Influence of Feeding Time on a Non-steroidal Anti-inflammatory Drug-induced Small Intestinal Injury Mouse Model

Abstract

Background/Aim

Non-steroidal anti-inflammatory drugs (NSAIDs), the most widely used pharmaceuticals, induce various adverse effects, including gastrointestinal injuries, such as ulcers and bleeding. Animal models of NSAID-induced small intestinal injury (NSI) have been extensively employed for the development of preventive and therapeutic agents. However, some experimental variations related to feeding times have been observed following NSI induction. This study aimed to investigate the impact of feeding time on an NSI mouse model.

Materials and Methods

The mice were divided into eight groups: normal, sham, and model groups (with feeding times of 2 h, 6 h, 10 h, 14 h, 18 h, and 22 h; n=10 in each group). The mice were fasted for 18 h before the injection of indomethacin (15 mg/kg, subcutaneously), except for the normal group. Food supply was halted at specific time points (2 h, 6 h, 10 h, 14 h, 18 h, and 22 h); however, the normal and sham groups were continuously fed throughout the experiment. The length of the small intestine was measured, and histological analysis was performed 24 h after induction.

Results

Up to 14 h after induction, NSI, indicated by small intestine shortening, remained consistent, with a reduction in length of approximately 10-20%. However, feeding for more than 14 h significantly exacerbated NSI, both anatomically and histologically.

Conclusion

The ulcerative changes observed in the small intestine 14 h after indomethacin injection may be closely associated with the influence of food on NSI.

Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most widely used medicines worldwide (1). With the increase in the geriatric population, the use of NSAIDs has gradually risen, and their side effects, such as gastrointestinal ulcers and bleeding, have become a clinical concern (2-4). Furthermore, NSAID-induced toxicity in the distal small intestine is gaining recognition as an important area of injury, alongside the stomach (5,6). However, there are currently no standard therapeutics available for preventing or treating NSAID-induced small intestinal injury (NSI). Consequently, various studies on NSI have been conducted using animal models to develop preventive and therapeutic agents (7).

NSI is closely associated with feeding conditions, including food characteristics and accessibility. Food antigens can act synergistically with NSAIDs to exacerbate intestinal inflammation (8). Additionally, the composition of consumed food may affect the interactions between NSAIDs and the small intestine. Insoluble dietary fiber (9) or a high-fat diet (10) has been shown to exacerbate indomethacin-induced small intestinal injury, while soluble dietary fiber (11-13) reduces lesion formation and prevents NSI.

However, the feeding times in reported experimental models for small intestine damage vary widely (14-16). To the best of our knowledge, no previous studies have compared the effects of feeding time after NSI induction. In this study, we compared the effects of various feeding times on an NSI mouse model after inducing NSI.

Materials and Methods

Animals. Male C57B/L mice (6 w, weighing 20±2 g) were obtained from Daehan Bio Link (Seoul, Republic of Korea). The mice were housed at an ambient temperature of 20-22˚C with 50±10% relative humidity and had ad libitum access to Purina extrusion regular rodent chow (EEGJ30060; Cargill Agri Purina, Seongnam, Republic of Korea) and water. The mice were acclimated for 5 d before the experiments. All experimental procedures were performed following the guidelines of the International Animal Ethical Committee of Kyung Hee University. The experimental protocol was approved by the committee (approval number: KHUASP (SE)-23-211).

Experimental design. NSI was induced using a previously described method with minor modifications (11). The mice were divided into eight groups: normal, sham, and model groups (2, 6, 10, 14, 18, and 22-h feeding groups, n=10 each). The mice fasted for 18 h and were then treated with indomethacin, except for those in the normal and sham groups. Indomethacin (Sigma, St. Louis, MO, USA) was dissolved in 0.01 M Na₂CO₃, sonicated at 40˚C for 90 min, and injected at a dose of 15 mg/kg (7.5 ml/kg, subcutaneously). The sham group received the same volume of the vehicle. Food supply was stopped at each time point; however, the normal and sham groups were fed until the end of the experiment (Figure 1).

Figure 1. Experimental plan and grouping.

Anatomical and histological analyses. The small intestine was isolated, and its length was measured 24 h after induction. Subsequently, the small intestine was dissected into two pieces, with the distal half being post-fixed in 4% paraformaldehyde for 24 h and then rolled using the Swiss roll method (17). For each roll, the paraffin block was sliced into 4-μm-thick sections and stained with periodic acid-Schiff using a previously described method (11). Three investigators, who were blind to the protocol, scored the histological lesions based on a modified scoring system from a previous study (18). The severity of inflammation or ulceration was graded on a scale of 0-4 (0=none, 1=mild, 2=moderate, 3=severe, and 4=very severe). The total score was calculated by adding the inflammation and ulceration scores.

Statistical analyses. All results are expressed as mean±standard error of the mean (SEM). Data were statistically analyzed using one-way analysis of variance followed by Tukey’s post-hoc test, with a significance level of p<0.05.

Results

Analysis of small bowel length. The normal and sham groups exhibited small bowel lengths of less than 35 cm, characterized by thin, narrow, and intact morphology (without edema or hemorrhage). In contrast, the model group showed a significant reduction in small bowel length when compared to that of the normal and sham groups. Specifically, the model group exhibited a 10-20% reduction from 2 to 14 h after treatment, with this shortening becoming more pronounced in the 18-h feeding group, reaching a 30% reduction by the 22-h feeding mark (Figure 2).

Figure 2. Changes in the small intestine length of the mouse model for indomethacin-induced small intestinal injury. The photo indicates the small intestine, and the graph shows comparison of the small intestine length between groups. ANOVA followed by Tukey’s post-hoc test was used for data analysis, and the data are expressed as mean±SEM. Bars with different letters are significantly different at p<0.05. NO is the normal group.

Histological analyses. Comparing the model groups to the normal and sham groups, we observed epithelial damage, alterations in villi structure, infiltration of inflammatory cells, mucosal and submucosal disruption, and thickening of the intestinal wall. Notably, the model group fed for 2 to 14 h exhibited milder inflammatory and ulcerative changes compared to the 18- and 22-h feeding groups. The 22-h feeding group displayed significant damage to villi and mucosal and muscle areas (Figure 3).

Figure 3. Changes in the scores for histological damage in the mouse model for indomethacin-induced small intestinal injury. The photos indicate the representative histological photos of the small intestine for each group (×100). The graphs indicate the inflammation, ulceration, and total scores. White arrows indicate intact villi, asterisks indicate ulceration, and black arrows indicate recruited inflammatory cells in damaged areas. The scale bar=200 μm. ANOVA followed by Tukey’s post-hoc test was used for data analysis, and the data are expressed as mean±SEM. Bars with different letters are significantly different at p<0.05.

Discussion

In this study, we investigated the impact of feeding duration on NSI. Our findings indicate that food deprivation reduces the severity of NSI. Within the first 14 h following indomethacin injection, the small intestine length decreased by 10-20%. However, extending the feeding duration beyond 14 h significantly exacerbated small intestine shortening, reaching a 30% reduction at the 22-h mark. Histological results mirrored this trend, with inflammatory and ulcer scores remaining similar between the 2-h and 14-h feeding groups but significantly increasing in groups fed for more extended periods. Additionally, the length of the small intestine in the 14-h feeding group decreased progressively as feeding time increased, correlating with rising histology scores.

NSAIDs decrease prostaglandin (PG) levels by inhibiting cyclooxygenases and causing damage to epithelial cell mitochondria (19). This PG deficiency and mitochondrial damage increase permeability, disrupt the barrier function, and facilitate the infiltration of enteric bacteria, bile acid, and food antigens, ultimately exacerbating inflammation in the small intestine (5). These factors trigger macrophage-mediated activation of the innate immune system, leading to neutrophil infiltration, intestinal ulceration, and bleeding (20).

Food deprivation following NSI induction is a well-established factor that mitigates NSI. As early as 1982, Satoh et al. reported a lower ulcer index in the food deprivation group compared to groups that were fed shortly after indomethacin injection (21). Additionally, Satoh and Urushidani found that non-fasted groups exhibited higher lesion indexes in the small intestine compared to groups fasted for 24 h after indomethacin injection (13).

Considering the pathogenesis of NSI and the results of this study, it appears that ulcers in the small intestine may exacerbate NSI after the 14-h mark. In a rat model of NSI induced by diclofenac, Reuter et al. reported increased small intestine permeability 12 h after NSAID injection, even though ulcerative changes had not yet occurred (22). Despite variations in experimental models, it is plausible that ulceration may occur at least 12 h after NSAID injection. Furthermore, several studies have suggested that mechanical irritation of the mucosa caused by food consumption can worsen NSI (21,23). The time-dependent increase in NSI from the 14-h to 22-h feeding groups, as indicated by small intestine length and histology scores, aligns with the findings of previous studies. Therefore, this study suggests that ulceration following a 14-h period exacerbates NSI by rendering the small intestine more susceptible to food-related irritation, resulting in more severe damage.

Conclusion

Feeding regular chow pellets to NSI mouse models significantly impacts the outcomes of experiments. Moreover, the 14-h period following NSAID injection appears to be a critical time point for small intestine deterioration in NSI mouse models. Thus, researchers should carefully consider feeding duration in their experimental design to ensure greater consistency in NSI animal models. Further research into the pathological mechanisms is essential to gain a deeper understanding of the effects of food on NSI.

Funding

This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean Government (MSIT) (No. 2022R1C1C1004937).

Conflicts of Interest

The Authors declare no conflicts of interest in relation to this study.

Authors’ Contributions

S-JK and YB contributed to conceptualization, project administration, and methodology; JB and J-HK contributed to data curation and formal analysis; S-JK contributed to funding acquisition and writing the original draft; J-WP contributed to investigation, resources, and validation; YB contributed to supervision, visualization, and writing–review and editing. All Authors contributed to the manuscript preparation and approved the final paper.