Mice
Male C57Bl/6 mice (10/group), eight to twelve-weeks-old, weighing 20–25 g have been maintained for many generations in the Animal Breeding Unit (Biotério da Universidade Federal do Maranhão, Sao Luis, MA, Brazil) under standard conditions. The animals were kept in well cross ventilated room at 26 ± 2°C, relative humidity 44–56%, light and dark cycles of 12 h. The animals had free access to sterilized food and acidified water. All procedures described were reviewed and approved by the Animal Ethics Committee in accordance with COBEA (Brazilian College of Animal Experimentation).
Plant material
Leaves of Syzygium jambolanum DC. (Myrtaceae) were collected and identified at the Ático Seabra Herbarium of the Universidade Federal do Maranhão (São Luís, MA, Brazil) (voucher specimen N° 1087). The fresh leaves (200 g) were extracted with 1 L of ethanol (70%) and mixed every 8 h during 24 h. The same procedure was repeated four times. After this period the hydroalcoholic crude extract (HCE) was filtered using a cotton funnel and it was concentrated under low pressure. The yield obtained was 5.1% (w/w). Finally, the HCE was dried and the remainder was later lyophilized. For the experiments, the lyophilized dry residue was diluted in an isotonic phosphate buffered saline (PBS) at a concentration of 1 mg/mL. The animals were then weighed to adjust the dose of HCE to 5 and 50 mg/Kg (mg of dried plant material/Kg of body weight). These doses were chosen based in pilot experiments of neutrophil recruitment.
Experimental design
Polymicrobial sepsis was induced using cecal ligation and puncture (CLP) according to previously described methods [8]. Briefly, under deep anesthesia, a laparotomy was performed and the cecum was mobilized and ligated below the cecal valve, punctured 8× with an 18-gauge needle to induce the lethal sepsis. The cecum was replaced into peritoneal cavity and the abdomen was closed in two layers. Saline (0.5 mL/10 g body weight) was given subcutaneously to CLP animals for fluid resuscitation.
The animals were initially divided into 4 groups that were treated by subcutaneous route 6 hours before the CLP induction. In group 1 (Control) the mice received a control vehicle (saline solution). In groups 2 and 3 the mice received the HCE treatment at the doses of 5 (HCE 5) or 50 mg/Kg (HCE 50), respectively. In group 4 (Sham), the cecum was not perforated and the mice were not treated.
To evaluate the lifespan the mortality of the animals was recorded every 12 h until the 5th day. The mice which remained alive were followed by one month. In all the subsequent assays, the blood of anesthetized mice was collected 12 h after the CLP, and the animals immediately sacrificed.
Blood Glucoses Concentration
The quantification of glucoses was made in peripheral blood using a digital glucosimeter (Advantage II – Roche) with specific material. The values obtained represent the concentration of glucoses (mg/dL).
TNF bioassay
The serum TNF was measured by a modification of the standard L929 in vitro cytotoxicity assay using actinomycin D-treated target cells as previously described method [9].
Determination of serum nitrite concentration
Serum NO levels were determined by the measurement of nitrite and nitrate after enzymatic reduction of nitrate with nitrate reductase, as previously described [10].
Peritoneal cell harvesting
The peritoneal cell harvesting and the assays to evaluate the spreading, the hydrogen release and the nitric oxide production by peritoneal cells were performed according to previously described methods [11].
Platelets, spleen, lymph node and bone marrow's cells counting
The platelets count and the lymphoid cells quantification were performed according to previously described methods [12].
Colony forming units (CFU) determination
The mice were killed 12 hours after the CLP. The skin of the abdomen was cut open in the midline after thorough disinfection and without injury to the muscle and the peritoneal cavity was washed with 2 mL of sterile phosphate buffered solution (PBS). Aliquots of serial log dilutions of the peritoneal fluid obtained were plated on Mueller-Hinton agar dishes (Difco Laboratories, Detroit); colony-forming units were counted after overnight incubation at 37°C, and the results were expressed as log10 of the number of colony-forming units per peritoneal cavity.
Histopathology
To evaluate the inflammatory infiltrate to the cecum walls from mice submitted to CLP, 3 animals from each group were killed 12 h after surgery; cecum fragments were removed, fixed in 10% phormol for 24 h, dehydrated in alcohol, and embedded in paraffin. Serial 5-mm sections were stained with hematoxylin-eosin for analysis of the inflammatory response.
Statistical Analysis
Results are expressed as the mean ± standard error of mean (SEM) deviation from 10 animals per group. Statistical evaluation was done by ANOVA test followed by Neuman-Keuls. Mice lifespan was demonstrated using the Kaplan-Meier curve and the log-rank statistical test was applied to compare the curves. Differences were considered significant at P = 0.05 and are represented by an asterisk. All experiments were repeated for at least two times.
