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.

Early reperfusion injury is associated to MMP2 and IL-1ß elevation in cortical neurons of rats subjected to middle cerebral artery occlusion.

The pathophysiological processes implicated in ischemic brain damage are strongly affected by an inflammatory reaction characterized by activation of immune cells and release of soluble mediators, including cytokines and chemokines. The pro-inflammatory cytokine interleukin (IL)-1ß has been implicated in ischemic brain injury, however, to date, the mechanisms involved in the maturation of this cytokine in the ischemic brain have not been completely elucidated. We have previously suggested that matrix metalloproteinases (MMPs) may be implicated in cytokine production under pathological conditions. Here, we demonstrate that significant elevation of IL-1ß occurs in the cortex as early as 1h after the beginning of reperfusion in rats subjected to 2-h middle cerebral artery occlusion (MCAo). At this early stage, we observe increased expression of IL-1ß in pericallosal astroglial cells and in cortical neurons and this latter signal colocalizes with elevated gelatinolytic activity. By gel zymography, we demonstrate that the increased gelatinolytic signal at 1-h reperfusion is mainly ascribed to MMP2. Thus, MMP2 seems to contribute to early brain elevation of IL-ß after transient ischemia and this mechanism may promote damage since pharmacological inhibition of gelatinases by the selective MMP2/MMP9 inhibitor V provides neuroprotection in rats subjected to transient MCAo.

Understanding the multifaceted role of inflammatory mediators in ischemic stroke.

The evolution of ischemic brain damage is strongly affected by an inflammatory reaction that involves soluble mediators, such as cytokines and chemokines, and specialized cells activated locally or recruited from the periphery. The immune system affects all phases of the ischemic cascade, from the acute intravascular reaction due to blood flow disruption, to the development of brain tissue damage, repair and regeneration. Increased endothelial expression of adhesion molecules and blood-brain barrier breakdown promotes extravasation and brain recruitment of blood-borne cells, including macrophages, neutrophils, dendritic cells and T lymphocytes, as demonstrated both in animal models and in human stroke. Nevertheless, most anti-inflammatory approaches showing promising results in experimental stroke models failed in the clinical setting. The lack of translation may reside in the redundancy of most inflammatory mediators, exerting both detrimental and beneficial functions. Thus, this review is aimed at providing a better understanding of the dualistic role played by each component of the inflammatory/immune response in relation to the spatio-temporal evolution of ischemic stroke injury.

Neuroprotection by leptin in a rat model of permanent cerebral ischemia: effects on STAT3 phosphorylation in discrete cells of the brain.

In addition to its effects in the hypothalamus to control body weight, leptin is involved in the regulation of neuronal function, development and survival. Recent findings have highlighted the neuroprotective effects of leptin against ischemic brain injury; however, to date, little is known about the role performed by the signal transducer and activator of transcription (STAT)-3, a major mediator of leptin receptor transduction pathway in the brain, in the beneficial effects of the hormone. Our data demonstrate that systemic acute administration of leptin produces neuroprotection in rats subjected to permanent middle cerebral artery occlusion (MCAo), as revealed by a significant reduction of the brain infarct volume and neurological deficit up to 7 days after the induction of ischemia. By combining a subcellular fractionation approach with immunohistofluorescence, we observe that neuroprotection is associated with a cell type-specific modulation of STAT3 phosphorylation in the ischemic cortex. The early enhancement of nuclear phospho-STAT3 induced by leptin in the astrocytes of the ischemic penumbra may contribute to a beneficial effect of these cells on the evolution of tissue damage. In addition, the elevation of phospho-STAT3 induced by leptin in the neurons after 24 h MCAo is associated with an increased expression of tissue inhibitor of matrix metalloproteinases-1 in the cortex, suggesting its possible involvement to the neuroprotection produced by the adipokine.

The protective role of catalase against cerebral ischemia in vitro and in vivo.

The present study aims to assess the protective role of the antioxidant enzyme catalase (CAT) with relation to hydrogen peroxide (H(2)O(2)) degradation in oxygen plus water on electrophysiological and fluorescence changes induced by in vitro ischemia and on brain damage produced by transient in vivo ischemia. Neuroprotective effects of CAT were determined by means of electrophysiological recordings and confocal fluorescence microscopy in the hippocampal slice preparation. Ischemia was simulated in vitro by oxygen/glucose deprivation (OGD). In vivo ischemia was produced by transient middle cerebral artery occlusion (MCAo). A protection of the rat CA1 field excitatory postsynaptic potential (fEPSP) loss caused by a prolonged OGD (40 min) was observed after exogenous CAT (500 U/mL) bath-applied before a combined exposure to OGD and H(2)O(2) (3 mM). Of note, neither H(2)O(2) nor exogenous CAT alone had a protective action when OGD lasted for 40 min. The CAT-induced neuroprotection was confirmed in a transgenic mouse model over-expressing human CAT [Tg(CAT)]. In the presence of H(2)O(2), the hippocampus of Tg(CAT) showed an increased resistance against OGD compared to that of wild-type (WT) animals. Moreover, CAT treatment reduced for about 50 min fEPSP depression evoked by repeated applications of H(2)O(2) in normoxia. A lower sensitivity to H(2)O(2)-induced depression of fEPSPs was also indicated by the rightward shift of concentration-response curve in Tg(CAT) compared to WT mice. Noteworthy, Tg(CAT) mice had a reduced infarct size after MCAo. Our data suggest new strategies to reduce neuronal damage produced by transient brain ischemia through the manipulation of CAT enzyme.

Methylprednisolone treatment delays remote cell death after focal brain lesion.

Glucocorticoids have a prominent role in the treatment of CNS injuries. However, the cellular consequences of glucocorticoid treatment on remote degenerative responses after focal brain lesions have been poorly investigated. Here we examine the effectiveness of a high dose (50 mg/kg) of methylprednisolone sodium succinate (MPSS) in reducing neuronal loss, glial response and glial-derived inflammatory mediators in inferior olive and pontine nuclei after lesion of the contralateral cerebellar hemisphere using immunohistochemistry and Western blot techniques. Quantitative analysis demonstrated that MPSS treatment significantly improved the survival of neurons in remote precerebellar stations. This survival was accompanied by reduction in the postlesional activation of microglia, astrocytes and interleukin-1 beta (IL-1beta). Cell death resumed after suspension of MPSS treatment and this delayed wave of cell loss was paralleled by reactivation of the inflammatory markers analyzed. The present study confirms the importance of inflammatory events in inducing remote cell death and that this type of degeneration can be delayed by MPSS treatment. Furthermore, the sustained effect of MPSS treatment, up to 28 days postlesion, and the reactivation of the degenerative phenomena after its suspension, support the hypothesis that glucocorticoid treatment, although capable of delaying cell death mechanisms, is not effective in blocking the cascade of remote degenerative events started by the primary lesion.

Brain regional and cellular localization of gelatinase activity in rat that have undergone transient middle cerebral artery occlusion.

Matrix metalloproteinases (MMPs) have been implicated in the pathophysiology of ischemic stroke. In particular, the gelatinases MMP-2 and MMP-9 contribute to disruption of the blood-brain barrier and hemorrhagic transformation following ischemic injury. In addition to extracellular matrix degradation, MMPs may directly regulate neuronal cell death through mechanisms that are not completely understood. Here we describe the spatio-temporal distribution of activated MMP-2 and MMP-9 in the brain of rats subjected to 2 h middle cerebral artery occlusion (MCAo) followed by different periods of reperfusion (15 min, 2 h, 6 h and 22 h). By in situ zymography we have observed that gelatinases become activated 15 min and 2 h after the beginning of reperfusion in the ischemic core and penumbra, respectively. In situ zymography signal broadly co-localized with NeuN-positive cells, thus suggesting that proteolysis mainly occurs in neurons. Gelatinolytic activity was mainly detected in cell nuclei, marginally appearing in the cytosol only at later stages following the insult; we did not detect variations in gelatinolysis in the extracellular matrix. Finally, we report that pharmacological inhibition of MMPs by N-[(2R)-2-(hydroxamidocarbonyl-methyl)-4-methylpenthanoyl]-L-tryptophan methylamide (GM6001) significantly reduces brain infarct volume induced by transient MCAo. Taken together our data underscore the crucial role of gelatinases during the early stages of reperfusion and further extend previous observations documenting the detrimental role of these enzymes in the pathophysiology of brain ischemia.

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.

Outcomes of a pharmacoepidemiological survey on the antibiotic treatment of uncomplicated acute cystitis in community.

We carried out a community-based survey in order to emphasize the importance of therapeutic appropriateness of antibiotic prescription by local physicians and the close connection between pharmacotherapy and pharmacoeconomics. Twenty general practitioners belonging to the local sanitary firm of Paola (CS, Italy) provided information, including their prescription, regarding 64 patients, both male and female, presenting clinical symptoms of uncomplicated acute cystitis. The data collected were compared with those of a previous trial performed in the same setting and documenting the effectiveness and advantages associated with the use of amoxicillin against community-acquired uncomplicated urinary tract infections (UTI). By comparing the prescriptive behaviour of physicians between the first and the present survey, we detected a significant increase in the use of amoxicillin (from 0 to 26.56%), paralleled by a decrease in prescribing aminoglycosides (from 18.18 to 1.56%). In addition, this resulted in a significant reduction in the costs of treatment (from 23.06 to 12.75 euros). Therefore, given the vast consensus concerning the adoption of empirical treatment for the eradication of UTI, the present survey underlines the crucial role of local antibiotic resistance monitoring in order to optimize the use of these drugs. Moreover, we have also observed a significant reduction in treatment costs associated with an appropriate and effective treatment of UTI.

17beta-estradiol reduces neuronal apoptosis induced by HIV-1 gp120 in the neocortex of rat.

The human immunodeficiency virus type 1 (HIV-1) coat glycoprotein gp120 represents a likely contributor to the development of HIV-1 associated dementia (HAD), a neurological syndrome often observed in AIDS patients and characterised by significant neuronal loss in the neocortex. Since recent studies have highlighted that female sex hormones represent potential neuroprotective agents against damage produced by acute and chronic injuries in the adult brain, we have investigated whether estrogens exert protection in a rat model of gp120 neurotoxicity. Our results demonstrate that systemic administration of 17beta-estradiol (E2, 0.02-0.2 mg/kg) significantly reduces apoptotic cell death observed in the neocortex of rat following subchronic i.c.v. administration of gp120 (100 ng/rat/day). Furthermore, both tamoxifen and ICI182,780, two selective antagonists of estrogen receptors (ER) in the brain, reverted the neuroprotective effect of E2. The molecular mechanism of estrogenic neuroprotection does not appear to involve modulation of the antiapoptotic Bcl-2 or the proapoptotic Bax since we failed to observe changes in the levels of the two proteins in the neocortical tissue after gp120 and/or E2 treatment. However, we detected increased levels of IL-1beta in the neocortex of rats injected with gp120, as early as 6h after drug administration, and this effect was potentiated following pretreatment with E2. Taken together, our results demonstrate that E2 exerts neuroprotection against gp120 neurotoxicity in vivo through a mechanism involving ER activation and, possibly, via modulation of neocortical levels of IL-1beta.

Caspase-1 inhibitors abolish deleterious enhancement of COX-2 expression induced by HIV-1 gp120 in human neuroblastoma cells.

The human CHP100 neuroblastoma cell line has been shown to provide an useful in vitro model to elucidate the mechanisms underlying HIV-1 gp120 neurotoxicity. Here we report western blotting evidence demonstrating that exposure to a cytotoxic concentration of the viral coat protein up-regulates expression of the inducible isoform of cyclooxygenase (COX-2) in neuroblastoma cells and this seems to be due to the previously observed increase in secreted IL-1beta. In fact, here we show that acetyl-Tyr-Val-Ala-Asp-chloromethylketone (Ac-YVAD-CMK) and t-butoxycarbonyl-L-aspartic acid benzyl ester-chloromethylketone (Boc-Asp-(OBzl)-CMK), two inhibitors of Interleukin-1 Converting Enzyme (ICE; also referred to as caspase-1), abolish COX-2 expression enhanced by gp120 and consequent cell death. In addition, NS-398, a selective inhibitor of COX-2 activity, affords neuroprotection strengthening the role of COX-2 in the mechanisms of death. In conclusion, the present data support the notion that IL-1beta is the signal through which gp120 elevates COX-2 expression and the latter is strongly implicated in the mechanisms underlying cytotoxicity.