Preparation of C. acnes suspensions

The strain C. acnes ATCC 6919 (belongs to the phylotype IA-1 and ribotype 1 subgroup) was obtained from the Pasteur Institute of Iran, and was stored as a freeze-dried culture. Before commencing the study, the strain underwent revival and maintenance in accordance with established microbiological approaches. The C. acnes ATCC 6919 strain was sub-cultured on blood agar plates (Merck, Germany) containing 5% defibrinated sheep blood before being transferred to the solution. A single colony was carefully chosen from the plate and inoculated into a liquid culture medium, Reinforced Clostridial Medium (RCM) broth, to facilitate targeted bacterial growth. Subsequently, the bacterial culture within the RCM broth was subjected to anaerobic incubation at 37 °C for a duration of 72 h, accompanied by gentle agitation at 120 rpm, until it reached the mid-log phase. This process was executed within an anaerobic incubator (Anoxomat MARK II system) utilizing a gas blend comprising 80% N2, 10% CO2, and 10% H2. McFarland standard was used as a reference to adjust the turbidity of bacterial suspensions. Serial dilution was prepared as 1.4 × 108 colony-forming units (CFU)/ml of bacteria.

Experimental design and groups

A group of healthy adult male Wistar rats, who were four months old and weighed between 220 and 250 g, were obtained from the Institute for Cognitive Science Studies. The rats were kept in a controlled environment at 22 ± 2 °C, 60 ± 5% humidity, and a 12-hour dark/light cycle. They had access to food and water at all times. All experimental methods meet the National Institute of Health Care and Use of Laboratory Animals Guideline and conformity with the regional guidelines. This study was approved by Medical Ethics Committee at Shahid Beheshti University of Medical Sciences, Tehran, Iran (Approved no: IR.SBMU.AEC.1401.022). In this study, 24 rats were randomly divided into the following 3 groups (8 rats in each group): (1) Control group, after incisions in the scalp to expose the skull, burr holes were drilled at the right lateral cerebral ventricle and punctured by the needle without injecting any solution. (2) Sham group, intracerebroventricular (ICV) injection of 2 µL normal saline, (3) C. acnes treated group, ICV injection of 2 µL bacteria with a concentration of 1.4 × 108 CFU/ml (2.8 × 10^5 CFU) into the right lateral cerebral ventricle. In the previous study, we identified the cognition impairment in behavioral tests, and between 3 doses of C. acnes, 1.4 × 108 CFU/ml showed the higher impairment [19]. In order to identify any structural changes in the neuronal cells that were related to bacterial infection in the hippocampus, an immunohistochemistry analysis was conducted to evaluate the expression of Aβ, β-tubulin, and MAP2. Additionally, western blot was conducted to evaluate Tau and p-Tau to identify changes in axons. The workflow of this study is presented in Fig. 1.

Fig. 1

Work flow of study contain 3 parts, preparation of C. acnes, injection ICV to animals and behavioral assess, and finally molecular study including IHC and western blot techniques

Surgery and intracerebroventricular (ICV) injection

The animals were first anesthetized with a mixture of 100 mg/kg Ketamine hydrochloride and 25 mg/kg Xylazine (Sigma) after 12 h of fasting and 4 h of water deprivation. They were then positioned in a stereotaxic instrument, and a cannula (22 Gauge) was inserted into the right ICV region as per Paxinos and Watson’s atlas (Paxinos, 2007). The cannula was positioned at ML: 1.4 mm right of the midline, DV: 2.4 mm ventral, and AP: 0.8 mm posterior to bregma to the superior surface of the skull, and was fixed with jeweler’s acrylic cement. The tip of the cannula was 1 mm above the microinjection site. After the surgery, the rats were allowed to recover from anesthesia and placed in a clean room. After 7 days, PBS and different doses of C. acnes solutions was injected using a set of polyethylene tubes (Single lumen PE50 tubing), a microsyringe (ILS, Stuetzerbach, Germany), and a dental needle (27 Gauge). The drug flow was established by the movement of an air bubble inside the polyethylene tube that attached the microsyringe and then to the dental needle. The solution was injected gently for over 1 min, and the needle was kept in position for an additional 60 s to prevent the backflow of the solution. After 7 days of C. acnes or PBS inoculation, some animals in each group were sacrificed for molecular studies, and the rest were assayed through behavioral test to evaluate the bacterial infection effect on memory performance.

Passive avoidance learning (PAL) test

The step-through passive avoidance apparatus was applied for measuring avoidance memory retention in rats [20]. The apparatus consists of two compartments, including a lighted chamber and a dark chamber with same size (20 cm x 20 cm x 30 cm) and separated by a guillotine door that blocks light. The chamber floors consist of stainless steel rods (3 mm diameter) that are spaced 10 mm apart. The bottom of the dark chamber is linked to an apparatus that generates electric shocks [21]. The test involved three steps, including a habituation step, an acquisition training step and a retrieval test. In the first step, animals were located for 10 min within the apparatus with the opened door and gate due to habituation. The acquisition trial was performed on the second day. In this step, the rats were allowed to acclimate in the light chamber for a short while. After one minute, the door to the chamber was raised, allowing the rats to move into the dark compartment. Once inside, the door was closed, and a three-second delay occurred before an electrical shock (1 mA, 50 Hz) was delivered for three seconds. After 20 s, we moved the rats back to their cages. Two minutes later, we placed the rats in the light compartment. The acquisition trial was considered successful if the rat did not enter the dark compartment within 120 s. However, if the rat entered the dark compartment before the 120 s were up, it would receive the same shock again. Each rat was limited to a maximum of three foot shocks. On the third day, the retention trial was performed according to the same procedure as the acquisition trial but without electrical shock. In both the acquisition and retrieval trials, entry latencies time (sec) into the dark compartment were recorded for each Rat. The time spent in the dark compartment (TDC) was also noted in retrieval trial. Animals that failed to enter the dark compartment during the acquisition trial were excluded from the study.

Immunohistochemistry (IHC)

The brains of rats that were deeply anesthetized were removed from their skulls. They were then fixed with 4% paraformaldehyde and embedded in paraffin. We used three brain sections per animal. To prepare the brain tissue sections for staining, they were deparaffinized with fresh xylene and rehydrated using a series of graded ethanol. To efficiently expose epitopes to the antibodies, phosphate-buffered saline (PBS, pH = 7.4) was used for antigen retrieval. The slides were then treated with 3% H2O2 for 10 min at 37℃ to reduce endogenous peroxidase activity. To avoid non-specific protein binding, the slides were blocked using 10% normal goat serum at room temperature for 1 h. The primary antibodies and their corresponding secondary antibodies (conjugated to HRP), including Aβ1-42, (1:500, sc-28365 - Santa Cruz); MAP2, (1:500, Sc-74421- Santa Cruz); and β-tubulin, NBP1-62416 (1:400) were then incubated with the slides. Finally, the sections were subjected to staining procedures utilizing 3, 3′-diaminobenzidine (DAB, Zhongshan Biotech Co., Ltd, Beijing, China) for 5 min followed by a re-staining process with hematoxylin for 2 min. In addition, to visualize nuclei, cell nuclei in the sections were stained with DAPI dye at a dilution of 1:500 (Sigma-Aldrich). The stained sections were assayed using Labomed TCM 400 fluorescence microscope coupled with a digital camera. For each brain section, three images were taken from the hippocampus region. Investigation of protein expression was carried out using ImageJ Fiji software. For Aβ plaques, a threshold was applied to convert the images to binary, and the “Analyze Particles” function was used to count the plaques. For MAP2-positive neurons, the cell counter plugin was employed to manually count the neurons with visible MAP2 staining.

Western blot

Brain tissues were immediately removed, and both hippocampi were dissected and lysed. Proteins were separated by gradient SDS polyacrylamide gel electrophoresis (15%) and after which they were transferred to an Immobilon-P membrane by electroblotting according to the manufacturer’s instruction (Invitrogen). Primary antibodies (purchased from Santa Cruz Biotechnology, United States) against Phospho-Tau (p-Tau) (PHF-13, sc32275) and Tau (sc32274), were incubated with the membranes overnight at 4 °C. After this step, the membranes were treated with Anti-Rabbit secondary antibody (1:3000, BA1054-2-bosterbio) for 2 h at room temperature. To ensure consistency in sample loading and protein transfer, and to standardize the levels of p-Tau and tau proteins, GAPDH was utilized.

Statistical analysis

GraphPad PRISM software (version 10) and Excel were utilized for statistical analysis. The data is presented as mean ± SD. For the statistical analysis of parametric data, one-way ANOVA was performed to compare several groups, and the Tukey test was used for comparing two groups. To analyze the non-parametric data, the Kruskal-Wallis test was used to compare multiple groups and Dunn’s multiple comparisons test was used to compare two groups. A significance level of P < 0.05 was considered statistically significant.