Pretreatment with pentoxifylline attenuates lung injury induced by intestinal ischemia / reperfusion in rats

PURPOSE: To investigate the protective effect of pentoxifylline against the lung injury observed after intestinal ischemia (I) followed by a period of reperfusion (R). METHODS: Twenty-eight male Wistar rats were equally divided into 4 experimental groups and operated under ketamine-xylazine anesthesia. (1) Sham: falsely-operated animals; (2) SS+IR: intestinal ischemia was accomplished by clipping the superior mesenteric artery during 60 minutes, with an administration of a standard volume of saline solution (SS) 5 min before the end of the ischemia period; the clip was then releases or a 120-min period of reperfusion; (3) I+PTX+R: ischemia as above, PTX was administered (25 mg/ kg) and the gut reperfused as above; (4) PTX+I+PTX+R: Five minutes before arterial occlusion PTX was administered; the superior mesenteric artery was then clipped for 60 minutes. After 55-min ischemia, an additional dosis of PTX was administered; the clip was removed for reperfusion as above. At the 60th min of reperfusion a third dosis of PTX was administered. RESULTS: PTX markedly attenuated lung injury as manifested by significant decreases (all P<0.001 as compared with the SS+IR group) of pulmonary wet/dry tissue weight ratio, total protein content, myeloperoxidase activity and tumor necrosis factor-alpha. Moreover, it was apparent that in the group PTX+I+PTX+R the improvements have been even more significant. CONCLUSION: PTX exerted a protective effect on the lung from the injuries caused by intestinal ischemia/reperfusion.


Introduction
Regardless the cause, an intestinal ischemia insult is a serious and growing clinical problem, with an unacceptable mortality rate over 60% 1 .
Intestinal ischemia and reperfusion (I/R) leads to an acute local inflammatory response, resulting in increased microvascular permeability.Intestinal I/R is a common medical event associated with both clinical and experimental distant organ injury.In particular, the lung tissue appears to be susceptible to injury resulting from systemic oxidative stress and inflammatory mediators activation.Despite decades of research in this area, I/R injury remains a clinically challenging problem [2][3][4][5][6][7] and particularly susceptible to the damaging effects of increased neutrophil activation following intestinal I/R 8 .Consequently, many cases of intestinal I/R progress into shock, multiple organ failure, and death 9,10 .
Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) is a consequence of host response to a variety of direct or indirect stimuli leading to pulmonary inflammation.
Many of the clinical conditions associated with the development of acute respiratory failure include periods of ischemia [11][12][13] .Pentoxifylline (PTX), a methylxanthine derivative known for many years for its haemorheological properties, has proven to be a potent inhibitor of tumor necrosis factor (TNF) production 14 .It has been suggested that PTX can enhance the chemotactic response of neutrophils, but may inhibit phagocytosis and the superoxide production by neutrophils and monocytes 15 .It has also been shown that this drug has beneficial effects in patients with lung ischemia/reperfusion injury 16 .The potential local effects are important for acute lung injury manifestation by inflammatory mediators that are blood-and lymph-borne x,y,z , and oxygen-free radical activations 17,18 .
Based on these literature data, herein we put forward the hypothesis that the pre-treatment with PTX may reduce lung injury in rats submitted to intestinal ischemia/reperfusion.For this purpose we used an animal model of intestinal I/R in which a superior mesenteric artery occlusion-reperfusion simulates the I/R injury that may occur in clinical situations, and investigated the effects of PTX on the systemic (pulmonary) repercussions of this insult.

Surgical procedure and experimental design
All animal procedures were approved by the Institutional Research Committee at Federal University of Sao Paulo (1551/09).
It was designed as an experimental, randomized, controlled study with blind assessment to the outcome.
Twenty-eight adult male Wistar rats (n=7 for every group; body weights were 270 to 300 g) were fasted overnight with free access to water.Anesthesia was induced by a single intraperitoneal injection of ketamine (80 mg/kg) plus xylazine (10 mg/kg), and the abdomen and groin were shaved and washed with 10% povidone iodine.A PE-50 tubing catheter was inserted in the femoral vein for saline or PTX (Pentox ® , Farmasa, SP, Brazil) solution infusion.PTX was suspended in Ringer lactate solution to a concentration of 25 mg/kg as described previously [19][20][21][22][23][24][25][26] .Upon a 2-cm midline laparotomy, the superior mesenteric artery was isolated at its origin and occluded with a Bulldog ® atraumatic microvascular clamp (Harvard Apparatus, MA, USA) during 60 min.Mesenteric ischemia was confirmed by cessation of the mesenteric pulsations and a palish appearance of the intestines.At the end of ischemia period the clamp was released and reperfusion (during 120 min) was confirmed by restoration of the pulsations and the normal gut color.The animals were randomly assigned to one of four groups, as follows.Rats of the Sham (falsely operated) group were anesthetized and subjected only to laparotomy and visceral manipulation.Group SS+I/R: animals subjected to I/R and injected with 0.1 ml of saline solution (SS) 5 minutes before reperfusion.Group I+PTX+R: ischemia procedure as above and, 5 minutes before reperfusion, PTX was administered (25 mg/kg in 0.1 ml).Group PTX+I+PTX+R: pre-treatment with PTX 5 minutes before ischemia, then another PTX dosis 5 minutes before the end of ischemia and a final dosis of PTX at the 60th minute of the reperfusion period.Body temperature was maintained to 37°C (37 ± 0.6 o C) with a heating pad.
At the end of experiment the animals were euthanized (T-61 Euthanasia Solution ® , Schering-Plough, SP, Brazil).

Bronchoalveolar lavage (BAL) fluid for protein assay
Upon thoracotomy, bronchoalveolar lavages (BALs) were taken by flushing three times the right lung with sterile saline via an intratracheal cannula, using a Miniplus 3 ® peristaltic pump (Gilson, WI, USA).The recovery rate was >85%.The collected BALs were centrifuged at 300 x g for 4 min at 4ºC and the supernatant was used for protein determination by the method of Lowry et al. 27 .

Determination of lipid derived oxidation products
The thiobarbituric acid reactive substancemalondiadehyde (TBARS-MDA) content of lung tissue was determined using the method described by Ohkawa et al. 28 .

Myeloperoxidase (MPO) activity
Homogenates were centrifuged at 15,000 x g for 10 min at 4°C.A 100 mL-aliquot of supernatant was mixed with 900 mL of 50 mmol•L -1 phosphate buffer (pH= 6.0) containing 0.167 mg•mL -1 of o-dianisidine dihydrochloride and 0.0005% hydrogen peroxide.One unit of peroxidase activity is taken as the amount of enzyme decomposing 1 mmol of hydrogen peroxide per minute at 25°C.Decomposition of hydrogen peroxide was calculated from the oxidation of o-dianisidine by using an absorption coefficient of 11.3 mmol•L -1 •cm -1 at 460 nm 29 .

Cytokine assay
Tissue TNF-α content was measured in lung homogenates using commercially available, rat-specific enzyme immunoassay (ELISA) kits (Quantikine, R&D Systems, MN, USA).Results were expressed as pg/mL.Absorbance was determined at 450 nm using a microplate reader (Bio-Tek Instruments, VT, USA).

Morphology
The middle lobe of the right lung was fixed in buffered formalin.After embedding in paraffin, 4-μm sections of the tissues were stained with hematoxylin-eosin (HE) for light microscopy coupled to a video camera (Axiolab Standart 2.0 and AxionCam, Zeiss, Jena, Germany, respectively).The slides were evaluated blindly by an independent consultant histopathologist.

Wet to dry tissue weight ratio
The lower lobe of the right lung was isolated and immediately weighed (wet weight) before being dried for 48 h at 80°C and weighed again (dry weight).The wet/dry weight ratio was then calculated.

Statistical analysis
Statistical analyses were carried out with a SPSS 11.0 statistical software (SPSS Inc.Software, IL, USA).Data were analyzed by 1-way analysis of variance (ANOVA) with a LSD post-hoc test to determine comparisons and differences between groups, respectively.All values are expressed as mean ± standard deviation with P<0.05 being considered significant.

Results
No deaths were recorded throughout the experiment.The protein content in BALs (Figure 1) increased in the group SS+IR (62.71 ± 4.61 mg/mL), and decreased in the groups treated with PTX (I+PTX+R: 50.14 ± 3.93 mg/mL; PTX+I+PTX+R: 36.28 ±   The levels of TNF-alpha (Figure 4) in lung homogenates were higher in all groups when compared with those of the Sham group.The has been an increase of TNF-alpha levels in the group SS+IR (67.14 ± 6.82 ρg/mL), and a reduction in PTX-treated groups, I+PTX+R (55.86 ± 2.27 pg/mL) and PTX+I+PTX+R (47.57± 2.64 pg/mL).directly translate to a decreased level of cytokines in the lung [37][38][39] .

Pretreatment with pentoxifylline attenuates lung injury induced by intestinal ischemia/reperfusion in rats
Our studies corroborated with these authors and expanded of the result.In our study, the TNF-alpha levels in homogenate lung tissue were higher in the saline-treated group (SS+IR) and reduced in the group I+PTX+R, but the decreased was more significant in the group treated with three doses of pentoxifylline (PTX+I+PTX+R).Our study demonstrated that pentoxifylline had preventive effects and therapeutic potential in I/R-induced lung injury, but our results must be considered carefully.The clinical relevance of this manuscript refers to the previous use of pentoxifylline in situations requiring procedures ischemia with reperfusion to reduce or prevent distant organs damage.Therefore, the long-term effect of pentoxifylline warrants further investigation.
In conclusion, our study showed that pentoxifylline decreased lung edema, ameliorated lung histological change, reduced the production of inflammatory mediators, and neutrophils in intestinal I/R-induced lung injury.Pentoxifylline has been given Pretreatment with pentoxifylline attenuates lung injury induced by intestinal ischemia/reperfusion in rats Acta Cirúrgica Brasileira -Vol.26 (6) 5011 -443 safely in previous clinical studies.Therefore, the administration of pentoxifylline may be a useful prophylactic or adjunct drug therapy for intestinal I/R-induced lung injury.

FIGURE 1 -
FIGURE 1 -Protein content in BALs from rats falsely operated (Sham) or subjected to mesenteric artery clamping for intestinal ischemia during 60 min, followed by clamp release for a 120-min period of reperfusion (IR).Control IR rats were treated i.v. with 0.1 mL saline solution (group SS+IR).Other IR rats were treated with pentoxifylline (PTX): group I+PTX+R received i.v. 25 mg/kg of PTX 5 min before starting the reperfusion period; the group PTX+I+PTX+R received i.v.three doses of PTX: one 5 min before ischemia, a second one 5 min before starting reperfusion, and a third one at the 60th min of reperfusion.Data are mean ± SD. *P<0.001compared with all other groups.

FIGURE 2 -
FIGURE 2 -Effect of pentoxifylline treatment on lipid-derived oxidation products as determined by the levels of thiobarbituric acid reactive substance-malondiadehyde (TBARS-MDA) in rat lung homogenates.Groups are as in the legend to Figure 1.Data are mean ± SD. *P<0.001regarding all other groups.

FIGURE 3 -
FIGURE 3 -Effect of pentoxifylline treatment on the myeloperoxidase (MPO) activity in rat lung homogenates.Groups are as in the legend to Figure 1.Data are mean ± SD. *P<0.001regarding all other groups.

FIGURE 4 -
FIGURE 4 -Effect of pentoxifylline treatment on the tumor necrosis factor (TNF)-alpha content in rat lung homogenates.Groups are as in the legend to Figure 1.Data are mean ± SD. *P<0.001regarding all other groups.

Figure 5
Figure 5 shows typical lung histological features from

FIGURE 5 -
FIGURE 5 -Histological lung sections of lungs from sham-operated rats [Sham] show a normal pulmonary architecture.In the group SS+IR it can be seen massive alveolar congestion and neutrophil infiltration.In the groups treated with pentoxifylline, ([I+PTX+R] and [PTX+I+PTX+R]) the interstitial congestion was minimal, and the leukocyte (neutrophil) infiltration was strongly reduced.Hematoxylin and eosin staining.Scale bar: 20 µm.(H.E -200X).
Morphological examination indicate the severe impact in the lung tissue submitted to I/R non-treated, by other hand the treatment with PTX reduced the lung damage, especially in the group treated with three doses of PTX.