Formula feeding, hypoxia and hypothermia are considered risk factors for the development of NEC. The mortality rate of the disorder varies from 10 to 50% among newborns treated in neonatal intensive care units 9,10. In this study, the mortality rate after NEC induction in neonatal rats was 35%, compared with a 38% mortality rate observed by other authors who used the same experimental model11.
Contrary to expectations, group C, which received only l-arginine, obtained the highest mortality rate and the lowest survival rate in relation to the other groups. It is difficult to determine which mechanisms are responsible for the harmful effect of l-arginine. We wondered if the substrate supply for NO synthesis, with its consequent increased production, would be the aggressor factor of tissue injury.
Group B, which only received sildenafil, had the lowest mortality rate. On the other hand, group D had the highest survival rate among NEC groups, which concomitantly received sildenafil and l-arginine. This difference could be due, above all, to the increase in cGMP intracellular levels, which is responsible for stimulating NO production.
We observed that the final body weight of the groups submitted to the NEC protocol was significantly lower in comparison to the negative control group, which received maternal milk. Other authors have obtained similar results, showing weight loss among animals fed formula milk9,11. An exception is a study by Dvorak et al.9, that detected an increase in bodyweight in the groups submitted to the NEC protocol.
Regarding the degree of histological injury, the results were similar to those reported in other experimental studies. The NEC induced microscopic changes were such as villous flattening, reduction in the number of villi and thinning of the intestinal wall12,13. As shown in Fig. 5, our study did not find villous edema, edema in layers, separation of layers, and peeling of the villi as other studies9,14. The probable explanation for this fact would be the immediate fixation of the jejunum and the terminal ileum in 10% buffered formaldehyde, which we performed shortly after the removal of the material. This fixation could have avoided slide artifacts, which could have been considered as injury findings in other studies.
We observed that the intestinal injuries presented high intensity and structural damage in the animals of group E, which were submitted exclusively to the conditions of hypoxia, formula milk, and hypothermia. Hence, the accuracy of the protocol in inducing NEC was confirmed.
The experimental model also demonstrated a higher proliferative index in the groups that received the interventional drugs compared with the positive control group. However, the use of sildenafil and l-arginine has not been able to prevent the development of NEC. One hypothesis is that the intestine of newborns rats would be unable or immature to synthesize NO. Another rationale is that there might not be enough eNOS available nor able to synthesize NO when the supply was high. Sukhotnik and Cols found that exposure of rats to l-arginine does not prevent the intestine from ischemic damage, but reasonably accelerates the repair of the damaged intestinal mucosa15,16,17. Another study showed that l-arginine is not able to inhibit the occurrence of intestinal injuries produced by hypoxia/hypothermia. It has rather shown a higher level of NO and a lower degree of morphological changes in the intestinal wall of animals8.
The present study also observed whether there would be any additive effect when concomitantly administering sildenafil and l-arginine to neonatal rats. As verified through the results of group D, we can affirm that there was a reduction in intestinal injury (with a mean of 26.66) and a higher proliferative rate (68.33%) compared with the other groups submitted to the protocol. However, such a reduction was not statistically significant. Therefore, it would not justify the association of substances.
In conclusion, results from the present study demonstrate that the administration of sildenafil and l-arginine is not able to prevent the occurrence of intestinal injuries produced by hypoxia, formula feeding, and hypothermia. Nevertheless, we have evidenced a reduction in the morphological changes in the intestinal wall of animals that received both substances concomitantly. The intervention drugs likely play a decisive role in the number of metabolically active cells, considering evidence from the groups that received the drugs in isolation or combination.
Our study is relevant both for its innovative character of testing sildenafil as a preventive strategy for NEC, as well as for adding new data on l-arginine literature. We were also able to prove the reproducibility of the NEC protocol and its effectiveness 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 l-arginine.