Bożena Gabryel

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.

Piracetam and vinpocetine exert cytoprotective activity and prevent apoptosis of astrocytes in vitro in hypoxia and reoxygenation

The aim of the present study was to establish whether piracetam (2-pyrrolidon-N-acetamide; PIR) and vinpocetine (a vasoactive vinca alkaloid; VINP) are capable of protecting astrocytes against hypoxic injury. Using the model of astrocyte cell culture we observed the cells treated with PIR and VINP during and after in vitro simulated hypoxia. Cell viability was determined by Live/Dead Viability/Cytotoxicity Assay Kit, LDH release assay and MTT conversion test. Apoptotic cell death was distinguished by a method of Hoechst 33342 staining underfluorescence microscope and caspase-3 colorimetric assay. In addition the intracellular levels of ATP and phosphocreatine (PCr) were evaluated by bioluminescence method. Moreover, the effect of the drugs on the DNA synthesis was evaluated by measuring the incorporation of [3H]thymidine into DNA of astrocytes. PIR (0.01 and 1 mM) and VINP (0.1 and 10 microM) were added to the medium both during 24 h normoxia, 24 h hypoxia or 24 h reoxygenation. Administration of 1 mM PIR or 0.1 microM VINP to the cultures during hypoxia significantly decreases the number of dead and apoptotic cells. The antiapoptic effects of drugs in the above mentioned concentrations was also confirmed by their stimulation of mitochondrial function, the increase of intracellular ATP, and the inhibition of the caspase-3 activity. The prevention of apoptosis was accompanied by the increase in ATP and PCr levels and increase in the proliferation of astrocytes exposed to reoxygenation. The higher concentration of VINP (10 microM) was detrimental in hypoxic conditions. Our experiment proved the significant cytoprotective effect of 1 mM PIR and 0.1 microM VINP on astrocytes in vitro.

Chlorpromazine and loxapine reduce interleukin-1β and interleukin-2 release by rat mixed glial and microglial cell cultures

The cytokines IL-1beta and IL-2 are released from activated glial cells in the central nervous system and they are able to enhance catecholaminergic neurotransmission. There is no data concerning influence of antipsychotics on glial cell activity. Antipsychotics reaching the brain act not only on neurons but probably also on glial cells. The aim of this study was to evaluate the effect of chlorpromazine and loxapine on release of IL-1beta and IL-2 by mixed glial and microglial cell cultures. Chlorpromazine in concentrations 2 and 20 muM, and loxapine 0.2, 2 and 20 microM reduced IL-1beta secretion by LPS-activated mixed glia cultures after 1 and 3 days of exposure. Chlorpromazine in concentrations of 0.2, 2 and 20 microM reduced the IL-2 secretion in mixed glial cultures after 3 days of exposure. Loxapine in concentrations of 0.2, 2 and 20 microM reduced IL-2 secretion in mixed glia cultures after 1 and 3 days of exposure, and additionally loxapine decreased IL-1beta and IL-2 secretion in LPS-induced microglia cultures in concentrations of 2, 10 and 20 muM. Quinpirole-a D2 dopaminergic agonist increased LPS-induced IL-1beta and IL-2 secretion in mixed glia cultures only in the highest dose of 20 microM. These findings suggest the absence of functional dopamine receptors on cortical microglial cells. Mixed glia cultures deprived of microglia (by shaking and incubating with L-leucine methyl ester) did not release IL-1beta and IL-2. This observation suggests that microglia can be a source of assessed cytokines. Results of the present study support the view that antipsychotics act not only on neurons but also on glial cells. However, the clinical significance of these observations still remains unclear.

Neuronal autophagy in cerebral ischemia--a potential target for neuroprotective strategies?

Although many attempts have been made, stroke treatment options are still extremely limited and brain ischemia remains the leading cause of death and disability worldwide. Two major strategies for ischemic stroke, reperfusion and neuroprotection, are currently being evaluated. Autophagy is a bulk protein degradation system that is involved in multiple cellular processes. Increasing data suggest that activation of autophagy in ischemic brain may contribute to neuroprotection. However, it should also be noted that there are evidences that autophagy is a process involved in neurodegeneration. Targeting signaling pathways related to autophagy might be a promising option in the treatment of cerebral ischemia, but the exact role of autophagy activation due to ischemic episodes and its potential applications in pharmacotherapy are still to be determined. In this paper we review recent evidences for cerebral ischemia-induced autophagy, briefly discuss mechanisms and signaling pathways that lead to this activation and we analyze its potential roles.

Rapamycin induces of protective autophagy in vascular endothelial cells exposed to oxygen-glucose deprivation.

The protective potential of rapamycin has been reported in a few experimental models of brain ischemia, both in vivo and in vitro. Although the precise cellular processes underlying the neuroprotective effects of rapamycin in experimental models of stroke remain unknown, the current experimental data suggest that the mechanism of action of the drug may result from the mTOR-mediated autophagy induction. However, it is unclear whether the activation of autophagy acts as a pro-death or pro-survival factor in vascular endothelial cells in ischemic brain damage. It seems to be very important, since stroke affects not only neurons and astrocytes but also microvessels. In the present study, we used human umbilical vein endothelial cells (HUVEC) subjected to ischemia-simulating conditions (combined oxygen and glucose deprivation, OGD) for 6h to determine potential effect of rapamycin-induced autophagy on HUVEC damage. The drug at concentrations of 100 and 1000nM increased the expression of Beclin 1 and LC3-II together with a significant increase in the p62 degradation in ischemic HUVEC. Treatment with rapamycin in OGD significantly increased the cell viability, indicating that the drug exerts cytoprotective effect. The inhibition of Beclin 1 by siRNAs significantly attenuated the expression of autophagy-related proteins and reduced HUVEC viability following OGD and rapamycin treatment. Our findings demonstrated that toxicity of simulated ischemia conditions were enhanced in HUVEC when autophagy was blocked, and that rapamycin effectively prevented OGD-evoked damage by induction of protective autophagy via inhibition of the mTOR pathway.

Role of astrocytes in pathogenesis of ischemic brain injury

Astrocytes play an important role in the homeostasis of the CNS both in normal conditions and after ischemic injury. The swelling of astrocytes is observed during and several seconds after brain ischemia. Then ischemia stimulates sequential morphological and biochemical changes in glia and induces its proliferation. Reactive astrocytes demonstrate stellate morphology, increased glial fibrillary acidic protein (GFAP) immunoreactivity, increased number of mitochondria as well as elevated enzymatic and non-enzymatic antioxidant activities. Astrocytes can re-uptake and metabolize glutamate and in this way they control its extracellular concentration. The ability of astrocytes to protect neurons against the toxic action of free radicals depends on their specific energy metabolism, high glutathione level, increased antioxidant enzyme activity (catalase, superoxide dismutase, glutathione peroxidase) and overexpression of antiapoptoticbcl-2 gene. Astrocytes produce cytokines (TNF-±, IL-1, IL-6) involved in the initiation and maintaining of immunological response in the CNS. In astrocytes, like in neurones, ischemia induces the expression of immediate early genes:c-fos, c-jun, fos B, jun B, jun D, Krox-24, NGFI-B and others. The protein products of these genes modulate the expression of different proteins, both destructive ones and those involved in the neuroprotective processes.

Metformin has adenosine-monophosphate activated protein kinase (AMPK)-independent effects on LPS-stimulated rat primary microglial cultures

The results of recent studies suggest that metformin, in addition to its efficacy in treating type 2 diabetes, may also have therapeutic potential for the treatment of neuroinflammatory diseases in which reactive microglia play an essential role. However, the molecular mechanisms by which metformin exerts its anti-inflammatory effects remain largely unknown. Adenosine-monophosphate-activated protein kinase (AMPK) activation is the most well-known mechanism of metformin action; however, some of the biological responses to metformin are not limited to AMPK activation but are mediated by AMPK-independent mechanisms. In this paper, we attempted to evaluate the effects of metformin on unstimulated and LPS-activated rat primary microglial cell cultures. The presented evidence supports the conclusion that metformin-activated AMPK participates in regulating the release of TNF-α. Furthermore, the effects of metformin on the release of IL-1β, IL-6, IL-10, TGF-β, NO, and ROS as well as on the expression of arginase I, iNOS, NF-κB p65 and PGC-1α were not AMPK-dependent, because pretreatment of LPS-activated microglia with compound C, a pharmacological inhibitor of AMPK, did not reverse the effect of metformin. Based on the present findings, we propose that the shift of microglia toward alternative activation may underlie the beneficial effects of metformin observed in animal models of neurological disorders.

Aniracetam attenuates apoptosis of astrocytes subjected to simulated ischemia in vitro.

The aim of the present study was to establish whether aniracetam is capable of protecting cultured rat astrocytes against ischemic injury. Treatment of the cultures with aniracetam (1, 10 and 100 mM) during 24 h ischemia simulated in vitro significantly decreased the number of apoptotic cells. The antiapoptotic effects of the drug were confirmed by the increase of intracellular ATP and phosphocreatine (PCr) levels and the inhibition of the caspase-3 activity. Aniracetam also attenuated cellular oxidative stress by decreased production of reactive oxygen species (ROS). These effects were associated with the decrease in levels of c-fos and c-jun mRNA in primary astrocyte cultures exposed to 24 h ischemia. When cultured astrocytes were incubated during 24 h simulated ischemia with wortmannin, a phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor or PD98059, a mitogen-activated protein (MAP)/extracellular signal regulated kinase (ERK) (MEK) inhibitor the cell apoptosis was accelerated. This effect was antagonized by adding 100 mM aniracetam to the culture medium. These findings suggest that the protective effect of aniracetam is mediated by PI 3-kinase and MEK pathways in the downstream mechanisms.

AICAR (5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside) increases the production of toxic molecules and affects the profile of cytokines release in LPS-stimulated rat primary microglial cultures

AICAR (5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside, Acadesine, AICA riboside) is an activator of AMP-activated protein kinase (AMPK). The results of recent studies suggest that AICAR, in addition to its application for treating metabolic disorders, may also have therapeutic potential for treating neuroinflammatory diseases where reactive microglia play an etiological role. However, the molecular mechanisms of action by which AICAR exerts its anti-inflammatory effects still remain unclear or controversial. In this paper we attempt to evaluate the effects of AICAR on non-stimulated and LPS-activated rat primary microglial cell cultures. The presented evidence supports the conclusion that AMPK activated by AICAR is involved in regulation of ROS and cytokine production (IL-1 beta, TNF-alpha (6h), IL-10 and TGF-beta) as well as arginase I and PGC-1alpha expression. Furthermore, we found that the effects of AICAR on IL-6 and TNF-alpha (12, 24h) release and on the expression of iNOS and NF-kappaB p65 are not AMPK-dependent because the pre-treatment of LPS-activated microglia with compound C (a pharmacological inhibitor of AMPK) did not reverse the effect of AICAR. The results of the presented study provide additional data about AMPK-dependent and -independent mechanisms whereby AICAR may modulate inflammatory response of microglia.

Activation of cPLA2 and sPLA2 in astrocytes exposed to simulated ischemia in vitro

Both cytosolic PLA2 (cPLA2) and secretory PLA2 (sPLA2) have been implicated in pathology of cerebral ischemia. However, which of PLA2 isoforms in astrocytes is responsible for arachidonic acid (AA) release contributing to their ischemic injury remains to be determined. The aim of the present study was to investigate the time‐dependent activation of cPLA2 and sPLA2 in astrocytes exposed to combined oxygen glucose deprivation (OGD) as well as to evaluate the effectiveness of their pharmacological blockage as a method of preventing ischemic damage of the glial cells. It was shown that exposure of cultured astrocytes to OGD (0.5–24 h) causes an increase in cPLA2 and sPLA2 expression and activity. The role of AA liberated mainly by cPLA2 in the process of apoptosis was also demonstrated. To confirm the specific role of cPLA2 and sPLA2 in the mechanism of cells injury by OGD exposure, the effect of AACOCF3 as cPLA2 inhibitor and 12‐epi‐scalaradial as sPLA2 inhibitor on AA release was examined. It was proved that simultaneous pharmacological blockade of enzymatic activity of cPLA2 and sPLA2 during OGD by AACOCF3 and 12‐epi‐scalaradial substantially improves survival of ischemic injured glial cells.