This study was approved by the Animal Experimental Committee of Duzce University Graduate School of Medicine Animal care and all procedures were performed according to guide for the care and use the National Institute of Heat’s. Rats weighed between 250 and 350 gr and a total of 28 male Spraque-Dawley rats were used for the experiment. The animals were fed a standard rat chow using the same nutrition protocol and were then studied. None of them had any neurological disorders before operation. Hemochron Jr signature plus (Keller Medical GmbH, Bad Soden, Germany) was used to detect ACT level.

Animals were divided into four groups. Sham group (n=7) was appointed as Group-1. The operation was performed in the same way, but without aortic occlusion and heparin administration. Only laparatomy was performed in this group. Non- heparinized control group (n=7) was appointed as Group 2. In this group animals’ abdominal aortas were clamped for 45 minutes by an aneurysm clip, approximately 1cm below renal arteries and 1cm above the iliac bifurcation. Heparin treated group (n=7) was appointed as Group 3. 400 IU/kg of heparin was administered via intraperitoneal to all animals immediately before the procedure. High dose heparin treated group (n=7) was appointed as Group-4. In this group heparin dose was increased 800 IU/kg for ACT level of 600 sec immediately before the procedure. All cross-clamped groups were reperfused after spinal cord ischemia.

Rats were premedicated with ketamine (50 mg/kg) and xylazine (5 mg/kg) intraperitoneally. The maintenance of anesthesia was established with intermittent delivery of ketamine, without endotracheal intubation and mechanical ventilation. Intraperitoneal cephalosporine (10 mg/kg) was administered before skin incision. Postoperative analgesia was provided with tramadol per 12 hours. Temperature probe was inserted into the rectum.

After the surface cleaning of the surgical area and standard midline laparatomy, the abdominal aorta was explored trough a transperitoneal approach retracting the intestines. The abdominal aorta was clamped with mini aneurysm clamp between below renal arteries above the iliac bifurcation. Temperature was maintained between 36,5 and 37,5°C during the spinal procedure with heating lamp. Artery blood pressure and heartbeat were monitored continuously the left carotid artery cannula.

After administrating study protocol in all rats, aortic clamps were removed and anterior abdominal wall was sutured by using 5/0 polyprolene suture.

At 48^th hour the all animals were anesthetized with penthobarbital (20 mmg/kg) and sacrificed. The lungs and heart were dissected totally and fixed in buffered formalin for 7 days.

In this experiment the production of the inflammatory markers IL-6, HSP-70, MPO were studied in four groups of animals. Blood samples were obtained at the end of 48 h. At the time of blood sampling the chest wall was cleansed with cholorohexidine in spirit, and a sterile 10ml syringe was then used to obtain a blood sample by direct cardiac puncture. Blood samples for cytokine assay were collected into heparinized (20 unit/ml blood) sterile tubes and immediately transferred on ice to be centrifuged at 2000 rpm (at 4°C) for 10 minutes and stored (−70°C) until the time of assay for IL-6, MPO and HSP-70.These markers level obtained not only from blood samples but also from lungs and heart tissue.

A midline laparotomy was performed to all animals immediately following blood sampling and total lobe of the lung and heart were removed and fixed in 10% formalin for histopathologic examination. Paraffin sections (4 μm thickness) were prepared. The slides were evaluated under light microscopy (Olympus BX51; Olympus Corp., Tokyo, Japan) at 400x magnification. 5 μm thick sections were placed on polylysine-coated slides and stained with hematoxylin and eosin (H & E).

Statistical analysis and calculations were performed by using SPSS 15 for Windows (Chicago,IL). Results were expressed as the mean (standard error mean). Kruskal-Wallis analysis of variance was used to detect differences between groups and statistical comparisons were made using the Mann–Whitney U test. A p value of < .01 was considered statistically significant. A p value of < .05 was considered statistically less significant.

The lung tissue was evaluated. Ischemic control group of the examination compared with sham group showed more edema, inflammation and cell degeneration according to histopathologic evaluation. Edema, inflammation and cellular degeneration differed significantly between the study groups (p value 0.032, 0.012 and < 0.0001, respectively). However level of congestion was similar (p=0.124) Therefore there was significantly more edema, inflammation and cellular degeneration in the ischemic control group (Table 1 and Figure 1).

Immunohistochemical markers as, MPO, HSP-70 and IL-6 parameters were studied from the lung tissue samples. Statistical difference was found among the groups at 48 hours postoperatively with MPO and hsp-70 stains (p value 0.019 and 0.05, respectively), whereas there was no difference among the groups with IL-6 stain (p=0.089) (Figure 2). Significant difference was seen in the Sham group when compared with the other groups. Intensity of staining with MPO was greatest in the ischemic control group followed by heparin group. Also, control group showed the most intense staining with Hsp-70 (Table 2) (Figure 3). The lowest HSP-70 level was measured in the sham group, after heparinized groups while the highest HSP-70 level was measured in the ischemic control group.

Examination of the heart tissue revealed that ischemic control group showed more degeneration according to histopathologic evaluation (p=0.005); whereas there was no difference between the groups according to the level of edema, inflammation and congestion (p values, 0.613, 0.499 and 0.558, respectively (Figure 4). Degeneration was worst in the ischemic control group followed by heparin and high dose-heparin groups (Table 3).

Comparison of immunohistochemical markers of the heart tissue revealed that there was a significant difference with MPO staining (p=0.023) which was strongest in the control group, where as IL-6 and Hsp-70 levels did not differ among the groups (p values, 0.483 and 0.4 respectively) (Figure 5).

Grade 1 and 2 staining with HSP-70 was strongest in the control group (n=4 and n=1, respectively) where as it was weakest in the high dose heparinized group. Grade 1 staining was strongest with IL-6 in the control group (n=6) where as it was again weakest in the high dose heparinized group (Figure 6) These qualitative differences did not reach statistical significance possibly due to the low number of samples (Table 4).