Enteral antioxidants in ischemia / reperfusion injuries in rats

Objective: To evaluate the role of pre-treatment with dietary antioxidants in an experimental model of intestinal injury of ischemia-reperfusion (I/R) in rats. Methods: Ninety adult male Wistar rats were used. An intestinal segment was isolated based on its vascular pedicle. A control biopsy was performed and the pedicle was sectioned and sutured again, ensuring a time of 60 minutes of ischemia followed by reperfusion. Sequential biopsies were performed at the end of the ischemic period and every 15 minutes during reperfusion. The treatment consisted of saline, vitamin C, vitamin E or a combination of the latter two. Quantitative and qualitative assessments of the biopsies were performed. Results: The groups treated with vitamin E alone or vitamin E combined with vitamin C showed a statistically significant attenuation of ischemia-reperfusion, with reduced loss of height of the villi and lower neutrophilic infiltration at the end of the study when compared to the control and vitamin Cexclusive groups. Conclusion: In this experimental model of ischemia-reperfusion, pre-treatment with vitamin E attenuated the I/R injury in the small intestine of Rats, demonstrated by reduced loss of height of the villi and the attenuation of neutrophil

Enteral antioxidants in ischemia/reperfusion injuries in rats Enteral antioxidants in ischemia/reperfusion injuries in rats Enteral antioxidants in ischemia/reperfusion injuries in rats Enteral antioxidants in ischemia/reperfusion injuries in rats Enteral antioxidants in ischemia/reperfusion injuries in rats INTRODUCTION

INTRODUCTION INTRODUCTION INTRODUCTION INTRODUCTION
T he restoration of blood flow to an ischemic tissue can lead to greater harm than that originally caused by ischemia.This event is called ischemia and reperfusion (I/R) injury.The small intestine is particularly susceptible to injury from I/R 1 and its occurrence is associated with high morbidity and mortality 2 .Although the mechanisms involved are not fully elucidated, it is believed that oxidative stress mediators, such as reactive oxygen species (ROS), polymorphonuclear neutrophils (PMN) and nitric oxide (NO) play an important role 3 .Due to the involvement of ROS in I/R injury, various antioxidants have been tested, among them vitamin C (ascorbic acid) and vitamin E. Ascorbic acid is an water-soluble antioxidant with chelating and reducing properties and it is used to prevent I/R injury 4 .Vitamin E (á-tocopherol) is a nonenzymatic, lipid soluble antioxidant that acts against oxidative stress by stabilizing membrane unsaturated fatty acids.Thus, tissues treated with vitamin E have an increased capacity to reduce ROS and be protected against membrane lipid peroxidation 5 .Vitamins C and E can act synergistically because ascorbic acid is unable to reduce the peroxyl radical, but it can regenerate á-tocopherol from the tocoferoxil radical, recycling the átocopherol 6 .
I/R can occur in a variety of clinical situations, one being the microsurgical transfer of jejunal segments.The jejunal flap was first described in 1957, and has been used for reconstruction of the pharynx and esophagus.During the transfer, an isolated segment of jejunum is removed from the abdomen and transferred to the neck 7 .In the clinical setting, I/R injury can be seen ten minutes after reperfusion, with gradual recovery until the 28th day 8 .
The objective of this study is to evaluate the impact of pre-treatment with vitamins C and E in an experimental model of jejunal flap using morphometric criteria to assess I/R injury.

Model of jejunal ischemia-reperfusion Model of jejunal ischemia-reperfusion Model of jejunal ischemia-reperfusion Model of jejunal ischemia-reperfusion Model of jejunal ischemia-reperfusion
Was used 90 adult male Wistar rats between 224 and 261 grams of weight (average of 237.5g and standard deviation (SD) of 12.3g) in the experiment.After anesthesia with intraperitoneal ketamine and xylazine, the animals were placed in the supine position on a warm table and Rev. Col. Bras.Cir.2011; 38(6): 422-428 underwent laparotomy.Body temperature was monitored with a rectal probe to avoid hypothermia.The superior mesenteric artery and vein were dissected and isolated from their sources in sufficient length to allow placement of vascular clamps and performance of sutures.The study area was then selected according to the arterial supply to the intestine.The first choice for biopsy was the sixth loop from the ileocecal valve, the subsequent seventh loop being an alternative.One fragment with a total thickness of the intestinal wall was removed and sent for biopsy control (BC).In order to avoid variations in height of villi throughout the intestine, a 3cm jejunal segment was dissected based on its vascular pedicle and isolated by sectioning of any collateral circulation to it.All the biopsy fragments were obtained from this segment.In order to simulate a microsurgical flap, a microvascular clamp was placed in the mesenteric vessels that, soon after, were sectioned.A microvascular anastomosis was then performed between the stumps by using magnification Zeiss microscope and 10-0 nylon suture.After one hour of ischemia, during which vascular anastomosis was performed, a second fragment (ischemic biopsy -BI) was extracted and the clamps removed, restoring blood flow.After removing the clamps, there were four successive tissue removals: at 15 minutes of reperfusion (RB15); at 30 minutes of reperfusion (RB30); at 45 minutes of reperfusion (RB45); and at 60 minutes of reperfusion (RB60).Each sample had a length of approximately 0.4 cm and included the entire circumference of the jejunal segment.To simulate the conditions of a microvascular transfer, the segment chosen for the study was kept outside the abdominal cavity, at room temperature (24°C).All animals were euthanized after the procedure.
This experiment was approved by the Ethics Committee on Animal Research of the Institute of Biology, Universidade Estadual de Campinas.

Treatment groups Treatment groups Treatment groups Treatment groups Treatment groups
The animals were randomly divided into the following groups, each with fifteen animals: Group T underwent laparotomy with preparation of the flap without ischemia and reperfusion; Control Group underwent laparotomy, preparation of the flap, ischemia and reperfusion, with collection of samples in predetermined intervals, but received no treatment; Group C was submitted to the same surgical procedure of the Control Group, associated with the intake of ascorbic acid, 250mg/kg, via enteral feeding for four days before the operation; Group E underwent the same surgical procedure of the Control Group, but received vitamin E at a dose of 60mg/kg in the four days preceding the operation; Group C + E underwent the same surgical procedure of the control group and received both drugs, the same dose and Groups C and E (250mg/kg of vitamin C and 60mg/kg vitamin E) for four days; Validation Group C -Same as Group C, except that during preparation of the drug , the active principle (ascorbic acid) was removed; Validation Group E -Same as Group E, with the active principle (a-tocopherol) removed during preparation of the drug.
The purpose of Group T is to validate the model and show that intestinal injury is not due to surgical manipulation, but due to ischemia-reperfusion. Validation Groups C and E are intended to demonstrate that the effect of vitamins C and E was due to their active principles and not to other components of the formula, which were kept in the drugs administered to these animals as well as the diluents.

Parameters of injury Parameters of injury Parameters of injury Parameters of injury Parameters of injury
Both qualitative and quantitative analyses were used in the assessment of I/R injury.The height of the villi and neutrophilic infiltration were evaluated in histological sections of intestinal biopsies.All samples were evaluated by the same histologist who was blinded to which group or time of biopsy the animal belonged.The sections were stained with routine histological techniques using hematoxylin and eosin.The classification proposed by Chiu et al. 9 was used for qualitative assessment.The height of villi was determined in ten random fields using an optical system that allowed the measurement of height of the villus in micrometers (mm).The neutrophil infiltration was assessed by counting cells in 20 random fields.

Statistical analysis Statistical analysis Statistical analysis Statistical analysis Statistical analysis
Data are presented as mean and standard deviation (SD).Spearman and Pearson tests were used to evaluate the correlation between variables.A comparison of multiple means was performed using analysis of variance (ANOVA).If variances were equal, the Tukey test was used, while the Dunnett test was applied to different variances.A p value <0.05 was considered statistically significant.The distribution of continuous variables was compared with the normal one.

Qualitative analysis
Qualitative analysis Qualitative analysis Qualitative analysis Qualitative analysis A progressive increase of I/R injury was observed in all groups, with a significant migration from degree zero to five over time, a statistically significant correlation between time and Chiu score being demonstrated (Figure 1).

Height of villi Height of villi Height of villi Height of villi Height of villi
The height of the villi showed a progressive decrease over the study period (Figure 2).A statistically significant correlation was found between the biopsy intervals and height of villi in all groups.The correlation coefficient and p values are shown in table 1.
Comparisons of height of the villi were performed within each group at different intervals and also between different groups within the same time interval.Initially, the Köhler Köhler Köhler Köhler Köhler Enteral antioxidants in ischemia/reperfusion injuries in rats Control Group was compared with those of validation.There was no statistically significant difference between them at any interval (Table 2).In the Control Group, the height of villi decreased from 363.55 + / -35.89 in BC to 129.16 + / -27.17 in RB60, and this decrease was statistically significant (p <0.001).The first statistically significant difference was observed in comparison with RB15, which showed height of 296.87 + / -46.13, p = 0.004 (Figure 2).
For Group C, a similar pattern was observed, with the height of villi ranging from 336.61 + / -44.63 in BC and 134.05 + / -36.22 in RB60 (p <0.001), the RB15 being the first biopsy presenting a significant difference, p <0.001 (Figure 2).
The animals treated with vitamin E had average vilar height of 343.93 + / -46.79 in CB and 154.12 + / -31.88 in RB60 (p <0.001), but unlike the other, the first significant difference was with BI, p <0.001 (Figure 2).
Animals treated with vitamins C + E repeated the pattern observed in Group E. The average height of villi    ranged from 332.37 + / -48.96 in CB to 156.23 + / -29.63 in RB60 (p <0.001) and the first statistically significant difference occurred in BI, p <0.001 (Figure 2).In the control biopsy, there was no statistically significant difference between groups, with average height of the villi ranging from 363.55 + / -35.89 in the Control Group and 332.37 + / -42.00 in Group E+C (p = 0.235, Figure 2).In BI, significant differences could be observed in groups E (249.00 + / -35.34, p <0.001) and C + E (240.07 + /-44.26) when compared to the Control Group (338.53 + / -45.08), p <0.001.In RB15, the only significant difference occurred between the control group (296.87 + / -46.13) and Group C + E (230.99 + / -46.38), p = 0.029.In RB30, the animals in Groups C + E and E showed statistically significant difference when compared to the Control Group (p <0.001), and this difference remained significant in RB45 (p = 0.020) for Group C + E and for Group E (p = 0.037), and in RB60 for Group E (p = 0.032) and for Group C + E (p = 0.025).These data are displayed in figure 2.

Neutrophilic infiltration Neutrophilic infiltration Neutrophilic infiltration Neutrophilic infiltration Neutrophilic infiltration
The number of infiltrating neutrophils showed a progressive increase during the reperfusion period, with no statistically significant difference between the Control and Validation Groups (Table 2).The number of infiltrating neutrophils in the mucosa and submucosa was compared for different time intervals and treatment.We found a statistically significant correlation between the times of the samples and neutrophilic infiltration.The correlation coefficient and p values for each group are shown in table 1.  Table 3 -Table 3 -Table 3 -Table 3 -Table 3  In all groups there was a progressive increase in the number of infiltrating neutrophils.In groups Control and E this increase was statistically significant when compared to the control biopsy from RB30, while in Groups C and C + E, this difference occurred from RB15 on (Figure 2).
In BI and BC there was no statistically significant difference between groups.There was a significant difference between Groups C and C + E when compared to Groups E and Control in RB15 and RB30 (Table 3).In RB45, there was no significant difference between groups (p = 0.2087).In RB60, a significant difference was observed between Groups C, E and C + E (Table 3).

DISCUSSION
DISCUSSION DISCUSSION DISCUSSION DISCUSSION Vitamin C has been extensively studied in I/R injury due to its antioxidant capacity.A study using an experimental model in rabbits with vitamin C, mannitol and N-acetylcysteine has been shown that the superior mesenteric artery flow was improved by the three drugs, but only vitamin C and mannitol had direct protective action on gut mucosa at the histopathological exam of the small intestine, with significant reduction of ischemia and reperfusion injury 10 .These results were confirmed by another article that showed morphological improvement of the lesion after administration of ascorbic acid 11 .Another study evaluated the role of non-enzymatic antioxidants using the measurement of levels of malondialdehyde (MDA) and glutathione in the intestinal mucosa, as well as a qualitative histopathologic score.It demonstrated a significant protective effect of vitamin C and mannitol, a decrease of mucosal damage and accumulation of MDA.However, the use of vitamin E showed no difference in these parameters.The authors suggested that vitamin C and mannitol may be useful in the prevention of intestinal I/R injury due to its effectiveness and ease of use 12 .Both articles used vitamin C intravenously and demonstrated an appropriate protective effect.A key difference is how the injury was evaluated, because both used qualitative rather than quantitative measurements.We can also point a significant difference in the doses used in this work and in Byrka-Owczarek's study 10 (250mg/kg) from that used by Günel 12 (10mg/kg).This difference in dosage may explain the contrasting results.Nevertheless, glycine has the same protective profile of action when used in different doses 13 .
Security in the administration of vitamin C has also been questioned by its contradictory role in I/R injury.In a model of liver damage by I/R, ascorbic acid was shown to have anti-and pro-oxidant properties.After cold I/R injury, a decreased ratio of reduced glutathione oxidation and increased levels of lipid peroxidation and mitochondrial swelling can be prevented by exposure to ascorbic acid at a dose of 0.5mM, whereas a dose of 2.0mM increases tissue injury 14 .
In a study of patients undergoing surgical repair of abdominal aortic aneurysm ou infrainguinal grafts, the preoperative administration of sodium ascorbate increased the arteriovenous difference of lipid hydroperoxides (LH), interleukin 6 and vascular endothelial growth factor during ischemia.This was reflected in an increase of LH, vascular endothelial growth factor and creatine phosphokinase during reperfusion.The findings of these authors suggest that sodium ascorbate can promote lipid oxidative damage induced by iron during the ischemic phase of the operation 15 .Perhaps the ascorbic acid may, at therapeutic doses, have a negative effect on ischemia-reperfusion injury by increasing inflammatory activity, being then indicated in smaller quantities.The use of ascorbic acid proved to be ineffective in preventing lipid peroxidation in serum and intestinal mucosa 16 .
The administration of á-tocopherol prior to ischemia showed both biochemical and histopathological protective effects against I/R injury in rat livers 17 .
The preoperative administration of drugs has also been demonstrated as an effective method for reducing I/ R injury.Selenium, taurine and á-tocopherol were administered for four days before the occlusion and reperfusion of visceral arteries in rats.The animals treated with selenium or a combination of the three drugs showed better mean arterial pressure of the visceral arteries and reperfusion than the control group or other treatments 18 .Oral supplementation of glutamine for a period of 48 hours before I/R injury rendered prevention of intestinal damage and recovery improvement 19 .In a clinical study 20 the preoperative use of vitamin E administered intravenously significantly reduced liver I/R injury, with a good safety profile, and reduction in liver enzyme concentration and time of intensive care unit stay 20 .The natural antioxidant resveratrol, administered preoperatively, also displayed efficacy in the treatment of intestinal I/R injury in an experimental model 21 .
In humans, both vitamins C and E reduce oxidative stress situations.In patients with myocardial infarction there is a significant decrease in the levels of vitamin E after reperfusion, which does not occur in failures of reperfusion.The serum level of vitamin C, however, has shown no difference between these two situations.This provides evidence for the consumption of vitamin E during the reperfusion phase after myocardial infarction, this being a sign of reperfusion 22 .
The number of infiltrating neutrophils in the mucosa increases during the reperfusion period.Polymorphonuclear neutrophils have been implicated in I/R injury and their depletion has been tested in order to improve intestinal damage 3 .Their simple counting using optical microscopy can be an indicator of I/R injury.Both vitamins C and E were shown to decrease lung I/R injury, but not prevent it, because they inhibit the activation of PMN, but not the production of reactive oxygen species from other sources 23 .The association of vitamins C and E showed a Rev. Col. Bras.Cir.2011; 38(6): 422-428 beneficial effect on I/R injury in rat epigastric flaps, increasing significantly the viability of the flap 24 .The role of neutrophil infiltration was also demonstrated by the improvement in I/ R injury with ventilation with 100% oxygen 25 .In this study, groups E and C + E had higher villi height and less neutrophil infiltration, pointing thus to a correlation between mucosal damage and neutrophil infiltration.
Administration of drugs before the onset of the episode of ischemia also showed good results.Pretreatment with oral vitamin C showed attenuation of I/R injury in an experimental model.Its use for five days improved the ability of muscle contraction, reduced neutrophil infiltration, tissue edema and the burst of the neutrophils' respiratory cycle 26 .The preoperative use for a long period (five days) of Larginine and methyl nitro-L-arginine attenuated ischemia and reperfusion in an experimental model in rats, with reduced leukocyte infiltration, as assessed by biochemical markers, such as the serum level of nitric oxide 27 , this effect being observed with their use in the immediate pre-ischemic period 28 .
In the present study, there was a progressive decrease in height of villi and an increase in neutrophil infiltration during reperfusion, as well as a significant reduction of mucosal damage in animals treated with vitamin E or C + E. This benefit could not be demonstrated in animals treated with vitamin C alone.This improvement in the height of villi was not previously demonstrated in other works and may be due to the form of administration of vitamin E. In most studies vitamin E is administered during the ischemic period by subcutaneous injection.In this work, we used the enteral route, which may have allowed an increase in deposits of vitamin E in tissues and its more effective action during reperfusion as retainer of ROS.Regarding vitamin C, the experiment showed no significant benefit of its administration alone.In this study, a significant increase in neutrophil infiltration was observed in all groups, but again with different responses to treatment.The groups treated with vitamin E or C + E showed significantly fewer neutrophils infiltrating the mucosa at the end of the study period.
It is inferred, therefore, that preoperative nutrition with vitamin E decreases I/R injury of the small intestine of rats submitted to an experimental model of microvascular flap of jejunum.Its administration is easy and safe.No benefit could be demonstrated in this model for the use of vitamin C.

Figure 2 2 -
Figure 2 -Figure 2 -Figure 2 -Figure 2 -Figure 2 -Average height of villi in the different treatment groups and time intervals.

Figure 3 3 -
Figure 3 -Figure 3 -Figure 3 -Figure 3 -Figure 3 -Comparison of the number of infiltrating neutrophils in the different treatment groups and time intervals.

Table 1 -Table 1 -Table 1 -Table 1 -
Correlation between the time of biopsy and the height of villi and the number of infiltrating neutrophils in the study groups.

Table 2 -Table 2 -Table 2 -Table 2 -Table 2 -
Analysis of variance comparing the height of villi between the Control Group and the Validation Groups C and E in different time intervals.