OBJECTIVETo study the influence and mechanism of acute ethanol intoxication (AEI) on rat neuronal apoptosis after severe traumatic brain injury (TBI).

METHODSNinety-six Sprague-Dawley rats were randomly divided into four groups: normal control, AEI-only, TBI-only and TBI+AEI (n equal to 24 for each). Severe TBI model was developed according to Feeney's method. Rats in TBI+AEI group were firstly subjected to AEI, and then suffered head trauma. In each group, animals were sacrificed at 6 h, 24 h, 72 h, and 168 h after TBI. The level of neuronal apoptosis and the expression of Bcl-2 protein were determined by TUNEL assay and immunohistochemical method, respectively.

RESULTSApoptotic cells mainly distributed in the cortex and white matter around the damaged area. Neuronal apoptosis significantly increased at 6 h after trauma and peaked at 72 h. Both the level of neuronal apoptosis and expression of Bcl-2 protein in TBI-only group and TBI+AEI group were higher than those in control group (P less than 0.05). Compared with TBI-only group, the two indexes were much higher in TBI+AEI group at all time points (P less than 0.05).

CONCLUSIONOur findings suggest that AEI can increase neuronal apoptosis after severe TBI.

Animals ; Apoptosis ; drug effects ; Brain Injuries ; Cerebral Cortex ; cytology ; Disease Models, Animal ; Ethanol ; poisoning ; Immunohistochemistry ; In Situ Nick-End Labeling ; Male ; Neurons ; physiology ; Prosencephalon ; cytology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Sprague-Dawley

Reactive oxygen species (ROS) contribute to the development of a number of neuronal diseases including ischemia. DJ-1, also known to PARK7, plays an important role in transcriptional regulation, acting as molecular chaperone and antioxidant. In the present study, we investigated whether DJ-1 protein shows a protective effect against oxidative stress-induced neuronal cell death in vitro and in ischemic animal models in vivo. To explore DJ-1 protein's potential role in protecting against ischemic cell death, we constructed cell permeable Tat-DJ-1 fusion proteins. Tat-DJ-1 protein efficiently transduced into neuronal cells in a dose- and time-dependent manner. Transduced Tat-DJ-1 protein increased cell survival against hydrogen peroxide (H2O2) toxicity and also reduced intracellular ROS. In addition, Tat-DJ-1 protein inhibited DNA fragmentation induced by H2O2. Furthermore, in animal models, immunohistochemical analysis revealed that Tat-DJ-1 protein prevented neuronal cell death induced by transient forebrain ischemia in the CA1 region of the hippocampus. These results demonstrate that transduced Tat-DJ-1 protein protects against cell death in vitro and in vivo, suggesting that the transduction of Tat-DJ-1 may be useful as a therapeutic agent for ischemic injuries related to oxidative stress.
Animals ; Blood-Brain Barrier/metabolism ; Brain Ischemia/*metabolism/pathology/prevention & control ; CA1 Region, Hippocampal/drug effects/metabolism/pathology ; Cell Line, Tumor ; Cell Survival/drug effects ; Gerbillinae ; Intracellular Signaling Peptides and Proteins/*administration & dosage/biosynthesis/pharmacokinetics ; Lipid Peroxidation ; Malondialdehyde/metabolism ; Mice ; Neuroprotective Agents/*administration & dosage/pharmacokinetics ; Oncogene Proteins/*administration & dosage/biosynthesis/pharmacokinetics ; *Oxidative Stress ; Prosencephalon/drug effects/metabolism/pathology ; Rats ; Recombinant Fusion Proteins/*administration & dosage/biosynthesis/pharmacokinetics ; tat Gene Products, Human Immunodeficiency Virus/*administration & dosage/biosynthesis/pharmacokinetics

It has been known that the glutamate transmission system and N-methyl-D-aspartate receptor (NMDA-R) were possibly related to anxiety processes. Although anxiety symptom can be relieved by NMDA-R antagonists and partial agonists treatment, the functions of NMDA-R and its subunits in anxiety behaviors remain unclear. We used forebrain specific NR2B over-expression mice to examine whether the increase of NR2B subunit level would induce anxiety behaviors. The results indicated that the juvenile (3-5 months old), middle-aged (8-10 months old) and old (19-22 months old ) NR2B transgenic mice showed no significant difference in open field test and elevated plus maze test as compared with the control mice. Capillary electrophoresis of monoamine neurotransmitter in subregions of forebrain revealed no significant difference between transgenic and control mice of 16-18 months age. These results suggest that the increase of NR2B expression and followed NR1 and NR2A expression augmentations in the forebrain have no significant effect on anxiety-related behaviors in mice.
Animals ; Anxiety ; metabolism ; Mice ; Mice, Transgenic ; Prosencephalon ; metabolism ; Receptors, N-Methyl-D-Aspartate ; metabolism

The present experiments were done to determine the effectiveness of a non-specific nitric oxide synthase inhibitor, N-nitro-L-arginine methyl ester (L-NAME), on oxidative stress parameters induced by aluminium chloride (AlCl3) intrahippocampal injections in Wistar rats. Animals were sacrificed 3 h and 30 d after treatments, heads were immediately frozen in liquid nitrogen and forebrain cortices were removed. Crude mitochondrial fraction preparations of forebrain cortices were used for the biochemical analyses: nitrite levels, superoxide production, malondialdehyde concentrations, superoxide dismutase (SOD) activities and reduced glutathione contents. AlCl3 injection resulted in increased nitrite concentrations, superoxide anion production, malondialdehyde concentrations and reduced glutathione contents in the forebrain cortex, suggesting that AlCl3 exposure promoted oxidative stress in this brain structure. The biochemical changes observed in neuronal tissues showed that aluminium acted as a pro-oxidant. However, the non-specific nitric oxide synthase (NOS) inhibitor, L-NAME, exerted anti-oxidant actions in AlCl3-treated animals. These results revealed that NO-mediated neurotoxicity due to intrahippocampal AlCl3 injection spread temporally and spatially to the forebrain cortex, and suggested a potentially neuroprotective effect for L-NAME.
Aluminum Compounds/*toxicity ; Animals ; Chlorides/*toxicity ; Glutathione/metabolism ; Male ; Malondialdehyde ; NG-Nitroarginine Methyl Ester/*pharmacology ; Nitric Oxide Synthase/*antagonists & inhibitors ; Nitrites/chemistry/metabolism ; Prosencephalon/*drug effects ; Rats ; Rats, Wistar ; Superoxide Dismutase/metabolism ; Superoxides/metabolism

Several studies have demonstrated that ischemic preconditioning increases superoxide dismutase activity, but it is unclear how ischemic preconditioning affects events downstream of hydrogen peroxide production during subsequent severe ischemia and reperfusion in the hippocampus. To answer this question, we investigated whether ischemic preconditioning in the hippocampal CA1 region increases the activities of antioxidant enzymes glutathione peroxidase and catalase, resulting in a decrease in the level of hydroxyl radicals during subsequent severe ischemia-reperfusion. Transient forebrain ischemia was induced by four-vessel occlusion in rats. Ischemic preconditioning for 3 min or a sham operation was performed and a 15-min severe ischemia was induced three days later. Ischemic preconditioning preserved the CA1 hippocampal neurons following severe ischemia. The concentration of 2,3-dihydroxybenzoic acid, an indicator of hydroxyl radical, was measured using in vivo microdialysis technique combined with HPLC. The ischemia-induced increase in the ratio of 2,3-dihydroxybenzoic acid concentration relative to baseline did not differ significantly between preconditioned and control groups. On the other hand, activities of the antioxidant enzymes glutathione peroxidase-1 and catalase were significantly increased at 3 days after ischemic preconditioning in the hippocampus. Our results suggest that, in preconditioned rats, while hydrogen peroxide is generated from severe ischemia, the activity of catalase and glutathione peroxidase-1 is correspondingly increased to eliminate the excessive hydrogen peroxide. However, our results show that the enhanced activity of these antioxidant enzymes in preconditioned rats is not sufficient to decrease hydroxyl radical levels during subsequent severe ischemia-reperfusion.
Animals ; Antioxidants/*metabolism ; Catalase/metabolism ; Enzyme Activation ; Glutathione Peroxidase/metabolism ; Hippocampus/*blood supply ; Hydrogen Peroxide/metabolism ; Hydroxybenzoic Acids/metabolism ; Hydroxyl Radical/*metabolism ; Ischemic Attack, Transient/*metabolism/physiopathology/prevention & control ; *Ischemic Preconditioning ; Male ; *Prosencephalon ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury/metabolism/prevention & control

Glutamate is the major fast excitatory transmitter in the central nervous system. While normal synaptic transmission is mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors, N-methyl-D-aspartate (NMDA) receptors are thought to selectively contribute to plasticity. Genetically enhancing NMDA receptor functions enhances animal behavior in normal physiological learning and enhances their sensitivity in the case of tissue injury. One major mechanism for NMDA receptors is synaptic long-term potentiation (LTP). Here we present evidence that NMDA receptors not only contribute to normal synaptic responses induced by stimulation of local layer V or white matters, but also contribute to generation of action potentials induced by a depolarizing step applied to the soma. Calcium-calmodulin sensitive adenylyl cyclase 1 and cAMP signal pathways likely mediate these effects. Considering the importance of cingulate neurons in nociception and pain, our results provide a new mechanism for NMDA receptor contributing to neuronal synaptic transmission, spiking properties in forebrains, and possible forebrain-related behavioral nociceptive responses and pain.
Action Potentials ; Adenylyl Cyclases ; metabolism ; Animals ; Cyclic AMP ; metabolism ; Mice ; Prosencephalon ; physiology ; Receptors, N-Methyl-D-Aspartate ; physiology

Animals ; Cerebral Cortex ; metabolism ; Disease Models, Animal ; Male ; Mice ; Mice, Inbred Strains ; Morphine Dependence ; metabolism ; Neurotrophin 3 ; metabolism ; Prosencephalon ; metabolism

AIM AND METHODSUsing double immunohistochemical method for Fos and tyrosine hydroxylase(TH) to examine the effects of intracerebroventricular (icv) administration of adrenomedullin (AM) on catecholaminergic neurons and the expression of c-fos gene in rat brain nuclei involved in cardiovascular regulation in order to define whether the effects of central administration of adrenomedullin (AM) were induced by activating the catecholaminergic neurons.

RESULTS(1) Following icy administration of AM (3 nmol/kg), Fos-like immunoreactivity neurons were markedly increased in several brain areas of the rat, including the brainstem, the hypothalamus and the forebrain. (2) Following icy administration of AM (3 nmol/kg), double-labeled neurons for Fos and TH increased significantly in the area postrema (AP), the nucleus of the solitary tract (NTS), the nucleus paragigantocellularis lateralis (PGL) and the locus coeruleus (LC). (3) Pretreatment with calcitonin gene-related peptide receptor antagonism CGRP (8-37) (30 nmol/kg) significantly reduced the action of AM (3 nmol/kg) in the brain.

CONCLUSIONAM activates the nuclei involved in cardiovascular regulation in the forebrain, the hypothalamus and the brainstem, and that the central actions of AM are induced by activating the catecholaminergic neurons of brainstem nuclei involved in cardiovascular regulation. CGRP receptor can mediate the effects of AM in brain.

Adrenomedullin ; administration & dosage ; pharmacology ; Animals ; Brain Stem ; drug effects ; Calcitonin Gene-Related Peptide ; metabolism ; Hypothalamus ; drug effects ; Male ; Neurons ; drug effects ; metabolism ; Peptide Fragments ; metabolism ; Prosencephalon ; drug effects ; metabolism ; Proto-Oncogene Proteins c-fos ; metabolism ; Rats ; Rats, Sprague-Dawley ; Tyrosine 3-Monooxygenase ; metabolism

The 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), a protein of unknown function in vivo, is abundantly expressed in myelinating glia in two isoforms, CNP1 and CNP2. In this study, immunoblot analysis showed that CNP1 is the major isoform in adult forebrain, and that both isoforms are included in the postsynaptic density (PSD) fraction and tyrosine-phosphorylated at the basal level. However, subcellular distribution and detergent extraction data showed that CNP is nonspecifically associated with the PSD fraction. Immunocytochemistry revealed that CNP is detected, in a weak but punctate pattern, in dissociated rat hippocampal neurons of 3 days to 2 weeks in vitro. The CNP-positive punctae were distributed throughout soma and dendrites, and distinct from PSD95-positive ones. Immunoblot analysis indicated that CNP is also expressed in neuronal stem cell lines, HiB5 and F11. Interestingly, in addition to the known two isoforms, a new CNP isoform of MW 45 kDa was expressed in these cell lines and was the major type of isoform in F11 cells. Taken together, our data suggest that CNP is expressed in the early stage of in vitro development and nonspecifically included in the adult rat PSD fraction.
2',3'-Cyclic-Nucleotide Phosphodiesterases/*metabolism ; Aging/physiology ; Animals ; Cells, Cultured ; Hippocampus/cytology/metabolism ; Immunohistochemistry ; Nerve Tissue Proteins/*metabolism ; Neurons/metabolism ; Phosphotyrosine/metabolism ; Prosencephalon/cytology/*metabolism ; Rats ; Rats, Sprague-Dawley ; Substrate Specificity

It has been suggested that propofol has the protective effect on cerebral ischemia-reperfusion injury. The aim of this study is to evaluate the effect of propofol pretreatment on incomplete forebrain ischemia-reperfusion injury in rats. Thirty Sprague-Dawley rats were anesthetized with isoflurane in oxygen and randomly allocated into propofol group (n=13) and saline group (n=17). In propofol group, propofol was pretreated in a step-down scheme before inducing forebrain ischemia by occlusion of both common carotid arteries and arterial hypotension. After ischemia (20 min) and reperfusion (30 min), rats were decapitated. Brain was sliced to obtain coronal slices of 4-12 mm from frontal pole, which were reacted with 2% 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) for 10 min to differentiate the damaged tissues from normal tissues. Median (interquartile range) values of the average percent infarct area were 0.0 (8.6)% and 20.1 (41.2)% in propofol and saline groups, respectively. There was significant difference between the groups. In conclusion, propofol may have a protective effect on incomplete forebrain ischemia-reperfusion injury.
Animal ; Brain Ischemia/prevention & control* ; Brain Ischemia/pathology ; Cerebral Infarction/prevention & control ; Cerebral Infarction/pathology ; Disease Models, Animal ; Free Radical Scavengers/pharmacology* ; Mitochondria/enzymology* ; Neuroprotective Agents/pharmacology* ; Oxidative Phosphorylation ; Propofol/pharmacology* ; Prosencephalon/metabolism ; Prosencephalon/injuries ; Prosencephalon/drug effects* ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury/prevention & control* ; Reperfusion Injury/pathology ; Tetrazolium Salts