Anna M. Bielecka

Quantification of metformin by the HPLC method in brain regions, cerebrospinal fluid and plasma of rats treated with lipopolysaccharide

Recently, it has been reported that metformin may attenuate inflammation and directly act on the central nervous system. Using the HPLC method, in Wistar rats, we assessed the changes in metformin concentrations in various brain regions (pituitary gland, olfactory bulb, hypothalamus, cerebellum, hippocampus, striatum, frontal cortex), cerebrospinal fluid and plasma after single and chronic oral administration, in the model of systemic inflammation induced by lipopolysaccharide (ip). Regarding the influence of systemic inflammation on metformin distribution, the pituitary gland demonstrated the highest its level after single and chronic administration (28.8 ± 3.5 nmol/g and 24.9 ± 3.2 nmol/g, respectively). We concluded that orally-dosed metformin rapidly crosses the blood-brain barrier and differently accumulates in structures of the central nervous system.

Imipramine and fluoxetine inhibit LPS-induced activation and affect morphology of microglial cells in the rat glial culture

BACKGROUND Recent evidence has suggested that antidepressants evoke neuroprotective and immunomodulatory effects in the brain, partly at least, by inhibiting glia activation. This study has been conducted on the lipopolysaccharide (LPS)-stimulated primary rat mixed glial cell culture in order to better recognize the influence of imipramine (a tricyclic antidepressant) and fluoxetine (a selective serotonin reuptake inhibitor) on the important balance between pro- and anti-inflammatory cytokines produced by the glial cells. Moreover, microscopic observations were made to describe the morphological alterations in the studied cell cultures exposed to the drugs. METHODS The effect of both antidepressants on TNF-α, IL-1β and IL-10 levels was determined by ELISA. The mRNA levels of mentioned cytokines were evaluated by qRT-PCR assay. Moreover, drug influence on the LPS-stimulated level of NF-κB p65 subunit in nuclear fraction was determined by the colorimetric transcription factor assay. RESULTS After LPS-stimulation both drugs decreased concentration of TNF-α and IL-1β in culture medium and expression of TNF-α and IL-1β mRNAs in cellular extracts. They also diminished the LPS-induced nuclear translocation of NF-κB p65 subunit. In contrast, imipramine and fluoxetine induced a few-fold weaker suppressing effect on the levels of IL-10. Parallelly to the inhibition of the LPS-induced inflammatory response, the antidepressants prevented the morphological alterations of cells elicited by LPS. Moreover, in unstimulated cultures imipramine but not fluoxetine caused transformation of microglia cells into cells with neuron-like morphology. CONCLUSIONS Imipramine and fluoxetine, by modulating glia activation, may exert anti-inflammatory effects in the CNS. It also seems that microglia cells are important target particularly for imipramine.

Moclobemide exerts anti-inflammatory effect in lipopolysaccharide-activated primary mixed glial cell culture

An increasing body of evidence indicates that glial activation and neuroinflammation play an important role in the pathogenesis of psychiatric and neurodegenerative diseases. Activated glial cells secrete various cytokines that influence neurotransmission, hypothalamus–pituitary–adrenal axis activity, neuronal plasticity and neurogenesis. It has been suggested that alterations in cytokine networks are involved in the mechanism of action of antidepressant drugs. Until now, only a few studies demonstrated that some tricyclic antidepressants and selective serotonin reuptake inhibitors reduced production of pro-inflammatory cytokines in brain glia cells. We have investigated for the first time whether the antidepressant, moclobemide (a reversible selective inhibitor of monoamine oxidase-A) has an influence on pro-inflammatory cytokines [interleukin (IL)-1β and tumor necrosis factor (TNF)-α] and anti-inflammatory cytokine (IL-10) in primary rat mixed glial cell cultures stimulated by lipopolysaccharide (LPS). Our results showed that moclobemide used in a wide range of concentrations diminished LPS-stimulated IL-1β and TNF-α mRNAs expression in cellular extracts and remarkably reduced the levels of both pro-inflammatory cytokines in culture medium. In opposite to this, the drug had no influence on IL-10 mRNA and slightly reduced IL-10 concentration. Moreover, moclobemide decreased LPS-stimulated translocation of NFκB p65 subunit into cellular nuclei. These results suggest that moclobemide exerts anti-inflammatory effect in the central nervous system because it affects the balance between pro- and anti-inflammatory cytokines (IL-1β, TNF-α/IL-10) in primary mixed glial cell cultures.

Atorvastatin and fenofibric acid differentially affect the release of adipokines in the visceral and subcutaneous cultures of adipocytes that were obtained from patients with and without mixed dyslipidemia

In this study, we compared the effects of atorvastatin and fenofibric acid, which were administered alone or in combination, on the secretory function of human adipocytes that were obtained from the visceral and subcutaneous adipose tissues of 19 mixed dyslipidemic patients and 19 subjects with a normal lipid profile. The adipocytes were incubated in vitro in the presence of atorvastatin and/or fenofibric acid. The secretory function of the cells was determined using ELISA assays. The visceral adipocytes released significantly more adiponectin and IL-6 and less PAI-1 than those that were obtained from subcutaneous tissue. The levels and patterns of adipokine release differed between the patients with or without lipid abnormalities and between the adipocytes that were obtained from visceral or subcutaneous adipose tissue. The culture that contained hypolipidemic drugs resulted in the significant changes of the release of adipokines. The effects of atorvastatin and fenofibric acid on the hormonal function of human adipocytes may be, in part, responsible for the clinical efficacy of these drugs in the prevention and treatment of dyslipidemia-related cardiovascular and metabolic disorders. The study supports the concept that the pleiotropic effects of fenofibrate and atorvastatin may be, in part, a result of their impact on the secretory function of adipocytes.

Immunosuppressant cytoprotection correlates with HMGB1 suppression in primary astrocyte cultures exposed to combined oxygen-glucose deprivation

The protective potential of immunosuppressants has been reported in many experimental models of ischemia both in vivo and in vitro, suggesting a novel therapeutic application of these drugs. Because high-mobility group box 1 (HMGB1) protein has recently been reported to be involved in ischemic brain injury, the purpose of the present study was to determine whether treatment with immunosuppressants could decrease the expression and release of HMGB1 in astrocytes exposed to simulated ischemic conditions (combined oxygen-glucose deprivation, OGD). We also investigated whether immunosuppressive drugs could attenuate necrosis in astrocyte cultures exposed to OGD. Finally, we studied the influence of immunosuppressants on the expression of NFκB, inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2). Cells were treated with cyclosporine A, FK506 and rapamycin (all drugs at concentrations of 0.1, 1 and 10 μM). Our study provides evidence that immunosuppressants decrease the expression and release of HMGB1 in ischemic astrocytes. Our data suggest that HMGB1 release may be partly an active process triggered by oxidative stress because the antioxidant N-acetylcysteine (NAC) clearly attenuated HMGB1 expression and release. Furthermore, we show that the immunosuppressants, at the same concentrations that significantly suppressed HMGB1 expression and release, were also able to prevent the necrosis of ischemic astrocytes and inhibit the expression of inflammatory mediators (NFκ, iNOS and COX-2). These results provide further information about the cytoprotective mechanisms of immunosuppressants on ischemic astrocytes, especially in relation to the pathophysiology of ischemic brain injury. It appears that the protective effects of immunosuppressants can be mediated in part by the suppressing the expression and release of HMGB1 in astrocytes, which leads to the attenuation of ischemia-induced necrosis and neuroinflammation.

Chronic social instability stress enhances vulnerability of BDNF response to LPS in the limbic structures of female rats: a protective role of antidepressants.

The aim of the present study was to estimate the influence of antidepressants given chronically on brain-derived neurotrophic factor (BDNF) alterations induced by lipopolysaccharide (LPS) in the amygdala and hippocampus of female rats subjected to chronic social instability stress (CSIS) for 29-30 days. CSIS was used as a paradigm known to be more stressful for females because stress induces affective disorders more frequently in women than men. An increased relative adrenal weight and a tendency towards the enhanced plasma corticosterone concentration were found in the stressed rats. Sucrose preference was not changed. On the last experimental day, the rats in the estrus phase were injected ip with LPS (1mg/kg). In the stressed rats, LPS administration decreased BDNF mRNA levels in both limbic structures. Desipramine (10mg/kg), fluoxetine (5mg/kg) or tianeptine (10mg/kg) given ip once daily reversed the effect of the combined stress and LPS, and tianeptine induced the strongest effects. These results indicate that chronic stress enhances vulnerability of BDNF response to deleterious influence of neuroinflammation in the examined limbic structures, what may account for its role in triggering neuropsychiatric diseases. The observed effect of antidepressants may be of significance for their therapeutic effects in the stress-induced affective disorders in females.

Antidepressant drugs as a complementary therapeutic strategy in cancer.

In the last decade, it has been increasingly recognized that antidepressant drugs may exert a range of effects, in addition to their well-documented ability to modulate neurotransmission. Although as a group they act on monoaminergic systems and receptors in different ways, a number of studies have demonstrated that at least some antidepressants might have other properties in common, including immunomodulatory, cyto/neuroprotective, analgesic and anti-inflammatory activities. These properties are partly related to the influence of antidepressants on glial cell function. Recently, emerging information about the possible anticancer properties of antidepressants has sparked increased interest within scientific community, and there is now evidence that these drugs affect the key cellular mechanisms of carcinogenesis. This review examines the putative cellular targets for the anticancer action of antidepressant drugs, and presents examples of the interaction between antidepressants and anticancer drugs. By reviewing the current state of research in this area, we hope to focus the attention of oncologists and researchers engaged in the study of cancer on the role that antidepressant drugs could play in the complementary therapy of cancer.

Chronic Physiological Hypoxia and High Glucose Concentration Promote Resistance of T98G Glioblastoma Cell Line to Temozolomide

In spite of the many studies carried out on glioblastoma multiforme (GBM) in recent years and the implementation of promising new therapeutic strategies, median survival rates in GBM patients have not been improved. A key issue in the fight against a tumor is learning its biology which is only possible in laboratory conditions that accurately reproduce its physiological microenvironment. GBM is among the most hypoxic and glycolytic tumors of the central nervous system. The most recent data suggest that these two metabolic features should be recognized as the most important markers of the GBM aggressive phenotype associated with its resistance to chemo- and radiotherapy. However, until now, the effect of modulation of the tumor’s microenvironment on drug efficacy has been evaluated only in a few studies. Since hypoxia inside a tumor creates protection for GBM cells from chemotherapy and the spatial arrangement of hypoxia in GBM is a dynamic process (the level of pO2 inside a tumor is from 0% to 5%), our experiments performed on the T98G glioblastoma cell line were carried out in a range of oxygen availability conditions and then compared to standard laboratory conditions. Moreover, the influence of high or standard glucose concentration or its deprivation in the culture medium on temozolomide effect (the standard chemotherapeutic for GBM) was estimated. We examined the influence of temozolomide on cell viability, division and apoptosis as well as on migration of tumor cells by using the time-lapse real-time observations. Our study showed that glioblastoma cells cultured in conditions of chronic hypoxia were almost completely resistant to the effect of the drug. Moreover, we observed that increased glucose availability in the GBM microenvironment was associated with the enhanced resistance of the GBM to the cytotoxic effect of temozolomide in hypoxic conditions which was manifested by an increased cellular viability and division and a diminished percentage of apoptotic cells.

Affect of antidepressants on the in vitro differentiation of rat bone marrow mesenchymal stem cells into neuronal cells

BACKGROUND Bone marrow is a valuable source of mesenchymal stem cells (MSCs) that can be used in regenerative medicine. MSCs are able to differentiate into cells from all three germ layers under specific conditions. The aim of the current work was to study the differentiation of rat MSCs (rMSCs) into neuron-like cells. NEW METHOD We investigated how the antidepressants imipramine, desipramine, fluoxetine and tianeptine affect the differentiation of rMSCs. Furthermore, we present differentiation cocktails using a cortex astrocyte-conditioned medium (CACM) separately or in conjunction with each of the antidepressants and investigated their additive effect on the efficiency of differentiation. We also observed how various differentiation conditions affect the number of primary dendrites and branching dendrites per cell. RESULTS Gene expression for an early neuronal marker (β-III-tubulin) and markers that are typical for adult neurons such as Th, Htr2A and Slc6a4 were observed. The Tubb3 and Htr2A gene expression were up-regulated, Th decreased slightly and Slc6a4 was down-regulated after differentiation We observed a two-fold higher percentage of β-III-tubulin positive cells after treatment with antidepressants and two-fold increase of neuron-like cells after using CACM with imipramine or fluoxetine simultaneously. Differentiation using imipramine or in conjunction with CACM and desipramine or fluoxetine simultaneously increased the number of cell dendrites. COMPARISON WITH EXISTING METHODS The results that were obtained are completely new and need further investigations in the nearest future. CONCLUSIONS These results suggest that antidepressants improve differentiation efficiency of rMSCs and may be useful in the preparation of rMSCs for transplantation. Differentiation efficiency is higher after long-term exposure to antidepressants, than after a 24-h exposure. Nearly additive effect of CACM and imipramine or fluoxetine suggests a beneficial role of antidepressants after transplantation.

Desipramine administered chronically inhibits lipopolysaccharide-stimulated production of IL-1β in the brain and plasma of rats.

Nowadays, it is assumed that therapeutic efficacy of antidepressants depends, at least partly, on their anti-inflammatory properties. The present study investigated for the first time the effect of 21-day oral administration of desipramine on the lipopolysaccharide (LPS)-stimulated IL-1β concentration in the olfactory bulb, hypothalamus, frontal cortex, hippocampus and plasma of rats, and on the LPS-induced IL-1β mRNA level in the olfactory bulb. Desipramine (15mg/kg/day) reduced significantly the LPS (250 μg/kg i.p.)-induced IL-1β concentration in the olfactory bulb, hypothalamus and in plasma, and diminished the LPS effect on IL-1β mRNA in the olfactory bulb. Plasma concentration of desipramine was comparable to its therapeutic range. By using the α1/α2-adrenoceptor antagonist prazosin and the unspecific β-adrenoceptor antagonist propranolol given prior to LPS, we found that the effect of desipramine on LPS-induced IL-1β production was partially mediated by both adrenoceptors in the olfactory bulb and plasma, and that β-adrenoceptors contributed also to its effect on the stimulated IL-1β concentration in the hypothalamus. The effect of LPS on the cerebral IL-1β levels was, in part, mediated by β-adrenoceptors and, in a region-specific manner, by α1/α2-adrenoceptors. The findings provide evidence for central and peripheral anti-inflammatory activity of desipramine and confirm the impact of the noradrenergic system on IL-1β production induced by an immunostimulatory challenge.