S. pneumoniae (serotype 3) was cultured overnight in 10 ml of Todd Hewitt broth, diluted in fresh medium and grown to logarithmic phase. This culture was centrifuged for 10 min at (5,000×g) and resuspended in sterile saline to the concentration of 5×109 cfu/ml. The size of the inoculum was confirmed by quantitative cultures [16, 17].
Animal model of meningitis
Adult male Wistar rats (250–350 g body weight), from our breeding colony were used for the experiments. All procedures were approved by the Animal Care and Experimentation Committee of UNESC, Brazil, and followed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23) revised in 1996. All surgical procedures and bacterial inoculations were performed under anesthesia, consisting of an intraperitoneal administration of ketamine (6.6 mg/kg), xylazine (0.3 mg/kg), and acepromazine (0.16 mg/kg) . Rats underwent a cisterna magna tap with a 23-gauge needle. The animals received either 10 μl of sterile saline as a placebo or an equivalent volume of S. pneumoniae suspension. At the time of inoculation, animals received fluid replacement and were subsequently returned to their cages [19, 20]. Eighteen hours later, the meningitis was documented by a quantitative culture of 5 μl of CSF obtained by puncture of the cisterna magna . The animals were randomly in three different groups sham (control group), meningitis with daptomicyn treatment and meningitis with ceftriaxone treatment. The daptomycin (Cubicin®; 50 mg/kg body weight) and ceftriaxone (100 mg/kg body weight) were administered subcutaneously [s.c.]) during 7 days.
After 10 days, the animals were free from infection. We performed blood cultures that were all negative in this period. Thus, the animals were separately undergone to four behavioral tasks: a) open field; b) object recognition; c) step-down inhibitory avoidance task (single-training) and d) continuous multiple-trials step-down inhibitory avoidance task.
The animals underwent separately to four behavioral tasks: habituation to an open field, step-down inhibitory avoidance task, continuous multiple-trials step-down inhibitory avoidance task and object recognition. All behavioral procedures were conducted between 13:00 and 16:00 p.m. in a sound-isolated room, and each animal performed only one behavior test. All behavioral tests were recorded by the same person who was blind to the animal group.
Open field test
The behavior was assessed in the open field apparatus in order to evaluate both locomotor and exploratory activities. The apparatus is a 40 cm × 60 cm open field surrounded by 50 cm high walls made of brown plywood with a frontal glasswall. The floor of the open field is divided into 9 rectangles by black lines. The animals were gently placed on the left rear quadrant and then left alone to explore the arena for 5 min (training session). Immediately after this procedure, the animals were taken back to their home cage and 24 h later they were submitted again to a similar open-field session (test session). Every cross of the black lines and rearing performed in both sessions were counted for 5 min. The decrease in the number of crossings and rearings between the two sessions was taken as a measure of the retention of habituation memory .
This task evaluates the non-aversive and non-spatial memory. The apparatus and procedures for the object recognition task have been described elsewhere . Briefly, the task took place in a 40 × 50 cm open field surrounded by 50 cm high walls made of plywood with a frontal glass wall. The floor of the open field was divided into 12 equal rectangles by black lines. All animals were submitted to a habituation session where they were allowed to freely explore the open field for 5 min. No objects were placed in the box during the habituation trial. The total number of crossings of the black lines and rearings performed in this session were evaluated as locomotors and exploratory activity, respectively. The training was conducted by placing individual rats for 5 min in the field, in which two identical objects (objects A1 and A2, both being cubes) were positioned in two adjacent corners, 10 cm from the walls. In a short-term recognition memory test given 1.5 h after training, the rats explored the open field for 5 min in the presence of one familiar (A) and one novel (B, a pyramid with a square-shaped base) object. All objects had similar textures (smooth), colors (blue), and sizes (weight 150–200 g), but distinctive shapes. A recognition index calculated for each animal is reported as the ratio TB/(TA + TB) (TA = time spent exploring the familiar object A; TB = time spent exploring the novel object B). In a long-term recognition memory test given 24 h after training, the same rats were allowed to explore the field for 5 min in the presence of the familiar object A and a novel object C (a sphere with a square shaped base). Recognition memory was evaluated using the same method of the short-term memory test. Exploration was defined as sniffing (exploring the object 3–5 cm away from it) or touching the object with the nose and/or forepaws.
Step-down inhibitory avoidance task
This task evaluates aversive memory. The apparatus and procedures have been described in previous reports [23, 24]. Briefly, the training apparatus was a 50 × 25 × 25 cm acrylic box (Albarsch, Porto Alegre, Brazil) which the floor was consisted of parallel caliber stainless steel bars (1 mm diameter) spaced 1 cm apart from each other. A 7 cm-wide, 2.5 cm-high platform was placed on the floor of the box against the left wall. In the training trial, animals were placed on the platform and their latency to step down on the grid with all four paws was measured with an automatic device. Immediately after stepping down on the grid, the animals received a 0.4 mA, 2.0 s foot shock and returned to their home cage. A retention test trial was performed 24 h after training (long-term memory). The retention test trial was procedurally identical to the training, except that no foot shock was presented. The retention test step-down latency (maximum, 180 s) was used as a measure of inhibitory avoidance retention. Reactivity to the foot shock was evaluated in the same apparatus used for inhibitory avoidance, except that the platform was removed. Each animal was placed on the grid and allowed 1 min for habituation period prior to the start of a series of shocks (0.5 s) delivered at 10 s intervals. The shock intensities ranged from 0.1 to 0.5 mA in 0.1 mA increments. The adjustments in the shock intensity were made in accordance to each animal’s response. The intensity was raised by 1 unit when no response occurred and lowered by 1 unit when a response was made. A “flinch” response was defined as the withdrawal of one paw from the grid floor, and a “jump” response was defined as rapid withdrawal of three or four paws. Two measurements of the “flinch” threshold were made and then two measurements of the “jump” threshold were made. For each animal the mean of the two scores for the flinch and the jump thresholds were calculated [25, 26].
Continuous multiple-trials step-down inhibitory avoidance task
This task evaluates aversive memory in the test section and learning when analyzing the number of training trials required for the acquisition criterion (see below). It was performed in the same step-down inhibitory avoidance apparatus; however, in the training session, the animal was placed on the platform and immediately after stepping down on the grid, received a 0.3 mA, 2.0 s foot shock. This procedure continued until the rat remained on the platform for 50 s. The animal was then returned to the home cage. The number of training trials required to reach the acquisition criterion of 50 s on the platform was recorded. The retention test was performed 24 h later (long-term memory) .
Data from the habituation to an open field task is reported as mean ± SEM, and it was analyzed by the paired Student’s t test and ANOVA post-hoc Tukey. Data from the object recognition task and inhibitory avoidance task are reported as median and interquartile ranges, and comparisons among groups were performed using Mann–Whitney U tests. The within-individual groups were analyzed by Wilcoxon’s tests. Data from continuous multiple-trials step-down inhibitory avoidance task the trials were reported as mean ± SD, and were analyzed by the ANOVA post-hoc Tukey. Data from the latency time was reported as median and interquartile ranges, and comparisons among groups were performed using Mann–Whitney U tests. In all comparisons, p<0.05 indicated statistical significance.