Joanna Slusarczyk

Beneficial properties of maraviroc on neuropathic pain development and opioid effectiveness in rats

Targeting chemokine signaling pathways is crucial in neuropathy development. In this study, we investigated the influence of chronic administration of maraviroc (CCR5 antagonist) on nociception and opioid effectiveness during neuropathy, which develops as a result of chronic constriction injury (CCI) of the sciatic nerve. To investigate the mechanism of action of maraviroc, we measured the expression of glial cell markers, CCR5 and certain CCR5 ligands (CCL3, CCL4, CCL5, CCL7, CCL11), in the spinal cord and dorsal root ganglia (DRG) of vehicle- and maraviroc-treated, CCI-exposed rats. Our results demonstrate that chronic intrathecal administration of maraviroc diminished neuropathic pain symptoms on day 7 post-CCI. Western blot analysis showed that maraviroc diminished protein level of Iba-1 and GFAP and reversed the up-regulated CCR5 expression observed in spinal cord and DRG after CCI. Additionally, using qRT-PCR, we demonstrated that CCR5 and some of its pronociceptive ligands (CCL3, CCL4, CCL5) increased in the spinal cord after nerve injury, and maraviroc effectively diminished those changes. However, CCL11 spinal expression was undetectable, even after injury. In vitro primary culture studies showed that CCL3, CCL4, CCL5 and CCL7 (but not CCL11) were of microglial and astroglial origin and were up-regulated after LPS stimulation. Our results indicate that maraviroc not only attenuated the development of neuropathic pain symptoms due to significant modulation of neuroimmune interactions but also intensified the analgesic properties of morphine and buprenorphine. In sum, our results suggest the pharmacological modulation of CCR5 by maraviroc as a novel therapeutic approach for co-treatment of patients receiving opioid therapy for neuropathy.

The impact of prenatal stress on insulin-like growth factor-1 and pro-inflammatory cytokine expression in the brains of adult male rats: The possible role of suppressors of cytokine signaling proteins

Stress, inflammation and the reduced expression of neurotrophic factors are risk factors for depression. The objective of this study was to determine if prenatal stress affects IGF-1 - cytokine interactions by influencing suppressors of cytokine signaling (SOCS) in the brains of adult rats, in basal conditions and after acute lipopolysaccharide (LPS) treatment. We demonstrated that prenatal stress leads to depression-like behavior, decreased IGF-1, increased IL-1β, TNF-α and IFN-γ release and disturbed SOCS-1, SOCS-2 and SOCS-3 expression in the hippocampus and frontal cortex of adult offspring. Furthermore, prenatal stress enhances the brain response to LPS-evoked inflammatory challenges.

Prenatal stress affects insulin-like growth factor-1 (IGF-1) level and IGF-1 receptor phosphorylation in the brain of adult rats

It has been shown that stressful events occurring in early life have a powerful influence on the development of the central nervous system. Insulin-like growth factor-1 (IGF-1) promotes the growth, differentiation and survival of both neurons and glial cells and is thought to exert antidepressant-like activity. Thus, it is possible that disturbances in the function of the IGF-1 system may be responsible for disturbances observed over the course of depression. Prenatal stress was used as a valid model of depression. Adult male offspring of control and stressed rat dams were subjected to behavioural testing (forced swim test). The level of IGF-1 in the blood and the expression of IGF-1, IGF-1R, and IRS-1/2 in the hippocampus and frontal cortex using RT-PCR, ELISA and western blotting were measured. In addition the effect of intracerebroventricularly administered IGF-1 and/or the IGF-1R receptor antagonist JB1 in the forced swim test was studied. Prenatally stressed rats showed depressive like behaviour, including increased immobility time as well as decreased mobility and climbing. Intracerebroventricular administration of IGF-1 reversed these effects in stressed animals, whereas concomitant administration of the IGF-1R antagonist JB1 completely blocked the effects. Biochemical analysis of homogenates from the hippocampus and frontal cortex revealed decreases in IGF-1 level and IGF-1R phosphorylation along with disturbances in IRS-1 phosphorylation. These findings reveal that prenatal stress alters IGF-1 signalling, which may contribute to the behavioural changes observed in depression.

Direct and indirect pharmacological modulation of CCL2/CCR2 pathway results in attenuation of neuropathic pain — In vivo and in vitro evidence

The repeated administration of microglial inhibitor (minocycline) and CCR2 antagonist (RS504393) attenuated the neuropathic pain symptoms in rats following chronic constriction injury of the sciatic nerve, which was associated with decreased spinal microglia activation and the protein level of CCL2 and CCR2. Furthermore, in microglia primary cell cultures minocycline downregulated both CCL2 and CCR2 protein levels after lipopolysaccharide-stimulation. Additionally, in astroglia primary cell cultures minocycline decreased the expression of CCL2, but not CCR2. Our results provide new evidence that modulation of CCL2/CCR2 pathway by microglial inhibitor as well as CCR2 antagonist is effective for neuropathic pain development in rats.

Prenatal Stress Enhances Excitatory Synaptic Transmission and Impairs Long-Term Potentiation in the Frontal Cortex of Adult Offspring Rats

The effects of prenatal stress procedure were investigated in 3 months old male rats. Prenatally stressed rats showed depressive-like behavior in the forced swim test, including increased immobility, decreased mobility and decreased climbing. In ex vivo frontal cortex slices originating from prenatally stressed animals, the amplitude of extracellular field potentials (FPs) recorded in cortical layer II/III was larger, and the mean amplitude ratio of pharmacologically-isolated NMDA to the AMPA/kainate component of the field potential—smaller than in control preparations. Prenatal stress also resulted in a reduced magnitude of long-term potentiation (LTP). These effects were accompanied by an increase in the mean frequency, but not the mean amplitude, of spontaneous excitatory postsynaptic currents (sEPSCs) in layer II/III pyramidal neurons. These data demonstrate that stress during pregnancy may lead not only to behavioral disturbances, but also impairs the glutamatergic transmission and long-term synaptic plasticity in the frontal cortex of the adult offspring.

Prenatal stress leads to changes in IGF-1 binding proteins network in the hippocampus and frontal cortex of adult male rat.

Depression is a mental disorder of still unknown origin. Currently, much attention is paid to the potential influence of disturbances in the functioning of neurotrophic factors on the onset of this disease. Insulin-like growth factor 1 (IGF-1) is one of the most important growth agents affecting processes that are crucial for brain development. To date, there are no data showing the impact of prenatal stress on the family of six IGF binding proteins (IGFBP 1-6) that regulate IGF-1 bioactivity. The goal of this study was to investigate whether the decreased expression of IGF-1 in the frontal cortex (FCx) and hippocampus (Hp) of adult male rats following a prenatal stress procedure is related to changes in the IGFBP family. Our results show that rats exposed prenatally to stressful stimuli displayed depression-like behavior based on sucrose preference and elevated plus maze tests. In both cases, in the adult rat brain structures that were examined after the prenatal stress procedure, the IGF-1 protein level was reduced. Moreover, we observed changes of varying degrees in the levels of IGFBPs in stressed animals. A decrease in IGFBP-2 and IGFBP-3 accompanied by an increase in the IGFBP-4 concentration in the Hp and the FCx was detected. There were no differences in IGFBP-1 and IGFBP-6 brain levels between the stressed and control animals, whereas IGFBP-5 concentration was decreased in the Hp of prenatally stressed animals. This study demonstrated that stress during pregnancy may lead not only to behavioral disturbances but also to a decrease in IGF-1 level and the dysregulation of the IGF-1 binding protein network in adult rat offspring.

Anti-inflammatory properties of tianeptine on lipopolysaccharide-induced changes in microglial cells involve toll-like receptor-related pathways.

Accumulating evidence suggests that activation of microglia plays a key role in the pathogenesis of depression. Activated microglia produce a wide range of factors whose prolonged or excessive release may lead to brain disorders. Thus, the inhibition of microglial cells may be beneficial in the treatment of depressive diseases. Tianeptine is an atypical antidepressant drug with proven clinical efficacy, but its mechanism of action remains still not fully understood. In the present study, using microglial cultures we investigated whether tianeptine modifies microglial activation after lipopolysaccharide (LPS) stimulation and which intracellular pathways are involved in the activity of this antidepressant. Our study shows that tianeptine attenuated the LPS‐evoked inflammatory activation of microglia by decreasing the expression of proinflammatory cytokines such as IL‐1β, IL‐18, IL‐6 and tumor necrosis factor α (TNF‐α), the release of nitric oxide (NO) and reactive oxygen species (ROS) as well as the expression of inducible nitric oxide synthase. Analyses of signaling pathways demonstrate that tianeptine led to the suppression of LPS‐induced TLR4 expression and ERK1/2 phosphorylation. Furthermore, our study reveals the inhibitory impact of tianeptine on caspase‐3‐induced PKCδ degradation and consequently on the activation of NF‐κB factor in microglial cells. Taken together, present results show anti‐inflammatory properties of tianeptine in microglial cultures stimulated by LPS. This study provides evidence that the inhibition of microglial activation may underlie the therapeutic activity of tianeptine.

Chronic mild stress influences nerve growth factor through a matrix metalloproteinase-dependent mechanism

Stress is generally a beneficial experience that motivates an organism to action to overcome the stressful challenge. In particular situations, when stress becomes chronic might be harmful and devastating. The hypothalamus is a critical coordinator of stress and the metabolic response; therefore, disruptions in this structure may be a significant cause of the hormonal and metabolic disturbances observed in depression. Chronic stress induces adverse changes in the morphology of neural cells that are often associated with a deficiency of neurotrophic factors (NTFs); additionally, many studies indicate that insufficient NTF synthesis may participate in the pathogenesis of depression. The aim of the present study was to determine the expression of the nerve growth factor (NGF) in the hypothalamus of male rats subjected to chronic mild stress (CMS) or to prenatal stress (PS) and to PS in combination with an acute stress event (AS). It has been found that chronic mild stress, but not prenatal stress, acute stress or a combination of PS with AS, decreased the concentration of the mature form of NGF (m-NGF) in the rat hypothalamus. A discrepancy between an increase in the Ngf mRNA and a decrease in the m-NGF levels suggested that chronic mild stress inhibited NGF maturation or enhanced the degradation of this factor. We have shown that NGF degradation in the hypothalamus of rats subjected to chronic mild stress is matrix metalloproteinase-dependent and related to an increase in the active forms of some metalloproteinases (MMP), including MMP2, MMP3, MMP9 and MMP13, while the NGF maturation process does not seem to be changed. We suggested that activated MMP2 and MMP9 potently cleave the mature but not the pro- form of NGF into biologically inactive products, which is the reason for m-NGF decomposition. In turn, the enhanced expression of Ngf in the hypothalamus of these rats is an attempt to overcome the reduced levels of m-NGF. Additionally, the decreased level of m-NGF together with the increased level of pro-NGF can decrease TrkA-mediated neuronal survival signalling and enhance the action of pro-NGF on the p75(NTR) receptor, respectively, to evoke pro-apoptotic signalling. This hypothesis is supported by elevated levels of the caspase-3 mRNA in the hypothalamus of rats subjected to chronic mild stress.

Stimulatory effect of desipramine on lung metastases of adenocarcinoma MADB 106 in stress highly-sensitive and stress non-reactive rats

The effect of antidepressant drugs on tumor progress is very poorly recognized. The aim of the present study was to examine the effect of individual reactivity to stress and 24-day desipramine (DES) administration on the metastatic colonization of adenocarcinoma MADB 106 cells in the lungs of Wistar rats. Wistar rats were subjected to stress procedure according to the chronic mild stress (CMS) model of depression for two weeks and stress highly-sensitive (SHS) and stress non-reactive (SNR) rats were selected. SHS rats were more prone to cancer metastasis than SNR ones and chronic DES treatment further increased the number of lung metastases by 59% and 50% in comparison to vehicle-treated appropriate control rats. The increase in lung metastases was connected with DES-induced skew macrophage activity towards M2 functional phenotype in SHS and SNR rats. Moreover, during 24h after DES injection in healthy rats, the decreased number of TCD8+ and B cells in SHS and SNR rats as well as NK cell cytotoxic activity in SNR rats could be attributed to the lowered capacity to defend against cancer metastasis observed in chronic DES treated and tumor injected rats.

A new potential mechanism of action of tianeptine – the effect on microglial cell activation

Tianeptine is an atypical antidepressant drug with proven efficacy, but still not fully understood mechanism of action. Recently it has been suggested that tianeptine may modulate inflammatory processes, however there is a lack of data on its influence on microglia - the main source of pro-inflammatory cytokines in the brain. Therefore this project aimed to investigate whether tianeptine can influence activation of microglial cells. We conducted our study in two experimental models: in vivo – in the hippocampus and frontal cortex of adult rats and in vitro in microglial cultures. Pregnant rats were subjected daily to 3 stress sessions from 14th day of pregnancy until delivery. Control pregnant females were left undisturbed in their homecages. Microglial cells were pre-treated for 30min with different concentrations of tianeptine and stimulated with LPS (100ng/ml). Next, expression of microglial activation markers and pro-inflammatory cytokines were evaluated. In the second part of experiments at 3 months of age, after behavioral verification, control and prenatally stressed rats were injected with tianeptine (10mg/kg i.p.) for 14 days. Next, biochemical studies were carried out on hippocampus and frontal cortex. We observed that in microglial pre-treatment with tianeptine (1-10uM) reduced the expression of microglial activation markers (CD40 and MHCII) and production of pro-inflammatory cytokines. Moreover, in adult animals subjected to prenatal stress (an animal model of depression) chronic tianeptine treatment inhibited microglial activation (decreased CD40 and CD68 expression) in both examined structures. In conclusion, our results show that tianeptine exerts anti-inflammatory properties suppressing microglial activation in both in vitro and in vivo experimental models.