Introduction

Lung injury is known to occur after temporary occlusion of the aorta and subsequent ischemia/reperfusion (IR) of the lower extremities. ₁,_{2 3} Polymorphonuclear neutrophil leucocytes (PMN) have been shown to have a central role in lung injury caused by IR of the lower extremities, and their depletion exerts a protective effect on the lungs under these conditions. ₃ In clinical settings, temporary ischemia of the lower extremities may result in shock and acute lung injury that requires inotropic and ventilatory support.₄

Tenoxicam is a nonsteroidal antiinflammatory (NSAI) drug that inhibits cyclooxygenase (resulting in similar inhibition of endothelial prostacyclin and platelet tromboxane production), but it also appears to have inhibitory actions on neutrophil function in vitro.₅

Dexamethasone has been shown to inhibit pulmonary inflammation in endotoxin shock and asthma by inhibiting nitric oxide synthase (iNOS)._{6 7} We studied the effect of pharmacologic interventions with these two antiinflamatory drugs on acute lung injury.

Methods

Thirty-five New Zealand white rabbits (2.5 to 3 kg) were used for the
study. All animals received humane care in compliance with the European
Convention on Animal Care. The study was approved by the Institutional
Ethics Committee.

During the surgical procedures, anesthesia was induced and then maintained
with intraperitoneal ketamine (30 mg/kg) and xylazin (6 mg/kg) fractionally
as needed. During surgical procedures, body temperature was maintained with
a water-filled heating pad. The animals were placed in a nose cone to
breathe oxygen at a rate of 0.5 l/minute. Rectal temperature was monitored
and maintained close to 38ºC under a warming light. A jugular venous line
was established for intravenous fluid infusion through the neck incision.
The animals were then given heparin (1000 units/kg) via the right jugular
vein.

The abdominal aorta was exposed through a midline abdominal incision, and
the aorta was exposed just above the iliac bifurcation. A bulldog clamp was
used for the aortic occlusion. Reperfusion was confirmed visually and by
doppler assessment in the femoral region.

The animals were randomized into four groups. In IR group (n=10), the aorta
was cross-clamped 3 hours, followed by 2 hours of reperfusion. In Dxm group
(n=10), animals were pretreated with 1 mg/kg dexamethasone via jugular vein
before aortic cross-clamping. In Tnx group (n=10), animals were pretreated
with 10 mg/kg tenoxicam before 3 hours of ischemia and 2 hour of
reperfusion. In the control group (n=5), the abdomen was left open at the
same period and pretreated with equal volume of saline.

At the end of 5 hours, both lungs and trachea were harvested. The left main
bronchus was cannulated and secured. Saline (15 ml) was then injected as 3
aliquots of 5 ml each. Each aliquot was injected quickly and then withdrawn
slowly 3 times to obtain BAL specimen. Fluid recovery was routinely 90% or
greater. Combined aliquots of BAL fluid were spun at 1000g for 10 minutes to
remove cells. The cell pellet was resuspended in 1 ml of saline, and PMN
rate in the 100 cell was counted.

After removing the right lung, it was inflated and fixed with 10% formalin. Fixed specimens were paraffin-embedded, sectioned in 6 µm pieces, and stained with routine hematoxylin-eosin stain. The specimens were examined by the same pathologist who was blinded to the study. At least two different sections of each specimen were examined to accurately determine the degree of injury. Lung injury was rated with a semiquantitative scoring system described by Tassiopoulos et al.₈, based on congestion, interstitial edema, PMN infiltration, and airspace hemorrhage, as follows: 0, no changes; 1+, focal, mild, subtle changes; 2+, multifocal mild changes; 3+, multifocal prominent changes; and 4+, extensive prominent changes.

The parametric data (the rate of PMN in BAL fluid) were expressed as mean ±
standard deviation, and compared with student t test. Nonparametric values
of lung injury scores were analyzed with Mann Whitney U test. A p-value of
less than 0.05 was considered significant.

Results

In the control group, there was no congestion and neutrophil infiltration in
the lung histology (Figure 1). In the BAL cytology, there were no
neutrophils; the dominating cells were macrophages.

The groups exposed to aortic occlusion showed significant differences in the
degree of lung injury (Figure 2,3,4). IR and Tnx groups had lesions ranging
from 2 to 4+, with average injury scores of 2.6+ and 2.5+, respectively
(p>0.05). The score of Dxm group had significantly lower than the others
(ranging from 1+ to 3+ and mean 1.7+, p<0.05).

In the BAL cytology, the rate of PMN was also significantly lower in Dxm
group (10.8±3.7%, p<0.05) than Tnx (23.4±6.5%) and IR groups (27.9±5.5%).
There is no difference between Tnx and IR groups according to their lung
injury scores and, BAL cytology (p>0.05).

Discussion

It was demonstrated that acute ischemia of the lower extremities in rats results in a significant lung injury._{1 8} According to the Stallone's work ₁ the lung changes were seen even in the animal that was killed with the aortic clamp still in place. The lung injury process begins once the blood supply to the lower extremities is interrupted and aggravated during reperfusion.₈ In our model only the arterial blood flow to the lower extremities was blocked, whereas the venous and lymphatic return were maintained open during ischemia, both ischemia and reperfusion contribute to injury process in the lung. I/R of lower extremity causes lung injury by PMN sequestration in pulmonary microvasculature, increased endothelial permeability, and interstitial edema.₂ The injuries are PMN-dependent and can be attenuated by prior depletion of circulating PMNs. _{3 9}

It was demonstrated that acute ischemia of the lower extremities in rats results in a significant increase in serum TNF concentration and a subsequent increase in NO production from the lung; TNF and NO are significant determinants of the lung injury process that is caused by lower extremity I/R. _{1 2 8 10} The administration of dexamethasone results in decreased production of NO by both a direct and an indirect decreasing TNF production._{6 8 11} Because of the lesser degree of neutrophil accumulation in Dxm group, the pre-treatment with dexamethasone before IR is associated with lesser degrees of lung injury.

Has椬ik et al suggest that tenoxicam is a potent inhibitor of neutrophil chemotaxis.₁₂ In the study of Angelis et al., ₁₃ tenoxicam is likely to possess anti-inflammatory properties which are independent from effects on cyclooxygenase.

Hellewell find that neutrophil accumulation induced by C5a attenuated by ibuprofen in cutaneous microcircullation, but leucocyte infiltration in pulmonary airspace increased in the same animal.₁₄ Similarly, pulmonary neutrophil accumulation was not decreased by tenoxicam pretreatment, probably caused by inhibition of tromboxane production, which under normal circumstances serves to decrease local blood flow and keep the inflammatory response localized and regulated.

In conclusion, lower extremity ischemia and reperfusion cause a significant
lung injury. It can be attenuated by dexamethasone pretreatment.

Figure 1

Figure 1: Normal histologic appearance of rabbit lung. (Control group, hematoxylin-eosin stain, orginal magnification X20, injury score:0+)

Figure 2

Figure 2: Histologic picture of the lung, minimal vascular congestion and cellularity of lung interstitium. (Dxm group, hematoxylin-eosin stain, orginal magnification X20, injury score:1+)

Figure 3

Figure 3: Histologic picture of the lung, increased vascular congestion and cellularity in the lung interstitium. (Tnx group, hematoxylin-eosin stain, orginal magnification X20, injury score:3+)

Figure 4

Figure 4: Histologic picture of the severe lung injury. There is alveolar flooding, indicating pulmonary edema. (IR group, hematoxylin-eosin stain, orginal magnification X20, injury score:4+)