Use of sildenafil and l-arginine in an experimental rat model for the prevention of necrotizing enterocolitis neonatal

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Formula feeding, hypoxia, and hypothermia are considered risk factors for the development of NEC. The mortality rate of the disease varies from 10 to 50% in newborns treated in neonatal intensive care units12.13. In this study, the mortality rate after NEC induction in newborn rats was 35%, compared to a mortality rate of 38% observed by other authors who used the same experimental model.14.

Contrary to expectations, Group C, which received only I-arginine, had the highest mortality rate and the lowest survival rate compared to the other groups. It is difficult to determine which mechanisms are responsible for the adverse effect of I-arginine. We wondered if the substrate supply for NO synthesis, with its consequent increased production, would be the stressor of tissue damage.

A recent study by Alexandre’s group defined a dual effect of nitric oxide and therefore arginine. Septic guinea pigs supplemented with low-dose arginine recovered better than controls, while supplementation with high doses of arginine led to catastrophic results15. The dose of I– the arginine used in this study: was it excessive as a preventive measure and therefore harmful or was it too weak to favor the reducing effect on the intestinal lesions? In our study, 0.1 mL of 10% I-the arginine solution was used intraperitoneally, as in the study by Cintra et al.ten However, Cintra et al.ten described a protective effect as they show a lower degree of hystological lesions in the intestine of rats that received I-arginine, contrary to the results found in our study. Our study method differs from that of Cintra et al.tenwe did not promote reoxygenation (100% O2) and heating (22°C) after episodes of hypoxia and hypothermia16.17. Perhaps reoxygenation and warming potentiate the protective action of I-arginine in intestinal lesions of NEC. Hypoxia and hypothermia associated with repeated trauma from intraperitoneal administration of I-arginine possibly leading to intestinal perforation.

Group B, which received only sildenafil, had the lowest mortality rate among the groups undergoing drug intervention, which was statistically significant. This difference could be due, above all, to the increase in intracellular levels of cGMP, responsible for the stimulation of NO production. However, it should be noted that group E, undergoing the NEC protocol but not receiving the intervention drug, obtained the highest lifespan among the NEC groups. Therefore, our study contradicts the current literature by obtaining a higher mortality rate in the NEC groups who received intervention measures with sildenafil and I-arginine. Perhaps the explanation for this fact can be the administered dose of sildenafil and I-arginine, which may have been toxic to newborn rats or the many intraperitoneal applications may have induced a very intense metabolic and endocrine immunological response to trauma that could progress to death. Could it be that if the doses of sildenafil and I– the arginine had lower concentrations or if the intraperitoneal applications were made with an interval greater than 12 hours or if the mode of administration of the drugs was different from that intraperitoneal, the results would be different?

We observed that the final body weight of the groups subjected to the NEC protocol was significantly lower compared to the negative control group, which received breast milk. Other authors have obtained similar results, showing weight loss in formula-fed animals.12.14. An exception is a study by Dvorak et al.12which detected an increase in body weight in the groups subjected to the NEC protocol.

Regarding the degree of histological damage, the results were similar to those reported in other experimental studies. The microscopic changes induced by NEC were such as flattening of the villi, reduction in the number of villi and thinning of the intestinal wall18.19. As shown in Figure 5, our study did not find villous edema, layered edema, layer separation, and villus desquamation like other studies.12.20. The likely explanation for this would be the immediate fixation of the jejunum and terminal ileum in 10% buffered formaldehyde, which we performed shortly after material removal. This fixation could have avoided slide artifacts, which could have been considered injury findings in other studies.

We observed that intestinal lesions exhibited high intensity and structural damage in group E animals, which were subjected exclusively to hypoxia, infant formula, and hypothermia conditions. Therefore, the accuracy of the protocol in the induction of NEC was confirmed.

The experimental model also demonstrated a higher proliferation index in the groups that received the interventional drugs compared to the positive control group. However, the use of sildenafil and I-arginine was unable to prevent the development of NEC. One hypothesis is that the intestine of newborn rats is incapable or immature of synthesizing NO. Another reason is that there might not be enough eNOS available or able to synthesize NO when the supply was high. Sukhotnik and Cols found that exposure of rats to I-arginine does not prevent the intestine from ischemic damage, but reasonably accelerates the repair of the damaged intestinal mucosa21,22,23. Another study showed that I– arginine is not able to inhibit the occurrence of intestinal lesions produced by hypoxia/hypothermia. Rather, it showed a higher level of NO and a lower degree of morphological changes in the gut wall of the animals.ten.

The present study also observed whether there would be an additive effect when co-administering sildenafil and I-arginine to newborn rats. As verified by the results of group D, we can affirm that there was a reduction in intestinal lesions (with an average of 26.66) and a higher proliferation rate (68.33%) compared to the other groups subject to protocol. However, such a reduction was not statistically significant. Therefore, this would not justify the combination of substances.

In conclusion, the results of the present study demonstrate that the administration of sildenafil and I– arginine is not able to prevent the appearance of intestinal lesions produced by hypoxia, formula feeding and hypothermia. Nevertheless, we found a slight reduction in morphological changes in the intestinal wall of animals given the two substances simultaneously, but no statistically significant change in body weight and intestinal lesions between the groups receiving sildenafil and I-arginine combined or individually compared to the NEC control group. Perhaps the timing of drug administration was ineffective in observing significant improvement in bowel lesions, although Cintra et al.ten also took a 12 hour break between I– the administrations of arginine made for 3 days, or the NEC induction model was so effective that it caused a very serious intestinal lesion which could not be prevented by a dose previously tested in other studies . The difference between our study and the experimental model of Jilling et al. and adapted by Gonçalves et al.16.17 it concerns the time of exposure to 100% nitrogen and the administration of the enteral diet. In our study, rats were kept under 100% nitrogen for 70 seconds and fed 0.1 mL of Esbilac® every 4 hours, and the experimental model of Jilling et al. and adapted by Gonçalves et al.16.17 left the rats under 60 seconds in an atmosphere of 100% nitrogen and fed with 0.1 mL of Esbilac® every 3 hours.

Our study is relevant both for its innovative character of testing sildenafil with I-arginine as a preventative strategy for NEC, as well as adding new data on I-the literature on arginine, contradicting previously published studies, which point to these drugs as preventive measures for necrotizing enterocolitis. We were also able to prove the reproducibility of the NEC protocol and its efficacy in inducing the disease. However, further studies are needed to better understand the highest mortality and lowest survival rate in the group with isolated use of I-arginine. In addition, other studies would be interesting to test lower concentrations of sildenafil and I-arginine or to test another route of drug administration to avoid repeated trauma from intraperitoneal applications.

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