Low FODMAP Diet Relieves Visceral Hypersensitivity and Is Associated with Changes in Colonic Microcirculation in Water Avoidance Mice Model

(1) Background: Irritable bowel syndrome (IBS) is a global public health problem, the pathogenesis of which has not been fully explored. Limiting fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAP) can relieve symptoms in some patients with IBS. Studies have shown that normal microcirculation perfusion is necessary to maintain the primary function of the gastrointestinal system. Here, we hypothesized that IBS pathogenesis might be related to abnormalities in colonic microcirculation. A low-FODMAP diet could alleviate visceral hypersensitivity (VH) by improving colonic microcirculation; (2) Methods: C57BL/6 mice were raised to establish an IBS-like rodent model using water avoidance (WA) stress or SHAM-WA as a control, one hour per day for ten days. The mice in the WA group were administered different levels of the FODMAP diet: 2.1% regular FODMAP (WA-RF), 10% high FODMAP diet (WA-HF), 5% medium FODMAP diet (WA-MF), and 0% low FODMAP diet (WA-LF) for the following 14 days. The body weight and food consumption of the mice were recorded. Visceral sensitivity was measured as colorectal distention (CRD) using the abdominal withdrawal reflex (AWR) score. Colonic microcirculation was assessed using laser speckle contrast imaging (LCSI). Vascular endothelial-derived growth factor (VEGF) was detected using immunofluorescence staining; (3) Results: The threshold values of CRD pressure in the WA-RF, WA-HF, and WA-MF groups were significantly lower than those in the SHAM-WA group. Moreover, we observed that colonic microcirculation perfusion decreased, and the expression of VEGF protein increased in these three groups of mice. Interestingly, a low-FODMAP dietary intervention could reverse this situation. Specifically, a low-FODMAP diet increased colonic microcirculation perfusion, reduced VEGF protein expression in mice, and increased the threshold of VH. There was a significant positive correlation between colonic microcirculation and threshold for VH; (4) Conclusions: These results demonstrate that a low-FODMAP diet can alter VH by affecting colonic microcirculation. Changes in intestinal microcirculation may be related to VEGF expression.


Introduction
Irritable bowel syndrome (IBS) is a functional gastrointestinal disease with a global impact [1,2], affecting 3.8-4.8% of the general population [3,4]. Chronic, recurrent abdomi-and intuitive technique. Moreover, we analyzed critical protein signaling pathways in intestinal vasculogenesis and angiogenesis to understand the changes in potential colonic microcirculation mechanisms.

Animals
Adult male C57/BL6 mice (20-22 g) were purchased from SLAC Laboratory Animal Co., Ltd. (Shanghai, China). The animals were raised in polypropylene cages with a light-dark cycle (light was turned off at 9:00 am, turned on at 9:00 pm, and then turned off automatically). Each cage housed 4-6 animals at a humidity of 50 ± 5% and a room temperature of 25-26 • C. Food and water were provided ad libitum. The animal research ethics committee approved the animal research protocol of Zhejiang University.

WA Stress Protocol and Grouping
Similar to the previous experiment [32,33], the customized test device included a transparent plastic tank (45 cm length × 32 cm width × 26 cm height) and a cuboid acrylic (3 cm length × 3 cm width × 9 cm height) fixed at the bottom center. The tank was filled with fresh room temperature water (25 • C) up to 1 cm below the acrylic bar. This was done according to the chronic WA stress protocol for one hour per day for ten consecutive days. The experimental animals were divided into SHAM-WA, WA-RF, WA-HF, WA-MF, and WA-LF groups, with 8-9 animals in each group ( Figure 1).
Similar to the previous experiment [32,33], the customized test device included a transparent plastic tank (45 cm length × 32 cm width × 26 cm height) and a cuboid acrylic (3 cm length × 3 cm width × 9 cm height) fixed at the bottom center. The tank was filled with fresh room temperature water (25 °C) up to 1 cm below the acrylic bar. This was done according to the chronic WA stress protocol for one hour per day for ten consecutive days. The experimental animals were divided into SHAM-WA, WA-RF, WA-HF, WA-MF, and WA-LF groups, with 8-9 animals in each group ( Figure 1).

Visceral Hypersensitivity Measurement
In mice, the abdominal withdrawal reflex (AWR) score was assessed using colorectal distension (CRD). We invented a novel and exquisite distension balloon (length 1 cm, diameter 0.5 cm), which was published previously [34]. After 30 min, the mice adapted to the environment. The balloon was lubricated with Vaseline, inserted into the rectum for 1 cm, and appropriately fixed under 2% isoflurane (RWD Life Science, Shenzhen, China) anesthesia. In the first stage, gas was continuously injected into the balloon slowly, the reaction of the mice was observed, and the corresponding pressure value was recorded. In the second stage, the balloon was promptly inflated to constant pressure (on the order of 10,20,30,40,50,60,70,80, and 90 mmHg pressure), and the AWR value of the mouse colorectum under different pressures was measured. AWR scoring standard was set as 0: no response; 1: slight head movement and no body movement; 2: abdominal muscle contraction; 3: abdominal uplift; 4: body arch-back elevation. The AWR score was measured by two independent observers using a double-blind method. Each experiment was repeated thrice to obtain an average value, and the mice were allowed to rest for 15-20 min between the 2 stages.

Visceral Hypersensitivity Measurement
In mice, the abdominal withdrawal reflex (AWR) score was assessed using colorectal distension (CRD). We invented a novel and exquisite distension balloon (length 1 cm, diameter 0.5 cm), which was published previously [34]. After 30 min, the mice adapted to the environment. The balloon was lubricated with Vaseline, inserted into the rectum for 1 cm, and appropriately fixed under 2% isoflurane (RWD Life Science, Shenzhen, China) anesthesia. In the first stage, gas was continuously injected into the balloon slowly, the reaction of the mice was observed, and the corresponding pressure value was recorded. In the second stage, the balloon was promptly inflated to constant pressure (on the order of 10, 20, 30, 40, 50, 60, 70, 80, and 90 mmHg pressure), and the AWR value of the mouse colorectum under different pressures was measured. AWR scoring standard was set as 0: no response; 1: slight head movement and no body movement; 2: abdominal muscle contraction; 3: abdominal uplift; 4: body arch-back elevation. The AWR score was measured by two independent observers using a double-blind method. Each experiment was repeated thrice to obtain an average value, and the mice were allowed to rest for 15-20 min between the 2 stages.

Immunofluorescence Staining
Frozen sections of 8 µm thickness were dried at 37 • C in an oven, then washed twice with phosphate-buffered saline (PBS) for 10 min each time, and twice with 0.3%Triton (Triton X-100, FuDebio Science, Hangzhou, China) for 10 min each time. After blocking with 2% bovine serum albumin (A1933, Sigma Aldrich, Saint Louis, MO, USA) for 1 h at room temperature, the sections were incubated with primary antibodies (anti-VEGF CAT#A0280, ABclonal, Wuhan, China) at 4 • C overnight. Next, the sections were washed with PBST 4 times for 10 min each and incubated with secondary antibodies (1:1000, Dylight594, Goat anti-rabbit, FuDebio Science) for 1.5 h at room temperature. After that, the sections were washed with PBST 4 times, 10 min each time, and PBS 2 times, 5 min each time in sequence. Antifade mounting medium with 4 ,6-diamidino-2-phenylindole (DAPI, Coolaber, Beijing, China) was added to the sections, and the slides were covered. Fluorescence signals were observed using a fluorescence microscope (Olympus VS200, Tokyo, Japan) under a 20× objective. The results were quantified using ImageJ software.

Laser Speckle Contrast Imaging
Colonic microcirculation blood flow was assessed directly using LSCI [35][36][37] (Laser Speckle Imaging system; RFLSI III; RWD Life Science, Shenzhen, China). Before initiating the study, parameters were corrected according to the manufacturer's recommendations. An approximate infrared laser source with a wavelength of 785 nm was used to irradiate the surface of the tissue of interest, with a depth of 1 mm. The movement of particles in tissues (such as red blood cells) leads to speckle patterns randomly [38]. The sliding spatial mode with a high signal-to-noise ratio was adopted, with a 5 ms exposure time. The average microcirculatory blood flow (mFlux) results of the region of interest (ROI) were recorded using a CMO camera (image resolution 2048 × 2048-pixel, 10 frames/s) for 10 s. Finally, the mean mFlux within the ROI was calculated using the laser speckle blood flow imaging system v5.0.
The specific procedures were as follows: after isoflurane gas anesthesia was administered, the mice were placed in a supine position on a black background, the limbs were fixed with double-sided adhesive tape, the abdominal hair was removed with an electric razor, and a longitudinal incision was cut in the midline. All the intestinal segments were completely exposed. On the premise of not changing the original position of the mouse intestinal tube, each intestinal segment of the mouse was skillfully identified. To maintain moisture, exposed intestinal segments must be coated with normal saline. The laser probe was fixed 15 cm above the area to be measured. mFlux of the proximal colon was measured, and then the intestine was unfolded into a fan shape, and the mFlux of the proximal colon and the mesentery of the colon was investigated again.

Statistic Analyses
Statistical analyses were performed using SPSS23.0 (IBM, Armonk, NY, USA) and GraphPad Prism9.0 (GraphPad Software Inc., San Diego, CA, USA). All data are expressed as the mean ± SEM. Verifying that the original data had a normal distribution, body weight, food intake, and CRD measurements were analyzed using two-way ANOVA followed by Sidak's multiple comparison tests. The remaining results were analyzed using a one-way ANOVA followed by Bonferroni's post-hoc test. Correlation coefficients (r) were calculated using Pearson's correlation coefficient. p < 0.05 was considered statistically significance.

Body Weight Change and Dietary Consumption
We observed fluctuations in the weight of the mice during WA. The weight of DAY0 is the baseline, and DAYn-DAY0 expresses the weight change. The results showed no statistical difference between the control and model groups during the WA stress ( Figure 2A). During the diet intervention, body weight and feed consumption were measured every three days. Average food consumption = (original amount − residual amount)/number of days/number of mice in each cage. The results showed no significant differences in body weight and average feed consumption among the groups ( Figure 2B,C).
During the diet intervention, body weight and feed consumption were measured every three days. Average food consumption = (original amount − residual amount)/number of days/number of mice in each cage. The results showed no significant differences in body weight and average feed consumption among the groups ( Figure 2B,C). WA, Water avoidance; RF, regular FODMAP; HF, high FODMAP; MF, medium FODMAP; LF, low FODMAP. n = 8~9. Data were expressed as mean ± SEM by two-way ANOVA followed by Sidak's multiple comparisons tests.
Under the pressure of 10-90 mmHg order, the AWR scores in the SHAM-WA, WA-RF, WA-HF, WA-MF, and WA-LF groups were evaluated. The data were analyzed using two-way ANOVA (diet × pressure) followed by Sidak's multiple comparisons test, interaction: F(32, 180) = 240.8, p < 0.0001. It had a significant main effect on dietary intervention: F(8, 180) = 240.8, p < 0.0001; moreover, it also had a significant main effect on pressure: F(4, 180) = 68.20, p < 0.0001. There was no significant difference at 80 and 90

Colonic Microcirculation Blood Flow Decreased in the WA-RF, WA-HF, and WA-MF Groups, and Was Reversible in the WA-LF Group
Due to physiological hypoxia in healthy individuals, intestinal blood flow is vulnerable to microcirculation disorders and intestinal ischemia [39]. Laser speckle blood flow is a powerful noninvasive tool [40], which can be used as a visual method to dynamically measure changes in intestinal and mesenteric microcirculation blood flow in vivo [41]. This study presents the first data on the use of live real-time imaging tools in mice to focus on the changes in colon blood flow in IBS model mice and FODMAP diet intervention.
In the original position, the results of the colonic mFlux in SHAM-WA, WA-RF, WA-HF, WA-MF, and WA-LF groups were 618.97 ± 24.03, 352.04 ± 40.07, 348.32 ± 46.73, 359.80 ± 36.69, and 654.66 ± 50.70 Perfusion Unit (PU) (Figure 5A,B). In the fan-shaped position, the results of mesenteric vessels mFlux were 594.74 ± 15.14, 575. 23 Figure 5C-E). The results showed that the colonic mFlux in the WA-RF, WA-HF, and WA-MF groups was lower than that in the SHAM-WA group in the original and fan-shaped positions. The low-FODMAP diet intervention could reverse this situation. There was no significant difference in the mFlux of the mesenteric vessels supplying the colon among the groups. Therefore, the decrease in colonic microcirculation blood flow perfusion after WA stress and FODMAP diet intervention may have been caused by local colon lesions.

Colonic Microcirculation Blood Flow Decreased in the WA-RF, WA-HF, and WA-MF Groups, and Was Reversible in the WA-LF Group
Due to physiological hypoxia in healthy individuals, intestinal blood flow is vulnerable to microcirculation disorders and intestinal ischemia [39]. Laser speckle blood flow is a powerful noninvasive tool [40], which can be used as a visual method to dynamically measure changes in intestinal and mesenteric microcirculation blood flow in vivo [41]. This study presents the first data on the use of live real-time imaging tools in mice to focus on the changes in colon blood flow in IBS model mice and FODMAP diet intervention.
In  (Figure 5A,B). In the fan-shaped position, the results of mesenteric vessels mFlux were 594.74 ± 15.14, 575. 23 Figure 5C-E). The results showed that the colonic mFlux in the WA-RF, WA-HF, and WA-MF groups was lower than that in the SHAM-WA group in the original and fan-shaped positions. The low-FODMAP diet intervention could reverse this situation. There was no significant difference in the mFlux of the mesenteric vessels supplying the colon among the groups. Therefore, the decrease in colonic microcirculation blood flow perfusion after WA stress and FODMAP diet intervention may have been caused by local colon lesions.

VEGF Fluorescence Intensity Increased in the WA-HF Group and Decreased in the WA-LF Group
We observed the colonic submucosa layer, which accounts for 70% of the intestinal blood flow, and the immunofluorescence intensity of VEGF protein in the SHAM-WA, WA-RF, WA-HF, WA-MF, and WA-LF groups were 92.75 ± 5.03, 130.83 ± 9.39, 145.01 ± 7.37, 128.06 ± 6.11, 108,48 ± 6.65. The result showed that the expression of VEGF in the WA-RF, WA-HF, and WA-MF groups was significantly higher than that in the SHAM-WA group. However, the level in the WA-LF group was significantly lower than that in the WA-HF group ( Figure 6A,B).

Colonic Microcirculation Blood Flow Was Positively Correlated with the Threshold of VH
When the AWR scores were 1, 2, 3, or 4, there was a positive correlation between colonic microcirculation blood flow and the threshold of VH, with 95% confidence bands displayed by dashed lines (r = 0.632, 0.637, 0.467, and 0.657; p= 0.0007, 0.0003, 0.0002, and 0.0004, respectively) ( Figure 7A

Discussion
In this study, we provided evidence that regular FODMAP, high FODMAP, and middle FODMAP diet interventions can aggravate VH, while low FODMAP can alleviate WA stress in mice. In addition, colonic microcirculation blood flow decreased in the WA-RF, WA-HF, and WA-MF groups, leading to colonic microcirculation dysfunction, while the expression of VEGF protein increased. Moreover, we found that a low-FODMAP diet can increase colonic blood perfusion and reduce VEGF protein expression.
There is evidence that diet is closely related to the balance of intestinal microecology. Dietary restriction may be essential in altering microbiota composition in people with IBS. However, the results are inconsistent. Some studies demonstrated that compared with low FODMAP and high FODMAP intervention, there was no difference in α-diversity and β-diversity [42]. In contrast, other studies showed that the total abundance of low FOD-MAP decreased [43]. Short-term restriction of the FODMAP diet can increase the abundance of Bacteroides, Firmicutes, Clostridium, and Actinobacteria while the abundance of Bifidobacteria decreases [44,45]. Among them, Bacteroides is a kind of bacteria related to the degradation of sugars [46].
VH is one of the most critical characteristics of IBS pathogenesis [47]. VH for mechanical stimulation of the colon is common among patients with IBS. Utilizing the rectal balloon distention method, evidence showed lower thresholds for visceral discomfort and

Discussion
In this study, we provided evidence that regular FODMAP, high FODMAP, and middle FODMAP diet interventions can aggravate VH, while low FODMAP can alleviate WA stress in mice. In addition, colonic microcirculation blood flow decreased in the WA-RF, WA-HF, and WA-MF groups, leading to colonic microcirculation dysfunction, while the expression of VEGF protein increased. Moreover, we found that a low-FODMAP diet can increase colonic blood perfusion and reduce VEGF protein expression.
There is evidence that diet is closely related to the balance of intestinal microecology. Dietary restriction may be essential in altering microbiota composition in people with IBS. However, the results are inconsistent. Some studies demonstrated that compared with low FODMAP and high FODMAP intervention, there was no difference in α-diversity and β-diversity [42]. In contrast, other studies showed that the total abundance of low FODMAP decreased [43]. Short-term restriction of the FODMAP diet can increase the abundance of Bacteroides, Firmicutes, Clostridium, and Actinobacteria while the abundance of Bifidobacteria decreases [44,45]. Among them, Bacteroides is a kind of bacteria related to the degradation of sugars [46].
VH is one of the most critical characteristics of IBS pathogenesis [47]. VH for mechanical stimulation of the colon is common among patients with IBS. Utilizing the rectal balloon distention method, evidence showed lower thresholds for visceral discomfort and pain in the majority of patients with IBS [48][49][50]. It has been shown that WA is an effective model of inducing VH in animals [33,51]. Similar to the previous experiment, there was no statistical difference between the groups in terms of weight and feed consumption in the present study, eliminating other factors affecting VH. However, the mechanism underlying VH in IBS is complex. It is now believed that intestinal barrier destruction, abnormal activation of silent gastrointestinal nociceptors, peripheral intestinal nerves, and central nervous system sensitization are also involved [52,53]. Here, we found that VH significantly correlated with colonic microcirculation.
To the best of our knowledge, the present study is the first to reveal the changes in colonicmicrocirculation in an IBS mouse model and directly after FODMAP diet intervention. Two potential central mechanisms may support these results. First, regional intestinal blood-flow-regulating factors affect intestinal blood flow. Second, changes in blood flow result from altered autonomic nervous system function and neurotransmitters. In this experiment, we confirmed that there was no statistical difference in the blood flow of the mesenteric artery supplying the proximal colon among the groups, which suggests that regional intestinal segment lesions caused changes in the blood flow of the colonic microcirculation. Nevertheless, the causal relationship between colonic microcirculation reduction and IBS pathogenesis in the WA-HF group remains to be determined. Interestingly, this phenomenon can be ameliorated by a low-FODMAP diet by dilating microcirculation and improving blood infusion.
VEGF is a powerful factor in vasculogenesis and angiogenesis processes in both development and pathological conditions, mainly targeting endothelial cells; it is also known as VEGF-A [54]. Moreover, VEGF is considered an inflammatory factor that can affect vascular permeability and regulate the recruitment of inflammatory cells in colitis diseases [55]. Low-grade mucosal inflammation is considered one of the causes of IBS [56].
Here, we verified that the expression of VEGF protein was significantly higher in the HF group and significantly reduced in the LF group, indicating that the number of microvessels might increase in the HF group. A small-sample study showed that the serum VEGF level in IBS patients was higher than in ordinary people [57]. Another double-blinded randomized trial discovered that the severity of IBS symptoms in patients decreased, and circulating levels of proinflammatory cytokines, including IL-6, IL-17, and interferon-γ were significantly reduced, as well as VEGF, after turanicum wheat dietary intervention [58]. Christina et al. focused on the treatment of stress-related and psychological IBS. They found that after the one-year experience with choirs, VEGF levels were higher in both choir and nonchoir groups [59]. Experiments in other tissues confirmed that in the brain and retina, VEGF signaling is closely related to leukocyte adhesion and capillary blockage [60,61]. Nevertheless, there remains a knowledge gap associated with the mechanism of the VEGF pathway signaling and tissue microcirculation, and more research needs to be conducted.
IBS is believed to be linked to disruptions in autonomic nervous system regulation, which can damage vascular homeostasis and cause IBS symptoms and exaggerated responses to stress [62,63]. Tanaka et al. measured finger blood perfusion using laser doppler blood flow, an indicator of sympathetic nerve function. It was found that the blood flow response of IBS patients decreased, indicating excessive sympathetic nerve activity [64]. A separate study showed that rectal blood flow was decreased in IBS due to excitation of the extrinsic colonic vagus nerve and a deficit in cholinergic activity [65]. Based on this evidence, it is reasonable to speculate that sympathovagal imbalance or dysfunction may result in altered colonic microcirculatory flow in IBS patients.
Currently, diet management is considered one of the most critical decisions in IBS management strategies, especially FODMAP food restriction [1]. The research has shown that FODMAP aggravates VH by increasing the water volume in the small intestine and gas production in the colon [14]. Zhou et al. revealed that a FODMAP diet modulates barrier dysfunction, thus activating visceral pain [18]. In the meantime, too much gas and/or water fermented by intestinal bacteria can lead to an intraluminal pressure increase. Boley et al. showed that colonic blood flow decreased stepwise with increasing cavity pressure [66], although the mesenteric blood pressure remained unchanged. Different diameters of the local intestinal cavities lead to a stretch of the intestinal cavity at the expansion site [67], which may potentially induce abdominal pain. In the present study, the threshold for VH in the WA-HF group was lower than that in the WA-RF group. However, we did not observe that colonic microcirculation in the WA-HF group was less than that in the RF group.
This study had several limitations. First, the results of this study are limited to a mouse model of WA stress, although it is a classic model simulating the preclinical experiment of IBS. The mechanism underlying low FODMAP requires further investigation in clinical practice. Second, the side effects of FODMAP in the body require further research and confirmation. Third, the study's investigation of intestinal microcirculation focused on the colon. Further research is required to determine the effects of different doses of FODMAP on microcirculation in other regions of the intestine.

Conclusions
Our study showed that a low-FODMAP diet could alleviate VH by improving colonic microcirculation. Colonic microcirculation blood flow was positively correlated with the threshold of VH. This may provide a theoretical basis for limiting the FODMAP diet for IBS management. Institutional Review Board Statement: The animal study protocol was approved by the Ethics Committee for Animal Research at Zhejiang University (19420) and included efforts to minimize suffering and distress.