Female albino Wistar rats (200–300 g) were used in these experiments (4–10 animals per experimental group). Rats were housed at 22 ± 1°C and 60% relative humidity, under a 12-h light–dark cycle, with free access to food and water.

We employed an experimental UC model based on the oral administration of DSS (molecular weight 36–50 kDa; PanReac AppliChem, Spain. Batch No. 9J013322) at a concentration of 5% (w/v) in drinking water. In this paradigm, female rats received DSS ad libitum for 1 week, following the method previously described by Okayasu (13). This was followed by 2 weeks of tap water and a final week of DSS administration, resulting in a total treatment duration of 28 days. Control animals received tap water throughout. A timeline of the DSS treatment and the parameters assessed is shown in Figure 1A.

Typical effects of DSS exposure, including rectal bleeding, diarrhea and weight loss, were evident by the end of treatment. In addition to these clinical signs, histopathological alterations in the gut resembled those observed in human UC. At the conclusion of the treatment period, animals were euthanized, and distal colons and brains were collected. The left and right hemispheres of each brain were analyzed separately. All animal procedures were conducted in accordance with the Guidelines of the European Union Directive (2010/63/EU) and Spanish regulations (BOE 34/11370-421, 2013) governing the use of laboratory animals. The study was approved by the Scientific Committee of the University of Seville.

The DSS treatment and subsequent measurements in female rats were conducted in parallel with those performed in male rats. Data obtained from the male cohort were previously published (9). Due to the severity of the UC-inducing treatment and in accordance with the 3R principles of replacement, reduction, and refinement, the study was not repeated in males. Instead, the previously published male data were used to calculate fold changes (FC DSS/water), which were then compared with the results from female rats to investigate potential sex differences.

The cylinder test is an ipsilateral-specific behavioral assay in which rats are placed inside a glass cylinder (19 cm in diameter, 35 cm in height) for 5 min without prior habituation. During spontaneous exploration, rats make repeated contacts with the cylinder walls using their forepaws. The number of wall touches made with each forepaw is recorded, and a right/left paw use ratio is calculated. All sessions were video-recorded for subsequent analysis. An imbalance in right versus left paw touches is indicative of ipsilateral motor impairment, making this test one of the most appropriate and reliable tools for detecting lateralized motor deficits, particularly those involving subtle hemispheric differences (14).

For histological assessment, paraffin-embedded sections of the distal colon were stained with hematoxylin/eosin. Analysis was performed in a blinded manner using a validated method (15). Colon damage was graded on a scale of 0–3 based on epithelial destruction, crypt dilatation, loss of goblet cells, inflammatory cell infiltration, oedema, and crypt abscesses. Each parameter was scored from 0 to 3, and the individual scores were summed to generate a cumulative damage score.

Animals used for TH immunohistochemistry underwent 28 days of treatment before being perfused via the heart under deep anesthesia (isofluorane) with 150–200 ml of saline, followed by 150–200 ml of 4% paraformaldehyde in phosphate buffer (pH 7.4). After perfusion-fixation, brains were carefully removed and cryoprotected sequentially in sucrose solutions prepared in phosphate-buffered saline (PBS, pH 7.4): first in 10% sucrose for 24 h, and then in 30% sucrose until the brain sank (2–5 days). Brains were subsequently frozen in isopentane at -80°C. All incubations and washes were performed at room temperature (RT) in either Tris-buffered saline (TBS) or PBS, pH 7.4. Primary and secondary antibodies used are listed in Table 1. Coronal sections (20 µm) were cut on a cryostat at -20°C and thaw-mounted onto gelatin-coated slides. Sections were washed and then treated with 0.3% hydrogen peroxide in methanol for 20 min, followed by additional washes. Sections were then incubated in a humid chamber for 1 h in a blocking solution containing PBS, 0.1% Triton-X-100, 1% bovine serum albumin (BSA; Sigma), and 2% goat serum. Slides were drained and incubated with the primary antibody in a solution containing PBS, 0.5% Triton-X-100, 1% BSA, and 2% goat serum for 12 h at 4°C in a humid chamber. Following primary antibody incubation, sections were washed three times for 10 min each and then incubated with a biotinylated secondary IgG for 2 h. The secondary antibody was diluted in PBS containing 0.5% Triton X-100, 1% BSA, and 2% goat serum. Sections were subsequently incubated with VECTASTAIN^®-Peroxidase solution (Vector; 1:100). Peroxidase activity was visualized using a standard diaminobenzidine (DAB)/hydrogen peroxide reaction for 5 min. Finally, slides were mounted in DPX (BDH Laboratories).

For immunofluorescence, animals also underwent 28 days of treatment before being perfused. Immunofluorescence staining for α-syn, MAP-2, and TH was performed on coronal brain sections. Sections were permeabilized with 1% Triton X-100 in PBS for 1 h, blocked with 5% (w/v) BSA and 1% Triton X-100 in PBS for 1 h, and then incubated overnight at 4°C with the primary antibodies diluted in 1% (w/v) BSA and 1% Triton X-100 in PBS. The following day, sections were rinsed for 1 h in PBS containing 0.1% Triton X-100 and subsequently incubated for 1 h with the corresponding secondary antibodies diluted in 1% (w/v) BSA and 0.1% Triton X-100 in PBS. Sections were then washed again in 0.1% Triton X-100 in PBS for 1h to remove excess of secondary antibodies. Finally, sections were counterstained with Hoechst 33258 (1:3000 dilution; Invitrogen) to visualize cell nuclei and mounted in 50% glycerol. Primary and secondary antibodies used are listed in Table 1.

For immunofluorescence detection of P-α-syn and UCHL-1 in distal colon samples, 5-μm-thick tissue sections were cut from paraffin blocks, dewaxed in xylene, and rehydrated through a series of graded alcohols. Sections were permeabilized by boiling in 0.01 M sodium citrate (pH 6) for 10 min. After blocking with 3% BSA, 3% fetal calf serum, and 0.1% Triton X-100 in PBS for 1h at RT, slides were incubated overnight at 4°C with the primary antibodies. Antibody binding was visualized using the appropriate fluorescent secondary antibodies. Nuclei were counterstained with Hoechst 33258 (1:3000 dilution; Invitrogen), and sections were mounted in 50% glycerol. Primary and secondary antibodies used are listed in Table 1.

For the analysis of immunofluorescence in colon sections, images were acquired using an Olympus BX61 microscope equipped with an Olympus DP73 camera. Quantification was performed using Image-J software. Five immunolabeled, non-consecutive, distal colon sections per rat were analyzed, with 9–15 fields examined per section to cover representative areas of each colon layer. An average of all measurements for each rat was calculated.

The number of TH-positive neurons in the SNpc was estimated using a fractionator sampling design (16). Because unilateral tremor is one of the earliest motor symptoms of PD, the left and right sides were analyzed separately. Counts were performed at regular predetermined intervals (x = 150 µm, y = 200 µm) within each section. The counted region spanned a thickness of 840 µm, corresponding to plates 38–42 in the atlas of Paxinos and Watson (17). Systematic sampling of the SNpc area was initiated from a random starting point. An unbiased counting frame of known area (40 µm x 25 µm = 1000 µm²) was superimposed on each tissue section image under a 60x objective, yielding an area sampling fraction of 1000 µm²/(150 µm x 200 µm) = 0.033. The entire z-dimension of each section (20 µm for all animals) was sampled, giving a section thickness sampling fraction of 1. Sections spaced 100 µm apart were analyzed (one section out of every five), resulting in a fraction of sections sampled of 20/100 (0.20). Figure 1B shows a schematic of the process. The total number of TH-positive neurons in the SNpc was estimated by multiplying the number of neurons counted within the sample regions by the reciprocals of the area sampling fraction and the fraction of sections sampled. To extrapolate the total number of neurons in the structure from the sampled volume using the dissector method, the software applies the Cavalieri principle, ensuring an unbiased final estimate.

For immunofluorescence quantification in the SNpc, the fluorescence intensity of TH-positive neurons was analyzed in six regions of interest (ROIs), three per hemisphere, in sections from six control females and seven females subjected to the DSS model. Images were acquired using an inverted ZEISS LSM 7 DUO confocal laser scanning microscope under identical laser intensity and gain settings. All images were captured at the same Z-plane to ensure consistent optical depth. Quantification was performed using ImageJ software (public domain: http://rsb.info.nih.gov/ij/download.html) without any additional image processing. To control for potential variability in tissue permeability across sections, Hoechst staining was also analyzed.

Left and right SN and distal colon samples were dissected from each rat after 28 days of treatment, snap-frozen in liquid nitrogen, and stored at −80°C. Total RNA was extracted using the RNeasy^® kit (Qiagen). Complementary DNA (cDNA) was synthesized from 1 μg of total RNA using the QuantiTect^® reverse transcription kit (Qiagen) in 20 μl reaction volume, following the manufacturer instructions. Real-time PCR was performed using iQ™SYBR^® Green Supermix (Bio-Rad Laboratories) with 0.4 μM primers and 1 μl of cDNA. Negative controls were run without cDNA. Amplification was carried out on a Mastercycler^® ep realplex (Eppendorf) thermal cycler with an initial denaturation at 94°C for 3 min, followed by 35 cycles of 94°C for 30 s, 55°C for 45 s, and 72°C for 45 s, and a final elongation step at 72°C for 7 min. A melting curve analysis was performed immediately afterward by heating the reactions from 65 to 95°C in 1°C increments while monitoring fluorescence, confirming the presence of a single PCR product at the expected melting temperature. β-actin served as the reference gene for normalization. Primer sequences for IL-1β, TNF, IL-6, ICAM-1, arginase, IL-10, and β-actin are provided in Table 2. The cycle threshold (Ct) for each sample was determined, and the triplicate values for each cDNA were averaged. Relative quantification was performed using the comparative Ct method integrated into the Bio-Rad System Software.

Statistical analyses were performed using GraphPad Prism 8.4.0. Prior to analysis, all data were assessed for normality using the Shapiro-Wilk test; some datasets passed the normality test, while others did not. To ensure consistency, comparisons were performed using either Mann-Whitney U test for two independent groups or two-way ANOVA followed by Sidak’s multiple comparisons test for analyses involving more than two groups. Sidak’s test includes a correction for multiple comparisons. Data are presented as mean ± standard deviation (SD). The study of statistical power and n-value was performed using the G*Power 3.1.9.4 software.

To calculate the FC, the average of the control group (water) and the treated group (DSS) is found, and the treated average is divided by the control average. The Log2 of the FC has also been calculated. Figure 2 shows a volcano plot summarizing the statistical significance of the FCs described throughout the text.

The statistical significance of each FC (DSS/water) was assessed using the Mann-Whitney U test. Differences in FCs between females and males were studied using two-factor ANOVA, where the factors were sex and treatment. The level of statistical significance of the interaction between these two factors indicates whether there are differences between the FCs of females and males for each parameter studied.

The study of statistical power and n-value was performed using the G*Power 3.1.9.4 software. Due to the severity of the UC-inducing treatment and in accordance with the 3R principles of replacement, reduction, and refinement, the number of animals per group was minimized taking into account the effect size observed for the different parameters in our previous study in males. The statistical power of the analysis of the parameters showing a significant difference between the control and DSS-treated animals ranged from 0.75 to 1. The parameters without statistical significance, showing a very small effect size (Cohen’s d), showed low powers (between 0.05 and 0.67). The a priori study using Cohen’s d to calculate the n-value needed to achieve a power of 0.8 for these parameters yielded values so high (hundreds of animals) that they cannot be used.

Female rats received DSS or normal drinking water according to the experimental design illustrated in Figure 1A. First, we aimed to determine whether the experimental UC model induced inflammation and damage in the colon of female rats and whether the response differed from that in males (data previously published in (9)). To assess this, histological analysis was performed on distal colon sections stained with hematoxylin and eosin from DSS-treated and untreated (control) female rats. A total histological score was calculated as the sum of six parameters (each scored from 0 to 3, 0 = healthy, 3 = severely damaged): epithelial/glandular destruction, crypt dilatation, goblet cells loss, inflammatory cell infiltration, edema, and crypt abscesses. Representative images in Figure 3A illustrate the loss of crypt architecture and infiltration of inflammatory cells in the mucosa and submucosa of the colon in both female and male DSS-treated rats. Rats with colitis exhibited significant colonic damage compared to controls, as indicated by an increased total histological score (Figure 3B; p < 0.0001; n = 6-10. See also Figure 2 for the significance of the FCs), with no difference observed between sexes (Figures 3B, C). Analysis of each parameter individually (Figure 3C) further demonstrated that DSS treatment induced comparable colon injury in male and female rats.

In addition, we assessed the relative mRNA expression of the pro-inflammatory cytokines tumor necrosis factor (TNF), interleukin (IL)-1β, and IL-6 in the colon of these animals (n= 4-5) using RT-qPCR. In female DSS-treated rats, TNF expression was significantly increased (231.0% of controls, p= 0.0286; Figure 4A), as was IL-1β expression (935.8% of controls, p= 0.0079; Figure 4B); whereas IL-6 expression showed no significant changes (Figure 4C; See also Figure 2). The comparison of FCs of the relative expression of cytokines showed no significant sex differences (Figures 4D-F).

We next investigated whether our UC model induces pathological α-syn aggregates in the colon. Immunofluorescence analysis of distal colon sections was performed to assess the accumulation of phosphorylated α-syn (P-α-syn) in female DSS-treated rats. This region was selected because DSS-induced pathology is largely restricted to the distal colon, and its histological features closely mirror those observed in human UC. Co-localization of P-α-syn with the pan-neuronal marker ubiquitin C-terminal hydrolase (UCHL)-1 demonstrated the presence of P-α-syn within the enteric nervous system, including both the myenteric and submucosal plexuses of rats with UC (Figures 5A, B). P-α-syn was detected in mucosal nerve fibers surrounding the crypts, as well as in neuronal somas and fibers within the submucosal and muscular externa layers. P-α-syn immunoreactivity was also observed in non-neuronal cells, likely of immune or glial origin. Quantification of fluorescence intensity (n = 6-10) revealed a significant increase in P-α-syn signal in the mucosa (523%; p= 0.0095) and muscularis externa (326%; p= 0.0214) of DSS-treated female compared with controls, but not in the submucosa (100%) (Figure 5C; see also Figure 2). FC analysis (DSS/water) performed for each colon layer showed the same pattern in male rats, with no sex-related differences (Figure 5D).

Because our DSS treatment induces inflammatory signs and symptoms, as well as the accumulation of toxic α-syn aggregates in the colon of female rats, we next investigated whether this peripheral inflammation could extend to the SN, as previously reported in males (9). To assess neuroinflammation in the SN of DSS-treated females, we performed RT-qPCR for several pro- and anti-inflammatory cytokines, including the pro-inflammatory markers TNF, IL-1β, and IL-6 (Figures 6A-C), and the anti-inflammatory markers arginase-1 and IL-10 (Figures 6G, H). We also examined expression of the intercellular adhesion molecule (ICAM)-1 due to its role in immune-cell infiltration (Figure 6I). No significant differences in the expression of any of these genes (n = 6-9) were detected compared with water-treated controls. FC (DSS/water) for mRNA expression in female rats were non-significant (Figure 2).

However, FC values were consistently higher in males (Figure 2) than in females across all markers examined (Figures 6D-F, K, L), with the exception of arginase-1 (Figure 6J), which showed no sex-related difference.

We performed immunofluorescence analyses on brain sections to assess the intensity of α-syn, microtubule-associated protein (MAP)-2, and TH staining in the SNpc of female DSS-treated and control rats, and found no significant differences (Figures 7A-C, E-H). As a control for potential tissue-processing variability, we quantified Hoechst mean fluorescence intensity across all sections (Figure 7D), which showed no significant differences (Figure 7H; n = 6-7). FC analysis revealed lower MAP-2 (39.5%) and TH (38.6%) signal intensities in female rats compared with males (Figure 7I; see also Figure 2).

We also employed an unbiased stereological approach to quantify TH-positive neurons in the SNpc of female animals following DSS treatment. The analysis revealed no significant differences between DSS-treated and control groups (control: 6823 ± 860 and 6762 ± 1005 TH-positive cells in the left and right SNpc, respectively; DSS: 6752 ± 723 and 6656 ± 870 TH-positive cells in the left and right SNpc, respectively; Figures 8A-C; n = 6-9). These findings are consistent with motor-behavior assessments using the cylinder, which showed no significant differences between DSS-treated and control females (Figure 8D; n = 6).

FC analysis showed no differences in behavioral performance (Figure 8F; n = 6; see also Figure 2). However, it revealed a significant reduction of TH-positive neurons in the right SNpc of males (40%, p = 0.0021; Figure 8E; n = 6-9).