Eight-week-old male Sprague Dawley (SD) rats (SLC, Inc., Shizuoka, Japan) were used. They were housed in plastic cages at a room temperature of 23 $\pm$ 2 °C, 12-hr light/12-hr dark cycle (lights on from 7:00–19:00), and 55 $\pm$ 5% humidity with free access to food and water. One week before the start of the experiment, the rats underwent a handling procedure once daily to decrease the experimental stress. The handling method consisted of lifting the rat with a gloved hand and massaging it for 10 minutes. All experimental procedures were conducted and approved by the Shimane University Animal Ethics Committee (Authorization No: IZ3-50).

Pg-LPS was purchased from InvivoGen (catalog no. tlrl-pglps, version no. 14F18-MM, San Diego, CA, USA). Pg-LPS was dissolved in endotoxin-free water.

We randomly divided the SD rats into a behavioral test group and an immunohistochemistry group. Each group was further assigned to the sham injection and Pg-LPS injection groups. It has been documented that the stress intensity of the forced swimming test (FST) is very strong [21], and the FST stress has the possibility of affecting brain structure and function [22]. In order to avoid such influence on immunohistochemistry results, we divided rats into the group undergoing behavior tests only and the group undergoing the immunohistochemical analysis only. The number of individuals in each group was 5 or 6. Investigators were not blinded to the group allocation at any stage.

The rats in the Pg-LPS subgroup received Pg-LPS at gradually increasing doses by daily i.p. injection for seven consecutive days (Fig. 1). The Pg-LPS dose was increased as follows: day 1: 0.5, day 2: 0.5, day 3: 0.75, day 4: 0.75, day 5: 1.0, day 6: 1.0, and day 7: 1.0 (mg/kg of body weight (BW)). Repeated daily administration of increasing LPS doses has been shown to induce depressive-like behavior more robustly, compared to administration of consistent LPS dose, with avoidance of LPS tolerance [15].

Fig. 1.

The experimental schedule. Pg, Porphyromonas gingivalis; LPS, Lipopolysaccharide; i.p., intraperitoneal; IHC, immunohistochemistry; FST, forced swimming test.

The sham injection group received an equivalent volume of saline intraperitoneally and daily for seven consecutive days. In the behavioral test group, the Y-maze test was performed 2 hr after the last i.p. injection. 2 hr after the Y-maze test, habituation of the forced swimming test (FST) was carried out. In the immunohistochemistry group, brain samples were collected 2 hr after the last injection (Fig. 1).

The forced swimming test (FST) was performed as described [23]. Briefly, the rat was placed in a cylinder of water for 6 min, and its behavior was recorded by a video camera. The time that the rat spent in an immobile state (i.e., the immobility time) throughout the test was manually counted based on the recorded video. The rat was judged immobile when it persisted motionless and floating without struggling and when it moved merely to maintain its head above the water.

The Y-maze test assessed the spatial working memory after the last Pg-LPS injection following a described protocol [23]. Briefly, the rat was placed at the terminus of one arm of the maze apparatus (catalog no.YM-03R, Muromachi Kikai, Tokyo, Japan) and allowed to move freely across the maze for 8 min. The entry was regarded as complete when the tail of the rat was entirely within the arm. Alternation behavior was regarded as successive entries into all three arms of the maze on successive occasions. The percentage of spontaneous alternation behavior (SAB) was estimated by the formula mentioned below.

$$\%\mathrm{SAB}=\frac{\text{The number of alternations}}{\text{The total number of arms entries}-2}\times 100\%$$

At 2 hr after the last administration of the Pg-LPS treatment, the rats in the immunohistochemistry group were sacrificed under deep intraperitoneal anesthesia with a mixture of three drugs: medetomidine (Domitor, Nippon Zenyaku Kogyo, Fukushima, Japan) at 0.15 mg/kg BW/rat, midazolam (Dormicum, Astellas Pharma, Tokyo, Japan) at 2 mg/kg BW/rat and butorphanol (Vetorphale, Meiji Seika Pharma, Tokyo, Japan) at 2.5 mg/kg BW/rat. Saline (Otsuka Pharmaceutical Factory, Tokushima, Japan) was then added up to adjust the mixture to a volume of 0.5 mL/100 g BW/rat. The rats were perfused transcardially with 500 mL of physiological saline and then 500 mL of 4% of paraformaldehyde (PFA) in phosphate buffer solution (Fujifilm, Wako, Osaka, Japan). After the perfusion, the brains were quickly removed and fixed with 4% of paraformaldehyde in phosphate buffer solution for 4 hr at room temperature (RT). The brains were submersed in 10% sucrose solution overnight at 4 °C and were subsequently immersed in 20% sucrose solution for 24 hr at 4 °C. The brains were sectioned at 40-µm thickness in the frontal plane by using a microtome (Microm HM 430, Thermo Scientific, Walldorf, Germany).

The free-floating brain sections were incubated in 1% H${}_2$O${}_2$ at RT for 30 min, followed by blocking incubation with 0.2% Triton-X and 1.5% and normal horse serum in 0.1 M phosphate buffer at RT for 1 hr. The brain sections were incubated overnight at RT with rabbit anti-ionized calcium-binding adapter molecule (Iba)-1 antibody (1:2000, catalog no. 019-19741, Wako, Osaka, Japan) and goat anti-glial fibrillary acidic protein (GFAP) antibody (1:2000, Abcam, Cambridge, UK). The next day, the sections were treated with biotinylated anti-rabbit or anti-goat IgG antibody (1:1000, catalog no. PK-4000, standard ABC kit, Vector Laboratories, Newark, CA, USA) at RT for 1 hr. The sections were incubated at RT for 1 hr with an avidin-biotin-peroxidase complex in phosphate-buffered saline (PBS). The immunoreactivity in the brain tissues was developed by incubating in PBS comprising 0.5% diaminobenzidine (DAB) and 0.03% H${}_2$O${}_2$ for 10 min. To halt the DAB reaction, the brain sections were washed with PBS for 30 min.

The intensity of microglial and astrocytic immunoreactivity in DAB-stained images was measured by a computer-assisted image analysis program (ImageJ 1.52a, National Institutes of Health, Bethesda, MD, USA) described [24]. The images were captured by using a BZ-X700 all-in-one microscope (Keyence, Osaka, Japan) with a 20$\times$ objective lens. The regions of interest consisted of three areas within the hippocampus, i.e., the dentate gyrus (DG), the cornu ammonis 1 (CA1), and the CA3 and two areas within PFC, i.e., the prelimbic (PrL) and the infralimbic (IL) areas. Twenty images were captured bilaterally from each area of the hippocampus (10 images from the left hemisphere and 10 images from the right hemisphere). Sixteen images were captured bilaterally from each area of the PFC (8 images from the left hemisphere and 8 images from the right hemisphere). A total of 92 images (60 images of hippocampus and 32 images of PFC) were analyzed on every rat (n = 5). The software automatically translated all immunolabeled components beyond the threshold range into black pixels and translated the rest of the image into white pixels. The software estimated the percentage of black pixels for statistical analysis.

All the data are expressed as the mean $\pm$ standard deviation (S.D.). The experimental unit of analysis is the SD rat. For two group comparisons, we performed the independent sample t-test. The primary outcome is the % of SAB in the Y-maze test. The secondary outcomes are the immobility time in the FST and % of black pixel area in immunohistochemical analysis. The minimum sample size was calculated as n = 5 with the Power*G software (version.3.1.9.2, Heinrich-Heine University, Dusseldorf, Germany). The parameters, which comprise effect size d = 2.437, $\alpha$ error prob = 0.05; power (1-$\beta$ error prob) = 0.9, and allocation ratio = 1, were put into the software. Using the Shapiro-Wilks test, we confirmed that the data met the statistical assumptions. The statistical analysis was conducted by using the software SPSS (version. 23.0, IBM Corp., Tokyo, Japan). A p-value less than 0.05 was considered significant.

The behavioral tests were conducted to examine whether peripheral injection of Pg-LPS can cause depressive-like behavior, such as behavioral despair and impaired spatial working memory. In the FST, learned helplessness in the rats was evaluated by measuring the immobility time, which was 108.73 $\pm$ 29.33 sec in the sham group (n = 6) and 181.54 $\pm$ 49.20 sec in the Pg-LPS group (n = 5) (Fig. 2A). The immobility time in the Pg-LPS group was significantly longer than that in the sham group, suggesting that the i.p. injection of Pg-LPS induce behavioral despair (Table 1).

Fig. 2.

Behavioral tests of Pg-LPS-injected rats. (A) Effects of Pg-LPS injection on immobility time in the forced swimming test. (B) Effect of Pg-LPS injection on spontaneous alternation behavior (SAB) in the Y-maze test. *p 0.05. Each value is the mean $\pm$ S.D. Sham group (n = 6), Pg-LPS group (n = 5).

We evaluated the rat’s spatial working memory in the Y-maze test by calculating the percentage of SAB. As shown in Fig. 2B. the percentage of SAB was 72.83 $\pm$ 12.29% in the sham group (n = 6) and 57.43 $\pm$ 4.75% in the Pg-LPS group (n = 5). A significant reduction in the % SAB was detected in the Pg-LPS group compared to the sham group. This result suggests that the i.p. injection of Pg-LPS impaired spatial working memory (Table 1).

We investigated the immunoreactivity of microglial Iba-1 in the hippocampus (CA1, CA3, and DG regions; Fig. 3A,B,D,E,G,H) and the PFC (PrL and IL regions; Fig. 3J,K,M,N). Among these regions, the CA3 region showed significantly higher immunoreactivity for Iba-1 in the Pg-LPS-injection group (18.05 $\pm$ 2.49%, n = 5) compared to the sham injection group (13.95 $\pm$ 1.57%, n = 5) (Fig. 3I). Also, the PrL region showed significantly higher immunoreactivity for Iba-1 in the Pg-LPS-injection group (18.05 $\pm$ 1.73%, n = 5) compared to the sham injection group (13.49 $\pm$ 2.31%, n = 5) (Fig. 3L). In the other regions, the Pg-LPS group (n = 5) showed a non-significant trend for higher Iba-1 immunoreactivity compared with the sham group (n = 5) (Fig. 3C,F,O) (Table 2).

Fig. 3.

Immunoreactivity for Iba1 in the hippocampus and prefrontal cortex. Representative images of 0.5% diaminobenzidine (DAB) staining in the dentate gyrus (DG) of Sham (A) and Pg-LPS rats (B), the cornu ammonis (CA)1 of Sham (D), and Pg-LPS rats (E), and CA3 of the Sham (G) and Pg-LPS rats (H), prelimbic (PrL) area of Sham (J) and Pg-LPS rats (K), and the infralimbic (IL) area of Sham (M), and Pg-LPS rats (N). Scale bars, 100 µm; Quantification data of Iba1-immunoreactivity in the DG (C), CA1 (F), CA3 (I), PrL (L), and IL (O). *p 0.05. N.S., not significant. Each value is the mean $\pm$ S.D. (n = 5).

We also evaluated the astrocytic GFAP immunoreactivity in the hippocampus (CA1, CA3 and DG regions; Fig. 4A,B,D,E,G,H) and the PFC (PrL and IL regions; Fig. 4J,K,M,N). The hippocampal CA1 regions showed significantly higher immunoreactivity for GFAP in the Pg-LPS-injection group (27.98 $\pm$ 2.69%, n = 5) compared to the sham injection group (21.75 $\pm$ 4.88%, n = 5) (Fig. 4F) and CA3 regions showed significantly higher immunoreactivity for GFAP in the Pg-LPS-injection group (21.47 $\pm$ 1.13%, n = 5) compared to the sham injection group (14.41 $\pm$ 3.21%, n = 5) (Fig. 4I). Also the DG region showed significantly higher immunoreactivity for GFAP in the Pg-LPS-injection group (31.19 $\pm$ 2.10%, n = 5) compared to the sham injection group (22.59 $\pm$ 4.67%, n = 5) (Fig. 4C). On the other hand, in the PFC PrL and IL regions, we observed no significant difference in GFAP immunoreactivity between the Pg-LPS (n = 5) and sham groups (n = 5) (Fig. 4L,O) (Table 3).

Fig. 4.

Immunoreactivity for GFAP in the hippocampus and the prefrontal cortex. Representative images of DAB staining in the DG of Sham (A) and Pg-LPS rats (B), CA1 of Sham (D) and Pg-LPS rats (E), CA3 of Sham (G) and Pg-LPS rats (H), PrL of Sham (J) and Pg-LPS rats (K), and IL of Sham (M), and Pg-LPS rats (N). Scale bars, 100 µm; Quantification data of Iba1-immunoreactivity in the DG (C), CA1 (F), CA3 (I), PrL (L), and IL (O). *p 0.05. N.S., not significant. Each value is the mean $\pm$ S.D. (n = 5). GFAP, glial fibrillary acidic protein.