Neurotox Res DOI 10.1007/s12640-013-9422-3

ORIGINAL ARTICLE

In Vivo Molecular Markers for Pro-inflammatory M1 Stage and Resident Microglia in Trimethyltin-Induced Hippocampal Injury

C. A. McPherson • B. A. Merrick • G. J. Harry

Received: 5 June 2013 / Revised: 13 August 2013 / Accepted: 20 August 2013 Ó Springer Science+Business Media New York (outside the USA) 2013

Abstract Microglia polarization to the classical M1 a decrease in neurotrophin 3, glutamate receptor subunit activation state is characterized by elevated pro-inflam- epsilon (Grin)-2b, and neurotrophic tyrosine kinase matory ; however, a full profile has not been receptor, type 3. The M2 anti-inflammatory marker, generated in the early stages of a sterile inflammatory transforming growth factor beta-1 (Tgfb1) was elevated. response recruiting only resident microglia. We charac- mRNAs associated with early stage of injury-induced terized the initial M1 state in a hippocampal injury model neurogenesis including fibroblast growth factor 21 and dependent upon tumor necrosis factor (TNF) receptor sig- Mki67 were elevated. In the ‘‘non-injured’’ temporal cortex naling for dentate granule cell death. Twenty-one-day-old receiving projections from the hippocampus, Lynx1, Grm3, CD1 male mice were injected with trimethyltin (TMT and Grin2b were decreased and Gfap increased. Formalin 2.3 mg/kg, i.p.) and the hippocampus was examined at an fixed-paraffin-embedded tissue did not generate a compa- early stage (24-h post-dosing) of neuronal death. Glia rable profile. activation was assessed using a custom quantitative nuclease protection assay. We report elevated mRNA Keywords M1 microglia Neuroinflammation levels for glia response such as ionizing calcium-binding Trimethyltin Tumor necrosis factor Microglia adapter molecule-1 and glial fibrillary acidic protein polarization (Gfap); Fas, hypoxia inducible factor alpha, complement component 1qb, TNF-related (Tnf, Tnfaip3, Tnfrsfla); interleukin-1 alpha, Cd44, chemokine (C–C motif) ligand Introduction (Ccl)2, Cc14, integrin alpha M, lipocalin (Lcn2), and secreted phosphoprotein 1 (Spp1). These changes occurred As resident of the brain, microglia serve a in the absence of changes in matrix metalloproteinase 9 and key role in brain injury and neurodegenerative disorders. 12, neural cell adhesion molecule, metabotropic glutamate Within injured tissue, microglia exist in various states of receptor (Grm)3, and Ly6/neurotoxin 1 (Lynx1), as well as, reactivity/activation and retain their capability to shift their functional phenotype during specific stages of the inflam- matory response (Stout et al. 2005; Graeber 2010). This C. A. McPherson G. J. Harry (&) flexibility ensures a well-maintained glial-inflammatory Neurotoxicology Group, Division of National Toxicology response and return to . In vivo, the transfor- Program, National Institute of Environmental Health Sciences, mation of resident microglia into a phagocytic phenotype is National Institutes of Health, P.O. Box 12233, MD E1-07, strictly regulated and occurs in response to stimuli such as, Research Triangle Park, NC 27709, USA e-mail: [email protected] cell death, accumulated debris, excess aberrant protein, or the presence of viral or bacterial pathogens (Kettenmann B. A. Merrick et al. 2011; Sierra et al. 2013). Upon the removal of such Biomolecular Screening Branch, Division of National stimuli, or in the presence of a down-regulating signal, Toxicology Program, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, microglia commonly return to a ramified, non-reactive NC, USA phenotype (Kettenmann et al. 2011). It is likely that the 123 Neurotox Res diverse morphological phenotypes of microglia observed different stimuli such as invading microorganisms versus over the course of an injury response are driven by the neuronal dysfunction or death. Such data will provide stage of participation in the cytotoxic response, immune important information on changes as they occur with envi- regulation, or injured-tissue resolution. In efforts to char- ronmental or disease-associated neurodegeneration and acterize functional changes of microglia and activation address the need to identify specific targets or molecular state, staging, as based upon peripheral acti- triggers to regulate the early pro-inflammatory response vation, has been proposed as a method to describe the (Carson et al. 2004; Patel et al. 2013). different stages that brain macrophages undergo in an Glial reactivity and neuroinflammation often accompany injury response (Colton 2009; Colton and Wilcock 2010). brain injury and neurodegenerative disorders and, while the For example, polarization of microglia to the classical causal nature of the response remains in question, the activation state (M1) is characterized by high expression reactivity, per se, has been considered as a surrogate marker levels of the pro-inflammatory cytokines [interleukin (IL)- for neurotoxicity (Kreutzberg 1996; Eng et al. 2000; 1b, IL-12, IL-23, and tumor necrosis factor alpha (TNFa)], O’Callaghan and Sriram 2005; Panickar and Norenberg as well as inducible nitric oxide synthase (iNOS), CD40, 2005; Sofroniew 2005; Streit et al. 2005). Results from major histocompatibility complex (MHC) I, and MHC II studies examining the cascade of responses occurring across (Colton and Wilcock 2010; Durafourt et al. 2012; Chhor different brain regions with multiple types of injury suggest et al. 2013; Jang et al. 2013). Transition or induction of an the occurrence of a common set of injury-induced changes M2 state, in which macrophages release anti-inflammatory associated with glial-inflammatory reactions (Sofroniew cytokines and growth factors and is associated with the 2009; Zhang et al. 2010; Little et al. 2012). Using a model repair and remodeling activities of macrophages (Colton of chemical-induced brain injury localized to the hippo- and Wilcock 2010; Miron et al. 2013; Pathipati et al. 2013). campus, we now provide data on the M1 polarization Thus, it is likely that the diverse phenotypes of microglia occurring during early stages of neuronal death and are driven by their participation in the cytotoxic response, microglia activation. immune regulation, or injured-tissue resolution. In the mouse, an acute systemic injection of the hippo- Much of this proposed M1/M2 characterization of campal toxicant, trimethyltin (TMT), induces hippocampal microglia is based upon classical activation by bacterial or damage that is characterized by localized dentate granule viral fragments, invading microorganisms, and following cell death with a sparing of Cornu Ammonis (CA) pyra- direct application of specific cytokines. Activation of midal neurons (Reuhl and Cranmer 1984; Bruccoleri et al. microglia to an M1 or M2 phenotype has been demonstrated 1998; Geloso et al. 2002; Harry et al. 2008). The associated in vitro (Michelucci et al. 2009; Chhor et al. 2013) and across microglia response coincides with an elevation of mRNA various conditions such as, aging (Lee et al. 2013), spinal levels for the M1 associated cytokines, Tnfa and Il1a cord injury (Kigerl et al. 2009), ischemia (Hu et al. 2012), (Bruccoleri et al. 1998; Harry et al. 2002, 2008; Figiel and and traumatic brain injury (Kumar et al. 2013). The identi- Dzwonek 2007; Funk et al. 2011), and Ccl2 (Bruccoleri fication that two distinct subsets of macrophages can have et al. 1998). This response occurs in the absence of ele- divergent effects on the nervous system following injury has vated interferon-c expression and iNOS activation (Bru- promoted work to further characterize and distinguish ccoleri et al. 1998; Bruccoleri and Harry 2000). There is between M1 and M2 activation state biomarkers as they supporting evidence that resident microglia are the primary relate to biological processes of tissue injury or repair (Kigerl component in this model rather than infiltrating blood- et al. 2009; De Simone et al. 2013; Boche et al. 2013). As borne monocytes (Funk et al. 2011). In this study, there characterized in peripheral macrophages, inflammation, in was an absence of bone marrow-derived green fluorescent the absence of microorganisms, has been termed ‘‘sterile protein positive monocytes observed within the brain inflammation’’ (Chen and Nunez 2010). Not unlike a clas- parenchyma in bone-marrow chimera mice following sical activation, the sterile inflammatory response involves exposure to TMT. A causal role for the pro-inflammatory macrophage recruitment and production of pro-inflamma- cytokine, TNFa, and TNF receptor activation in the neu- tory cytokines, TNFa and IL-1, and a likely activation of ronal apoptosis induced by TMT has been demonstrated iNOS (Chen and Nunez 2010). With an absence of micro- using various pharmacological interventions and geneti- organisms in the brain, one could consider that inflammatory cally modified mice (Harry and d’Hellencourt 2003; Harry responses associated with neurodegenerative disease, et al. 2008). With this injury model, a quantitative nuclease trauma, or chemical-induced injury are representative of a protection assay (qNPA) was used to assess changes sterile inflammation that may or may not display a unique in the mouse hippocampus occurring during the time of profile of cellular changes. The ability to distinguish between neuronal death and TNFa elevation as previously identified classical and sterile inflammation may offer a greater (Bruccoleri et al. 1998; Harry et al. 2002, 2008). A primary understanding of microglia responses as initiated by advantage of the qNPA is its multiplex format to hybridize 123 Neurotox Res specific mRNA probes to many different target transcripts decapitated, and the brain excised. No morbidity was within one sample aliquot without requiring RNA extrac- observed under these dosing conditions. All procedures tion, cDNA synthesis, or gene amplification. The assay complied with a protocol approved by the Institutional allows for measurement of mRNA transcripts by direct 1:1 Animal Care and Use Committee (IACUC) at the National binding of a complementary DNA sequence and quantita- Institute of Environmental Health Sciences. tion by chemiluminescence (Pechhold et al. 2009). A tar- geted qNPA containing mRNA probes for genes associated Confirmation of Hippocampal Damage and Glial with various aspects of cellular responses to injury was Response developed for this study and included genes related to cell death, glutamate activation, inflammation, glial activation, The TMT model of hippocampal damage has been well and development/cell adhesion. The selection was made established in the literature. However, to confirm the with the expectation that the profile generated would help severity of damage and the associated glia response to identify underlying mechanisms and affected cellular occurring in the same cohort as animals used for molecular processes. With TMT-induced injury, the qNPA identified analysis, a randomly selected subset of the dosing cohort elevations in structural-related genes associated with glia (two vehicle control and six TMT dosed) was selected for and in inflammatory genes associated with microglia histological examination at 24 h post-injection. The reactivity that had not been previously characterized as excised brains were dissected in the mid-sagittal plane and M1-related. The profile of change observed in the hippo- immersion fixed in 4 % paraformaldehyde (PFA)/phos- campus showed region specificity when compared to phate buffer (PB; pH 7.2) overnight at room temperature changes observed in the temporal cortex. Since the qNPA (RT). After rinsing in PB, tissue was processed for paraffin platform has been used for molecular classification of B embedding. Rehydrated and ethanol cleared 8 lm paraffin cell lymphomas in formalin fixed-paraffin embedded tissue sections through the hippocampus were collected and (FFPE; Rimsza et al. 2011), we examined the feasibility of mounted on SuperfrostÒ Plus slides (Fisher Scientific; using this method for generating a molecular profile from Pittsburgh, PA). fixed brain tissue. We found significant limitations that Cells undergoing apoptotic cell death were identified by raised questions regarding the applicability of this method immunohistochemistry of cleaved caspase 3 (active cas- for such assessments. pase 3). Rehydrated and ethanol cleared sections were

incubated with 3 % H2O2 for 15 min at RT, washed at RT in two changes of 19 wash buffer (Biocare Medical; Concord, CA) for 5 min each, and subjected to heat- Methods induced epitope retrieval (HIER) in 19 citrate buffer using a decloaking chamber (Biocare Medical). Slides were then Animals and Dosing washed at RT in two changes of 19 wash buffer for 5 min each and incubated in 10 % normal donkey serum (Jackson At postnatal day 20, pathogen-free CD-1 male mice ImmunoResearch; West Grove, PA) for 20 min at RT. (Charles River Laboratories, Raleigh, NC) were singular Sections were incubated for 1 h at RT with a rabbit poly- housed in plastic cages with shaved hardwood bedding that clonal antibody for cleaved caspase 3 (1:100; Biocare included NestlettÒ nesting material and one-third of ori- Medical; 1 % BSA in Tris–buffered salts) and rinsed in two ginal bedding to minimize stress. Animals were maintained changes of 19 wash buffer (5 min RT). Donkey anti-rabbit within a semi-barrier animal facility (21 ± 2 °C; IgG secondary antibody (1:500; Jackson ImmunoResearch) 50 ± 5 % humidity; 12-h light/dark cycle; 6:00–18:00). was applied to each section for 30 min at RT then rinsed Food (NIH 31) and deionized, distilled drinking water were with two changes of 19 Wash Buffer. Vectastain R.T.U. available ad libitum. On PND 21, mice received a single Elite label (Vector Laboratories, Burlingame, CA) was intraperitoneal (i.p.) injection of either vehicle (sterile applied to each section for 30 min at RT then rinsed with 0.9 % NaCl) or 2.3 mg/kg trimethyltin hydroxide (TMT; two changes of 19 wash buffer. Active caspase 3 positive Alfa Products, Danvers, MA) in a dosing volume of 2 ml/ cells were visualized with 3,30-diaminobenzidine (DAB)– kg body weight using a 50 ll Hamilton syringe. The H2O2 (Dako Corporation, Carpinteria, CA) following a injection was delivered 4 h into the light period of a 12-h 5-min incubation. light/dark cycle. This dose level of TMT has been well Astrocytes were labeled with an antibody to glial characterized to induce apoptotic death of dentate granule fibrillary acidic protein (GFAP). Sections were treated with neurons and microglia activation within 24 h post-injection 3%H2O2 for 10 min then subjected HIER in 1X Reveal (Harry et al. 2008). Thus, at this time point (24 h post- Decloaker using a decloaking chamber (Biocare Medical), injection) mice were deeply anesthetized with CO2, allowed to cool to RT, and washed with PB for 20 min at 123 Neurotox Res

RT. Sections were blocked with 2 % normal goat serum/ CA) at RT for 18 h, rinsed in PB, and processed for par- PB for 30 min at RT prior to 1 h incubation at RT to a affin embedding using a RHS-1 Microwave Vacuum Histo- polyclonal rabbit anti-cow GFAP (1:200; Dako Corpora- Processor (Milestone Medical Technologies, Inc, Kalama- tion). Sections were washed for 20 min with PB at RT, zoo MI). Serial 5 lm paraffin sections through the entire incubated with a secondary IgG antibody (20 min RT), hippocampus were collected on non-coated microscope rinsed, incubated for 30 min at RT in avidin–biotin com- slides from a 38 °C waterbath. Prior to sectioning, the plex (ABC) reagent (Vectastain Elite, Vector Laborato- water-bath was washed with RNase Away (Life Technol- ries), and rinsed with PB for 20 min at RT. The reaction ogies, Durham, NC), maintained under a UV light for 12 h, was developed with DAB–H2O2 for 5 min at RT. and filled with RNase-free water for section collection. The Microglia were identified by lectin binding using the technician maintained RNase-free conditions throughout method of Streit (1990) with slight modification. HIER tissue sectioning, including the use of personal items such sections were pre-incubated for 20 min at RT with PB as, latex gloves, facial mask, and hair covering. A cleaning containing CaCl2 MgCl2, MnCl2 (0.1 mm each) and 0.1 % and decontamination procedure was conducted each day. Triton-X 100 (Sigma-Aldrich; St. Louis, MO). Sections Slides were shipped to HTG for processing and analysis. were incubated overnight at 4 °C with horseradish per- For each section, paraffin around the tissue was etched and oxidase-conjugated lectin (1:100; Bandeiraea simplifolia sections were scraped from the slides into a microcentri- BS-I, IB4; Sigma-Aldrich) in PB containing cations and fuge tube in a manner to minimize collection of paraffin. Triton-X 100. Following overnight incubation, sections Multiple sections from each individual animal were pooled were rinsed in PB for 20 min at RT and sites containing into a tube for a final concentration of 0.12 cm2 of the bound peroxidase–lectin conjugates were developed with 5 lm tissue sections/ml. A 25 ll aliquot was added to each

DAB–H2O2 following incubation for 5 min at RT. Fol- well. Denaturation oil (500 ll) was added to each sample lowing DAB, sections were rinsed in dH2O for 3 min at and incubated at 95 °C for 15 min. Proteinase K (Sigma- RT and counterstained with Harris hematoxylin for 30 s, Aldrich) was added to a final concentration of 1 mg/ml of rinsed in tap water, dehydrated by an ethanol gradient, and lysate and incubated at 50 °C for 30 min, followed by cover slipped using Vecta MountTM permanent mounting incubation at 95 °C for 15 min to destroy the proteinase K medium (Vector Laboratories). Immunohistochemical activity. stained tissues were imaged at 409 magnification using an Aperio Scanscope T2 Scanner (Aperio Technologies, Inc. qNPA Array Vista, CA) and viewed using Aperio Imagescope v. 6.25.0.1117. A specialized array of 50-mer probes was designed for the detection of 44 selected genes of interest in neural tissue Tissue Collection and Preparation for HTG qNPA samples using a 96-well plate format for a qNPA (Table 1). The qNPA assay provides a multiplex format to hybridize Hippocampi were dissected from each hemisphere of specific mRNA probes to many different target transcripts excised brains from mice previously injected with either within one sample aliquot (HTG, Tucson, AZ). This saline vehicle or TMT (n = 7 per group). From one method is based upon the direct ribonuclease protection hemisphere, the dissected hippocampi were frozen and assay approach and allows for optimization of conditions to stored at -80 °C. The temporal cortex was collected to detect low-copy signals as can occur in the brain with represent a brain region in which TMT-induced neuronal inflammatory related genes. To identify genes of interest death was not observed but one that receives projections for inclusion on the qNPA array, various sources of data from the hippocampus. Samples were shipped to High were evaluated: (1) data from other Throughput Genomics (HTG; Tucson, AZ) for analysis models of brain injury, (2) protein and gene expression by qNPA. Tissue was weighed, placed in lysis buffer for studies conducted on hippocampal tissue from TMT-dosed a final tissue concentration of 50 mg/ml and ground animals, and (3) pilot data from an Agilent Mouse Oligo using a plastic pestle. The tissue was heated to 95 °C array (Agilent Technologies, Palo Alto, CA) of the hip- for 15–30 min until the tissue lysate was no longer pocampus at 24 h post-TMT. The qNPA probe selection viscous. Samples were diluted to a concentration of represented numerous aspects of inflammatory responses, 4 ng/ml and subjected to HTG Molecular’s qNPATM both pro-inflammatory and anti-inflammatory, and inclu- (Tucson, AZ). ded many of the ILs, chemokines, matrix metalloprotein- From the contralateral hemisphere of the same brain ases (MMP), and growth factors. Aspects of the cell-injury used for fresh tissue analysis, the dissected hippocampi response were represented with the inclusion of probes for were immersion fixed in 10 % formalin (in DEPC treated genes associated with glia, heat shock proteins, oxidative RNase-free water; Phoenix Biotechnologies; Encinitas, stress, hypoxia, and apoptosis. For a number of these genes, 123 Neurotox Res

Table 1 Hippocampal qNPA mRNA expression levels

Function Gene Fresh FFPE

Saline TMT Saline TMT

Inflammation C1qb 1.027 ± 104 6.500 ± 326 **** 1.708 ± 421 6.069 ± 361 **** Ccl2 252 ± 91 222 ± 21 454 ± 189 Ccl4 538 ± 103 240 ± 28 451 ± 68 Cd44 328 ± 39 3.937 ± 425 **** 521 ± 171 2.522 ± 412 *** Ifng 64 ± 40 281 ± 16 395 ± 46 b Il10 69 ± 0 223 ± 490± 20 * Il1a 66 ± 15 136 ± 11 ** 511 ± 242 389 ± 83 b Il1r1 48 ± 063± 6 112 ± 33 217 ± 53 b Il6 79 ± 048± 10 319 ± 146 180 ± 42 Itgam 252 ± 18 452 ± 35 *** 392 ± 83 603 ± 59 * Lcn2 105 ± 27 6.440 ± 337 **** 521 ± 242 7.176 ± 2.087 ** Mmp12 91 ± 056± 7 487 ± 216 335 ± 67 b Mmp9 44 ± 041± 7 189 ± 83 125 ± 38 Tgfb1 180 ± 17 695 ± 50 **** 504 ± 132 731 ± 84 Tnf 42 ± 3 302 ± 42 111 ± 21 **b Tnfaip3 112 ± 14 181 ± 28 * 426 ± 154 366 ± 52 b Tnfrsf1a 285 ± 29 1.557 ± 61 **** 582 ± 120 1.638 ± 243 ** Tnip3 220 ± 13 105 ± 32 Cell death Atf3 682 ± 176 457 ± 235 807 ± 172 Atf4 1.615 ± 58 3.560 ± 810 * 2.133 ± 201 3.222 ± 744 Casp3 463 ± 32 994 ± 265 653 ± 134 1.026 ± 204 Cdr2 150 ± 10 372 ± 118 324 ± 73 543 ± 119 Ddit3 662 ± 35 3.273 ± 1.093 * 937 ± 236 2.385 ± 575 * Fas 52 ± 18 131 ± 10 ** 166 ± 10 116 ± 15 Fasl 39 ± 083± 070± 23 Hif1a 2.704 ± 113 3.994 ± 442 * 3.229 ± 386 3.619 ± 290 Hspa1b 73 ± 590± 6 133 ± 20 a 214 ± 18 **b Hsp1b 312 ± 51 3.412 ± 485 * 507 ± 104 4.963 ± 2.397 Development Dclk3 296 ± 19 221 ± 23 * 486 ± 124 362 ± 24 b Epha3 336 ± 26 277 ± 23 525 ± 90 323 ± 25 * Fgf21 1.156 ± 1.048 322 ± 150 987 ± 662 Mki67 75 ± 7 340 ± 40 **** 669 ± 311 620 ± 108 Ncam1 4.993 ± 194 5.354 ± 756 4.211 ± 282 4.566 ± 625 Nefh 6.384 ± 312 5.341 ± 335 * 5.416 ± 384 4.029 ± 289 *b Ntf3 349 ± 36 191 ± 25 ** 1.059 ± 354 534 ± 92 b Ntrk3 5.271 ± 200 4.095 ± 110 *** 4.671 ± 351 3.726 ± 194 * Sema3c 246 ± 21 229 ± 11 338 ± 75 318 ± 20 b Synapse/ Grin2b 6.621 ± 248 5.375 ± 259 ** 7.622 ± 471 5.932 ± 672 receptor Grm3 3.021 ± 142 2.594 ± 289 3.507 ± 605 2.231 ± 285 Lynx1 7.121 ± 395 6.522 ± 407 8.365 ± 407 5.914 ± 413 ** Syt7 1.038 ± 53 944 ± 81 1.367 ± 164 1.041 ± 86 Glia Aif1 723 ± 65 1.411 ± 143 *** 1.165 ± 338 1.260 ± 97 Gfap 3.812 ± 374 15.142 ± 1.060 **** 3.166 ± 231 19.721 ± 2.230 **** Spp1 151 ± 23 11.430 ± 1.233 **** 726 ± 277 8.994 ± 2.739 **

Data represents mean ± SEM. Absence of data indicates absence of signal. Asterisks indicate significant difference (* p \ 0.05; ** p \ 0.01; *** p \ 0.001; **** p \ 0.0001) between saline and TMT treated mice within each of the tissue preparation, Student t test. Bold asterisks indicate genes that failed to reach significance following Benjamini– Hochberg adjustment for false discovery rate 0.05 a Significant difference between saline fresh and saline FFPE with Benjamini–Hochberg adjustment b Significant difference between TMT fresh and TMT FFPE with Benjamini–Hochberg adjustment

123 Neurotox Res our previous work provided and existing database on Results mRNA and protein changes to allow for comparison. The assays were conducted at HTG (Tucson, AZ) using Confirmation of Hippocampal Injury methods as previously reported (Rimsza et al. 2011). Briefly, 25 ll sample lysates representing 100 ng tissue Prior to molecular analysis, hippocampal damage was were incubated with a mixture of gene specific probes to confirmed in the TMT-dosed mice. Histological evaluation form probe–mRNA duplexes. Non-hybridized probes were of the hippocampus confirmed a localization and level of digested by S1 nuclease followed by alkaline hydrolysis to severity of dentate granule cell death in the experimental eliminate mRNA in duplexes. The remaining intact qNPA cohort within 24 h following TMT injection. The injury probes were proportional to the abundance of specific pattern displayed was consistent with previous reports mRNA in neural lysates. Samples were transferred for (Bruccoleri et al. 1998; Harry et al. 2008; McPherson et al. probe detection to array plates (96 well) in which each well 2011). At this age, no immunoreactivity for active caspase contained plate-bound 50-mer oligonucleotides specifically 3 was detected in the vehicle control hippocampus, while a designed to capture each specific gene probe. The plate pronounced immunoreactivity was observed in the dentate contained both fresh and fixed hippocampus from both granule cell layer (GCL) of mice dosed with TMT saline and TMT injected mice (n = 7 per group) for equal (Fig. 1a). The microglia response to TMT was similar to representation of all conditions. what has been previously described for this model (Bru- Each well contained an array of 47 probes consisting ccoleri et al. 1998; Harry et al. 2008). In the hippocampus of 44 genes of interest, two housekeeping genes, and one of control mice, IB4? microglia displayed few cell bodies negative control gene (a plant gene, ANT). After binding within the GCL but showed evidence of fine processes of the detection probes with horseradish peroxidase and throughout the area (Fig. 1b). With injury induced by the addition of F substrate, a chemiluminescent signal TMT, microglia showed a shift in morphology and, at 24 h, was detected for quantification. Signals were simulta- the cells displayed either thickened processes with stellate neously imaged from the plate bottom with a CCD-based morphology or an amoeboid morphology (Fig. 1b). Omix Imager (HTG) and analyzed using Vuescript soft- GFAP? astrocytes displayed long and thin processes ware (HTG). Based upon a pilot study examining 30 throughout the hippocampus in control animals; while, in potential housekeeping genes, Actb (beta actin) and Ppia TMT-dosed mice, astrocytes displayed signs of hypertro- [peptidylprolyl isomerase A (cyclophilin A)] were selec- phy with a thickening of cell processes and increased ted on the basis of expression level and low variance in GFAP immunoreactivity evident in the GCL (Fig. 1c). brain tissue across various ages. mRNA levels of the genes of interest were normalized to housekeeping genes qNPA for fold-change comparisons. The mean, standard devia- tion, and coefficient of variance (CV) were determined Hippocampus across triplicate wells of each sample. CV for each gene ranged between 2 and 40 %, with an average of 13 % for By 24 h post-TMT, a distinct profile of elevated mRNA both groups. Individual genes showing a CV of [30 % levels was demonstrated (Table 1). Prominent and signifi- were normally associated with low signal intensity. The cant changes were observed in genes associated with each means of sample triplicates were used for statistical of the classifications of inflammation, cell death, glial analysis. response, and development. Elevated inflammatory genes included the complement factor, C1qb; TNF-related genes Statistical Analysis (Tnfaip3, Tnfrsfla); the pro-inflammatory cytokine inter- leukin-1a (Il1a), the cell surface glycoprotein, Cd44; mRNA levels of each gene, were analyzed by Student’s integrin alpha M (Itgam); secreted phosphoprotein 1 t tests for comparisons between saline/FFPE and saline/ (Spp1), as well as the gene for microglia/macrophage fresh or between TMT/FFPE and TMT/fresh tissue marker Iba1 (Aif1—allograft inflammatory factor 1). In the groups. A Benjamini–Hochberg (Benjamini and Hochberg TMT-dosed animals, signals were detected for chemokine 1995) procedure was applied separately to the fresh tissue (C–C motif) ligand (Ccl)2 and Ccl4; Tnf, interferon-c group and to the FFPE tissue group, using a false dis- (Ifng), and the anti-inflammatory cytokine, IL-10 that were covery rate (FDR) of 0.05. Student t tests were used to not observed in control tissue. Transforming growth factor determine the influence of tissue preparation with tests run b1(Tgfb1) was elevated in TMT-dosed tissue while, Il6 within each dose group for comparisons between fresh was decreased. Elevated cell death-related genes included and FFPE tissue and a Benjamini–Hochberg procedure those associated with apoptosis (caspase 3), inflammatory applied. mediated apoptosis (Fas and Fasl), ischemia [hypoxia 123 Neurotox Res

(Lcn2), a protein known to induce an M1 phenotype, was significantly elevated in the TMT dose group. Gene tran- script levels for metabotropic glutamate receptor 3 (Grm3) and Ly6/neurotoxin 1 (Lynx1) remained within control levels. Following a Benjamini–Hochberg adjustment (FDR of 0.05) changes in Atf4, Ddit3, Hsp1b, Dclk3, and Nefh were no longer found to reach statistical significance.

Temporal Cortex

To evaluate the specificity of the response in the hippo- campus, fresh tissue samples of the temporal cortex were examined by qNPA. mRNA levels for immune-related and cell death-related factors remained within normal control levels. Following a Benjamini–Hochberg adjustment (FDR of 0.05), fresh tissue samples of the cortex indicated a significant decrease (\1.5-fold) in Lynx1, Grm3, and Grin2b. A significant increase ([1.5-fold) was observed for Gfap (data not shown). Fig. 1 Histopathology of the suprapyramidal blade of the hippocam- pal dentate GCL 24 h following a single injection of TMT hydroxide Comparison Between FFPE and Fresh Frozen (2.3 mg/kg body wt, i.p., 2 ml/kg). Consistent with previous reports (Harry et al. 2008; McPherson et al. 2011), the current dosing cohort Hippocampus displayed dentate granule cell death with a concurrent microglia and astrocyte response. a Representative image of active caspase 3 In the overall evaluation of the array data, the chemilu- immunoreactivity showing dark and collapsed cells (arrow) indicative minescent levels detected for mRNA transcripts in FFPE of apoptosis of dentate granule neurons with TMT and no indication of apoptosis in the control. b Representative images of Bandeiraea tissue were slightly higher than those observed in fresh simplifolia BS-I, IB4? microglia. Microglia in the saline control saline control tissue obtained from the same animal. showed small cell bodies and elongated distal ramified processes When the FFPE saline control tissue was compared to the throughout the GCL (arrow). At 24 h post-TMT, microglia displayed matched fresh tissue, eight transcripts were detected for an amoeboid morphology (arrow head) indicative of phagocytic activity. c Representative image of GFAP? astrocytes. Cells which there was no detection level in the fresh tissue displayed thin elongated processes (arrow) throughout the GCL in (Table 1). One gene Hspa1b showed a significantly higher saline control mice. Following TMT, astrocytes showed an increased level in the FFPE tissue as compared to fresh tissue from immunoreactivity of GFAP within the blade. Immunoreactivity was the saline control group. In the TMT-dosed animals, a detected with 3,30-diaminobenzidine (DAB) and sections counter- stained with Harris hemotoxylin. Scale bar 50 lm comparison of the FFPE tissue with fresh tissue indicated that 11 genes were significantly different with 10 higher and one lower in tissue that had undergone fixation and inducible factor 1a (Hif1a)], heat shock (Hspa1b, Hspb1), embedding (Table 1). While not all genes were found to activating transcription factor 3 (Atf3) and Atf4. No chan- be significantly higher in the FFPE tissue, the pattern was ges were observed in matrix metalloproteinase (Mmp)9 or indicative of an overall higher chemiluminescent signal. Mmp12. A number of genes were also elevated that However, the most important comparison was whether or reflected the injury-induced hippocampal neurogenesis that not the profile of gene changes observed in fresh tissue as has been reported with TMT injury (Harry et al. 2004; a function of TMT could be recapitulated from FFPE Ogita et al. 2005; McPherson et al. 2011). These included samples. To maximize the comparison, data prior to genes for the proliferative protein Ki67 (Mki67) and neu- adjustment for FDR were used to determine the number of rofilament heavy polypeptide (Nefh), with a signal detected possible shared genes (Fig. 2a). Of the genes initially for fibroblast growth factor (Fgf) 21 that was not observed indicated in the fresh hippocampal tissue to be altered by in saline control tissue. However, decreases were observed TMT, only 11 were also indicated in the FFPE tissue. The in double cortin-like kinase 3 (Dclk3), neurotrophin 3 fold-change in the TMT-dosed tissue, relative to saline (Ntf3), G protein-regulated inducer of neurite outgrowth 2 controls, was generally lower in the FFPE tissue than that (Grin2b; glutamate receptor subunit epsilon-2b), and neu- seen in fresh tissue (Fig. 2b). In addition, data from the rotrophic tyrosine kinase, receptor, type 3 (Ntrk3). mRNA FFPE tissue indicated a decrease in four genes with TMT levels for the astrocyte structural protein, GFAP were exposure that were not found changed in fresh tissue elevated in the TMT dose group. In addition, lipocalin (Fig. 2a). 123 Neurotox Res

consistent with earlier work, the profile did not support these as primary mechanisms of neuronal death induced by TMT. In agreement with earlier studies demonstrating a TMT-induced pro-inflammatory environment, we now provide data supporting the pro-inflammatory M1 activa- tion state as an early event for neuronal death. The regional specificity of this response was demonstrated by the con- trasting molecular pattern obtained from the temporal cortex. As a neuronal projection site from the hippocam- pus, inflammation was not implicated in the cortex by the gene profile but rather the data suggested activity-related events for down-regulation and remodeling that are possi- bly related to a M2 activation phenotype. The characteristic features of neuronal death, astrocyte reactivity, and microglia activation seen with TMT hip- pocampal injury were reflected by elevations in related gene transcripts on the qNPA. Previous in vivo and in vitro studies established the hypothesis that TMT-induced neu- ronal death in the mouse was a TNF receptor-mediated event (Bruccoleri et al. 1998; Harry et al. 2008). In the mouse, TMT results in the induction of TNFa production by microglia (Bruccoleri et al. 1998; Figiel and Dzwonek 2007; Harry et al. 2002) and activation of TNF receptors (Figiel and Dzwonek 2007; Harry et al. 2008). Further confirmation of the contributory role for TNFa was dem- onstrated by a diminished neuronal death with the neu- tralization of TNFa signaling (Harry et al. 2002, 2008). Consistent with these reports, the qNPA demonstrated that Fig. 2 Students t tests were used to identify genes differentially expressed in the hippocampus of mice 24 h post-injection of either elevated gene transcripts were primarily associated with saline or trimethyltin hydroxide (TMT; 2.3 mg/kg body wt, i.p.). M1 pro-inflammatory processes such as chemokine and Tissue was generated either as fresh frozen (fresh) or sections from cytokine signaling. The data also suggested that an alter- hippocampus rapidly fixed in formalin and embedding in paraffin native common neuronal death mechanism such as ex- (FFPE). a Venn diagram representing the distribution of genes either increased (up arrow) or decreased (down arrow) 24 h post-injection citotoxicity was not initiated. This is in contrast with the unique to and shared between fresh and FFPE samples (p \ 0.05; microarray gene profile generated for TMT-induced hip- non-adjusted for false detection). b Representation of mean (±SEM) pocampal injury in the rat (Little et al. 2012). In that case, fold-change of the shared genes identified in A as compared to saline the localized loss of CA3-4 pyramidal neurons, rather than control samples for each tissue preparation dentate granule neurons, and the associated microglia activation was not accompanied by an elevation in pro- Discussion inflammatory related genes such as TNFa. It was proposed that the difference in an inflammatory response between The TMT model of hippocampal damage has been used mice and rats was due to differences in the localization of over the last few decades as a model to examine hippo- neuronal damage and microglia heterogeneity (Lawson campal function (Harry and d’Hellencourt 2003) and has et al. 1990). However, earlier studies reported that TMT been employed more recently as a model of Alzheimer’s exposure in the rat did result in elevations in mRNA levels disease-associated hippocampal dysfunction (Andjus et al. for Tnfa (Jahnke et al. 2001; Maier et al. 1995) and TNF 2009; Gasparova et al. 2012). Early reports on TMT elevations have been seen with in vitro studies using rat neurotoxicity examined a number of potential neuronal mixed glia cultures (Figiel and Dzwonek, 2007; Harry et al. death mechanisms and, in general, excluded the involve- 2002; Viviani et al. 2001). While the elevation in TNF- ment of ischemia/hypoxia and the contribution of excito- related genes seen in the mouse differs from that reported toxicity; while, later reports suggested a specific TNFa- for the rat (Little et al. 2002; Little et al. 2012), we did receptor-mediated apoptosis of dentate granule cells (Harry observe an elevation in mRNA levels for Ccl2 following et al. 2008). In this study, genes associated with hypoxia/ TMT that is in agreement with elevations observed in the ischemia or glutamate activation were not elevated and rat (Little et al. 2002). The discrepancy in the 123 Neurotox Res pro-inflammatory contribution across species may be a inflammatory cytokines but also as an effort to protect the factor of timing, the toxicokinetics of the compound, hippocampal network from the injury-related stress. Such a severity of the damage, or method of detection. However, it mechanism could be linked to the elevation observed in the does offer an interesting comparison for the future to anti-apoptotic pathway gene, heat shock protein 1b determine common underlying biomarkers of neurotoxicity (Hsp1b). Elevations in Ddit3 following TMT were indi- and neuroinflammation. cated prior to conducting a FDR adjustment and were no In the mouse hippocampus, the highest level of eleva- longer statistically significant after adjustment, however, tion following TMT was observed in Spp1, secreted given the level of fold-change it remains of interest. It is a phosphoprotein 1. SPP1 is a member of the osteopontin key regulator of the mammalian target of rapamycin superfamily. It functions to regulate the expression of the (mTOR) pathway and serves as an important signaling M1-associated cytokines, interferon-c and interleukin-12, pathway in the hippocampus contributing to translational in the peripheral (Santamaria and Corral control and synaptic plasticity (Hoeffer and Klann 2010; 2013). It has been associated with microglia upregulation Polman et al. 2012). One could propose that an elevation in following transient forebrain ischemia (Choi et al. 2007), in Ddit3 might represent the induction of signaling processes a subset of amoeboid microglia in the hippocampus fol- necessary for dealing with injury-induced stress and lowing kainic acid-induced injury (Kim et al. 2002), and in maintaining synaptic integrity. peri-arterial areas following TMT (Morita et al. 2008). Its In considering the specificity of the response in the elevation is consistent with the microglia response hippocampus, changes were observed in the cortex of mice observed at 24 h following TMT. Also, related to the exposed to TMT. In general, the response was minimal as microglia response were elevations in Itgam, one protein compared to the hippocampus. Three genes, Lynx1, subunit of macrophage-1 antigen also known as cluster of Grin2b, and Grm3, were significantly decreased. Both differentiation molecule 11b (Cd11b), and Cd44, a receptor Grin2b and Grm3 are associated with glutamate receptors. for hyaluronic acid that can interact with osteopontin and Grin2b, or the NR2 subunit, acts as the agonist-binding site MMP and is upregulated in microglia upon focal stroke for glutamate and Grm3 encodes the metabotropic gluta- (Wang et al. 2001). ITGAM is involved in the complement mate receptor 3. Lynx1 is expressed in large projection system and has been implicated in several immune pro- neurons in the hippocampus and cortex and binding cesses (Solovjov et al. 2005). The elevation observed in the appears to be correlated with the distribution of nicotinic hippocampus was likely related to the elevation observed acetylcholine receptors (nAChRs) (Miwa et al. 1999). in the complement gene C1qb and possibly to synaptic Lynx1 has been demonstrated to enhance desensitization of remodeling (Yuzaki 2010). nAChRs and modulate their functional properties (Ibanez- Similar to what has been reported with kainic acid- Tallon et al. 2002) suggesting that the down-regulation of induced hippocampal damage, we observed a significant these genes represents an adaptive and protective response elevation in lipocalin 2 (Lcn2). Lipocalin is a member of a of cortical neurons upon stimulation from the injured hip- diverse family of small soluble proteins, many which are pocampus. Such stimulation may also be the basis for the secreted extracellularly and implicated in apoptosis and elevation observed in Gfap suggestive of an astrocyte inflammation (Kim et al. 2002; Yang et al. 2009). Recently, response to early changes in the neuronal projections from secreted Lcn2 was demonstrated to promote microglial the hippocampus. polarization to the M1 state in vitro. Upon further exami- Inflammation as a mechanism for initiating neuronal nation in vivo, Lcn2 was shown to enhance M1 microglia death or exacerbating neuronal injury and degeneration polarization in the hippocampus following lipopolysac- has gained a significant level of interest and enthusiasm; charide injection (Jang et al. 2013). In response to kainic however, understanding the nature of any such response acid, Lcn2 is elevated in the hippocampus and localized to is necessary for interpretation of any biological impact. reactive astrocytes (Chia et al. 2011). In cultured astro- In this study, we utilized a specific set of probes that cytes, inflammatory stimulation elevates expression and were designed to detect changes in genes associated with secretion of Lcn2 (Lee et al. 2009). Thus, the combined pro-inflammatory events, neuronal death processes, and elevation in Lcn2 and Gfap would provide a molecular repair mechanisms. Using this approach, we have now indicator of the early astrocyte response or an interaction identified a number of gene transcripts that may be uti- between microglia and astrocytes occurring with TMT lized as specific markers of an M1 microglia state or M1 injury (Fig. 1c). Lcn2 has also been identified to modulate environment. While there are numerous methods that the effects of stress on the hippocampus by altering neu- would suffice for analyzing fresh tissue, we were also ronal spine formation (Mucha et al. 2011). Thus, it is likely interested in determining if we could obtain a reliable that the upregulation of Lcn2 mRNA is not only associated molecular profile from tissue that had been processed for with the injury in the hippocampus as a response pro- histological analysis. Quite often, the initial assessments 123 Neurotox Res of neurotoxicity rely heavily on an anatomical demon- References stration of neuronal death or other forms of neuropa- thology. If such lesions could then be evaluated at a Andjus PR, Bataveljic D, Vanhoutte G, Mitrecic D, Pizzolante F, Djogo molecular level an evaluation of region specific changes N, Nicaise C, Kengne FG, Gangitano C, Michetti F, van der Linden A, Pochet R, Bacic G (2009) In vivo morphological changes in could be conducted to characterize the neurotoxicity by animal models of amyotrophic lateral sclerosis and Alzheimer’s- identifying changes preceding or maintained after neu- like disease: MRI approach. 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Biochim Biophys Acta 1832:650–659 Durafourt BA, Moore CS, Zammit DA, Johnson TA, Zaguia F, Guiot Acknowledgments The authors thank the NIEHS Microarray Core MC, Bar-Or A, Antel JP (2012) Comparison of polarization for their expert assistant with the initial microarray analysis used for properties of human adult microglia and blood-derived macro- selection of qNPA probes, Dr. Grace Kissling for statistical exper- phages. Glia 60:717–727 tise, and Drs. Stephanie L. Smith-Roe and Chad Blystone of NIEHS Eng LF, Ghirnikar RS, Lee YL (2000) Glial fibrillary acidic protein: for reviewing the final manuscript. This study was funded by the GFAP-thirty-one years (1969–2000). Neurochem Res Division of Intramural Research and the Division National Toxi- 25:1439–1451 cology Program, NIEHS/NIH (1Z01ES101623). The qNPA was Figiel I, Dzwonek K (2007) TNFalpha and TNF receptor 1 expression in conducted at HTG under an NTP contract with technical expertise the mixed neuronal-glial cultures of hippocampal dentate gyrus from Dr John Luecke. The statements, opinions, or conclusions exposed to glutamate or trimethyltin. Brain Res 1131:17–28 contained within this manuscript do not necessarily represent the Funk JA, Gohlke J, Kraft AD, McPherson CA, Collins JB, Harry GJ statements, opinions, or conclusions of NIEHS, NIH, or the United (2011) Voluntary exercise protects hippocampal neurons from States Government. trimethyltin injury: possible role of interleukin-6 to modulate tumor necrosis factor receptor-mediated neurotoxicity. Brain Conflict of interest The authors declare no conflict of interest Behav Immun 6:1063–1077 associated with this study. 123 Neurotox Res

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