Spatial metabolomics reveals key features of hippocampal lipid changes in rats with postoperative cognitive dysfunction

Abstract

Postoperative cognitive dysfunction (POCD) is a common complication after cardiac surgery. Numerous evidence suggest that dysregulation of lipid metabolism is associated with cognitive impairment; however, its precise role in the development of POCD is still obscure. In this study, we established a cardiopulmonary bypass (CPB) model in rats and employed the Barnes maze to assess cognitive function, selecting POCD rats for subsequent experimentation. Utilizing mass spectrometry imaging, we detected plenty of lipids accumulates within the hippocampal CA1in the POCD group. Immunofluorescence staining revealed a significant reduction in the fluorescence intensity of calcium-independent phospholipases A2 (iPLA2) in the POCD group compared to the control, while serine palmitoyl transferase (SPT) was markedly increased in the POCD group. Transmission electron microscopy revealed that the number of synapses in hippocampal CA1decreased significantly and postsynaptic density became thinner in POCD group. Furthermore, after reversing the metabolic disorders of iPLA2 and SPT in the rat brain with docosahexaenoic acid and myriocin, the incidence of POCD after CPB was significantly reduced and the disrupted lipid metabolism in the hippocampus was also normalized. These findings may offer a novel perspective for exploring the etiology and prevention strategies of POCD after CPB.

Keywords

Postoperative cognitive dysfunction cardiopulmonary bypass hippocampus spatial metabolomics lipid

Introduction

Postoperative cognitive dysfunction (POCD) is a severe complication after cardiac surgeries, with advanced age, the effects of anesthetic agents, and pre-existing cognitive impairments serving as prominent risk factors for the development of POCD.¹ The main clinical manifestations of POCD include impaired learning and memory ability, inattention and even mental disorder, some patients with severe conditions may develop into personality changes. Previous studies revealed that about 30–65% of patients presented cognitive decline after cardiac surgery, and the symptoms in about 20–40% of patients were still not fully remission at 6 months after surgery.^2,3 POCD not only impose the financial and mental burden to their family, and even associate with increased risk of complications and mortality.⁴ Currently, available evidence indicated that cardiopulmonary bypass (CPB) during cardiac surgery seriously interfered with the physiological state of patients, leading to severe systemic inflammatory response, oxidative stress, and abnormal cerebrovascular regulatory function.^5
–8 However, the mechanisms of POCD after cardiac surgery remain elusive due to the complex pathophysiological changes induced by CPB.⁹

Hippocampus is one of remarkable brain region that provides the foundation for the formation of cognitive ability and memory,^10,11 and has been confirmed to be vulnerable and susceptible to various pathological lesions, such as ischemia and hypoxia during major surgery.^12,13 Lipids, as crucial constituents of the hippocampus, play a pivotal role in the normal function of the hippocampus through the maintenance of metabolic homeostasis, and the metabolism disorders have been proven to play a key role in the pathogenesis of cognitive dysfunction and mental disease.^14
–16 Studies have demonstrated that exposure to a high-fat diet disrupts lipid metabolism within the hippocampus, which in turn compromises the plasticity of hippocampal neurons and synaptic function, ultimately resulting in cognitive impairment.^17,18 Additionally, the disturbance of lipid homeostasis induced by dysregulated lipid metabolism leads to the production of toxic lipids. The lipotoxicity arising from such metabolic abnormalities commonly results in abnormal brain function, a key factor in the pathogenesis of Alzheimer’s disease (AD).^19,20

Although liquid chromatography/gas chromatography mass spectrometry (LC/GC-MS) metabolomics strategies can detect subtle changes of metabolites in disease state, analyzing the spatial distribution of metabolites will help to better understand the regional heterogeneity of abnormal tissue. The recent spatial metabolomics technique can not only respond to subtle alterations in biological metabolites, but also display their spatial information. Using this technique, it is feasible to visually observe the distribution of drugs in the cerebral cortex, so as to describe the spatial map of drug metabolism in the brain.²¹ In an investigation exploring the influence of caffeine on memory and cognitive function, the researchers employed spatial metabolic techniques revealed that prolonged caffeine intake notably alters hippocampal metabolism, with lipid alterations constituting the most substantial proportion of these changes.²²

Considering that CPB seriously interferes with cerebral blood perfusion in patients undergoing cardiac surgery and may lead to cerebral metabolic disorders, the purpose of this study was to detect the spatial-specific lipid metabolite changes in the hippocampal region of rat after CPB procedure by airflow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI) technique. Furthermore, by matching differential metabolites to metabolic pathways, we revealed the key enzymes that lead to metabolic changes. Integration of spatial information on metabolic enzymes and corresponding downstream metabolites will expand our understanding of metabolic disturbances in the hippocampus after CPB procedure and further facilitate the exploration in the pathogenesis of POCD.

Materials and methods

Animals

The protocol of this animal study was approved by the Ethics Committee for Animal Care and Use of the First Affiliated Hospital of Nanjing Medical University. The experimental procedures for this study were conducted in compliance with the Guide for the Care and Use of Laboratory Animals, and the reporting was done in accordance with the ARRIVE guidelines.²³ 18-months old male Sprague-Dawley (SD) rats were purchased from the Pizhou Oriental husbandry company limited (Xuzhou, China). All rats were housed in SPF environmental cages in a temperature- and humidity-regulated room with a 12-h light/dark cycle and were allowed free access to food and water. Rats were randomly assigned to non- surgical group (NSG, not being exposed to CPB, n = 20) or CPB group (rats were performed CPB, n = 20). The experiment protocol was shown in Figure 1.

Anesthesia and surgical procedure

The rats of experimental group were anesthetized with 3% isoﬂurane in a plastic box with subsequent intubation and mechanical ventilation via a 50% O2-balanced air mixture. During the anesthesia and surgical procedure, pulse oxygen and body temperature were monitored routinely.

After skin preparation and surgical field disinfection, the right external jugular vein and the right internal carotid artery were exposed through a longitudinal incision in the neck, and the right femoral artery was exposed through an incision in the right groin. The right femoral artery was intubated and connected with a pressure sensor to detect the blood pressure of the rats. Then the right carotid artery was intubated with a 20 G venous indent needle for blood return, and the right external jugular central venous catheter was used to establish venous access for blood output.

Cardiopulmonary bypass procedure

Heparin was injected 500 IU/kg through the right external jugular vein for whole-body heparinization, then all the CPB pipe was connected. The connection sequence was right external jugular vein-blood storage tank-peristaltic pump-rat membrane oxygenator-rat heat exchanger (connected to circulating water bath for heat preservation)-right carotid artery. Priming fluid: 10 ml of allogenic SD rat blood, 10 ml of replacement plasma (HES, Hospira, America), 1 ml of 5% NaHCO₃ (Shanghai Kanglang Biotechnology Co., Ltd., China), 1 ml of 20% mannitol (Shanghai Kanglang Biotechnology Co., Ltd., China), and 1 ml of heparin sodium (250 IU, Shanghai Kanglang Biotechnology Co., Ltd., China) were used for pipeline prefilling. After the beginning of CPB, the perfusion flow was gradually increased according to the volume of drainage and the fluid level of the blood storage chamber until it reached 70–80 ml/(kg·min), and was gradually stopped after 60 min. After the end of CPB, 2 ml (2 mg/ml) protamine was injected intravenously to neutralize heparin. Then the rats were placed on thermal insulation electric blankets.

Intervention

The reagents utilized in the experimentation were freshly prepared prior to use. Both Docosahexaenoic acid (DHA, Catalog No. C4188) and myriocin (Catalog No. B6064) were sourced from APExBIO (USA). Rats were randomly allocated into two study parts to undergo distinct interventions. DHA, an omega-3 fatty acid, has been previously demonstrated to enhance the expression of the enzyme iPLA₂ when supplemented.²⁴ In the first experiment, rats were randomly assigned to three groups: DHA vehicle group (only received injections of DHA vehicle), DHA vehicle + CPB group (underwent CPB surgery and received injections of DHA vehicle), and DHA + CPB group (underwent CPB surgery and DHA supplementation). DHA supplementation was administered intraperitoneally to the rats, commencing 3 days prior to CPB and continuing until 7 days post-CPB, at a dosage of 80 mg/kg per day, dissolved in a vehicle (10% dimethyl sulfoxide + 45% PEG300 + 45% PBS). DHA vehicle and DHA vehicle + CPB groups were administered an equal volume of vehicle injection using the same vehicle.

Myriocin is a selective inhibitor of SPT, and numerous studies have demonstrated that supplementation with myriocin can reduce the levels of myriocin.^25,26 In the second experiment, rats were randomly allocated to three groups: myriocin vehicle group (only received injections of myriocin vehicle), myriocin vehicle + CPB group (underwent CPB surgery and received injections of myriocin vehicle), and myriocin + CPB group (underwent CPB surgery and myriocin supplementation). Myriocin supplementation was administered intraperitoneally to the rats, beginning 3 days prior to CPB and continuing until 7 days post-CPB, at a dosage of 0.3 mg/kg per day, dissolved in a vehicle (10% dimethyl sulfoxide + 45% PEG300 + 45% PBS). The myriocin vehicle group and the myriocin vehicle + CPB group were both administered an equal volume of vehicle injection using the same vehicle.

Prior to drug administration, cognitive baseline assessments were conducted for all rats. Seven days after CPB, all rats underwent the Barnes maze test to ascertain whether there was a decline in cognitive function. After the evaluation of cognitive function, the rats were euthanized, and the brain tissue were procured for spatial metabolomic imaging and immunofluorescence staining. The experimental protocol is illustrated in Figure 6(a).

Behavioral test

All rats were returned to their cages after CPB procedure and had free access to food and water. The open field test was evaluated at 7 days after CPB procedure. Then, the Barnes maze test was performed after the end of the open field test.

Open field test

The open field test was applied to identify the differences in exercise ability between two groups in this study. As previously described, the apparatus consisted of a black base (100 cm × 100 cm) with black walls (40 cm) surrounding it, and the interior was divided into four zones.²⁷ The rats were placed in a box for 10 min and allowed to move freely, and their behavior was observed and recorded by video camera. After each test, the test box was cleaned with 75% ethanol to remove olfactory cues. The total traveling distance and number of movements were used to measure locomotor activity.

Barnes maze test

Based on the rodents’ nature of avoiding light and loving to explore, the Barnes maze was designed to test hippocampus-related learning and memory ability.^28,29 Before each trial, the maze was randomly rotated, save for the target hole, which remains fixed. Rats were positioned at the maze’s center, amidst 18 holes, one of which leads to a dark chamber serving as the goal. The latency of rats tempted to explore any hole, the total completion time, and the number of errors (defined as investigatory or head-turning responses to non-target holes) were recorded. The assessment spans four days, with daily 9:00 a.m. tests, and concludes with the maze’s thorough sanitization using 70% ethanol.

Hematoxylin and eosin staining

Hematoxylin and eosin (H&E) staining was performed on brain tissue from sacrificed rats. Tissue was extracted and placed in liquid nitrogen for freezing, then sectioned using cryostat microtome (Leica CM 1950, Leica Microsystem, Germany). Slices were air-dried for 5 minutes, followed by staining with Mayer’s hematoxylin (Sigma-Aldrich) for 5 minutes, tap water rinsing for 5 minutes, and 1% eosin Y solution (Sigma-Aldrich) for 1 minute. Slices were then dehydrated, cleared, and mounted with a synthetic mounting medium (Sigma-Aldrich). Stained sections were visualized under an light microscope (Olympus), with representative images captured using an digital camera (Olympus).

Nissl staining

For Nissl staining, the tissue sections were placed at room temperature for 5 minutes and then immersed in a 0.1% cresyl violet solution (Sigma-Aldrich) for 10 minutes at room temperature. The slides were rinsed in distilled water and differentiated in 95% ethanol containing 0.5% glacial acetic acid for 1–2 minutes. The sections were further dehydrated in 100% ethanol, cleared in xylene, and mounted with a coverslip using a synthetic mounting medium (Sigma-Aldrich). The Nissl-stained sections were examined under a light microscope (Olympus), and representative images were captured using a digital camera (Olympus).

AFADESI-MSI data acquisition

For AFADESI-MSI data collection, three rats per group were sacrificed post-neurological assessment, with brain tissues from the control and POCD groups rapidly frozen in liquid nitrogen and stored at −80°C. Tissue sections (10 μm thick) were generated using a cryostat microtome (Leica CM 1950, Leica Microsystem, Germany) and thaw-mounted on positive charge desorption plates (Thermo Scientific, USA). They were desiccated at −20°C for 1 h and then at room temperature for 30 minutes before mass spectrometry imaging (MSI) analysis.

The analyses were performed as previously reported.³⁰ In brief, the study utilized an AFADESI-MSI platform (Beijing Victor Technology Co., LTD, Beijing, China) coupled with a Q-Orbitrap mass spectrometer (Q Exactive, Thermo Scientific, USA) for data collection. Mass spectrometry imaging was performed via line scanning, with horizontal sections scanned at 0.2 mm/s and vertical spacing at 0.1 mm. The spray solvent for positive ion mode was an 80:20 acetonitrile (MS grade, Thermo Scientific, USA): water (Watsons distilled water, Watsons Group) mixture with 0.1% formic acid (HPLC grade, Merck, Germany), while the negative ion mode used the same solvent without formic acid. The MS settings included a resolution of 70,000, a mass range of 70–1000 Da, and a capillary temperature of 350°C. The MSI experiment involved continuous scanning at 0.2 mm/s in the x-direction and a 100 μm step in the y-direction.

Data processing and analysis

The collected “.raw” files were converted into “.imzML” format using imzMLConverter³¹ and then imported into MSiReader (an open-source interface to view and analyze high resolving power MS imaging files on Matlab platform) for ion image reconstructions after background subtraction using the Cardinal software package.³² The ions detected by AFADESI were annotated by the pySM pipeline and an in-house SmetDB database (Lumingbio, Shanghai, China).³³ The data obtained by scanning was not only contained the mass and charge data, but also contained the spatial site information. All MS images were normalized using total ion count normalization (TIC) in each pixel.³⁴ Region-specific MS profiles were precisely extracted by matching high-spatial resolution H&E images.

The discriminating endogenous molecules of different tissue microregions were screened by a supervised statistical analytical method: orthogonal partial least squares discrimination analysis (OPLS-DA). The variables’ contributions to group separation were assessed via their variable importance in projection (VIP) values. A two-tailed Student’s T-test confirmed the significance of metabolite differences between groups. Differential metabolites were identified with VIPs > 1.0 and p-values < 0.05. Spatial shrunken centroids clustering (SSCC) was then employed to cluster MSI data, allowing the differentiation of cell clusters based on ion abundance at each pixel. As previous study reported,³² the function of SSCC is to present a heatmap-like effect by clustering mass spectrometry imaging data through comparative analysis of mass spectrometry data variations. It merely groups similar mass spectrometry information and labels them through various means without altering the actual mass spectrometry localization information.

Immunofluorescence staining

The protocol for acquiring cryosections of brain tissue was as previously outlined, then the frozen sections were drying at room temperature, stabilization in cold acetone, and washing with PBS. After microwave assisted EDTA antigen repair, the sections were blocked with 10% donkey serum, incubated with primary antibodies against iPLA₂ (calcium-independent Phospholipases A₂; 1: 100, Boosen Biotechnology, Beijing, China) or SPT (Serine palmitoyl transferase; 1: 100, Boosen Biotechnology, Beijing, China), and then with secondary antibodies at room temperature for 2 h. The DAPI was added for nuclear staining and incubated away from light. The immunofluorescence images were visualized under the laser scanning confocal microscope (Fluoview 1000, Olympus) and the fluorescence intensity analysis of iPLA₂ and SPT in the hippocampal CA1was conducted using ImageJ 1.5 software (version 1.50 b).

Transmission electron microscopy

Immediately after behavioral testing, targeted hippocampal tissue (1 mm × 1 mm × 1 mm) was excised and fixed with 2.5% glutaraldehyde. After dehydration, epoxy resin was added, and followed by embedding treatment with acetone and embedding solution. The slices were sliced by ultramicrotome (Leica UC 7) with a thickness of 70–90 nm. Used 3% uranium acetate and lead citrate for double staining.

The synapses of hippocampal CA1 region were observed under JEOL transmission electron microscopy (JEM-1230, Japan) at 10000×. For the enumeration of synapses, we counted the hippocampal tissue from a total of 6 rats (3 for each group), selecting two tissue blocks from each rat, and at least three sections from each block, observing and counting under a magnification of 10,000 times. The thickness of postsynaptic density (PSD) was assessed using ImageJ software (version 1.50 b) by measuring the length of a perpendicular line traced from the postsynaptic membrane to the most convex part of the synaptic complex.³⁵ In the manner previously described for the statistical analysis of synaptic number, we conducted counts of hippocampal tissue from a total of 6 rats, with 3 rats in each group. Each rat was selected to provide 2 tissue blocks, and from each block, at least 3 sections were sampled. Observation and counting were performed under a magnification of 10,000 times.

Statistical analysis

Statistical analyses were carried out using SPSS software (IBM SPSS Statistics 26). And the results were presented by prism (GraphPad Prism 8.0.2). All image processing involved in the experiment, including thickness measurement and fluorescence intensity detection, was performed using ImageJ 1.5 software (version 1.50 b). For the experimental data within this study, the Shapiro-Wilk test was applied to evaluate the normality of distribution. The data were statistically characterized using the mean ± standard deviation, with an unpaired t-test employed to establish the statistical significance between the two groups under investigation. The data from the Barnes maze test between groups were tested by two-way repeated measures analysis of variance followed by the Bonferroni’s test. P < 0.05 was considered statistically significant.

Results

The cognitive function was impaired in CPB-induced POCD rats

Following CPB, 11 rats in the CPB group exhibited significant cognitive decline (Supplementary Table 1), and these were selectively identified to constitute the POCD group. An equivalent number of rats from the non-surgical group were randomly chosen to serve as the Control group for subsequent experiment. The methodology for diagnosing POCD in rats and the outcomes of cognitive ability assessments are detailed in the Construction of CPB-induced POCD Rats Model section of supplementary material. The physical prowess of rat after CPB procedure was assessed by open field test. As depicted in Figure 2(a) and (b), control rats covered an average distance of 1423.11 ± 256.95 cm, while POCD rats covered 1371.84 ± 286.53 cm. There was no statistically significant difference between the two groups (t = 0.44; P > 0.05). The number of movements were 134.00 ± 15.74 in control rats and 129.73 ± 20.24 in POCD rats, with no statistically significant difference between the two groups (t = 0.55; P > 0.05). As shown in Figure 2(c), motion trails were similar. These findings suggesting CPB did not significantly affect rat movement.

The cognitive function was assessed by the Barnes maze test. Our results showed that the total completion time, the latency to explore any hole and the number of errors decreased over days in both groups (Figure 2(d) to (f)). The total completion time in control group was 76.18 ± 13.33 s on the first day, 55.73 ± 8.49 s on the second day, 25.36 ± 7.16 s on the third day, and 11.36 ± 2.94 s on the fourth day, respectively. In contrast, the total completion time in POCD group was 81.27 ± 10.12 s on the first day, 75.64 ± 9.43 s on the second day, 25.36 ± 7.16 s on the third day, and 31.27 ± 5.42 s on the fourth day, respectively (Figure 2(d)). Statistical analysis revealed that the CPB procedure significantly prolonged the total completion time (F = 56.48, P < 0.001). The subsequent inter-group analysis revealed significant differences in the total completion time in day 2 (t = −5.20, P < 0.001), day 3 (t = −6.84, P < 0.001) and day 4 (t = −10.70, P < 0.001). In the control group, the latency to explore any hole was 28.45 ± 6.98 s on the first day, 18.91 ± 3.48 s on the second day, 11.45 ± 2.94 s on the third day, and 4.27 ± 1.35 s on the fourth day. In contrast, the latency to explore any hole in POCD group was 30.91 ± 4.87 s on the first day, 22.09 ± 6.39 s on the second day, 20.36 ± 7.98 s on the third day, and 9.91 ± 2.66 s on the fourth day. Statistical analysis revealed a significant difference in the latency to explore any hole between the two groups (F = 17.99, P < 0.001). The subsequent inter-group analysis revealed significant differences in the latency to explore any hole in day 3 (t = −3.47, P < 0.01) and day 4 (t = −6.26, P < 0.001). Besides, the number of errors in control group was 12.00 ± 3.07 on the first day, 8.45 ± 1.75 on the second day,4.27 ± 1.35 on the third day, and 2.00 ± 1.34 on the fourth day, respectively. For POCD group, the number of errors was 10.73 ± 2.20 on the first day, 11.73 ± 2.61 on the second day, 10.91 ± 2.47 on the third day, and 7.91 ± 1.51 on the fourth day, respectively. Statistical analysis showed that the CPB procedure significantly increased the number of errors of rats entering the target hole (F = 101.03, P < 0.001). The subsequent inter-group analysis revealed significant differences in the number of errors in day 2 (t = −3.45, P < 0.01), day 3 (t = −7.83, P < 0.001) and day 4 (t = −9.69, P < 0.001). Taken together, these findings revealed that cognitive dysfunction related to learning and memory was appeared in CPB-induced POCD rats.

Spatial profiling of hippocampal metabolites in CPB-induced POCD rats

Initially, we conducted spatial metabolomics analysis on entire coronal brain slices of rats and found that lipid metabolites showed the most significant changes after CPB, accounting for 25.6% of the total metabolic changes (Supplementary Figure 2A–F). Additionally, SSCC distinctly separated different brain regions in rats, and we observed that the hippocampus, which is closely related to cognition, could be divided into different categories, indicating the presence of unique biochemical processes within the hippocampus (Supplementary Figure 2 G). Based on these results, we focused on the lipid metabolism status in the rat hippocampal region. For the study of metabolism in hippocampal region, we initially conducted SSCC analysis on the hippocampus, unveiling that a unique metabolic profile in the hippocampal CA1 region, a key area sensitive to damage (Figure 3(a)). Next, the OPLS-DA analysis of two groups identified significant metabolic differences, with 112 distinct metabolites were found (Figure 3(b)). Lipids and lipid-like metabolites were predominantly differentially expressed, comprising 35.7% of changed metabolites (Figure 3(d) and (f)). Notably, phospholipids and sphingolipids showed the most substantial changes. After CPB, the levels of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), crucial for membrane function and nerve impulse conduction, were significantly increased in the hippocampus, especially in the CA1 region. Among the specific metabolites, PC (36:4), PC (38:4), PC (38:6), and PE (40:7) showed notable elevations. Additionally, sphingolipids such as SM (d36:1) and Cer (d36:1) were increased post-CPB in the hippocampal CA1 region (Figure 3(c) and (e)), indicating that CPB profoundly disrupted hippocampal metabolism, causing abnormal lipid accumulation.

The changes in the activity of lipid metabolic enzymes in hippocampus of CPB-induced POCD rats

Following CPB, hippocampal metabolite profiling revealed differential expression of phospholipid and sphingolipid metabolism-related compounds. To investigate the underlying regulatory mechanisms, immunofluorescence assays were performed on key enzymes in these metabolic pathways. As shown in Figure 4(a), a schematic diagram of phospholipid synthesis and catabolism was presented. The iPLA₂ is a kind of hydrolase that responsible for the enzyme hydrolysis of phospholipids in the brain. The fluorescence intensity of iPLA₂ in the control group was 50.89 ± 8.38 a.u. In comparison, the fluorescence intensity of iPLA₂ in the POCD group was significantly decreased at 31.84 ± 2.72 a.u. (t = 3.74, P < 0.05, Figure 4(b) and (c)). A schematic diagram of sphingolipid metabolism was shown in the Figure 4(d), the SPT plays a crucial role in sphingolipid initiation synthesis.³⁶ The fluorescence intensity of SPT in control group was 39.42 ± 1.96 a.u., and the fluorescence intensity of SPT was distinctly up-regulated of rats in the POCD group at 61.27 ± 10.08 a.u. (t = −3.69, P < 0.05, Figure 4(e) and (f)). The above results suggested that the lipid metabolism was disturbed a lot in the CA1 region of hippocampus and the key enzymes in metabolic pathways changed accordingly, after CPB procedure.

The ultrastructure in hippocampal CA1 region was damaged in CPB-induced POCD rats

Nissl-staining showed no significant damage in the hippocampus of two groups (Figure 5(a) and (b)), suggesting that CPB procedure did not cause obviously neuronal apoptosis in the hippocampus. To investigate whether abnormal lipid metabolism induces synaptic damage in the hippocampal CA1 region, transmission electron microscopy was subsequently employed for observation. Within the same magnification, the POCD group exhibited a reduction in the number of synapses and the thickness of the postsynaptic density (PSD) (Figure 5(c) to (h)). The control group exhibited a synaptic count of 6.44 ± 1.82 in the hippocampus, which was significantly greater compared to the POCD group, which displayed a synaptic count of 3.67 ± 1.37 (t = 5.17, P < 0.001). Additionally, the PSD thickness in the hippocampal CA1of the control group was measured at 58.50 ± 4.20 nm, whereas the POCD group demonstrated a notably reduced PSD thickness of 45.26 ± 3.16 nm (t = 7.56, P < 0.001).

DHA or myristicin ameliorate cognitive deficits induced by CPB

As shown in Figure 6(a), the experimental interventions were performed on the rats as previously described. Seven days after CPB, the Barnes maze test revealed a significant difference in cognitive performance between the DHA vehicle group and the DHA vehicle + CPB group on the third and fourth days (Figure 6(b)). On the second day of the Barnes maze test, the total completion time in the DHA + CPB group was significantly shorter than that in the DHA vehicle + CPB group (Figure 6(b)). The Z-score revealed that no rats in the DHA vehicle group developed cognitive function decline, whereas in the DHA vehicle + CPB group, cognitive impairment occurred in 5 rats, and in the DHA + CPB group, cognitive impairment was observed in 1 rat following CPB (Supplementary Table 2). The findings uncovered that supplementation with DHA could significantly decrease the incidence of cognitive impairment following CPB. Subsequently, the POCD rats in DHA vehicle + CPB group and an equivalent number of rats from the other two groups without POCD were selected for subsequent spatial metabolomics imaging and immunofluorescent staining experiments. The spatial metabolomics analysis of lipids in the hippocampus further validated that the POCD rats had accumulations of lipids such as PC (36:1) and PE (40:7) within the hippocampal CA1 (Figure 6(c)), and these lipid accumulations may contribute to the decline in cognitive function. As shown in Figure 7(a) and (b), fluorescence staining experiments revealed that the fluorescence intensity of iPLA₂ in the DHA vehicle + CPB group was significantly decreased compared to the DHA vehicle and DHA + CPB groups, while no significant difference was observed between the DHA vehicle and DHA + CPB groups (P < 0.001). This further confirms the critical role of the iPLA₂ in the disruption of lipid metabolism in the rat hippocampus by CPB.

As illustrated in Figure 6(d), the Barnes maze test revealed a significant discrepancy in the total completion time during the third and fourth days between the myriocin vehicle group and the myriocin vehicle + CPB group. On the second and third day of the Barnes maze test, the total completion time in the myriocin + CPB group was significantly shorter than that in the myriocin vehicle + CPB group. The Z-score revealed that no rats in the myriocin vehicle group developed cognitive function decline, whereas 7 rats in the myriocin vehicle + CPB group, 2 rats in the myriocin + CPB group developed cognitive impairment (Supplementary Table 3). The results elucidated that supplementation with myriocin could significantly decrease the incidence of cognitive impairment following CPB. Following this, 5 POCD rats in myriocin vehicle + CPB group and an equivalent number of rats from the other two groups without POCD, were subsequently selected for further spatial metabolomics imaging and immunofluorescent staining experiments. The spatial metabolomic analysis has provided further evidence that the POCD rats had accumulations of lipids such as Cer (d36:1) and SM (36:1) within the rat hippocampal CA1(Figure 6(e)), which may potentially result in the decline of cognitive function. As shown in Figure 7(c) and (d), the fluorescence staining experiments revealed that the fluorescence intensity of SPT in the myriocin vehicle + CPB group was significantly increased compared to the myriocin vehicle and myriocin + CPB groups, while no significant difference was observed between the myriocin vehicle and the myriocin + CPB groups (P < 0.001). Immunofluorescence staining for the SPT has further corroborated the close relationship between the lipid accumulation in the hippocampus after CPB and the level of SPT expression.

Discussion

Despite decades of clinical technological advancements, making CPB relatively safe for a broad patient population, the procedure still elicits marked systemic stress responses, leading to a systemic inflammatory response. Additionally, the sustained absence of pulsatile blood flow during CPB, compounded by the disruption of cerebral blood flow autoregulation due to hypothermia and fast rewarming during the surgery, may elevate the risk of postoperative complications in patients.³⁷ Numerous studies have now demonstrated that CPB can induce significant alterations in vascular reactivity, and the changes in cerebral blood flow and cerebrovascular perfusion have been linked to cognitive dysfunction in patients.^38
–40 Moreover, the alterations induced by CPB can lead to cerebral metabolism disorder, which result in abnormalities brain function. This may explain why patients with POCD exhibit no apparent cerebral lesion yet demonstrate cognitive deficits.

Current clinical investigation has established a close relationship between lipid metabolism disturbances and the onset of POCD. The higher levels of PCs and SMs in the plasma were associated with poorer clinical performance and biomarker measurements in patients with mild cognitive impairment or early-stage dementia.⁴¹ Our recent clinical studies demonstrated that lipid metabolism was disturbed in the peripheral circulation of patients suffering from post-operative delirium and delayed neurocognitive recovery after cardiac surgery, as determined by liquid chromatography mass spectrometry (LC/MS). The most pronounced differences in metabolites were observed in phosphatidylinositol (PI), sphingomyelin (SM), and phosphatidylglycerol (PG).^42,43 A recent basic study revealed that several lipids exhibited significant increase in fetal sheep brain after extracorporeal circulation, such as PC (14:1e/3:0), PC (2:0/16:0), PC (18:3e/2:0), and PE (18:0/22:5). Moreover, glycerophospholipids accounted for 38.94% of the identified brain metabolites that differed between the two groups, indicating that extracorporeal circulation can greatly disrupt lipid metabolism in the brain.⁴⁴ Therefore, profiling the early changes in brain lipids after cardiac surgery under CPB procedure is important to increase our understanding of POCD.

Advances in spatial metabolomics enhanced our grasp of disease progression heterogeneity. A recent study employed this technique to analyze lipid metabolism across different brain regions in ischemic mice, providing new insights into the alterations in lipid metabolism following ischemia and reperfusion and their effects on symptoms.⁴⁵ In the present investigation, we conducted SSCC analysis on the entire coronal brain section and hippocampus revealing that the distinctiveness of the hippocampal CA1 region’s metabolic profile. Lipid metabolites in the hippocampal CA1were particularly affected by CPB, altering enzyme expression and causing metabolic disruption, potentially leading to ultrastructural damage and cognitive impairment. Therefore, we proposed a hypothesis that CPB may induced profound lipid metabolism disorders within the hippocampal area, especially hippocampal CA1 region, culminating in destruction of the hippocampal ultrastructure and subsequent cognitive dysfunction.

Different types of neurons in the hippocampal subregion leads to significant differences in susceptibility to injury. Numerous studies have demonstrated that granule neuron in the hippocampal dentate gyrus (DG) region exhibit greater resistance to various negative conditions than pyramidal neurons in the CA1 region, such as stress, hypoxia, transient cerebral ischemia, obesity, and hypothyroidism.^46
–50 In addition, the hippocampal CA1contains a large number of cells but with lower capillary density and circulating blood volume compared that in other cortices. This further enhanced the neuronal damage in CA1 region caused by short-tern ischemic reperfusion.^51,52 In this study, we identified significant alterations in hippocampal lipid metabolite after CPB, particularly phospholipids and sphingolipids, with pronounced spatial distribution. Notable accumulations of PCs (e.g., PC (36:4), PC (38:4), PC (38:6)), PEs (e.g., PE (40:7)), Cers (e.g., Cer (d36:1)), and SMs (e.g., SM (d36:1)) were observed in the hippocampal CA1 pyramidal cell layer of POCD rats. We considered that altered blood perfusion during CPB leads to lipotoxicity in the CA1 region, potentially causing ultrastructural damage and the occurrence of POCD.

The metabolism of phospholipids and sphingolipids in the brain is strictly regulated. The iPLA₂ belongs to the PLA₂ superfamily and functions to catalyze the hydrolysis of unsaturated fatty acids in the phosphatidylglycerol portion of SN-2. The PLA₂ superfamily generally includes cytosolic PLA₂ (cPLA₂), calcium-independent PLA₂ (iPLA₂), plasmalogen-selective PLA₂ (PLsEtn-PLA₂), and secretory PLA₂ (sPLA₂). Studies have shown that iPLA₂ expression in various regions of the rat brain is significantly higher than that of cPLA₂. Research using selective iPLA₂ inhibitors such as bromoenol lactone or antisense oligonucleotides has shown that iPLA₂ is an important “housekeeping” enzyme under normal conditions.⁵³

Previous studies have shown that the activity of iPLA₂ in the brain, such as the cortex and hippocampus, is reduced in patients with AD.⁵⁴ Furthermore, cognitive training that stimulates iPLA₂ activity or supplementation with arachidonic acid, a product of iPLA₂, has been shown to alleviate cognitive impairment in experimental animals or human subjects.⁵⁵ Based on the important role of iPLA₂, our study investigated the changes in iPLA₂ using immunofluorescence after the CPB procedure and found that compared with the control group, the enzymatic metabolism activity in the hippocampal CA1 of POCD group was significantly inhibited. Moreover, our study also discovered that supplementation with DHA, which has been shown to upregulate iPLA₂ level,²⁴ can significantly reduce the incidence of CPB-induced POCD, and normalize lipid metabolism in the hippocampal CA1 region, as well as normalize iPLA₂ levels in the hippocampal CA1 region. These findings indicated that the downregulation of iPLA₂ induced by CPB procedure was the main factor contributing to the substantial accumulation of PCs and phosphatidylethanolamines PEs within the hippocampal CA1 region. Regulating iPLA₂ may be a potential target for preventing cognitive dysfunction after cardiac surgery.

In addition, previous study has shown that an increase in neuronal sphingomyelin levels leads to motor and memory impairments, while supplementation with enzymes that break down sphingomyelin can alleviate these symptoms.⁵⁶ Moreover, the increase in Cers can be considered one of the key factors in the pathophysiology of AD, and its elevated levels are closely associated with the activation of neuroinflammatory pathways.⁵⁷ As a key enzyme for the initiation of sphingolipid synthesis, the expression of SPT showed a significant up-regulation in the hippocampal CA1 after CPB procedure, and the acceleration of sphingolipid synthesis may be the reason for the significant increase in sphingolipids such as in the hippocampal CA1 in this study. Additionally, the administration of the SPT inhibitor myriocin to CPB rats could significantly reduce the incidence of POCD. Furthermore, myriocin restored SPT in the hippocampal CA1 of the rats to more normal levels and eliminated the abnormal accumulation in lipid metabolism within the hippocampal CA1. This finding corroborates to the hypothesis that a pathological upregulation of SPT enzyme level leads to a substantial increase in sphingolipids within the hippocampus, thereby causing cognitive dysfunction. Under normal physiological conditions, the regular activity of SPT ensures the constant production of sphingolipids, maintaining the homeostasis of sphingolipids in the hippocampus. However, in pathological conditions, the abnormal elevation of SPT level leads to excessive generation of sphingolipids, which may accumulate in the hippocampal CA1and disrupt its normal function, ultimately causing a decline in cognitive abilities.

There are two major limitations in this study that could be addressed in future research. First, all rats included in this study were male, and we did not include female rats for comparative analysis to explore the influence of sex differences. Numerous investigations have established dissimilarities in vascular reactivity and regional cerebral blood flow between males and females.^58,59 Moreover, gender differences markedly affect the prognosis or disease progression in ischemic stroke or cognitive disorders such as Alzheimer’s disease.^60,61 Second, the study failed to dynamically delineate the temporal changes in lipid metabolism within the hippocampus of CPB-induced POCD rat, thereby limiting our insight into the underlying mechanisms of POCD evoked by CPB. Our subsequent experiments will provide a more detailed discussion on the impact of CPB on gender-specific lipid metabolism in rat hippocampus, along with continuous dynamic observations and recordings of intracerebral lipid metabolic changes.

In summary, our findings indicate that rats undergoing CPB exhibit significant accumulation of various phospholipids and sphingolipids in the hippocampal CA1 region, accompanied by altered expression levels of iPLA₂ and SPT within the metabolic pathways. Furthermore, reversing the dysregulation of iPLA₂ and SPT could normalize lipid metabolism in the rat hippocampus and reduce the incidence of POCD after CPB. The metabolic disruption of lipids is most pronounced in the hippocampal CA1 region, further confirming its vulnerability of hippocampal CA1 region to abnormal cerebral perfusion. These discoveries enhance our understanding of the potential mechanisms underlying POCD after cardiac surgery and provide valuable insights into the exploration of potential therapeutic avenues for POCD in the future.

Supplemental Material

sj-pdf-1-jcb-10.1177_0271678X241261949 - Supplemental material for Spatial metabolomics reveals key features of hippocampal lipid changes in rats with postoperative cognitive dysfunction

Supplemental material, sj-pdf-1-jcb-10.1177_0271678X241261949 for Spatial metabolomics reveals key features of hippocampal lipid changes in rats with postoperative cognitive dysfunction by Zheng Lei, Jie Wan, Jing-jing Han, Chun-Yan Zhang, Hao-Tian Wang, Ding-jie Zhou, Yu Chen and He Huang in Journal of Cerebral Blood Flow & Metabolism

Footnotes

Availability of data and materials

All datasets on the spatial metabolomics data used and analyzed in the current study are available upon reasonable request by the corresponding authors.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: National Natural Science Foundation of China (grant number: 81901100); Jiangsu Health Development Research Center open project (grant number: JSHD2021006).

Acknowledgements

The authors gratefully acknowledge Shanghai Lu Ming Biotech Co., Ltd. (Shanghai, China) for their assistance on the AFADESI spatial-resolved metabolomics. And we are also grateful to Jiangxi Zvast-Biotechnology Co., Ltd. for their guidance in CPB model building.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Authors’ contributions

All authors contributed to the study conception and design. Material preparation, animal feeding and data collection were performed by Zheng Lei, Jing-jing Han, Chun-yan Zhang and Hao-tian Wang. Immunofluorescence were performed by Jie Wan. Data analysis and interpreted was performed by He Huang, Yu Chen, Ding-jie Zhou and Zheng Lei. The manuscript was written by Zheng Lei.

Supplementary material

Supplemental material for this article is available online.

ORCID iD

Ding-jie Zhou

References

Rudolph

Marcantonio

ER.

Review articles: postoperative delirium: acute change with long-term implications. Anesth Analg 2011; 112: 1202–1211.

Hogue

Jr. Palin

Arrowsmith

JE.

Cardiopulmonary bypass management and neurologic outcomes: an evidence-based appraisal of current practices. Anesth Analg 2006; 103: 21–37.

Kok

Koerts

Tucha

, et al. Neuronal damage biomarkers in the identification of patients at risk of long-term postoperative cognitive dysfunction after cardiac surgery. Anaesthesia 2017; 72: 359–369.

Steinmetz

Christensen

Lund

, ISPOCD Groupet al. Long-term consequences of postoperative cognitive dysfunction. Anesthesiology 2009; 110: 548–555.

Abrahamov

Levran

Naparstek

, et al. Blood-Brain barrier disruption after cardiopulmonary bypass: diagnosis and correlation to cognition. Ann Thorac Surg 2017; 104: 161–169.

Abu-Omar

Balacumaraswami

Pigott

, et al. Solid and gaseous cerebral microembolization during off-pump, on-pump, and open cardiac surgery procedures. J Thorac Cardiovasc Surg 2004; 127: 1759–1765.

Day

Taylor

KM.

The systemic inflammatory response syndrome and cardiopulmonary bypass. Int J Surg 2005; 3: 129–140.

Rajaram

Milej

Suwalski

, et al. Optical monitoring of cerebral perfusion and metabolism in adults during cardiac surgery with cardiopulmonary bypass. Biomed Opt Express 2020; 11: 5967–5981.

Bhushan

Huang

, et al. Progress of research in postoperative cognitive dysfunction in cardiac surgery patients: a review article. Int J Surg 2021; 95: 106163.

10.

Rothschild

Eban

Frank

LM.

A cortical-hippocampal-cortical loop of information processing during memory consolidation. Nat Neurosci 2017; 20: 251–259.

11.

Biane

Ladow

Stefanini

, et al. Neural dynamics underlying associative learning in the dorsal and ventral hippocampus. Nat Neurosci 2023; 26: 798–809.

12.

Cao

Wang

, et al. Postoperative cognitive deficits and neuroinflammation in the hippocampus triggered by surgical trauma are exacerbated in aged rats. Prog Neuropsychopharmacol Biol Psychiatry 2010; 34: 1426–1432.

13.

Wan

, et al. Postoperative impairment of cognitive function in rats: a possible role for cytokine-mediated inflammation in the hippocampus. Anesthesiology 2007; 106: 436–443.

14.

Sanjana

Delgorio

Hiscox

, et al. Blood lipid markers are associated with hippocampal viscoelastic properties and memory in humans. J Cereb Blood Flow Metab 2021; 41: 1417–1427.

15.

Zhang

, et al. Large-scale functional network connectivity mediates the associations between lipids metabolism and cognition in type 2 diabetes. J Cereb Blood Flow Metab 2024; 44: 384–396.

16.

Iadecola

Smith

Anrather

, et al. The neurovasculome: key roles in brain health and cognitive impairment: a scientific statement from the American Heart Association/American Stroke Association. Stroke 2023; 54: e251–e271.

17.

Zhuang

Yao

, et al. Long-term high-fat diet consumption by mice throughout adulthood induces neurobehavioral alterations and hippocampal neuronal remodeling accompanied by augmented microglial lipid accumulation. Brain Behav Immun 2022; 100: 155–171.

18.

Calvo-Ochoa

Hernández-Ortega

Ferrera

, et al. Short-term high-fat-and-fructose feeding produces insulin signaling alterations accompanied by neurite and synaptic reduction and astroglial activation in the rat hippocampus. J Cereb Blood Flow Metab 2014; 34: 1001–1008.

19.

Murdock

Tsai

LH.

Insights into Alzheimer’s disease from single-cell genomic approaches. Nat Neurosci 2023; 26: 181–195.

20.

Chen

Wang

Zhao

, et al. Lipid-accumulated reactive astrocytes promote disease progression in epilepsy. Nat Neurosci 2023; 26: 542–554.

21.

Pang

Gao

, et al. Mapping metabolic networks in the brain by ambient mass spectrometry imaging and metabolomics. Anal Chem 2021; 93: 6746–6754.

22.

Paiva

Cellai

Meriaux

, et al. Caffeine intake exerts dual genome-wide effects on hippocampal metabolism and learning-dependent transcription. J Clin Invest 2022; 132: e149371.

23.

Percie Du Sert

Hurst

Ahluwalia

, et al. The ARRIVE guidelines 2.0: updated guidelines for reporting animal research. Br J Pharmacol 2020; 177: 3617–3624.

24.

Kokacya

Inanir

Copoglu

, et al. The antipsychotic effects of omega-3 fatty acids in rats. Am J Med Sci 2015; 350: 212–217.

25.

Dasgupta

Ray

SK.

Ceramide and sphingosine regulation of myelinogenesis: Targeting serine palmitoyltransferase using microRNA in multiple sclerosis. Int J Mol Sci 2019; 20: 5031.

26.

Wadsworth

Clarke

McMahon

, et al. The chemical basis of serine palmitoyltransferase inhibition by myriocin. J Am Chem Soc 2013; 135: 14276–14285.

27.

Zhang

Liu

Zhao

, et al. Depression and anxiety behaviour in a rat model of chronic migraine. J Headache Pain 2017; 18: 27.

28.

Harrison

Hosseini

McDonald

MP.

Endogenous anxiety and stress responses in water maze and Barnes maze spatial memory tasks. Behav Brain Res 2009; 198: 247–251.

29.

Sharma

Rakoczy

Brown-Borg

Assessment of spatial memory in mice. Life Sci 2010; 87: 521–536.

30.

Luo

Chen

, et al. Air flow-assisted ionization imaging mass spectrometry method for easy whole-body molecular imaging under ambient conditions. Anal Chem 2013; 85: 2977–2982.

31.

Race

Styles

Bunch

Inclusive sharing of mass spectrometry imaging data requires a converter for all. J Proteomics 2012; 75: 5111–5112.

32.

Bemis

Harry

Eberlin

, et al. Cardinal: an R package for statistical analysis of mass spectrometry-based imaging experiments. Bioinformatics 2015; 31: 2418–2420.

33.

Palmer

Phapale

Chernyavsky

, et al. FDR-controlled metabolite annotation for high-resolution imaging mass spectrometry. Nat Methods 2017; 14: 57–60.

34.

Wang

Heijs

Kostidis

, et al. Analyzing cell-type-specific dynamics of metabolism in kidney repair. Nat Metab 2022; 4: 1109–1118.

35.

Shi

Wang

Zheng

, et al. Supplement of microbiota-accessible carbohydrates prevents neuroinflammation and cognitive decline by improving the gut microbiota-brain axis in diet-induced obese mice. J Neuroinflammation 2020; 17: 77.

36.

Hoffman

Palioura

Kyriazis

, et al. Cardiomyocyte Kruppel-like factor 5 promotes De novo ceramide biosynthesis and contributes to eccentric remodeling in ischemic cardiomyopathy. Circulation 2021; 143: 1139–1156.

37.

Bukauskiene

Sirvinskas

Lenkutis

, et al. Impact of blood flow velocity changes during cardiac surgery with cpb to postoperative cognitive functions. Journal of Cardiothoracic and Vascular Anesthesia 2019; 33: S99–S100.

38.

Wei

JLK

Leng

, et al. Heart-brain mapping: Cardiac atrial function is associated with distinct cerebral regions with high free water in older adults. J Cereb Blood Flow Metab 2024. DOI: 10.1177/0271678X241229581.

39.

Weijs

Oudegeest-Sander

Hopman

, et al. Cerebrovascular CO(2) reactivity and dynamic cerebral autoregulation through the eighth decade of life and their implications for cognitive decline. J Cereb Blood Flow Metab 2024; 44: 712–725.

40.

Moraca

Lin

Holmes

, et al. Impaired baseline regional cerebral perfusion in patients referred for coronary artery bypass. J Thorac Cardiovasc Surg 2006; 131: 540–546.

41.

Toledo

Arnold

Kastenmuller

, et al. Metabolic network failures in Alzheimer’s disease: a biochemical road map. Alzheimers Dement 2017; 13: 965–984.

42.

Han

Huang

Lei

, et al. Association between the early serum lipid metabolism profile and delayed neurocognitive recovery after cardiopulmonary bypass in cardiac surgical patients: a pilot study. J Cardiovasc Transl Res 2022; 16: 662–673.

43.

Huang

Han

, et al. Early serum metabolism profile of post-operative delirium in elderly patients following cardiac surgery with cardiopulmonary bypass. Front Aging Neurosci 2022; 14: 857902.

44.

Teng

Tian

, et al. Tissue-specific metabolomic profiling after cardiopulmonary bypass in fetal sheep. Front Cardiovasc Med 2022; 9: 1009165.

45.

Chen

, et al. Spatiotemporal lipidomics reveals key features of brain lipid dynamic changes after cerebral ischemia and reperfusion therapy. Pharmacol Res 2022; 185: 106482.

46.

Alkadhi

KA.

Cellular and molecular differences between area CA1 and the dentate gyrus of the hippocampus. Mol Neurobiol 2019; 56: 6566–6580.

47.

Gerges

Stringer

Alkadhi

KA.

Combination of hypothyroidism and stress abolishes early LTP in the CA1 but not dentate gyrus of hippocampus of adult rats. Brain Res 2001; 922: 250–260.

48.

Hsu

Zhang

Takagi

, et al. Decreased expression and functionality of NMDA receptor complexes persist in the CA1, but not in the dentate gyrus after transient cerebral ischemia. J Cereb Blood Flow Metab 1998; 18: 768–775.

49.

Coupe

Nadal

, et al. Distinct hippocampal subfields atrophy in older people with vascular brain injuries. Stroke 2021; 52: 1741–1750.

50.

Gerges

Alkadhi

KA.

Hypothyroidism impairs late LTP in CA1 region but not in dentate gyrus of the intact rat hippocampus: MAPK involvement. Hippocampus 2004; 14: 40–45.

51.

Imdahl

Hossmann

KA.

Morphometric evaluation of post-ischemic capillary perfusion in selectively vulnerable areas of gerbil brain. Acta Neuropathol 1986; 69: 267–271.

52.

Bartsch

Dohring

Reuter

, et al. Selective neuronal vulnerability of human hippocampal CA1 neurons: lesion evolution, temporal course, and pattern of hippocampal damage in diffusion-weighted MR imaging. J Cereb Blood Flow Metab 2015; 35: 1836–1845.

53.

Ong

Farooqui

AA.

Involvement of cytosolic phospholipase A(2), calcium independent phospholipase A(2) and plasmalogen selective phospholipase A(2) in neurodegenerative and neuropsychiatric conditions. Curr Med Chem 2010; 17: 2746–2763.

54.

Gattaz

Maras

Cairns

, et al. Decreased phospholipase A2 activity in Alzheimer brains. Biol Psychiatry 1995; 37: 13–17.

55.

Schaeffer

Forlenza

Gattaz

WF.

Phospholipase A2 activation as a therapeutic approach for cognitive enhancement in early-stage Alzheimer disease. Psychopharmacology (Berl) 2009; 202: 37–51.

56.

Arroyo

Camoletto

Morando

, et al. Pharmacological reversion of sphingomyelin-induced dendritic spine anomalies in a Niemann pick disease type a mouse model. EMBO Mol Med 2014; 6: 398–413.

57.

Ferre-Gonzalez

Balaguer

Roca

, et al. Brain areas lipidomics in female transgenic mouse model of Alzheimer’s disease. Sci Rep 2024; 14: 870.

58.

Koep

Bond

Barker

, et al. Sex modifies the relationship between age and neurovascular coupling in healthy adults. J Cereb Blood Flow Metab 2023; 43: 1254–1266.

59.

Panerai

Davies

Clough

, et al. The effect of hypercapnia on the directional sensitivity of dynamic cerebral autoregulation and the influence of age and sex. J Cereb Blood Flow Metab 2024; 44: 272–283.

60.

Jia

Lovins

Malone

, et al. Female-specific neuroprotection after ischemic stroke by vitronectin-focal adhesion kinase inhibition. J Cereb Blood Flow Metab 2022; 42: 1961–1974.

61.

Demetrius

Eckert

Grimm

Sex differences in Alzheimer’s disease: metabolic reprogramming and therapeutic intervention. Trends Endocrinol Metab 2021; 32: 963–979.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.52 MB