inhibition of BCR-ABL1 oncogenic tyrosine kinase and PAK1/2 serine/threonine kinase exert better anti-CML effect than that of individual treatments. PAK1 was inhibited by small-molecule inhibitor IPA-3 (p21-activated kinase inhibitor III), PAK2 was downregulated by specific short hairpin RNA (shRNA), and BCR-ABL1 tyrosine kinase was inhibited by imatinib (IM). The studies were conducted by using (i) primary CML-CP stem/early progenitor cells and normal hematopoietic counterparts isolated from the bone marrow of newly diagnosed patients with CML-CP and from healthy donors, respectively, (ii) CML-blast phase cell lines (K562 and KCL-22), and (iii) from BCR-ABL1-transformed 32Dcl3 cell line. Herein, we show that inhibition of the activity of PAK1 and/or PAK2 enhanced the effect of IM against CML cells without affecting the normal cells. We observed that the combined use of IM with IPA-3 increased the inhibition of growth and apoptosis of leukemia cells. To evaluate the type of interaction between the two drugs, we performed median effect analysis. According to our results, the type and strength of drug interaction depend on the concentration of the drugs tested. Generally, combination of IM with IPA-3 at the 50% of the cell kill level (EC50) generated synergistic effect. Based on our results, we hypothesize that IM, a BCR-ABL1 tyrosine kinase inhibitor, combined with a PAK1/2 inhibitor facilitates eradication of CML-CP cells.

The activation of group II metabotropic glutamate receptors protects neonatal rat brains from oxidative stress injury after hypoxia-ischemia.

Birth asphyxia resulting in brain hypoxia-ischemia (H-I) can cause neonatal death or lead to persistent brain damage. Recent investigations have shown that group II metabotropic glutamate receptor (mGluR2/3) activation can provide neuroprotection against H-I but the mechanism of this effect is not clear. The aim of this study was to investigate whether mGluR2/3 agonists applied a short time after H-I reduce brain damage in an experimental model of birth asphyxia, and whether a decrease in oxidative stress plays a role in neuroprotection. Neonatal H-I in 7-day-old rats was used as an experimental model of birth asphyxia. Rats were injected intra peritoneally with mGluR2 (LY 379268) or mGluR3 (NAAG) agonists 1 h or 6 h after H-I (5 mg/kg). The weight deficit of the ischemic brain hemisphere, radical oxygen species (ROS) content levels, antioxidant enzymes activity and the concentrations of reduced glutathione (GSH) were measured. Both agonists reduced weight loss in the ischemic hemisphere and mitigated neuronal degeneration in the CA1 hippocampal region and cerebral cortex. Both agonists reduced the elevated levels of ROS in the ipsilateral hemisphere observed after H-I and prevented an increase in antioxidant enzymes activity in the injured hemisphere restoring them to control levels. A decrease in GSH level was also restored after agonists application. The results show that the activation of mGluR2 and mGluR3 a short time after H-I triggers neuroprotective mechanisms that act through the inhibition of oxidative stress and ROS production. The prevention of ROS production by the inhibition of glutamate release and decrease in its extracellular concentration is likely the main mechanism involved in the observed neuroprotection.

Pretreatment with Group II Metabotropic Glutamate Receptor Agonist LY379268 Protects Neonatal Rat Brains from Oxidative Stress in an Experimental Model of Birth Asphyxia.

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 LY379268 24 or 1 h before H-I (5 mg/kg). LY379268 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.

Combining hypobaric hypoxia or hyperbaric oxygen postconditioning with memantine reduces neuroprotection in 7-day-old rat hypoxia-ischemia.

BACKGROUND: Perinatal hypoxia-ischemia causes brain injury in neonates, but a fully successful treatment to prevent changes in the brain has yet to be developed. The aim of this study was to evaluate the effect of combining memantine treatment with HBO (2.5 ATA) or HH (0.47 ATA) on neonatal hypoxia-ischemia brain injury. METHODS: 7-day old rats were subjected to hypoxia-ischemia (H-I) and treated with combination of memantine and HBO or HH. The brain damage was evaluated by examination of infarct area and the number of apoptotic cells in CA1 region of hippocampus. Additionally, the level of reactive oxygen species (ROS) was measured. RESULTS: Memantine, HBO or HH postconditioning applied at short time (1-6h) after H-I, and repeated for two subsequent days, resulted in significant neuroprotection. The reduction in ipsilateral hemisphere weight deficit and in the size of infarct area was observed 14days after H-I. A reduction in apoptosis and ROS level was also observed. Combining memantine with HBO or HH resulted in a loss of neuroprotection. CONCLUSIONS: Our results show that, combining HBO or HH postconditioning with memantine produce no additive increase in the neuroprotective effect. On the contrary, combining the treatments resulted in lower neuroprotection in comparison to the effects of memantine, HBO or HH alone.

Hyperbaric oxygen and hyperbaric air preconditioning induces ischemic tolerance to transient forebrain ischemia in the gerbil.

Ischemic preconditioning with sublethal stress triggers defensive mechanisms against ischemic brain damage; however, such manipulations are potentially dangerous and, therefore, safe stimuli have been sought. Hyperoxia preconditioning by administration of hyperbaric (HBO) or normobaric oxygen (NBO) may have neuroprotective potential. The aim of this study was to determine whether preconditioning with HBO and air (HBA) applied at 2.5 absolute pressure (ATA) or NBO preconditioning induces ischemic tolerance in the brain of gerbils subjected to 3min transient cerebral ischemia. Neuronal cell survival, changes in brain temperature, the generation of factors involved in neurodegeneration and basic behavior in nest building were all tested. Hyperoxic preconditioning prevented ischemia-induced neuronal cell loss, reduced the number of TUNEL positive cells in the CA1 region of the hippocampus and improved the nest building process compared to untreated ischemic animals. Preconditioning also suppressed the production of reactive oxygen species and increased Bax expression normally observed after an ischemic episode. Only HBO preconditioning inhibited ischemia-evoked increases in brain temperature. Our results show that hyperoxic preconditioning results in induction of ischemic tolerance and prevents ischemia-induced neuronal damage in the gerbil brain. Pressurized air preconditioning was as effective as HBO or NBO preconditioning in providing neuroprotection. The observed neuroprotection probably results from mild oxidative stress evoked by increased brain tissue oxidation and activation of antioxidant and antiapoptotic defenses.