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Glial activation with concurrent up-regulation of inflammatory mediators in trimethyltin-induced neurotoxicity in mice
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Glial activation with concurrent up-regulation of inflammatory mediators in trimethyltin-induced neurotoxicity in mice Juhwan Kim a,b,1 , Miyoung Yang a,1 , Yeonghoon Son a , Hyosun Jang a , Dongwoo Kim a , Jong-Choon Kim a , Sung-Ho Kim a , Man-Jong Kang c , Heh-In Im b , Taekyun Shin d,∗ , Changjong Moon a,∗ a Department of Veterinary Anatomy, College of Veterinary Medicine and Animal Medical Institute, Chonnam National University, Gwangju 500-757, South Korea b Center for Neuroscience, Korea Institute of Science and Technology (KIST), Seoul 136-791, South Korea c Department of Animal Science, College of Agriculture and Life Science, Chonnam National University, Gwangju 500-757, South Korea d Department of Veterinary Anatomy, College of Veterinary Medicine, Jeju National University, Jeju 690-756, South Korea
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Article history: Available online xxx Keywords: Trimethyltin Hippocampus Neuroinflammation Glial activation Cytokine
a b s t r a c t 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]1, IL-6 and tumor necrosis factor [TNF]-␣) were significantly increased in the mouse hippocampus following TMT treatment. In BV-2 microglia, iNOS, IL-1, TNF-␣, 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-1, TNF-␣, 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. © 2014 Elsevier GmbH. All rights reserved.
Introduction Organotin compounds are used in a wide range of industrial applications as plastic stabilizers owing to their stability and transparency (Smitiene et al., 2014). Among the organotin compounds, trimeththyltin (TMT) has been considered as the main contributor for acute neurotoxic poisoning (Tang et al., 2013). TMT induces specific neuronal cell loss in the limbic system of the brain (Walsh et al., 1982). TMT intoxication is accompanied by a syndrome characterized by cognitive and mood dysfunction, seizures, nystagmus, and ataxia (Geloso et al., 2011). Mice exposed to TMT showed
∗ Corresponding authors at: College of Veterinary Medicine, Chonnam National University, 300 Yongbong-Dong, Buk-Gu, Gwangju 500-757, South Korea. E-mail addresses: [email protected] (T. Shin), [email protected] (C. Moon). 1 These authors equally contributed to this work.
characteristic clinical symptoms, such as seizure/tremor and cognitive dysfunction, accompanied by neuronal degeneration in the hippocampus (Kim et al., 2013b; Yang et al., 2012). Thus, TMT has been regarded as a useful tool for the study of chemically induced neurodegeneration and neurobehavioral impairment in experimental animals (Geloso et al., 2011; Ishikawa et al., 1997). However, the precise mechanism of neuronal cell death in TMTinduced neurotoxicity is not fully understood. Neuroinflammation plays a major role in diverse neurodegenerative disorders, including Alzheimer’s disease, prion disease, and stroke (Ceulemans et al., 2010; Eikelenboom et al., 2002). The inflammatory response in the central nervous system (CNS) is characterized by astroglial and microglial activation, and these processes are known to play a role in neurodegenerative diseases (Rathke-Hartlieb et al., 1999). Activated macrophages/microglia can be classified into two subtypes: classically activated (M1) macrophages/microglia and alternatively
http://dx.doi.org/10.1016/j.acthis.2014.09.003 0065-1281/© 2014 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Kim J, et al. Glial activation with concurrent up-regulation of inflammatory mediators in trimethyltininduced neurotoxicity in mice. Acta Histochemica (2014), http://dx.doi.org/10.1016/j.acthis.2014.09.003
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activated (M2) macrophages/microglia (Gordon, 2003). M1 macrophages/microglia are activated by T-helper-1-type lymphocytes, natural killer cells, and interferon-␥, and are characterized by production of high levels of pro-inflammatory cytokines, nitric oxide (NO), and reactive oxygen species (ROS) (Kigerl et al., 2009; Perego et al., 2011). Thus, M1 macrophages/microglia strongly induce neuroinflammation and thereby mediate detrimental effects on neuronal cells (Mantovani et al., 2002). In contrast, M2 macrophages/microglia, activated by IL-4 and IL-13, may resolve inflammation and facilitate wound healing (Gordon, 2003). Chemically induced neurodegeneration is usually accompanied by an inflammatory reaction, as indicated by elevated levels of inflammatory cytokines (Jung et al., 2006; Rizzi et al., 2003), and glial activation, as indicated by an increase in the number and size of microglial and astroglial cells (Cabrera et al., 2013; Lee et al., 2014; Yang et al., 2012). Furthermore, pro-inflammatory cytokines derived from activated glial cells in various neurodegenerative conditions may contribute toward brain dysfunction, including decreased hippocampal neurogenesis and the onset of depression (Monje et al., 2003; Nelson et al., 2012; Raison et al., 2006). However, the dynamics of inflammatory cytokine expression and glial activation in the hippocampus after TMT treatment have not been definitively established. In the present study, we examined temporal changes in glial activation and mRNA expression of inflammatory signals, including inflammatory cytokines and markers for M1 and M2 microglia, in the mouse hippocampus after TMT treatment to elucidate the involvement of inflammatory processing in chemically induced neurodegeneration.
Fluoro-Jade C staining and semi-quantitative analysis Staining with Fluoro-Jade C (FJC; Millipore, Billerica, MA, USA), a high-affinity fluorescence marker for the localization of neuronal degeneration, was performed according to a method described previously (Schmued et al., 2005). In brief, 5-m-thick sagittal sections were transferred to a solution of 0.06% potassium permanganate and then to a 0.0004% FJC staining solution. After washing, the sections were counterstained with 0.005 mg/mL 4 ,6-diamidino-2phenylindole, 2HCl (DAPI; Thermo Fisher Scientific, Waltham, MA, USA) before being mounted. The FJC-stained sections were examined by immunofluorescence microscopy using a BX-40 microscope (Olympus, Tokyo, Japan) with an eXcope X3 digital camera (DIXI Optics, Daejeon, South Korea). The FJC intensity in the hippocampus was calculated as the mean optical density using ImageJ software (NIH, Bethesda, MD, USA). JPEG images were converted to 8-bit grey scale, and the colors were inverted. The threshold of the image was adjusted until FJC-negative cells were no longer detectable, and the same threshold was used for all images. In mice, TMT treatment causes neuronal degeneration, especially in the hippocampal dentate gyrus (DG), which is characterized by localized dentate granule cell death with sparing of Cornu ammonis (CA) pyramidal cells (Fiedorowicz et al., 2001; Kim et al., 2013b; Lee et al., 2014). Thus, areas of analysis containing the DG of the sagittal brain sections (at ∼1.44 mm lateral to the medial border of the brain) were outlined individually and measured using the freehand selection tool. The pixel intensities for each section were summed and divided by the selected area to obtain the mean FJC intensity. Immunohistochemistry
Materials and methods Animals Male C57BL/6 mice at 8 weeks of age were obtained from a specific pathogen-free colony at Daehan Biolink (Daejeon, Korea). Procedures for the care and handling of animals conformed to current international laws and policies (NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85-23, 1985, revised 1996). The Institutional Animal Care and Use Committee of Chonnam National University (Gwangju, Korea) approved the protocols used in this study (CNU IACUC-YB-2012-18). All of the experiments minimized the number of animals used and any suffering caused by the procedures. TMT treatment and tissue sampling TMT hydroxide (Wako, Osaka, Japan) was dissolved in sterile 0.9% saline immediately before use. Mice received a single intraperitoneal (i.p.) injection of 2.6 mg/kg TMT or vehicle (0.9% saline), and seizure/tremor activity was observed for 1–8 days after injection (n = 10 mice/group). Tremor/seizure tests were performed in brightly lit areas (40 × 40 cm, 250 lx). Behavioral changes were scored as follows: (1) aggression; (2) weak tremor; (3) systemic tremor; (4) tremor and spasmodic gait; and (5) death (Yang et al., 2012; Yoneyama et al., 2008). For the preparation of protein and RNA extracts, six mice per group were sacrificed at 1, 2, 4, and 8 days after TMT administration, and the hippocampi were dissected and stored at −80 ◦ C. For histological and Western blot analysis, brains were processed for paraffin wax embedding after fixation in 4% paraformaldehyde in phosphate buffered saline (PBS, pH 7.4) using routine protocols (n = 3 mice/group) and at 1, 2, 4, and 8 days after TMT administration.
Sagittal 5 m thick brain sections were cut using a rotary microtome (Leica 820; Leica Microsystems, Wetzlar, Germany). The brains were sectioned laterally at approximately 1.44 mm. For immunohistochemistry, the endogenous peroxidase in sagittal sections was deactivated with 0.03% hydrogen peroxide in distilled water and then sections were blocked with 10% normal goat serum (Vector Laboratories, Burlingame, CA, USA) in PBS with 0.2% Tween 20 (PBS-T). Sections were then incubated overnight at 4 ◦ C with primary antibodies, including polyclonal rabbit anti-ionized calcium binding adaptor molecule 1 (Iba1; 1:1000 dilution, Wako) and anti-glial fibrillary acidic protein (GFAP; 1:2000 dilution, Dako, Glostrup, Denmark) in PBS-T. After three washes, the sections were incubated with biotinylated goat anti-rabbit IgG (Vector ABC Elite Kit) for 1 h at room temperature (RT). After three washes, the sections were incubated for 1 h at RT with an avidin–biotin peroxidase complex prepared according to the manufacturer’s instructions (Vector ABC Elite Kit). After three washes, the peroxidase reaction was developed for 3 min using a diaminobenzidine substrate prepared according to the manufacturer’s instructions (DAB kit; Vector Laboratories). Each experiment included sections from which primary antibodies were omitted, as a negative control (data not shown). The stained specimens were visualized using a BX-40 microscope (Olympus) with an eXcope X3 digital camera (DIXI Optics). Western blot analysis Mouse hippocampi were individually immersed immediately in buffer H (50 mM -glycerophosphate, 1.5 mM ethylene glycol tetraacetic acid, 0.1 mM Na3 VO4 , 1 mM dithiothreitol, 10 g/mL aprotinin, 2 g/mL pepstatin, 10 g/mL leupeptin, 1 mM phenylmethanesulfonylfluoride, pH 7.4), and sonicated for 10 s. SDS sample buffer (×4) was added to each homogenized sample, and the samples were heated at 100 ◦ C for 10 min. The
Please cite this article in press as: Kim J, et al. Glial activation with concurrent up-regulation of inflammatory mediators in trimethyltininduced neurotoxicity in mice. Acta Histochemica (2014), http://dx.doi.org/10.1016/j.acthis.2014.09.003
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samples were then separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a nitrocellulose membrane. Membranes were blocked in 1% normal goat serum and 0.5% bovine serum albumin in PBS-T for 1 h at RT and incubated in primary antibody against polyclonal rabbit anti-Iba1 (1:1000 dilution) and monoclonal mouse anti-GFAP (1:1000 dilution, Sigma-Aldrich, St. Louis, MO, USA) in PBS-T overnight at 4 ◦ C. After extensive washing and subsequent incubation with either a horseradish peroxidase (HRP)-conjugated antirabbit or anti-mouse secondary antibody (1:10,000 dilution; Thermo Fisher Scientific, Waltham, MA, USA), signals were visualized using a chemiluminescence kit (SuperSignal West Pico; Thermo Fisher Scientific). For the normalization of Iba1 and GFAP expression, the membranes were re-probed with monoclonal mouse antibody to -actin (1:20,000 dilution; Sigma-Aldrich). Several exposure times were used to obtain signals in the linear range. The bands were quantified using ImageJ software (NIH).
Cell culture
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at a ratio of 1:4 onto six-well-plates and grown to 80% confluence prior to use. To examine concentration-dependent effects, we used 0, 1, 5, 10 M of TMT and harvested 3, 6 and 12 h after TMT treatment to examine time-dependent effects in BV-2 cells and astrocytes. RNA extraction and cDNA synthesis Total RNA was isolated from hippocampal tissue samples and cultured BV2 and astroglial cells using a RNAeasy® Lipid Tissue Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The concentration of RNA samples was ascertained by measuring optical density using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). Then, firststrand complementary DNA (cDNA) was prepared using random primers (Takara Bio, Tokyo, Japan) with Superscript II reverse transcriptase (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. cDNA was diluted to 8 ng/L with RNase-free water and stored at −80 ◦ C. Real-time reverse transcription PCR analysis
The mouse microglial cell line BV-2 was a generous gift of Dr. Jung (Advanced Radiation Technology Institute, KAERI, Jeongeup, Korea). Cells were plated onto 6-well plates and maintained in Dulbecco’s modified medium (DMEM; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 g/mL streptomycin in a 37 ◦ C humidified CO2 incubator (5% CO2 and 95% air). The cells were grown to about 80% confluence prior to use. Primary astrocytes cultures were prepared from 2-day-old C57BL/6 mice pups by modification of previously described methods (McCarthy and De Vellis, 1980; Wormser et al., 2012). Briefly, after removal of the meninges, the cerebral cortices from the mice were transferred into a Petri dish containing DMEM/F-12 (Invitrogen), chopped, and digested with 10 units/mL papain (Worthington, Freehold, NJ, USA) and 1000 units/mL DNase I (Roche, Basel, Switzerland) in DMEM/F-12 at 37 ◦ C for 25 min. The cell suspension was triturated and transferred to T75 culture flasks at a density of 100,000 cells/mL in 10 mL culture medium per flask (DMEM/F12 with 10% fetal bovine serum, 100 U/mL penicillin, and 100 g/mL streptomycin). Cultures were maintained in a 37 ◦ C humidified 5% CO2 incubator. The culture medium was changed within 24 h and then twice a week until the cells became confluent. At this time, the flasks were shaken overnight at 150 rpm to remove microglia and oligodendrocytes. Astrocytes were washed three times with PBS and incubated with trypsin/EDTA (0.05% trypsin, 0.53 mM EDTA) for 8 min at 37 ◦ C. The detached cells were replated
The primer sequences are described in Table 1. Real-time PCR amplification was performed using TOPrealTM qPCR 2X PreMIX (Enzynomics, Daejeon, Korea) on a Stratagene MX3000P instrument (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions. The thermal cycling profile consisted of a pre-incubation step at 94 ◦ C for 10 min, followed by 45 cycles of denaturation (94 ◦ C, 15 s), annealing (55–57 ◦ C, 30 s), and elongation (72 ◦ C, 20 s). A melt curve was generated to verify that only one product was amplified. Amplification curves from each of the real-time PCR reactions were generated within the software, and threshold cycle values were determined. GAPDH was used as a housekeeping gene for normalization to an internal control for each sample. Results were expressed as a mean fold change over the vehicle-treated control for each transcript using the 2−CT method (Kim et al., 2014; Livak and Schmittgen, 2001). Statistical analysis The data are reported as means ± standard error (SE). A one-way analysis of variance (ANOVA) was used for analysis of FJC-positive cell counting, Western blots, and gene expression after TMT treatment in vivo. A two-way ANOVA was used to establish the effects of time (3, 6, 12 h after TMT treatment) and concentration (0, 1, 5, 10 M) and the interactions between them. Holm–Sidak’s post hoc test was used to test for multiple comparisons when warranted by
Table 1 Primer sequences for quantitative real-time PCR analysis. Gene
Accession number
Primer sequence
iNOS
NM 010927.2
CD206
NM 008625.1
Arginase-1
NM 007482.2
IL-1
NM 008361.3
TNF-␣
NM 013693.2
IL-6
NM 031168.1
IL-10
NM 010548.2
GAPDH
NM 008084.2
Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse
5 -TTGAAATCCCTCCTGATCTTGT-3 5 -RTCACAGAAGTCTCGAACTCCAA-3 5 -ACGACAATCCTGTCTCCTTTGT-3 5 -TCAGCTTTGGTTGTAATGGATG-3 5 -TGAAAGGAAAGTTCCCAGATGT-3 5 -TGAAGGTCTCTTCCATCACCTT-3 5 -CTCGCAGCAGCACATCAACAAG-3 5 -CCACGGGAAAGACACAGGTAGC-3 5 -CATCTTCTCAAAATTCGAGTGACAA-3 5 -TGGGAGTAGACAAGGTACAACCC-3 5 -TGGAGTCACAGAAGGAGTGGCTAAG-3 5 -TCTGACCACAGTGAGGAATGTCCAC-3 5 -ACCTGGTAGAAGTGATGCCC-3 5 -ACACCTTGGTCTTGGAGCTT-3 5 -TCCATGACAACTTTGGCATT-3 5 -GTTGCTGTTGAAGTCGCAGG-3
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Table 2 Results of two-way ANOVA testing for the effects of concentration and time on the TMT-induced change in mRNA expression in each dependent variable. Gene Microglia iNOS CD206 Arginase-1 IL-1 TNF-␣ IL-6 IL-10 Astrocytes iNOS Arginase-1 IL-1 TNF-␣ IL-6 IL-10
Concentration
Time
Interaction
F (3, 60) = 16.20, p < 0.0001 F (3, 60) = 23.68, p < 0.0001 F (3, 60) = 60.23, p < 0.0001 F (3, 60) = 5.560, p = 0.0020 F (3, 60) = 23.32, p < 0.0001 F (3, 60) = 6.447, p = 0.0007 F (3, 60) = 2.010, p = 0.1222
F (2, 60) = 11.87, p < 0.0001 F (2, 60) = 18.14, p < 0.0001 F (2, 60) = 5.649, p = 0.0057 F (2, 60) = 9.204, p = 0.0003 F (2, 60) = 8.435, p = 0.0006 F (2, 60) = 2.191, p = 0.1207 F (2, 60) = 4.058, p = 0.0222
F (6, 60) = 8.304, p < 0.0001 F (6, 60) = 3.408, p = 0.0058 F (6, 60) = 2.907, p = 0.0148 F (6, 60) = 1.797, p = 0.1149 F (6, 60) = 4.406, p = 0.0009 F (6, 60) = 0.4861, p = 0.8162 F (6, 60) = 0.7182, p = 0.6365
F (3, 60) = 10.99, p < 0.0001 F (3, 60) = 19.12, p < 0.0001 F (3, 60) = 89.41, p < 0.0001 F (3, 60) = 35.73, p < 0.0001 F (3, 60) = 27.88, p < 0.0001 F (3, 60) = 6.375, p = 0.0008
F (2, 60) = 3.842, p = 0.0269 F (2, 60) = 71.30, p < 0.0001 F (2, 60) = 4.853, p = 0.0111 F (2, 60) = 4.407, p = 0.0164 F (2, 60) = 5.636, p = 0.0057 F (2, 60) = 3.779, p = 0.0284
F (6, 60) = 2.578, p = 0.0274 F (6, 60) = 12.15, p < 0.0001 F (6, 60) = 5.281, p = 0.0002 F (6, 60) = 1.882, p = 0.0988 F (6, 60) = 3.913, p = 0.0023 F (6, 60) = 2.383, p = 0.0394
a significant omnibus F-statistic. The ANOVA results are provided in Table 2, and the results of the test for multiple comparisons are provided in the relevant figures. In all analyses, a p-value of less than 0.05 was deemed to indicate statistical significance. All statistical analyses were conducted using the GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA).
Results Evaluation of TMT-induced seizure/tremor with hippocampal neurodegeneration As shown in Fig. 1A, we assessed and recorded clinical symptoms daily for 8 days after treatment with 2.6 mg/kg TMT. Mice exhibited hypersensitivity accompanied by seizure behavior from days 1 to 5 post-treatment, and recovered from seizure/tremor at day 6 posttreatment (n = 10 for each group). We also performed histopathological analyses to confirm that the clinical symptoms induced by TMT treatment were accompanied by neurodegeneration in the hippocampus, particularly in the DG. Extensive neuronal cell death characterized by eosinophilic cytoplasm, nuclear pyknosis, and karyolysis was detected in the granular cell layer of the DG under high magnification posttreatment (Fig. 1B, left panels). We performed further FJC staining to quantify TMT-induced neurodegeneration in the hippocampal DG post-treatment (n = 3 for each group). Semi-quantitative analysis of the FJC-positive intensity showed that TMT treatment resulted in a significant increase of degenerative neurons in the hippocampal DG (Fig. 1B and C).
Microglial and astroglial activation in the hippocampus after TMT treatment To test whether TMT treatment induced microglial and astroglial activation, we evaluated the expression of Iba-1, a microglia marker, and GFAP, an astrocyte marker, in the hippocampus post-treatment (n = 3 mice at each time point). The levels of Iba-1 expression increased significantly in the hippocampal DG of TMT-treated mice at 4 days post-treatment (Fig. 2A and B). Activated microglia of amoeboid morphology with thickened and less ramified processes were extensively shown in the hippocampus after TMT treatment (Fig. 2D, upper panels). The levels of GFAP expression gradually increased in the hippocampus of TMT-treated mice until 8 days post-treatment (Fig. 2A and C). After TMT treatment, GFAP-positive hypertrophied astrocytes were found extensively in the hippocampus (Fig. 2D, lower panels).
Expression of iNOS, CD206, arginase-1 and pro-inflammatory cytokine mRNA in the hippocampus To assess the activation of microglia after TMT treatment, we examined the mRNA expression levels of the M1 (classically activated) microglia marker inducible nitric oxide synthase (iNOS) and the M2 (alternatively activated) microglia markers CD206 and arginase-1 in the hippocampus using quantitative real-time PCR. The levels of both iNOS (Fig. 3A) and CD206 mRNA (Fig. 3B) were significantly increased in the hippocampus at 2 days post-treatment. However, there was no significant change in arginase-1 mRNA expression in the hippocampus after TMT treatment, although the expression peaked 2 days post-treatment (Fig. 3C). We also examined inflammatory cytokine mRNA expression in the mouse hippocampus, to establish whether glial activation in the hippocampus correlates with inflammatory cytokine expression. The expression levels of interleukin (IL)-1 (Fig. 3D), tumor necrosis factor (TNF)-␣ (Fig. 3E), and IL-6 mRNA (Fig. 3F) were significantly up-regulated at 2 days, 1–2 days, and 2 days post-treatment, respectively. However, TMT treatment did not induce a significant change in IL-10 mRNA expression, although the expression peaked at 1 day post-treatment (Fig. 3G). This suggests that TMT treatment induces a robust increase in the expression of the pro-inflammatory cytokines IL-1, TNF-␣, and IL-6, but not the anti-inflammatory cytokine IL-10 in the hippocampus.
Polarization and up-regulation of pro-inflammatory cytokine mRNA expression in BV-2 microglia after TMT treatment To examine the microglia subsets in BV-2 microglia after TMT treatment, we examined mRNA expression of iNOS, CD206, and arginase-1 using quantitative real-time PCR. Expression of iNOS was significantly increased after TMT treatment in a timedependent manner at 10 M of TMT concentration (Fig. 4A). In contrast, expressions of CD 206 (Fig. 4B) and arginase-1 (Fig. 4C) were significantly decreased after TMT treatment in time- and concentration-dependent manner. These results suggest that TMT toxicity leads to microglia polarization toward the M1 phenotype in BV-2 microglia. We also determined whether TMT treatment increased expression of inflammatory cytokine mRNA in BV-2 microglia. Expression of IL-1 (Fig. 4D), TNF-␣ (Fig. 4E), and IL-6 (Fig. 4F) was upregulated at 6–12 h, 3–12 h, and 6 h post-treatment, respectively. However, TMT treatment did not induce a significant change in IL10 mRNA expression (Fig. 4G). These results were consistent with our in vivo results; TMT treatment increases the mRNA expression
Please cite this article in press as: Kim J, et al. Glial activation with concurrent up-regulation of inflammatory mediators in trimethyltininduced neurotoxicity in mice. Acta Histochemica (2014), http://dx.doi.org/10.1016/j.acthis.2014.09.003
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Fig. 1. Clinical symptoms and histopathological examination of mice after trimethyltin (TMT) treatment. (A) Clinical scores of seizure behaviors in mice following TMT treatment based on a 0–5 scale. Animals were sacrificed at each time-point (1, 2, 4, and 8 days after treatment; arrows, n = 10 per group). (B) Representative microphotographs of hematoxylin and eosin (H&E) and Fluoro-Jade C (FJC) staining in the dentate gyrus (DG) of the hippocampus. Scale bars = 50 m. (C) Semi-quantitative analysis of FJCpositive cells in the DG of the hippocampus (n = 3 per group). The data are reported as means ± SE. * p < 0.05, *** p < 0.001; these values indicate statistically significant differences compared to vehicle-treated controls.
of the pro-inflammatory cytokines IL-1, TNF-␣, and IL-6, but not the anti-inflammatory cytokine IL-10.
Induction of iNOS, arginase-1 and pro-inflammatory cytokine mRNA expression in the primary astrocyte cultures after TMT treatment Because we observed contrasting mRNA expression patterns of microglia markers in the hippocampus and in BV-2 cells, we hypothesized that the markers were non-specific and being expressed by additional cell types. Therefore, we examined the expression of microglia markers in primary astrocyte cultures. The expression of iNOS mRNA was increased significantly at 6–12 h post-treatment (Fig. 5A). The expression of arginase1 mRNA was increased significantly at 3 h post-treatment, but decreased significantly at 6–12 h post-treatment following a high concentration of TMT (Fig. 5B). These results suggest the TMT treatment also induces changes in iNOS and arginase-1 expressions in astrocytes. Additionally, we investigated whether TMT treatment increases the expression of inflammatory cytokines in primary astrocyte cultures. IL-1 (Fig. 5C), TNF-␣ (Fig. 5D), and IL-6 (Fig. 5E) mRNA transcript levels were up-regulated at
3–12 h post-treatment, respectively, whereas expression of IL10 mRNA was significantly decreased at 12 h post-treatment, also in concentration-dependent manner (Fig. 5F). These results suggest that TMT treatment increases the expression of the proinflammatory cytokines IL-1, TNF-␣, and IL-6, but decreases the expression of the anti-inflammatory cytokine IL-10, in astrocytes.
Discussion The present study demonstrated that TMT-induced neurodegeneration is closely related to neuroinflammation in the hippocampus. Furthermore, this study is the first to provide complementary in vivo and in vitro evidence concerning the possible roles of activated glial cells in inflammatory cytokine expression in the mouse hippocampus following TMT treatment. Our in vitro study demonstrates that TMT increases the expression of pro-inflammatory cytokines. However, in the mouse hippocampus in vivo, TMT treatment increases simultaneously the expression of both pro- and anti-inflammatory mediators. This is accompanied by activation of microglia, by both classical and alternative pathways, and astroglia.
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Fig. 2. TMT treatment induced microglia and astrocyte activation in the mouse hippocampus. (A–C) TMT treatment induced a significant increase in the expression of a microglial marker (Iba-1) and an astroglial marker (GFAP) in the hippocampus. (D) Representative images of Iba-1-positive microglia and GFAP-positive astrocytes in the DG of adult hippocampus 4 days post-treatment. Scale bars = 150 m. The data are reported as means ± SE (n = 6 per group). ** p < 0.01, *** p < 0.001; these values indicate statistically significant differences compared to vehicle-treated controls.
Neuroinflammation, represented by neuronal cell death, glial cell activation, and up-regulation of pro-inflammatory cytokines, such as TNF-␣ and IL-1, is a prominent pathological feature of several neurodegenerative diseases (Cacabelos et al., 1994; Chapman et al., 2006). Previous studies show that TMT treatment induces selective neuronal cell death and enhances microglial and astroglial activation in the mouse hippocampal DG, accompanied by clinical symptoms, such as seizure/tremor and impaired learning and memory (Kim et al., 2013b; Yang et al., 2012). In the present study, we confirmed that a dose of 2.6 mg/kg TMT was sufficient to induce seizure/tremor concurrent with extensive neuronal cell death in the hippocampal DG within 4 days post-treatment. We also observed early activation of hypertrophied microglia clustered around damaged neurons in the hippocampal DG from post-treatment days 2–4 (the acute damage period). Furthermore, a significant increase in GFAP expression on post-treatment 4–8 days (the chronic recovery period) indicates that TMT may induce delayed astroglial activation throughout the hippocampus. These results confirm that TMT treatment induces selective neuronal cell death and glial activation in the mouse hippocampus. In addition, the dynamic changes in microglial and astroglial activation may play detrimental (e.g., induction of inflammation and phagocytosis) and/or beneficial (e.g., wound healing and resolution of inflammation) roles in the hippocampus under neurotoxic conditions. TMT-induced glial activation is largely associated with the upregulation of pro-inflammatory cytokines, suggesting that these
cytokines may act as modulators in several neurodegenerative models (Allan and Rothwell, 2001; Harry et al., 2002; Maier et al., 1995). However, as described earlier, there are conflicting reports regarding the expression of pro-inflammatory cytokines in the hippocampus during TMT toxicity. For example, Little et al. (2002) reported that TMT does not increase expression of proinflammatory cytokines in the rat hippocampus, because TMT causes neurodegeneration without disrupting the blood–brain barrier (BBB), which is thought to be the origin of cytokines from the periphery (Little et al., 2012). In contrast, Harry et al. (2008) reported early expression of pro-inflammatory cytokine mRNA in the mouse hippocampus at 1–2 days after TMT treatment. In the present study, we demonstrate that TMT induces an early increase in mRNA expression of the pro-inflammatory cytokines IL-1, IL-6, and TNF-␣. Furthermore, our preliminary study revealed no change in the plasma levels of inflammatory cytokines during TMT intoxication (data not shown). This is also supported by the observation that TMT-induced neurodegeneration is not accompanied by a breakdown of the BBB, and that blood–borne macrophages might not be recruited into the hippocampus (McCann et al., 1996). Therefore, we conclude that TMT might induce selective hippocampal cell death and activation of endogenous glial cells by directly stimulating the release of pro-inflammatory cytokines within the hippocampus. When interpreting these conflicting results, it is important to consider the different experimental conditions, such as the animal model used,
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Fig. 3. TMT treatment increased the gene expression of M1/M2 markers and inflammatory cytokines in the mouse hippocampus. A M1 microglia marker (A, iNOS) and M2 microglia markers (B, CD206; C, arginase-1) were examined in the mouse hippocampus. Expression of IL-1 (D), TNF-␣ (E), IL-6 (F) was significantly increased at 1–4 days post-treatment. (G) No significant change in IL-10 expression was observed after TMT treatment. The data are reported as means ± SE (n = 6 per group). * p < 0.05, ** p < 0.01, *** p < 0.001; these values indicate statistically significant differences compared to vehicle-treated controls.
route of administration of TMT, and methods for cytokine detection. Previous studies show that enhanced glial activation is a consequence of TMT intoxication (Fiedorowicz et al., 2001; Yang et al., 2012). However, the detrimental or beneficial role of the microglial response during TMT-induced neurodegeneration has not been fully elucidated. In an attempt to clarify the role of microglial activation in TMT intoxication, we classified microglia into M1 and M2 phenotypes using subset-specific markers. Although this classification system can be considered an oversimplification of the complex microglial response spectrum, it has proved to be a useful method for defining the microglial response to neurotoxic events (Chan et al., 2008; Kigerl et al., 2009; Mikita et al., 2011). In the present study, in vitro data demonstrate that TMT treatment significantly increases expression of the M1 markers TNF-␣, IL-1, IL-6 and iNOS and significantly decreases the expression of the M2 markers CD206 and arginase-1 in BV-2 microglia. This result indicates that TMT treatment might result in M1 polarization of BV-2 microglia. This is the first study to specifically examine M1 and M2 microglial subtypes in TMT neurotoxicity. A key
feature of polarized microglia is differential cytokine production (Mantovani et al., 2002). M1 microglia produce high levels of proinflammatory cytokines, whereas M2 microglia are characterized by the production of low levels of pro-inflammatory cytokines, and down-regulation of the inflammatory response (Kigerl et al., 2009; Mikita et al., 2011). In the present study, we observed increased mRNA expression of pro-inflammatory cytokines in BV-2 microglia and primary astrocyte cultures. Furthermore, a decrease in the mRNA encoding the anti-inflammatory cytokine IL-10 was detected in primary astrocyte cultures. Thus, our findings suggest that TMT treatment induces a shift toward the M1 microglial phenotype. This might play a detrimental role, particularly when considered in conjunction with the observed up-regulation of pro-inflammatory cytokines and astrocytic activation. However, contrary to our results in BV-2 microglia, TMT treatment significantly increases the expression of both iNOS and CD206 mRNA in the hippocampus in vivo. In addition, the expression of arginase-1 is increased at 2 days post-treatment, although this effect is not statistically significant. This suggests that there might be other factors (i.e., iNOS and arginase-1 derived from neurons and astrocytes) involved
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Fig. 4. TMT treatment induced changes in gene expression levels of M1/M2 markers and inflammatory cytokines in BV-2 microglia. (A) iNOS. (B) CD206. (C) arginase-1. (D) IL-1. (E) TNF-␣. (F) IL-6. (G) IL-10. The data are reported as means ± SE (n = 6 wells per condition). * p < 0.05, ** p < 0.01 and *** p < 0.001; these values indicate statistically significant differences in mRNA levels between TMT-treated BV-2 cells (1, 5, 10 M) and vehicle-treated controls at each time point. §§ p < 0.01 and §§§ p < 0.001 indicate statistically significant differences between TMT-treated BV-2 cells at 6 h post-treatment and TMT-treated BV-2 cells at 3 h post-treatment. † p < 0.05 and ††† p < 0.001; these values indicate statistically significant differences between TMT-treated BV-2 cells at 12 h post-treatment and TMT-treated BV-2 cells at 3 h post-treatment. ¶ p < 0.05, ¶¶ p < 0.01 and ¶¶¶ p < 0.001; these values indicate statistically significant differences between TMT-treated BV-2 cells at 12 h post-treatment and TMT-treated BV-2 cells at 6 h post-treatment.
in the expression of iNOS and arginase-1 in the hippocampus during TMT neurotoxicity. Therefore, we also examined the expression of iNOS and arginase-1 mRNA in primary astrocyte cultures. A high concentration of TMT induced an increase in arginase-1 expression at 3 h post-treatment, and a decrease in arginase-1 expression at 6–12 h post-treatment, in primary astrocyte cultures. Thus, our in vitro study suggests that astrocytes may be partly responsible for the increase in both iNOS and arginase-1 mRNA expression in the hippocampus after TMT treatment. Moreover, since our previous studies have shown that changes in arignase1 expression may influence cell survival, the increased expression
of arginase-1 at 3 h post-treatment suggests that activated astrocytes may have a protective function against neurotoxicity, and the decreased expression of arginase-1 at 6–12 h post-treatment may be indicative of a loss of this function (Ahn et al., 2012; Kim et al., 2013a). Our data therefore suggest that the activation of microglia by both classical and alternative mechanisms is indicative of the existence of concurrent protective (e.g., wound healing and resolution of inflammation) and detrimental (e.g., induction of inflammation and phagocytosis) mechanisms in vivo under neurotoxic conditions (Colton et al., 2006; Ohtaki et al., 2008; Perego et al., 2011). However, owing to the complexity of microglial and
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Fig. 5. TMT treatment induced changes in levels of iNOS, arginase-1 and inflammatory cytokine mRNA in primary astrocyte cultures. (A) iNOS. (B) arginase-1. (C) IL-1. (D) TNF-␣. (E) IL-6. (F) IL-10. The data are reported as means ± SE (n = 6 wells per condition). * p < 0.05, ** p < 0.01 and *** p < 0.001; these values indicate statistically significant differences in mRNA levels between TMT-treated astrocytes (1, 5, 10 M) and vehicle-treated controls at each time point. §§ p < 0.01 and §§§ p < 0.001 indicates statistically significant differences between TMT-treated astrocytes at 6 h post-treatment and TMT-treated astrocytes at 3 h post-treatment. † p < 0.05, †† p < 0.01 and ††† p < 0.001; these values indicate statistically significant differences between TMT-treated astrocytes at 12 h post-treatment and TMT-treated astrocytes at 3 h post-treatment. ¶ p < 0.05 and ¶¶¶ p < 0.001; these values indicate statistically significant differences between TMT-treated astrocytes at 12 h post-treatment and TMT-treated astrocytes at 6 h post-treatment.
astrocytic responses in diverse environments, determining the exact role of M1 and M2 microglia and reactive astrocytes in chemical-neurotoxicity requires further investigation. In summary, TMT treatment induces neuronal cell death in conjunction with microglial and astroglial cell activation in the mouse hippocampus. TMT also up-regulates mRNA expression of proinflammatory cytokines in activated glial cells, both in vivo and in vitro. Thus, TMT-induced neurodegeneration is closely linked to the inflammatory reaction in response to dynamic changes in inflammatory signals in vivo and in vitro. These results may contribute toward the establishment of an experimental model of chemically induced neuroinflammation. Acknowledgments This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF2012R1A1B4001262). The animal experiment in this study was
supported by the Animal Medical Institute of Chonnam National University.
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Glial activation with concurrent up-regulation of inflammatory mediators in trimethyltin-induced neurotoxicity in mice Juhwan Kim a,b,1 , Miyoung Yang a,1 , Yeonghoon Son a , Hyosun Jang a , Dongwoo Kim a , Jong-Choon Kim a , Sung-Ho Kim a , Man-Jong Kang c , Heh-In Im b , Taekyun Shin d,∗ , Changjong Moon a,∗ a Department of Veterinary Anatomy, College of Veterinary Medicine and Animal Medical Institute, Chonnam National University, Gwangju 500-757, South Korea b Center for Neuroscience, Korea Institute of Science and Technology (KIST), Seoul 136-791, South Korea c Department of Animal Science, College of Agriculture and Life Science, Chonnam National University, Gwangju 500-757, South Korea d Department of Veterinary Anatomy, College of Veterinary Medicine, Jeju National University, Jeju 690-756, South Korea
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Article history: Available online xxx Keywords: Trimethyltin Hippocampus Neuroinflammation Glial activation Cytokine
a b s t r a c t 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]1, IL-6 and tumor necrosis factor [TNF]-␣) were significantly increased in the mouse hippocampus following TMT treatment. In BV-2 microglia, iNOS, IL-1, TNF-␣, 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-1, TNF-␣, 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. © 2014 Elsevier GmbH. All rights reserved.
Introduction Organotin compounds are used in a wide range of industrial applications as plastic stabilizers owing to their stability and transparency (Smitiene et al., 2014). Among the organotin compounds, trimeththyltin (TMT) has been considered as the main contributor for acute neurotoxic poisoning (Tang et al., 2013). TMT induces specific neuronal cell loss in the limbic system of the brain (Walsh et al., 1982). TMT intoxication is accompanied by a syndrome characterized by cognitive and mood dysfunction, seizures, nystagmus, and ataxia (Geloso et al., 2011). Mice exposed to TMT showed
∗ Corresponding authors at: College of Veterinary Medicine, Chonnam National University, 300 Yongbong-Dong, Buk-Gu, Gwangju 500-757, South Korea. E-mail addresses: [email protected] (T. Shin), [email protected] (C. Moon). 1 These authors equally contributed to this work.
characteristic clinical symptoms, such as seizure/tremor and cognitive dysfunction, accompanied by neuronal degeneration in the hippocampus (Kim et al., 2013b; Yang et al., 2012). Thus, TMT has been regarded as a useful tool for the study of chemically induced neurodegeneration and neurobehavioral impairment in experimental animals (Geloso et al., 2011; Ishikawa et al., 1997). However, the precise mechanism of neuronal cell death in TMTinduced neurotoxicity is not fully understood. Neuroinflammation plays a major role in diverse neurodegenerative disorders, including Alzheimer’s disease, prion disease, and stroke (Ceulemans et al., 2010; Eikelenboom et al., 2002). The inflammatory response in the central nervous system (CNS) is characterized by astroglial and microglial activation, and these processes are known to play a role in neurodegenerative diseases (Rathke-Hartlieb et al., 1999). Activated macrophages/microglia can be classified into two subtypes: classically activated (M1) macrophages/microglia and alternatively
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activated (M2) macrophages/microglia (Gordon, 2003). M1 macrophages/microglia are activated by T-helper-1-type lymphocytes, natural killer cells, and interferon-␥, and are characterized by production of high levels of pro-inflammatory cytokines, nitric oxide (NO), and reactive oxygen species (ROS) (Kigerl et al., 2009; Perego et al., 2011). Thus, M1 macrophages/microglia strongly induce neuroinflammation and thereby mediate detrimental effects on neuronal cells (Mantovani et al., 2002). In contrast, M2 macrophages/microglia, activated by IL-4 and IL-13, may resolve inflammation and facilitate wound healing (Gordon, 2003). Chemically induced neurodegeneration is usually accompanied by an inflammatory reaction, as indicated by elevated levels of inflammatory cytokines (Jung et al., 2006; Rizzi et al., 2003), and glial activation, as indicated by an increase in the number and size of microglial and astroglial cells (Cabrera et al., 2013; Lee et al., 2014; Yang et al., 2012). Furthermore, pro-inflammatory cytokines derived from activated glial cells in various neurodegenerative conditions may contribute toward brain dysfunction, including decreased hippocampal neurogenesis and the onset of depression (Monje et al., 2003; Nelson et al., 2012; Raison et al., 2006). However, the dynamics of inflammatory cytokine expression and glial activation in the hippocampus after TMT treatment have not been definitively established. In the present study, we examined temporal changes in glial activation and mRNA expression of inflammatory signals, including inflammatory cytokines and markers for M1 and M2 microglia, in the mouse hippocampus after TMT treatment to elucidate the involvement of inflammatory processing in chemically induced neurodegeneration.
Fluoro-Jade C staining and semi-quantitative analysis Staining with Fluoro-Jade C (FJC; Millipore, Billerica, MA, USA), a high-affinity fluorescence marker for the localization of neuronal degeneration, was performed according to a method described previously (Schmued et al., 2005). In brief, 5-m-thick sagittal sections were transferred to a solution of 0.06% potassium permanganate and then to a 0.0004% FJC staining solution. After washing, the sections were counterstained with 0.005 mg/mL 4 ,6-diamidino-2phenylindole, 2HCl (DAPI; Thermo Fisher Scientific, Waltham, MA, USA) before being mounted. The FJC-stained sections were examined by immunofluorescence microscopy using a BX-40 microscope (Olympus, Tokyo, Japan) with an eXcope X3 digital camera (DIXI Optics, Daejeon, South Korea). The FJC intensity in the hippocampus was calculated as the mean optical density using ImageJ software (NIH, Bethesda, MD, USA). JPEG images were converted to 8-bit grey scale, and the colors were inverted. The threshold of the image was adjusted until FJC-negative cells were no longer detectable, and the same threshold was used for all images. In mice, TMT treatment causes neuronal degeneration, especially in the hippocampal dentate gyrus (DG), which is characterized by localized dentate granule cell death with sparing of Cornu ammonis (CA) pyramidal cells (Fiedorowicz et al., 2001; Kim et al., 2013b; Lee et al., 2014). Thus, areas of analysis containing the DG of the sagittal brain sections (at ∼1.44 mm lateral to the medial border of the brain) were outlined individually and measured using the freehand selection tool. The pixel intensities for each section were summed and divided by the selected area to obtain the mean FJC intensity. Immunohistochemistry
Materials and methods Animals Male C57BL/6 mice at 8 weeks of age were obtained from a specific pathogen-free colony at Daehan Biolink (Daejeon, Korea). Procedures for the care and handling of animals conformed to current international laws and policies (NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85-23, 1985, revised 1996). The Institutional Animal Care and Use Committee of Chonnam National University (Gwangju, Korea) approved the protocols used in this study (CNU IACUC-YB-2012-18). All of the experiments minimized the number of animals used and any suffering caused by the procedures. TMT treatment and tissue sampling TMT hydroxide (Wako, Osaka, Japan) was dissolved in sterile 0.9% saline immediately before use. Mice received a single intraperitoneal (i.p.) injection of 2.6 mg/kg TMT or vehicle (0.9% saline), and seizure/tremor activity was observed for 1–8 days after injection (n = 10 mice/group). Tremor/seizure tests were performed in brightly lit areas (40 × 40 cm, 250 lx). Behavioral changes were scored as follows: (1) aggression; (2) weak tremor; (3) systemic tremor; (4) tremor and spasmodic gait; and (5) death (Yang et al., 2012; Yoneyama et al., 2008). For the preparation of protein and RNA extracts, six mice per group were sacrificed at 1, 2, 4, and 8 days after TMT administration, and the hippocampi were dissected and stored at −80 ◦ C. For histological and Western blot analysis, brains were processed for paraffin wax embedding after fixation in 4% paraformaldehyde in phosphate buffered saline (PBS, pH 7.4) using routine protocols (n = 3 mice/group) and at 1, 2, 4, and 8 days after TMT administration.
Sagittal 5 m thick brain sections were cut using a rotary microtome (Leica 820; Leica Microsystems, Wetzlar, Germany). The brains were sectioned laterally at approximately 1.44 mm. For immunohistochemistry, the endogenous peroxidase in sagittal sections was deactivated with 0.03% hydrogen peroxide in distilled water and then sections were blocked with 10% normal goat serum (Vector Laboratories, Burlingame, CA, USA) in PBS with 0.2% Tween 20 (PBS-T). Sections were then incubated overnight at 4 ◦ C with primary antibodies, including polyclonal rabbit anti-ionized calcium binding adaptor molecule 1 (Iba1; 1:1000 dilution, Wako) and anti-glial fibrillary acidic protein (GFAP; 1:2000 dilution, Dako, Glostrup, Denmark) in PBS-T. After three washes, the sections were incubated with biotinylated goat anti-rabbit IgG (Vector ABC Elite Kit) for 1 h at room temperature (RT). After three washes, the sections were incubated for 1 h at RT with an avidin–biotin peroxidase complex prepared according to the manufacturer’s instructions (Vector ABC Elite Kit). After three washes, the peroxidase reaction was developed for 3 min using a diaminobenzidine substrate prepared according to the manufacturer’s instructions (DAB kit; Vector Laboratories). Each experiment included sections from which primary antibodies were omitted, as a negative control (data not shown). The stained specimens were visualized using a BX-40 microscope (Olympus) with an eXcope X3 digital camera (DIXI Optics). Western blot analysis Mouse hippocampi were individually immersed immediately in buffer H (50 mM -glycerophosphate, 1.5 mM ethylene glycol tetraacetic acid, 0.1 mM Na3 VO4 , 1 mM dithiothreitol, 10 g/mL aprotinin, 2 g/mL pepstatin, 10 g/mL leupeptin, 1 mM phenylmethanesulfonylfluoride, pH 7.4), and sonicated for 10 s. SDS sample buffer (×4) was added to each homogenized sample, and the samples were heated at 100 ◦ C for 10 min. The
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samples were then separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a nitrocellulose membrane. Membranes were blocked in 1% normal goat serum and 0.5% bovine serum albumin in PBS-T for 1 h at RT and incubated in primary antibody against polyclonal rabbit anti-Iba1 (1:1000 dilution) and monoclonal mouse anti-GFAP (1:1000 dilution, Sigma-Aldrich, St. Louis, MO, USA) in PBS-T overnight at 4 ◦ C. After extensive washing and subsequent incubation with either a horseradish peroxidase (HRP)-conjugated antirabbit or anti-mouse secondary antibody (1:10,000 dilution; Thermo Fisher Scientific, Waltham, MA, USA), signals were visualized using a chemiluminescence kit (SuperSignal West Pico; Thermo Fisher Scientific). For the normalization of Iba1 and GFAP expression, the membranes were re-probed with monoclonal mouse antibody to -actin (1:20,000 dilution; Sigma-Aldrich). Several exposure times were used to obtain signals in the linear range. The bands were quantified using ImageJ software (NIH).
Cell culture
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at a ratio of 1:4 onto six-well-plates and grown to 80% confluence prior to use. To examine concentration-dependent effects, we used 0, 1, 5, 10 M of TMT and harvested 3, 6 and 12 h after TMT treatment to examine time-dependent effects in BV-2 cells and astrocytes. RNA extraction and cDNA synthesis Total RNA was isolated from hippocampal tissue samples and cultured BV2 and astroglial cells using a RNAeasy® Lipid Tissue Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The concentration of RNA samples was ascertained by measuring optical density using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). Then, firststrand complementary DNA (cDNA) was prepared using random primers (Takara Bio, Tokyo, Japan) with Superscript II reverse transcriptase (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. cDNA was diluted to 8 ng/L with RNase-free water and stored at −80 ◦ C. Real-time reverse transcription PCR analysis
The mouse microglial cell line BV-2 was a generous gift of Dr. Jung (Advanced Radiation Technology Institute, KAERI, Jeongeup, Korea). Cells were plated onto 6-well plates and maintained in Dulbecco’s modified medium (DMEM; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 g/mL streptomycin in a 37 ◦ C humidified CO2 incubator (5% CO2 and 95% air). The cells were grown to about 80% confluence prior to use. Primary astrocytes cultures were prepared from 2-day-old C57BL/6 mice pups by modification of previously described methods (McCarthy and De Vellis, 1980; Wormser et al., 2012). Briefly, after removal of the meninges, the cerebral cortices from the mice were transferred into a Petri dish containing DMEM/F-12 (Invitrogen), chopped, and digested with 10 units/mL papain (Worthington, Freehold, NJ, USA) and 1000 units/mL DNase I (Roche, Basel, Switzerland) in DMEM/F-12 at 37 ◦ C for 25 min. The cell suspension was triturated and transferred to T75 culture flasks at a density of 100,000 cells/mL in 10 mL culture medium per flask (DMEM/F12 with 10% fetal bovine serum, 100 U/mL penicillin, and 100 g/mL streptomycin). Cultures were maintained in a 37 ◦ C humidified 5% CO2 incubator. The culture medium was changed within 24 h and then twice a week until the cells became confluent. At this time, the flasks were shaken overnight at 150 rpm to remove microglia and oligodendrocytes. Astrocytes were washed three times with PBS and incubated with trypsin/EDTA (0.05% trypsin, 0.53 mM EDTA) for 8 min at 37 ◦ C. The detached cells were replated
The primer sequences are described in Table 1. Real-time PCR amplification was performed using TOPrealTM qPCR 2X PreMIX (Enzynomics, Daejeon, Korea) on a Stratagene MX3000P instrument (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions. The thermal cycling profile consisted of a pre-incubation step at 94 ◦ C for 10 min, followed by 45 cycles of denaturation (94 ◦ C, 15 s), annealing (55–57 ◦ C, 30 s), and elongation (72 ◦ C, 20 s). A melt curve was generated to verify that only one product was amplified. Amplification curves from each of the real-time PCR reactions were generated within the software, and threshold cycle values were determined. GAPDH was used as a housekeeping gene for normalization to an internal control for each sample. Results were expressed as a mean fold change over the vehicle-treated control for each transcript using the 2−CT method (Kim et al., 2014; Livak and Schmittgen, 2001). Statistical analysis The data are reported as means ± standard error (SE). A one-way analysis of variance (ANOVA) was used for analysis of FJC-positive cell counting, Western blots, and gene expression after TMT treatment in vivo. A two-way ANOVA was used to establish the effects of time (3, 6, 12 h after TMT treatment) and concentration (0, 1, 5, 10 M) and the interactions between them. Holm–Sidak’s post hoc test was used to test for multiple comparisons when warranted by
Table 1 Primer sequences for quantitative real-time PCR analysis. Gene
Accession number
Primer sequence
iNOS
NM 010927.2
CD206
NM 008625.1
Arginase-1
NM 007482.2
IL-1
NM 008361.3
TNF-␣
NM 013693.2
IL-6
NM 031168.1
IL-10
NM 010548.2
GAPDH
NM 008084.2
Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse
5 -TTGAAATCCCTCCTGATCTTGT-3 5 -RTCACAGAAGTCTCGAACTCCAA-3 5 -ACGACAATCCTGTCTCCTTTGT-3 5 -TCAGCTTTGGTTGTAATGGATG-3 5 -TGAAAGGAAAGTTCCCAGATGT-3 5 -TGAAGGTCTCTTCCATCACCTT-3 5 -CTCGCAGCAGCACATCAACAAG-3 5 -CCACGGGAAAGACACAGGTAGC-3 5 -CATCTTCTCAAAATTCGAGTGACAA-3 5 -TGGGAGTAGACAAGGTACAACCC-3 5 -TGGAGTCACAGAAGGAGTGGCTAAG-3 5 -TCTGACCACAGTGAGGAATGTCCAC-3 5 -ACCTGGTAGAAGTGATGCCC-3 5 -ACACCTTGGTCTTGGAGCTT-3 5 -TCCATGACAACTTTGGCATT-3 5 -GTTGCTGTTGAAGTCGCAGG-3
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Table 2 Results of two-way ANOVA testing for the effects of concentration and time on the TMT-induced change in mRNA expression in each dependent variable. Gene Microglia iNOS CD206 Arginase-1 IL-1 TNF-␣ IL-6 IL-10 Astrocytes iNOS Arginase-1 IL-1 TNF-␣ IL-6 IL-10
Concentration
Time
Interaction
F (3, 60) = 16.20, p < 0.0001 F (3, 60) = 23.68, p < 0.0001 F (3, 60) = 60.23, p < 0.0001 F (3, 60) = 5.560, p = 0.0020 F (3, 60) = 23.32, p < 0.0001 F (3, 60) = 6.447, p = 0.0007 F (3, 60) = 2.010, p = 0.1222
F (2, 60) = 11.87, p < 0.0001 F (2, 60) = 18.14, p < 0.0001 F (2, 60) = 5.649, p = 0.0057 F (2, 60) = 9.204, p = 0.0003 F (2, 60) = 8.435, p = 0.0006 F (2, 60) = 2.191, p = 0.1207 F (2, 60) = 4.058, p = 0.0222
F (6, 60) = 8.304, p < 0.0001 F (6, 60) = 3.408, p = 0.0058 F (6, 60) = 2.907, p = 0.0148 F (6, 60) = 1.797, p = 0.1149 F (6, 60) = 4.406, p = 0.0009 F (6, 60) = 0.4861, p = 0.8162 F (6, 60) = 0.7182, p = 0.6365
F (3, 60) = 10.99, p < 0.0001 F (3, 60) = 19.12, p < 0.0001 F (3, 60) = 89.41, p < 0.0001 F (3, 60) = 35.73, p < 0.0001 F (3, 60) = 27.88, p < 0.0001 F (3, 60) = 6.375, p = 0.0008
F (2, 60) = 3.842, p = 0.0269 F (2, 60) = 71.30, p < 0.0001 F (2, 60) = 4.853, p = 0.0111 F (2, 60) = 4.407, p = 0.0164 F (2, 60) = 5.636, p = 0.0057 F (2, 60) = 3.779, p = 0.0284
F (6, 60) = 2.578, p = 0.0274 F (6, 60) = 12.15, p < 0.0001 F (6, 60) = 5.281, p = 0.0002 F (6, 60) = 1.882, p = 0.0988 F (6, 60) = 3.913, p = 0.0023 F (6, 60) = 2.383, p = 0.0394
a significant omnibus F-statistic. The ANOVA results are provided in Table 2, and the results of the test for multiple comparisons are provided in the relevant figures. In all analyses, a p-value of less than 0.05 was deemed to indicate statistical significance. All statistical analyses were conducted using the GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA).
Results Evaluation of TMT-induced seizure/tremor with hippocampal neurodegeneration As shown in Fig. 1A, we assessed and recorded clinical symptoms daily for 8 days after treatment with 2.6 mg/kg TMT. Mice exhibited hypersensitivity accompanied by seizure behavior from days 1 to 5 post-treatment, and recovered from seizure/tremor at day 6 posttreatment (n = 10 for each group). We also performed histopathological analyses to confirm that the clinical symptoms induced by TMT treatment were accompanied by neurodegeneration in the hippocampus, particularly in the DG. Extensive neuronal cell death characterized by eosinophilic cytoplasm, nuclear pyknosis, and karyolysis was detected in the granular cell layer of the DG under high magnification posttreatment (Fig. 1B, left panels). We performed further FJC staining to quantify TMT-induced neurodegeneration in the hippocampal DG post-treatment (n = 3 for each group). Semi-quantitative analysis of the FJC-positive intensity showed that TMT treatment resulted in a significant increase of degenerative neurons in the hippocampal DG (Fig. 1B and C).
Microglial and astroglial activation in the hippocampus after TMT treatment To test whether TMT treatment induced microglial and astroglial activation, we evaluated the expression of Iba-1, a microglia marker, and GFAP, an astrocyte marker, in the hippocampus post-treatment (n = 3 mice at each time point). The levels of Iba-1 expression increased significantly in the hippocampal DG of TMT-treated mice at 4 days post-treatment (Fig. 2A and B). Activated microglia of amoeboid morphology with thickened and less ramified processes were extensively shown in the hippocampus after TMT treatment (Fig. 2D, upper panels). The levels of GFAP expression gradually increased in the hippocampus of TMT-treated mice until 8 days post-treatment (Fig. 2A and C). After TMT treatment, GFAP-positive hypertrophied astrocytes were found extensively in the hippocampus (Fig. 2D, lower panels).
Expression of iNOS, CD206, arginase-1 and pro-inflammatory cytokine mRNA in the hippocampus To assess the activation of microglia after TMT treatment, we examined the mRNA expression levels of the M1 (classically activated) microglia marker inducible nitric oxide synthase (iNOS) and the M2 (alternatively activated) microglia markers CD206 and arginase-1 in the hippocampus using quantitative real-time PCR. The levels of both iNOS (Fig. 3A) and CD206 mRNA (Fig. 3B) were significantly increased in the hippocampus at 2 days post-treatment. However, there was no significant change in arginase-1 mRNA expression in the hippocampus after TMT treatment, although the expression peaked 2 days post-treatment (Fig. 3C). We also examined inflammatory cytokine mRNA expression in the mouse hippocampus, to establish whether glial activation in the hippocampus correlates with inflammatory cytokine expression. The expression levels of interleukin (IL)-1 (Fig. 3D), tumor necrosis factor (TNF)-␣ (Fig. 3E), and IL-6 mRNA (Fig. 3F) were significantly up-regulated at 2 days, 1–2 days, and 2 days post-treatment, respectively. However, TMT treatment did not induce a significant change in IL-10 mRNA expression, although the expression peaked at 1 day post-treatment (Fig. 3G). This suggests that TMT treatment induces a robust increase in the expression of the pro-inflammatory cytokines IL-1, TNF-␣, and IL-6, but not the anti-inflammatory cytokine IL-10 in the hippocampus.
Polarization and up-regulation of pro-inflammatory cytokine mRNA expression in BV-2 microglia after TMT treatment To examine the microglia subsets in BV-2 microglia after TMT treatment, we examined mRNA expression of iNOS, CD206, and arginase-1 using quantitative real-time PCR. Expression of iNOS was significantly increased after TMT treatment in a timedependent manner at 10 M of TMT concentration (Fig. 4A). In contrast, expressions of CD 206 (Fig. 4B) and arginase-1 (Fig. 4C) were significantly decreased after TMT treatment in time- and concentration-dependent manner. These results suggest that TMT toxicity leads to microglia polarization toward the M1 phenotype in BV-2 microglia. We also determined whether TMT treatment increased expression of inflammatory cytokine mRNA in BV-2 microglia. Expression of IL-1 (Fig. 4D), TNF-␣ (Fig. 4E), and IL-6 (Fig. 4F) was upregulated at 6–12 h, 3–12 h, and 6 h post-treatment, respectively. However, TMT treatment did not induce a significant change in IL10 mRNA expression (Fig. 4G). These results were consistent with our in vivo results; TMT treatment increases the mRNA expression
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Fig. 1. Clinical symptoms and histopathological examination of mice after trimethyltin (TMT) treatment. (A) Clinical scores of seizure behaviors in mice following TMT treatment based on a 0–5 scale. Animals were sacrificed at each time-point (1, 2, 4, and 8 days after treatment; arrows, n = 10 per group). (B) Representative microphotographs of hematoxylin and eosin (H&E) and Fluoro-Jade C (FJC) staining in the dentate gyrus (DG) of the hippocampus. Scale bars = 50 m. (C) Semi-quantitative analysis of FJCpositive cells in the DG of the hippocampus (n = 3 per group). The data are reported as means ± SE. * p < 0.05, *** p < 0.001; these values indicate statistically significant differences compared to vehicle-treated controls.
of the pro-inflammatory cytokines IL-1, TNF-␣, and IL-6, but not the anti-inflammatory cytokine IL-10.
Induction of iNOS, arginase-1 and pro-inflammatory cytokine mRNA expression in the primary astrocyte cultures after TMT treatment Because we observed contrasting mRNA expression patterns of microglia markers in the hippocampus and in BV-2 cells, we hypothesized that the markers were non-specific and being expressed by additional cell types. Therefore, we examined the expression of microglia markers in primary astrocyte cultures. The expression of iNOS mRNA was increased significantly at 6–12 h post-treatment (Fig. 5A). The expression of arginase1 mRNA was increased significantly at 3 h post-treatment, but decreased significantly at 6–12 h post-treatment following a high concentration of TMT (Fig. 5B). These results suggest the TMT treatment also induces changes in iNOS and arginase-1 expressions in astrocytes. Additionally, we investigated whether TMT treatment increases the expression of inflammatory cytokines in primary astrocyte cultures. IL-1 (Fig. 5C), TNF-␣ (Fig. 5D), and IL-6 (Fig. 5E) mRNA transcript levels were up-regulated at
3–12 h post-treatment, respectively, whereas expression of IL10 mRNA was significantly decreased at 12 h post-treatment, also in concentration-dependent manner (Fig. 5F). These results suggest that TMT treatment increases the expression of the proinflammatory cytokines IL-1, TNF-␣, and IL-6, but decreases the expression of the anti-inflammatory cytokine IL-10, in astrocytes.
Discussion The present study demonstrated that TMT-induced neurodegeneration is closely related to neuroinflammation in the hippocampus. Furthermore, this study is the first to provide complementary in vivo and in vitro evidence concerning the possible roles of activated glial cells in inflammatory cytokine expression in the mouse hippocampus following TMT treatment. Our in vitro study demonstrates that TMT increases the expression of pro-inflammatory cytokines. However, in the mouse hippocampus in vivo, TMT treatment increases simultaneously the expression of both pro- and anti-inflammatory mediators. This is accompanied by activation of microglia, by both classical and alternative pathways, and astroglia.
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Fig. 2. TMT treatment induced microglia and astrocyte activation in the mouse hippocampus. (A–C) TMT treatment induced a significant increase in the expression of a microglial marker (Iba-1) and an astroglial marker (GFAP) in the hippocampus. (D) Representative images of Iba-1-positive microglia and GFAP-positive astrocytes in the DG of adult hippocampus 4 days post-treatment. Scale bars = 150 m. The data are reported as means ± SE (n = 6 per group). ** p < 0.01, *** p < 0.001; these values indicate statistically significant differences compared to vehicle-treated controls.
Neuroinflammation, represented by neuronal cell death, glial cell activation, and up-regulation of pro-inflammatory cytokines, such as TNF-␣ and IL-1, is a prominent pathological feature of several neurodegenerative diseases (Cacabelos et al., 1994; Chapman et al., 2006). Previous studies show that TMT treatment induces selective neuronal cell death and enhances microglial and astroglial activation in the mouse hippocampal DG, accompanied by clinical symptoms, such as seizure/tremor and impaired learning and memory (Kim et al., 2013b; Yang et al., 2012). In the present study, we confirmed that a dose of 2.6 mg/kg TMT was sufficient to induce seizure/tremor concurrent with extensive neuronal cell death in the hippocampal DG within 4 days post-treatment. We also observed early activation of hypertrophied microglia clustered around damaged neurons in the hippocampal DG from post-treatment days 2–4 (the acute damage period). Furthermore, a significant increase in GFAP expression on post-treatment 4–8 days (the chronic recovery period) indicates that TMT may induce delayed astroglial activation throughout the hippocampus. These results confirm that TMT treatment induces selective neuronal cell death and glial activation in the mouse hippocampus. In addition, the dynamic changes in microglial and astroglial activation may play detrimental (e.g., induction of inflammation and phagocytosis) and/or beneficial (e.g., wound healing and resolution of inflammation) roles in the hippocampus under neurotoxic conditions. TMT-induced glial activation is largely associated with the upregulation of pro-inflammatory cytokines, suggesting that these
cytokines may act as modulators in several neurodegenerative models (Allan and Rothwell, 2001; Harry et al., 2002; Maier et al., 1995). However, as described earlier, there are conflicting reports regarding the expression of pro-inflammatory cytokines in the hippocampus during TMT toxicity. For example, Little et al. (2002) reported that TMT does not increase expression of proinflammatory cytokines in the rat hippocampus, because TMT causes neurodegeneration without disrupting the blood–brain barrier (BBB), which is thought to be the origin of cytokines from the periphery (Little et al., 2012). In contrast, Harry et al. (2008) reported early expression of pro-inflammatory cytokine mRNA in the mouse hippocampus at 1–2 days after TMT treatment. In the present study, we demonstrate that TMT induces an early increase in mRNA expression of the pro-inflammatory cytokines IL-1, IL-6, and TNF-␣. Furthermore, our preliminary study revealed no change in the plasma levels of inflammatory cytokines during TMT intoxication (data not shown). This is also supported by the observation that TMT-induced neurodegeneration is not accompanied by a breakdown of the BBB, and that blood–borne macrophages might not be recruited into the hippocampus (McCann et al., 1996). Therefore, we conclude that TMT might induce selective hippocampal cell death and activation of endogenous glial cells by directly stimulating the release of pro-inflammatory cytokines within the hippocampus. When interpreting these conflicting results, it is important to consider the different experimental conditions, such as the animal model used,
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Fig. 3. TMT treatment increased the gene expression of M1/M2 markers and inflammatory cytokines in the mouse hippocampus. A M1 microglia marker (A, iNOS) and M2 microglia markers (B, CD206; C, arginase-1) were examined in the mouse hippocampus. Expression of IL-1 (D), TNF-␣ (E), IL-6 (F) was significantly increased at 1–4 days post-treatment. (G) No significant change in IL-10 expression was observed after TMT treatment. The data are reported as means ± SE (n = 6 per group). * p < 0.05, ** p < 0.01, *** p < 0.001; these values indicate statistically significant differences compared to vehicle-treated controls.
route of administration of TMT, and methods for cytokine detection. Previous studies show that enhanced glial activation is a consequence of TMT intoxication (Fiedorowicz et al., 2001; Yang et al., 2012). However, the detrimental or beneficial role of the microglial response during TMT-induced neurodegeneration has not been fully elucidated. In an attempt to clarify the role of microglial activation in TMT intoxication, we classified microglia into M1 and M2 phenotypes using subset-specific markers. Although this classification system can be considered an oversimplification of the complex microglial response spectrum, it has proved to be a useful method for defining the microglial response to neurotoxic events (Chan et al., 2008; Kigerl et al., 2009; Mikita et al., 2011). In the present study, in vitro data demonstrate that TMT treatment significantly increases expression of the M1 markers TNF-␣, IL-1, IL-6 and iNOS and significantly decreases the expression of the M2 markers CD206 and arginase-1 in BV-2 microglia. This result indicates that TMT treatment might result in M1 polarization of BV-2 microglia. This is the first study to specifically examine M1 and M2 microglial subtypes in TMT neurotoxicity. A key
feature of polarized microglia is differential cytokine production (Mantovani et al., 2002). M1 microglia produce high levels of proinflammatory cytokines, whereas M2 microglia are characterized by the production of low levels of pro-inflammatory cytokines, and down-regulation of the inflammatory response (Kigerl et al., 2009; Mikita et al., 2011). In the present study, we observed increased mRNA expression of pro-inflammatory cytokines in BV-2 microglia and primary astrocyte cultures. Furthermore, a decrease in the mRNA encoding the anti-inflammatory cytokine IL-10 was detected in primary astrocyte cultures. Thus, our findings suggest that TMT treatment induces a shift toward the M1 microglial phenotype. This might play a detrimental role, particularly when considered in conjunction with the observed up-regulation of pro-inflammatory cytokines and astrocytic activation. However, contrary to our results in BV-2 microglia, TMT treatment significantly increases the expression of both iNOS and CD206 mRNA in the hippocampus in vivo. In addition, the expression of arginase-1 is increased at 2 days post-treatment, although this effect is not statistically significant. This suggests that there might be other factors (i.e., iNOS and arginase-1 derived from neurons and astrocytes) involved
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Fig. 4. TMT treatment induced changes in gene expression levels of M1/M2 markers and inflammatory cytokines in BV-2 microglia. (A) iNOS. (B) CD206. (C) arginase-1. (D) IL-1. (E) TNF-␣. (F) IL-6. (G) IL-10. The data are reported as means ± SE (n = 6 wells per condition). * p < 0.05, ** p < 0.01 and *** p < 0.001; these values indicate statistically significant differences in mRNA levels between TMT-treated BV-2 cells (1, 5, 10 M) and vehicle-treated controls at each time point. §§ p < 0.01 and §§§ p < 0.001 indicate statistically significant differences between TMT-treated BV-2 cells at 6 h post-treatment and TMT-treated BV-2 cells at 3 h post-treatment. † p < 0.05 and ††† p < 0.001; these values indicate statistically significant differences between TMT-treated BV-2 cells at 12 h post-treatment and TMT-treated BV-2 cells at 3 h post-treatment. ¶ p < 0.05, ¶¶ p < 0.01 and ¶¶¶ p < 0.001; these values indicate statistically significant differences between TMT-treated BV-2 cells at 12 h post-treatment and TMT-treated BV-2 cells at 6 h post-treatment.
in the expression of iNOS and arginase-1 in the hippocampus during TMT neurotoxicity. Therefore, we also examined the expression of iNOS and arginase-1 mRNA in primary astrocyte cultures. A high concentration of TMT induced an increase in arginase-1 expression at 3 h post-treatment, and a decrease in arginase-1 expression at 6–12 h post-treatment, in primary astrocyte cultures. Thus, our in vitro study suggests that astrocytes may be partly responsible for the increase in both iNOS and arginase-1 mRNA expression in the hippocampus after TMT treatment. Moreover, since our previous studies have shown that changes in arignase1 expression may influence cell survival, the increased expression
of arginase-1 at 3 h post-treatment suggests that activated astrocytes may have a protective function against neurotoxicity, and the decreased expression of arginase-1 at 6–12 h post-treatment may be indicative of a loss of this function (Ahn et al., 2012; Kim et al., 2013a). Our data therefore suggest that the activation of microglia by both classical and alternative mechanisms is indicative of the existence of concurrent protective (e.g., wound healing and resolution of inflammation) and detrimental (e.g., induction of inflammation and phagocytosis) mechanisms in vivo under neurotoxic conditions (Colton et al., 2006; Ohtaki et al., 2008; Perego et al., 2011). However, owing to the complexity of microglial and
Please cite this article in press as: Kim J, et al. Glial activation with concurrent up-regulation of inflammatory mediators in trimethyltininduced neurotoxicity in mice. Acta Histochemica (2014), http://dx.doi.org/10.1016/j.acthis.2014.09.003
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Fig. 5. TMT treatment induced changes in levels of iNOS, arginase-1 and inflammatory cytokine mRNA in primary astrocyte cultures. (A) iNOS. (B) arginase-1. (C) IL-1. (D) TNF-␣. (E) IL-6. (F) IL-10. The data are reported as means ± SE (n = 6 wells per condition). * p < 0.05, ** p < 0.01 and *** p < 0.001; these values indicate statistically significant differences in mRNA levels between TMT-treated astrocytes (1, 5, 10 M) and vehicle-treated controls at each time point. §§ p < 0.01 and §§§ p < 0.001 indicates statistically significant differences between TMT-treated astrocytes at 6 h post-treatment and TMT-treated astrocytes at 3 h post-treatment. † p < 0.05, †† p < 0.01 and ††† p < 0.001; these values indicate statistically significant differences between TMT-treated astrocytes at 12 h post-treatment and TMT-treated astrocytes at 3 h post-treatment. ¶ p < 0.05 and ¶¶¶ p < 0.001; these values indicate statistically significant differences between TMT-treated astrocytes at 12 h post-treatment and TMT-treated astrocytes at 6 h post-treatment.
astrocytic responses in diverse environments, determining the exact role of M1 and M2 microglia and reactive astrocytes in chemical-neurotoxicity requires further investigation. In summary, TMT treatment induces neuronal cell death in conjunction with microglial and astroglial cell activation in the mouse hippocampus. TMT also up-regulates mRNA expression of proinflammatory cytokines in activated glial cells, both in vivo and in vitro. Thus, TMT-induced neurodegeneration is closely linked to the inflammatory reaction in response to dynamic changes in inflammatory signals in vivo and in vitro. These results may contribute toward the establishment of an experimental model of chemically induced neuroinflammation. Acknowledgments This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF2012R1A1B4001262). The animal experiment in this study was
supported by the Animal Medical Institute of Chonnam National University.
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