Modulation of pro-survival and death-associated pathways under retinal ischemia/reperfusion: effects of NMDA receptor blockade.

Loss of retinal ganglion cells occurs in a variety of pathological conditions, including central retinal artery occlusion, diabetes and glaucoma. Using an experimental model of retinal ischemia induced by transiently raise the intraocular pressure (IOP), In this study, we report the original observation that ischemic retinal ganglion cells death is associated with the transient deactivation of the pro-survival kinase Akt and activation of GSK-3beta followed, during reperfusion, by a longer lasting, PI3K-dependent, activation of Akt and phosphorylation of GSK-3beta. Under these experimental conditions, retinal ischemia induced the expression of Bad, a pro-apoptotic protein, member of the Bcl-2 family. The detrimental effects yielded by the ischemic stimulus were minimized by intravitreal administration of the NMDA receptor antagonist, MK801, that reduced the expression of Bad and significantly increased Akt phosphorylation. In conclusion, our present results contribute to unravel the mechanisms underlying retinal damage by high IOP-induced transient ischemia in rat. In addition, these data implicate the pro-survival PI3K/Akt pathway and the observed reduced expression of Bad in the neuroprotection afforded by MK801.

Choline pivaloyl ester strengthened the benefit effects of Tacrine and Galantamine on electroencephalographic and cognitive performances in nucleus basalis magnocellularis-lesioned and aged rats.

The aim of the present work was the assessment of the effects produced on the electroencephalographic (EEG) activity and the cognitive and memory performances of nucleus basalis magnocellularis (NBM)-lesioned or aged rats by the combined treatment with [2-(2,2-dimethylpropionyloxy)ethyl]trimethylammonium 2,2-dimethylpropionate (choline pivaloyl ester) (CPE) and the Cholinesterase inhibitors (ChEIs) Tacrine (THA) and Galantamine (GAL). Intraperitoneal administration of CPE combined with THA or GAL to both NBM-lesioned or aged rats, produced EEG desynchronisation, and a significant decrease in the energy of the total EEG spectrum and the lower frequency bands (delta 0.25-3 and theta 4-7 Hz) lasting many minutes. Furthermore, drug associations reversed in aged rats the scopolamine (0.2 mg/kg, i.p.)-induced increase in EEG power, slow waves and high-voltage spindle (HVS). Furthermore, the combined administration of CPE and Cholinesterase inhibitors in both NBM-lesioned or aged animals, improved performances in all behavioural tasks, enhancing object discrimination, increasing locomotory activity and alternation choice in T-maze, ameliorating retention in passive avoidance and decreasing escape latency in Morris water maze. In all test, AChEIs and CPE combinations proved to be more effective than CPE, THA or GAL given alone. In conclusion, the present work shows the ability of choline pivaloyl ester in strengthening the positive cerebral activity of THA and GAL.

Effects of tetanus toxin after intracerebral microinjection are antagonized by drugs enhancing GABAergic transmission in adult fowls.

In adult fowls (Gallus domesticus), the behavioural effects of small doses of tetanus toxin, after unilateral microinjection into the nucleus mesencephalicus profundus (NMP), homologous to the mammalian substantia nigra, were studied. A rich pattern of stereotyped movements, vocalization, ipsilateral head-neck rotation, wing abduction, occasional ipsilateral circling and escape responses from the box were observed; the intensity of such symptomatology was dose-related and fully reversible within approx. 4 hr. Pretreatment with sodium valproate (100 and 200 mg/kg, i.m.) or with ethanolamine-sulphate (EOS, 1.6 mumol into the NMP) completely prevented the effects of tetanus toxin. The present findings show that fowls can represent an ideal species for studying acute central effects of tetanus toxin and more interestingly that drugs enhancing GABAergic mechanisms are able to prevent the effects of tetanus toxin.

Effects of apomorphine on glutamate decarboxylase activity in chick paleostriatum augmentatum.

In young chicks the effects of two different doses of apomorphine, a small dose, producing behavioural and electrocortical sleep and a larger one producing arousal, on GABA content, GAD and GABA-T activities in the paleostriatum augmentatum were studied. The small dose of apomorphine did not affect GABAergic mechanisms in this area, whereas the dose producing behavioural and electrocortical arousal significantly increased GAD activity and GABA content. The results of the present experiments are in favour of an interaction between dopaminergic and GABAergic mechanisms in the avian paleostriatum augmentatum.

Effects of compounds acting on GABA(B) receptors in the pentylenetetrazole kindling model of epilepsy in mice.

The involvement of GABA(B) receptors in the behavioural and epileptic electrocortical discharges occurring in chemical kindling induced by repeated treatments with a subconvulsant dose of pentylenetetrazole (25 mg/kg i.p.) has been investigated in CD1 mice. Behavioural and electrocorticographic epileptic seizures following kindling induced by pentylenetetrazole (25 mg/kg i.p.) were attenuated or completely antagonized in a dose-dependent manner by the GABA(B) receptor agonist R-baclofen (2 and 6 mg/kg) whilst the GABA(B) receptor antagonist 3-amino-propyl-diethoxy-methyl-phosphinic acid (CGP 35348, 25, 50 or 100 mg/kg) and 3-[1-(S)-(3, 4-dichloro-phenyl-ethyl]amino-2-(S)-hydroxy-propyl-benzyl-phosphinic acid (CGP 55845A, 10 or 20 mg/kg) produced a more rapid development of kindling and an increase in behavioural and electrocorticographic epileptic changes. In addition, all GABA(B) receptor antagonists were able to induce an increase in Fos and Jun protein expression in pentylenetetrazole (25 mg/kg i.p.) treated mice whilst the GABA(B) receptor agonist R-baclofen (2 or 6 mg/kg) attenuated the expression of Fos and Jun protein, at cortical and limbic structures. In order to study the persistence of changes induced by pentylenetetrazole kindling, different groups of mice were rechallenged with a kindling stimulus 15 or 30 days after withdrawal from the last injection of vehicle+pentylenetetrazole, R-baclofen+pentylenetetrazole or GABA(B) receptor antagonists+pentylenetetrazole. The groups receiving GABA(B) receptor antagonists+pentylenetetrazole showed a higher incidence of seizures following the kindling stimulus than mice receiving vehicle+pentylenetetrazole whilst animals treated with R-baclofen were protected from the kindling stimulus. The different effects observed following repeated treatment with the GABA(B) receptor agonist and antagonist used revealed that GABA(B) receptors are able to affect the development of the epileptic kindling state induced by pentylenetetrazole.

The effect of nitric oxide on cytokine-induced release of PGE2 by human cultured astroglial cells.

1. The role of the L-arginine-nitric oxide (NO) pathway on the formation of prostaglandin E2 (PGE2) by human cultured astroglial cells incubated with interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha) was investigated. 2. Incubation of T 67 astroglial cell line with IL-beta (10 ng ml(-1)) and TNF-alpha (500 u ml(-1)) produced a significant (P<0.05) increase of both nitrite (the breakdown product of NO), cyclic GMP and PGE2 levels in cell supernatants. N omega-nitro-L-arginine methyl ester (L-NAME; 20-300 microM), an inhibitor of NO synthase (NOS), inhibited the increase of cyclic GMP and nitrite levels found in supernatants of cytokine-treated astroglial cells and reduced the release of PGE2. The latter effect showed that the enhanced arachidonic acid (AA) metabolism subsequent to stimulation of astroglial cells with IL-1beta and TNF-alpha was, at least in part, induced by NO. This occurred also when sodium nitroprusside (SNP; 120 microM), an NO donor, was incubated with astroglial cells, an effect antagonized by oxyhaemoglobin (OxyHb; 10 microM). 3. The inhibition elicited by L-NAME on PGE2-release by cytokine-treated astroglial cells was reversed by adding AA (40 microM), showing that the effect of NO on cytokine-dependent PGE2 release occurred at the cyclo-oxygenase (COX) level. Furthermore, the release of PGE2 in cytokine-treated astroglial cells was inhibited by indomethacin (10 microM), a COX inhibitor as well as by preincubating cells with dexamethasone (20 microM), an inhibitor of inducible enzymes, showing that the inducible isoform of COX (COX-2) was involved. 4. On the other hand, pretreating astroglial cells with methylene blue (MB; 10 microM), an inhibitor of NO biological activity acting at the guanylate cyclase level, failed to affect PGE2 release in cytokine-treated astroglial cells, leading to the conclusion that cyclic GMP changes related to NO formation are not involved in the generation of AA metabolites. 5. The present experiments demonstrated that the release of PGE2 by astroglial cells pretreated with IL-1beta and TNF-alpha is due to enhanced COX-2 activity via activation of the L-arginine-NO pathway, and this may be relevant to the understanding of the pathophysiological mechanisms underlying neuroimmune disorders.

The HIV envelope protein gp120 in the nervous system: interactions with nitric oxide, interleukin-1beta and nerve growth factor signalling, with pathological implications in vivo and in vitro.

The neuronal loss often described at post-mortem in the brain neocortex of patients suffering from AIDS has been proposed to be responsible for the development of the AIDS dementia complex. Neuroinvasive strains of the HIV virus infect macrophages, microglial cells, and multinucleated giant cells, but not neurones. Processing of the virus by cells of the myelomonocytic lineage yields viral products known to initiate a complex network of events that may lead to the death of neurones and to the development of AIDS-associated neurological syndrome. The HIV-1 coat protein gp120, in particular, has been proposed as a likely etiologic agent of the described neuronal loss because it causes the death of neurones in culture. More recently, it has been shown that brain cortical cell death caused in rats by intracerebroventricular injection of gp120 occurs via apoptosis. This observation broadens our knowledge of the pathophysiology of the reported neuronal cell loss and opens a new avenue of experimental research for the development of novel therapeutic strategies for the treatment of patients suffering from AIDS-associated neurological syndrome.

Effects of glucocorticoids on activation of c-jun N-terminal, extracellular signal-regulated, and p38 MAP kinases in human pulmonary endothelial cells.

Mitogen-activated protein kinases (MAPK) play a central role in signal transduction by regulating many nuclear transcription factors involved in inflammatory, immune, and proliferative responses. The aim of this study was to investigate, in human pulmonary endothelial cells, the effects of synthetic glucocorticosteroids on activation of c-jun N-terminal kinases, extracellular signal-regulated kinases, and p38 subgroups of the MAPK family. Human microvascular endothelial cells from lung were stimulated for 2 h with either H(2)O(2) (2 mM), IL-1beta (10 ng/mL), or tumour necrosis factor-alpha (10 ng/mL). Under these conditions, a remarkable increase in the phosphorylation pattern of c-jun N-terminal kinases, extracellular signal-regulated kinases 1/2, and p38 was detected. Pretreatment for 12 h with dexamethasone (100 nM) was able to prevent phosphorylation-dependent MAPK activation in stimulated cells, without substantially affecting the expression levels of these enzymes. Our results suggest that inhibition of MAPK signaling pathways in human pulmonary endothelial cells may significantly contribute, by interfering with activation of several different transcription factors, to the antiinflammatory and immunosuppressive effects of glucocorticosteroids.

Evidence that behavioural and electrocortical sleep induced by guanfacine is due to stimulation of alpha 2-adrenoceptors.

The effects of guanfacine and other drugs acting at alpha 1- and alpha 2-adrenoceptors on behaviour, electrocortical activity and ECoG spectrum power were studied in chicks and rats. Guanfacine, given systematically in chicks, produced behavioural and electrocortical slow-wave sleep lasting 100-200 min, depending on the dose; these effects were prevented by yohimbine, a selective antagonist, at alpha 2-adrenoceptors and potentiated by prazosin, a selective antagonist, at alpha 1-adrenoceptors. Similar behavioural and electrocortical effects were obtained after systemic or intraventricular infusion of guanfacine in rats. In addition, a significant increase in total and in lower frequency band (0-4; 4-8 Hz) voltage power was observed. Behavioural and ECoG effects of guanfacine were prevented by phentolamine or yohimbine, whereas prazosin and propranolol were ineffective. Yohimbine itself, given systemically in chicks, produced behavioural stimulation, vocalization, increase in locomotor activity and ECoG desynchronization, with a significant fall in total and 0-3, 3-6, 6-9 and 9-12 Hz voltage power lasting approx. 3 h. Desipramine, an inhibitor of noradrenaline reuptake, produced in chicks behavioural and ECoG arousal, vocalization, pecking, escape responses and aggressive behaviour. In conclusion, the present experiments show that guanfacine sedative effects seem to be mediated predominantly via an activation of presynaptic alpha 2-adrenoceptors and suggest that arousal is due to stimulation of post-synaptic alpha 1-adrenoceptors.

Central cardiovascular responses induced by interleukin 1 beta and tumor necrosis factor alpha infused into nucleus tractus solitarii, nucleus parabrachialis medialis and third cerebral ventricle of normotensive rats.

The effect of IL-1 beta and TNF alpha infused into nucleus tractus solitari (NTS), nucleus parabrachialis medialis (NPBmed) and third cerebral ventricle of normotensive rats on blood pressure (BP) and heart rate (HR) was investigated. Microinfusion of IL-1 beta and TNF alpha into the third cerebral ventricle and NPBmed of normotensive rats produced a dose-dependent hypotensive and bradycardic response. A similar cardiovascular response was produced by infusion of IL1 beta into NTS but not by TNF alpha. When rats were pre-treated with Escherichia coli lipopolisaccharide (LPS), an enhancement of cardiovascular response elicited by IL-1 beta and TNF alpha was found. Thus, IL-1 beta and TNF alpha produce cardiovascular responses when infused into specific areas of the CNS. This effect is potentiated by LPS and this may explain the alteration in cardiovascular regulation which can be observed in diseases in which an excess of circulating endotoxins and cytokines may occur.

Neurodegeneration produced by intrahippocampal injection of paraquat is reduced by systemic administration of the 21-aminosteroid U74389F in rats.

The behavioural, electrocortical (ECoG) and neurodegenerative effects of intrahippocampal injection of paraquat, a well-known free radical producing agent, were studied in rats. Injection of paraquat (100 nmol) into one dorsal hippocampus produced limbic motor and ECoG seizures. These effects were accompanied at 24 h by severe damage to CA1, CA3 and CA4 hippocampal pyramidal neurones and dentate gyrus granule cells. In comparison to the cell number counted in control, untreated, side of the hippocampus, significant (P < 0.05) neuronal loss was observed in the CA1 and CA3 pyramidal cell layers of the treated hippocampus. A lower dose of the herbicide (10 nmol) did not produce consistent motor and ECoG effects and in no instance was significant neuronal loss observed. A pretreatment with U74389F [21-4-(2,6-di-l-pyrrodinyl-4-pyridinyl)-1-piperazinyl-pregna-1,4,9 (11)triene-3,20-dione monomethansulfonate] (30 mg/kg i.p., 15 min before paraquat) completely protected rats from motor and ECoG epileptogenic effects induced by intrahippocampal paraquat (100 nmol). This dose of U74389F also reduced the hippocampal damage typically produced by paraquat and no significant neuronal loss was reported in the CA1 and CA3 pyramidal cell layers. A lower dose of U74389F (10 mg/kg i.p.) did not afford any protection against the epileptogenic effects produced by paraquat (100 nmol); in these animals hippocampal damage was still evident though neuronal loss did not reach statistical significance. In conclusion, the present data show that systemic administration of U74389F possesses neuroprotective effects against seizures and neurodegeneration typically elicited by intrahippocampal injection of paraquat.

Enhanced behavioural, electrocortical and hyperthermic effects of serotonin-like agents after impairment of serotonin transmission in fowl brain.

In adult fowls and in young chicks (Gallus domesticus) the effects were studied on body temperature, behaviour and electrocortical activity of 5-HT and other direct or indirect serotonin agonists given into the III cerebral ventricle in conditions of prolonged impairment of serotoninergic transmission. In chicks pretreated with 5,6-dihydroxytryptamine the subsequent intraventricular injection of 5-HT produced more intense and longer-lasting hyperthermic response and behavioural and electrocortical sleep. On the other hand fenfluramine given intraventricularly in fowls pretreated with 5,6-DHT did not produce significant body temperature and behavioural changes. After 14 and 21 day treatment with methysergide the subsequent administration of 5-HT, fenfluramine and quipazine produced in fowls an hyperthermic response and behavioural sleep more marked and longer-lasting than in control animals. The present experiments show that in conditions of chronic impairment of 5-HT function there is an enhanced behavioural and body temperature response to 5-HT and drugs acting by releasing endogenous 5-HT or 5-HT agonists.

Epileptogenic effects of skin extracts from the Australian frog Pseudophryne coriacea after intracerebral microinfusion in rats.

The behavioural and electrocortical (ECoG) effects induced by a methanol extract of the skin of the Australian frog Pseudophryne coriacea, directly microinjected into several areas of the brain, were studied in freely moving rats. Administration of the P. coriacea extract (5, 10, 15 and 20 micrograms) into the dorsal hippocampus produced a dose-dependent and reversible behavioural stimulation and ECoG spikes lasting 20-140 min. Similar but less intense effects were elicited in rats receiving injections into the III cerebral ventricle, amygdala and caudate nucleus. In conclusion, the present experiments show that the skin extract of P. coriacea produces behavioural stimulation and ECoG spikes when injected into the rat brain, the most sensitive area being the hippocampus.

Comparative, behavioural and electrocortical effects of tumor necrosis factor-alpha and interleukin-1 microinjected into the locus coeruleus of rat.

The behavioural and electrocortical (ECoG) effects of human recombinant tumor necrosis factor-alpha (hrTNF-alpha) and various forms of interleukin-1 (IL-1) microinjected into the locus coeruleus (LC) of rats were studied. IL-1 induced a typical, dose-dependent, behavioural sedation and/or sleep which was associated with ECoG synchronization. IL-1 beta appeared more potent than IL-1 alpha. During sleep induced by the various forms of IL-1 a dose-dependent increase in total voltage power (0.25-16 Hz) as well as in the 3-6, 6-9 and sometimes 0.25-3 Hz frequency bands was observed. The behavioural and ECoG effects of IL-1 beta were blocked in rats pretreated with anti-IL-1 monoclonal antibodies. The microinjection of hrTNF-alpha into the LC produced a typical pattern characterized by a first short lasting (20-30 min) phase of behavioural arousal and ECoG desynchronization, followed by a longer lasting (45-80 min) phase of behavioural sedation and/or sleep and ECoG synchronization characterized by an increase in total voltage power as well as in the 3-6, 6-9 and sometimes 0.25-3 Hz frequency bands. The behavioural and ECoG effects of hrTNF-alpha were antagonized by a pretreatment (15 min before) with specific anti-TNF-alpha polyclonal antibodies. In addition, a pretreatment with anti-IL-1 receptor monoclonal antibodies was unable to significantly affect the stimulation of behaviour and ECoG desynchronization effects elicited by hrTNF-alpha whilst the same pretreatment completely prevent the sedative and ECoG synchronizing phase elicited by the microinjection of hrTNF-alpha into the LC. These results are consisted with the hypothesis that the sedative and/or soporific behavioural and ECoG changes of hrTNF-alpha are mediated, at LC level, through a local IL-1 release.

Neurobiological mediators of neuronal apoptosis in experimental neuroAIDS.

Neuronal loss has often been described at post-mortem in the brain neocortex of patients suffering from AIDS. Neuroinvasive strains of HIV infect macrophages, microglial cells and multinucleated giant cells but not neurones. Processing of the virus by cells of the myelomonocytic lineage yields viral products that, in conjunction with potentially neurotoxic molecules generated by the host, might initiate a complex network of events which leads neurones to death. In particular, the HIV-1 coat glycoprotein gp120 has been proposed as a likely aetiologic agent of the described neuronal loss because it causes death of neurones in culture. More recently, it has been shown that brain neocortical cell death is caused in rat by intracerebroventricular injection of a recombinant gp120 coat protein and this occurs via apoptosis. The latter observation broadens our knowledge in the pathophysiology of the reported neuronal cell loss and opens a new lane of experimental research for the development of novel therapeutic strategies to limit damage to the brain of patients suffering from HIV associated dementia.

Subacute cardiac rupture complicating myocardial infarction. A case report.

The authors have focused this study on the emergence of subacute ventricular free wall rupture in a seventy-six-year-old patient admitted to hospital for inferior acute myocardial infarction. After six days he showed clinical signs of bradycardia and hypotension evolving to electromechanical dissociation. Given an adequate pharmacologic therapy, the patient was submitted to echocardiography, which was believed to be consistent with myocardial rupture, showing a moderate to large pericardial effusion. Pericardiocentesis of 150 mL of bloody fluid resulted in a further improvement in his hemodynamics. The patient underwent cardiac surgery with repair of the myocardial rupture through a large diaphragmatic infarction by a Dacron polyester fiber graft and pacemaker placement. In conclusion the authors confirm the relevant role of clinical data such as persistent chest pain and hemodynamic instability and the value of echocardiography in identifying subacute myocardial free wall rupture after an episode of acute myocardial infarction.

Distribution of paraquat into the brain after its systemic injection in rats.

Paraquat concentrations were determined in specific brain areas of rats systemically treated with different doses of the herbicide and sacrificed at different time periods following the acute administration. The systemic treatment with paraquat gave rise to a regional differential distribution of the herbicide into the rat brain, the highest levels being detected in the prefrontal cortex and hypothalamus. The rate of paraquat elimination showed time-dependency in the prefrontal cortex and hypothalamus; by contrast, in the other regions studied the initial drop observed at 3 h was followed by a second phase of significant accumulation. The concentrations detected into the cortex may account for the neuronal cell death reported in rats following systemic injection of this herbicide.

Stimulatory effects on lactotrophs and crop-sac of interleukin-1 and interleukin-2 in pigeons (Columba livia).

The ultrastructural effects of human-recombinant interleukin-1 beta (IL-1 beta) and human-recombinant interleukin-2 (IL-2) on the crop-sac (the target organ for prolactin secretion in birds) and the anterior pituitary lactotrophs, were studied in pigeons (Columba livia). The intraventricular microinfusion of the two interleukins produced maximal crop-sac stimulation with milk-like secretion, as demonstrated by the observation of ultrastructural changes in the lactiferous areas through scanning and transmission electron microscopy of crop-sac mucosa. A marked activation of the anterior pituitary lactotrophs was also observed. Crop-sac and pituitary lactotrophs stimulatory effects were prevented by a previous intraventricular treatment with monoclonal antibodies for IL-1 beta and IL-2 receptors, but not by an intraperitoneal administration of naloxone. The present results show that interleukins possess in pigeons marked stimulatory effects on prolactin secretion and that these are mediated by specific receptors.

Calpain-mediated cleavage of Beclin-1 and autophagy deregulation following retinal ischemic injury in vivo.

Autophagy is the major intracellular degradation pathway that regulates long-lived proteins and organelles turnover. This process occurs at basal levels in all cells but it is rapidly upregulated in response to starvation and cellular stress. Although being recently implicated in neurodegeneration, it remains still unclear whether autophagy has a detrimental or protective role. In this study, we investigated the dynamics of the autophagic process in retinal tissue that has undergone transient ischemia, an experimental model that recapitulates features of ocular pathologies, including glaucoma, anterior ischemic optic neuropathy and retinal vessels occlusion. Retinal ischemia, induced in adult rats by increasing the intraocular pressure, was characterized by a reduction in the phosphatidylethanolamine-modified form of LC3 (LC3II) and by a significant decrease in Beclin-1. The latter event was associated with a proteolytic cleavage of Beclin-1, leading to the accumulation of a 50-kDa fragment. This event was prevented by intravitreal treatment with the non-competitive N-methyl-D-aspartate antagonist MK801 and calpain inhibitors or by calpain knockdown. Blockade of autophagy by pharmacological inhibition or Beclin-1 silencing in RGC-5 increased cell death, suggesting a pro-survival role of the autophagic process in this neuronal cell type. Altogether, our results provide original evidence for calpain-mediated cleavage of Beclin-1 and deregulation of basal autophagy in the rat retina that has undergone ocular ischemia/reperfusion injury.