Ischemia-reperfusion histopathology alterations of the rabbit intestinal wall with and without ischemic preconditioning

Purpose: To evaluate the histopathology alterations of the intestinal mucosa of rabbits submitted to mesenteric artery ischemia and reperfusion with and without ischemic preconditioning. Methods: Two groups of ten male New Zealand white rabbits body (weight 2.2– 3.0, average 2.5 kg). For mesenteric ischemia induction in all animals the small bowel and mesentery were cut 30cm and 60cm far from the gastroduodenal pyloric transition before the proximal mesenteric artery occlusion. In the Group 1 animals, the proximal mesenteric artery was occluded for 45 min with an atraumatic vascular clamp, followed by reperfusion for 30 min. In the Group 2 the 45 min ischemic phase was preceded by three cycles of ischemia (2 minutes each) alternated with three cycles of reperfusion (2 minutes each). For istopathology study small bowel biopsies were obtained before ischemia (control), after 45 min of mesenteric ischemia and at 30 min. of mesenteric artery reperfusion. Results: In the Group I animals, the followings histopathology grade results were observed: t1, mean 2,8; t2, mean 3,3. Using the Kruskal-Wallis non-parameter test, differences between t0 and t1 and t0 and t2 were significants (p<0.05), but not significant between t1 and t2 (p>0.05). In the Group 2 animals histopathology grade results were: t1 mean 2,6 and t2, mean 2,1. Differences between t0 and t1, t0 and t2 were significant (p<0.05). It was not observed differences (p>0.05) between results of t1 in both groups but histopathology injury observed in Group 1 t2 biopsies were higher (p<0.05) than observed in the same period (t2) of Group 2 animals. Conclusion: Microscopic examination of the biopsies revealed significant evidence of preconditioning protection against small bowel wall ischemia–reperfusion injury.


Introduction
In 1986, Parks and Granger 1 and Murry et al. 2 demonstrated by the first time that reperfusion can be more harmful than ischemia separately and the phenomenon of myocardial protection by the ischemic preconditioning with reduction of the myocardium ischemia-reperfusion injury in dogs.Yellon et al. 3 demonstrated that the ischemic preconditioning protection also occurs in the human myocardium which raised a great interest extending this study also to other organs, with the landmark study by Hotter et al. 4 and more recently Santos et al. 5 confirming the ischemic preconditioning protection Santos et al. 4 demonstrating the ischemic preconditioning protection of the small bowel of rats.Nowadays the ischemia-reperfusion stress is confirmed to affect with different intensity different animal species but with still little reports in the literature regarding rabbit intestinal mucosa response 5 .
The objective of this study is to evaluate the ischemic preconditioning effect in the protection of the histopathology lesions of the intestinal mucosa of rabbits submitted to mesenteric ischemia considering the particular anatomy standard 6 of the collateral mesenteric circulation in these animals.

Methods
This study was approved by the Ethics Committee for Animal Experimentation of our institutions and was conducted according to the guidelines for animal experimentation of the Brazilian College on Animal experimentationTwo groups of ten male New Zealand white rabbits body (weight 2.2-3.0,average 2.5 kg) were used in this study.After an overnight fast with unrestricted access to water, the animals were anesthetized with muscle injections of xilazine (15mg/kg bw) and ketamine (25mg/kg bw) repeated as necessary to maintain an adequate anesthetic plane.For mesenteric ischemia induction in all animals the small bowel and mesentery were cut 30cm and 60cm far from the gastroduodenal pyloric transition before the proximal mesenteric artery occlusion.
In the Group 1 animals, the proximal mesenteric artery was occluded for 45 min with an atraumatic vascular clamp, followed by reperfusion for 30 min.
In the Group 2 the 45 min ischemic phase was preceded by three cycles of ischemia (2 minutes each) alternated with three cycles of reperfusion (2 minutes each).In both groups small bowel biopsies were obtained before ischemia (t1-control), after 45 min of mesenteric ischemia (t2) and at 30 min. of mesenteric artery reperfusion (t3).
Between surgical interventions, the midline incision was sutured to minimize fluid losses.The animals were sacrificed with lethal intravenous dose of anesthetics.
The histopathology study was performed in the Pathology Anatomy Laboratory of the Minas Gerais Federal University School of Medicine and results described according to the following classification 7 , modified from Chiu, McArdle, Brown et al. 8 classification in rats: Grade 0: Normal mucosa histology; Grade 1: Small cytology alterations in the cell structure representation.Increased leucocytes presence and space widening between villosities; Grade 2: Cell alterations with focused lesions and presence of some cell lysis.There are destructions of the the villosities in at most 25% of their extension; Grade 3: Besides the cytology alterations, there are structural lesions in intermediate extension.Presence of dilated capillaries and higher quantity of inflamed cells.The destruction should be between 25% and 50% of the villosities extension; Grade 4: Structural destruction of the villosities, only traces of some villosities, formed by inflamed cells and necrotic material, with hemorrhage and basal glandular ulceration.The destruction should be between 50% and 75% of the villosities extension; Grade 5: destruction of all the mucosa, no glandular structure can be seen, only the amorphous material laying on the sub-mucosa tissue.The destruction should be between 75% and 100% of the villosities extension (Figure 1).
Results are reported as mean ± standard deviation (SD).The Kruskal-Wallis non parametric method was employed with statistical significance set when p<0.5.
It was not observed differences (p>0.05) between results of t1 in both groups but histopathology injury observed in Group 1 t2 biopsies were higher (p<0.05)than observed in the same period (t2) of Group 2 animals.

Discussion
The mesenteric blood flow reduction and the ischemiareperfusion injury plays an important role in the pathogenesis and survival of many clinical and surgical diseases [9][10][11] , with important research approach done [12][13][14][15] to understand the involved mechanisms aiming to obtain the best protection.Although confirmed in many researches, the pre and postconditioning failure to avoid the ischemia and ischemia-reperfusion lesions is also reported [16][17][18][19] , raising the respiratory dysfunction after cardiopulmonary bypass 20 and to brain stroke events when induced by proton pump Na + /H + ATPase 21 .
This relative contradictory scenario regarding results with ischemic and or drug induced preconditioning was consistently recently studied by Ramzy et al. 22 pointing the importance and benefits of new researches for best clinical results with pre and postconditioning.In this way it is very important to know the phenomenon behavior in different animal species opening new horizons to better understand the histology, physiology and biochemical mechanism of ischemia-reperfusion lesions and of the pre, post and remote preconditioning effect.Introduced in the medicine universe by Murry et al. 3 , discovering the ischemic preconditioning protection in hearts of dogs submitted to temporary occlusion and reported to occur also in humans by Yellon et al. 4 , the occlusion by selective clamping of the cranial mesenteric artery in rats being the routine procedure until now in most reported mesenteric circulation research.Mesenteric Ischemia-reperfusion study in rabbits is rare and as reported by Bretz et al. 23 with different and bad protective results than observed with the mesenteric ischemic pre and postconditioning in rats.
In the present investigation ,considering the particular anatomy of the mesenteric circulation in rabbits, previously reported 24 , for mesenteric ischemia induction in all animals the small bowel and mesentery were cut 30cm and 60cm far from the gastroduodenal pyloric transition before the proximal mesenteric artery occlusion thus avoiding the interference of the collateral circulation from the gastroduodenal arterial supply .
In the Group I animals, without ischemic preconditioning, the histopathology mean grade results were 2,8 after 45 minutes of mesenteric ischemia (t1) and mean 3,3 after 30 min. of reperfusion (t2) without statistical significance between them.In the Group 2 animals, with ischemic preconditioning, histopathology mean grade results were 2.7 in t1 samples, significantly different from t2 with mean value of 2,3.Difference between t1 results in both groups was not significant (p>0.05)but post ischemia reperfusion injury (t2) in Group 2 animals was significantly lower than observed in Group 1.
Although with limited results interpretation mainly by considering only one period of induced mesenteric ischemia and only one sequence of the ischemia and reperfusion periods for the preconditioning induction it was demonstrated by this investigation in rabbits best significant protection by the ischemic preconditioning against the ischemia reperfusion injury than observed with the ischemia before reperfusion beginning.

Conclusion
The obtained results leads to the conclusion that microscopic examination of the biopsies revealed significant evidence of preconditioning protection against small bowel wall ischemia-reperfusion injury.

TABLE 1 -
Histopathology of Group 1 animals with ischemia without ischemic preconditioning.

TABLE 2 -
Histopathology of Group 2 animals with ischemic preconditioning.