Acclimatization and Ethical Approval

Twenty-four adult male Sprague–Dawley (SD) rats weighing between 160 and 180 g were used in this study. During the entire experimental period, the animals were acclimatized for 7 days to normal environmental conditions, including a dark/light cycle (8:00 am-8:00 pm), temperature (22–25 °C), and relative humidity (60%). Baseline assessments of body weight and cognitive functions were conducted for all rats. The animals were housed in the animal facility at Cairo University's Faculty of Medicine.

All experimental procedures were performed following the"Institutional Animal Care and Use Committee"at Cairo University (CU-IACUC); IACUC Protocol Number: CU III-F-20–23.

Experimental Design and Animal Study

The animals were randomly classified into four groups (six rats each). Group 1 (the control group) received regular rat chow (10% kcal fat) [11]. Group 2 (HFD 2 W) was fed HFD (60% kcal fat) for 2 weeks and then sacrificed. Group 3 (HFD 4 W) received HFD (60% kcal fat) for 4 weeks before sacrifice. Group 4 (HFD 8 W) received HFD (60% kcal fat) for 8 weeks and was then sacrificed. Thus, we present the first report investigating the time-dependent effect of consuming 60% HFD.

Diet Composition

The rats received a rodent control diet containing 10% of their total calories from fat (D12450Ji, Research Diets) or a diet with 60% of their total calories from fat (Research Diets D12492) [15]. Each group of rats, either the control or the HFD group, received the control diet, which consisted of 10% kcal from fat, or the diet with 60% kcal from fat throughout the study until the time of sacrifice. The rat chow was provided ad libitum, and all rats had access to water (Supplementary File).

At the end of each time point, the rats were assessed for body weight changes and cognitive function. The rats in the control group were sacrificed at the end of the experiment after 8 weeks.

Evaluation of Cognitive FunctionMorris Water Maze Test for Testing the Spatial Memory

The Morris task [16] was conducted in a water tank (150 cm in diameter) filled to a depth of 30 cm. The water was rendered opaque by adding approximately 0.5 L of dried milk powder and maintained at 26 ± 1 °C. The rats underwent four trials per day for four consecutive days. The platform was submerged 1.5 cm below the water surface. Twenty-four hours after the last acquisition trial (on the fifth day), a probe trial was conducted to assess the rats'spatial memory of the submerged platform's location.

Acquisition of the platform location was assessed during the first four days. Each trial began by placing a rat into the water facing the tank wall. Each starting position (north, south, east, and west) was used once per day in a randomized order across the four trials. Each trial ended when the rat climbed onto the platform or after 2 min had elapsed. Once on the platform, the rat remained there for 30 s. After completing the fourth trial, rats were gently dried with a towel and returned to their home cages. The inter-trial interval was consistently maintained at 30 min.

During the probe trial, the platform was removed, and the rat was allowed to search the pool for 60 s before it was removed. During this period, rats were expected to spend more time searching in the quadrant where the platform had previously been located compared to the other three quadrants [16].

Standard protocols for the Morris water maze (acquisition and probe trials) were employed to assess spatial learning and reference memory, which are indicators of long-term memory in animals [17, 18]. The latency to the platform, the time each rat required to reach the platform, and the percentage of time spent in the target quadrant were recorded and analyzed using a video camera to detect latency to the platform [19].

The Y-Maze Spontaneous Alternation Test for the Evaluation of Working Memory

For this behavioral test, a wooden Y-maze apparatus was used, consisting of three arms of equal size (40 cm long × 15 cm wide × 35 cm high) extending from the center at 120° [20]. Each rat was placed in the maze for 5 min, starting from the center, and the sequence of arm choices was recorded. A successful cycle was defined as the rat choosing three successive different arms. The percentage of successful cycles was measured [21].

Modified Novel Object Test for the Evaluation of Episodic-Like Memory

We followed our previous protocol for recording novel object investigation time [22]. This test assesses learning and recognition memory. Each animal was allowed to explore the arena freely for 10 min to acclimate, then returned to its cage for 20 min of rest. The area was cleaned with 70% ethanol after each exploration. Two different objects were then placed diagonally in two corners, and each rat was allowed to explore the area freely for 10 min before returning to its cage for another 20 min of rest. Finally, one of the two objects was replaced with a novel object, and each rat was allowed to re-explore the area for 10 min. The animals were monitored using a video camera, and the total investigation time of the novel object was calculated for each rat. The following equation was used to calculate the percentage of total investigation time:

$$Percentage of total investigation time=\fracx 100$$

At the end of the study, under general anesthesia induced by intraperitoneal injection of pentobarbital sodium (50 mg/kg) [23]. Blood samples were collected from each rat by cardiac puncture after thoracotomy, followed by euthanasia via decapitation. Ten milliliters of blood were obtained from each rat, and the blood was centrifuged at 3000 rpm for 15 min to collect the serum, which was then stored at −20 °C until analysis.

Biochemical Evaluation

Blood samples were collected, and serum from all groups was used to estimate lipid profiles, Lipopolysaccharide (LPS) and I-FABP levels.

Lipid Profile

Estimation of lipid profiles (cholesterol and triglycerides) was done using the total cholesterol rat ELISA kit (Competitive ELISA, Cat No. MBS722885) and the rat triglyceride (TG) ELISA kit (Competitive ELISA, Cat No. MBS726298), respectively.

Briefly, 100 μL of the sample was added to coated wells and shaken, followed by the addition of 50 μL of conjugate mixed with the sample in each well, except for the control. Wells were covered and incubated for 1 h at 37 °C. After washing and removing liquid, substrates A and B were added, followed by a stop solution. Total cholesterol and triglyceride levels were measured at an optical density (O.D.) of 450 nm using a microplate reader.

Lipopolysaccharide (LPS)

Lipopolysaccharide (LPS) level was estimated using the rat LPS ELISA kit (Cat No. MBS268498). After adding LPS standards and samples to the corresponding wells, 100 μL of biotinylated antibody was added to each well. Wells were sealed and incubated at 37 °C for 1 h. Then, 100 μL of prepared enzyme conjugate was added to each well, followed by incubation at the same temperature for 30 min. Subsequently, 100 μL of prepared color reagent was added to each well and incubated until the highest standards exhibited a darker coloration and a color gradient appeared. Finally, 100 μL of color reagent C was added to each well, and results were read at an O.D. of 450 nm [24].

I-FABP

I-FABP level was estimated using the rat I-FABP ELISA kit (Cat No. MBS164325). 50 μL of the standard was added to the standard wells, and 40 μL of the sample was added to each well with 10 μL of anti-I-FABP antibody. Then, 50 μL of streptavidin-HRP was added to both sample and standard wells, mixed, and incubated for 1 h at 37 °C. After washing and removing the liquid, substrates A and B were added, followed by a stop solution. The levels of I-FABP were measured at an optical density of 450 nm using a microplate reader. All kits were obtained from MyBioSource, Inc., San Diego, CA 92195–3308.

Histological Evaluation

Following euthanasia, the right cerebral hemisphere, ileum, and colon were dissected and fixed in 10% formaldehyde solution for 24 h, then processed into paraffin blocks. To investigate structural alterations, five µm-thick sections from all three organs were cut using a microtome and mounted on glass slides for hematoxylin and eosin (H&E) staining [25].

Immunohistochemical Staining

The sections were mounted on ready-to-use, positively charged slides. After deparaffinization, the tissues were hydrated by gradually decreasing the alcohol concentration and then transferred to distilled water for 5 min. Antigen retrieval was performed by treating the sections with 10 mM citrate buffer (pH 6) in a microwave for 2 min, followed by a 20-min cooling period at room temperature. Sections were incubated overnight with an anti-beta-amyloid antibody (ab252816; rat monoclonal, dilution 1:100; Abcam, Cambridge, UK) and anti-malonaldehyde (anti-MDA) antibody (ab243066; mouse monoclonal, dilution 1:100; Abcam, Cambridge, UK). Next, two drops of streptavidin peroxidase were applied for 10 min, followed by two drops of biotinylated secondary antibody for 20 min. The reaction was visualized using Mayer's hematoxylin as a counterstain and diaminobenzidine (DAB) as a chromogen. Phosphate-buffered saline (PBS) was used instead of primary antibodies in the negative control sections.

Morphometric Study

The"Leica Qwin 500C"image analyzer computer system (Leica Imaging System Ltd., Cambridge, UK) was used to obtain results in the Medical Histology and Cell Biology Department at Cairo University's Faculty of Medicine. The image analyzer was made up of an Olympus colour video camera, a colored monitor, and an IBM personal computer's hard drive that was attached to the microscope and managed by"Leica Qwin 500 C"software.

Slides were examined using a light microscope, and binary mode was employed to measure parameters in ten non-overlapping, randomly chosen high-power fields (× 400) for each section:

a.

Optical density of amyloid and MDA positive immunoreactivity in immunostained sections.

b.

Mean area percentage of GFAP positive immunoreactivity in immunostained sections.

Statistical Methods

Data were coded and analyzed using SPSS version 28 (IBM Corp., Armonk, NY, USA) and expressed as mean ± standard deviation. Between-group comparisons were performed using one-way analysis of variance (ANOVA) followed by the Bonferroni post hoc test. Within-group comparisons between baseline and endpoint values were conducted using paired t-tests. Two-group comparisons were performed using unpaired t-tests [26]. Pearson correlation analysis was employed to assess linear relationships. A p-value < 0.05 was considered statistically significant.