Pharmacological Modulation of Excitotoxicity through the Combined Use of NMDA Receptor Inhibition and Group III mGlu Activation Reduces TMT-Induced Neurodegeneration in the Rat Hippocampus.

We studied the neuroprotective properties of the non-competitive NMDA receptor antagonist memantine, in combination with a positive allosteric modulator of metabotropic glutamate receptors of Group III, VU 0422288. The treatment was started 48 h after the injection of neurotoxic agent trimethyltin (TMT) at 7.5 mg/kg. Three weeks after TMT injection, functional and morphological changes in a rat hippocampus were evaluated, including the expression level of genes characterizing glutamate transmission and neuroinflammation, animal behavior, and hippocampal cell morphology. Significant neuronal cell death occurred in the CA3 and CA4 regions, and to a lesser extent, in the CA1 and CA2 regions. The death of neurons in the CA1 field was significantly reduced in animals with a combined use of memantine and VU 0422288. In the hippocampus of these animals, the level of expression of genes characterizing glutamatergic synaptic transmission (Grin2b, Gria1, EAAT2) did not differ from the level in control animals, as well as the expression of genes characterizing neuroinflammation (IL1b, TGF beta 1, Aif1, and GFAP). However, the expression of genes characterizing neuroinflammation was markedly increased in the hippocampus of animals treated with memantine or VU 0422288 alone after TMT. The results of immunohistochemical studies confirmed a significant activation of microglia in the hippocampus three weeks after TMT injection. In contrast to the hilus, microglia in the CA1 region had an increase in rod-like cells. Moreover, in the CA1 field of the hippocampus of the animals of the MEM + VU group, the amount of such microglia was close to the control. Thus, the short-term modulation of glutamatergic synaptic transmission by memantine and subsequent activation of Group III mGluR significantly affected the dynamics of neurodegeneration in the hippocampus.

The Effect of Dual Bioactive Compounds Artemisinin and Metformin Co-loaded in PLGA-PEG Nano-particles on Breast Cancer Cell lines: Potential Apoptotic and Anti-proliferative Action.

The most prevalent malignancy among women is breast cancer. Phytochemicals and their derivatives are rapidly being recognized as possible cancer complementary therapies because they can modify signaling pathways that lead to cell cycle control or directly alter cell cycle regulatory molecules. The phytochemicals' poor bioavailability and short half-life make them unsuitable as anticancer drugs. Applying PLGA-PEG NPs improves their solubility and tolerance while also reducing drug adverse effects. According to the findings, combining anti-tumor phytochemicals can be more effective in regulating several signaling pathways linked to tumor cell development. The point of the study was to compare the anti-proliferative impacts of combined artemisinin and metformin on cell cycle arrest and expression of cyclin D1 and apoptotic genes (bcl-2, Bax, survivin, caspase-7, and caspase-3), and also hTERT genes in breast cancer cells. T-47D breast cancer cells were treated with different concentrations of metformin (MET) and artemisinin (ART) co-loaded in PLGA-PEG NPs and free form. The MTT test was applied to assess drug cytotoxicity in T47D cells. The cell cycle distribution was investigated using flow cytometry and the expression levels of cyclin D1, hTERT, Bax, bcl-2, caspase-3, and caspase-7, and survivin genes were then determined using real-time PCR. The findings of the MTT test and flow cytometry revealed that each state was cytotoxic to T47D cells in a time and dose-dependent pattern. Compared to various state of drugs (free and nano state, pure and combination state) Met-Art-PLGA/PEG NPs demonstrated the strongest anti-proliferative impact and considerably inhibited the development of T-47D cells; also, treatment with nano-formulated forms of Met-Art combination resulted in substantial downregulation of hTERT, Bcl-2, cyclin D1, survivin, and upregulation of caspase-3, caspase-7, and Bax, in the cells, as compared to the free forms, as indicated by real-time PCR findings. The findings suggested that combining an ART/MET-loaded PLGA-PEG NP-based therapy for breast cancer could significantly improve treatment effectiveness.

Therapeutic Effects of Hydrogen Gas Inhalation on Trimethyltin-Induced Neurotoxicity and Cognitive Impairment in the C57BL/6 Mice Model.

Oxidative stress (OS) is one of the causative factors in the pathogenesis of various neurodegenerative diseases, including Alzheimer's disease (AD) and cognitive dysfunction. In the present study, we investigated the effects of hydrogen (H2) gas inhalation in trimethyltin (TMT)-induced neurotoxicity and cognitive dysfunction in the C57BL/6 mice. First, mice were divided into the following groups: mice without TMT injection (NC), TMT-only injection group (TMT only), TMT injection + lithium chloride-treated group as a positive control (PC), and TMT injection + 2% H2 inhalation-treated group (H2). The TMT injection groups were administered a single dosage of intraperitoneal TMT injection (2.6 mg/kg body weight) and the H2 group was treated with 2% H2 for 30 min once a day for four weeks. Additionally, a behavioral test was performed with Y-maze to test the cognitive abilities of the mice. Furthermore, multiple OS- and AD-related biomarkers such as reactive oxygen species (ROS), nitric oxide (NO), calcium (Ca2+), malondialdehyde (MDA), glutathione peroxidase (GPx), catalase, inflammatory cytokines, apolipoprotein E (Apo-E), amyloid ß (Aß)-40, phospho-tau (p-tau), Bcl-2, and Bcl-2- associated X (Bax) were investigated in the blood and brain. Our results demonstrated that TMT exposure alters seizure and spatial recognition memory. However, after H2 treatment, memory deficits were ameliorated. H2 treatment also decreased AD-related biomarkers, such as Apo-E, Aß-40, p-tau, and Bax and OS markers such as ROS, NO, Ca2+, and MDA in both serum and brain. In contrast, catalase and GPx activities were significantly increased in the TMT-only group and decreased after H2 gas treatment in serum and brain. In addition, inflammatory cytokines such as granulocyte colony-stimulating factors (G-CSF), interleukin (IL)-6, and tumor necrosis factor alpha (TNF-α) were found to be significantly decreased after H2 treatment in both serum and brain lysates. In contrast, Bcl-2 and vascular endothelial growth factor (VEGF) expression levels were found to be enhanced after H2 treatment. Taken together, our results demonstrated that 2% H2 gas inhalation in TMT-treated mice exhibits memory enhancing activity and decreases the AD, OS, and inflammatory-related markers. Therefore, H2 might be a candidate for repairing neurodegenerative diseases with cognitive dysfunction. However, further mechanistic studies are needed to fully clarify the effects of H2 inhalation on TMT-induced neurotoxicity and cognitive dysfunction.

Fucoidan-Rich Substances from Ecklonia cava Improve Trimethyltin-Induced Cognitive Dysfunction via Down-Regulation of Amyloid ß Production/Tau Hyperphosphorylation.

Ecklonia cava (E. cava) was investigated to compare the effect of polyphenol and fucoidan extract and mixture (polyphenol:fucoidan = 4:6) on cognitive function. The ameliorating effect of E. cava was evaluated using the Y-maze, passive avoidance and Morris water maze tests with a trimethyltin (TMT)-induced cognitive dysfunction model, and the results showed that the fucoidan extract and mixture (4:6) had relatively higher learning and memory function effects than the polyphenol extract. After a behavioral test, the inhibitory effect of lipid peroxidation and cholinergic system activity were examined in mouse brain tissue, and the fucoidan extract and mixture (4:6) also showed greater improvements than the polyphenol extract. Mitochondrial activity was evaluated using mitochondrial reactive oxygen species (ROS) content, mitochondrial membrane potential (MMP, ΔΨm), adenosine triphosphate (ATP) content, and mitochondria-mediated protein (BAX, cytochrome C) analysis, and these results were similar to the results of the behavioral tests. Finally, to confirm the cognitive function-related mechanism of E. cava, the amyloid-ß production and tau hyperphosphorylation-medicated proteins were analyzed. Based on these results, the improvement effect of E. cava was more influenced by fucoidan than polyphenol. Therefore, our study suggests that the fucoidan-rich substances in E. cava could be a potential material for improving cognitive function by down-regulating amyloid-ß production and tau hyperphosphorylation.

Trimethyltin (TMT) Reduces Testosterone Production in Adult Leydig Cells in Rats.

Trimethyltin (TMT) is widely used as a plastic heat stabilizer and can cause severe toxicity. Here, the effects of TMT on testosterone production by adult Leydig cells and the related mechanisms of action were investigated. Eighteen adult male Sprague Dawley rats (56 days old) were randomly divided into 3 groups and given intraperitoneal injection of TMT for 21 consecutive days at the doses of 0 (vehicle control), 5, or 10 mg/kg/d. After treatment, trunk blood was collected for hormonal analysis. In addition, related gene and protein expression in testes was detected. At 10 mg/kg, TMT significantly reduced serum testosterone levels but increased serum luteinizing and follicle-stimulating hormone levels. The messenger RNA and protein levels of luteinizing hormone/chorionic gonadotropin receptor, steroidogenic acute regulatory protein, cytochrome P450 17-hydroxylase/17,20-lyase, follicle-stimulating hormone receptor, and SRY box 9 were significantly lower in the TMT-treated testes than in controls. Immunohistochemical study showed that TMT decreased adult Leydig cell number. In conclusion, these findings indicate that TMT reduced adult Leydig cell testosterone production in vivo by directly downregulating the expression of steroidogenic enzymes and decreasing adult Leydig cell number in the testis.

NLRP3 inflammasome activation is involved in trimethyltin-induced neuroinflammation.

Trimethyltin (TMT), a neurotoxic organotin compound, is selectively localized within the limbic system. The mechanisms of TMT-induced hippocampal neurodegeneration include inflammatory responses, oxidative stress, and neuronal death. Increasing evidence shows that the inflammatory response, mediated by activated inflammasomes, is involved in apoptosis and cellular dysfunction during brain injury. This study aimed to assess the role of the nucleotide-binding oligomerization domain-like receptor pyrin-domain-containing protein 3 (NLRP3) inflammasome in TMT-induced central nervous system (CNS) injury. In addition, the mechanisms underlying TMT neurotoxicity are similar to those involved in the pathogenesis of multiple neurodegenerative diseases; hence, a study on TMT cytotoxicity may be informative for the understanding of human CNS diseases. Microglia were significantly activated in the rat hippocampal dentate gyrus after TMT treatment. The mRNA expression of pro-inflammatory cytokines, interleukin-1ß and interleukin-18, was induced both in vitro and in vivo. TMT treatment activated the NLRP3 inflammasome in the microglial cell line BV2. NLRP3 RNA interference significantly protected these cells from TMT-induced neuroinflammation. Our results demonstrate that the NLRP3 inflammasome is a key mediator of neuroinflammation and plays an important role in TMT-induced neuroinflammation.

Neuroprotective effect of clavulanic acid on trimethyltin (TMT)-induced cytotoxicity in PC12 cells.

Trimethyltin (TMT) is a short-chain trialkyltin with various applications in industry. In addition, it is a known neurotoxin, producing significant and selective neurodegeneration in the limbic system of both human and animals. Recently, effect of clavulanic acid (CA) in nervous system has been mentioned. Therefore, in this study, the role of CA in TMT-induced toxicity in PC12 cells was evaluated. For this study, PC12 cells were cultured and exposed to different concentrations of CA for 24 h. Then, TMT (20 µM) was added to cells. After that, MTT test was performed to assay cytotoxicity. Reactive oxygen species production (ROS) was determined using 2,7-dichlorofluorescein diacetate (DCFH-DA) method. Additionally, the levels of Bax, Bcl-2, caspase-3, CERB and p-CREB proteins were evaluated using Western blot analysis. The exposure of PC12 cells to TMT reduced cell viability, increased intracellular ROS production, elevated Bax/Bcl-2 ratio and enhanced the expression of caspase-3 (Pro and cleaved forms) protein. Pretreatment of cells with CA before TMT, significantly reduced ROS generation, diminished upregulation of proapoptotic Bax protein and attenuated caspase-3 protein expression. In conclusion, CA exhibited significant neuroprotective effects against neurotoxicity of TMT mainly throughout reduction of ROS production and regulation of proteins, which are involved in apoptosis pathway.

Anti-inflammatory effects of Ginkgo biloba extract against trimethyltin-induced hippocampal neuronal injury.

BACKGROUND: Despite the immense neuromodulatory potentials of Ginkgo biloba extract as a memory enhancer, its underlying mechanism seems inadequate particularly with regard to its anti-inflammatory properties. AIM: The objective of the present study is to investigate the protective potentials of Ginkgo biloba extract (GBE) against hippocampal neuronal injury induced by trimethyltin (TMT), a potent neurotoxicant. METHODS: Male SD rats were administered trimethyltin (8.5 mg kg-1 b.wt) single intraperitoneal (i.p.) injection, followed by Ginkgo biloba extract (100 mg kg-1 b.wt i.p) for 21 days. RESULTS: The co-administration of GBE with TMT showed marked improvement in cognitive functions. Concomitantly, there was a significant decrease in oxidative stress as evident by reduction in MDA and total ROS levels. In addition, there was a marked suppression of astrocyte activation (GFAP), transcription factor NFκB and proinflammatory cytokines (TNF-α, IL-1α, 1L-6), which were found to be elevated by TMT administration. Histopathological observations showed remarkable improvement in hippocampal neuronal injury in the conjunctive group. CONCLUSION: Therefore, it is suggested that Ginkgo biloba extract is an effective agent against trimethyltin-induced hippocampal neuronal loss owing to its antioxidative as well as anti-inflammatory properties.

Unravelling the effects of multiple experimental factors in metabolomics, analysis of human neural cells with hydrophilic interaction liquid chromatography hyphenated to high resolution mass spectrometry.

This work introduces a strategy for decomposing variable contributions within the data obtained from structured metabolomic studies. This approach was applied in the context of an in vitro human neural model to investigate biochemical changes related to neuroinflammation. Neural cells were exposed to the neuroinflammatory toxicant trimethyltin at different doses and exposure times. In the frame of an untargeted approach, cell contents were analysed using HILIC hyphenated with HRMS. Detected features were annotated at level 1 by comparison against a library of standards, and the 126 identified metabolites were analysed using a recently proposed chemometric tool dedicated to multifactorial Omics datasets, namely, ANOVA multiblock OPLS (AMOPLS). First, the total observed variability was decomposed to highlight the contribution of each effect related to the experimental factors. Both the dose of trimethyltin and the exposure time were found to have a statistically significant impact on the observed metabolic alterations. Cells that were exposed for a longer time exhibited a more mature and differentiated metabolome, whereas the dose of trimethyltin was linked to altered lipid pathways, which are known to participate in neurodegeneration. Then, these specific metabolic patterns were further characterised by analysing the individual variable contributions to each effect. AMOPLS was highlighted as a useful tool for analysing complex metabolomic data. The proposed strategy allowed the separation, quantitation and characterisation of the specific contribution of the different factors and the relative importance of every metabolite to each effect with respect to the total observed variability of the system.

Turmeric extract inhibits apoptosis of hippocampal neurons of trimethyltin-exposed rats.

OBJECTIVES: The aim of the present study was to reveal the possible antiapoptotic effect of turmeric (Curcuma longa Linn.) on the hippocampal neurons of rats exposed to trimethyltin (TMT). BACKGROUND: Oxidative damage in the hippocampus can induce the apoptosis of neurons associated with the pathogenesis of dementiaMETHODS. The ethanolic turmeric extract and a citicoline (as positive control) solution were administered to the TMT-exposed rats for 28 days. The body weights of rats were recorded once a week. The hippocampal weights and imumunohistochemical expression of caspase 3 proteins in the CA1 and CA2-CA3 regions of the hippocampi were examined at the end of the experiment. RESULTS: Immunohistochemical analysis showed that the injection of TMT increased the expression of caspase 3 in the CA1 and CA2-CA3 regions of hippocampus. TMT also decreased the body and hippocampal weights. Furthermore, the administration of 200 mg/kg bw dose of turmeric extract decreased the caspase 3 expression in the CA2-CA3 pyramidal neurons but not in the CA1 neurons. It also prevented the decrease of the body and hippocampal weights. CONCLUSION: We suggest that the 200 mg/kg bw dose of turmeric extract may exert antiapoptotic effect on the hippocampal neurons of the TMT-exposed rats (Tab. 1, Fig. 3, Ref. 49).

Enhanced expression of immediate-early genes in mouse hippocampus after trimethyltin treatment.

Immediate-early genes (IEGs) are transiently and rapidly activated in response to various cellular stimuli. IEGs mediate diverse functions during pathophysiologic events by regulating cellular signal transduction. We investigated the temporal expression of several IEGs, including c-fos, early growth response protein-1 (Egr-1), and activity-regulated cytoskeleton-associated protein (Arc), in trimethyltin (TMT)-induced hippocampal neurodegeneration. Mice (7 weeks old, C57BL/6) administered TMT (2.6mg/kg intraperitoneally) presented severe neurodegenerative lesions in the dentate gyrus (DG) and showed behavioral seizure activity on days 1-4 post-treatment, after which the lesions and behavior recovered spontaneously over time. c-fos, Egr-1, and Arc mRNA and protein levels significantly increased in the mouse hippocampus after TMT treatment. Immunohistochemical analysis showed that nuclear c-fos expression increased mainly in the DG, whereas nuclear Egr-1 expression was increased extensively in cornu ammonis (CA) 1, CA3, and the DG after TMT treatment. Increased Arc levels were detected in the cellular somata/dendrites of the hippocampal subregions after TMT treatment. Therefore, we suggest that increased IEGs are associated with TMT-induced pathological events in mouse hippocampus.

Trialkyltin Rexinoid-X Receptor Agonists Selectively Potentiate Thyroid Hormone Induced Programs of Xenopus laevis Metamorphosis.

The trialkyltins tributyltin (TBT) and triphenyltin (TPT) can function as rexinoid-X receptor (RXR) agonists. We recently showed that RXR agonists can alter thyroid hormone (TH) signaling in a mammalian pituitary TH-responsive reporter cell line, GH3.TRE-Luc. The prevalence of TBT and TPT in the environment prompted us to test whether they could also affect TH signaling. Both trialkyltins induced the integrated luciferase reporter alone and potentiated TH activation at low doses. Trimethyltin, which is not an RXR agonist, did not. We turned to a simple, robust, and specific in vivo model system of TH action: metamorphosis of Xenopus laevis, the African clawed frog. Using a precocious metamorphosis assay, we found that 1nM TBT and TPT, but not trimethyltin, greatly potentiated the effect of TH treatment on resorption phenotypes of the tail, which is lost at metamorphosis, and in the head, which undergoes extensive remodeling including gill loss. Consistent with these responses, TH-induced caspase-3 activation in the tail was enhanced by cotreatment with TBT. Induction of a transgenic reporter gene and endogenous collagenase 3 (mmp13) and fibroblast-activating protein-α (fap) genes were not induced by TBT alone, but TH induction was significantly potentiated by TBT. However, induction of other TH receptor target genes such as TRß and deiodinase 3 by TH were not affected by TBT cotreatment. These data indicate that trialkyltins that can function as RXR agonists can selectively potentiate gene expression and resultant morphological programs directed by TH signaling in vivo.

Trimethyltin Modulates Reelin Expression and Endogenous Neurogenesis in the Hippocampus of Developing Rats.

Reelin is an extracellular matrix glycoprotein involved in the modulation of synaptic plasticity and essential for the proper radial migration of cortical neurons during development and for the integration and positioning of dentate granular cell progenitors; its expression is down-regulated as brain maturation is completed. Trimethyltin (TMT) is a potent neurotoxicant which causes selective neuronal death mainly localised in the CA1-CA3/hilus hippocampal regions. In the present study we analysed the expression of reelin and the modulation of endogenous neurogenesis in the postnatal rat hippocampus during TMT-induced neurodegeneration (TMT 6 mg/kg). Our results show that TMT administration induces changes in the physiological postnatal decrease of reelin expression in the hippocampus of developing rats. In particular, quantitative analysis of reelin-positive cells evidenced, in TMT-treated animals, a persistent reelin expression in the stratum lacunosum moleculare of Cornu Ammonis and in the molecular layer of Dentate Gyrus. In addition, a significant decrease in the number of bromodeoxyuridine (BrdU)-labeled newly-generated cells was also detectable in the subgranular zone of P21 TMT-treated rats compared with P21 control animals; no differences between P28 TMT-treated rats and age-matched control group were observed. In addition the neuronal commitment of BrdU-positive cells appeared reduced in P21 TMT-treated rats compared with P28 TMT-treated animals. Thus TMT treatment, administrated during development, induces an early reduction of endogenous neurogenesis and influences the hippocampal pattern of reelin expression in a temporally and regionally specific manner, altering the physiological decrease of this protein.

Trimethyltin chloride inhibits neuronal cell differentiation in zebrafish embryo neurodevelopment.

Trimethyltin chloride (TMT) is a neurotoxicant widely present in the aquatic environment, primarily from effluents of the plastic industry. It is known to cause acute neuronal death in the limbic-cerebellar system, particularly in the hippocampus. However, relatively few studies have estimated the effects of TMT toxicity on neurodevelopment. In this study, we confirmed the dose-dependent effects of TMT on neurodevelopmental stages through analysis of morphological changes and fluorescence assays using HuC-GFP and olig2-dsRed transgenic zebrafish embryos. In addition, we analyzed the expression of genes and proteins related to neurodevelopment. Exposure of embryos to TMT for 4 days post fertilization (dpf) elicited a concentration-related decrease in body length and increase in axial malformation. TMT affected the fluorescent CNS structure by decreasing pattern of HuC-GFP and olig2-dsRed transgenic zebrafish. In addition, it significantly modulated the expression patterns of Sonic hedgehog a (Shha), Neurogenin1 (Ngn1), Embryonic lethal abnormal vision like protein 3 (Elavl3), and Glial fibrillary acidic protein (Gfap). The overexpression of Shha and Ngn1, and downregulation of Elavl3 and Gfap, indicate repression of proneural cell differentiation. Our study demonstrates that TMT inhibits specific neurodevelopmental stages in zebrafish embryos and suggests a possible mechanism for the toxicity of TMT in vertebrate neurodevelopment.

Progressive increase of lysosomal enzyme activities in hippocampus associated with reduction of population spike in a rat model of neurodegeneration.

OBJECTIVES: Extensive effort has been made to identify early markers of neurodegeneration as late stages have no chance of treatment. Recently, many experimental models have been used to study hallmarks of neuronal injury. One of them is the model of trimethyltin (TMT)-induced damage associated with cognitive decline, thus called a model of Alzheimer-like disease. OBJECTIVE AND METHODS: Our aim was to study neuronal transmission in hippocampal slices of male Wistar rats affected with a single dose of TMT (7.5 mg/kg, i.p.) during the first three weeks of its action. The monitored time periods after TMT administration were days 1-3; 8-10 and 15-17. At the same time periods, right hippocampi were collected for determination of changes in specific activities of two lysosomal enzymes. Electrophysiological measurements were based on stimulation of Schäffer collaterals and registration of evoked responses in the stratum pyramidale and the stratum radiatum at the CA3-CA1 synapse. Specific activities of N-acetyl-ß-D-glucosaminidase (NAGA) and cathepsin D (Cat D) were determined spectrophotometrically. RESULTS: During three weeks after i.p. TMT administration to rats, we found a time-dependent reduction of postsynaptic neuronal firing, expressed by diminished population spike (PoS) amplitude recorded in the stratum pyramidale accompanied with marked increase in specific activity of NAGA to respective 111%, 163% and 252% in the 1st, 2nd and 3rd week compared to unaffected rats. In the stratum radiatum, reduction of the slope of excitatory postsynaptic potential was not time-dependent but almost constantly reduced from the 1st to 3rd week after TMT administration (55-60%) compared to control rats. Specific activity of lysosomal enzyme Cat D was significantly increased in the 3rd week after TMT administration. CONCLUSION: This work demonstrates a time-dependent reduction of somatic response in the hippocampus of TMT affected rats during the first three weeks. This reduction of neuronal firing was later accompanied with increase of specific activity of NAGA and Cat D, supporting evidence that lysosomal dysfunction may be one of the primary contributors to TMT-induced neurodegeneration.

Effect of Chaenomeles sinensis Extract on Choline Acetyltransferase Activity and Trimethyltin-Induced Learning and Memory Impairment in Mice.

The aim of this study was to search for a novel choline acetyltransferase (ChAT) activator from plants traditionally grown in Korea. An ethanol extract from Chaenomeles sinensis Koehne showed the highest ChAT-activating effect in vitro in an assay that used human neuroblastoma cells and [(14)C]acetyl-CoA. The active compound was speculated to be stearic acid methyl ester (SAME). In an in vivo experiment, C. sinensis extract and SAME improved trimethyltin (TMT)-induced deficits in learning and memory in mice as assessed by a Y-maze behavioral test and a passive avoidance test. The C. sinensis extract might attenuate the TMT-induced brain disorder. This study suggests that SAME from C. sinensis might be useful in the treatment of Alzheimer's disease.

Trimethyltin-Induced Microglial Activation via NADPH Oxidase and MAPKs Pathway in BV-2 Microglial Cells.

Trimethyltin (TMT) is known as a potent neurotoxicant that causes neuronal cell death and neuroinflammation, particularly in the hippocampus. Microglial activation is one of the prominent pathological features of TMT neurotoxicity. Nevertheless, it remains unclear how microglial activation occurs in TMT intoxication. In this study, we aimed to investigate the signaling pathways in TMT-induced microglial activation using BV-2 murine microglial cells. Our results revealed that TMT generates reactive oxygen species (ROS) and increases the expression of CD11b and nuclear factor-κB- (NF-κB-) mediated nitric oxide (NO) and tumor necrosis factor- (TNF-) α in BV-2 cells. We also observed that NF-κB activation was controlled by p38 and JNK phosphorylation. Moreover, TMT-induced ROS generation occurred via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in BV-2 cells. Interestingly, treatment with the NADPH oxidase inhibitor apocynin significantly suppressed p38 and JNK phosphorylation and NF-κB activation and ultimately the production of proinflammatory mediators upon TMT exposure. These findings indicate that NADPH oxidase-dependent ROS generation activated p38 and JNK mitogen-activated protein kinases (MAPKs), which then stimulated NF-κB to release proinflammatory mediators in the TMT-treated BV-2 cells.

Glial activation with concurrent up-regulation of inflammatory mediators in trimethyltin-induced neurotoxicity in mice.

Trimethyltin (TMT), a potent neurotoxic chemical, causes dysfunction and neuroinflammation in the brain, particularly in the hippocampus. The present study assessed TMT-induced glial cell activation and inflammatory cytokine alterations in the mouse hippocampus, BV-2 microglia, and primary cultured astrocytes. In the mouse hippocampus, TMT treatment significantly increased the expression of glial cell markers, including the microglial marker ionized calcium-binding adapter molecule 1 and the astroglial marker glial fibrillary acidic protein. The expression of M1 and M2 microglial markers (inducible nitric oxide synthase [iNOS] and CD206, respectively) and pro-inflammatory cytokines (interleukin [IL]-1ß, IL-6 and tumor necrosis factor [TNF]-α) were significantly increased in the mouse hippocampus following TMT treatment. In BV-2 microglia, iNOS, IL-1ß, TNF-α, and IL-6 expression increased significantly, whereas arginase-1 and CD206 expression decreased significantly after TMT treatment in a time- and concentration-dependent manner. In primary cultured astrocytes, iNOS, arginase-1, IL-1ß, TNF-α, and IL-6 expression increased significantly, whereas IL-10 expression decreased significantly after TMT treatment in a time- and concentration-dependent manner. These results indicate that significant up-regulation of pro-inflammatory signals in TMT-induced neurotoxicity may be associated with pathological processing of TMT-induced neurodegeneration.

Nestin expression and glial response in the hippocampus of mice after trimethyltin treatment.

Nestin is a protein of embryonic intermediate filaments expressed by multipotent neural stem cells. In the present study, the nestin expression pattern in the mouse hippocampus 1, 2, 3, 4, and 8 days after treatment with trimethyltin (TMT) was examined to explore the possible role played by nestin in chemically induced hippocampal injury. TMT treatment (2.5mg/kg, intraperitoneally) selectively injured the dentate gyrus (DG) of the mouse hippocampus. The level of hippocampal mRNA encoding nestin increased significantly 2 and 3 days post-treatment and thereafter decreased (at 4 and 8 days post-treatment). The level of nestin protein significantly increased 2 - 4 days post-treatment, particularly in the injured region of the DG, and predominantly in glial fibrillary acidic protein-positive astrocytes in the hippocampal DG. Ki67-positive proliferating cells were increased following TMT treatment and co-localized with nestin-positive reactive astrocytes. Thus, we suggest that nestin contributes to remodeling of the chemically injured DG via glial scar formation and the alteration of neurogenesis.

The neurogenic effects of exogenous neuropeptide Y: early molecular events and long-lasting effects in the hippocampus of trimethyltin-treated rats.

Modulation of endogenous neurogenesis is regarded as a promising challenge in neuroprotection. In the rat model of hippocampal neurodegeneration obtained by Trimethyltin (TMT) administration (8 mg/kg), characterised by selective pyramidal cell loss, enhanced neurogenesis, seizures and cognitive impairment, we previously demonstrated a proliferative role of exogenous neuropeptide Y (NPY), on dentate progenitors in the early phases of neurodegeneration. To investigate the functional integration of newly-born neurons, here we studied in adult rats the long-term effects of intracerebroventricular administration of NPY (2 µg/2 µl, 4 days after TMT-treatment), which plays an adjuvant role in neurodegeneration and epilepsy. Our results indicate that 30 days after NPY administration the number of new neurons was still higher in TMT+NPY-treated rats than in control+saline group. As a functional correlate of the integration of new neurons into the hippocampal network, long-term potentiation recorded in Dentate Gyrus (DG) in the absence of GABAA receptor blockade was higher in the TMT+NPY-treated group than in all other groups. Furthermore, qPCR analysis of Kruppel-like factor 9, a transcription factor essential for late-phase maturation of neurons in the DG, and of the cyclin-dependent kinase 5, critically involved in the maturation and dendrite extension of newly-born neurons, revealed a significant up-regulation of both genes in TMT+NPY-treated rats compared with all other groups. To explore the early molecular events activated by NPY administration, the Sonic Hedgehog (Shh) signalling pathway, which participates in the maintenance of the neurogenic hippocampal niche, was evaluated by qPCR 1, 3 and 5 days after NPY-treatment. An early significant up-regulation of Shh expression was detected in TMT+NPY-treated rats compared with all other groups, associated with a modulation of downstream genes. Our data indicate that the neurogenic effect of NPY administration during TMT-induced neurodegeneration involves early Shh pathway activation and results in a functional integration of newly-generated neurons into the local circuit.