Antenatal Dexamethasone Treatment Induces Sex-dependent Upregulation of NTPDase1/CD39 and Ecto-5'-nucleotidase/CD73 in the Rat Fetal Brain.

Dexamethasone (DEX) is frequently used to treat women at risk of preterm delivery, but although indispensable for the completion of organ maturation in the fetus, antenatal DEX treatment may exert adverse sex-dimorphic neurodevelopmental effects. Literature findings implicated oxidative stress in adverse effects of DEX treatment. Purinergic signaling is involved in neurodevelopment and controlled by ectonucleotidases, among which in the brain the most abundant are ectonucleoside triphosphate diphosphohydrolase 1 (NTPDase1/CD39) and ecto-5'-nucleotidase (e5'NT/CD73), which jointly dephosphorylate ATP to adenosine. They are also involved in cell adhesion and migration, processes integral to brain development. Upregulation of CD39 and CD73 after DEX treatment was reported in adult rat hippocampus. We investigated the effects of maternal DEX treatment on CD39 and CD73 expression and enzymatic activity in the rat fetal brain of both sexes, in the context of oxidative status of the brain tissue. Fetuses were obtained at embryonic day (ED) 21, from Wistar rat dams treated with 0.5 mg DEX/kg/day, at ED 16, 17, and 18, and brains were processed and used for further analysis. Sex-specific increase in CD39 and CD73 expression and in the corresponding enzyme activities was induced in the brain of antenatally DEX-treated fetuses, more prominently in males. The oxidative stress induction after antenatal DEX treatment was confirmed in both sexes, although showing a slight bias in males. Due to the involvement of purinergic system in crucial neurodevelopmental processes, future investigations are needed to determine the role of these observed changes in the adverse effects of antenatal DEX treatment.

MMP-9/BDNF ratio predicts more severe COVID-19 outcomes.

COVID-19 clinically manifests from asymptomatic to the critical range. Immune response provokes the pro-inflammatory interactions, which lead to the cytokines, reactive oxygen/nitrogen species, peptidases, and arachidonic acid metabolites enlargement and activation of coagulation components. Matrix metalloproteinases (MMPs) contribute to tissue destruction in the development of COVID-19. Due to the endothelial, systemic course of the disease, VEGF A participates actively in COVID-19 development, while neurotrophic and metabolic effects of BDNF recommends for the prediction of complications in COVID-19 patients. Searching for a marker that would improve and simplify the ranking in COVID-19, the study intended to evaluate the relationship of MMP-9 with VEGF A, BDNF, and MMP-8 with the COVID-19 severity. Upon admission to the hospital and before the therapy administration, 77 patients were classified into a mild, moderate, severe, or critical group. Due to the inflammatory stage in COVID-19, a comparison between groups showed related differences in leukocytes, neutrophils, lymphocytes, and platelets counts as anticipated. Only in seriously ill patients, there is a significant increase in the serum concentration of MMP-9, MMP-8, and VEGF A, while BDNF values did not show significant variations between groups. However, all those parameters positively correlated with each other. The ratio of MMP-9/BDNF markedly decreased in the severe and critically patients compared to the mild group. Testing the capability of this ratio to predict the COVID-19 stage by ROC curves, we found the MMP-9/BDNF could be a suitable marker for differentiating stages I/II (AUC 0.7597), stage I/III (AUC 0.9011), and stage I/IV (AUC 0.7727). Presented data describe for the first time the high-level systemic MMP-9/BDNF ratio in patients with COVID-19. This parameter could contribute to a more precise determination of the phase of the disease.

Agmatine Mitigates Inflammation-Related Oxidative Stress in BV-2 Cells by Inducing a Pre-Adaptive Response.

Neuroinflammation and microglial activation, common components of most neurodegenerative diseases, can be imitated in vitro by challenging microglia cells with Lps. We here aimed to evaluate the effects of agmatine pretreatment on Lps-induced oxidative stress in a mouse microglial BV-2 cell line. Our findings show that agmatine suppresses nitrosative and oxidative burst in Lps-stimulated microglia by reducing iNOS and XO activity and decreasing O2- levels, arresting lipid peroxidation, increasing total glutathione content, and preserving GR and CAT activity. In accordance with these results, agmatine suppresses inflammatory NF-kB, and stimulates antioxidant Nrf2 pathway, resulting in decreased TNF, IL-1 beta, and IL-6 release, and reduced iNOS and COX-2 levels. Together with increased ARG1, CD206 and HO-1 levels, our results imply that, in inflammatory conditions, agmatine pushes microglia towards an anti-inflammatory phenotype. Interestingly, we also discovered that agmatine alone increases lipid peroxidation end product levels, induces Nrf2 activation, increases total glutathione content, and GPx activity. Thus, we hypothesize that some of the effects of agmatine, observed in activated microglia, may be mediated by induced oxidative stress and adaptive response, prior to Lps stimulation.

Maternal Dexamethasone Exposure Induces Sex-Specific Changes in Histomorphology and Redox Homeostasis of Rat Placenta.

As the mediator between the mother and fetus, the placenta allows the most appropriate environment and optimal fetal growth. The placenta of one sex sometimes has a greater ability over the other to respond to and protect against possible maternal insults. Here, we characterized sex differences in the placenta's morphological features and antioxidant status following dexamethasone (Dx) exposure. Pregnant rats were exposed to Dx or saline. The placenta was histologically and stereologically analyzed. The activity of the antioxidant enzymes, lipid peroxides (TBARS), superoxide anion and nitric oxide (NO) was measured. The decrease in placental zone volumes was more pronounced (p < 0.05) in female placentas. The volume density of PCNA-immunopositive nuclei was reduced (p < 0.05) in both sexes. The reduced (p < 0.05) antioxidant enzyme activities, enhanced TBARS and NO concentration indicate that Dx exposure triggered oxidative stress in the placenta of both fetal sexes, albeit stronger in the placenta of female fetuses. In conclusion, maternal Dx treatment reduced the size and volume of placental zones, altered placental histomorphology, decreased cell proliferation and triggered oxidative stress; however, the placentas of female fetuses exerted more significant responses to the treatment effects. The reduced placental size most probably reduced the transport of nutrients and oxygen, thus resulting in the reduced weight of fetuses, similar in both sexes. The lesser ability of the male placenta to detect and react to maternal exposure to environmental challenges may lead to long-standing health effects.

Compensatory Neuroprotective Response of Thioredoxin Reductase against Oxidative-Nitrosative Stress Induced by Experimental Autoimmune Encephalomyelitis in Rats: Modulation by Theta Burst Stimulation.

Cortical theta burst stimulation (TBS) structured as intermittent (iTBS) and continuous (cTBS) could prevent the progression of the experimental autoimmune encephalomyelitis (EAE). The interplay of brain antioxidant defense systems against free radicals (FRs) overproduction induced by EAE, as well as during iTBS or cTBS, have not been entirely investigated. This study aimed to examine whether oxidative-nitrogen stress (ONS) is one of the underlying pathophysiological mechanisms of EAE, which may be changed in terms of health improvement by iTBS or cTBS. Dark Agouti strain female rats were tested for the effects of EAE and TBS. The rats were randomly divided into the control group, rats specifically immunized for EAE and nonspecifically immuno-stimulated with Complete Freund's adjuvant. TBS or sham TBS was applied to EAE rats from 14th-24th post-immunization day. Superoxide dismutase activity, levels of superoxide anion (O2•-), lipid peroxidation, glutathione (GSH), nicotinamide adenine dinucleotide phosphate (NADPH), and thioredoxin reductase (TrxR) activity were analyzed in rat spinal cords homogenates. The severity of EAE clinical coincided with the climax of ONS. The most critical result refers to TrxR, which immensely responded against the applied stressors of the central nervous system (CNS), including immunization and TBS. We found that the compensatory neuroprotective role of TrxR upregulation is a positive feedback mechanism that reduces the harmfulness of ONS. iTBS and cTBS both modulate the biochemical environment against ONS at a distance from the area of stimulation, alleviating symptoms of EAE. The results of our study increase the understanding of FRs' interplay and the role of Trx/TrxR in ONS-associated neuroinflammatory diseases, such as EAE. Also, our results might help the development of new ideas for designing more effective medical treatment, combining neuropsychological with noninvasive neurostimulation-neuromodulation techniques to patients living with MS.

Extracellular ATP induces graded reactive response of astrocytes and strengthens their antioxidative defense in vitro.

It is widely accepted that adenosine triphosphate (ATP) acts as a universal danger-associated molecular pattern with several known mechanisms for immune cell activation. In the central nervous system, ATP activates microglia and astrocytes and induces a neuroinflammatory response. The aim of the present study was to describe responses of isolated astrocytes to increasing concentrations of ATP (5 µM to 1 mM), which were intended to mimic graded intensity of the extracellular stimulus. The results show that ATP induces graded activation response of astrocytes in terms of the cell proliferation, stellation, shape remodeling, and underlying actin and GFAP filament rearrangement, although the changes occurred without an apparent increase in GFAP and actin protein expression. On the other hand, ATP in the range of applied concentrations did not evoke IL-1ß release from cultured astrocytes, nor did it modify the release from LPS and LPS+IFN-γ-primed astrocytes. ATP did not promote astrocyte migration in the wound-healing assay, nor did it increase production of reactive oxygen and nitrogen species and lipid peroxidation. Instead, ATP strengthened the antioxidative defense of astrocytes by inducing Cu/ZnSOD and MnSOD activities and by increasing their glutathione content. Our current results suggest that although ATP triggers several attributes of activated astrocytic phenotype with a magnitude that increases with the concentration, it is not sufficient to induce full-blown reactive phenotype of astrocytes in vitro. © 2016 Wiley Periodicals, Inc.

Disulfiram moderately restores impaired hepatic redox status of rats subchronically exposed to cadmium.

Examination of cadmium (Cd) toxicity and disulfiram (DSF) effect on liver was focused on oxidative stress (OS), bioelements status, morphological and functional changes. Male Wistar rats were intraperitoneally treated with 1 mg CdCl2/kg BW/day; orally with 178.5 mg DSF/kg BW/day for 1, 3, 10 and 21 days; and co-exposed from 22nd to 42nd day. The co-exposure nearly restored previously suppressed total superoxide dismutase (SOD), catalase (CAT) and increased glutathione peroxidase (GPx) activities; increased previously reduced glutathione reductase (GR) and total glutathione-S-transferase (GST) activities; reduced previously increased superoxide anion radical (O2·-) and malondialdehyde (MDA) levels; increased zinc (Zn) and iron (Fe), and decreased copper (Cu) (yet above control value), while magnesium (Mg) was not affected; and decreased serum alanine aminotransferases (ALT) levels. Histopathological examination showed signs of inflammation process as previously demonstrated by exposure to Cd. Overall, we ascertained partial liver redox status improvement, compared with the formerly Cd-induced impact.

Methionine-choline deprivation alters liver and brain acetylcholinesterase activity in C57BL6 mice.

Choline and methionine are precursors of acetylcholine, whose hydrolysis is catalyzed by acetylcholinesterase (AChE). Considering the possibility of their common deficiency, we investigated the influence of methionine-choline deprivation on AChE activity in liver and various brain regions (hypothalamus, hippocampus, cerebral cortex and striatum) in mice fed with methionine-choline deficient (MCD) diet. Male C57BL/6 mice (n = 28) were randomly and equally divided into following groups: control group fed with standard diet for 6 weeks (C) and groups fed with MCD diet for 2 weeks (MCD2), 4 weeks (MCD4) and for 6 weeks (MCD6). After the diet, mice were sacrificied and AChE activity in liver and brain was determined spectrophotometrically. Hepatic AChE activity was higher in MCD2, MCD4 and MCD6 compared to control (p < 0.01), with most prominent increase in MCD6. AChE activity in hypothalamus was higher in MCD4 and MCD6 vs. control (p < 0.05 and p < 0.01, respectively), as well as in MCD6 compared to MCD4 (p < 0.01). In hippocampus, increase in AChE activity was shown in MCD6 compared to control (p < 0.01). In cortex and striatum, increase in AChE activity was noted in MCD6 compared to control (p < 0.05). Our findings indicate the increase of hepatic and brain AChE activity in mice caused by methionine-choline deprivation.

The Use of Agmatine Provides the New Insight in an Experimental Model of Multiple Sclerosis.

The aim of the study is to investigate the hypothesis that agmatine (AGM) enhances blood brain barrier (BBB) compounds properties in experimental autoimmune encephalomyelitis (EAE), which is an established animal model for studying multiple sclerosis (MS). Wild-type (WT) and knockout (KO) CBA/H iNOS-/- mice, 3 months old (15 ± 5 g) were used for EAE induction by myelin basic protein (MBP) dissolved in complete Freund's adjuvant (CFA). The animals were divided into control, CFA, EAE, EAE + AGM and AGM groups. After the development of full clinical remission, the animals were sacrificed and the immunohistochemical and biochemical examinations were performed in brain homogenates. We had noticed the increased expressions of occludin in WT and KO mice with EAE + AGM, compared to EAE groups in which these expressions were significantly decreased compared to the controls. The significant elevations of matrix metalloproteinases (MMPs)-MMP-3 and MMP-9 in WT and KO EAE animals were decreased during AGM treatment in both groups. AGM application post EAE in WT and KO mice caused decreased level of Iba-1 stain, compared to EAE groups. The obtained results suggest beneficial AGM effects in EAE on BBB components, which might be useful for novel therapeutic strategies in MS.

Protective effects of glucose-6-phosphate dehydrogenase on neurotoxicity of aluminium applied into the CA1 sector of rat hippocampus.

BACKGROUND & OBJECTIVES: Aluminum (Al) toxicity is closely linked to the pathogenesis of Alzheimer's disease (AD). This experimental study was aimed to investigate the active avoidance behaviour of rats after intrahippocampal injection of Al, and biochemical and immunohistochemical changes in three bilateral brain structures namely, forebrain cortex (FBCx), hippocampus and basal forebrain (BF). METHODS: Seven days after intra-hippocampal (CA1 sector) injection of AlCl3 into adult male Wistar rats they were subjected to two-way active avoidance (AA) tests over five consecutive days. Control rats were treated with 0.9% w/v saline. The animals were decapitated on the day 12 post-injection. The activities of acetylcholinesterase (AChE) and glucose-6-phosphate dehydrogenase (G6PDH) were measured in the FBCx, hippocampus and BF. Immunohistochemical staining was performed for transferrin receptors, amyloid ß and tau protein. RESULTS: The activities of both AChE and G6PDH were found to be decreased bilaterally in the FBCx, hippocampus and basal forebrain compared to those of control rats. The number of correct AA responses was reduced by AlCl3 treatment. G6PDH administered prior to AlCl 3 resulted in a reversal of the effects of AlCl3 on both biochemical and behavioural parameters. Strong immunohistochemical staining of transferrin receptors was found bilaterally in the FBCx and the hippocampus in all three study groups. In addition, very strong amyloid ß staining was detected bilaterally in all structures in AlCl3-treated rats but was moderate in G6PDH/AlCl3-treated rats. Strong tau staining was noted bilaterally in AlCl3-treated rats. In contrast, tau staining was only moderate in G6PDH/AlCl3-treated rats. INTERPRETATION & CONCLUSIONS: Our findings indicated that the G6PDH alleviated the signs of behavioural and biochemical effects of AlCl3-treatment suggesting its involvement in the pathogenesis of Al neurotoxicity and its potential therapeutic benefit. The present model could serve as a useful tool in AD investigations.

Influence of the green tea leaf extract on neurotoxicity of aluminium chloride in rats.

Aluminium may have an important role in the aetiology/pathogenesis/precipitation of Alzheimer's disease. Because green tea (Camellia sinensis L.) reportedly has health-promoting effects in the central nervous system, we evaluated the effects of green tea leaf extract (GTLE) on aluminium chloride (AlCl3 ) neurotoxicity in rats. All solutions were injected into the cornu ammonis region 1 hippocampal region. We measured the performance of active avoidance (AA) tasks, various enzyme activities and total glutathione content (TGC) in the forebrain cortex (FbC), striatum, basal forebrain (BFb), hippocampus, brain stem and cerebellum. AlCl3 markedly reduced AA performance and activities of cytochrome c oxidase (COX) and acetylcholinesterase (AChE) in all regions. It decreased TGC in the FbC, striatum, BFb, hippocampus, brain stem and cerebellum, and increased superoxide dismutase activity in the FbC, cerebellum and BFb. GTLE pretreatment completely reversed the damaging effects of AlCl3 on AA and superoxide dismutase activity, markedly corrected COX and AChE activities, and moderately improved TGC. GTLE alone increased COX and AChE activities in almost all regions. GTLE reduces AlCl3 neurotoxicity probably via antioxidative effects and improves mitochondrial and cholinergic synaptic functions through the actions of (-)-epigallocatechin gallate and (-)-epicatechin, compounds most abundantly found in GTLE. Our results suggest that green tea might be beneficial in Alzheimer's disease.

Sustained Systemic Antioxidative Effects of Intermittent Theta Burst Stimulation beyond Neurodegeneration: Implications in Therapy in 6-Hydroxydopamine Model of Parkinson's Disease.

Parkinson's disease (PD) is manifested by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and caudoputamen (Cp), leading to the development of motor and non-motor symptoms. The contribution of oxidative stress to the development and progression of PD is increasingly recognized. Experimental models show that strengthening antioxidant defenses and reducing pro-oxidant status may have beneficial effects on disease progression. In this study, the neuroprotective potential of intermittent theta burst stimulation (iTBS) is investigated in a 6-hydroxydopamine (6-OHDA)-induced PD model in rats seven days after intoxication which corresponds to the occurrence of first motor symptoms. Two-month-old male Wistar rats were unilaterally injected with 6-OHDA to mimic PD pathology and were subsequently divided into two groups to receive either iTBS or sham stimulation for 21 days. The main oxidative parameters were analyzed in the caudoputamen, substantia nigra pars compacta, and serum. iTBS treatment notably mitigated oxidative stress indicators, simultaneously increasing antioxidative parameters in the caudoputamen and substantia nigra pars compacta well after 6-OHDA-induced neurodegeneration process was over. Serum analysis confirmed the systemic effect of iTBS with a decrease in oxidative markers and an increase in antioxidants. Prolonged iTBS exerts a modulatory effect on oxidative/antioxidant parameters in the 6-OHDA-induced PD model, suggesting a potential neuroprotective benefit, even though at this specific time point 6-OHDA-induced oxidative status was unaltered. These results emphasize the need to further explore the mechanisms of iTBS and argue in favor of considering it as a therapeutic intervention in PD and related neurodegenerative diseases.

Intrinsic ecto-5'-Nucleotidase/A1R Coupling may Confer Neuroprotection to the Cerebellum in Experimental Autoimmune Encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is widely used animal model of multiple sclerosis (MS). The disease is characterized by demyelination and neurodegeneration triggered by infiltrated autoimmune cells and their interaction with astrocytes and microglia. While neuroinflammation is most common in the spinal cord and brainstem, it is less prevalent in the cerebellum, where it predisposes to rapid disease progression. Because the induction and progression of EAE are tightly regulated by adenosinergic signaling, in the present study we compared the adenosine-producing and -degrading enzymes, ecto-5'-nucleotidase (eN/CD73) and adenosine deaminase (ADA), as well as the expression levels of adenosine receptors A1R and A2AR subtypes in nearby areas around the fourth cerebral ventricle-the pontine tegmentum, the choroid plexus (CP), and the cerebellum. Significant differences in histopathological findings were observed between pontine tegmentum and cerebellum on the same horizontal section level. Reactive astrogliosis and massive infiltration of CD4 + cells and macrophages in CP and pontine tegmentum resulted in local demyelination. In cerebellum, there was no evidence of infiltrates, microgliosis and neuroinflammation at the same sectional level. In addition, Bergman glia showed no signs of reactive gliosis. As for adenosinergic signaling, significant upregulation of eN/CD73 was observed in all areas studied, but in association with different adenosine receptor subtypes. In CP and pons, overexpression of eN/CD73 was coupled with induction of A2AR, whereas in cerebellum, a modest increase in eN/CD73 in resident Bergman glia was accompanied by a strong induction of A1R in the same type of astrocytes. Thus, the presence of specialized astroglia and intrinsic differences in adenosinergic signaling may play a critical role in the differential regional susceptibility to EAE inflammation.

Intermittent theta burst stimulation attenuates oxidative stress and reactive astrogliosis in the streptozotocin-induced model of Alzheimer's disease-like pathology.

Introduction: Intracerebroventricularly (icv) injected streptozotocin (STZ) is a widely used model for sporadic Alzheimer's disease (sAD)-like pathology, marked by oxidative stress-mediated pathological progression. Intermittent theta burst stimulation (iTBS) is a noninvasive technique for brain activity stimulation with the ability to induce long-term potentiation-like plasticity and represents a promising treatment for several neurological diseases, including AD. The present study aims to investigate the effect of the iTBS protocol on the animal model of STZ-induced sAD-like pathology in the context of antioxidant, anti-inflammatory, and anti-amyloidogenic effects in the cortex, striatum, hippocampus, and cerebellum. Methods: Male Wistar rats were divided into four experimental groups: control (icv normal saline solution), STZ (icv STZ-3 mg/kg), STZ + iTBS (STZ rats subjected to iTBS protocol), and STZ + Placebo (STZ animals subjected to placebo iTBS noise artifact). Biochemical assays and immunofluorescence microscopy were used to evaluate functional and structural changes. Results: The icv STZ administration induces oxidative stress and attenuates antioxidative capacity in all examined brain regions. iTBS treatment significantly reduced oxidative and nitrosative stress parameters. Also, iTBS decreased Aß-1-42 and APP levels. The iTBS enhances antioxidative capacity reported as elevated activity of its enzymatic and non-enzymatic components. In addition, iTBS elevated BDNF expression and attenuated STZ-induced astrogliosis confirmed by decreased GFAP+/VIM+/C3+ cell reactivity in the hippocampus. Discussion: Our results provide experimental evidence for the beneficial effects of the applied iTBS protocol in attenuating oxidative stress, increasing antioxidant capacity and decreasing reactive astrogliosis in STZ-administrated rats.

Intermittent Theta Burst Stimulation Improves Motor and Behavioral Dysfunction through Modulation of NMDA Receptor Subunit Composition in Experimental Model of Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder characterized by the progressive degeneration of the dopaminergic system, leading to a variety of motor and nonmotor symptoms. The currently available symptomatic therapy loses efficacy over time, indicating the need for new therapeutic approaches. Repetitive transcranial magnetic stimulation (rTMS) has emerged as one of the potential candidates for PD therapy. Intermittent theta burst stimulation (iTBS), an excitatory protocol of rTMS, has been shown to be beneficial in several animal models of neurodegeneration, including PD. The aim of this study was to investigate the effects of prolonged iTBS on motor performance and behavior and the possible association with changes in the NMDAR subunit composition in the 6-hydroxydopamine (6-OHDA)-induced experimental model of PD. Two-month-old male Wistar rats were divided into four groups: controls, 6-OHDA rats, 6-OHDA + iTBS protocol (two times/day/three weeks) and the sham group. The therapeutic effect of iTBS was evaluated by examining motor coordination, balance, spontaneous forelimb use, exploratory behavior, anxiety-like, depressive/anhedonic-like behavior and short-term memory, histopathological changes and changes at the molecular level. We demonstrated the positive effects of iTBS at both motor and behavioral levels. In addition, the beneficial effects were reflected in reduced degeneration of dopaminergic neurons and a subsequent increase in the level of DA in the caudoputamen. Finally, iTBS altered protein expression and NMDAR subunit composition, suggesting a sustained effect. Applied early in the disease course, the iTBS protocol may be a promising candidate for early-stage PD therapy, affecting motor and nonmotor deficits.

Intermittent theta burst stimulation ameliorates cognitive impairment and hippocampal gliosis in the Streptozotocin-induced model of Alzheimer's disease.

Intracerebroventricularly (icv) injected streptozotocin (STZ) model of Alzheimer's disease (AD) is used to explore the effect of intermittent theta burst stimulation (iTBS) on astrocyte and microglia reactivity in selectively vulnerable brain regions and answer the question whether these changes are in the context of cognitive capacity. The iTBS is a non-invasive approach for stimulating neuronal and glial activity with the ability to induce long-term potentiation-like plasticity and represents a promising treatment for different neurological diseases, including AD. Male Wistar rats were assigned to five groups: 1. Control subjected to icv saline solution, 2. STZ subjected to icv-STZ (bilaterally, 3 mg/kg), 3. STZ+iTBS subjected to iTBS therapy after icv-STZ, 4. STZ+iTBS placebo subjected to noise artifact after icv-STZ and 5. Control+iTBS subjected to iTBS therapy after icv- saline solution. The RotaRod result showed that STZ did not alter motor function in rats. Eight arm radial maze test results showed that iTBS significantly improved cognitive impairment induced by STZ intoxication. Reactive gliosis in the hippocampus and periventricular area, manifested through elevated levels of Iba1+ and GFAP+/VIM+ following icv-STZ, was ameliorated after iTBS treatment. Our research identifies iTBS as an effective therapeutic candidate against STZ-induced neurotoxicity and AD-like changes. The beneficial effects of iTBS on cognitive dysfunction might be due to targeting microglia and astrocytes, as they exert a protective role in neurodegenerative and neuroinflammatory diseases. The results could provoke translation into clinical practice as an early/add-on non-invasive therapeutic intervention for cognitive impairment in AD.

Intermittent Theta Burst Stimulation Ameliorates Cognitive Deficit and Attenuates Neuroinflammation via PI3K/Akt/mTOR Signaling Pathway in Alzheimer's-Like Disease Model.

Neurodegeneration implies progressive neuronal loss and neuroinflammation further contributing to pathology progression. It is a feature of many neurological disorders, most common being Alzheimer's disease (AD). Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive stimulation which modulates excitability of stimulated brain areas through magnetic pulses. Numerous studies indicated beneficial effect of rTMS in several neurological diseases, including AD, however, exact mechanism are yet to be elucidated. We aimed to evaluate the effect of intermittent theta burst stimulation (iTBS), an rTMS paradigm, on behavioral, neurochemical and molecular level in trimethyltin (TMT)-induced Alzheimer's-like disease model. TMT acts as a neurotoxic agent targeting hippocampus causing cognitive impairment and neuroinflammation, replicating behavioral and molecular aspects of AD. Male Wistar rats were divided into four experimental groups-controls, rats subjected to a single dose of TMT (8 mg/kg), TMT rats subjected to iTBS two times per day for 15 days and TMT sham group. After 3 weeks, we examined exploratory behavior and memory, histopathological and changes on molecular level. TMT-treated rats exhibited severe and cognitive deficit. iTBS-treated animals showed improved cognition. iTBS reduced TMT-induced inflammation and increased anti-inflammatory molecules. We examined PI3K/Akt/mTOR signaling pathway which is involved in regulation of apoptosis, cell growth and learning and memory. We found significant downregulation of phosphorylated forms of Akt and mTOR in TMT-intoxicated animals, which were reverted following iTBS stimulation. Application of iTBS produces beneficial effects on cognition in of rats with TMT-induced hippocampal neurodegeneration and that effect could be mediated via PI3K/Akt/mTOR signaling pathway, which could candidate this protocol as a potential therapeutic approach in neurodegenerative diseases such as AD.

Trimethyltin Increases Intracellular Ca2+ Via L-Type Voltage-Gated Calcium Channels and Promotes Inflammatory Phenotype in Rat Astrocytes In Vitro.

Astrocytes are the first responders to noxious stimuli by undergoing cellular and functional transition referred as reactive gliosis. Every acute or chronic disorder is accompanied by reactive gliosis, which could be categorized as detrimental (A1) of beneficial (A2) for nervous tissue. Another signature of pathological astrocyte activation is disturbed Ca2+ homeostasis, a common denominator of neurodegenerative diseases. Deregulation of Ca+ signaling further contributes to production of pro-inflammatory cytokines and reactive oxygen species. Trimethyltin (TMT) intoxication is a widely used model of hippocampal degeneration, sharing behavioral and molecular hallmarks of Alzheimer's disease (AD), thus representing a useful model of AD-like pathology. However, the role of astrocyte in the etiopathology of TMT-induced degeneration as well as in AD is not fully understood. In an effort to elucidate the role of astrocytes in such pathological processes, we examined in vitro effects of TMT on primary cortical astrocytes. The application of a range of TMT concentrations (5, 10, 50, and 100 µM) revealed changes in [Ca2+]i in a dose-dependent manner. Specifically, TMT-induced Ca2+ transients were due to L-type voltage-gated calcium channels (VGCC). Additionally, TMT induced mitochondrial depolarization independent of extracellular Ca2+ and disturbed antioxidative defense of astrocyte in several time points (4, 6, and 24 h) after 10 µM TMT intoxication, inducing oxidative and nitrosative stress. Chronic exposure (24 h) to 10 µM TMT induced strong upregulation of main pro-inflammatory factors, components of signaling pathways in astrocyte activation, A1 markers, and VGCC. Taken together, our results provide an insight into cellular and molecular events of astrocyte activation in chronic neuroinflammation.

The Function of the Hypothalamic-Pituitary-Adrenal Axis During Experimental Autoimmune Encephalomyelitis: Involvement of Oxidative Stress Mediators.

Multiple sclerosis (MS) is an inflammatory, demyelinating disease with an unknown origin. Previous studies showed the involvement of the hypothalamic-pituitary-adrenal (HPA) axis to susceptibility to autoimmune diseases, including MS, and its best-characterized animal model, experimental autoimmune encephalomyelitis (EAE). During MS/EAE, innate immune cells are activated and release cytokines and other inflammatory mediators, leading to a vicious cycle of inflammation. In response to inflammation, the activated HPA axis modulates immune responses via glucocorticoid activity. Because the mechanisms involving oxidative stress to the HPA axis are relatively unrevealed, in this study, we investigate the inflammatory and oxidative stress status of HPA axis during EAE. Our results reveal an upregulation of Pomc gene expression, followed by POMC and ACTH protein increase at the peak of the EAE in the pituitary. Also, prostaglandins are well-known contributors of HPA axis activation, which increases during EAE at the periphery. The upregulated Tnf expression in the pituitary during the peak of EAE occurred. This leads to the activation of oxidative pathways, followed by upregulation of inducible NO synthase expression. The reactive oxidant/nitrosative species (ROS/RNS), such as superoxide anion and NO, increase their levels at the onset and peak of the disease in the pituitary and adrenal glands, returning to control levels at the end of EAE. The corticotrophs in the pituitary increased in number and volume at the peak of EAE that coincides with high lipid peroxidation levels. The expression of MC2R in the adrenal glands increases at the peak of EAE, where strong induction of superoxide anion and malondialdehyde (MDA), reduced total glutathione (GSH) content, and catalase activity occurred at the peak and end of EAE compared with controls. The results obtained from this study may help in understanding the mechanisms and possible pharmacological modulation in MS and demonstrate an effect of oxidative stress exposure in the HPA activation during the course of EAE.

Downregulation of CD73/A2AR-Mediated Adenosine Signaling as a Potential Mechanism of Neuroprotective Effects of Theta-Burst Transcranial Magnetic Stimulation in Acute Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) is a chronic neurodegenerative disease caused by autoimmune-mediated inflammation in the central nervous system. Purinergic signaling is critically involved in MS-associated neuroinflammation and its most widely applied animal model-experimental autoimmune encephalomyelitis (EAE). A promising but poorly understood approach in the treatment of MS is repetitive transcranial magnetic stimulation. In the present study, we aimed to investigate the effect of continuous theta-burst stimulation (CTBS), applied over frontal cranial bone, on the adenosine-mediated signaling system in EAE, particularly on CD73/A2AR/A1R in the context of neuroinflammatory activation of glial cells. EAE was induced in two-month-old female DA rats and in the disease peak treated with CTBS protocol for ten consecutive days. Lumbosacral spinal cord was analyzed immunohistochemically for adenosine-mediated signaling components and pro- and anti-inflammatory factors. We found downregulated IL-1ß and NF- κB-ir and upregulated IL-10 pointing towards a reduction in the neuroinflammatory process in EAE animals after CTBS treatment. Furthermore, CTBS attenuated EAE-induced glial eN/CD73 expression and activity, while inducing a shift in A2AR expression from glia to neurons, contrary to EAE, where tight coupling of eN/CD73 and A2AR on glial cells is observed. Finally, increased glial A1R expression following CTBS supports anti-inflammatory adenosine actions and potentially contributes to the overall neuroprotective effect observed in EAE animals after CTBS treatment.