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  • The Influence of G-CSF Addition to Antibiotic Treatment: MATERIALS AND METHODS

The experiment was performed in adherence with the National Institutes of Health guidelines on the use of experimental animals. The study was approved by the university’s animal care committee. Fifty Sprague-Dawley rats (10 weeks old) weighing 200-250 g were allowed to become acclimatized to laboratory conditions for five days before experimental use. The animals were housed at 2 with a 12-hour light-dark cycle and were allowed free access to tap water and standard rodent chow.

The rats were anesthetized by intramuscular injection of ketamine (30 mg/kg of body weight) and injected intraperitoneally with Pseudomonas aeruginosa (ATCC 27 8 5 3, 109 cfu/ml, 3 ml) in a total volume of 500 ц1 of sterile saline.


Recombinant human granulocyte colony-stimulating factor (rhG-CSF) with activity of approximately 8xl0 U/mg (50 U is equivalent to the activity capable of inducing granulocyte colonies from half of the exposed CFU granulocytes) (Neupogen, Roche, Turkey) was diluted in phosphate-buffered saline (PBS) immediately before use. Rats received 100 Lig/kg per day of G-CSF solution subcutaneously.

Experimental Protocol

After fasting overnight, the 50 rats were randomly divided into five groups. The first four groups received a bolus intraperitoneal injection of R aeruginosa (ATCC 27853, 109 cfu/ml, 3 ml). Additionally, the first group (SAG) was injected with intramuscular imipenem (Tienam 20 mg/kg/bid per day) followed by rhG-CSF (Neupogen, Roche) at 100 Lig/kg per day,
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Table 1. Groups and applied procedures


Procedure applied

Group SAG (n=10J

G-CSF + imipenem

Group SA (n=10)


Group SG (n=10)


Group S (n=10)

No G-CSF + no imipenem

Group С (n=10)


again injected subcutaneously. The second group (SA) was injected with intramuscular imipenem (Tienam 20 mg/kg/bid per day). The third group (SG) received rhG-CSF (Neupogen, Roche) at a dosage of 100 |iig/kg per day, injected subcutaneously. The fourth group (S) received no antibiotic medications or rhG-CSF. The fifth group (C) served as the control, receiving PBS (0.1 mL/rat) every day (Table 1).

The experiment was terminated after 120 hours. Peripheral leukocyte counts (PLC) and absolute neutrophil counts (ANC) (Abbott CELL-DYN 3500) were assessed in all groups prior to the inoculation of the infective agent, at the commencement of the experiment and at the 12th, 36th, 60th and 90th hours after inoculation. Survival was monitored up to the 120th hour. Animals surviving the five-day period were sacrificed by decapitation under ether anesthesia.
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Histological Examination

Rats were monitored for 120 hours, with survival defined as hours following sepsis and survival time being recorded for each animal. Rats were sacrificed at the 120th hour or immediately following death. The lung tissues were fixed in 10% formalin for 24 hours, and standard dehydration and paraffin-wax embedding procedures were used. Hematoxylin and eosin-stained slides were prepared using standard methods. The lung tissue in each slide preparation was evaluated without knowledge of the treatment group from which it came. The slides were reviewed at low magnification for an overview to exclude sections containing bronchi, connective tissue, large blood vessels and areas of confluent atelectasis, so that only regions reflecting the degree and state of parenchymal injury would be evaluated. Slides were assessed at high magnification (X400) and five high-power fields (HPF) were randomly sampled. Features of alveolar wall thickening, infiltration of the inflammatory cells, hemorrhage and pulmonary architecture were noted in each of the five HPFs (5HPF). Specifically, alveolar wall thickening, defined as >2 cell layers thick, was graded as 0 (absent) or 1 (present) in each field. Intraalveolar edema fluid, defined as homogenous or fibrillar proteinaceous staining within the alveoli, was graded as 0 (absent) or 1 (present) in each field. A total score/5HPF for alveolar wall thickening and intra-alveolar edema fluid was recorded for each animal. The total number of neutrophils was counted in each of the five HPFs and expressed as the total num-ber/5HPF for each animal. All data were expressed as minimum-maximum. The results were classified into four grades, with grade 1 representing normal histology; grade 2 indicating low neutrophil leukocyte sequestration, slight alveolar wall thickening and edema; grade 3 representing moderate neutrophil leukocyte sequestration, focal hemorrhage and ede­ma, and partial destruction of pulmonary architecture; and grade 4, including dense hemorrhages, massive neutrophil leukocyte sequestration and complete destruction of pulmonary architecture.
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Statistical Analysis

Data were expressed as minimum-maximum. In statistical analysis, the Kruskall-Wallis test and oneway ANOVA were used. Post hoc comparisons between pairs of means were made using the Mann Whitney U test, with a downward adjustment of the alpha level to compensate for multiple comparisons. Within-group comparisons of the groups’ PLC and ANC values for hours were made using the Wilcox-on test. P values <0.05 were considered significant. The log rank test was used to compare survival rates.

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