Knocking out Sema4B prevents anesthesia/surgery-induced cognitive decline in aged mice by suppressing hippocampal neuroinflammation

2.1 Animals

The initial heterozygous Sema4B mice were obtained from the Jackson Laboratory (United States). Homozygous (Sema4B^−/−) and wild-type (WT) littermates were generated through heterozygous intercrossing. Genotypes were identified by Polymerase Chain Reaction (PCR) analysis of genomic DNA extracted from auricular tissue. The WT Sema4B allele was detected using Primer 1 (5′-AGACATGGTGCTGGAGAGGT-3′), while the mutant Sema4B^−/− allele was confirmed using Primer 3 (5′-TGCACATGCTTTACGTGTG-3′).

Every animal procedure was carried out in compliance with the U.S. National Institutes of Health’s published standards for the use and care of laboratory animals. The Qingdao Chengyang People’s Hospital Animal Experimentation Ethics Committee gave its approval to the study protocol. A total of 24 male mice, including 12 Sema4B^−/− and 12 WT mice aged 18–22 months and weighing 26–33 g, were randomly assigned to four groups. Every mouse had unlimited access to food and water in a controlled environment (22–24 °C, 12/12 h light-dark cycle).

2.2 Animal groups and treatment

The mice were randomly divided into four groups (n = 6 per group): (1) WT control, (2) anesthesia/surgery (A/S), (3) Sema4B^−/− control, and (4) A/S+ Sema4B^−/− Mice in the WT and Sema4B^−/− groups did not undergo anesthesia or surgery. In the A/S and A/S + Sema4B^−/− groups, anesthesia was induced using 3.0% isoflurane and maintained with 1.5% isoflurane. A surgical incision was made along the lateral aspect of the tibia, after which a stainless-steel intramedullary fixation pin (0.8 mm diameter, 10 mm length) was inserted through the distal tibial plateau into the medullary cavity, traversing the fracture site to the distal end to complete internal fixation.

During the surgical procedure, the physiological parameters of the mice, including respiratory rate, heart rate and body temperature, were continuously monitored in real-time using dedicated respiratory monitors, heart rate monitors, and temperature probes to ensure physiological stability. Postoperative analgesia was administered by topical application of 2% lidocaine at the surgical incision site. Specifically, immediately after suturing the incision, a single dose of 2% lidocaine solution was applied to the lateral tibial incision and its surrounding area. Based on the observed pain-related behaviors, additional applications were performed at 2 hours and 4 hours postoperatively to sustain the analgesic effect [4].

2.3 Immunofluorescence

After 4% Paraformaldehyde (PFA) was used to preserve the hippocampal tissue, 40 μm cryosections were prepared. The sections were blocked in bovine serum albumin (BSA) for one hour at room temperature. After that, they were incubated with primary antibodies against Sema4B (1:500, ab118458, Abcam, Cambridge, UK) and Iba1 (1:500, ab178846, Abcam, Cambridge, UK). Following incubation, the sections were subjected to three Phosphate Buffered Saline (PBS) washes before being incubated for two hours at room temperature with secondary antibodies (goat anti-rabbit Immunoglobulin G (IgG), 1:500, Abcam, ab150080, Cambridge, UK). In order to prepare the sections for confocal imaging, they were lastly sealed and dried for 10 to 15 minutes at 30 to 32 °C (Thermo Fisher, USA).

2.4 Morris water maze (MWZ) test

The behavioral test was conducted in a circular pool measuring 120 cm in diameter and 50 cm in height, with the water temperature maintained at 22 ± 1 °C. Briefly, the mice were trained for five consecutive days beginning one week before surgery. During each training session, the mice were guided to locate a hidden platform, and the time taken to reach the platform was recorded. To evaluate spatial memory, the hidden platform was taken away on the sixth day, and the number of times the mice crossed the platform’s original location was noted [13].

2.5 Enzyme-linked immunosorbent assay (ELISA)

After the hippocampal tissue was homogenized, the supernatant was collected by centrifugation at 10,000 g for 10 minutes at 4 °C. According to the manufacturer’s instructions, the concentrations of TNF-α, IL-1β, and IL-6 (Elabscience) in the supernatant were measured using ELISA kits. Using an ELISA plate reader (Multiskan GO, Thermo Fisher Scientific, Waltham, MA, USA), the optical density (OD) at 450 nm was measured.

2.6 Western blotting

After hippocampal tissue lysis using Radioimmunoprecipitation Assay (R0278, Millipore, Burlington, MA, USA), equal amounts of protein (20 μg) were loaded onto an 8% Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) gel for electrophoresis and transferred to a Polyvinylidene Fluoride (PVDF) membrane (Millipore). The membrane was blocked with 5% skim milk at room temperature for two hours and then incubated overnight at 4 °C with the following primary antibodies: Sema4B (1:1000, ab118458, Abcam, Cambridge, UK), Phospho-Phosphatidylinositol 3-Kinase (p-PI3K, 1:1000, ab278545, Abcam, Cambridge, UK), Phospho-Protein Kinase B (p-AKT, 1:1000, ab8805, Abcam, Cambridge, UK), PI3K (1:1000, ab302958, Abcam, Cambridge, UK), AKT (1:1000, ab283852, Abcam, Cambridge, UK), and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH, 1:1000, ab8245, Abcam, Cambridge, UK). After primary incubation, the membranes were incubated with Horseradish Peroxidase (HRP)-conjugated secondary antibodies specific to rabbit or mouse IgG (1:5000, ab205718, ab6728, Abcam, Cambridge, UK) for one hour at room temperature. Chemiluminescent detection was performed using substrate (34580, Waltham, MA, USA) from Thermo Fisher, and band intensity was quantified using ImageJ software, with GAPDH used as an internal reference.

2.7 Statistical analysis

All statistical analyses were conducted using GraphPad Prism version 8.0 (GraphPad Software, Inc., San Diego, CA, USA). Data are presented as mean ± SD. The Student’s t-test was used to assess comparisons between two groups. Tukey’s post hoc test was used after one-way analysis of variance (ANOVA) for comparisons between different groups. A p-values below 0.05 were regarded as statistically significant.

3.1 Sema4B is upregulated in the aged mouse PND model

To determine the expression pattern of Sema4B in aged mice with PND, Western blot analysis was first performed to assess Sema4B protein levels in the hippocampus. The results revealed a significant increase in Sema4B expression in the hippocampus of mice subjected to A/S compared with the control group (Fig. 1A). Consistent with this, immunofluorescence staining demonstrated strong Sema4B fluorescence in the CA1 region of the hippocampus in the A/S group relative to the control group (Fig. 1B). These findings suggest that elevated Sema4B expression in the hippocampus may play a role in the development of anesthesia-induced cognitive impairment following surgery.

Fig. 1.Sema4B is upregulated in the aged mouse PND model. (A) Western blotting showing Sema4B protein expression in mice hippocampus tissues. (B) Immunofluorescence detection of Sema4B fluorescence expression in hippocampal tissue. Values are presented as mean ± SD. **: p < 0.01. n = 6. Sema4B: Semaphorin 4B; WT: wild-type; A/S: anesthesia/surgery; DAPI: 4′,6-Diamidino-2-phenylindole; GAPDH: Glyceraldehyde-3-Phosphate Dehydrogenase.

3.2 Knockout of Sema4B improves cognitive decline in PND aged mice

To investigate whether the increased hippocampal expression of Sema4B is associated with cognitive impairment, aged mice with Sema4B knockout were utilized. Western blot analysis confirmed that Sema4B expression was significantly reduced in the hippocampus of Sema4B^−/− mice (Fig. 2A), validating the effectiveness of the knockout. To assess the impact of Sema4B deficiency on cognitive function, the MWZ test was performed to evaluate spatial learning and memory in the A/S mouse model. A representative swimming trajectory on day six is presented in Fig. 2B. Compared with the WT group, the A/S group exhibited significantly longer escape latency, along with fewer platform crossings and a shorter duration spent in the target quadrant, indicating impaired spatial memory. Notably, these deficits were markedly reversed in the A/S + Sema4B^−/− group, suggesting that Sema4B deficiency mitigates surgery-induced cognitive decline. In contrast, Sema4B knockout in control mice did not produce significant changes in escape latency or other behavioral indicators (Fig. 2C–E). Taken together, these findings demonstrate that Sema4B knockout improves cognitive performance in aged mice with PND.

Fig. 2.Knockout of Sema4B improves cognitive decline in PND-aged mice. (A) Western blotting showing Sema4B protein expression in mice hippocampus tissues. (B) Swimming path detected by MWM. (C–E) Escape time, time spent in the target quadrant, and number of times the mouse crossed the original platform as measured by MWM. Values are presented as mean ± SD. **: p < 0.01. n = 6. Sema4B: Semaphorin 4B; WT: wild-type; A/S: anesthesia/surgery; Sema4B^−/−: Sema4B knockout; GAPDH: Glyceraldehyde-3-Phosphate Dehydrogenase.

3.3 Knockout of Sema4B inhibits hippocampal microglial activation and neuroinflammation in aged PND mice

Microglia serve as resident immune cells in the central nervous system, and their activation is a key contributor to neuroinflammation. Upon activation, microglia release a range of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6, which can compromise synaptic structure and function, ultimately resulting in cognitive dysfunction [14]. To determine whether Sema4B deletion influences neuroinflammation in the PND model, we assessed the expression of inflammatory markers and Iba1 in the hippocampus. Iba1 is a well-established and specific marker for microglial activation, which is considered an early and prominent feature of hippocampal neuroinflammation in PND [15]. Compared with the WT group, the A/S group exhibited significantly elevated levels of TNF-α, IL-1β, and IL-6 in the hippocampus. Notably, Sema4B knockout effectively suppressed the increase in these inflammatory cytokines (Fig. 3A). Consistently, immunofluorescence staining revealed increased Iba1 expression in the CA1 region of the hippocampus in the A/S group, whereas Sema4B deletion attenuated this elevation (Fig. 3B). Together, these findings suggest that Sema4B knockout reduces hippocampal neuroinflammation and microglial activation in aged mice with PND.

Fig. 3.Knockout of Sema4B inhibits hippocampal microglial activation and neuroinflammation in aged PND mice. (A) ELISA was performed to measure the levels of inflammatory factors TNF-α, IL-1β, and IL-6 in hippocampal tissue. (B) Immunofluorescence detection of Iba1 fluorescence expression in hippocampal tissue. Values are presented as mean ± SD. **: p < 0.01. n = 6. TNF-α: tumor necrosis factor-α; IL: interleukin; Sema4B: Semaphorin 4B; WT: wild-type; A/S: anesthesia/surgery; Sema4B^−/−: Sema4B knockout; Iba1: Ionized calcium-binding adapter molecule 1; DAPI: 4′,6-Diamidino-2-phenylindole.

3.4 Knockout of Sema4B activates the PI3K/AKT signaling pathway

To further explore the potential mechanisms through which Sema4B modulates PND in aged mice, we examined the PI3K/AKT signaling pathway. Western blot analysis revealed that the phosphorylation levels of PI3K and AKT were significantly reduced in the A/S group, indicating suppression of this pathway, but Sema4B knockout could reverse this effect and reactivate PI3K and AKT phosphorylation (Fig. 4), supporting the beneficial effect of Sema4B knockout in improving PND may be mediated, at least in part, through activation of the PI3K/AKT signaling pathway.

Fig. 4.Knockout of Sema4B activates the PI3K/AKT signaling pathway. Western blotting showing PI3K, p-PI3K, AKT, p-AKT protein expressions in mice hippocampus tissues. Values are presented as mean ± SD. **: p < 0.01. n = 6. WT: wild-type; A/S: anesthesia/surgery; Sema4B^−/−: Sema4B knockout; p-PI3K: Phospho-Phosphatidylinositol 3-Kinase; p-AKT: Phospho-Protein Kinase B; GAPDH: Glyceraldehyde-3-Phosphate Dehydrogenase.