Development and validation of an HPLC-DAD method for the quantification of cannabigerol, cannabidiol, cannabinol and cannabichromene in human plasma and mouse matrices.

Cannabigerol, cannabidiol, cannabinol and cannabichromene are non-psychoactive phytocannabinoids, highly present in Cannabis sativa, for which numerous therapeutical applications have been described. However, additional pre-clinical and clinical data, including toxicopharmacokinetic and pharmacodynamic studies, remain required to support their use in clinical practice and new therapeutic applications. To support these studies, a new high performance liquid chromatography technique (HPLC) with diode-array detection (DAD) was developed and validated to quantify these cannabinoids in human plasma and mouse matrices. Sample extraction was accomplished by protein precipitation and double liquid-liquid extraction. Simvastatin and perampanel were used as internal standards in human and mouse matrices, respectively. Chromatographic separation was achieved in 16 min on an InfinityLab Poroshell® 120 C18 column (4.6 mm × 100 mm, 2.7 µm) at 40 °C. A mobile phase composed of water/acetonitrile was pumped with a gradient elution program at 1.0 mL min-1. The technique revealed linearity in the defined concentration ranges with a determination coefficient of over 0.99. Intra and inter-day accuracy and precision values ranged from -14.83 to 13.97% and 1.08 to 13.74%, respectively. Sample stability was assessed to ensure that handling and storage conditions did not compromise analyte concentrations in different matrices. Carry-over was absent and recoveries were over 77.31%. This technique was successfully applied for the therapeutic monitoring of cannabidiol and preliminary pre-clinical studies with cannabigerol and cannabidiol. All samples were within calibration ranges, with the exception of cannabigerol after intraperitoneal administration. This is the first HPLC-DAD technique that simultaneously quantifies cannabinoids in these biological matrices, supporting future pre-clinical and clinical investigations.

Cystic fibrosis: Physiopathology and the latest pharmacological treatments.

Cystic fibrosis (CF) is a lethal autosomal recessive genetic disease, caused by a mutation in the cystic fibrosis transmembrane conductance regulator gene (CFTR), which primarily affects the lungs and digestive system. This gene encodes the CFTR protein, a distinctive membrane transporter of the ATP-binding cassette (ABC) superfamily. It functions as a chloride channel, allowing the balance and transport of chloride through the apical membrane of epithelial cells. Due to its ubiquitous location, mutations in the CFTR gene trigger multiple changes in ion transport and metabolic pathways, affecting various organs, as it will be herein explained. Pulmonary impairment is the most characteristic comorbidity of CF and respiratory failure is the main cause of death. This review presents the importance of an early diagnosis of CF to establish, as soon as possible, a primary therapy for symptomatic prevention and relief. It also mentions new therapeutic approaches that include CFTR modulators. They are correctors and/or potentiators of the deficient CFTR channel. In an attempt to overcome the disadvantages of CFTR modulators, the application of biotechnology techniques is addressed, such as gene therapy, gene editing, RNA therapy and therapeutic microRNAs. The potential of the intranasal administration route is another presented aspect.

Repairing blood-CNS barriers: Future therapeutic approaches for neuropsychiatric disorders.

Central nervous system (CNS) drug development faces significant difficulties that translate into high rates of failure and lack of innovation. The pathophysiology of neurological and psychiatric disorders often results in the breakdown of blood-CNS barriers, disturbing the CNS microenvironment and worsening disease progression. Therefore, restoring the integrity of blood-CNS barriers may have a beneficial influence in several CNS disorders and improve treatment outcomes. In this review, pathways that may be modulated to protect blood-CNS barriers from neuroinflammatory and oxidative insults are featured. First, the participation of the brain endothelium and glial cells in disruption processes is discussed. Then, the relevance of regulatory systems is analysed, specifically the hypothalamic-pituitary axis, the renin-angiotensin system, sleep and circadian rhythms, and glutamate neurotransmission. Lastly, compounds of endogenous and exogenous origin that are known to mediate the repair of blood-CNS barriers are presented. We believe that enhancing the protection of blood-CNS barriers is a promising therapeutic strategy to pursue in the future.

Teaching Chest Ultrasound in an Experimental Porcine Model.

OBJECTIVE: The objective of this study was to evaluate the efficacy of a short hands-on chest ultrasound course to detect normal lung pattern, pneumothorax (PTX), and pleural effusion (PE) in a porcine animal model. METHODS: Thirty-six trainees with no previous experience in chest ultrasound participated in the study. A 1.5-hour training course covering both theory and practice was developed. All static and dynamic signs of the normal lung parenchyma, PTX, and PE were analyzed. Four pigs were used. Approval by the local institutional animal care was obtained. An 8F drainage catheter was inserted into the pleural cavity under general anesthesia for injection of air or saline solution. A Vivid Q ultrasound with a 12L-RS linear probe was used. A baseline preintervention evaluation and 2 postintervention evaluations (one after theoretical class and the other after additional training with the animal model) were made. Sensitivity and specificity with the 95% confidence interval for recognition of the 3 patterns were analyzed, and results were compared with those obtained in the preintervention evaluation. RESULTS: All normal lung signs were detected, as these were signs of PE and PTX. Participants were able to diagnose a normal pattern (sensitivity, 100% [90%-100%]; specificity, 90% [84%-95%]), PE (sensitivity, 89% [75%-95%]; specificity, 95% [89%-98%]), and PTX (sensitivity, 82% [72%-89%]; specificity, 97% [90%-99%]) after 30 minutes of class and normal pattern (sensitivity, 95% [85%-98%]; specificity, 95% [85%-98%]), PE (sensitivity, 100% [88%-100%]; specificity, 98% [94%-99%]), and PTX (sensitivity, 90% [73%-96%]; specificity, 98% [92%-99%]) in the animal model. CONCLUSIONS: The porcine model is useful for ultrasound examination of the lung parenchyma and detection of pleural disease. Its use in the experimental laboratory is a major refinement that enables trainees to identify acute pulmonary complications.

H2O2- or l-DOPA-injured dopaminergic neurons trigger the release of soluble mediators that up-regulate striatal GDNF through different signalling pathways.

Glial cell line-derived neurotrophic factor (GDNF) is a potent neuroprotective molecule for dopaminergic neurons of the nigrostriatal pathway that degenerate in Parkinson's disease. We have previously shown that H2O2- or l-3,4-dihydroxyphenylalanine (l-DOPA)-challenged dopaminergic neurons trigger the release of soluble factors that signal ventral midbrain astrocytes to increase GDNF expression. In the present work, we evaluated whether the factors released by ventral midbrain-challenged cells were able to alter GDNF expression in striatal cells, the targets of dopaminergic neurons projecting from the substantia nigra, and investigated the signalling pathways involved. Our data showed that soluble mediators released upon H2O2- or l-DOPA-induced dopaminergic injury up-regulated GDNF in striatal cells, with different temporal patterns depending on the oxidative agent used. Conditioned media from H2O2- or l-DOPA-challenged midbrain astrocyte cultures failed to up-regulate GDNF in striatal cultures. Likewise, there was no direct effect of H2O2 or l-DOPA on striatal GDNF levels suggesting that GDNF up-regulation was mediated by soluble factors released in the presence of failing dopaminergic neurons. Both phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways were involved in striatal GDNF up-regulation triggered by H2O2-induced dopaminergic injury, while diffusible factors released in the presence of l-DOPA-challenged dopaminergic neurons induced GDNF expression in striatal cells through the activation of the MAPK pathway. These soluble mediators may constitute, in the future, important targets for the control of endogenous GDNF expression enabling the development of new and, hopefully, more efficient neuroprotective/neurorestorative strategies for the treatment of Parkinson's disease.

Intranasal administration of sertraline ensures sustained brain delivery and antidepressant effect in a mouse model of depression.

The pursuit of more potent and efficacious antidepressant therapies is of utmost significance. Herein, the intranasal (IN) route was investigated for sertraline brain delivery, encompassing a comparative pharmacokinetic study after a single-dose administration to mice by IN, intravenous (IV) (4.87 mg/kg) and oral (10 mg/kg) routes, and an efficacy/toxicity study to explore the therapeutic effect in mice subjected to the unpredictable chronic mild stress (UCMS) protocol. Neurotransmitters and melatonin were quantified in prefrontal cortex and plasma, respectively. A different drug biodistribution behavior was unveiled for a CNS-acting drug administered by means of the IN route. For the first time, IN administration of sertraline exhibited heightened systemic exposure (bioavailability = 166 %), and a sustained drug release into the brain, in opposition to IV and oral routes, avoiding drug fluctuation. The lower lung exposition (given by normalized area under the curve) observed after IN instillation envisions the reduction of sertraline pulmonary side effects and similarly other peripheral side effects. IN sertraline treatment displayed significant efficacy in ameliorating anhedonia after one week of administration while the 14-day IN treatment regimen translated into decreased immobility time and increased swimming time in the forced swimming test, suggesting an improvement of the depressive-like behavior displayed by the animal depressive-model. Remarkably, these effects were absent with oral sertraline, despite the higher used dose. Noteworthy neurotransmitter alterations were observed, with IN sertraline markedly reducing adrenaline in the prefrontal cortex, while serotonin and melatonin increased following both administration routes. With its sustained brain delivery and serotonin- and melatonin-enhancing potential, the innovative strategy of IN sertraline holds the potential not only to effectively address depressive symptoms but also to mitigate challenges inherent to classic treatments.

Intranasal irbesartan reverts cognitive decline and activates the PI3K/AKT pathway in an LPS-induced neuroinflammation mice model.

BACKGROUND: New strategies are urgently needed to manage and delay the development of Alzheimer's disease (AD). Neuroinflammation is a significant contributor to cognitive decline in neurodegenerative diseases, including AD. Angiotensin receptor blockers (ARBs) and angiotensin converting enzyme inhibitors (ACEIs) protect hypertensive patients against AD, but the cellular and molecular mechanisms underlying these effects remain unknown. In light of this, the protective effects of three ARBs and three ACEIs against neuroinflammation and cognitive decline were investigated through comprehensive pharmacologicalin vitro/in vivoscreening. METHODS: BV-2 microglia cells were exposed tolipopolysaccharide (LPS) and treated with ARBs and ACEIs to provide initial insights into the anti-inflammatory properties of the drugs. Subsequently, irbesartan was selected, and its efficacy was evaluated inC57/BL6 male miceintranasally administered with irbesartan and injected with LPS. Long-term memory and depressive-like behavior were evaluated; dendritic spines were measured as well as neuroinflammation, neurodegeneration and cognitive decline biomarkers. RESULTS: Irbesartan mitigated memory loss and depressive-like behavior in mice treated with LPS, probably because itincreased spine density, ameliorated synapsis dysfunction and activated the PI3K/AKT pathway. Irbesartan elevated the levels of hippocampalsuperoxide dismutase2 andglutathione peroxidaseandsuppressed LPS-induced astrogliosis. CONCLUSIONS: Overall, this study provides compelling evidence that multiple intranasal administrations of irbesartan can effectively prevent LPS-induced cognitive decline by activating pathways involved in neuroprotection and anti-inflammatory events. These findings underscore the potential of irbesartan as a preventive strategy against the development of AD and other neurodegenerative conditions associated with neuroinflammation.

"Lipopolysaccharide-induced animal models for neuroinflammation - An overview."

Neuroinflammation is a pathological mechanism contributing to neurodegenerative diseases. For in-depth studies of neuroinflammation, several animal models reported reproducing behavioral dysfunctions and cellular pathological mechanisms induced by brain inflammation. One of the most popular models of neuroinflammation is the one generated by lipopolysaccharide exposure. Despite its importance, the reported results using this model show high heterogeneity, making it difficult to analyze and compare the outcomes between studies. Therefore, the current review aims to summarize the different experimental paradigms used to reproduce neuroinflammation by lipopolysaccharide exposure and its respective outcomes, helping to choose the model that better suits each specific research aim.

23Na multiple quantum filtered NMR characterisation of Na+ binding and dynamics in animal cells: a comparative study and effect of Na+/Li + competition.

Double quantum and triple quantum filtered (23)Na nuclear magnetic resonance techniques were used to characterise in detail the isotropic and anisotropic binding and dynamics of intra- and extracellular Na(+) in different cellular systems, in the absence and presence of Li(+). The kinetics of Li(+) influx by different cell types was evaluated. At steady state, astrocytes accumulated more Li(+) than red blood cells (RBCs), while a higher intracellular Li(+) concentration was found in chromaffin than in SH-SY5Y cells. Anisotropic and isotropic motions were detected for extracellular Na(+) in all cellular systems studied. Isotropic intracellular Na(+) motions were observed in all types of cells, while anisotropic Na(+) motions in the intracellular compartment were only detected in RBCs. (23)Na triple quantum signal efficiency for intracellular Na(+) was SH-SY5Y > chromaffin > RBCs, while the reverse order was observed for the extracellular ions. (23)Na double quantum signal efficiency for intracellular Na(+) was non-zero only in RBCs, and for extracellular Na(+) the order RBCs > chromaffin > SH-SY5Y cells was observed. Li(+) loading generally decreased intracellular Na(+) isotropic movements in the cells, except for astrocytes incubated with a low Li(+) concentration and increased anisotropic intracellular Na(+) movements in RBCs. Li(+) effects on the extracellular signals were more complex, reflecting Li(+)/Na(+) competition for isotropic and anisotropic binding sites at the extracellular surface of cell membranes and also at the surface of the gel used for cell immobilisation. These results are relevant and contribute to the interpretation of the in vivo pharmacokinetics and sites of Li(+) action.

Single-Access Fetal Endoscopy (SAFE) for myelomeningocele in sheep model I: amniotic carbon dioxide gas approach.

BACKGROUND: This study aimed to assess the feasibility of single-access fetal endoscopy (SAFE) for the management of myelomeningocele (MMC) using intrauterine carbon dioxide as a distension medium in a sheep model. METHODS: This prospective experimental case-control study investigated 12 lamb fetuses that had a myelomeningocele-like defect surgically created on the 75th day of gestation. Four fetuses remained untreated (control group), and eight fetuses had MMC repair using two fetoscopic approaches with carbon dioxide used to distend the amniotic cavity. A collagen patch was placed over the defect and secured with surgical sealant. Four animals had a two-port fetoscopic procedure, and four animals had SAFE. Clinical and pathologic studies were performed after delivery. RESULTS: This study confirmed the validity of the animal MMC model. None of the control animals was able to stand or walk, and all had a significant defect in the lumbar area with continuous leakage of cerebrospinal fluid, ventriculomegaly, and a Chiari-II malformation. All the treated animals, independently of the number of ports used in the repair, were able to walk and had a closed defect with resolution of the Chiari malformation. CONCLUSIONS: The SAFE patch and glue coverage of surgically created fetal MMC is feasible and effective in restoring gross neurologic function in the fetal lamb model.

Under the umbrella of depression and Alzheimer's disease physiopathology: Can cannabinoids be a dual-pleiotropic therapy?

Depression and Alzheimer´s disease (AD) are two disorders highly prevalent worldwide. Depression affects more than 300 million people worldwide while AD affects 60-80% of the 55 million cases of dementia. Both diseases are affected by aging with high prevalence in elderly and share not only the main brain affected areas but also several physiopathological mechanisms. Depression disease is already ascribed as a risk factor to the development of AD. Despite the wide diversity of pharmacological treatments currently available in clinical practice for depression management, they remain associated to a slow recovery process and to treatment-resistant depression. On the other hand, AD treatment is essentially based in symptomatology relieve. Thus, the need for new multi-target treatments arises. Herein, we discuss the current state-of-art regarding the contribution of the endocannabinoid system (ECS) in synaptic transmission processes, synapses plasticity and neurogenesis and consequently the use of exogenous cannabinoids in the treatment of depression and on delaying the progression of AD. Besides the well-known imbalance of neurotransmitter levels, including serotonin, noradrenaline, dopamine and glutamate, recent scientific evidence highlights aberrant spine density, neuroinflammation, dysregulation of neurotrophic factor levels and formation of amyloid beta (Aß) peptides, as the main physiopathological mechanisms compromised in depression and AD. The contribution of the ECS in these mechanisms is herein specified as well as the pleiotropic effects of phytocannabinoids. At the end, it became evident that Cannabinol, Cannabidiol, Cannabigerol, Cannabidivarin and Cannabichromene may act in novel therapeutic targets, presenting high potential in the pharmacotherapy of both diseases.

Endogenous GDNF Is Unable to Halt Dopaminergic Injury Triggered by Microglial Activation.

Overactivation of microglial cells seems to play a crucial role in the degeneration of dopaminergic neurons occurring in Parkinson's disease. We have previously demonstrated that glial cell line-derived neurotrophic factor (GDNF) present in astrocytes secretome modulates microglial responses induced by an inflammatory insult. Therefore, astrocyte-derived soluble factors may include relevant molecular players of therapeutic interest in the control of excessive neuroinflammatory responses. However, in vivo, the control of neuroinflammation is more complex as it depends on the interaction between different types of cells other than microglia and astrocytes. Whether neurons may interfere in the astrocyte-microglia crosstalk, affecting the control of microglial reactivity exerted by astrocytes, is unclear. Therefore, the present work aimed to disclose if the control of microglial responses mediated by astrocyte-derived factors, including GDNF, could be affected by the crosstalk with neurons, impacting GDNF's ability to protect dopaminergic neurons exposed to a pro-inflammatory environment. Also, we aimed to disclose if the protection of dopaminergic neurons by GDNF involves the modulation of microglial cells. Our results show that the neuroprotective effect of GDNF was mediated, at least in part, by a direct action on microglial cells through the GDNF family receptor α-1. However, this protective effect seems to be impaired by other mediators released in response to the neuron-astrocyte crosstalk since neuron-astrocyte secretome, in contrast to astrocytes secretome, was unable to protect dopaminergic neurons from the injury triggered by lipopolysaccharide-activated microglia. Supplementation with exogenous GDNF was needed to afford protection of dopaminergic neurons exposed to the inflammatory environment. In conclusion, our results revealed that dopaminergic protective effects promoted by GDNF involve the control of microglial reactivity. However, endogenous GDNF is insufficient to confer dopaminergic neuron protection against an inflammatory insult. This reinforces the importance of further developing new therapeutic strategies aiming at providing GDNF or enhancing its expression in the brain regions affected by Parkinson's disease.

Astrocyte-derived GDNF is a potent inhibitor of microglial activation.

Neuroinflammation is recognized as a major factor in Parkinson's disease (PD) pathogenesis and increasing evidence propose that microglia is the main source of inflammation contributing to the dopaminergic degeneration observed in PD. Several studies suggest that astrocytes could act as physiological regulators preventing excessive microglia responses. However, little is known regarding how astrocytes modulate microglial activation. In the present study, using Zymosan A-stimulated midbrain microglia cultures, we showed that astrocytes secrete factors capable of modulating microglial activation, namely its phagocytic activity and the production of reactive oxygen species since both parameters were highly diminished in cells incubated with astrocytes conditioned media (ACM). Glial cell line-derived neurotrophic factor (GDNF), cerebral dopamine neurotrophic factor (CDNF) and brain-derived neurotrophic factor (BDNF), known to have a neuroprotective role in the nigrostriatal system, are among the candidates to be astrocyte-secreted molecules involved in the modulation of microglial activation. The effect of ACM on Zymosan A-induced microglial activation was abolished when the GDNF present in the ACM was abrogated using a specific antibody, but not when ACM was neutralized with anti-CDNF, anti-BDNF or with a heat-inactivated GDNF antibody. In addition, media conditioned by astrocytes silenced for GDNF were not able to prevent microglial activation, whereas supplementation of non-conditioned media with GDNF prevented the activation of microglia evoked by Zymosan A. Taken together, these results indicate that astrocyte-derived GDNF plays a major contribution to the control of midbrain microglial activation, suggesting that GDNF can protect from neurodegeneration through the inhibition of neuroinflammation.

Pharmacology of lacosamide: From its molecular mechanisms and pharmacokinetics to future therapeutic applications.

Epilepsy is one of the most common brain disorders, affecting more than 50 million people worldwide. Although its treatment is currently symptomatic, the last generation of anti-seizure drugs is characterized by better pharmacokinetic profiles, efficacy, tolerability and safety. Lacosamide is a third-generation anti-seizure drug that stands out due to its good efficacy and safety profile. It is used with effectiveness in the treatment of partial-onset seizures with or without secondary generalization, primary generalized tonic-clonic seizures and off-label in status epilepticus. Despite scarcely performed until today, therapeutic drug monitoring of lacosamide is proving to be advantageous by allowing the control of inter and intra-individual variability and promoting a successful personalized therapy, particularly in special populations. Herein, the pharmacology, pharmacokinetics, and clinical data of lacosamide were reviewed, giving special emphasis to the latest molecular investigations underlying its mechanism of action and therapeutic applications in pathologies besides epilepsy. In addition, the pharmacokinetic characteristics of lacosamide were updated, as well as current literature concerning the high pharmacokinetic variability observed in special patient populations and that must be considered during treatment individualization.

Is intranasal administration an opportunity for direct brain delivery of lacosamide?

Lacosamide is well-known as an effective and safe anticonvulsant drug. Nevertheless, there is also evidence of anti-epileptogenic, neuroprotective and antinociceptive properties of lacosamide. It is currently available as oral and intravenous (IV) formulations, and its brain concentrations and therapeutic effects depend on its passage across the blood-brain barrier (BBB). Therefore, to circumvent the restrictive BBB, we herein evaluated the intranasal (IN) administration of lacosamide. Nasal thermoreversible gels were screened in vitro for their influence on the viability of human nasal septum (RPMI 2650) and lung adenocarcinoma (Calu-3) cells. According to the Alamar Blue test, the in situ gel composed of Pluronic F-127 (22.5%, w/v) and Carbopol 974P (0.2%, w/v) did not affect cell viability, which remained higher than 85%, within the concentration range of lacosamide. The in situ gel was intranasally administered to healthy male CD-1 mice (8.33 mg/kg) to describe the pharmacokinetic profiles of lacosamide in plasma, brain, lung and kidney and compare them with those obtained after IV administration of the same dose. Accordingly, IN administration allowed a fast (tmax in plasma: 5 min) and complete systemic absorption of lacosamide (absolute bioavailability: 120.46%). Interestingly, IN lacosamide demonstrated higher exposure (given by the AUCt) in the brain (425.44 µg.min/mL versus 274.49 µg.min/mL), but lower exposure in kidneys (357.56 µg.min/mL versus 762.61 µg.min/mL), in comparison to IV administration. These findings, together with the tmax in brain of 15 min, a drug targeting efficiency (DTE) of 128.67% and a direct transport percentage of 22.28%, evidence that part of lacosamide reaches the brain directly after nasal administration, even though penetration into the brain from the systemic circulation seems to be the major determinant of brain exposure. Importantly, lacosamide concentrations found in lungs following IN administration were considerably higher than those observed after IV injection, until 30 min post-dosing (p < 0.05). Nevertheless, attained drug concentrations were lower than those tested in vitro in the Calu-3 cell line (1-100 µM), indicating that adverse effects are unlikely to occur in vivo. Hence, it seems that the proposed IN route has potential to be a suitable and valuable strategy for the brain delivery of lacosamide in emergency conditions and for the chronic treatment of epilepsy and other neurological diseases.

Encapsulated Escitalopram and Paroxetine Intranasal Co-Administration: In Vitro/In Vivo Evaluation.

Depression is a common mental disorder. Its treatment with selective serotonin reuptake inhibitors (SSRIs) is effective only in a fraction of patients, and pharmacoresistance is increasing steadily. Intranasal (IN) drug delivery to the brain stands out as a promising strategy to improve current therapeutic approaches by operating as a shuttle to overcome the blood-brain barrier. This work aimed to simultaneously administer escitalopram and paroxetine by IN route to mice. For this purpose, three nanostructured lipid carriers (NLC1, NLC2, and BorNLC) and one nanoemulsion (NE) were tested for drug loading. After their characterization, investigation of their impact on nasal cell viability and SSRI permeability assays were performed, using a human nasal RPMI 2650 cell line in air-liquid interface. In vitro assays demonstrated that NLCs, including borneol (BorNLC), significantly increased escitalopram permeability (p < 0.01) and paroxetine recovery values (p < 0.05) in relation to the other formulations and non-encapsulated drugs. IN and intravenous (IV) pharmacokinetic studies performed in vivo with a single dose of 2.38 mg/kg demonstrated similar results for escitalopram brain-to-plasma ratios. IN administrations delayed escitalopram peak concentrations in the brain for 15-60 min and no direct nose-to-brain delivery was detected. However, encapsulation with BorNLC considerably decreased escitalopram exposure in the lungs (124 µg min/g) compared with free escitalopram by IN (168 µg min/g) and IV (321 µg min/g) routes. Surprisingly, BorNLC IN instillation increased concentration levels of paroxetine in the brain by five times and accelerated brain drug delivery. Once again, lung exposure was considerably lower with BorNLC (AUCt = 0.433 µg min/g) than that with IV administration (AUCt = 1.01 µg min/g) and non-encapsulated IN formulation (AUCt = 2.82 µg min/g). Direct nose-to-brain delivery was observed for paroxetine IN administration with a direct transport percentage (DTP) of 56.9%. If encapsulated, it increases to 74.2%. These results clearly emphasize that nose-to-brain delivery and lung exposure depend on the formulation and on the characteristics of the drug under investigation. NLCs seem to be an advantageous strategy for nose-to-brain delivery of lipophilic molecules, since they reduce systemic and lung exposure, thereby decreasing adverse effects. For hydrophilic compounds, NLCs are particularly important to decrease lung exposure after IN administration.

Microglia of rat ventral midbrain recovers its resting state over time in vitro: let microglia rest before work.

Cortical or total brain cultures of microglia are commonly used as a model to study the inflammatory processes in Parkinson's disease. Here we characterize microglia cultures from rat ventral midbrain and evaluate their response to zymosan A. We used specific markers of microglia and evaluated the morphology, the phagocytic activity and reactive oxygen species (ROS) levels of the cells. During the first 10 days in vitro (DIV), cultures presented predominantly cells with a round morphology, expressing CD68 and with high phagocytic activity and ROS production. After 13 DIV, this tendency was reversed, with cultures showing higher number of ramified cells and fewer CD68(+) cells along with lower phagocytic and ROS production capability, suggesting that microglia must be kept in vitro for at least 13 days to recover its resting state. The exposure of cultures with less than 10 DIV to zymosan A significantly decreased cell viability. Exposure of cultures with 13 DIV to zymosan A (0.05, 0.5, or 5 microg/ml) increased the total cell number, the percentage of CD68(+) cells, and the phagocytic activity. Concentrations of zymosan A higher than 5 microg/ml were also effective in activating microglia but significantly decreased the number of viable cells. In summary, microglial cells remain in the activated state for several days after the isolation process and, thus, stimulation of microglia recently isolated can compromise interpretation of the results. However, upon 13 DIV, cells achieve properties of nonactivated microglia and present a characteristic response to a proinflammatory agent.

Mechanisms underlying Li+ effects in glutamatergic and GABAergic neurotransmissions in the adult rat brain and in primary cultures of neural cells as revealed by 13C NMR.

We investigated by (13)C nuclear magnetic resonance (NMR) the mechanisms underlying Li(+) effects on glutamatergic and GABAergic neurotransmission systems in the adult rat brain and in primary cultures of cortical neurons and astrocytes during the metabolism of (1-(13)C) glucose or (2-(13)C) acetate. Adult male rats receiving a single dose of Li(+) intraperitoneally (7 mmol/kg) were infused 2 hr later, for 60 min, with (1-(13)C) glucose (80 mumol/min/kg) or (2-(13)C) acetate (240 micromol/min/kg). High-resolution (13)C NMR spectra of brain extracts prepared after the infusion revealed that Li(+) significantly decreased the incorporation of (13)C in glutamate and GABA (gamma-aminobutyric acid) carbons from (1-(13)C) glucose, but not from (2-(13)C) acetate. To complement the in vivo approach, primary cultures of cortical neurons or astrocytes were incubated with 1 mM uniformly (13)C-labeled glucose or 5 mM (2-(13)C) acetate, in the absence and presence of increasing Li(+) concentrations up to 15 mM. Under these conditions, Li(+) significantly decreased neuronal glucose uptake in a concentration-dependent manner without apparent effects on astrocytic acetate uptake. Extracts prepared at the end of the incubations showed that Li(+) significantly decreased the incorporation of (13)C labeling into GABA carbons from its precursor glutamate in neurons, but such a decrease into glutamine carbons in astrocytes was not statistically significant. Our results indicate that the effects of Li(+) are mediated through a reduction of neuronal glucose uptake, resulting in a decrease of glutamatergic and GABAergic neurotransmission without apparent effects on astrocytic metabolism.

Development of a bioreactor system for cytotoxic evaluation of pharmacological compounds in living cells using NMR spectroscopy.

INTRODUCTION: The evaluation of drug's cytotoxicity is a crucial step in the development of new pharmacological compounds. 31P NMR can be a tool for toxicological screening, as it enables the study of drugs' cytotoxicity and their effect on cell energy metabolism in a real-time, in a non- invasive and non-destructive way. This paper details a step-by-step protocol to implement a bioreactor system able to maintain cell viability during NMR acquisitions, at high cell densities and for several hours, enabling toxicological evaluation of pharmacological compounds in living cells. METHOD: HeLa cells were immobilized in agarose gel threads and continuously perfused with oxygenated medium inside a 5 mm NMR tube. Signals corresponding to intracellular high-energy phosphorous compounds were continuously monitored by 31P NMR to assess cell energy levels, intracellular pH and intracellular free Mg2+ concentrations ([Mg2+]f) under control and in the presence of two different cytotoxic drugs, calix-NH2 or 5-fluorouracil (5-FU). RESULTS: The bioreactor system was effective in maintaining cell energy levels as well as intracellular pH and [Mg2+]f along time, with a good 31P NMR signal to noise ratio. Calix-NH2 and 5-FU decreased cell energy levels by 35% and 39%, respectively, with a negligible increase in intracellular [Mg2+]f, and without affecting intracellular pH. DISCUSSION: The immobilization and perfusion system here detailed, along with 31P NMR, is useful in toxicological evaluation of new pharmacological compounds, enabling the continuous assessment of drugs' effect on energy levels, intracellular pH and [Mg2+]f in intact cells, for several hours without compromising cell viability.

Compassion fatigue among nurses working on an adult emergency and urgent care unit. / Fadiga por compaixão em enfermeiros de urgência e emergência hospitalar de adultos.

OBJECTIVE: to assess compassion fatigue levels among nurses and its variation according socio-demographic and professional characteristics. METHOD: quantitative, descriptive and cross-sectional study, with 87 nurses from an emergency and urgent care unit for adults from a university hospital. A socio-demographic and professional questionnaire, along with the Professional Quality of Life Scale 5 were used. Data analysis was performed using descriptive and inferential statistics. RESULTS: compassion satisfaction presents the highest means, followed by burnout and secondary traumatic stress. Among the participants, 51% presented a high level of compassion satisfaction, 54% a high level of burnout, and 59% a high level of secondary traumatic stress. Older participants presented higher score of compassion satisfaction, and younger nurses, women, nurses having less job experience and nurses without leisure activities showed higher means of secondary traumatic stress. CONCLUSION: we found compassion fatigue, expressed in the large percentage of nurses with high levels of burnout and secondary traumatic stress. Fatigue is related to individual factors such as age, gender, job experience and leisure activities. Doing research and understanding this phenomenon allow the development of health promotion strategies at work.