Effect of ibuprofen on regional eicosanoid production and neuronal injury after forebrain ischemia in rats

Post-ischemic metabolism of arachidonic acid by cyclooxygenase results in the elaboration of numerous eicosanoids and in the generation of free radicals. Accordingly, the effect of cyclooxygenase inhibition by ibuprofen on post-ischemic eicosanoid production and delayed neuronal death was evaluated in Wistar-Kyoto rats subjected to incomplete forebrain ischemia. In control (C) and ibuprofen-treated groups (n = 5 each), pre- and post-ischemic eicosanoid production in the caudate nucleus (CN) and dorsal hippocampus (HPC) were evaluated by microdialysis. The ibuprofen-treated animals were given ibuprofen, 15 mg/kg i.v., prior to insertion of microdialysis probes. Forebrain ischemia was induced by bilateral carotid artery occlusion (BCAO) for 10 min with simultaneous hypotension to 35 Torr. The concentrations of thromboxane B₂ (TxB₂), 6-keto-PGF_1α and PGF_2α in the microdialysate were measured by radioimmunoassay. In two additional concurrent groups of rats (>n = 10 each), neuronal injury in the HPC, CN and cortex (parietal, temporal and entorhinal regions) was evaluated histologically three days after 10 min of forebrain ischemia with and without pre-ischemic ibuprofen administration. In the control microdialysis group, levels of TxB₂, 6-keto-PGF_1α and PGF_2α increased in both CN and HPC after probe insertion. These probe related increases were substantially reduced in the ibuprofen group. After ischemia and reperfusion in the control group, the levels of TxB₂ and PGF_2α increased in both CN and HPC. Levels of 6-keto-PGF_1α increased in the CN but not in the HPC. The administration of ibuprofen substantially reduced post-ischemic TxB₂ and PGF_2α levels in both CN and HPC and decreased 6-keto-PGF_1α levels in the CN. The results of these initial microdialysis studies left the possibility that, in the ibuprofen group, the reduction in eicosanoid levels after probe penetration might have influenced the subsequent post-ischemic eicosanoid production. Therefore, in an additional group of animals (n = 5), ibuprofen was administered after probe insertion. Only PGF_2α levels were measured in this group. Increased levels of PGF_2α comparable to the original control group were detected after probe penetration. Nonetheless, after ibuprofen administration, the pre- and post-ischemic levels of PGF_2α were again significantly reduced. In the histologic evaluation groups, overall neuronal injury was significantly less in the ibuprofen treated animals. This protective effect of ibuprofen was most clearly evident in the CA3 sector of the HPC. The data suggest that metabolism of arachidonic acid by cylooxygenase may contribute to post-ischemic neuronal injury, though the relative contributions of eicosanoids per se and of free radicals remains undefined.     

Transient cerebral ischemia increases CA1 pyramidal neuron excitability

In human and experimental animals, the hippocampal CA1 region is one of the most vulnerable areas of the brain to ischemia. Pyramidal neurons in this region die 2–3 days after transient cerebral ischemia whereas other neurons in the same region remain intact. The mechanisms underlying the selective and delayed neuronal death are unclear. We tested the hypothesis that there is an increase in post-synaptic intrinsic excitability of CA1 pyramidal neurons after ischemia that exacerbates glutamatergic excitotoxicity. We performed whole-cell patch-clamp recordings in brain slices obtained 24 h after in vivo transient cerebral ischemia. We found that the input resistance and membrane time constant of the CA1 pyramidal neurons were significantly increased after ischemia, indicating an increase in neuronal excitability. This increase was associated with a decrease in voltage sag, suggesting a reduction of the hyperpolarization-activated non-selective cationic current (I_h). Moreover, after blocking I_h with ZD7288, the input resistance of the control neurons increased to that of the post-ischemia neurons, suggesting that a decrease in I_h contributes to increased excitability after ischemia. Finally, when lamotrigine, an enhancer of dendritic I_h, was applied immediately after ischemia, there was a significant attenuation of CA1 cell loss. These data suggest that an increase in CA1 pyramidal neuron excitability after ischemia may exacerbate cell loss. Moreover, this dendritic channelopathy may be amenable to treatment.     

Anticonvulsant action of intranigral γ-vinyl-GABA: role of noradrenergic neurotransmission

Intranigral γ-vinyl-GABA (GVG) suppresses electroshock seizures (ES). This anticonvulsant action was blocked by systemic treatment with the α₂-antagonist idazoxan. Consequently, we tested the idea that intranigral GABA mimetics suppress ES by increasing noradrenergic (NE) neuronal activity. Contrary to our hypothesis. GVG decreased NE turnover. This result indicates that while the seizure-suppressant effect of intranigral GVG requires α₂-mediated NE neurotransmission, the mechanism of this anticonvulsant action is not by increasing NE neuronal activity.     

α-Tocopherol and ubiquinones in rat brain subjected to decapitation ischemia

Levels of α-tocopherol (α-Toc), reduced ubiquinones (QH₂) and oxidized ubiquinones (Q) were assayed in rat forebrain subjected to decapitation ischemia. Post-decapitation levels of α-Toc decreased by 16% at 3 min and 20% at 15 min. Increases in Q₉H₂ (83%) and in Q₁₀H₂ (107%) were observed immediately following decapitation; thereafter their levels began to decrease and approached to the pre-ischemic values at 15 min. In contrast, Q₉ and Q₁₀ tended to increase continuously during ischemia. The data indicate that complete ischemia results in distinct changes in the recebral content of lipid-soluble antioxidants. The decrease of α-Toc may make the brain prone to peroxidative attack when cerebral tissue is subsequently reoxygenated.     

Vasodilator effects of sulforaphane in cerebral circulation: A critical role of endogenously produced hydrogen sulfide and arteriolar smooth muscle KATP and BK channels in the brain

We investigated the effects of sulforaphane (SFN), an isothiocyanate from cruciferous vegetables, in the regulation of cerebral blood flow using cranial windows in newborn pigs. SFN administered topically (10 µM–1 mM) or systemically (0.4 mg/kg ip) caused immediate and sustained dilation of pial arterioles concomitantly with elevated H₂S in periarachnoid cortical cerebrospinal fluid. H₂S is a potent vasodilator of cerebral arterioles. SFN is not a H₂S donor but it acts via stimulating H₂S generation in the brain catalyzed by cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS). CSE/CBS inhibitors propargylglycine, β-cyano-L-alanine, and aminooxyacetic acid blocked brain H₂S generation and cerebral vasodilation caused by SFN. The SFN-elicited vasodilation requires activation of potassium channels in cerebral arterioles. The inhibitors of K_ATP and BK channels glibenclamide, paxilline, and iberiotoxin blocked the vasodilator effects of topical and systemic SFN, supporting the concept that H₂S is the mediator of the vasodilator properties of SFN in cerebral circulation. Overall, we provide first evidence that SFN is a brain permeable compound that increases cerebral blood flow via a non-genomic mechanism that is mediated via activation of CSE/CBS-catalyzed H₂S formation in neurovascular cells followed by H₂S-induced activation of K_ATP and BK channels in arteriolar smooth muscle.     

大鼠脑缺血再灌注后星形胶质细胞反应性变化差异与海马CA1区的迟发性神经元死亡

BACKGROUND： Blood supply to the hippocampus is not provided by the middle cerebral artery. However, previous studies have shown that delayed neuronal death in the hippocampus may occur following focal cerebral ischemia induced by middle cerebral artery occlusion.
OBJECTIVE： To observe the relationship between reactive changes in hippocampal astrocytes and delayed neuronal death in the hippocampal CA1 region following middle cerebral artery occlusion.
DESIGN, TIME AND SETTING： The immunohistochemical, randomized, controlled animal study was performed at the Laboratory of Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, from July to November 2007.
MATERIALS： Rabbit anti-glial fibrillary acidic protein （GFAP） （Neomarkers, USA）, goat anti-rabbit IgG （Sigma, USA） and ApoAlert apoptosis detection kit （Biosciences Clontech, USA） were used in this study. METHODS： A total of 42 healthy adult male Wistar rats, aged 3–5 months, were randomly divided into a sham operation group （n = 6） and a cerebral ischemia/reperfusion group （n = 36）. In the cerebral ischemia/reperfusion group, cerebral ischemia/reperfusion models were created by middle cerebral artery occlusion. In the sham operation group, the thread was only inserted into the initial region of the internal carotid artery, and middle cerebral artery occlusion was not induced. Rats in the cerebral ischemia/reperfusion group were assigned to a delayed neuronal death （＋） subgroup and a delayed neuronal death （–） subgroup, according to the occurrence of delayed neuronal death in the ischemic side of the hippocampal CA1 region following cerebral ischemia.
MAIN OUTCOME MEASURES： Delayed neuronal death in the hippocampal CA1 region was measured by Nissl staining. GFAP expression and delayed neuronal death changes were measured in the rat hippocampal CA1 region at the ischemic hemisphere by double staining for GFAP and TUNEL.
RESULTS： After 3 days of ischemia/reperfusion, astrocytes with abnormal morphology were detected in the rat hippocampal CA1 region in the delayed neuronal death （＋） subgroup. No significant difference in GFAP expression was found in the rat hippocampal CA1 region at the ischemic hemisphere in the sham operation group, delayed neuronal death （＋） subgroup and delayed neuronal death （–） subgroup （P 〉 0.05）. After 7 days of ischemia/reperfusion, many GFAP-positive cells, which possessed a large cell body and an increased number of processes, were activated in the rat hippocampal CA1 region at the ischemic hemisphere. GFAP expression in the hippocampal CA1 region was greater in the delayed neuronal death （＋） subgroup and delayed neuronal death （–） subgroup compared with the sham operation group （P 〈 0.01）. Moreover, GFAP expression was significantly greater in the delayed neuronal death （–） subgroup than in the delayed neuronal death （＋） subgroup （P 〈 0.01）. After 30 days of ischemia/reperfusion, GFAP-positive cells were present in scar-like structures in the rat hippocampal CA1 region at the ischemic hemisphere. GFAP expression was significantly greater in the delayed neuronal death （＋） subgroup and delayed neuronal death （–） subgroup compared with the sham operation group （P 〈 0.05）. GFAP expression was significantly lower in the delayed neuronal death （–） subgroup than in the delayed neuronal death （＋） subgroup （P 〈 0.05）. The delayed neuronal death rates were 42% （5/12）, 33% （4/12） and 33% （4/12） at 3, 7 and 30 days, respectively, followingischemia/reperfusion. No significant differences were detected at various time points （χ2 = 0.341, P 〉 0.05）.
CONCLUSION： The activation of astrocytes was poor in the hippocampal CA1 region during the early stages of ischemia, which is an important reason for delayed neuronal death. Glial scar formation aggravated delayed neuronal death during the advanced ischemic stage.     

Hypothalamic sites of neuronal histamine action on food intake by rats

To identify sites of histaminergic modulation of food intake, histamine H₁-receptor antagonist was microinfused into the rat hypothalamus, the ventromedial hypothalamus (VMH), the lateral hypothalamus (LHA), the paraventricular nucleus (PVN), the dorsomedial hypothalamus (DMH), or the preoptic anterior hypothalamus (POAH), during the early light period. Feeding, but not drinking, was elicited in 100% of the rats (P<0.01) that were bilaterally microinfused with 26 nmol chlorpheniramine into the VMH. Unilateral infusion into the VMH did not affect food intake at doses of 26 or 52 nmol. Feeding was also induced by bilateral microinfusion into the PVN, but only the 52 nmol dose was effective. Bilateral infusions into the LHA, the DMH or the POAH did not affect ingestive behavior. Feeding induced by an H₁-antagonist was completely abolished in all 7 rats tested when endogenous neuronal histamine was decreased by pretreatment with α-fluoromethylhistidine (100 mg/kg). The findings suggest that H₁-receptors in the VMH and the PVN, but not in the LHA, the DMH or the POAH, may be involved in histaminergic suppression of foof intake.     

Neuronal protective role of PBEF in a mouse model of cerebral ischemia

Pre-B-cell colony-enhancing factor (PBEF) (also known as nicotinamide phosphoribosyltransferase) is a rate-limiting enzyme in the salvage pathway for mammalian biosynthesis of nicotinamide adenine dinucleotide (NAD⁺). By synthesizing NAD⁺, PBEF functions to maintain an energy supply that has critical roles in cell survival. Cerebral ischemia is a major neural disorder with a high percentage of mortality and disability. Ischemia leads to energy depletion and eventually neuronal death and brain damage. This study investigated the role of PBEF in cerebral ischemia using a photothrombosis mouse model. Using immunostaining, we initially determined that PBEF is highly expressed in neurons, but not in glial cells in the mouse brain. To study the role of PBEF in ischemia in vivo, we used PBEF knockout heterozygous (Pbef+/−) mice. We showed that these mice have lower PBEF expression and NAD⁺ level than do wild-type (WT) mice. When subjected to photothrombosis, Pbef+/− mice have significantly larger infarct volume than do age-matched WT mice at 24 hours after ischemia. Higher density of degenerating neurons was detected in the penumbra of Pbef+/− mice than in WT mice using Fluoro-Jade B staining. Our study shows that PBEF has a neuronal protective role in cerebral ischemia presumably through enhanced energy metabolism.     

Functional recovery after delayed treatment of ischemic stroke with melanocortins is associated with overexpression of the activity-dependent gene Zif268

Melanocortin peptides afford strong neuroprotection and improve functional recovery in experimental ischemic stroke; they also have established neurotrophic actions. The expression of the immediate early gene Zif268 is dependent on synaptic activity and is involved in injury repair and memory formation. Here, we investigated the role of Zif268 in learning and memory recovery after delayed treatment of ischemic stroke with the melanocortin analog [Nle⁴, d-Phe⁷]α-MSH (NDP-α-MSH). A 10-min period of global cerebral ischemia was induced by occluding both common carotid arteries in gerbils. Treatment with a nanomolar dose of NDP-α-MSH (every 12 h for 11 days) was performed starting 3 h or 9 h after stroke induction; where indicated, gerbils were pretreated with the melanocortin MC₄ receptor antagonist HS024. Animals were subjected to the Morris water-maze test (four sessions from 4 to 50 days after the ischemic episode). Fifty days after stroke, histological damage and Zif268 expression were investigated in the hippocampus. Treatment with NDP-α-MSH significantly reduced hippocampal damage, including neuronal death, and improved learning and memory recovery. This protective effect was long-lasting (50 days, at least) and associated with Zif268 overexpression, with both schedules of NDP-α-MSH treatment. Pharmacological blockade of MC₄ receptors prevented these effects. Our data indicate that MC₄ receptor-mediated actions of melanocortins could trigger repair mechanisms able to improve neuronal functionality and synaptic plasticity, and to promote long-lasting functional recovery from ischemic stroke with Zif268 gene involvement.     

Subtype-specificity of the presynaptic α2-adrenoceptors modulating hippocampal norepinephrine release in rat

In vivo brain microdialysis and high-performance liquid chromatography with electrochemical detection were used to study the effect of different selective α₂-antagonists on hippocampal norepinephrine (NE) release in freely moving awake rat. Systemic administration (0.5 mg/kg i.p.) of either the α_2AD-antagonist BRL 44408 or the α-_2BC-antagonist ARC 239 did not significantly change the basal release of NE. At a higher dose (5 mg/kg i.p.) ARC 239 was still ineffective, whereas BRL 44408 caused a significant increase of the extracellular level of NE. Similar results were obtained from in vitro perfusion experiments. Rat hippocampal slices were loaded with [³H]NE and the electrical stimulation-evoked release of [³H]NE was determined. The α₂-antagonists were applied in a concentration range of 10⁻⁸ to 10⁻⁶ M. ARC 239 was ineffective, whereas BRL 44408 significantly increased the electrically induced release of [³H]NE. In agreement with the data of microdialysis and perfusion experiments. BRL 44408 displaced [³H]yohimbine from hippocampal and cortical membranes of rat brain with high affinity whereas ARC 239 was less effective. The pK_i values of eight different α₂-adrenergic compounds showed a very good correlation (r = 0.98, slope = 1.11 P < 0.0001) in hippocampus and frontal cortex where the α₂-adrenoceptors have been characterized as α_2D-subtype. Our data indicate that hippocampal NE release in rat is regulated by α_2D-adrenoceptors, a species variation of the human α_2A-subtype.     

Cerebral vessels express interleukin 1β after focal cerebral ischemia

Rapid and marked increased levels of expression of interleukin 1β (IL-1β) mRNA have been detected in animal models of cerebral ischemia. However, the protein production of IL-1β and the cellular sources of IL-1β are largely undefined after cerebral ischemia. In the present study, we have measured the cellular localization of IL-1β protein in brain tissue from non-ischemic and ischemic mice using immunohistochemistry. Male C57B/6J (n=45) mice were subjected to middle cerebral artery (MCA) occlusion by a clot or a suture. The mice were sacrificed at time points spanning the period from 15 min to 24 h after onset of the MCA occlusion. Non-operated and sham-operated mice were used as control groups. A monoclonal anti-IL-1β antibody was used to detect IL-1β. In the non-operated and sham-operated mice, a few IL-1β immunoreactive cells were detected scattered throughout both hemispheres. IL-1β immunoreactive cells increased in the ischemic lesion as early as 15 min and peaked at 1 h to 2 h after MCA occlusion. IL-1β immunoreactivity was detected in the cortex of the contralateral hemisphere 1 h after ischemia. By 24 h after onset of ischemia, IL-1β immunoreactivity was mainly present adjacent to the ischemic lesion and in the non-ischemic cortex. IL-1β immunoreactivity was found on endothelial cells and microglia. This study demonstrates an early bilateral expression of IL-1β on endothelium after MCA occlusion in mice.     

α2-Adrenergic receptor activation inhibits calcitonin gene-related peptide expression in cultured dorsal root ganglia neurons

Calcitonin gene-related peptide (CGRP), a potent vasodilator, is produced in dorsal root ganglia (DRG) neurons which extend nerves peripherally to blood vessels and centrally to the spinal cord. We previously reported that neuronal CGRP expression is significantly reduced in the spontaneously hypertensive rat (SHR) which could contribute to the elevated BP. Other studies suggest that the enhanced activity of the sympathetic nervous system in the SHR may mediate, at least in part, this reduction in neuronal CGRP expression via activation of α₂-adrenoreceptors (α₂-AR) on DRG neurons. To test this hypothesis in vitro we employed primary cultures of adult rat DRG neurons. Neuronal cultures were initially exposed (24 h) to either the α₂-AR agonist UK 14,304 (10⁻⁶ M) or vehicle; however, no changes in CGRP mRNA content or immunoreactive CGRP (iCGRP) release were observed. Using the rationale that in vivo DRG neurons receive a continuous supply of target tissue derived nerve growth factor (NGF), which stimulates CGRP synthesis, the cultured neurons were treated (24 h) with either vehicle, NGF (25 ng/ml) alone, or NGF plus UK. NGF treatment increased CGRP mRNA accumulation 5.5±0.9-fold (p<0.001) and iCGRP release 2.9±0.4-fold (p<0.001) over control levels. The stimulatory effects of NGF were markedly attenuated, but not abolished, by UK (NGF+UK vs. control, CGRP mRNA, 2.9±0.4-fold, p<0.05; iCGRP, 1.7±0.2-fold, p<0.05). These values were also significant (p<0.05) when compared to NGF treatment alone. Experiments performed using the α₂-antagonist yohimbine confirmed that the effects of UK were mediated by the α₂-AR. These results, therefore, demonstrate that α₂-AR activation attenuates the stimulatory effects of NGF on CGRP expression in DRG neurons.     

Immunohistochemical detection of oxidative DNA damage induced by ischemia–reperfusion insults in gerbil hippocampus in vivo

There is much evidence to suggest that ischemic injury occurs during the reperfusion phase of ischemia–reperfusion insults, and that the injury may be due to reactive-oxygen-species (ROS)-mediated oxidative events, including lipid peroxidation and DNA damage. However, oxidative DNA damage has until now not been examined in situ. In the present study, we report for the first time observation of cell type- and region-specific oxidative DNA damages in 5 min transient ischemic model by immunohistochemical methods, using monoclonal antibody against 8-hydroxy-2′-deoxyguanosine (8-OHdG), an oxidative DNA product. The cell types containing 8-OHdG immunoreactivity were neurons, glia and endothelial cells in the hippocampus. The 8-OHdG immunoreactivity was present in the nucleus but not the cytoplasm of these cells. The level of 8-OHdG in CA1 increased significantly (P<0.05) at the end of 30 min after ischemia, but there was no increase within CA2 and CA3 areas. The 8-OHdG levels in the hippocampus increased significantly (about fourfold) after 3 h of reperfusion and remained significantly (P<0.01) elevated for at least 12 h. At 4 days after ischemia, 8-OHdG levels in the CA2 and CA3 areas decreased to levels of the sham without neuronal loss, while disappearance of 8-OHdG immunoreactivity in the CA1 coincided with neuronal death in this area. These findings strongly suggest that ischemia-induced DNA damage evolves temporally and spatially, and that oxidative DNA damage may be involved in delayed neuronal death in the CA1 region.     

Late expression of Na+/H+ exchanger 1 (NHE1) and neuroprotective effects of NHE inhibitor in the gerbil hippocampal CA1 region induced by transient ischemia

Although acidosis may be involved in neuronal death, the participation of Na⁺/H⁺ exchanger (NHE) in delayed neuronal death in the hippocampal CA1 region induced by transient forebrain ischemia has not been well established. In the present study, we investigated the chronological alterations of NHE1 in the hippocampal CA1 region using a gerbil model after ischemia/reperfusion. In the sham-operated group, NHE1 immunoreactivity was weakly detected in the CA1 region. Two and 3 days after ischemia/reperfusion, NHE1 immunoreactivity was observed in glial components, not in neurons, in the CA1 region. Four days after ischemia/reperfusion, NHE1 immunoreactivity was markedly increased in CA1 pyramidal neurons as well as glial cells. These glial cells were identified as astrocytes based on double immunofluorescence staining. Western blot analysis also showed that NHE protein level in the CA1 region began to increase 2 days after ischemia/reperfusion. The treatment of 10 mg/kg 5-(N-ethyl-N-isopropyl) amiloride, a NHE inhibitor, significantly reduced the ischemia-induced hyperactivity 1day after ischemia/reperfusion. In addition, NHE inhibitor potently protected CA1 pyramidal neurons from ischemic damage, and NHE inhibitor attenuated the activation of astrocytes and microglia in the ischemic CA1 region. In addition, NHE inhibitor treatment blocked Na⁺/Ca²⁺ exchanger 1 immunoreactivity in the CA1 region after transient forebrain ischemia. These results suggest that NHE1 may play a role in the delayed death, and the treatment with NHE inhibitor protects neurons from ischemic damage.     

Selegiline treatment after transient global ischemia in gerbils enhances the survival of CA1 pyramidal cells in the hippocampus

Selegiline (l-deprenyl) has shown neuroprotective effects in a variety of degenerative processes. The present experiments were designed to test whether post-ischemia administered selegiline would alleviate delayed neuronal death of the gerbil hippocampal pyramidal cells following transient global ischemia. Common carotid arteries were occluded for 5 min. Saline or selegiline, 0.25 mg/kg s.c., was administered 2 h after the ischemia followed by a daily injection for either 3 or 7 days. After decapitation, the delayed death of the hippocampal CA1 pyramidal cells was assessed using Nissl-stained sections. In situ hybridization was used to reveal the expression of hsp70 mRNA 1, 3 or 7 days after the ischemia. Animals treated with selegiline for 7 days showed significantly lower damage score (scale 0–3: 0, normal; 1, <10% of the neurons damaged; 2, 10–50% damaged; 3, >50% damaged) compared to the saline-treated animals 1.73±0.18 and 2.41±0.16 (mean±S.E.M., P=0.0133), respectively. A similar trend was found in animals after the 3-day treatment: 1.68±0.32 vs. 2.06±0.25 (P>0.5). The expression of hsp70 mRNA in the CA1 pyramidal cell layer was strong still 3 days after the ischemic insult but vanished by 7 days. Densitometric measurements using ¹⁴C-plastic standards showed that the intensity of the CA1a hsp70 signal on the 3rd day correlated negatively to the cell-damage score (r=−0.72, P<0.001), suggesting that hsp70 does not serve as a quantitative marker for CA1 neuronal injury in this model. Instead, the hsp70 expression was associated with improved neuronal survival lasting often longer in selegiline-treated animals (P>0.5). The results show that a low dose of selegiline can alleviate the delayed hippocampal neuronal death in gerbils when administered 2 h after an ischemic insult.     

α1-Adrenergic agonists act on the ventromedial hypothalamus to cause neuronal excitation and lordosis facilitation: electrophysiological and behavioral evidence

To see if activation of central α₁-adrenergic receptors can cause facilitation of lordosis in rats, the behavioral effects of centrally administered α₁-agonist, methoxamine (MA) and phenylephrine (PhE), and related agents were studied. In ovariectomized rats treated with estrogen, infusion of MA, PhE, or a β-agonist isoproterenol, into the lateral ventricle, or bilateral infusions of MA or PhE into the ventromedial hypothalamus (VMH) facilitated lordosis. Conversely, intra-VMH infusion of the α₁-antagonist prazosin (PZ) inhibited lordosis. Intra-VMH infusion of isoproterenol or an α₂-agonist clonidine, had no effect. Neither was the intra-VMH infusion of MA effective if: (i) the rats were not primed with estrogen; (ii) the tips of the cannulae were outside the VMH; or (iii) it was preceded by an intra-VMH infusion of the α_1b-antagonist, chloroethylclonidine (CEC). These results not only verify implications from recent studies that α₁-receptors in the hypothalamus are important for lordosis facilitation, but further show that the adrenergic facilitatory effect are: (i) mediated specifically by α_1b-subtype of the α₁-receptor, (ii) estrogen-dependent, and (iii) site-specific to VMH. To investigate neural mechanisms potentially underlying the lordosis-facilitating effect of α₁-activation, the actions of MA and PhE on the electrical activity of single neurons of the ventromedial nucleus of the hypothalamus (VMN) in vitro were studied. As in the behavioral study, the neuronal actions of the agonists are similar to each other: (i) both are excitatory — MA affected 78% of the 65 units tested, all by excitation, and PhE affected 74% of 65 VMN units, predominantly with excitation; (ii) the excitatory actions of MA and PhE affected the same population of VMN neurons: and (iii) the excitatory actions of both agonists were blocked by both PZ and CEC, indicative of mediation by α_1b-receptors. Behavioral/electrophysiological parallels suggest that act α₁-agonists through α_1b-receptors to excite VMN neurons and thereby facilitate lordosis. Since α_1b-receptors are known to be coupled to phosphoinositide (PI) second messenger system, and since the behavioral effect is estrogen-dependent, the facilitatory effect of α₁-agonists may require estrogen modulation of α_1b-receptors and/or the coupled PI system in the VMN.     

Inhibition of -homocysteic acid and buthionine sulphoximine-mediated neurotoxicity in rat embryonic neuronal cultures with α-lipoic acid enantiomers

In the present report, we have set out to investigate the potential capacity of both the oxidised and reduced forms of RS-α-lipoic acid, and its separate R-(+) and S-(−)enantiomers, to prevent cell death induced with -homocysteic acid ( -HCA) and buthionine sulphoximine (BSO) in rat primary cortical and hippocampal neurons. -HCA induced a concentration-dependent neurotoxic effect, estimated by cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) reduction, in primary neurons, but was significantly more toxic for hippocampal (EC₅₀=197 μM) compared with cortical neurons (EC₅₀=1016 μM) whereas -HCA demonstrated only moderate (<20%) toxicity. On the other hand, cortical and hippocampal cultures were equally susceptible (341 and 326 μM, respectively) to the neurotoxic action of BSO. Antioxidants including butylated hydroxyanisole, propyl gallate and vitamin E protected cells against the neurotoxic effect of -HCA and BSO. However, N-acetyl-cysteine and tert-butylphenyl nitrone, although capable of abrogating -HCA-mediated cell death showed no protective effect against BSO-mediated toxicity. RS-α-lipoic acid, RS-α-dihydrolipoic acid and the enantiomers R-α-lipoic acid and S-α-lipoic acid protected cells against -HCA-mediated toxicity with EC₅₀ values between 3.1–8.3 μM in primary hippocampal neurons and 2.6–16.8 μM for cortical neurons. However, RS-α-lipoic acid, RS-α-dihydrolipoic acid, and S-α-lipoic acid failed to protect cells against the degeneration induced by prolonged exposure to BSO, whereas the natural form, R-α-lipoic, was partially active under the same conditions. The present results indicate a unique sensitivity of hippocampal neurons to the effect of -HCA-mediated toxicity, and suggest that RS-α-lipoic acid, and in particular the R-α-enantiomeric form is capable of preventing oxidative stress-mediated neuronal cell death in primary cell culture.     

The effects of histamine H3-receptor antagonists on amygdaloid kindled seizures in rats

The effects of histamine H₃-receptor antagonists, thioperamide, and clobenpropit on amygdaloid kindled seizures were investigated in rats. Both intracerebroventricular (i.c.v.) and intraperitoneal (i.p.) injections of H₃-antagonists resulted in a dose-related inhibition of amygdaloid kindled seizures. An inhibition induced by thioperamide was antagonized by an H₃-agonist [(R)-α-methylhistamine] and H₁-antagonists (diphenhydramine and chlorpheniramine). On the other hand, an H₂-antagonist (cimetidine and ranitidine) caused no antagonistic effect. Metoprine, an inhibitor of N-methyltransferase was also effective in inhibiting amygdaloid kindled seizure, and this effect was augmented by thioperamide treatment.     

Comparative neuroprotective properties of the benzodiazepine receptor full agonist diazepam and the partial agonist PNU-101017 in the gerbil forebrain ischemia model

Recent studies have demonstrated the neuroprotective properties of the novel imidazoquinoline benzodiazepine receptor partial agonist, PNU-101017, in the gerbil forebrain ischemia model. The compound effectively reduces delayed post-ischemic (5 min bilateral carotid occlusion) hippocampal CA₁ neuronal degeneration even when its administration is witheld until 4 h after reperfusion and the effect is unrelated to hypothermia. The purpose of the present study was to determine the comparative abilities of PNU-101017 versus the full agonist diazepam to attenuate post-ischemic CA₁ damage. Male gerbils were treated either 30 min before ischemia induction or immediately after reperfusion with an initial dose of PNU-101017 (30 mg/kg i.p.) or diazepam (10 mg/kg i.p.) with a second dose being given at 2 h after reperfusion. Possible hypothermic effects of either compound were prevented by external heating. In vehicle (0.05 N HCl)-treated gerbils, the loss of hippocampal CA₁ neurons at 5 days was 85%. PNU-101017 pretreatment reduced the loss to 50% (p<0.05 vs. vehicle) whereas pretreatment with diazepam attenuated damage to only 17% (p<0.001 vs. vehicle). Delaying treatment with PNU-101017 until just after reperfusion still resulted in a reduction in CA₁ degeneration statistically that was indistinguishable from that seen with pretreatment. In contrast, diazepam post-treatment did not significantly decrease CA₁ neuronal loss. These results suggest that a benzodiazepine receptor partial agonist may have greater neuroprotective practicality than a full agonist for the treatment of global cerebral ischemia. The mechanistic basis for this difference may relate to the partially pro-excitatory neuronal response to endogenous GABA before and after neuronal insult.