3-n-Butylphthalide suppresses neuroinflammation through BDNF signaling in postoperative cognitive dysfunction in mice

doi:10.21203/rs.3.rs-3334803/v1

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3-n-Butylphthalide suppresses neuroinflammation through BDNF signaling in postoperative cognitive dysfunction in mice

https://doi.org/10.21203/rs.3.rs-3334803/v1

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Aims: Effects of 3-n-Butylphthalide (NBP) on neuroinflammation in postoperative cognitive dysfunction (POCD) have not been well studied. This study aimed to investigate the effects of NBP treatment on surgery-induced cognitive dysfunction in mice.

Methods: Male C57BL/6 mice, aged ten months, were subjected to tibialfracture surgery while under isoflurane anesthesia in order to mimic orthopedic surgery performed on humans. Tests to assess behavior were conducted at the specified time intervals. We observed structural changes in hippocampal neurons, mitochondria and Golgi apparatus by transmission electron microscopy. The expression levels of IBA1, TNF-α, IL-1β, and apoptotic proteins were determined using Western blot and ELISA.

Results: The results indicated that NBP treatment ameliorates surgery-induced cognitive impairment. Moreover, NBP treatment inhibits microglial activation and reverses neuronal, mitochondrial and Golgi damage in the hippocampus of POCD mice. Furthermore, NBP decreases neuroinflammation in the hippocampus of mice with POCD.

Conclusion: Our research indicates that NBP could be a promising target for therapeutic intervention in POCD.

Postoperative cognitive dysfunction

3-n-Butylphthalide

neuroinflammation

microglia

blood-brain barrier

Postoperative cognitive dysfunction (POCD), mainly deficits in attention, memory, executive function and learning, is commonly observed in elderly patients. The condition has the potential to endure for an extended period and potentially evolve into a severe disorder of the central system. ¹Ongoing worries exist among the public regarding the possibility of surgery leading to lasting cognitive decline, which can result in negative consequences such as prolonged hospitalization, diminished quality of life, impaired self-care, heightened burden on families and society, and elevated mortality rates.^2,3Unfortunately, there are currently no successful interventions to delay or stop the progression of this ailment.

Previous research has indicated that POCD is linked to neuroinflammation. ^4,5The surgical procedure activates the innate immune system, resulting in the release of pro-inflammatory substances like tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-1β. ⁴Neuroinflammation is associated with these inflammatory factors, which can cause inflammation through direct neural pathways or by crossing a disrupted blood-brain barrier (BBB). ⁶The brain's increased levels of proinflammatory cytokines lead to the overactivation of microglia, initiating a chain reaction of inflammatory factor release and perpetuating a cycle of neuroinflammation. ⁷Overactivated microglia can generate a neurotoxic response, leading to neuronal damage and impairment, ultimately resulting in cognitive decline.⁸ Research has demonstrated that tibial surgery can induce cognitive impairment after surgery by triggering inflammation in the hippocampus. ⁹Therefore, we conducted a study using a mouse model of tibial fracture-induced POCD to investigate the role of neuroinflammation. Our investigation focused on behavioral tests, hippocampal neuron analysis, mitochondrial morphology, and the expression of inflammatory factor proteins in mice.

In 2002, the US Food and Drug Administration approved 3-n-butylphthalide (NBP), a compound extracted from celery seeds, for treating ischemic stroke.¹⁰ Numerous studies have explored the neuroprotective properties and potential mechanisms of action of this small-molecule compound. Furthermore, NBP not only helps in the prevention of ischemic brain injury but also enhances vascular cognitive impairment in patients with dementia, encompassing Alzheimer's disease and Parkinson's disease.^11–13 Although the precise pharmacological mechanisms of NBP remain unidentified, a growing body of research indicates potential neuroprotective benefits. Hence, we devised this study to examine its contribution to POCD and its possibility for practical implementation.

2.1 Animals used in experiments

For this study, we employed male C57 mice (Beijing Vital River Laboratory Animal Technology Co) that were 10 months old and had a weight ranging from 25 to 30 g. These mice were divided into three groups: a control group (Con), a surgical group (POCD), and a surgical + NBP group (with 10 mice in each group), using random allocation. Prior to tibial fracture surgery, mice in the NBP group received intraperitoneal injections of NBP (40 mg/kg). The animals were kept in a controlled environment with a consistent room temperature of 23 ± 1.5℃, humidity ranging from 50–60%, and a 12-hour cycle of light and darkness. Each cage contained approximately 3 to 4 mice, and they were given standard rodent chow and water. After the animals had adapted to their surroundings, the experiments commenced. The Ethics Committee of Qingdao University (Approval number AHQU-MAL20220323) granted approval for the experiments, which were carried out following the Ethical Guidelines for Animal Experiments of Qingdao University and the IASP Ethical Guidelines.

2.2 Anesthesia and surgery

Based on prior research, the surgical procedure involved a sterile incision on the tibia to treat a fracture, followed by intramedullary fixation while the mice was under isoflurane anesthesia. The mice were given 4.0% isoflurane to induce anesthesia, which was then followed by 1.5% isoflurane for maintenance. The left hind paw was shaved and disinfected. A longitudinal incision was then made and a 0.38 mm steel nail was inserted into the intramedullary canal. The periosteum was removed and an osteotomy was performed. After cleaning the wound, the skin was stitched together. For postoperative pain relief, the surgical cut was encircled by 0.2% lidocaine infiltration, while the sutures were sterilized using iodophor. To facilitate postoperative recovery, the mice were positioned on a warmed blanket, ensuring a constant temperature of 36 ± 1°C. After regaining full consciousness, they were brought back to the rearing cage and closely monitored while being fed a regular diet. (Fig. 1).

2.3 Behavioral assessments

2.3.1 Open field test

The investigators were blind to animal grouping. The purpose of the open field test was to assess the mice's movement and activity levels. Motor activity was assessed on the 13th day post-surgery by conducting the open field test inside a black opaque plastic chamber measuring 40cm*40cm*40cm. A video tracking system automatically recorded the activity of the mice for 10 minutes as they were placed in the middle of an open field. In order to prevent the existence of smell signals following each examination, the test area's floor was sanitized using a solution of 75% ethanol.

2.3.2 Morris water maze (MWM) test

The MWM is specifically created to evaluate the spatial learning abilities of rodents. The mice underwent training four times daily for four consecutive days, with a 2-hour gap between each session, and each trial lasted for 60 seconds. The mice began each trial from a distinct quadrant, and the video camera recorded the duration it took for them to reach the platform (known as escape latency). The water maze software was utilized to analyze the digital images. After 4 days of testing, the platform was taken away, allowing the mice to swim without restraint for 60 seconds in order to locate the missing platform. The count of platform crossings was documented.

2.4 Transmission electron microscopy

Transmission electron microscopy was used to observe the ultrastructure of mitochondria in the hippocampus of mice. Mice were killed, the hippocampus was removed, cut into 1mm*1mm sections and fixed in electron microscopy solution. The sections were left at ambient temperature for a duration of two hours and subsequently transferred to a refrigerator set at a temperature of 4℃ for the entire night. The sections were embedded and observed as described above.

2.5 Immunofluorescence

Mice were anesthetized using 2% sodium pentobarbital (60 mg/kg, injected into the peritoneum) and then perfused with saline through the heart. Subsequently, the hippocampal tissue was collected. The tissue was preserved in 4% paraformaldehyde for 12 hours. After preservation, the tissue was encased in paraffin, sliced using a microtome, treated to remove the paraffin, and then rehydrated. Following three rinses using PBS, the sections were treated with 3% BSA for 30 minutes at room temperature to fix them. Subsequently, they were exposed to the primary antibody anti-IBA1 in 1% BSA at 4°C overnight. The sections were treated with goat anti-rabbit antibody at room temperature for a duration of 1 hour. Following the rinsing of secondary antibodies, the sections were subjected to incubation with 4,6-diamidino-2-phenyl-indole (DAPI) to facilitate nuclear staining. Confocal microscopy was used to capture images of fluorescence.

2.6 Enzyme-linked Immunosorbent Assay (ELISA)

Tumour necrosis factor-alpha (TNF-alpha) and interleukin-1β (IL-1β) were measured using ELISA kits. Blood was obtained from the abdominal aorta of the mouse, left at room temperature for a duration of 2 hours, then centrifuged at a force of 10000g for 10 minutes to separate the serum. A standard curve was generated using the reference cytokine concentrations provided. The instructions were followed and specific procedures were executed.

2.7 Measurement of MDA and SOD

Sodium pentobarbital was used to anesthetize the mice. The median line was incised to expose the abdominal skin, fascia, and muscle. The abdominal aorta was then isolated and blood was collected. The collected blood was centrifuged to obtain serum, and the contents were analyzed using SOD and MDA kits.

2.8 Western blot

Mouse hippocampal tissues were obtained as described above and homogenized in RIPA lysis buffer containing protease inhibitors (PMSF). After being centrifuged at a speed of 12,000 revolutions per minute for a duration of 10 minutes at a temperature of 4 degrees Celsius, the liquid portion was gathered. The BCA assay was used to determine the concentration of proteins, which were then denatured in SDS sample buffer and separated using SDS-polyacrylamide gel electrophoresis. Protein was transferred to a PVDF membrane using a wet transfer device. The membranes were treated with 5% skim milk powder dissolved in TBST for 2 hours at room temperature. Subsequently, the membranes were incubated with various primary antibodies overnight (12–16 hours) at 4°C.Rabbit anti-brain-derived trophic factor (BDNF), rabbit anti-Bax, rabbit anti-BCL2, and rabbit anti-caspase3 were the primary antibodies employed. Following the inclusion of goat anti-rabbit secondary antibody (1:5000) for a duration of 60 minutes, the protein bands on the membrane were identified utilizing an enhanced chemiluminescence kit, and the images were captured using the Automatic Chemiluminescence Image Analysis System. The analysis of the data was conducted using Image J software.

2.9 Statistical analysis

The figures display the average ± standard errors of the mean for each individual data point (GraphPad Prism 9). The variances in escape latencies observed during the Morris water maze experiment were examined using a two-way ANOVA followed by the Tukey test to compare multiple groups. Significant variations among the three groups in terms of Western blot, immunofluorescence, ELISA, and reagent kit data were assessed using one-way ANOVA followed by the Tukey test. A significance level of less than 0.05 was deemed statistically significant.

3.1 NBP treatment ameliorates surgery-induced cognitive impairment

We conducted behavioral tests to investigate if surgery for tibia fracture under isoflurane anesthesia can result in cognitive deficits in mice and whether NBP treatment can enhance surgery-induced cognitive impairment. Spatial learning and memory in mice were evaluated using the Morris water maze experiment, and the training resulted in a significant decrease in the time taken to reach the platform (Fig. 2A-B). Groups experienced significant variations in the time taken to reach the platform during the training phase. In comparison to control mice, POCD mice required more time to reach the plateau, and the latency period was significantly decreased by NBP treatment (Fig. 2C). During the exploration period, we observed that POCD mice made fewer trips across the platform. On the other hand, mice treated with NBP exhibited improved memory in comparison to the POCD group, as indicated by a notable rise in the occurrence of platform crossing (Fig. 2D). Nonetheless, there was no notable disparity in the swimming velocity among the various sets of mice throughout both the training and exploration phases. (Fig. 2E-F).

Simultaneously, we conducted an open field experiment to evaluate the mice's locomotion. No significant variations in overall distances were observed among the control, POCD, and NBP treatment groups (Fig. 2G).

The findings suggest that the use of isoflurane anesthesia during tibial fracture surgery could have a negative impact on cognitive function, specifically learning and memory, in mice. However, it does not seem to have any effect on their mobility. This indicates that a successful mouse model for POCD has been developed, and the administration of NBP treatment shows potential in improving the surgery-induced deficits in learning and memory.

3.2 NBP treatment inhibits microglial activation

To assess the level of microglial activation, we conducted immunofluorescence labeling of the microglial marker IBA-1, which is crucial for hippocampal neuroinflammation in POCD (Fig. 3A).¹⁴ In comparison to the control mice, there was an augmentation in microglial activation in the hippocampus of POCD mice, which was alleviated by the administration of NBP (Fig. 3B).

The findings indicate that NBP therapy has the potential to suppress the activation of microglia, leading to a decrease in neuroinflammation.

3.3 NBP reverses neuronal, mitochondrial and Golgi damage in the hippocampus of POCD mice

Cognitive decline is significantly influenced by neurodegeneration, which is linked to alterations in neurons, mitochondria, and the Golgi apparatus. ¹⁵In POCD mice, we observed structural modifications in hippocampal neurons, mitochondria, and the Golgi apparatus. Figure 4A shows that mice in the control group exhibited typical neuronal morphology, distinct membrane structure, evenly distributed chromatin, regular mitochondrial morphology, and compact cristae. In contrast, hippocampal neurons from POCD mice exhibited degenerative damage, including membrane rupture, chromatin condensation, mitochondrial swelling and vacuolization, cristae fracture and Golgi pool fragmentation(Figure 4B).NBP treatment rescued these injuries (Fig. 4C).

The findings indicate that NBP can counteract neuronal degeneration and repair mitochondrial and Golgi impairments in the hippocampus of mice with POCD. Thus, NBP reduces neuroinflammation.

In the hippocampus of mice with POCD, NBP effectively counteracts neuronal destruction, as well as mitochondrial and Golgi damage, as shown in Figure.4. Electron micrographs of hippocampal neurons were taken for the control group (A), POCD group (B), and POCD + NBP group (C). Neurons of normal mice exhibit sleek nuclear envelopes (thick arrows), elliptical mitochondria (short-thin arrows), and consistent Golgi reservoirs (long-thin arrows). (B) Neurons from POCD mice show folding and disruption of the nuclear membrane, fewer and enlarged mitochondrial cristae, and rupture of the Golgi pool. (C) Treatment with NBP can reverse these injuries. n = 3.

3.4 NBP decreases neuroinflammation in the hippocampus of mice with POCD.

To evaluate if NBP treatment could improve surgery-induced neuroinflammation, we assessed the levels of apoptosis-related proteins like caspase3, Bcl2, and Bax in the hippocampus of POCD mice. In the hippocampus of POCD mice, the expression levels of Bcl-2 protein were notably decreased when compared to the control group. On the other hand, the levels of Caspase3 and Bax protein expression in the hippocampus of mice with POCD were notably higher when compared to the control mice. The condition was enhanced by NBP therapy (Fig. 5A-E).

In order to further illustrate the impact of NBP on neuroinflammation in the hippocampus of mice with POCD, we analyzed the levels of TNF-α, IL-1β, MDA, and SOD expression. In comparison to the control group, the hippocampus of mice in the POCD group exhibited significantly elevated levels of TNF-α, IL-1β, and MDA, whereas the levels of SOD were notably reduced. The NBP treatment improved this condition (Fig. 5F-I).

The findings indicate that NBP therapy decreased neuroinflammation in the hippocampus of mice with POCD.

In POCD mice, the administration of NBP treatment decreases neuroinflammation in the hippocampus, as shown in Fig. 5. Illustrative western blot images showcasing Bax and Bcl2 proteins.(B) Bax (C) Bcl2 (D) Western blot images showing caspase3 protein.(E) Caspase3 (F) TNF-α (G) IL-1β (H) MDA (I) SOD with a sample size of 3.P < 0.05, **P < 0.01, ***P < 0.001 signifies a comparison between the control group and the POCD group; #P < 0.05, ##P < 0.01, ###P < 0.001 signifies a comparison between the POCD + NBP group and the POCD group.

3.5 The BDNF/TrkB pathway might be involved in the potential ability of NBP to relieve neuroinflammation in the hippocampus of mice with POCD.

Past findings indicate that BDNF functions as a neurotrophic factor, fostering the growth of immature neurons and enhancing both neuronal survival and synaptic plasticity. The impact of NBP treatment on BDNF levels in the hippocampus was examined, as BDNF can produce its influence through interaction with the receptor tyrosine kinase TrkB.¹⁶ In the hippocampus of POCD mice, we observed a significant decrease in BDNF protein expression levels compared to control mice. However, this reduction was reversed by NBP treatment, as shown in Fig. 6. The findings indicate that NBP has the potential to reduce neuroinflammation and enhance neurogenesis and neuronal plasticity in the hippocampus of mice with POCD by activating the BDNF/TrkB signaling pathway. The exact mechanism of action is not known.

According to Fig. 6, the BDNF/TrkB signalling pathway could be responsible for the alleviation of neuroinflammation in the hippocampus of mice with POCD by NBP. (A) Representative pictures of western blot analysis for BDNF protein (B) BDNF n = 3.P values less than 0.05, 0.01, and 0.001 indicate a comparison between the control group and the POCD group. P values less than 0.05, 0.01, and 0.001 indicate a comparison between the POCD + NBP group and the POCD group.

Cognitive decline occurs as a consequence of surgery, which brings about alterations in the structure and function of the central nervous system. The extent of cognitive decline is linked to neuroinflammation, ^9,17and it is crucial to understand the precise molecular mechanisms and discover logical and efficient approaches for treatment. In this current investigation, we observed cognitive impairment and decreased ability to learn and remember in mice following the surgical procedure. The primary molecular mechanisms implicated are neuroinflammation and the presence of oxidative stress.

NBP is commonly utilized in the management of ischemic stroke.^18–20 Numerous investigations have demonstrated that NBP possesses notable neuroprotective properties in cerebral ischemia, potentially because of its capacity to diminish cerebral oedema, thus impeding oxidative stress and diminishing neuronal demise and inflammation.^21–23NBP has also been documented to enhance cognitive function in Alzheimer's disease and cerebral ischemia-reperfusion injury.^24–28Furthermore, prior research has indicated that NBP ameliorates cognitive function in Alzheimer's disease and cerebral ischemia-reperfusion injury. ^6,29–31Furthermore, prior research has indicated that NBP effectively mitigates cognitive impairments in mice, as demonstrated by shorter escape latencies and increased plateau crossings, which is consistent with our current study findings.

The brain's hippocampus, which is divided into three regions known as CA1, CA3, and the dentate gyrus (DG), plays a crucial role in brain function. The CA1 area of the hippocampus plays a role in controlling short-term memory, learning, executive function, and attention. ^22,32Cognitive deficits are linked to structural alterations in neurons. ^33,34To investigate this, we specifically examined the hippocampus and observed neuronal destruction, as well as mitochondrial and Golgi damage in mice with POCD using electron microscopy. However, the administration of NBP improved the neuronal changes in the hippocampus.

Deficits in cognitive function are linked to neuroinflammation and oxidative stress, which are connected to many neurological disorders.^4,35–42A neuroinflammatory response, characterized by microglial activation, increased expression of inflammatory factors and apoptotic proteins, and decreased expression of anti-apoptotic proteins, was observed following surgical stimulation. Oxidative stress is known to cause oxidative damage to biological macromolecules. Elevated MDA is regarded as a distinctive indicator of lipid peroxidation in the presence of oxidative harm. Impairment of antioxidant defense mechanisms like SOD is additionally a contributing element in the occurrence of oxidative harm.^43–45Earlier investigations have demonstrated that NBP mitigates the deterioration of cognitive abilities induced by neuroinflammation through its inhibition in rats with diabetes.³⁵Nevertheless, the previous studies have not provided a comprehensive explanation of the potential mechanisms through which NBP enhances postoperative cognitive dysfunction. Our hypothesis is that neuroinflammation and oxidative stress could potentially serve as the primary molecular mechanisms underlying POCD, and we propose that NBP treatment has the potential to mitigate the occurrence of POCD. During this investigation, it was discovered that performing surgery for tibial fractures with isoflurane anesthesia led to the occurrence of neuroinflammation and oxidative stress. Additionally, there was an activation of microglial cells, an elevation in inflammatory substances, apoptotic proteins, and lipid peroxides, as well as a reduction in anti-apoptotic proteins and antioxidant enzymes.

Importantly, NBP was able to reverse these changes. The findings indicate that NBP could have a substantial impact on the development and advancement of postoperative cognitive dysfunction through its ability to suppress neuroinflammation and oxidative stress.

Cognitive decline can be delayed or stopped by stimulating neurotrophic factors, which are crucial in neurogenesis, as neurogenic dysfunction is seen as a significant cause of cognitive impairment.⁴⁶ In our present investigation, it was observed that POCD inhibited the expression of BDNF in the hippocampus, while NBP reversed this alteration. BDNF, a crucial neurotrophic factor, plays a significant role in enhancing synapse formation and cognitive function.^35,47This indicates that NBP might relieve neuroinflammation in the hippocampus of POCD mice through the BDNF/TrkB signaling pathway.

While the beneficial role of NBP in POCD progression is clearly explained by these findings, the specific targets of NBP are still unknown. The permeability of the blood-brain barrier (BBB) becoming higher is a significant neurovascular complication of T2D that has a negative impact on the balance and operation of the central nervous system.^48,49 Dysfunction of the BBB might be a crucial initiator for the intricate series of molecular occurrences during POCD. According to earlier research, NBP has been found to be effective in restoring the permeability of the BBB following traumatic brain injury and cerebral ischemia-reperfusion injury. ^50–55This suggests that the BBB could be a crucial focus for NBP in the context of POCD. To understand the various potential molecular mechanisms, further research is required to uncover their anti-inflammatory and angiogenic effects.

To summarize, our research demonstrates that NBP has significant neuroprotective benefits by restoring hippocampal structure, decreasing neuronal cell death, and alleviating cognitive decline in POCD. Research has indicated that the suppression of oxidative stress and neuroinflammation plays a crucial role in how NBP functions in POCD. Hence, these discoveries propose that NBP could serve as a promising remedy for managing POCD.

Acknowledgments

We thank the Medical Research Center of Qingdao University for providing the experimental platform.

Ethical Approval

The study was approved by The Ethics Committee of Qingdao University (Approval number AHQU-MAL20220323).

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author Contributions

Zangong Zhou and Ning Yu designed research; Xiaofei Miao performed research; Xiaofei Miao and Wei Feng contributed new reagents or analytic tools; Xiaofei Miao analyzed data; Xiaofei Miao wrote the paper. All authors had reviewed the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (81771415).

Availability of data and materials

not applicable

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英文缩写	英文全称	中文全称
Bax	Bcl2-associated X protein	BCL-2相关X蛋白
BBB	blood-brain barrier	血脑屏障
BCA	bicinchoninicacid	二喹啉甲酸
Bcl-2	B-cell lymphoma-2	B淋巴细胞瘤-2基因
BDNF	brain-derived neurotrophic factor	脑源性神经营养因子
BSA	Bovine Serum Albumin	牛血清白蛋白
Caspase3	cysteinyl aspartate specific proteinase 3	含半胱氨酸的天冬氨酸蛋白水解酶-3
DAPI	4',6-diamidino-2-phenylindole	4’，6-二脒基-2-苯基吲哚
DG	dentate gyrus	齿状回
ELISA	enzyme-linked immunosorbent assay	酶联免疫吸附试验
IBA-1	ionized calcium binding adapter molecule-1	离子化钙结合适配分子-1
IL-1β	cytokine interleukin-1beta	白细胞介素-1β

MDA	malondialdehyde	丙二醛
MWM	Morris water maze	莫里斯水迷宫
NBP	3-n-Butylphthalide	3-N-丁基苯酞
PMSF	Phenylmethanesulfonyl fluoride	苯甲基磺酰氟
POCD	Postoperative cognitive dysfunction	术后认知功能障碍
PVDF	polyvinylidene fluoride	聚偏二氟乙烯膜
RIPA	Radio Immunoprecipitation Assay	放射免疫沉淀法
SDS	sodium dodecyl sulfate	十二烷基硫酸钠
SOD	Superoxide dismutase	超氧化物歧化酶
TBST	Tris-Buffered Saline and Tween 20	Tris-吐温20缓冲盐溶液
TNF-α	Tumor necrosis factor alpha	肿瘤坏死因子-α
β-actin	beta-actin	β-肌动蛋白

Table.1 list of abbreviations

No competing interests reported.

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3-n-Butylphthalide suppresses neuroinflammation through BDNF signaling in postoperative cognitive dysfunction in mice

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Abstract

Figures

1. Introduction

2. Experimental procedures and techniques

2.1 Animals used in experiments

2.2 Anesthesia and surgery

2.3 Behavioral assessments

2.3.1 Open field test

2.3.2 Morris water maze (MWM) test

2.4 Transmission electron microscopy

2.5 Immunofluorescence

2.6 Enzyme-linked Immunosorbent Assay (ELISA)

2.7 Measurement of MDA and SOD

2.8 Western blot

2.9 Statistical analysis

3. Results

3.1 NBP treatment ameliorates surgery-induced cognitive impairment

3.2 NBP treatment inhibits microglial activation

3.3 NBP reverses neuronal, mitochondrial and Golgi damage in the hippocampus of POCD mice

3.4 NBP decreases neuroinflammation in the hippocampus of mice with POCD.

4 Discussion

Declarations

References

Tables

Additional Declarations

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