Liu et al. Journal of Neuroinflammation(2021) 18:41
https://doi.org/10.1186/s12974-021-02089-z

RESEARCH

Open Access

Sirtuin 3 protects against anesthesia/
surgery-induced cognitive decline in aged
mice by suppressing hippocampal
neuroinflammation
Qiang Liu1, Yi-Man Sun1, Hui Huang1, Chen Chen1, Jie Wan1, Lin-Hui Ma1, Yin-Ying Sun1, Hui-Hui Miao2* andYu-Qing Wu1*

Abstract
Background: Postoperative cognitive dysfunction (POCD) is a very common complication that might increase themorbidity and mortality of elderly patients after surgery. However, the mechanism of POCD remains largely
unknown. The NAD-dependent deacetylase protein Sirtuin 3 (SIRT3) is located in the mitochondria and regulates
mitochondrial function. SIRT3 is the only sirtuin that specifically plays a role in extending lifespan in humans and isassociated with neurodegenerative diseases. Therefore, the aim of this study was to evaluate the effect of SIRT3 on
anesthesia/surgery-induced cognitive impairment in aged mice.
Methods: SIRT3 expression levels were decreased after surgery. For the interventional study, an adeno-associatedvirus (AAV)-SIRT3 vector or an empty vector was microinjected into hippocampal CA1 region before anesthesia/
surgery. Western blotting, immunofluorescence staining, and enzyme-linked immune-sorbent assay (ELISA) wereused to measure the oxidative stress response and downstream microglial activation and proinflammatory
cytokines, and Golgi staining and long-term potentiation (LTP) recording were applied to evaluate synaptic
plasticity.
Results: Overexpression of SIRT3 in the CA1 region attenuated anesthesia/surgery-induced learning and memory
dysfunction as well as synaptic plasticity dysfunction and the oxidative stress response (superoxide dismutase [SOD]and malondialdehyde [MDA]) in aged mice with POCD. In addition, microglia activation (ionized calcium binding
adapter molecule 1 [Iba1]) and neuroinflammatory cytokine levels (tumor necrosis factor-alpha [TNF-α], interleukin[IL]-1β and IL-6) were regulated after anesthesia/surgery in a SIRT3-dependent manner.
Conclusion: The results of the current study demonstrate that SIRT3 has a critical effect in the mechanism of POCD
in aged mice by suppressing hippocampal neuroinflammation and reveal that SIRT3 may be a promisingtherapeutic and diagnostic target for POCD.
Keywords: SIRT3, Postoperative cognitive dysfunction, Mitochondrial oxidative stress, Neuroinflammation, Microglia,
Synaptic plasticity* Correspondence: iverymhh@hotmail.com; xzmcyqwu@163.com
2
Department of Anesthesiology, Beijing Friendship Hospital, Capital Medical
University, Beijing 100050, P.R. China
1
Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical
University, Tongshan Road 209, Xuzhou 221004, P.R. China© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate ifchanges were made. The images or other third party material in this article are included in the article's Creative Commonslicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to thedata made available in this article, unless otherwise stated in a credit line to the data.

Liu et al. Journal of Neuroinflammation(2021) 18:41

Background
Postoperative cognitive dysfunction (POCD) is one of themost common postoperative complications in aged patients[1]. Evidence from clinical research has revealed that POCD
may contribute to increased mortality and decreased qualityof life as well as prolonged hospitalization [2]. Nevertheless,the mechanism of POCD in aged patients is still unclear.
Mitochondrial damage, neuroinflammation, and synaptic plasticity dysfunction have been demonstrated to
be involved in the mechanism of POCD [3–9]. Netto
et al. reported oxidative damage in the hippocampus anddecreased activity of the antioxidant enzyme superoxide
dismutase (SOD) in aged rats with tibial fracture [10]. In
addition, anesthesia/surgery may induce microglial activation, resulting in the release of inflammatory factors,
such as tumor necrosis factor-alpha (TNF-α), interleukin(IL)-1β, and IL-6 [11, 12]. Furthermore, the hippocampal
CA1 region is critical for cognition [13], and overactivated microglia and neuroinflammation create a neurotoxic response and cause synaptic dysfunction, resulting
in cognitive dysfunction [14].
The NAD-dependent deacetylase protein Sirtuin 3(SIRT3) is related to mitochondrial function and oxidative stress regulation [15]. SIRT3 is found mostly in the
mitochondria and is the only sirtuin that specificallyplays a role in extended lifespan in humans [16, 17].
Several SIRT3 substrates have been reported in the past
few years that are involved in pathological mechanisms
dependent on mitochondrial biogenesis and dynamism
pathways [18]. It was reported that SIRT3 overexpression increased the expression of SOD2 [19]. Tyagi et al.
found increased IL-1β levels and microglial activation in
SIRT3−/− mouse brains [20]. In addition, the mitochondriarelated inflammatory response was also shown to be mediated by SIRT3 [21, 22]. SIRT3−/− mice exhibited poor remote memory [23], suppressed long-term potentiation(LTP), and decreased neuronal number [23]. However, lessis known about the role of SIRT3 in POCD in aged mice.
Consequently, the aim of our current study was todetermine the effect of SIRT3 on anesthesia/surgery-induced cognitive impairment and the related molecularmechanism. We propose that SIRT3 may attenuate postoperative cognitive dysfunction by regulating the hippocampal neuroinflammation pathway.
Materials and methods
Animals

All experiments were approved by the Animal Care and
Use Committee of Xuzhou Medical University and inaccordance with the Guide for the Care and Use ofLaboratory Animals of National Research Council. Aged
(18 months old, weighing 28–32 g) male C57BL/6J mice
were ordered from the Model Animal Research Centerof Nanjing University. Five mice per cage were group-

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housed (22–25°C) with food and water available ad libitum under a 12-h light/dark cycle. The animals wererandomly grouped and underwent experiments in an
unbiased double-blind manner during the daytime.
POCD mouse model

Tibial fracture procedures were performed under isoflurane anesthesia to establish the POCD model as
described in other studies [3]. In the first experiment,
the mice were randomly placed into 2 groups: the control group (C) and the anesthesia/surgery group (A/S).
The mice in group C received 100% oxygen without any
anesthesia/surgery procedure [3]. For the anesthesia/surgery group, mice received 3.0% isoflurane for anesthesia
induction followed by 1.5% isoflurane for maintenance.
Next, the incision was made lateral to the tibia, exposing
the bone. An intramedullary fixation pin was implanted
into the spinal canal from the hole drilled at the trochanter of the tibia. Osteotomy was performed in the
middle and distal tibia, and the incision was continuously sutured. The mouse body temperature was monitored and kept between 36 and 37°C by a heating pad
during the operation. After the mice recovered fromanesthesia/surgery, they were returned to their home
cages. Lidocaine (2%) was locally applied to treat postoperative pain. For the interventional study, the mice wererandomly divided into 4 groups: the control + control
adeno-associated virus (AAV) vehicle (VEH) group (C +VEH), the anesthesia/surgery + control AAV-VEH group(A/S + VEH), the control + AAV-SIRT3 vector group (C+ SIRT3), and the anesthesia/surgery + AAV-SIRT3
vector group (A/S + SIRT3). A schematic diagram of theexperimental procedure is shown in Fig. 1.
Viral microinjection

The construct was packaged into a chimeric AAV2/8
vector, and the rAAV-mNeonGreen vector without
SIRT3 was used as a control AAV vehicle (AAV-VEH).
The virus constructs were generated by OBiO Technology (Shanghai, China). A rAAV-CMV-SIRT3-IRES2mNeonGreen vector (AAV-SIRT3) and a control rAAVCMV-MCS-IRES2-mNeonGreen vehicle (AAV-VEH)(titers > 1.0 × 1012) were delivered by bilateral stereotactic injections into the CA1 region of the brain (0.5 μl/side) following the mouse brain atlas (AP 2.00 mm,
lateral ± 1.50 mm, DV-1.40 mm). The mice were allowed
to recover from the virus injection for 4 weeks before
anesthesia/surgery. The location of virus transfection
was confirmed by fluorescence.
Behavioral tests

The behavioral tests included the open field test (OFT)and fear conditioning test (FCT). We used the OFT tomeasure the locomotor activity of the mice at different

Liu et al. Journal of Neuroinflammation(2021) 18:41

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Fig. 1 Schematic diagram of the experimental procedure. a The mice received behavior training at day − 1 and behavioral tests on days 1, 3, and7 after anesthesia/surgery. Anesthesia/surgery (A/S) was performed on day 0. Hippocampal tissues were harvested at the end of the behavioral
tests on days 1, 3, and 7. Western blotting analysis of SIRT3 expression was performed on days 1, 3, and 7. The levels of SOD, MDA, TNF-α, IL-1β,
and IL-6 were measured on days 1 and 3. Immunofluorescence and western blotting analysis for Iba1 were performed on day 1. Golgi staining
and LTP of synaptic function were measured on postoperative day 7. b AAV-SIRT3/VEH virus was microinjected into the CA1 region of the
hippocampus 28 days before anesthesia/surgery. The mice received behavioral training at day − 1 and behavioral tests on days 1, 3, and 7 after
anesthesia/surgery. Western blotting analysis of SIRT3 expression was performed on days 1, 3, and 7. The levels of SOD, MDA, TNF-α, IL-1β, and IL6 were measured on days 1 and 3. Immunofluorescence and western blotting analysis for Iba1 were performed on day 1. Golgi staining and LTPof synaptic function were measured on postoperative day 7

time points. The mice were allowed to freely move inthe square field (40 × 40 × 40 cm) for 5 min. A video
camera was used to automatically track and record
movement traces of the mice with the ANY-maze software system (ANY-maze, Stoelting Co., IL, USA). The
total distance traveled was used to assess the locomotor
activity of the mice. The installation was wiped with 75%
ethanol after each test to avoid olfactory cues.
The FCT was employed following the protocol
described in previous studies [3]. During the training
session before anesthesia/surgery, the mice were placedin the chamber to acclimate for 2 min, and then conditional (20-s, 70-dB tone conditional stimulus, 25-s interval) and unconditional (2-s and 0.70-mA footshock)stimuli were applied in six pairs. The intervals between
the pairs were random and ranged from 45 to 60 s. For
the test session, the context test was used to measure
hippocampus-dependent memory, and the tone test wasused to measure hippocampus-independent memory.
The mice were allowed in the conditioning chamber
without any stimulus for 5 min during the context test,
and 2 h later, the tone test was performed. The mice
were transferred into a novel chamber with the soundstimulus (70 dB, 3 min) but not the footshock stimulus
for 5 min. The freezing time during each test wasrecorded and analyzed by the system software (MedAssociates, Inc., USA).
Western blotting analysis

Western blotting was performed as described before[24]. Briefly, we measured the protein concentrations in
the CA1 region of the hippocampus by the bicinchoninicacid (BCA) Protein Assay Kit (Beyotime, P0010,
Shanghai, China). Then, SDS-PAGE was used to separate the proteins, which were subsequently transferred to
PVDF membranes (Merck Millipore, ISEQ00010, USA).
Five percent nonfat milk with 0.1% Tween-20 in theta
burst stimulation (TBS) was used to block the membranes, and the membranes were then incubated at 4 °C
overnight in a cold room. The following primary antibodies were used: SIRT3 (1:1000, 5490, Cell Signaling
Technology, USA), Iba1 (1:1000, ab178847, Abcam, UK),and β-actin (1:2000, AC004, ABclonal, China). Horseradish peroxidase-conjugated antibodies (1:2000, Beyotime)
were used as secondary antibodies. An ECL detection
system (Beyotime) and the ImageJ software were used toquantify the protein bands. The raw image files of thewestern blots are shown in the supplementary information (Additional file 1).
Immunofluorescence staining of ionized calcium binding
adapter molecule 1 (Iba1)

Immunofluorescence staining of the microglial marker
Iba1 was used to study microglial morphology. Mice
were perfused transcardially with 0.9% saline followed by
4% paraformaldehyde in 0.1 M phosphate buffer (pH7.4). The brain tissues were removed and postfixed in
4% paraformaldehyde overnight and cryoprotected in
30% sucrose as we reported before [25]. Sections (30 μm
thick) were prepared with a freezing microtome(VT1000S, Leica Microsystems). The brain tissue wasblocked with 5% goat serum albumin for 30 min andthen incubated with the primary antibody for Iba1 (1:100, ab178847, Abcam) in a cold room at 4 °C overnight.

Liu et al. Journal of Neuroinflammation(2021) 18:41

The brain tissue was incubated with goat anti-rabbit
Alexa 594 (1:500, ab150080, Abcam) as the secondary
antibody in the dark at room temperature for 2 h. The
fluorescence intensity was visualized under a confocal
microscope (Zeiss, LSM880, Germany). The fluorescenceintensities from three slides (three visual fields per slide)
were averaged for each animal and then normalized tothose of the control group as we reported before [25].
Enzyme-linked immunosorbent assay (ELISA)

Hippocampal homogenate was obtained on days 1 and 3
after anesthesia/surgery and centrifuged at 10000g at
4 °C for 10 min to collect the supernatant. TNF-α (Cat.
No. E-EL-M0049c, Elabscience), IL-1β (Cat. No. E-ELM0037c, Elabscience), and IL-6 (Cat. No. E-EL-M0044c,
Elabscience) were measured according to the manufacturer’s protocols. The optical density (OD) at 450 nm
was obtained with an ELISA plate reader (Multiskan
GO, Thermo Scientific, USA).
Oxidative activity evaluation

SOD and malondialdehyde (MDA) were measured byspectrophotometric assay kits (Elabscience, Wuhan,
China). A Varioskan LUX microplate reader was used todetermine SOD activity by measuring the absorbance at
450 nm (U/mg protein), and MDA activity was determined by measuring the absorbance at 532 nm (nmol/
mg protein).
Golgi-Cox staining and dendritic spine counting

Golgi-Cox staining was used to visualize synaptic structural plasticity. The HITO Golgi-Cox OptimStain PreKit(Hitobiotec Corp, Kingsport, TN, USA) was employedaccording to the manufacturer’s instructions. Specifically, the brain tissues were immersed in solutions 1 and
2 for 24 h, the solutions were changed, and the tissues
were stored in the dark at room temperature for 2
weeks. Next, solution 3 was used, and the brains werestored in the dark at 4 °C for another 72 h. A cryotome(chamber temperature − 22 °C) was used to prepare
brain sections (100 μm). An Olympus BX53 was used forthe imaging and analysis of the dendrites and dendritic
spines within the CA1 region of the hippocampus.
Multichannel field potential recording

LTP recordings were used to evaluate functional synaptic
plasticity, and the 64-channel recording system (MED64,
Panasonic Alpha-Med Sciences) was used as described
previously [26, 27]. One of 64 microelectrodes was
selected as a stimulating electrode within the CA1 regionof the hippocampus. The evoked field excitatory postsynaptic potentials (fEPSPs) were used for recording from theother channels. Theta burst stimulation (TBS) (5 trains ofbursts with 4 pulses at 100 Hz, repeated 5 times at 10-s

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intervals) was used in this study. Averaged fEPSP slopes
over the last 20 min of the recording were used foranalysis.
Statistical analysis

Statistical analyses were performed by GraphPad Prism
6.0 (GraphPad Software, Inc.). Data are presented as themean ± SEM. The differences between the C and A/Sgroups were compared by unpaired t test. Behavioral
and other tests comprising four groups were analyzed by
one-way ANOVA with a Tukey post hoc test for
multiple comparisons. A significant difference wasdefined as p < 0.05.

Results
Effects of anesthesia/surgery on the OFT and FCT of aged
mice

The OFT was employed to assess the locomotor activityof aged mice. We found that the total distance traveledby the mice was not significantly changed between thecontrol (C) and anesthesia/surgery (A/S) groups at baseline (1 day before anesthesia/surgery) (p > 0.05, Fig. 2a)or postoperation (1, 3, and 7 days after anesthesia/surgery) (p > 0.05, Fig. 2b–d). In the FCT, context tests
were used to examine hippocampus-dependent learningand memory, while tone tests were used to examine
hippocampus-independent learning and memory [3].
During the training sessions, we found that the freezingtime was not significantly different between the C and
A/S groups (p > 0.05, Fig. 2e), which means that the cognitive condition of the aged mice was comparable before
the operation. In the FCT context test, the freezing time
was significantly decreased at 1, 3, and 7 days after
anesthesia/surgery in the A/S group compared to the Cgroup (*p < 0.05, Fig. 2f–h). Nevertheless, in the FCT
tone test, the freezing time of the A/S group did not
obviously differ from that of the C group after theoperation (p > 0.05, Fig. 2i–k). Taken together, theseresults indicated that anesthesia/surgery induced
hippocampus-dependent cognitive decline but did not
induce hippocampus-independent cognitive decline or
locomotor activity impairment in aged mice.
Effects of anesthesia/surgery on SIRT3 expression and themitochondrial oxidative stress response in the
hippocampus of aged mice

SIRT3 expression levels in the CA1 region of the
hippocampus were measured in the control andanesthesia/surgery groups. Compared with that in
the control mice, the SIRT3 expression in the A/Smice was obviously decreased at postoperative days
1, 3, and 7 (*p < 0.05, Fig. 3a–f). To evaluate themitochondrial oxidative stress response, the levels of
SOD and MDA in the CA1 region of aged miceLiu et al. Journal of Neuroinflammation(2021) 18:41

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Fig. 2 Hippocampus-dependent memory, but not hippocampus-independent memory or locomotor activity, in mice was impaired byanesthesia/surgery. The OFT was conducted to assess the total distance traveled by the mice at 1 day preoperatively and at postoperative days 1,
3, and 7 (a–d). The percent of freezing time during the training session at 1 day preoperatively (e) in the context test (f–h) and tone test (i–k)
were assessed at postoperative days 1, 3, and 7. The data are presented as the means ± standard error of the mean for each group (n = 10). *p <0.05 compared with the control groupwere determined by ELISA. Anesthesia/surgery induced decreased SOD levels in the hippocampal CA1
region on postoperative days 1 and 3 in aged mice(*p < 0.05, Fig. 3g). Meanwhile, anesthesia/surgery
significantly increased the level of MDA in the CA1
region of the hippocampus compared with the control group (*p < 0.05, Fig. 3h). These results showedthat anesthesia/surgery induced the downregulationof SIRT3 and increased the oxidative stress responsein the CA1 region of the hippocampus of aged mice.
Effects of anesthesia/surgery on hippocampal microglia
activation and neuroinflammation in aged mice withPOCD

Microglia activation is an essential early-phase change inhippocampal neuroinflammation in POCD. First, wefound by immunofluorescence staining that Iba1 expression was increased in the CA1 region of the hippocampus on postoperative day 1 (**p < 0.01, Fig. 4a, b).
Correspondingly, immunoblotting for Iba1 revealed avisible increase in Iba1 expression in the CA1 region of
the hippocampus of aged mice on postoperative day 1(**p < 0.01, Fig. 4c, d). Then, the TNF-α, IL-1β and IL-6
levels were used to assess the level of neuroinflammationin aged mice. As shown in Fig. 4e–j, the levels of TNF-α,
IL-1β, and IL-6 in the CA1 were increased in theanesthesia/surgery group compared with the control
group on postoperative days 1 and 3 (*p < 0.05). These
data indicated that anesthesia/surgery induced microglia
activation and neuroinflammation in the CA1 region of
the hippocampus of aged mice.
Effects of anesthesia/surgery on synaptic plasticity in the
hippocampus of aged mice with POCD

Previous studies have revealed that synaptic plasticity
is a critical molecular mechanism for learning andmemory [28, 29]. First, synaptic structural plasticity
was investigated by Golgi-Cox staining. The morphology of the CA1 neurons and camera tracings fromthe control and anesthesia/surgery groups are shownin Fig. 5a. Sholl analysis was used to measure dendritic branching and spine density in the CA1 regionof the hippocampus. The total number of dendritic
intersections at 70–130 μm from the soma was significantly reduced in the anesthesia/surgery group (**p <0.01, Fig. 5d). Additionally, the total dendritic lengthand number of spines were also reduced after
anesthesia/surgery (**p < 0.01, Fig. 5b, c and e, f).
Then, synaptic functional plasticity was measured by electrophysiological methods. LTP in the CA1 region of the
hippocampus was evaluated using a 64-channel multielectrode (MED64) system. As shown in Fig. 5g–i, LTP

Liu et al. Journal of Neuroinflammation(2021) 18:41

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Fig. 3 SIRT3, SOD, and MDA levels in the hippocampal CA1 region of aged mice after anesthesia/surgery. Representative western blots of SIRT3
at days 1, 3, and 7 after anesthesia/surgery (a–c). Quantitative analysis of SIRT3 levels compared with the control group (d–f). Analysis of SOD (g)and MDA (h) levels in the hippocampal CA1 region on postoperative days 1 and 3. The data are presented as the means ± standard error of themean for each group (n = 6). *p < 0.05, **p < 0.01 compared with the control groupinduced by TBS in the CA1 region of aged mice was attenuated in the A/S group on postoperative day 7. These
results indicated that anesthesia/surgery induced both
structural and functional synaptic plasticity dysfunction in
the CA1 region of the hippocampus of aged mice.
SIRT3 overexpression in the hippocampal CA1 region ofaged mice with POCD

To demonstrate the role and mechanism of SIRT3 in
POCD in aged mice, AAV-SIRT3 vector (which carries
the SIRT3 gene) was locally injected into the CA1 region of aged mice 4 weeks before anesthesia/surgery,and AAV-VEH vector was injected as a control (Fig. 6a,b). The SIRT3 expression level was increased in the C+ SIRT3 group but decreased in the A/S + VEH groupcompared with that of the control group, while theoverexpression of SIRT3 rescued the decreased SIRT3
level in the A/S + SIRT3 group compared with that of
the A/S + VEH group at postoperative days 1, 3, and 7of aged mice (p < 0.05, Fig. 6c–h). As shown in Fig. 6i–n, SIRT1 expression was markedly decreased after
anesthesia/surgery. However, the expression level of
SIRT1 in the A/S + SIRT3 group was not statisticallydifferent from that in the A/S + VEH group on postoperative days 1, 3, and 7. Similarly, there was also no
statistic difference in SIRT1 expression between the C+ SIRT3 group and the C + VEH group. Then, theSOD and MDA levels were measured in the 4 groups.
We found that the SOD level was significantly decreased in the A/S + VEH group compared with thecontrol group on postoperative days 1 and 3 (p < 0.05,
Fig. 6o). Meanwhile, the level of SOD in the A/S +SIRT3 group was significantly increased compared withthat in the A/S + VEH group. Conversely, comparedwith that of the C + VEH group, the level of MDA (p <0.05, Fig. 6p) was significantly increased in the A/S +VEH group on postoperative days 1 and 3. Furthermore, preoperative injection of AAV-SIRT3 in the CA1
significantly attenuated the upregulation of MDA.
These data indicated that the anesthesia/surgery-induced mitochondrial oxidative stress response in the
CA1 region of the hippocampus of aged mice with
POCD was attenuated in a SIRT3-dependent manner.
Effects of SIRT3 overexpression on microglia activationand neuroinflammation in the hippocampus of aged micewith POCD

To determine the downstream signaling pathway andmolecules involved in SIRT3-mediated POCD in aged
mice, we detected microglia activation and neuroinflammation in the 4 groups by both immunofluorescenceand western blotting. We found obviously increased

Liu et al. Journal of Neuroinflammation(2021) 18:41

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Fig. 4 Anesthesia/surgery induced microglial activation and neuroinflammation in the hippocampus of aged mice. a Representative images ofIba1 (a marker of microglia) in the CA1 region at day 1 after anesthesia/surgery. b Quantification of Iba1 fluorescence. c Representative western
blots of Iba1 at day 1 after anesthesia/surgery. d Quantitative analysis of Iba1 levels compared with the control group. Analysis of TNF-α, IL-1β,
and IL-6 levels in the hippocampal CA1 region on postoperative days 1 (e–g) and 3 (h–j). The data are presented as the means ± standard errorof the mean for each group (n = 6). *p < 0.05, **p < 0.01 compared with the control group

Liu et al. Journal of Neuroinflammation(2021) 18:41

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Fig. 5 Hippocampal synaptic plasticity changes on day 7 after anesthesia/surgery. a A hippocampal profile image of Golgi staining and camera
tracings of hippocampal CA1 neurons. b Sample images of Golgi-Cox staining of CA1 neurons for spine counting. c Representative dendritic
spine density of hippocampal CA1 neurons. d Quantitation of dendritic intersections (n = 18). e Quantitation of the total dendritic length (n =18). f Quantitation of the dendritic spine density (n = 18). g Sample images showing the location of the MED64 probe in the CA1 region. h LTP
recording in the hippocampal CA1 region. Arrows indicate the time point of TBS application. I. Averaged fEPSP slope during the last 20 min. The
data are presented as the means ± standard error of the mean for each group (n = 6). *p < 0.05, **p < 0.01 compared with the control group.
Scale bar = 10 μm

fluorescence intensity and protein expression of Iba1 in
the A/S + VEH group compared to the C + VEH group(**p < 0.01, Fig. 7a–d). Of note, the A/S + SIRT3 group
had significantly reduced fluorescence intensity andprotein expression of Iba1 compared to those of theA/S + VEH group (Fig. 7a–d, #p < 0.05). Next, theTNF-α, IL-1β, and IL-6 levels were measured in the
4 groups. Compared with those in the C + VEH
group, the levels of TNF-α, IL-1β, and IL-6 (p <0.05, Fig. 7e–j) were significantly increased in the A/
S + VEH group on postoperative days 1 and 3. Furthermore, preoperative injection of AAV-SIRT3 in
the CA1 significantly attenuated the upregulation of
TNF-α, IL-1β, and IL-6 levels. Thus, these data indicated that microglia activation and neuroinflammation may be involved in the pathogenesis of SIRT3mediated POCD in aged mice.

Effects of SIRT3 overexpression on synaptic plasticity inthe hippocampus of aged mice with POCD

Regarding synaptic structural plasticity, Sholl analysis
showed that the total number of dendritic intersections at 50–130 μm from the soma in the hippocampal
CA1 region was reduced significantly in the A/S +VEH group compared to the C + VEH group (*p <0.05, Fig. 8a, d). SIRT3 overexpression significantly
upregulated the total number of dendritic intersections in the A/S + SIRT3 group compared to the A/S+ VEH group (#p < 0.05, Fig. 8a, d). Additionally,
SIRT3 overexpression also significantly upregulated
the total dendritic length and number of spines in the
A/S + SIRT3 group compared to the A/S + VEH group(#p < 0.05, Fig. 8b, c, e–f). Regarding synaptic functional plasticity, as shown in Fig. 8g, h, LTP in the
CA1 region of aged mice was suppressed in the A/S +Liu et al. Journal of Neuroinflammation(2021) 18:41

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Fig. 6 Overexpression of SIRT3 in CA1 region of aged mice. a The location for viral microinjection with a rAAV-CMV-SIRT3-IRES2-mNeonGreen
vector (AAV-SIRT3) or a control rAAV-CMV-MCS-IRES2-mNeonGreen vehicle (AAV-VEH). b Fluorescence images showing efficient expression of
AAV-SIRT3 vector in CA1 region. Representative images of Western blots showing that AAV-SIRT3 injection increased SIRT3 expression in CA1 cells(c–e). Quantification of SIRT3 expression among the 4 groups (f–h). Representative images of Western blots and quantification of SIRT1 expression
among the 4 groups (i–n). Analysis of SOD and MDA levels in the hippocampal CA1 region on postoperative days 1 and 3 (o, p). The data arepresented as the means ± standard error of the mean for each group (n = 6). *p < 0.05, **p < 0.01 compared with the C + VEH group, #p < 0.05
compared with the A/S + VEH group

VEH group compared to the C + VEH group on postoperative day 7, but SIRT3 overexpression significantly
attenuated the loss of LTP in the A/S + SIRT3 groupcompared to the A/S + VEH group (Fig. 8g, h). These
results indicated that SIRT3 overexpression may protect aged mice from anesthesia/surgery-induced synaptic plasticity dysfunction.

Effects of SIRT3 overexpression on the OFT and FCT ofaged mice with POCD

In the OFT, the total distance traveled was not significantly different among the 4 groups during the preoperative (p > 0.05, Fig. 9a) or postoperative period (p >0.05, Fig. 9b–d). These results showed that the locomotor ability of the mice was not impaired by the viralLiu et al. Journal of Neuroinflammation

Fig. 7 (See legend on next page.)(2021) 18:41

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Liu et al. Journal of Neuroinflammation(2021) 18:41

Page 11 of 16(See figure on previous page.)
Fig. 7 Microglial activation and neuroinflammation in the hippocampal CA1 region among the 4 groups. a Representative images of Iba1 (amarker of microglia) in the CA1 region. b Quantification of Iba1 fluorescence. c Representative Western blots of Iba1 among the 4 groups at day
1 after anesthesia/surgery. d Quantitative analysis of Iba1 levels compared with the control group. Analysis of TNF-α, IL-1β, and IL-6 levels in the
hippocampal CA1 region on postoperative days 1 and 3 (e–g and h–j). The data are presented as the means ± standard error of the mean foreach group (n = 6). *p < 0.05, **p < 0.01 compared with the C + VEH group, #p < 0.05 compared with the A/S + VEH groupinjection prior to the operation or by the anesthesia/surgery procedure. In the FCT training session, no significant difference in freezing time was found among the 4groups of aged mice (p > 0.05, Fig. 9e), showing that
their cognition was comparable before the anesthesia/
surgery procedure. Next, we found that the freezing time
was decreased on postoperative days 1, 3, and 7 in the
A/S + VEH group compared to the C + VEH group in
the context test (*p < 0.05, Fig. 9f–h). Moreover, SIRT3
overexpression increased the freezing time during the
postoperative period (postoperative days 1, 3, and 7) in
the A/S + SIRT3 group compared to the A/S + VEH
group in the context test (#p < 0.05, Fig. 9d–f). Additionally, the freezing time was not significantly different
among the 4 groups on postoperative days 1, 3, and 7 in
the tone test (p > 0.05, Fig. 9i–k). Collectively, these results indicated that SIRT3 overexpression attenuatedanesthesia/surgery-induced hippocampus-dependent cognitive decline in aged mice.

Discussion
The purpose of the current study was to assess the effect of SIRT3 in anesthesia/surgery-induced cognitive
decline in aged mice with hippocampal inflammationrelated molecular mechanisms. We found that SIRT3
expression was obviously decreased in the hippocampal
CA1 region of aged mice with POCD. In addition,
SIRT3 overexpression prevented anesthesia/surgery-induced cognitive decline and synaptic plasticity dysfunction in aged mice. Importantly, SIRT3 overexpression
ameliorated the anesthesia/surgery-induced mitochondrial oxidative stress response, microglia activation andneuroinflammation. Hence, these results demonstrated
the critical effect of SIRT3 in the treatment ofanesthesia/surgery-induced neuroinflammation andcognitive dysfunction in aged mice.
Studies on patients with cognitive decline after
anesthesia/surgery have been conducted for morethan 100 years. In 2018, the term “perioperative neurocognitive disorders” (PNDs) was recommended by
the Nomenclature Consensus Working Group as an
overarching term for cognitive impairments identified
in the preoperative or postoperative period [30–34],
which includes cognitive decline diagnosed before operation, acute postoperative delirium, and delayed
neurocognitive recovery. Since we focused on

postoperative cognitive function, the term “POCD”
was used in our current study.
The mitochondrial oxidative stress response contributes to postoperative cognitive dysfunction [10].
Recently, SIRT3 has been shown to be strongly involvedin the regulation of oxidative stress-related mitochondrial damage in neurodegenerative diseases, such as Parkinson’s disease (PD) and Alzheimer’s disease (AD) [35].
For example, SIRT3 has been shown to be downregulated in AD patients [36]. Apolipoprotein E4 (APOE4) isa major genetic factor related to late-onset AD, andSIRT3 has been reported to be decreased in the frontal
cortices of APOE4-carrying individuals compared to
non-APOE4-carrying individuals [17]. Additionally, it
has been reported that SIRT3 overexpression suppressedoxidative stress and therefore extended neuronal longevity in primary hippocampal cultures [37]. This suggeststhat SIRT3 upregulation can reverse brain oxidative
stress, which is implicated in cognitive decline. Specifically, SIRT3 is known to regulate the activity of mitochondrial antioxidant enzymes. Therefore, the levels of
SOD and MDA were measured in this study, and we
found decreased SOD and increased MDA levels after
anesthesia/surgery that could be ameliorated by SIRT3
overexpression. These findings suggest that the oxidative
stress response of hippocampal mitochondria in aged
mice with POCD is likely to be SIRT3-dependent. To
address the potential impact of SIRT3 overexpression onother Sirt proteins, we then measured the expression
level of SIRT1, another important Sirt protein in the
nervous system. SIRT1 expression was markedly decreased after anesthesia/surgery, which was consistent
with the results of previous studies [38, 39]. We also
found that the expression level of SIRT1 in the A/S +SIRT3 group was not significantly changed comparedwith that in the A/S + VEH group on days 1, 3, and 7
after anesthesia/surgery. Similarly, the expression levelof SIRT1 in the C + SIRT3 group was also not markedly
different from that in the C + VEH group. These results
indicated that the overexpression of SIRT3 did not significantly change the SIRT1 expression level, which suggested that SIRT1 may not be involved in the SIRT3mediated neuroprotective effect in POCD. Verma et al.
reported that SIRT3 KO might cause a compensatory
rise in SIRT1 expression, which was responsible for neuroprotection in a stroke model [40]. However, we did
not find the corresponding result that SIRT3

Liu et al. Journal of Neuroinflammation

Fig. 8 (See legend on next page.)(2021) 18:41

Page 12 of 16

Liu et al. Journal of Neuroinflammation(2021) 18:41

Page 13 of 16(See figure on previous page.)
Fig. 8 Hippocampus synaptic plasticity among the 4 groups. a Camera tracings of hippocampal CA1 neurons among the different groups. b
Sample images of Golgi-Cox staining of CA1 neurons for spine counting. c Representative dendritic spine density of hippocampal CA1 neurons. d
Quantitation of the dendritic intersections. e Quantitation of the total dendritic length. f Quantitation of the dendritic spine density. g LTP
recording in the hippocampal CA1 region. Arrows indicate the time point of TBS application. h Averaged fEPSP slope during the last 20 min. The
data are presented as the means ± standard error of the mean for each group (n = 6). *p < 0.05, **p < 0.01 compared with the C + VEH group,#p < 0.05 compared with the A/S + VEH group. Scale bar = 10 μm

overexpression causes a compensatory decrease in
SIRT1 expression. The different changes in SIRT1 expression (compensatory rise vs no change) may be dueto the different disease models (stroke vs POCD) andthe different techniques used (genetic knockout vs virus
injection). Further study is still needed to fully clarify thepotential interaction of SIRT3 and other Sirt proteins in
POCD.
The enzymes of the redox pathway maintain thebalance between the production and elimination ofoxygen radicals by a well-regulated system [41]. Uncontrolled oxidative stress promotes the inflammatory
response [42]. We found that the levels of the inflammatory mediators TNF-α, IL-1β, and IL-6 in the CA1
region were significantly increased at postoperativedays 1 and 3 and that these changes were accompanied by changes in SOD and MDA levels, whereas
SIRT3 overexpression attenuated these phenomena,suggesting a potential link between the mitochondrial
oxidative stress response and neuroinflammation. Previous studies have focused on the mechanism bywhich cytokines generated in the periphery cross the
blood–brain barrier (BBB) and induce secondary inflammation in the brain [43, 44]. We propose amechanism in which neuroinflammation is triggered
through SIRT3-dependent mitochondrial damage inthe brain. Moreover, local SIRT3 overexpression in
the hippocampal CA1 region ameliorated theanesthesia/surgery-induced neuroinflammation andthus confirmed the causal relationship.
In the present study, we also observed the postoperative proliferation of microglia in the CA1 region ofaged mice. These results are consistent with previous
studies. Anesthesia/surgery can induce the innate
immune system response [45] and microglia activation, leading to the synthesis and release of

Fig. 9 SIRT3 overexpression alleviated anesthesia/surgery-induced hippocampus-dependent cognitive dysfunction in aged mice. The total
distance traveled in the OFT among the 4 groups at 1 day before and 1, 3, and 7 days after anesthesia/surgery (a–d). The percent freezing time
was assessed during the training session on 1 day preoperatively (e) as well as in the context test (f–h) and tone test (i–k) at postoperative days
1, 3, and 7. The data are presented as the means ± standard error of the mean for each group (n = 10). *p < 0.05, **p < 0.01 compared with the
C + VEH group, #p < 0.05 compared with the A/S + VEH group

Liu et al. Journal of Neuroinflammation(2021) 18:41

inflammatory cytokines (e.g., TNF-α, IL-1β, and IL-6)[46, 47] and cognitive decline [48]. It was recently reported that pharmacological activation of SIRT3 significantly reduced the susceptibility of microglial cells
to inflammatory stress [49]. Consistent with these results, preoperative injection of AAV-SIRT3 in the
CA1 region attenuated microglial activation and hippocampal neuroinflammation. Therefore, these findings also indicated that microglia activation andhippocampal neuroinflammation are involved in theeffects of SIRT3 in POCD in aged mice.
Microglial synaptic pruning contributes to neuronal
network establishment [50]. Microglia serve as housekeepers and constantly monitor the brain environment. Microglia contact with synapses is dynamic.
Microglia activation and neuroinflammation are also
involved in aging-related cognitive decline and theprogression of neurodegenerative diseases, including
AD [51]. Furthermore, alterations in microglia activation and neuroinflammation have been associatedwith synaptic plasticity dysfunction in neurodegenerative diseases, such as PD [52]. Previous studies havedemonstrated that the structural and functional
synaptic plasticity of the hippocampus plays a critical role in the development of neurological disorders [53, 54]. It has been shown that the density ofdendritic spines is impaired and the maintenance of
LTP is suppressed in the hippocampus of AD mice
[29, 55–57]. It has also been suggested that thedensity of dendritic spines of CA1 pyramidal neurons is strongly involved in memory and learning[58–60]. Qiu et al. reported anesthesia/surgery-induced changes in structural synaptic plasticity withincreased neuroinflammation in aged mice by Golgi
staining [4], while we measured functional synaptic
plasticity by LTP recording as well. LTP, which isan increase in synaptic efficacy following highfrequency stimulation, is widely considered a molecular model for learning and memory [61–63].
LTP is also involved in the local remodeling of dendritic spines and synapses [28, 64]. We found thatboth structural and functional synaptic plasticity in
the CA1 region were impaired after anesthesia/surgery in aged mice, while SIRT3 overexpression reversed these synaptic changes, providing animportant clue regarding the effect of SIRT3 in
POCD in aged mice.
Considering the negative regulation of SIRT3 in hippocampal neuroinflammation, we speculated thatanesthesia/surgery induced a mitochondrial oxidative
stress response in a SIRT3-dependent manner, resulting
in microglia activation and hippocampal neuroinflammation and thus reducing synaptic plasticity in the CA1 region and postoperative cognition in aged mice.

Page 14 of 16

There are several limitations in our study. First, in thecurrent work, we only focused on cognitive function in
the early postoperative period; therefore, the effect onlong-term outcomes of brain cognitive function should
be investigated in further studies. Second, different kindsof anesthesia and surgical treatment procedures may
also influence SIRT3 expression, the inflammatory response, and cognitive performance in mice. However, inthe current project, we did not include a separate
anesthesia control group. We reasoned that because apatient who undergoes an operation will experience allthese procedures, all of these factors may serve to imitate the whole treatment plan in clinical management.
Other perioperative factors related to SIRT3 expression
and neuroinflammation in the hippocampus of aged
mice should be determined in further studies. Third, theresults of fear conditioning and open field tests in mice
do not directly correlate with cognitive function inhumans. Fourth, although there are three types of SOD
enzymes (CuZn-SOD [SOD1], Mn-SOD [SOD2], and
EC-SOD [SOD3]), we measured the total SOD enzymatic activity to study oxidative stress in the hippocampus
of aged mice under anesthesia/surgery. We found that
Giuseppa Mudo et al. also employed total SOD enzymatic activity measurements in a rat model of AD [65].
Whether the SOD enzyme activity dysfunction induced
by anesthesia/surgery is dependent on SOD enzyme typeremains to be further investigated. Finally, we could not
fully exclude other potential molecules that might be involved in SIRT3-mediated POCD in aged mice.

Conclusion
In conclusion, the current study indicated that SIRT3
may attenuate postoperative cognitive decline by preventing the anesthesia/surgery-induced neuroinflammatory response in the hippocampal CA1 region of aged
mice. SIRT3 may be a potential therapeutic and diagnostic target for the management of POCD in aged patients.
Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s12974-021-02089-z.
Additional file 1. Supplementary information.

Abbreviations
SIRT3: Sirtuin 3; POCD: Postoperative cognitive dysfunction; SOD: Superoxide
dismutase; MDA: Malondialdehyde; Iba1: Ionized calcium binding adapter
molecule 1; TNF-α: Tumor necrosis factor-alpha; IL-1β: Interleukin (IL)-1β; IL6: Interleukin (IL)-6; BBB: Blood–brain barrier; AAV: Adeno-associated virus;
OFT: Open field test; FCT: Fear conditioning test; BCA: Bicinchoninic acid;
ELISA: Enzyme-linked immune-sorbent assay; OD: Optical density;
fEPSPs: Field excitatory postsynaptic potentials; TBS: Theta burst stimulation;
PNDs: Perioperative neurocognitive disorders; PD: Parkinson’s disease;
AD: Alzheimer’s disease; LTP: Long-term potentiation

Liu et al. Journal of Neuroinflammation(2021) 18:41

Acknowledgements
Not applicable.
Authors’ contributions
QL, YMS, HH, and CC carried out the molecular tests. QL, JW, and LHM
carried out the behavior tests. QL and YYS performed the morphologic tests.
QL and HHM performed statistical analysis and drafted the manuscript. HHM
and YQW designed the study and supported the funding. All authors
reviewed and approved the final manuscript.
Funding
This work was supported by the National Natural Science Foundation ofChina (81701040), Beijing Talents Fund (2017000021469G258), awarded to
HHM, and by the Natural Science Foundation of Jiangsu Province(BK20191464), awarded to YQW.
Availability of data and materials
The data supporting the findings of this study are presented within the
manuscript.
Ethics approval and consent to participate
All animals were treated in accordance with the International Guidelines foranimal research. All experimental procedures involving animals wereapproved by the Animal Care and Use Committee of Xuzhou Medical
University.
Consent for publication
Not applicableCompeting interests
The authors declare that they have no competing interest.
Received: 14 August 2020 Accepted: 19 January 2021

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