Ethical statement
All experimental procedures strictly adhered to the guidelines established by the European Communities Council (86/609/EEC/2 and 2010/63 Directives of European Community). The protocol was approved by the Institutional Animal Care and Use Committee of the Institute of Experimental Medicine, Budapest, under permit number PEI/001/29-4/2013. This study is reported in accordance with ARRIVE guidelines.
Animals
Male FVB/Ant mice (80-120 days old) were sourced from the Medical Gene Technology Unit, Institute of Experimental Medicine, Budapest. Mice were kept in a Specific Pathogen Free (SPF) environment at the Minimal Disease (MD) level, with 3-5 mice per cage. The housing conditions were carefully controlled, maintaining a temperature of 21°C±1°C, humidity at 65%, and a 12-hour light-dark cycle. Mice were provided with unrestricted access to food and water.
Murine DSS-colitis model and L-clodronate treatments
Male FVB/Ant mice (100-120 days old) were treated with drinking water containing 3% Dextran sulphate sodium (DSS; MP Biomedicals, #160110) for 7 days. During treatment, DAI (Disease Activity Index) and weight were monitored. At the end of the experiment, animals were sacrificed by cervical dislocation followed by colon length measurements. Distal colon samples were fixed in 4% PFA for histopathological assessment and immunostainings.
For clodronate treatments, DSS-treated mice and simultaneously, control animals were injected via tail vein with 200μl of liposomal clodronate suspension (Clodronate Liposomes dissolved in PBS; LMS Consult GmbH&Co. KG) on the fourth day of the protocol to deplete infiltrating macrophages. Animals were relocated to the animal facility, where DSS administration continued for the DSS-clodronate-treated group for 4 additional days. Animals in the DSS-vehicle experimental group were injected with 200 μl of control liposomes as vehicle (Control (empty) Liposomes dissolved in PBS; LMS Consult GmbH&Co. KG). Experimental design of the study is shown in Figure 1A.
Motility measurements
Experimental mice were placed individually in new bedding-free cages and monitored for 3 hours after. Measurements for all mice started after one hour with no ad libitum food or water source as baseline point. Fecal pellets were collected and weighed every 30 min. Feces were dried at 50 °C for 6 hours and re-weighed to collect dry fecal pellet weight data.
Assessment of whole gut transit time was carried out as previously described [26]. Briefly, mice were gavaged with 200 μL of the non-absorbable Evans Blue dye (Sigma-Aldrich). Period from the time of the gavage to the appearance of the first blue-colored fecal pellet was measured for every mice. Tests were ended at 600 min, mice that produced no Evans blue dyed feces until this time point were given 600 min as measurement value.
RNA-seq library preparation and sequencing
RNA extraction from whole colon samples were prepared with the RNeasy Mini Kit (Qiagen) RNA Extraction and Isolation Kit. RNA integrity numbers (RINs) were determined with the BioAnalyzer Total RNA Nano6000 kit (Agilent, Santa Clara, CA, USA). Total RNA from the distal colon samples were converted into RNA-Seq libraries with the TrueSeq Stranded mRNA HT Sample Preparation kit (Illumina, San Diego, CA, USA). Sequencing was performed on an Illumina NextSeq500 instrument using the NextSeq500/550 High Output Kit v2.5.
Quantitative real-time PCR
For qPCR analysis, the muscularis externa layer of the distal colon were separated mechanically from mucosal layers and were washed in DEPC-PBS and stored in -80C freezer. Frozen tissue samples were homogenized in TRI reagent solution (Ambion), and total RNA was extracted using a QIAGEN RNeasy minikit (QIAGEN), as directed by the manufacturer. DNase I treatment was employed to remove genomic DNA contamination, and 100mL of RNase-free DNase I (1 U of DNase) solution (Thermo Scientific) was added. NanoDrop 2000 was used to perform sample quality control and quantitative analysis. (Thermo Scientific). There was no evidence of amplification in the RT-minus controls. A high-capacity cDNA reverse transcription kit was used to create the cDNA. (Applied Biosystems). The Primer Ex-press 3.0 tool and Primer-BLAST software were used to create primers for the comparative Ct assays. Supplemental Table 1 shows the primers used in the real-time PCR reaction with Fast EvaGreen qPCR master mix (Biotium) on an ABI Ste-pOnePlus instrument (Applied Biosystems). The ABI StepOne 2.3 software was used to evaluate gene expression. Melt curve analysis on an ABI StepOnePlus PCR equipment was used to evaluate the amplicon. GADPH expression was used to standardize the results of the experiments.
Histological procedures
Colon samples from experimental mice were fixed in 4% paraformaldehyde in PBS (PFA) for 24 hours for immunofluorescence studies. The distal colon samples were obtained by removing the last 2 cm from the abdominal part of the large intestine in each mouse. Subsequently, the samples were thoroughly washed in PBS and then immersed in a medium containing 7.5% gelatin and 15% sucrose at 37 °C for 2 hours. The tissues were rapidly frozen at -60 °C using isopentane (Sigma). To prepare frozen sections for laser scanning confocal microscopy, the sections were cut at a thickness of 10 μm and collected on poly-L-lysine-coated slides (Sigma).
Immunofluorescence and image analysis
For immunofluorescence staining, primary antibodies were diluted in 1% PBS-BSA. Frozen sections were incubated with primary antibodies for Agrin (R&D Systems, #AF550), Hu (abcam, #96474), F4/80(BM8) (Invitrogen, #41-4801-82) and Col4 (abcam, #236640) overnight at 4 °C, followed by secondary antibodies (Alexa Fluor 647 conjugated anti-rabbit IgG; Alexa Fluor 555 conjugated anti-goat IgG; Alexa fluor 488 conjugated anti-rat; Invitrogen) for 1 h. Cell nuclei were visualized by DAPI. Sections were covered with aqueous Poly/Mount (Polyscience Inc.) and examined with a Zeiss LSM 780 laser-scanning confocal microscope (Zeiss). Images were compiled by ImageJ and Adobe Photoshop CS6 software package.
Cell counting and morphometry
Cell counting and morphometry analyses were conducted on images of sections obtained from distal segments of mouse colons using a Zeiss LSM 780 laser-scanning confocal microscope. To determine cell counts, tile scans of 9 images (3 horizontal, 3 vertical) at 20× magnification with a resolution of 2 megapixels (2MP) were compiled using the ZEN software package. This approach encompassed the entire cross-sectional area of the observed gut sample, as previously described in Dora et al. [16]. Morphometric analysis for cell density measurements was performed using the ZEN software package, with manual annotation of the measured areas. For cell counting of F4/80+ macrophages in the mucosa and muscularis layers, a systematic quantitative method was employed. This method involved software-assisted, manual cell counting performed by two independent observers, aided by the "cell counter" plugin of the ImageJ software package. The obtained cell counts were then compiled. To facilitate statistical analyses of cell density parameters, square micrometers (μm²) were converted to square millimeters (mm²).
Statistical analyses
To assess normality we used the Shapiro-Wilk test and Bartlett’s test was used for testing homogeneity of variances. When comparing 3 groups, we used the Kruskal-Wallis test followed by uncorrected Dunn’s multiple comparison test. P-values < 0.05 indicate the significance and all P-values were two-sided. Differential gene expression panels were filtered for genes included in GO biological processes Response to hypoxia, Response to ROS, Neuron apoptotic process, Neuroinflammatory response and Neuron death and compiled with ggrepel (0.8.2), whereas volcano plot visualization was generated with EnhancedVolcano (1.8.0) R packages. Data were analyzed and graphs were generated with GraphPad Prism 9.1.1 for Windows, GraphPad Software, San Diego, CA.