Detrimental effect of apoptosis of lymphocytes at an early time point of experimental abdominal sepsis

The study received license permit K/8980/11-12-2006 form the Ethics Committee for Animal Experiments of the Perfecture of Athens. Permit was given in conformance with the following regulations: a) the Greek Presidential approval 30/1996; b) the ministerial decision 167/1997; c) the laws 1197/1981 and 2015/1992 about the rights and about protection of laboratory animals; and d) the directive 160/1991 of the European Union about performance of experiments in laboratory animals.

The experiments were performed in two sets. In the first set, 35 animals were studied. The study endpoint of the initial set of experiments was the relationship between apoptosis of lymphocytes at 4 and 24 hours with 7-day survival. Animals were initially sedated by the intramuscular injection of 25 mg/kg of ketamine and 5 mg/kg of xylazine. Anesthesia was maintained by the intramuscular administration of 15 mg/kg of xylazine at 30-minute time intervals. In 31 rabbits, after an upper midline abdominal incision, the peritoneal cavity was entered and the intestines were displaced to the left. The cecum was recognized and ligated with a 3.0 suture. Three holes were performed in the wall of the cecum just above the suture with a 3.0 needle followed by light massage of the cecum. The peritoneal cavity and the abdominal wall were then closed in layers. Animal resuscitation was done by the continuous intravenous infusion of normal saline at a rate of 30 ml/hour through a catheter connected with the vein of the right ear. Four animals were sham-operated i.e. they were subject only to abdominal incision and closure. All experiments were performed on separate days by the same surgeons.

A volume of 5 ml of blood was sampled from the vein of the left ear of each animal under aseptic conditions before the operation, and at 4, 24 and 48 hours. Three ml were collected into heparin-coated tubes for flow cytometry and stimulation assays. One ml was collected into pyrogen-free tubes and centrifuged. Serum was kept refrigerated at -70°C for the measurement of tumor necrosis factor-alpha (TNFα). Another ml was added into tubes containing 4 ml of blood culture medium (Becton Dickinson, Cockeysville Md) and incubated at 35°C for seven days.

After the end of the operation, animals were transported to their cages. Survival was recorded every 12 hours for a total period of follow-up of 7 days. Every effort was done to minimize animal suffering. This was done by the administration of paracetamol suppositories twice daily starting two hours after the end of the operation. Within one hour after death autopsy was performed; under sterile conditions, segments from the right kidney, liver, spleen and lower lobe of the right lung were taken and placed into separate sterile plastic containers for quantitative cultures and biopsy. Animals remaining alive after 7 days were sacrificed. This was done after initial sedation followed by the rapid intravenous infusion of phenobarbital. Tissue cultures were drawn from these animals as described above.

In the second set of experiments, 40 rabbits were studied. In all animals, peritonitis was induced as described above; blood was sampled at 4 hours after induction of peritonitis. In 20 animals, single shots of ciprofloxacin and metronidazole were administered intravenously by one right ear vein at 4 hours after induction of peritonitis. The dose of ciprofloxacin was 30 mg/kg (Bayer, Germany) and that of metronidazole 25 mg/kg (Sanofi Aventis, France), as reported elsewhere [8, 9]. Survival was recorded for 7 days.

The study endpoint was the relationship between apoptosis of lymphocytes at 4 and 24 hours with 7-day survival. To this end, Ethics Committees licensed the study since it was unavoidable to investigate the impact of apoptosis on survival without 7-day survival as an endpoint.

PBMCs were isolated after gradient centrifugation of heparinized whole blood over Ficoll (Biochrom, Berlin, Germany). After three consecutive washings in ice-cold phosphate buffered saline pH 7.2 (Biochrom), PBMCs were counted in a Neubauer chamber after trypan blue exclusion of dead cells. Half of PBMCs were stained with the protein ANNEXIN-V at the flurochrome fluorescein isothiocyanate (FITC, emission 525 nm, Immunotech, Marseille, France) and with propidium iodine (PI) (emission 575 nm, Immunotech). Cells were analyzed after running through the EPICS XL/MSL flow cytometer (Beckman Coulter Co, Miami, Florida) with separate gating for lymphocytes and for monocytes based on their characteristic FS/SS scattering. Cells staining positive for ANNEXIN-V and staining negative for PI were considered apoptotic. Indicative gating on lymphocytes and on monocytes and staining for ANNEXIN-V and PI are shown in Figure 1.

The remaining half of PBMCs were distributed into wells of 96-well plate of a final volume of 0.2 ml per well with RMPI 1640 enriched with 10% Fetal Bovine Serum (Biochrom), 2 mM glutamine and 10 mM pyruvate at a density of 2 × 10⁶ PBMCs/ml. They were stimulated without/with 10 ng/ml of LPS of Escherichia coli O155:H5 which is a TLR4 ligand (Sigma Co, St. Louis, USA) or without/with 5 μg/ml of Pam3Cys-SKKK (EMC Microcollections, Tübingen, Germany) which a TLR2 ligand. The plates were incubated for 24 hours at 37°C in 5% CO₂. After incubation, plates were centrifuged and the supernatants were collected and stored at -70°C until assayed for TNFα. All stimulation assays were performed in duplicate.

TNFα was measured by a bioassay on L929 fibrosarcoma cell line, as already described [10, 11]. Briefly, confluent cells were thoroughly washed with Hank' s solution and harvested with 0.25% thrypsin/0.02% EDTA (Biochrom). Cells were centrifuged, re-suspended in RMPI 1640 supplemented with 10% Fetal Bovine Serum and 2 mM of glutamine and distributed into a 96-well cell culture plate at a density of 1 × 10⁵ cells/well. The final volume of fluid into each well was 0.05 ml. After incubation for 2 hours at 37°C at 5% CO₂, 0.06 ml of supernatants or of serum or of standard dilutions of known concentrations of human TNFα (Sigma, range 5.75-375.00 pg/ml) were added into each well followed by 0.05 ml of a 0.3 mg/ml dilution of cycloheximide (Sigma) to inhibit de novo protein biosynthesis. After over-night incubation, the supernatant of each well was discarded by aspiration and 0.1 ml of a 0.5 mg/ml methylene blue solution in methanol 99% was added. After ten minutes, the dye was removed and wells were thoroughly washed three times with 0.9% sodium chloride. Wells were left to dry and remnants of the dye in each well became soluble by the addition of 0.1 ml of 50% glacial acetic acid (Merck, Darmstadt, Germany). Optical density in each well was read at 495 nm (Hitachi Spectophotometer, Tokyo, Japan) against blank wells and control wells without added serum. Concentrations of TNFα were estimated by the reduction of the optical density of control wells by unknown samples applying a standard curve generated by standard concentrations. All determinations were performed in quadruplicate. The inter-day variation of the assay was 13.75%.

Tissue segments were weighted and homogenized; one 0.1 ml aliquot was diluted 1:10 into sterile sodium chloride four consecutive times. Another aliquot of 0.1 ml of each dilution was plated onto MacConkey agar and incubated at 35°C for a total period of three days. Plates were incubated at 35°C and the number of viable colonies were counted into each dilution and multiplied by the appropriate dilution factor. Identification of bacteria was performed by the API20E and the API20NE systems (bioMerieux, Paris, France). The lower detection limit was 30 cfu/g. Bacterial cells were expressed by their log₁₀ value.

Results were expressed by their mean (± SE). Comparisons between groups were performed by Mann-Whitney U test. Survival was estimated by Kaplan-Meier analysis; groups were compared by the log-rank test. Bacterial growth was assessed separately for animals that died and separately for animals that were sacrificed after 7 days. To define if the time until death may have an impact on the results of tissue cultures, correlation according to Spearman's rank of order was done between time until death and tissue bacterial growth. Any value of p below 0.05 was considered significant after adjustment for multiple comparisons.