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.

Neuroprotective agents in the management of glaucoma.

Glaucoma is an optic neuropathy, specifically a neurodegenerative disease characterized by loss of retinal ganglion cells (RGCs) and their axons. The pathogenesis of RGC loss in glaucoma remains incompletely understood and a broad range of possible mechanisms have been implicated. Clinical evidence indicates that lowering intraocular pressure (IOP) does not prevent progression in all patients; therefore, risk factors other than those related to IOP are involved in the disease. The need for alternative, non-IOP-lowering treatments focused at preventing progression, that is, neuroprotectants, has become of interest to both the patient and the physician. Experimental evidence accumulated during the past two decades lend a great deal of support to molecules endowed with neuroprotective features. However, translation to the clinic of the latter drugs results unsuccessful mostly because of the lack of reliable in vivo measure of retinal damage, thus hampering the good therapeutic potential of neuroprotective agents given alone or as adjuvant therapy to IOP-lowering agents. Further research effort is needed to better understand the mechanisms involved in glaucoma and the means to translate into clinic neuroprotective drugs.

Cell signaling pathways in the mechanisms of neuroprotection afforded by bergamot essential oil against NMDA-induced cell death in vitro.

BACKGROUND AND PURPOSE: The effects of bergamot essential oil (BEO; Citrus bergamia, Risso) on excitotoxic neuronal damage was investigated in vitro. EXPERIMENTAL APPROACH: The study was performed in human SH-SY5Y neuroblastoma cells exposed to N-methyl-D-aspartate (NMDA). Cell viability was measured by dye exclusion. Reactive oxygen species (ROS) and caspase-3 activity were measured fluorimetrically. Calpain I activity and the activation (phosphorylation) of Akt and glycogen synthase kinase-3beta (GSK-3beta) were assayed by Western blotting. KEY RESULTS: NMDA induced concentration-dependent, receptor-mediated, death of SH-SY5Y cells, ranging from 11 to 25% (0.25-5 mM). Cell death induced by 1 mM NMDA (21%) was preceded by a significant accumulation of intracellular ROS and by a rapid activation of the calcium-activated protease calpain I. In addition, NMDA caused a rapid deactivation of Akt kinase and this preceded the detrimental activation of the downstream kinase, GSK-3beta. BEO (0.0005-0.01%) concentration dependently reduced death of SH-SY5Y cells caused by 1 mM NMDA. In addition to preventing ROS accumulation and activation of calpain, BEO (0.01%) counteracted the deactivation of Akt and the consequent activation of GSK-3beta, induced by NMDA. Results obtained by using specific fractions of BEO, suggested that monoterpene hydrocarbons were responsible for neuroprotection afforded by BEO against NMDA-induced cell death. CONCLUSIONS AND IMPLICATIONS: Our data demonstrate that BEO reduces neuronal damage caused in vitro by excitotoxic stimuli and that this neuroprotection was associated with prevention of injury-induced engagement of critical death pathways.

Rational Basis for the Use of Bergamot Essential Oil in Complementary Medicine to Treat Chronic Pain.

In complementary medicine, aromatherapy uses essential oils to improve agitation and aggression observed in dementia, mood, depression, anxiety and chronic pain. Preclinical research studies have reported that the essential oil obtained from bergamot (BEO) fruit (Citrus bergamia, Risso) modifies normal and pathological synaptic plasticity implicated, for instance, in nociceptive and neuropathic pain. Interestingly, recent results indicated that BEO modulates sensitive perception of pain in different models of nociceptive, inflammatory and neuropathic pain modulating endogenous systems. Thus, local administration of BEO inhibited the nociceptive behavioral effect induced by intraplantar injection of capsaicin or formalin in mice. Similar effects were observed with linalool and linalyl acetate, major volatile components of the phytocomplex, Pharmacological studies showed that the latter effects are reversed by local or systemic pretreatment with the opioid antagonist naloxone hydrochloride alike with naloxone methiodide, high affinity peripheral µ-opioid receptor antagonist. These results and the synergistic effect observed following systemic or intrathecal injection of an inactive dose of morphine with BEO or linalool indicated an activation of peripheral opioid system. Recently, in neuropathic pain models systemic or local administration of BEO or linalool induced antiallodynic effects. In particular, in partial sciatic nerve ligation (PSNL) model, intraplantar injection of the phytocomplex or linalool in the ipsilateral hindpaw, but not in the contralateral, reduced PSNL-induced extracellularsignal- regulated kinase (ERK) activation and mechanical allodynia. In neuropathic pain high doses of morphine are needed to reduce pain. Interestingly, combination of inactive doses of BEO or linalool with a low dose of morphine induced antiallodynic effects in mice. Peripheral cannabinoid and opioid systems appear to be involved in the antinociception produced by intraplantar injection of ß -caryophyllene, present in different essential oils including BEO. The data gathered so far indicate that the essential oil of bergamot is endowed with antinociceptive and antiallodynic effects and contribute to form the rational basis for rigorous testing of its efficacy in complementary medicine.

Rational use of antibiotics in acute uncomplicated cystitis: a pharmaco-epidemiological study.

Uncomplicated community-acquired urinary tract infections are among those most commonly found in clinical practice, resulting in significant morbidity and health care costs. Current management is usually empirical because of the narrow and predictable spectrum of etiologic agents that cause acute cystitis and their susceptibility patterns. However, since antimicrobial resistance is increasing, the use narrow-spectrum, inexpensive antimicrobial agents becomes less feasible. In our study we have evaluated the effectiveness of amoxicillin, a narrow-spectrum, inexpensive and non toxic drug, against non-complicated acute cystitis in 34 patients, and compared the results with the antibiotic therapy previously employed by the physicians of the Health Care Unit of Paola (CS), Italy. Amoxicillin was found to be effective for the treatment of community-acquired cystitis, thus suggesting that the development of bacterial resistance does not represent a limit to its use. Furthermore, our study demonstrates that besides providing an effective alternative to broad-spectrum antibiotics, the use of amoxicillin significantly reduced health care costs.

Some pharmacological characteristics of the guinea pig ileum opioid system activated by cholecystokinin.

Naloxone, added after contractions induced by CCK-8 on the guinea pig ileum preparation, elicited a contraction attributed to the release of endogenous opioid which could inhibit the excitatory action of the peptide. With large concentrations of CCK-8, the preparation gave reproducible responses with time. Naloxone, added before the peptide, protracted the excitatory response to CCK-8, but not its height. Morphine decreased the response to CCK-8 but simultaneously raised the response to naloxone. The latter effect appeared very similar to the withdrawal contraction observed after brief exposure of the opioid in the guinea pig ileum to opioids. Clonidine, and alpha-2 adrenoceptor agonist, and nifedipine, a calcium channel antagonist, both known to interfere with tolerance and physical dependence, affected the excitatory response to CCK-8 and the subsequent response to naloxone in a different way.

Hippocampal extracellular amino acids and EEG spectral analysis in a genetic rat model of absence epilepsy.

Absence seizures have a clearly defined thalamocortical origin. However, there is evidence from a genetic rat model of absence epilepsy, GAERS, that the underlying cellular and molecular abnormalities may also manifest themselves in other brain regions. As enhanced learning has previously been associated with this rat model, we have studied extracellular amino acid levels and EEG spectra in the hippocampus of these rats, this being a brain region associated with memory and learning. We report significantly higher levels of basal extracellular glutamate within the hippocampus of GAERS, together with transient increases in citrulline and glycine following aggravation of the absence seizures with the GABA(B) agonist, (-)baclofen. Furthermore, there is a reduction in the relative power of the EEG theta frequencies in GAERS, and a slowing of the EEG following administration of (-)baclofen which is not evident in control animals. Administration of a GABA(B) antagonist, CGP 56999, at a dose which blocks absence seizures in GAERS, caused a shift to faster frequencies of the EEG in both GAERS and control rats. It is speculated that the mechanisms underlying absence seizures in GAERS may manifest themselves in other functions modulated by thalamocortical oscillations such as cognitive processing.

Mu and kappa opioid system interactions in the expression of acute opioid dependence in isolated guinea-pig ileum.

In vivo studies have suggested that the kappa opioid system can partially inhibit the development of physical dependence to mu agonists. Vice versa, activation of mu receptors may inhibit the expression of physical dependence to kappa agonists. We studied mu-kappa interactions in the isolated guinea-pig ileum (GPI). In the isolated GPI briefly exposed to mu or kappa agonists the addition of the respective antagonists precipitated a withdrawal contracture. After a first withdrawal response, however, some tissues failed to exhibit subsequent mu or kappa withdrawal contractures. A withdrawal contracture to the selective mu antagonist, cyprodime, after repeated exposures to a selective mu agonist, dermorphin, was restored by nor-binaltorphimine (BNI), a selective kappa antagonist. Vice versa, after repeated exposures to the kappa agonist, U-50,488H, cyprodime restored tissue responsiveness to BNI. Tissues repeatedly exposed to dermorphin and washed after each exposure contracted to the addition of BNI. Tissues repeatedly exposed to U-50,488H contracted on the addition of cyprodime. These findings strongly suggest that exogenous agonist-elicited stimulation of the mu (or kappa) opioid system indirectly activates the endogenous kappa (or mu) system. The indirectly-activated endogenous system inhibits the withdrawal response to the exogenously-stimulated opioid system. In isolated GPI the mu and kappa opioid systems thus appear to interact, regulating each other.

Manifestations of acute opiate withdrawal contracture in rabbit jejunum after mu-, kappa- and delta-receptor agonist exposure.

1. Following a 5 min in vitro exposure to morphine (1.3 x 10(-7) M), U-50,488H (2.5 x 10(-8) M) and deltorphin (1.6 x 10(-8)-6.5 x 10(-9) M), the rabbit isolated jejunum exhibited a precipitated contracture after the addition of naloxone (2.75 x 10(-7) M). 2. The precipitated responses to U-50,488H and deltorphin but not to morphine were reproducible in the same tissue. 3. The precipitated contractures were blocked completely by tetrodotoxin (3 x 10(-7) M), partially by atropine (1.5 x 10(-7) M) and not affected by hexamethonium (1.4 x 10(-5) M). 4. Naloxone administration (2.75 x 10(-7) M) before the agonist prevented the development of the adaptive response to morphine and U-50,488H but not to deltorphin. 5. The selective antagonists norbinaltorphimine (2.7 x 10(-8)-2.7 x 10(-9) M) and naltrindole (1.1 x 10(-7) M) prevented the adaptive response development only to the respective agonists. 6. The opioid agonists partially inhibited the spontaneous activity of the tissue. This study has shown that independent activation of mu-, kappa- and delta-opioid receptors can induce dependence in this isolated tissue. Rabbit jejunum is a suitable tissue for studying the acute effects of opioids on the adaptative processes determined by their administration.

Withdrawal contractures of guinea-pig isolated ileum after acute activation of kappa-opioid receptors.

1. The present study was undertaken to investigate firstly whether a brief exposure for 5 min of guinea-pig isolated ileum to the kappa-opioid agonist, U-50,488H produced a withdrawal contracture on addition of naloxone and secondly to ascertain whether the response was due to the activation of kappa-opioid receptors. 2. Naloxone (10(-6) M) did not elicit a response in preparations exposed to U-50,488H (5 x 10(-7) M-2 x 10(-6) M). However, after exposure to U-50,488H (5 x 10(-7) M), naloxone (10(-6) M) produced a strong contracture if the agonist was washed out 1 min before the addition of the antagonist. 3. The addition of naloxone (10(-6) M) to the ileum preparation exposed to U-50,488H (10(-7) M or lower) caused a response of similar intensity irrespective of whether the agonist had been washed out. 4. The selective kappa-opioid antagonist, nor-binaltorphimine (2.7 x 10(-9) M and 2.7 x 10(-7) M), injected before the opioid agonists, prevented the naloxone-induced contracture after exposure to U-50,488H (8 x 10(-8) M) but did not affect the contracture after exposure to morphine (5 x 10(-7) M). 5. Nor-binaltorphimine (2.7 x 10(-9) M) caused a contraction of the ileum preparation when injected 5 min after exposure to U-50,488H (8 x 10(-8) M) but not after morphine (5 x 10(-7) M). 6. The alpha 2-adrenoceptor agonist, clonidine (3 x 10-8 M) and the calcium channel blocker, nifedipine(3 x 10-8 M), injected 1 min before naloxone, blocked the ileum contraction to naloxone after exposure to U-50,488H (8 x 10-8 M). The results demonstrate that the stimulation of Kappa-opioid receptors can induce a similar dependence in guinea-pig ileum to that produced by activation of micro receptors.

Acute withdrawal after bremazocine and the interaction between mu- and kappa-opioid receptors in isolated gut tissues.

1. This study was undertaken to investigate whether, after a brief exposure of guinea-pig isolated ileum and rabbit jejunum to bremazocine, a kappa-opioid agonist also possessing antagonist activity at mu-opioid receptors, the addition of opioid antagonists produced withdrawal contractures. Our aim was to verify in these tissues the existence of an interaction between the mu- and kappa-opioid systems. 2. In guinea-pig ileum preparations previously exposed for 5 min to bremazocine at 5.7 x 10(-7) M and 5.7 x 10(-8) M, naloxone (5 x 10(-7) M) elicited no response whereas in tissues exposed to a lower bremazocine concentration (5.7 x 10(-9) M), naloxone (5 x 10(-7) M) and the selective kappa-opioid antagonist, nor-binaltorphimine (3.4 x 10(-8) M) both produced a strong contracture. 3. Bremazocine (5.7 x 10(-7) M) administered to guinea-pig isolated ileum, previously exposed for 5 min to morphine (10(-7) M), induced a withdrawal contracture. In contrast, lower bremazocine concentrations (1.4 and 7.1 x 10(-8) M) did not elicit a withdrawal contracture. 4. Naloxone (5 x 10(-7) M), added to the bath after a 5 min exposure of guinea-pig ileum to morphine (10(-7) M), elicited the characteristic withdrawal contracture. Bremazocine (1.4-7.1 x 10(-8) M) added 1 min before naloxone (5 x 10(-7) M) inhibited the naloxone withdrawal contracture in a dose-related way whereas naloxone 5 x 10(-8) M added 1 min before naloxone 5 x 10(-7) M, did not affect the withdrawal response. 5. In the rabbit jejunum, bremazocine (1.4-7.1 x 10-8 M) caused a decrease in amplitude in the spontaneous tissue activity. In tissues exposed to these bremazocine concentrations, naloxone(5 x 10-7 M) elicited a marked contracture. A similar contracture occurred when nor-binaltorphimine(3.4 x 10-8 M) was added in place of naloxone. These effects were dose-related to the bremazocine concentration. The specific K-agonist, U-50,488H (5 x 10-8 M), elicited the same effects as bremazocine.6. These findings show that stimulation of K-opioid receptors induces a state of dependence that is not prevented by blocking the pi-opioid system. The observation that low bremazocine concentrations inhibit the morphine-induced withdrawal contractures, indicates an interaction between the micro- and K-opioid system in guinea-pig isolated ileum, similar to that observed in the whole animal.

Interactions between cholecystokinin and opioids in the isolated guinea-pig ileum.

1. Although cholecystokinin octapeptide sulphate (CCK-8) activates the opioid system of isolated guinea-pig ileum (GPI) whether it activates the mu- or kappa-system, or both, remains unclear. Neither is it known whether CCK-8 influences the withdrawal responses in GPI preparations briefly exposed to opioid agonists. This study was designed to clarify whether CCK-8 activates mu- or kappa-opioid systems or both; and to investigate its effect on the withdrawal contractures in GPI exposed to mu- or kappa-agonists and on the development of tolerance to the withdrawal response. 2. In GPI exposed to CCK-8, the selective kappa-antagonist nor-binaltorphimine elicited contractile responses that were concentration-related to CCK-8 whereas the selective mu-antagonist cyprodime did not. 3. In GPI preparations briefly exposed to the selective mu-agonist, dermorphin, or the selective kappa-agonist, U-50, 488H, and then challenged with naloxone, CCK-8 strongly enhanced the withdrawal contractures. 4. During repeated opioid agonist/CCK-8/opioid antagonist tests tolerance to opioid-induced withdrawal responses did not develop. 5. These results show that CCK-8 preferentially activates the GPI kappa-opioid system and antagonizes the mechanism(s) that control the expression of acute dependence in the GPI.

Effects of alpha-dendrotoxin and dendrotoxin K on extracellular excitatory amino acids and on electroencephalograph spectral power in the hippocampus of anaesthetised rats.

Dendrotoxins, important pharmacological tools for studying K(+) channels, are potently convulsant in the central nervous system and evidence suggests that different members of the dendrotoxin family may act at pre- or post-synaptic sites. Using a combination of intrahippocampal infusion, microdialysis and electroencephalograph (EEG) recording, we have compared the effects of alpha-dendrotoxin and dendrotoxin K on extracellular levels of excitatory amino acids in anaesthetised rats. Our findings show that although infusion of 35 pmol of both peptides was associated with elevated extracellular aspartate and glutamate, these increased levels were more sustained with dendrotoxin K. Furthermore, there was EEG evidence of an associated transient functional change consistent with an action on pre-synaptic K(+) channels. In contrast, infusion of alpha-dendrotoxin produced only a brief effect on amino acid levels and no evidence of a functional consequence.

Inducible nitric oxide synthase is involved in the mechanisms of cocaine enhanced neuronal apoptosis induced by HIV-1 gp120 in the neocortex of rat.

Cocaine, often abused by human immunodeficiency virus (HIV) infected patients, has been suggested to worsen the HIV associated dementia via unknown mechanisms. Here we report that subchronic treatment with a dose of cocaine (30 mg/kg i.p.), unable per se to cause neuronal death, increases the number of apoptotic cells typically observed in the neocortex of rats treated with HIV-1 gp120 (100 ng given i.c.v.). A pre-treatment with MK801 (0.3 mg/kg i.p.), a NMDA receptor antagonist, L-NAME (10 mg/kg i.p.) and 7-nitroindazole (50 mg/kg i.p.), two specific inhibitors of NOS, or with 1400 W (1 mg/kg s.c.), a selective inhibitor of inducible NOS (iNOS), minimized neurotoxicity by combined administration of cocaine and gp120 thus implicating iNOS. This conclusion is supported by the evidence that cocaine increases brain neocortical citrulline, the co-product of NO synthesis.

Dapiprazole, a selective alpha-1 adrenoceptor antagonist, inhibits diuresis but not polydipsia produced by amphetamine in rats.

Chronic amphetamine administration has been found to produce diuresis and polydipsia in rats. We have found that dapiprazole acutely suppresses the diuretic, but not the ingestive, effects of amphetamine. To see whether diuresis is the physiological stimulus driving amphetamine-mediated polydipsia, we injected rats daily with d,l-amphetamine and the alpha-1 adrenergic antagonist dapiprazole. Throughout 19 days of treatment, dapiprazole completely prevented the increased urine output produced by amphetamine, but did not affect the development of polydipsia. This finding rules out a renal site of the primary action for amphetamine-mediated polydipsia and proposes water and electrolyte imbalance produced by chronic amphetamine administration as a model of the polydipsia and hyponatremia that develop in some psychotic patients.

Effects of trazodone and m-chlorophenylpiperazine (m-CPP) on acute dependence in mice.

The antidepressant trazodone and its main metabolite, m-CPP, having an antiserotoninergic and serotoninergic activity respectively, were studied in an acute dependence model in mice, to establish whether 5-hydroxytryptaminergic systems are involved in the manifestations of acute opiate dependence and in its development. When drugs were administered 15 min before naloxone, all signs of abstinence decreased, with the exception of teeth chattering that was increased by m-CPP and unaffected by trazodone. When injected 15 min before morphine, jump episodes were decreased by the highest doses of both drugs, while teeth chattering was decreased by m-CPP only. When administered 1 h before morphine, trazodone increased paw and head shakes and mCPP decreased teeth chattering and both left the other signs unaffected. Serotoninergic systems seem to have a significant role in events involved in the withdrawal syndrome and a minor one in those leading to the development of dependence.

Ocular absorption and distribution of bendazac after topical administration to rabbits with different vehicles.

Ocular and systemic absorption of bendazac was investigated after topical administration to rabbits of 0.5% solutions of bendazac lysine in different polysaccharide vehicles. The results show that the drug is absorbed into the retina-choroid via an extracorneal, or sclero-conjunctival route; the iris and the ciliary body are presumably supplied via both the transcorneal and the extracorneal pathways. The extent of absorption via the extracorneal route is not related to vehicle viscosity but rather to the chemical features of vehicle. The transcorneal penetration appears to be hindered by the binding of the drug to corneal tissues.

Reproducible withdrawal contractions of isolated guinea-pig ileum after brief morphine exposure: effects of clonidine and nifedipine.

Guinea-pig ileum stored for 30 min in Krebs solution and then mounted in Tyrode solution gave reproducible contracture responses to naloxone after brief exposure to morphine. The preparation lasted for several hours and a variety of pharmacological tests could be made. Clonidine, an alpha 2-adrenoceptor agonist, and nifedipine, a calcium channel antagonist, both known to interfere with tolerance and physical dependence, inhibited naloxone withdrawal contractures in a dose related way. Their action seemed to be receptor-mediated since yohimbine and Bay k 8644, respectively, reversed their inhibitory effect.

Apoptosis in the mechanisms of neuronal plasticity in the developing visual system.

Visual experience during early postnatal life is essential for normal development of synaptic connections in the visual system. In fact, altered visual experiences such as monocular deprivation (MD) or abnormal visual stimulation (e.g. strabismus, anisometropia) during this period disrupt the physiologic organization of the visual pathway, leading to loss of visual responses in cortical neurons and reduction in visual acuity of the affected eye, so that it becomes amblyopic. The authors review the main functional and morphologic changes induced by altered visual experiences in the developing visual system and focus on the recent discovery that MD induces apoptotic cell death in the lateral geniculate nucleus of newborn rats. Particular attention is given to the authors' studies documenting that, during development, MD leads retinal terminals to release excessive glutamate in the lateral geniculate nucleus where it elevates nitric oxide and causes DNA fragmentation. The latter event is known to activate poly-(ADP-ribose) polymerase, which in turn may trigger apoptosis. Better understanding of the mechanisms underlying the morphologic changes induced by altered visual experiences during development may open new venues for studying novel neuroprotective strategies for amblyopia and, more generally, for the treatment of ophthalmic diseases associated with neuronal apoptosis.

Aspirin-like drugs inhibit neuronally evoked responses in isolated guinea-pig ileum.

The acute effects of non steroidal anti-inflammatory drugs (NSAIDs) were studied on guinea-pig ileum isolated preparation. Two cyclo-oxygenase inhibitors, aspirin and indomethacin, and two NSAID devoid of this effect, salicylic acid and benzydamine, were injected into the bath 1 min before PGE1 or CCK-8. All drugs tested elicited a dose-related inhibition of the neuronally evoked contractile responses to submaximal dose of PGE1 and CCK-8. These drugs depressed also the opioid system(s) activated by PGE1 or CCK-8. These results indicate that the inhibition of neuronally evoked response is a common mechanism of NSAIDs. This mechanism may have an important role on analgesic and anti-inflammatory action of these drugs.