Gene expression profiles of neuroinflammatory responses in broad brain regions in rats repeatedly administered with N-methyl-N-nitrosourea for 28 days

Abstract

N-methyl-N-nitrosourea (MNU) exposure impairs hippocampal neurogenesis in rats. The present study investigated the gene expression profiles that were commonly up or downregulated across different brain substructures in response to repeated MNU administration in rats. Five-week-old rats were orally administered MNU at 0, 5, 15 mg/kg body weight/day for 28 days and subjected to gene expression microarray analysis in the hippocampal dentate gyrus, corpus callosum, cerebral cortex and cerebellar vermis. MNU at 15 mg/kg revealed multiple functional clusters of upregulated genes related to immune and inflammatory responses in all brain regions, and also clusters of up or downregulated genes related to regulation of apoptotic process in several regions. Specifically, the upregulated genes commonly found in all four regions were enriched in clusters of “immune response” and/or “inflammatory response” (Cd74, Ccl3, Fcgr3a, Serping1, Lgals3, Fcgr2b, Hcst, Kcnn4, Tnf, Gpr18, Tyrobp and Cyba) and “metal-binding proteins” (Mt1, Mt2A and Apobec1). Meanwhile, downregulated genes common to all four regions (Bmp4, Vcan and Fhit) were included in clusters of “cell proliferation”, “glial cell migration” and “nucleotide metabolism”. Immunohistochemical analysis of representative gene products revealed that in all brain regions examined, MNU treatment increased metallothionein-I/II ⁺ cells and galectin-3⁺ cells co-expressing Iba1, and also increased Iba1⁺ and CD68⁺ cells. These results suggest that repeated MNU administration in rats causes neuroinflammation and oxidative stress accompanied by apoptosis of neural cell components in the brain, as well as concurrent anti-inflammatory responses for neuroprotection from MNU exposure, involving activation of microglia producing metallothionein-I/II and galectin-3 on these responses.

INTRODUCTION

N-methyl-N-nitrosourea (MNU) is a DNA-alkylating agent that induces cell death of neural stem/progenitor cells in the mouse brain (Komada et al., 2010). We have recently shown that repeated 28-day oral administration of MNU to postpubertal rats causes lethal damage to proliferating neural stem/progenitor cells in the subgranular zone (SGZ; Watanabe et al., 2017a), a hippocampal subregion of the dentate gyrus (DG) that continues to produce new neurons during the postnatal period (Kempermann et al., 2004). The hippocampus plays an important role in cognitive function, and dysfunction of the hippocampus induced by chemotherapy in cancer patients, such as by carmustine, a nitrosourea compound like MNU, appears to be associated with decreased neurogenesis (Seigers et al., 2013). As well as the disruption of neurogenesis, a growing body of literature suggests that cytocidal agents disrupt various neurobiological processes and induce cognitive deficits in animal models (Seigers et al., 2013). The mechanisms underlying chemotherapy-induced neurotoxicity include apoptosis of mature oligodendrocytes (Dietrich et al., 2006), gliogenesis (Dietrich et al., 2006), toxic neurotransmitter/monoamine release (Madhyastha et al., 2002), disruption of blood vessel density and blood supply (Seigers et al., 2010), cellular redox dynamics and production of reactive oxygen species (Aluise et al., 2011), and neuroinflammatory responses (Tangpong et al., 2011).

Microarray-based gene expression profiling provides a global view of tissue-specific alterations in the mechanisms underlying disease or toxicity development following chemical exposure and offers an opportunity to obtain new cellular markers applicable to toxicity testing. The central nervous system (CNS) is anatomically elaborate, with region-specific differences in the distribution of neuronal and glial cell populations. To assess the neurotoxicity of chemicals in the brain, it is reasonable to analyze as many brain regions as possible. However, for the purpose of gene expression profiling, it is necessary to select representative brain regions in terms of anatomical structure, cell types, or specific function undertaken. To this end, we previously established a high-throughput tissue sampling method that allows molecular analysis of RNA and polypeptides in anatomically-specific brain regions after whole brain fixation with methacarn solution (Akane et al., 2013). By applying this method, we have revealed gene expression profiles and immunohistochemical marker molecules in response to developmental or postpubertal exposure to glycidol (Akane et al., 2014a, 2014b), cuprizone (Abe et al., 2016a, 2016b), 6-propyl-2-thiouracil (Shiraki et al., 2014; Shiraki et al., 2016a, 2016b), or valproic acid (Ojiro et al., 2022; Watanabe et al., 2017b, 2019)

In this study, we performed microarray-based global gene expression analysis in several different brain regions of postpubertal rats treated with repeated oral administration of MNU for 28 days in order to capture the mechanisms underlying chemotherapy-induced neurotoxicity. For this purpose, we examined common gene expression profiles across different brain regions, including both cortical and white matter tissues. The retrosplenial to parietal association cortex was selected to represent the cerebral cortex, and the hippocampal DG to represent the unique structure that conducts neurogenesis, both of which are part of the limbic system. We also selected the corpus callosum to represent cerebral white matter and cerebellar vermis to represent the cerebellum. Based on the gene expression profiles obtained, the cellular localization of representative gene products showing altered gene expression was examined immunohistochemically in different brain regions.

MATERIALS AND METHODS

Chemicals and animals

MNU (CAS No. 684-93-5) was purchased from Toronto Research Chemicals Inc. (Toronto, Canada). Four-week-old male Crl:CD^®(SD) rats were purchased from The Jackson Laboratory Japan, Inc. (Yokohama, Japan) and were housed three or four animals per cage in polycarbonate cages with paper chip bedding. Animals were provided ad libitum with pelleted basal diet (CRF-1; Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water throughout the experimental period. These animals were maintained in an air-conditioned animal room (temperature: 23 ± 2°C, relative humidity: 55 ± 15%) with a 12/12-hr light/dark cycle.

Experimental design

The present animal experiment and samples were identical to those previously reported (Watanabe et al., 2017a). After one-week acclimation, 5-week-old animals were divided into three groups of 24–30 animals by stratified randomization according to body weight (BW) and were orally administered MNU at 0, 5 or 15 mg/kg BW in vehicle water by gavage using feeding needles for 28 days (30 animals each for 0 mg/kg controls and 15 mg/kg group; and 24 animals for 5 mg/kg group). Based on the previous study that revealed lethal dose 50 values at 110 mg/kg in rats (International Agency for Research on Cancer, 1978), we conducted a preliminary experiment consisting of three groups of MNU doses at 0, 15, and 30 mg/kg BW for 28 days. As a result, MNU at 30 mg/kg decreased BW, and 15 mg/kg dose revealed only suppression of BW gain. Therefore, the high dose was set at 15 mg/kg and low dose was at 5 mg/kg, using a common ratio of 3 that is recommended in OECD Guidelines for the Testing of Chemicals, Test No. 407 (Organization for Economic Co-operation and Development, 2007).

During the experimental period, all animals were examined for general conditions regarding nutritional state and signs of abnormal gait and behavior in their home cage in gross observation. BW and consumption of food and water were measured once or twice per week throughout the administration period. On the next day of the last MNU treatment, 11 animals per group were subjected to perfusion fixation for brain immunohistochemistry through the left cardiac ventricle with ice-cold 4% (w/v) paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4) at a flow rate of 35 mL/min under CO₂/O₂ anesthesia. For gene expression analysis of the transcript levels, 6 animals per group were euthanized by exsanguination from the abdominal aorta under CO₂/O₂ anesthesia and subjected to necropsy, and then brain samples were prepared. The remaining brain samples from 7 or 13 animals were prepared for other purposes of analysis.

All procedures of this study were conducted in compliance with the Guidelines for Proper Conduct of Animal Experiments (Science Council of Japan, June 1, 2006) and according to the protocol approved by the Animal Care and Use Committee of Tokyo University of Agriculture and Technology (approved No.: 27-10). All efforts were made to minimize animal suffering.

Tissue sampling of specific brain regions for gene expression analysis

For microarray and real-time reverse transcription PCR (RT-PCR) analyses, removed whole brains were fixed in methacarn solution at 4°C for 5 hr with agitation (Akane et al., 2013). Fixed brains were dehydrated for 1 hr three times in fresh 99.5% ethanol, and stored overnight at 4°C with agitation. Two 2-mm-thick coronal slices of the cerebrum and two 2-mm-thick sagittal slices of the cerebellum were prepared. Coronal slices were prepared by cutting laterally at the positions of –1.0 mm, –3.0 mm and –5.0 mm from the bregma using the brain-matrix cast (Muromachi Kikai Co., Ltd., Tokyo, Japan). Portions of the hippocampal DG and retrosplenial to parietal association cortex (as cerebral cortex) were punched from the posterior cerebral slice (Supplementary Fig. 1). Portions of the corpus callosum and cerebellar vermis were similarly punched from two cerebral slices and two cerebellar slices, respectively (Supplementary Fig. 1). Sampling was made using punch-biopsy devices with a pore size of 1 mm (BP-10F; Kai Industries Co., Ltd., Gifu, Japan). Punched-out tissue samples were stored at –80°C in 99.5% ethanol.

Gene expression microarray analysis

Isolation of total RNA from each tissue sample was performed using QIAzol Lysis Reagent (Qiagen, Hilden, Germany) together with RNeasy Mini kit (Qiagen) according to the manufacturer’s protocol. Gene expression analysis was conducted using Agilent Whole Rat Genome array Toxplus 60Kx8 (Design ID: 035170) with 61,657 probes for known genes and expressed sequence tags (Agilent Technologies, Inc., Santa Clara, CA, USA) in each brain region of the animals (N = 3/group). For sample preparation and array processing, the Agilent protocol “One-Color Microarray-Based Gene Expression Analysis” was used. Briefly, the recommended volume of control RNAs (Agilent One-Color RNA Spike-In Kit; Agilent Technologies, Inc.) was added to 100 ng of total RNA from the 0 mg/kg controls, 5 and 15 mg/kg groups. Thereafter, Cy3-labeled cRNA was produced using Agilent Low Input Quick Amp Labeling (one-color), purified with the RNeasy Mini kit (Qiagen), fragmented using the in situ Hybridization Kit (Agilent Technologies, Inc.) and subjected to hybridization by incubation in a hybridization oven (Agilent Technologies, Inc.). Hybridized slides were scanned (G4900DA scanner, Agilent Technologies, Inc.), and data were obtained using Agilent Feature Extraction software (version 11.7.1.1) with defaults for all parameters. Microarray data analysis was performed using GeneSpring GX (version 11.5.1) software (Agilent Technologies, Inc.). Expression values of less than 1 were substituted by 1, and 75^th percentile normalization was performed using GeneSpring normalization algorithms. Reliability of each expression value was represented by a flag based on default setting of GeneSpring (Detected, Marginal and Not Detected). Gene ontology-based functional annotation clustering of gene expression was performed using the Database for Annotation, Visualization and Integrated Discovery, abbreviated as DAVID, v6.8 (Huang et al., 2009a, 2009b) to clarify biological functions of all genes differentially expressed at least 1.50-fold in magnitude from the 0 mg/kg controls.

Real-time RT-PCR analysis

Analysis of the mRNA levels for genes selected from expression microarray data (listed in Supplementary Table 1) was performed using real-time RT-PCR and 1.0 μg of total RNA from each brain region obtained from the 0 mg/kg controls and 15 mg/kg group (N = 6/group). First-strand cDNA was synthesized in the presence of dithiothreitol, deoxynucleoside triphosphate, random primers, RNaseOUT and SuperScript^® III Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) in a 20 μL total reaction mixture. Real-time RT-PCR with Power SYBR^® Green PCR Master Mix (Thermo Fisher Scientific) was performed using a StepOnePlus^TM Real-Time PCR System (Thermo Fisher Scientific) according to the manufacturer’s protocol. The PCR primers for each gene shown in Supplementary Table 1 were designed using Primer Express software (version 3.0; Thermo Fisher Scientific). The relative differences in gene expression were calculated using threshold cycle (C_T) values that were first normalized to those of the Hprt1 (encoding hypoxanthine phosphoribosyltransferase 1) or Gapdh (encoding glyceraldehyde-3-phosphate dehydrogenase) gene as the endogenous control in the same sample, and then relative to a control C_T value by the 2^−ΔΔC_T method (Livak and Schmittgen, 2001).

Immunohistochemical analysis

Brain tissues from 10 animals per group were subjected to immunohistochemical analysis. The perfusion-fixed brains were additionally fixed with 4% PFA-buffer solution overnight at 4°C. For analysis of the hippocampal DG, corpus callosum, and cerebral cortex, coronal slices of the cerebral hemisphere were prepared at −3.5 mm from the bregma of the brains. For analysis of the cerebellum, sagittal slices were prepared. Tissue slices were further fixed with 4% PFA-buffer solution routinely processed for paraffin embedding and were sectioned into 3-μm-thick sections.

Immunohistochemistry of the gene products identified from gene expression microarray analysis and following real-time RT-PCR analysis was performed using primary antibody against metallothionein-I/II (MT-I/II), galectin-3 (GAL3), and versican (VCAN) shown in Supplementary Table 2. Immunohistochemistry of glial cell subpopulations was performed using primary antibodies against ionized calcium-binding adaptor molecule 1 (Iba1) or cluster of differentiation 68 (CD68) expressed in microglia (Ito et al., 1998; Walker and Lue, 2015), and glial fibrillary acidic protein (GFAP) expressed in astrocytes. For some antibodies, antigen retrieval treatment on deparaffinized tissue sections was performed as shown in Supplementary Table 2. Before applying primary antibodies, endogenous peroxidase was quenched by incubating tissue sections with 0.3% hydrogen peroxide solution in absolute methanol at room temperature for 30 min. Blocking was conducted with 1.5% normal horse or goat serum in phosphate-buffered saline (pH 7.4) at room temperature for 30 min for mouse and rabbit primary antibodies, respectively. Incubation with each primary antibody was performed overnight at 4°C, followed by incubation with secondary antibody at room temperature for 30 min. Immunodetection was carried out using a Vectastain^® Elite ABC kit (Vector Laboratories Inc., Burlingame, CA, USA) with 3,3′-diaminobenzidine/H₂O₂ as the chromogen. Sections were then counterstained with hematoxylin and cover slipped for microscopic examination.

Immunoreactive cells were counted in one section per animal and were analyzed by an investigator blinded to the treatment conditions. In each brain region for examination, immunoreactive cells, i.e., MT-I/II ⁺ cells, GAL3⁺ cells, VCAN⁺ cells, Iba1⁺ cells, CD68⁺ cells and GFAP⁺ cells, were counted and normalized per area unit of the hilar region of the hippocampal DG, corpus callosum, retrosplenial to parietal association cortex (as cerebral cortex) or flocculonodular lobe of the cerebellum (as cerebellar vermis) (Supplementary Fig. 2). In case of MT-I/II ⁺ cells in the hippocampal DG, immunoreactive cells in the granule cell layer were counted and normalized per length of the SGZ of the hippocampal DG.

The number of each immunoreactive cellular population was manually counted under microscopic observation using a BX53 microscope (Evident Corporation, Tokyo, Japan). The length of the SGZ and the areas of the DG hilus, corpus callosum, cerebral cortex or cerebellar vermis were measured in microscopic images at ×40-fold magnification by applying the DP2-BSW image analysis software package (ver. 2.1; Evident Corporation).

To examine cellular identity of MT-I/II⁺ cells and GAL3⁺ cells in each brain region of MNU-treated animals, double immunohistochemistry was performed with primary antibodies (listed in Supplementary Table 2) against Iba1, a microglia marker, GFAP, an astrocyte marker, oligodendrocyte lineage transcription factor 2 (Olig2), a total oligodendrocyte marker that is expressed broadly in oligodendrocyte lineage maturation (Bercury et al., 2014), neural/glial antigen 2 (NG2), expressed in oligodendrocyte progenitor cells (Dawson et al., 2000), and neuronal nuclei (NeuN), a postmitotic neuron marker (Mullen et al., 1992). For double immunohistochemistry of MT-I/II or GAL3 with Iba1, GFAP, Olig2, NG2, or NeuN, 3,3′-diaminobenzidine was applied to visualize Iba1, GFAP, Olig2, NG2, and NeuN, and Vectastain^® ABC-AP kit (Vector Laboratories Inc.) with Vector Red Alkaline Phosphate Substrate Kit I (Vector Laboratories Inc.) was applied to visualize MT-I/II and GAL3.

Statistical analysis

In microarray analysis, the statistically significant difference in gene expression between two groups was determined using Aspin–Welch’s t-test. The expression levels of each gene were computed by the ratio of gene expression in the 5 or 15 mg/kg group to the 0 mg/kg controls. Genes significantly altered expression were selected by following criteria: (i) Significance level is 0.05 (P < 0.05). Gene expression ratio is > 1.50 or < 0.67. It does not include the ‘Not Detected’ flag of all samples of the two groups for comparison. (iia) Significance level is 0.01. Gene expression ratio is > 4. Not including the ‘Not Detected’ flag in all samples in the MNU-treated group for comparison, and including the ‘Not Detected’ flag in one or more samples of the 0 mg/kg controls. (iib) Significance level is 0.01. Gene expression ratio is < 0.25. Not including the ‘Not Detected’ flag in all samples in the 0 mg/kg controls, and including the ‘Not Detected’ flag in one or more samples of the respective MNU-treated groups.

Numerical data are expressed as the mean ± SD. Differences between the 0 mg/kg controls and each MNU-exposed group were evaluated as follows. Data were analyzed by Bartlett’s test for homogeneity of variance. If the variance was homogenous, numerical data were assessed using Dunnett’s test to compare the 0 mg/kg controls and each of the MNU-exposed groups. For heterogeneous data, Steel’s test was used. Numerical data consisting of two sample groups were analyzed by the F-test for homogeneity of variance. Student’s t-test was applied when the variance was homogenous between the groups, and Aspin–Welch’s t-test was performed when the variance was heterogeneous. All analyses were performed using the Excel Statistics 2010 software package (Social Survey Research Information Co., Ltd., Tokyo, Japan).

RESULTS

Clinical signs, BW, food and water consumption, and necropsy data

No deaths occurred and there were no abnormalities in clinical observations in any group during the MNU treatment period. BW was significantly decreased from day 7 to day 28 at ≥ 5 mg/kg compared with the 0 mg/kg controls (Supplementary Table 3). Food consumption was significantly decreased from day 3 to day 21 at ≥ 5 mg/kg and on day 28 at 15 mg/kg compared with the 0 mg/kg controls. Water consumption was significantly decreased from day 14 to day 28 at 15 mg/kg compared with the 0 mg/kg controls (Supplementary Table 4). Upon necropsy, terminal BW was significantly decreased at ≥ 5 mg/kg, and brain weight was significantly decreased at 15 mg/kg compared with the 0 mg/kg controls (Supplementary Table 5).

Microarray analysis in anatomically defined brain regions

With regard to the number of genes with altered expression in the hippocampal DG, a total of 328 genes (143 genes upregulated; 185 genes downregulated) and 103 genes (53 genes upregulated; 50 genes downregulated) were identified as those showing altered expression in the 15 and 5 mg/kg groups, respectively (Supplementary Tables 6 and 7). In the corpus callosum, a total of 1144 genes (672 genes upregulated; 472 genes downregulated) and 54 genes (46 genes upregulated; 8 genes downregulated) were identified as those showing altered expression in the 15 and 5 mg/kg groups, respectively (Supplementary Tables 8 and 9). In the cerebral cortex, a total of 317 genes (204 genes upregulated; 113 genes downregulated) and 29 genes (17 genes upregulated; 12 genes downregulated) were identified as those showing altered expression in the 15 and 5 mg/kg groups, respectively (Supplementary Tables 10 and 11). In the cerebellar vermis, a total of 1386 genes (975 genes upregulated; 411 genes downregulated) and 138 genes (54 genes upregulated; 84 genes downregulated) were identified as those showing altered expression in the 15 and 5 mg/kg groups, respectively (Supplementary Tables 12 and 13).

Result of gene ontology clustering of genes significantly up or downregulated with regard to immune and inflammatory responses and nervous system by MNU treatment at 15 mg/kg in four brain regions are shown in Table 1 and Supplementary Table 14, respectively. The hippocampal DG revealed several numbers of functional gene clusters in upregulated genes. Upregulated gene clusters enriched under the gene ontology terms included “immune response”, “innate immune response”, “inflammatory response” and “positive regulation of inflammatory response”. Downregulated gene clusters included “neural crest cell migration”. In the corpus callosum, diverse functional gene clusters were involved in both upregulated and downregulated genes, and upregulated gene clusters included “immune response” and “inflammatory response”, as well as “positive regulation of cell migration”, and downregulated gene clusters included “glial cell migration” and “negative regulation of neuron apoptotic process”. In the cerebral cortex, representative gene clusters were only observed in upregulated genes, and upregulated gene clusters included “inflammatory response” and “immune response”, as well as both “positive and negative regulation of apoptotic processes”. In the cerebellar vermis, diverse functional gene clusters were involved in upregulated genes, such as “tumor necrosis factor-mediated signaling pathway” and “positive regulation of inflammatory response”. Downregulated gene clusters included “neuron projection”.

Table 1. Representative gene ontology terms of genes showing up- or downregulation with regard to immune and inflammatory responses in four brain regions of male rats after 28-day treatment with MNU at 15 mg/kg.

Brain region		Gene ontology term	No. of genes	P-value
Hippocampal dentate gyrus
	≥1.50 fold	Immune response	10	5.40E-05
		Innate immune response	9	2.70E-04
		Inflammatory response	9	6.40E-04
		Positive regulation of inflammatory response	4	7.60E-03
Corpus callosum
	≥1.50 fold	Positive regulation of cell migration	17	3.70E-04
		Angiogenesis	15	1.10E-03
		Positive regulation of angiogenesis	11	2.30E-03
		Immune response	18	2.40E-03
		Positive regulation of interferon-gamma production	7	3.60E-03
		Positive regulation of vascular endothelial growth factor production	5	5.70E-03
		Tumor necrosis factor-mediated signaling pathway	6	6.00E-03
		Inflammatory response	18	6.90E-03
		Immunoglobulin-mediated immune response	4	8.80E-03
	≤0.67 fold	Angiogenesis	14	2.30E-04
		Positive regulation of cell migration	13	2.00E-03
		Vascular endothelial growth factor receptor signaling pathway	4	1.00E-02
Cerebral cortex
	≥1.50 fold	Inflammatory response	15	2.30E-06
		Immune response	13	2.00E-05
		Positive regulation of tumor necrosis factor production	7	2.20E-05
		Cellular response to tumor necrosis factor	9	6.70E-05
		Positive regulation of apoptotic process	13	1.90E-04
		Tumor necrosis factor-mediated signaling pathway	5	7.20E-04
		Negative regulation of apoptotic process	15	8.70E-04
		Apoptotic signaling pathway	5	2.40E-03
		Regulation of immune response	4	2.80E-03
		Positive regulation of extrinsic apoptotic signaling pathway	4	4.20E-03
		Positive regulation of cell migration	8	5.20E-03
		Immunoglobulin-mediated immune response	3	1.00E-02
Cerebellar vermis
	≥1.50 fold	Tumor necrosis factor-mediated signaling pathway	10	8.90E-05
		Positive regulation of inflammatory response	11	7.50E-04
		Positive regulation of cell migration	22	8.40E-04
		Positive regulation of apoptotic process	31	8.60E-04
		Tumor necrosis factor-activated receptor activity	6	2.00E-03
		Negative regulation of tumor necrosis factor production	8	4.50E-03
		Angiogenesis	18	7.20E-03
		Apoptotic process	29	9.90E-03

Abbreviation: MNU, N-methyl-N-nitrosourea.

The numbers of genes that showed statistically significant up or downregulation commonly across the multiple brain regions are shown in a Venn diagram (Supplementary Figs. 3 and 4). Genes showing upregulation commonly across the four brain regions, i.e., Cd74, Ccl3, Fcgr3a, Serping1, Lgals3, Fcgr2b, Lsp1, Hcst, Kcnn4, Tspo, Tnf, Mt1, Capg, Gpr18, Ckap2, Mt2A, Hlx, Tyrobp, Isg20, Pla1a, Cyba, Apobec1, Ret, Eva1c and Rhbdf2, were obtained in the 15 mg/kg group compared with the 0 mg/kg controls (Table 2). Specifically, Cd74, Ccl3, Fcgr3a, Serping1, Lgals3, Fcgr2b, Hcst, Kcnn4, Tnf, Gpr18, Tyrobp and Cyba were included in the functional clusters of “immune response” and/or “inflammatory response”, and Mt1, Mt2A and Apobec1 were included in the functional cluster of “metal-binding proteins”. Meanwhile, genes showing downregulation commonly among the four brain regions, i.e., Bmp4, Vcan and Fhit, as those included in the functional clusters of “cell proliferation”, “glial cell migration” and “nucleotide metabolism”, were obtained in the 15 mg/kg group compared with the 0 mg/kg controls (Table 2).

Table 2. Representative genes with known functional annotations that were up- (≥1.50 fold) or downregulated (≤0.67 fold) commonly in the four brain regions of rats after 28-day treatment with 15 mg/kg MNU as determined by microarray analysis.

Accession No.	Gene title	Gene symbol	Fold change
			Hi	Co	Ce	Cv
NM_013069	Cd74 molecule, major histocompatibility complex, class II invariant chain	Cd74	8.15	10.49	11.23	7.01
NM_013025	Chemokine (C-C motif) ligand 3	Ccl3	4.60	3.33	3.99	4.32
NM_207603	Fc fragment of IgG, low affinity IIIa, receptor	Fcgr3a	4.19	2.96	2.66	2.65
NM_199093	Serpin peptidase inhibitor, clade G (C1 inhibitor), member 1	Serping1	3.85	2.53	2.42	2.09
NM_031832	Galectin 3	Lgals3	3.82	3.43	2.25	3.94
NM_175756	Fc fragment of IgG, low affinity IIb, receptor (CD32)	Fcgr2b	3.79	2.27	2.65	1.99
NM_001025420	Lymphocyte-specific protein 1	Lsp1	3.70	3.66	4.42	1.67
NM_001005900	Hematopoietic cell signal transducer	Hcst	3.50	2.40	2.68	2.04
NM_023021	Potassium intermediate/small conductance calcium-activated channel, subfamily N, member 4	Kcnn4	3.29	3.01	2.56	2.78
NM_012515	Translocator protein	Tspo	3.23	1.94	2.82	1.69
NM_012675	Tumor necrosis factor	Tnf	3.08	2.04	2.82	2.10
NM_138826	Metallothionein 1	Mt1	3.06	2.34	2.21	2.82
NM_001013086	Capping protein (actin filament), gelsolin-like	Capg	2.93	1.98	2.41	1.78
NM_001079710	G protein-coupled receptor 18	Gpr18	2.90	1.90	2.47	1.77
NM_001169139	Cytoskeleton associated protein 2	Ckap2	2.75	1.82	2.99	2.02
NM_001137564	Metallothionein 2A	Mt2A	2.48	1.76	2.00	2.15
NM_001077674	H2.0-like homeobox	Hlx	2.39	1.88	2.36	2.06
NM_212525	Tyro protein tyrosine kinase binding protein	Tyrobp	2.31	1.71	1.51	1.65
NM_001008510	Interferon stimulated exonuclease gene 20	Isg20	2.27	2.17	1.98	1.94
NM_138882	Phospholipase A1 member A	Pla1a	2.23	1.54	2.05	1.77
NM_024160	Cytochrome b-245, alpha polypeptide	Cyba	2.04	1.67	2.07	1.59
NM_012907	Apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1	Apobec1	2.02	2.00	2.28	2.03
NM_001110099	Ret proto-oncogene	Ret	1.96	1.70	6.21	2.75
XM_001073261	Eva-1 homolog C	Eva1c	1.73	1.77	1.74	2.06
NM_001107067	Rhomboid 5 homolog 2 (Drosophila)	Rhbdf2	1.66	1.53	1.75	1.76
NM_012827	Bone morphogenetic protein 4	Bmp4	0.42	0.28	0.42	0.33
NM_053663	Versican	Vcan	0.48	0.36	0.39	0.61
NM_021774	Fragile histidine triad	Fhit	0.66	0.60	0.62	0.52

Abbreviations: Ce, cerebral cortex; Co, corpus callosum; Cv, cerebellar vermis; Hi, hippocampal dentate gyrus; MNU, N-methyl-N-nitrosourea.

Table 3 shows the upregulated genes related to M1 phenotype of macrophages, i.e., Cd74, Tnf, Ccl3, Il1b, Cxcl10 and Cxcl16, or M2 phenotype of macrophage, i.e., Tgfb1 and Tgfb3, in the four brain regions examined. In the hippocampal DG, transcript levels of Cd74, Tnf, Ccl3 and Tgfb3 were significantly increased in the 15 mg/kg group compared with the 0 mg/kg controls. In the corpus callosum, transcript levels of Cd74, Il1b, Tnf and Ccl3 were significantly increased in the 15 mg/kg group compared with the 0 mg/kg controls. In the cerebral cortex, transcript levels of Cd74, Tnf, Ccl3, Cxcl16, Tgfb1 and Tgfb3 were significantly increased in the 15 mg/kg group compared with the 0 mg/kg controls. In the cerebellar vermis, transcript levels of Cd74, Tnf, Ccl3, Cxcl10 and Tgfb1 were significantly increased in the 15 mg/kg group compared with the 0 mg/kg controls.

Table 3. Expression levels of genes related to M1 or M2 phenotype of macrophages in the four brain regions of rats after 28-day treatment with 15 mg/kg MNU as determined by microarray analysis.

Brain region	Accession No.	Gene title	Gene symbol	Fold change
Hippocampal dentate gyrus
	NM_013069	Cd74 molecule, major histocompatibility complex, class II invariant chain	Cd74	8.15*
	NM_012675	Tumor necrosis factor	Tnf	3.08*
	NM_013025	Chemokine (C-C motif) ligand 3	Ccl3	4.60**
	NM_013174	Transforming growth factor, beta 3	Tgfb3	1.79**
Corpus callosum
	NM_013069	Cd74 molecule, major histocompatibility complex, class II invariant chain	Cd74	10.49*
	NM_031512	Interleukin 1 beta	Il1b	2.55*
	NM_012675	Tumor necrosis factor	Tnf	2.04**
	NM_013025	Chemokine (C-C motif) ligand 3	Ccl3	3.33*
Cerebral cortex
	NM_013069	Cd74 molecule, major histocompatibility complex, class II invariant chain	Cd74	11.23*
	NM_012675	Tumor necrosis factor	Tnf	2.82*
	NM_013025	Chemokine (C-C motif) ligand 3	Ccl3	3.99**
	NM_001017478	Chemokine (C-X-C motif) ligand 16	Cxcl16	2.18**
	NM_021578	Transforming growth factor, beta 1	Tgfb1	1.64*
	NM_013174	Transforming growth factor, beta 3	Tgfb3	1.68**
Cerebellar vermis
	NM_013069	Cd74 molecule, major histocompatibility complex, class II invariant chain	Cd74	7.01*
	NM_012675	Tumor necrosis factor	Tnf	2.10*
	NM_013025	Chemokine (C-C motif) ligand 3	Ccl3	4.32**
	NM_139089	Chemokine (C-X-C motif) ligand 10	Cxcl10	3.47*
	NM_021578	Transforming growth factor, beta 1	Tgfb1	2.40*

Abbreviation: MNU, N-methyl-N-nitrosourea.

*P < 0.05, **P < 0.01, significantly different from the 0 mg/kg controls.

Real-time RT-PCR analysis of genes commonly altered expression across the brain regions

Among the genes showing commonly altered expression across the four brain regions in the 15 mg/kg group, we focused on those functionally related to immune response, inflammation, apoptosis, cellular proliferation and differentiation, and signal transduction, to validate gene expression changes using real-time RT-PCR (Supplementary Table 15). In the hippocampal DG, transcript levels of Mt1, Mt2A, Cd74, Tnf, Ret and Lgals3 were significantly increased and transcript levels of Bmp4 and Vcan were significantly decreased in the 15 mg/kg group compared with the 0 mg/kg controls. In the corpus callosum, the transcript level of Vcan was significantly decreased in the 15 mg/kg group compared with the 0 mg/kg controls. In the cerebral cortex, transcript levels of Mt2A, Cd74, Tnf, Ret and Lgals3 were significantly increased and transcript levels of Bmp4 and Vcan were significantly decreased in the 15 mg/kg group compared with the 0 mg/kg controls. In the cerebellar vermis, transcript levels of Mt1, Mt2A, Cd74 and Ret were significantly increased and transcript levels of Bmp4 and Vcan were significantly decreased in the 15 mg/kg group compared with the 0 mg/kg controls.

Distribution changes of MT1/2⁺, GAL3⁺ and VCAN⁺ cells in brain regions

Immunohistochemically, the glial cell population was positive for MT-I/II in all brain regions examined. The number of MT-I/II ⁺ cells was significantly increased in the cerebral cortex at ≥ 5 mg/kg, and in the hippocampal DG, corpus callosum and cerebellar vermis at 15 mg/kg, compared with the 0 mg/kg controls (Fig. 1).

Fig. 1

Distribution of immunoreactive cells for (A) metallothionein-I/II (MT-I/II), (B) galectin-3 (GAL3) and (C) versican (VCAN) in the hippocampal dentate gyrus (granule cell layer or hilar region), corpus callosum, cerebral cortex and cerebellar vermis of rats after 28-day repeated oral dose treatment with N-methyl-N-nitrosourea (MNU). Representative images from the 0 mg/kg (controls; left) and 15 mg/kg MNU groups (right). Arrowheads indicate immunoreactive cells. Magnification: dentate gyrus (granule cell layer): ×400, scale bar 50 μm; dentate gyrus (hilar region): ×200, scale bar 100 μm; corpus callosum: ×400, scale bar 50 μm; cerebral cortex: ×400, scale bar 50 μm; cerebellar vermis: ×200, scale bar 100 μm. Values are expressed as mean + SD. N = 10 for all groups. *P < 0.05, **P < 0.01, significantly different from the 0 mg/kg controls by Dunnett’s or Steel’s test.

Immunohistochemically, the glial cell population was positive for GAL3 in all brain regions examined. The number of GAL3⁺ cells was significantly increased in all brain regions at 15 mg/kg compared with the 0 mg/kg controls.

Immunohistochemically, both of the neuronal and glial cell populations were positive for VCAN in the brain regions examined. The number of VCAN⁺ cells was not changed in all brain regions at both 5 and 15 mg/kg compared with the 0 mg/kg controls.

Distribution changes of Iba1⁺, CD68⁺ and GFAP⁺ cells in brain regions

The number of Iba1⁺ cells was significantly increased in all brain regions at 15 mg/kg compared with the 0 mg/kg controls (Fig. 2). The number of CD68⁺ cells was significantly increased in all brain regions at 15 mg/kg compared with the 0 mg/kg controls. The number of GFAP⁺ cells was significantly increased in the corpus callosum at 15 mg/kg compared with the 0 mg/kg controls. In the hippocampal DG, cerebral cortex, and cerebellar vermis, there was no change in the number of GFAP⁺ cells at both 5 and 15 mg/kg compared with the 0 mg/kg controls.

Fig. 2

Distribution of immunoreactive cells for (A) ionized calcium binding adaptor molecule 1 (Iba1), (B) cluster of differentiation 68 (CD68) and (C) glial fibrillary acidic protein (GFAP) in the hippocampal dentate gyrus (hilar region), corpus callosum, (C) cerebral cortex and (D) cerebellar vermis of rats after 28-day repeated oral dose treatment with N-methyl-N-nitrosourea (MNU). Representative images from the 0 mg/kg (controls; left) and 15 mg/kg MNU groups (right). Arrowheads indicate immunoreactive cells. Magnification: dentate gyrus: A–C ×200, scale bar 100 μm; corpus callosum: A, B ×200, scale bar 100 μm, C ×400, scale bar 50 μm; cerebral cortex: A, B ×200, scale bar 100 μm, C ×400, scale bar 50 μm; cerebellar vermis: A–C ×200, scale bar 100 μm. Values are expressed as mean + SD. N = 10 for all groups. *P < 0.05, **P < 0.01, significantly different from the 0 mg/kg controls by Dunnett’s or Steel’s test.

Double immunohistochemistry of MT-I/II or GAL3 vs. Iba1, GFAP, NG2, Olig2 and NeuN in brain regions

Co-localization of MT-I/II or GAL3 with Iba1 and GFAP was observed in the hippocampal DG, corpus callosum, cerebral cortex, and cerebellar vermis of the 15 mg/kg group (Fig. 3). In contrast, co-localization of MT-I/II or GAL3 with Olig2, NG2, and NeuN was not detected in any brain regions in animal samples of the 15 mg/kg group (data not shown).

Fig. 3

Cellular identity of metallothionein-I/II (MT-I/II) ⁺ cells and galectin-3 (GAL3)⁺ cells in analysis of double immunohistochemistry. (A) MT-I/II ⁺ cells (red) vs. ionized calcium binding adaptor molecule 1 (Iba1) or glial fibrillary acidic protein (GFAP) (brown), (B) GAL3⁺ cells (red) vs. Iba1 or GFAP (brown). Representative images from the hippocampal dentate gyrus or cerebral cortex in animals treated with N-methyl-N-nitrosourea (MNU) at 15 mg/kg. Magnification: ×600; scale bar 300 μm.

DISCUSSION

In the present study, functional annotation clustering based on gene ontology of gene expression microarray data revealed that MNU at 15 mg/kg for 28 days increased upregulated gene clusters related to immune and inflammatory responses in the four brain regions examined. Specifically, genes related to immune and inflammatory responses, i.e., Cd74, Ccl3, Fcgr3a, Serping1, Lgals3, Fcgr2b, Hcst, Kcnn4, Tnf, Gpr18, Tyrobp and Cyba, and metal-binding proteins, i.e., Mt1, Mt2A and Apobec1, were commonly upregulated in the four regions. In contrast, downregulated genes commonly detected in the four regions, i.e., Bmp4, Vcan and Fhit, were associated with cell proliferation, glial cell migration and nucleotide metabolism. These results suggest that repeated MNU treatment for 28 days targets glial cell populations in addition to neural stem/progenitor cells in the hippocampal neurogenic niche as previously reported (Watanabe et al., 2017a). Among the genes with altered expression in common in the brain regions examined, we selected Mt1 and Mt2A (encoding MT-I/II), Lgals3 (encoding GAL3), and Vcan (encoding VCAN) for analysis of distribution changes in immunoreactive cell populations in different brain regions after repeated MNU administration.

Immunohistochemically, we found an increase in the number of MT-I/II ⁺ cells in all four brain regions examined after MNU administration in this study. This result was paralleled by increased gene expression of Mt1 and Mt2A in the same brain regions in the 15 mg/kg MNU group. It has been reported that MTs are induced in the hippocampus of rats exposed to carmustine (Helal et al., 2009). Therefore, the increase in MT-I/II ⁺ cells might be the reflection of the action of MNU as an alkylating nitrosourea on brain tissue. Of note, MTs are intracellular proteins containing the highest amount of thiol groups within the cytoplasm, and these thiol groups are able to bind to several chemotherapeutic cytocidal agents, such as platinum compounds and alkylating agents (Doz et al., 1993). The administered cytocidal agents are bound by MT-I/II after competition with zinc and copper; these metals are known as cofactors of more than 200 metalloenzymes, some of which are involved in the metabolism of nucleic acids (Doz et al., 1993). The increased MT levels could provide a zinc cofactor reserve that increases the cellular reparative potential when faced with DNA damage by cytocidal agents (Doz et al., 1993). As discussed below, activated microglia are considered to represent a population of MT-I/II⁺ cells increased in response to MNU administration in the present study. Given that microglia can elevate the levels of DNA repair enzymes and antioxidant enzymes (Huang et al., 2006), microglia may have a neuroprotective function against chemotherapeutic alkylating agents by producing MT-I/II.

In the present study, double-immunohistochemical analysis revealed that MT-I/II⁺ cells co-expressed Iba1 and GFAP in all of the brain regions in the 15 mg/kg MNU treatment group, but did not co-express Olig2, NG2, or NeuN in any brain region. In the CNS, MT-I/II is known to be expressed in astrocytes and activated microglia, as well as in leptomeningeal cells, ependymal and choroid plexus epithelium (Hidalgo et al., 2001). In the present study, Iba1⁺ microglia were increased in numbers after MNU treatment in all four brain regions examined, while GFAP⁺ cells were increased only in the corpus callosum. These results suggest that microglia may be the major population of MT-I/II⁺ cells increased in response to MNU treatment in the brain. Activated microglia have been reported to promote MT-I/II synthesis in relation to their putative antioxidant functions as well as zinc and/or copper metabolism (Penkowa et al., 1999). Although oxidative stress parameters were not measured in the present study, these results suggest that repeated administration of MNU in rats induces an increase in MT-I/II in activated microglia to exert antioxidant effects in response to oxidative stress in the brain. According to the gene expression profiles obtained in this study, we observed a significant upregulation of Cyba in all brain regions examined. Cytochrome b-245 alpha chain (encoded by Cyba) is a critical subunit involved in the assembly and activation of NADPH oxidase and helps transfer electrons from NADPH to molecular oxygen, resulting in the production of superoxide anions. Therefore, overexpression of Cyba can lead to oxidative stress and subsequently promote cell apoptosis (Chéret et al., 2008; Wu et al., 2016).

Oxidative stress and neuroinflammation are interrelated, as oxidative stress increases in inflamed tissues and can lead to cell death (Corona, 2020). In this study, we observed up or downregulated gene clusters related to the regulation of apoptotic processes in multiple brain regions. These results suggested that repeated administration of MNU to rats induces an inflammatory response and oxidative stress accompanied by apoptosis of neural cell components in the brain. The increase in MT-I/II⁺ cells might be a signature of concurrent anti-inflammatory responses.

In this study, we found upregulation in the transcript levels of Cd74 and Tnf, M1 pro-inflammatory microglia markers of neuroinflammation (Hwang et al., 2017; Tang and Le, 2016), in all four brain regions after repeated administration of MNU. We also found upregulation of Ccl3, which encodes a chemokine necessary for M1 polarization of microglia (Cassol et al., 2009), in all brain regions by MNU treatment. In addition, other M1 phenotype-related genes such as Il1b, Cxcl10 and Cxcl16 were also upregulated in some brain regions. In contrast, upregulation of M2 anti-inflammatory phenotype-related genes such as Tgfb1 and Tgfb3 was also observed in some brain regions. These results suggest that repeated administration of MNU caused an increase in the microglial population that underwent polarization to M2 phenotype as well as to M1 phenotype. In accordance with the gene expression profiles of these chemical mediator genes, in all brain regions we examined, 15 mg/kg MNU administration resulted in an increase in the number of CD68⁺ microglia, reflecting activated M1/M2 microglia and peripheral macrophages (Walker and Lue, 2015).

In the present study, increased GAL3⁺ cell counts and upregulation of Lgals3 gene expression were observed in all examined brain regions in the 15 mg/kg MNU group. Double-immunohistochemical analysis revealed that GAL3⁺ cells co-expressed GFAP and Iba1 in all four brain regions in the 15 mg/kg MNU group, but did not co-express Olig2, NG2, and NeuN in any brain regions. It has also been reported that GAL3 is abundant in microglia and also in astrocytes (Ge et al., 2022; Pasquini et al., 2011). As mentioned above, Iba1⁺ and CD68⁺ microglia were increased by MNU administration in all brain regions examined, whereas GFAP⁺ astrocytes were increased only in the corpus callosum in this study. These results suggest that activated microglia may be the major population of GAL3⁺ cells increased in the brain in response to repeated MNU administration.

GAL3 is a secreted protein that functions as an extracellular molecule that activates a variety of cells, including monocytes/macrophages, lymphocytes, mast cells, and neutrophils (Sano et al., 2000). GAL3 has numerous proven functions and is considered a versatile multifunctional molecule involved in several biological processes. Of particular relevance is the role of GAL3 in the extracellular space that regulates inflammatory responses (Hara et al., 2020). In the CNS, extracellular GAL3 plays an important role in microglia-mediated diseases, such as multiple sclerosis, CNS infection, Alzheimer’s disease and brain stroke, by activating their disease-specific signaling cascades (Ge et al., 2022). For instance, GAL3 released by activated microglia acts as an endogenous ligand for Toll-like receptor 4, thus regulating the severity of Toll-like receptor-mediated inflammatory response following injury or infection. Moreover, several studies have demonstrated that GAL3 can regulate the expression and secretion of matrix metallopeptidase 2, a neurotoxic molecule produced by activated microglia in the brain, which contributes to neuronal cell death (Kaptan et al., 2017; Lindhout et al., 2021). In the current study, multiple brain regions after repeated MNU administration showed upregulation of Mmp2, concurrent with the gene expression profiles suggestive of activation of apoptotic processes. Therefore, these findings suggest that repeated administration of MNU in the present study increased GAL3 secretion from microglia to cause pro-inflammatory responses as well as neural cell apoptosis. Of note, we also found upregulation of Lgals3bp, which encodes a GAL3-binding protein that plays an important role in modulating galectin-mediated biological effects (Cibor et al., 2019), in the cerebral cortex after repeated administration of MNU in this study. As mentioned before, neuroinflammation might be causally involved in chemotherapy-related cognitive dysfunction (Cleeland et al., 2003). Therefore, GAL3 overproduction from activated microglia may be responsible for chemotherapy-related cognitive dysfunction.

In the present study, downregulation of Vcan transcript level was observed in all brain regions examined in the 15 mg/kg MNU group. However, the number of VCAN⁺ cells was not altered by MNU administration in the corresponding brain regions examined. VCAN is one of the hippocampal proteoglycans that is associated with spatial memory in rats (Saroja et al., 2014). Given that adult neurogenesis in the hippocampal DG regulates spatial memory (Lieberwirth et al., 2016), it is suggested that MNU decreases VCAN expression in association with suppression of hippocampal neurogenesis as we have previously reported using the same animal samples examined in this study (Watanabe et al., 2017a). Because VCAN is an extracellular matrix glycoprotein, analysis of VCAN⁺ cell population may not be suitable for analysis of expression changes.

In conclusion, repeated administration of MNU at 15 mg/kg for 28 days to postpubertal rats revealed multiple clusters of upregulated genes related to immune and inflammatory responses in all brain regions examined, and also clusters of up or downregulated genes related to regulation of apoptotic processes in several brain regions. Genes commonly upregulated in all four regions were identified as those related to immune and inflammatory responses and metal-binding proteins. Meanwhile, genes commonly downregulated in all four regions were identified as those related to cell proliferation, glial cell migration and nucleotide metabolism. Based on the gene expression profiles obtained common to the brain regions examined, immunohistochemical analysis of representative gene products revealed that MNU treatment increased activated microglia expressing MT-I/II or GAL3 in all brain regions examined. The results obtained indicate that repeated MNU administration in rats causes neuroinflammation and oxidative stress accompanied by apoptosis of neural cell components in the brain, as well as concurrent anti-inflammatory responses for neuroprotection from MNU exposure, involving activation of microglia producing MT-I/II and GAL3 on these responses.

ACKNOWLEDGMENTS

Xinyu Zou received the scholarship from the China Scholarship Council for study at the University of Tokyo University of Agriculture and Technology. This work was supported by a grant from the Ministry of Economy, Trade and Industry (METI), Japan. The authors thank Yayoi Kohno for her technical assistance in preparing the histological specimens.

Conflict of interest

The authors declare that there is no conflict of interest.

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