2.5.2. Histology

Six months post-NIMP exposure, mice were deeply anaesthetized with pentobarbital and transcardially perfused with 10 mL of cold NaCl (0.9%), followed by 30 mL of 4% paraformaldehyde (PFA) in phosphate buffer.

• Microglia staining

> Brains were quickly removed and immersed in cold 4% PFA for overnight post-fixation. Brains were cryoprotected for 24–48 h in cold 20% sucrose and then frozen in −40 ◦C isopentane. All brains were sliced in 14-μm coronal sections using a cryostat, and slices were sequentially mounted on Superfrost + slides (VWR, Radnor, PA, USA). Immunofluorescent labeling with rabbit anti-IBA1 (1/1000, Wako, USA), detected by anti-rabbit 555 (1/500, Invitrogen, Waltham, MA, USA), was performed on one slide per animal to analyze microglial reactivity. Fluorescent labeling of the amygdala was imaged using an automated Leica DM6000 B research microscope (Leica Microsystems, Wetzlar, Germany), and the two hemispheres of three different slices were analyzed for each animal. All acquisitions were performed using the same acquisition setup. Stereotaxic consistency between animals was maintained with the help of a reference mouse brain atlas [24]. Analyses were performed using the ImageJ software. To quantify Iba1 staining, we performed an area fraction analysis. Briefly, after setting a threshold, the pixels in the image with values inside this range were converted to white, whereas pixels with values outside this range were converted to black. The threshold was determined to obtain a clear area representing IBA1 labeling in CTL animals. The same threshold was applied to each image of all animal groups. This analysis measured the area of labeling in regions of interest.

• Gut histology

> The Swiss-rolling technique was used to examine complete colonic sections. This technique helps in the histological assessment of the complete colonic sections examined. The result is an intestinal roll which allows for the scanning of a large part of the intestine. The intestinal villi and the epithelial lining remain intact despite being rolled up. Following PFA fixation, the organs were rinsed with distilled water

and dehydrated. Samples were cut at a thickness of 4 μm on a rotary microtome (LEICA®). Hematoxylin-eosin-saffron staining was performed on successive sections for structural and functional analysis of the colon. The length of villi, number of goblet cells and area of immune infiltrates were quantified manually using Histolab software (GT Vision, UK).

### *2.6. Gut Microbiota Modification*

### 2.6.1. Gram+/Gram− Ratio Determination

To observe the bacterial microflora, a fecal smear of each animal was performed using the Gram staining technique. For this, the colon was cut lengthwise and the feces were directly removed, diluted in 500 μL of 1X PBS (Gibco) and spread on a slide. Differential staining of Gram+ bacteria from Gram− bacteria was performed using the Gram−Hucker R Kit (RAL Diagnostics). The Gram+/Gram− ratio was determined for the entire smear by microscopy using a 40× objective.

### 2.6.2. Assessment of Gut Microbiota Composition by High-Throughput Sequencing

DNA extraction, 16S rRNA gene amplification, 16S rDNA amplicon library preparation and sequencing were carried out by SMALTIS (http://www.smaltis.fr/, accessed on 26 September 2019). DNA was extracted from 200 mg of distal colonic luminal content using the QIAamp Fast DNA stool Mini Kit (Qiagen) according to the manufacturer's recommendations (isolation of DNA from stool for pathogen detection). The V3–V4 region of the 16S rRNA gene was amplified using AccuStart™ II PCR ToughMix (QuantaBio) and the following primers: V3F "CTTTCCCTACACGACGCTCTTCCGATC-TACGGRAGGCAGCAG" (344F) and V4R "GGAGTTCAGACGTGTGCTCTTCCGATCT-TACCAGGGTATCTAATCCT" (802R). The thermocycler was programmed with an initial DNA denaturing step at 95 ◦C for 2 min followed by 30 cycles at 95 ◦C for 1 min, 65 ◦C for 1 min, 72 ◦C for 1 min and a final extension step at 72 ◦C for 10 min. Ligation of MiSeq sequencing adapters was performed by PCR using the MTP™ Taq DNA Polymerase (Sigma) and the following thermocycler conditions: 94 ◦C for 1 min followed by 12 cycles at 95 ◦C for 1 min, 65 ◦C for 1 min, 72 ◦C for 1 min and a final extension step at 72 ◦C for 10 min. Sequencing was performed on a MiSeq device using the 2 × 250bp V3 kit. The remaining adapter/primer sequences were trimmed, and reads were checked for quality (≥20) and length (≥200 bp) using cutadapt [25]. Reads were further corrected for known sequencing errors using SPAdes [26] and then merged using PEAR [27]. Operational taxonomic units (OUTs) were identified using a Vsearch pipeline [28] set up to dereplicate (–derep\_prefix –minuquesize 2), cluster (–unoise3) and chimera check (uchime3\_denovo) the merged reads. OTU taxonomical classification was performed using a classifier from the RDPTools suit [29].
