Abstract

   Perioperative neurocognitive disorders (PND) are common complications
   observed in surgical patients, but there are no effective treatments
   and the detailed mechanisms remain largely unknown. In this study,
   transcriptome analysis was performed to investigate the hippocampal
   changes after surgery and underlying molecular mechanisms of PND.
   Tibial fracture surgery was performed in 3–4 months old C57BL/6J mice
   to mimic human orthopedic surgery. We demonstrated that memory
   consolidation of the hippocampal-dependent trace-fear conditioning task
   was significantly impaired. By using ELISA, a significant elevated IL-6
   was observed both in circulating system and central nervous system and
   peaked at 6 h post-surgery, but transiently returned to baseline
   thereafter. Hippocampus were collected at 6 h post-surgery then
   processed for RNA-Seq. A total of 268 genes were screened
   differentially expressed between the Surgery and Control group,
   including 170 up-regulated genes and 98 down-regulated genes. By
   functional enrichment analysis of differently expressed genes, several
   KEGG pathways involved in inflammatory mediator regulation of TRP
   channels, neuroactive ligand-receptor interaction and cholinergic
   synapse were overrepresented. Quantitative real-time PCR confirmed 15
   dysregulated genes of interest. These results provide a comprehensive
   insight into global gene expression changes during the acute presence
   of hippocampal inflammation and a better understanding on early stage
   of PND.

   Keywords: cognitive dysfunction, surgery, hippocampus,
   neuroinflammation, RNA-Seq

Introduction

   Cognitive impairments are frequent complications among surgical
   patients. These impairments are now termed as Perioperative
   neurocognitive disorders (PND), including acute postoperative delirium
   and long-lasting postoperative cognitive dysfunction ([33]1). PND are
   becoming increasing concerns as they are associated with various
   adverse outcomes, including prolonged hospitalization, increased
   complications and mortality, and decreased quality of life ([34]2,
   [35]3).

   Surgery trauma itself provokes release of damage-associated molecular
   patterns (DAMPs), leading to innate immune activation and increasing
   proinflammatory cytokines, both in peripheral and central. Appearance
   of inflammatory cytokines in cerebral spinal fluid (CSF) has been
   repeatedly observed in surgical patients ([36]4–[37]8). Additionally,
   in a remarkable clinical study, a profound immune activity change in
   the human brain after surgery was found by positron emission tomography
   (PET) and associated with their performance on mental test ([38]9).
   Growing preclinical data from rodent models suggest neuroinflammation,
   which featured as proinflammatory cytokines accumulation and glial
   activation in hippocampus, plays a key role in the pathogenesis and
   progression of PND. Increased proinflammatory cytokines serve as
   immune-to-brain signaling which are capable of disrupting synaptic
   plasticity ([39]10), a neurochemical foundation of learning and memory
   ([40]11). Preserving cognitive function can be achieved by attenuating
   the inflammatory signaling pathways ([41]12, [42]13). Accumulating
   evidences suggest that interleukin-6 (IL-6), one of the main elevated
   proinflammatory cytokines in PND, plays a critical role in cognitive
   decline after surgery ([43]14–[44]16).

   Hippocampus is widely recognized as playing important roles in learning
   and memory which is also vulnerable to neuroinflammation after surgery.
   Several studies have reported deficits in trace-fear conditioning (TFC)
   test after structure lesions that input into hippocampus or blocking
   receptor functions in hippocampus by pharmacological agents
   ([45]17–[46]20), indicating that it is hippocampal-dependent. Moreover,
   TFC test correlates well with performance in a Morris water maze
   ([47]21), a well-validated assay of learning and memory often used in
   clinical models of cognitive dysfunction. Therefore, TFC test assessed
   in rodents within days following surgery has been widely used in PND
   studies ([48]14, [49]22).

   Most previous studies focused on the long-term effects of PND, little
   is known about early events of PND. In fact, administration of LPS
   causes synaptic transmission change as early as 2 h, which is mediated
   by rapid response of pericytes ([50]23). Gene expression profiling in
   brain tissue during acute stage could provide a more comprehensive
   insight into the pathophysiology of PND.

   In this study, we determined perioperative dynamic levels of IL-6 and
   applied high-throughput RNA-Seq technology to identify dysregulated
   genes during the acute presence of neuroinflammation. We also employed
   functional gene set enrichment to identify classes of differentially
   regulated genes which may associate with PND. Finally, we used
   quantitative real-time PCR (qRT-PCR) to confirm expression of 15
   dysregulated genes of interest. These findings may further our
   understanding of biological mechanisms of PND, especially during the
   early stage of the disease.

Materials and Methods

Animals

   Three to four months old male C57BL/6J mice were purchased from
   Shanghai SLAC laboratory animal Co., Ltd. (Shanghai, China). All animal
   experiments were carried out with the approval of the Animal Care
   Committee at Zhejiang University. Mice were housed in polypropylene
   cages and maintained at 25°C under reverse phase 12 h light-dark cycle
   with ad libitum access to water and rodent chow. Animals were tagged
   and randomly allocated to each group before any procedure (For RNA-seq
   and qRT-PCR, 4 mice were used in control and surgery groups; for ELISA,
   5–8 mice were used in control and surgery groups).

Experiment Paradigm

   Since almost undetectable IL-6 changes were observed by ELISA at 3 days
   post-surgery, at the time when TFC test was performed, mice were
   divided into 2 separate cohorts (presented in [51]Figure 1).

Figure 1.

   Figure 1
   [52]Open in a new tab

   Experiment Paradigm. Mice were divided into 2 cohorts. (A) One cohort
   for behavior experiment includes the training session of the memory
   test performed 30 min before tibial fracture and TFC test session; (B)
   the other cohort for neuroinflammation assessment complete tibia
   surgery without TFC training and test session and collect serum/ tissue
   at time point of 6/24/72 h.

Tibial Fracture Surgery

   Surgery was consisted of an aseptic open tibial fracture with
   intramedullary fixation performed under general anesthesia, including
   5% sevoflurane (Sevorane™, Abbott, Switzerland) in 500 ml/min O[2]
   using a rodent inhalation anesthesia apparatus (MIDTRX VIP2000,
   Midmark, Dayton, OH, USA) and Tramadol (Merck & Co., Inc., Kenilworth,
   NJ, USA) as previously described with minor modifications ([53]22,
   [54]24). Briefly, the left hind paw was disinfected. A median paw
   incision was then performed, followed by the insertion of a 28 G needle
   (~0.38 mm) in the tibial intramedullary canal. Then osteotomy was
   performed at the junction of the middle and distal thirds of the tibia.
   The fixation needle remained in situ and be cut flush with the tibial
   cortex. After producing the fracture, skin was closed with 5–0 Vicryl
   sutures (Monocryl; Ethicon Inc, Somerville, NJ); thereafter, animals
   were allowed to recover spontaneously from the anesthetic. Analgesia
   (s.c., Tramadol, 30 mg/kg) was administered after anesthetic induction
   and before skin incision. Control (sham-treated) mice received
   anesthesia and analgesia.

Trace-Fear Conditioning Test

   TFC test was used to assess learning and memory as previously published
   ([55]22). Freezing behavior is an indicator of aversive memory which is
   evoked by contextual cues related to a fear-inducing stimulus-response
   pairing learned previously. The extent of this learning (contextual
   testing) was recorded in a TFC chamber (Shanghai XinRuan Information
   Technology Co., Ltd, Shanghai, China). Memory impairment is indicated
   by a decrease in freezing time.

Training

   On the training day, mice were placed in the TFC chamber and allowed to
   explore it for 100 s. Mice were then exposed to an auditory cue (75–80
   Db, 5 kHz, conditional stimulus) for 20 s, followed 20 s later by a 2-s
   foot shock (0.8 mAmp; unconditional stimulus). The tone and foot-shock
   pairing was repeated with an inter-trial interval of 100 s. After
   training is completed, allow the mice to remain in the testing chamber
   for 1 min before removing.

Contextual Testing

   Three days after the training session, the mice were placed back into
   the same TFC chamber but without any tone or shock for 5 min. Video
   tracking system recorded the time spent freezing as a fraction of total
   time in the chamber.

Cytokine Measurement

   Blood was transcardially collected, then perfused with saline and
   hippocampus were then rapidly extracted, stored at −80°C until assayed.
   Blood samples were centrifuged at 3,000 rcf per minute for 15 min and
   serum were collected and stored at −20°C until assayed. ELISA assays
   (MultiSciences, Hangzhou, China) were performed to measure IL-6 levels
   in the serum and hippocampus, following manufacturer's instructions.

RNA-Seq Analysis

   Total RNA was extracted from hippocampus of mice in the Control and
   Surgery groups (four mice in each group) using TRIzol Reagent
   (Invitrogen) and treated with DNase I at 37°C for 20 min to digest DNA.
   Then, the mRNA was purified using poly-T oligo-attached magnetic beads
   and followed by fragmented into small pieces using divalent cations
   under elevated temperature. The cleaved RNA fragments were copied into
   first strand cDNA using reverse transcriptase and random primers. Then
   second strand cDNA synthesis was synthesized by using DNA polymerase I
   and retained mRNAs were removed by RNase H. These cDNA fragments then
   have the addition of a single “A” base and subsequent ligation of the
   adapter. Sequencing was performed on the BGISEQ-500RS developed by BGI
   Co., Ltd. (China). The raw RNA-Seq data were filtered into clean reads,
   followed by mapping to the mouse reference genome (mm10) using HISAT.
   The gene quantification was analyzed using RSEM quantification tool,
   and the expression level of each gene was calculated in fragments per
   kilobase of exon per million fragments mapped. The NOISeq method was
   used to screen the differentially expressed genes (DEGs) between the
   control and surgery groups according to the criteria of fold change ≥ 2
   and diverge probability ≥ 0.8. The gene ontology (GO) annotation mapped
   all DEGs to GO terms in the database
   ([56]http://www.geneontology.org/). The public KEGG database was used
   to perform the pathway enrichment analysis ([57]25).

qRT-PCR

   qRT-PCR was performed on a CFX 96 real-time PCR thermocycler (Bio-Rad
   Laboratories, Hercules, CA, USA) using same RNA samples used for
   RNA-Seq. Primer sequences were listed in supplemented file [58]Table
   S1. For amplification, SYBR Premix Ex Taq II (Takara Bio Inc., Otsu,
   Japan) was used as instructed in the manufacturer's manual. Relative
   changes of mRNA expression were analyzed with the 2^−ΔΔCt method and
   GAPDH was serving as an internal reference. These standardized data
   were used to calculate fold changes in gene expression. All real-time
   PCR amplifications were performed in triplicate.

Statistics

   qRT-PCR and ELISA data were expressed as means ± standard error of the
   mean (SEM). Graphical and statistical analyses were performed using the
   Prism 6.0 (GraphPad Software Inc., CA, USA). Comparisons between two
   groups were performed using two-tailed Student's t-test, assuming equal
   variance. P < 0.05 was considered significant.

Results

Transient Period of IL-6 Elevation After Surgery

   To investigate the mechanisms of PND, an aseptic tibial fracture with
   internal fixation surgery model was established and memory of
   association learned preoperatively was assessed in a trace-fear
   conditioning (TFC) paradigm ([59]Figure 1).

   Compared with anesthesia-matched sham procedure, Surgery group showed a
   significant reduction of freezing time at 72 h ([60]Figure 2A),
   indicating cognitive decline was induced by surgery. Meanwhile, IL-6
   was increased promptly post-surgery both in serum and hippocampus and
   reached a plateau at 6 h, then falls thereafter ([61]Figure 2B). These
   findings consistent with previous studies ([62]22, [63]26) and
   demonstrate a mild and transient period of hippocampal inflammation
   after surgery.

Figure 2.

   [64]Figure 2
   [65]Open in a new tab

   Surgery induces cognitive decline and inflammation. (A) Freezing in
   response to contextual cues, showed as a percentage of total time in
   the testing environment prior to surgery. (B) Serum ELISA, showing the
   significant elevated IL-6 level after surgery in serum. (C) Tissue
   ELISA, showing the postoperative elevated IL-6 level in hippocampus (P
   < 0.01 at 6 h). Data represent mean ± SEM; n = 5–8/group. NS, no
   significant; ^***P < 0.001; ^**P < 0.01 for Surgery vs. Control.

Transcriptome Profile in Hippocampus

   To gain a more comprehensive insight into the gene expression changes
   of hippocampus during the early presence of neuroinflammation, RNA-Seq
   and DEGs screening analyses were performed on 8 samples (four mice in
   Control and Surgery group, respectively).

   The sequencing generated, on average 21.69 M clean reads and mean clean
   reads ratio of 98.87%. Of 2,05,782 genes in the mice reference genome,
   18,771 mRNAs were identified; of those, 268 genes were differentially
   expressed following surgery ([66]Table S2). The majority (170) were
   increased (Fold Change ≥ 2, q-Value ≤ 0.001), while 98 were decreased
   (Fold Change ≤ 2, q-Value ≤ 0.001) ([67]Figure 3).

Figure 3.

   [68]Figure 3
   [69]Open in a new tab

   Scatter plots of all expressed genes in each pairwise. Blue represents
   downregulated genes, orange represents upregulated genes and brown
   represents non-regulated gene. Fold change ≥2, q-value ≤ 0.001 used as
   screening threshold.

Gene Ontology (GO) and Enriched KEGG Pathway Analysis

   In GO analysis, DEGs were classified into biological process (BP),
   cellular component (CC), and molecular function (MF). Among BP classes,
   cellular process (179 genes) contained the largest group of genes, and
   genes involved in biological regulation constituted the second largest
   group with 143 genes. The third largest group included 128 genes
   involved with regulation of biological process. Additionally, 117 genes
   were identified that were involved in multicellular organismal process,
   112 genes were also identified involved in response to stimulus
   ([70]Figure 4).

Figure 4.

   [71]Figure 4
   [72]Open in a new tab

   GO functional classification on DEGs for each pairwise. X-axis
   indicates the number of DEGs (the number is presented by its square
   root value). Y-axis represents GO terms. All GO terms are grouped into
   three ontologies: blue is for biological process, green is for cellular
   component and red is for molecular function.

   In the CC category, the genes expressed were mostly enriched for cell
   (195 genes), cell part (195 genes), and organelle (159 genes).
   Additional DEGs were related to membrane (127 genes), membrane part
   (107 genes), extracellular region (90 genes), extracellular region part
   (80 genes), organelle part (69 genes), and macromolecular complex (52
   genes) ([73]Figure 4).

   Finally, in MF classes, binding (150 genes) and catalytic activity (54
   genes) were top two enriched terms. Additional DEGs were involved in
   molecular transducer activity (29 genes), transcription regulator
   activity (29 genes), transporter activity (29 genes), and signal
   transducer activity (28 genes), suggesting an active and vigorous
   response provoked in hippocampus ([74]Figure 4).

   Next, we performed KEGG pathway enrichment analysis to further
   understand gene biological functions of DEGs. Surprisingly, several
   neurogenic disease-associated pathways were identified to be in the top
   20 enriched pathway terms, including neuroactive ligand-receptor
   interaction, cholinergic synapse, Parkinson's disease, inflammatory
   mediator regulation of TRP channels and cocaine addiction ([75]Figure
   5). Moreover, some other pathways involved in cell communication or
   metabolism were also displayed ([76]Figure 5), such as cell adhesion
   molecules (CAMs), tyrosine metabolism, ECM-receptor, tryptophan
   metabolism, pentose and glucuronate interconversions, and porphyrin and
   chlorophyll metabolism.

Figure 5.

   [77]Figure 5
   [78]Open in a new tab

   Scatter plot of the top 20 KEGG enrichment results of DEGs in each
   pairwise comparison. Rich Factor is the ratio of DEG numbers annotated
   in this pathway term to all gene numbers annotated in this pathway
   term. A large Rich Factor represents greater intensiveness. The Q-value
   ranges from 0 to 1, and a lower Q-value represents greater intensity.

Validation of RNA-Seq Results by qRT-PCR

   To verify the results of RNA-Seq analyses, 15 DEGs of interest were
   selected for qRT-PCR ([79]Table 1). Gene expressions measured by
   qRT-PCR exhibited similar changes to the gene expression analyses
   ([80]Figure 6), with 10 genes (Lcn2, Cdkn1a, Serpina3f, Trim29, Ch25ch,
   Trpa1, Lao1, Ly6g6e, Bcl3, Rtp1) up-regulated and 5 genes (Six3, Gkn3,
   Ccdc153, Inmt, Opalin) down-regulated. Among these genes, Ch25h, Trpa1,
   Cdkn1a, Ly6g6e, Six3, and Opalin are potentially associated with
   neurological diseases; Lcn2, Serpina3f, Lao1, Trim29, bcl3, and Gkn3
   are associated with inflammatory response. Other genes of interest for
   the qRT-PCR analysis included Rtp1, Ccdc153 and Inmt, which are
   involved in metabolism and signal transduction and showed large up- or
   down-regulation by both RNA-Seq and qRT-PCR.

Table 1.

   Functional properties of genes selected for qRT-PCR analysis.
   Gene ID Symbol log2Ratio Up/Down-regulation Pathway and related
   function
   16819 Lcn2 3.29 Up Immune system; IL-17 signaling pathway
   238393 Serpina3f 2.63 Up Serine protease inhibitor
   12642 Ch25h 1.74 Up Lipid metabolism; risk factor of AD; contributes to
   cerebral Inflammation.
   277328 Trpa1 1.71 UP Inflammatory mediator regulation of TRP channels;
   contributes to AD developments.
   100470 Lao1 1.62 Up Amino acid metabolism.
   72169 Trim29 1.40 Up Negatively regulates innate immunity.
   239766 Rtp1 1.33 Up Receptor transporter.
   12575 Cdkn1a 1.22 Up Signaling transduction;
   cell growth and death.
   12051 bcl3 1.17 Up Signal transduction;
   TNF signaling pathway.
   70274 Ly6g6e 1.05 Up nAChR signaling.
   20473 Six3 −1.94 Down Wnt signaling; brain development.
   68888 Gkn3 −1.43 Down Immune system;
   NOD-like receptor signaling pathway.
   270150 Ccdc153 −1.29 Down /
   21743 Inmt −1.25 Down Amino acid metabolism.
   226115 Opalin −1.00 Down oligodendrocytic myelin protein;
   regulation of the oligodendroglial actin cytoskeleton.
   [81]Open in a new tab

Figure 6.

   [82]Figure 6
   [83]Open in a new tab

   qRT-PCR validation of selected DEGs identified by RNA-seq analyses.(A)
   Relative expression of up-regulated genes. (B) Relative expression of
   down-regulated genes. After normalized to GAPDH, fold changes were
   calculated by comparing the normalized copy number in each sample to
   the mean of Control group. Data represent mean ± SEM; n = 4/group. ^*P
   < 0.05; ^**P < 0.01; ^***p < 0.001; ^****p < 0.0001.

Discussion

   In clinical practice, 10–26% of patients retain subtle but persistent
   learning and cognitive deficits, referred as PND ([84]1, [85]27),
   resulting in higher risk for further morbidity and permanent dementia
   ([86]28). In this study, we determined dynamic levels of IL-6 in serum
   and hippocampus, then isolated hippocampus for RNA-Seq to investigate
   the differentially expressed gene signature when IL-6 reached at
   highest level post-surgery. A total of 268 genes were screened
   differentially expressed, including 170 up-regulated genes and 98
   down-regulated genes (Surgery vs. Control group). Gene ontology (GO)
   and KEGG pathway enrichment analyses were performed to explore the
   principal functions of dysregulated genes. It is the first report to
   comprehensively analyze gene expression profile during acute phase of
   hippocampal inflammation in PND, and may lead to further investigations
   on the mechanisms and resolutions of PND.

   IL-6 is a late-phase proinflammatory cytokine, and its circulating
   level negatively correlates with cognitive performance in humans
   ([87]29, [88]30). It consistently elevates in both the circulating and
   CNS following surgical trauma in rodents ([89]14) and surgical patients
   ([90]8, [91]31). Administering exogenous IL-6 inhibits
   mitogen-activated protein kinase ERK and Long-term potentiation (LTP)
   in a hippocampal slice ([92]32–[93]34), ultimately impairs cognitive
   performance ([94]14). Surgery-induced decline in cognitive function
   could be attenuated by blocking IL-6 signals or rescued in IL-6
   deficient mice ([95]14–[96]16). These findings demonstrated that
   dysregulated IL-6 levels play a key role in hippocampal inflammatory
   responses that negatively affect cognitive processing.

   Hippocampus plays important roles in consolidation of information from
   short-term memory to long-term memory, and it is particularly
   susceptible to the effects of inflammation ([97]35, [98]36). In fact,
   hippocampus is one of the few areas of the CNS outside the hypothalamus
   that expresses high levels of IL-1β, IL-6, and TNF-α receptors ([99]37,
   [100]38). Consistent with this enhanced sensitivity, peripheral surgery
   has been shown to alter neuroglial metabolic coupling ([101]39) and
   neurotransmission in hippocampus ([102]26, [103]39).

   In the present study, we employed RNA-Seq technology to analyze gene
   expression across the entire mice genome to find differentially
   expressed genes during early stage of PND. A number of differential
   expressed genes were identified by DEGs analysis. These genes are
   involved in various functions, including cellular process, biological
   regulation and regulation of biological process. Though lots of them
   contribute to neural functions in various neurodegenerative disease
   models, most of their dysregulations are revealed for the first time in
   PND. For example, Lcn2, one of the most up-regulated genes, given its
   features as an iron regulator and inflammatory protein in the CNS, has
   emerged as a new player in neurodegenerative diseases ([104]40).
   Investigations into these genes' expression changes and functions would
   greatly improve our understanding for pathophysiology of PND.

   In addition, KEGG pathway analysis of DEGs showed potential underlying
   mechanisms of PND, including neuroactive ligand-receptor interaction,
   cell adhesion molecules (CAMs), cholinergic synapse, inflammatory
   mediator regulation of TRP channels, and ECM-receptor interaction were
   severely affected. Some of them are critical for neuronal transmission,
   and dysfunction of these pathways may ultimately lead to disrupted
   neuronal plasticity and memory deficits. Specially, TRP channels can be
   activated by thermal and chemical stimuli and play a central role in
   pain sensation in peripheral nervous system ([105]41), but it has also
   been proved that some of TRP channels may induce Long-term depression
   (LTD) in hippocampus ([106]42), the opposite of LTP. Moreover,
   neuroactive ligand-receptor interaction which mediates the transduction
   of signals from the extracellular environment into cells may be
   involved in modulation of synaptic plasticity. Besides, cholinergic
   neurotransmission acts as a signal-to-noise ratio modulator of sensory
   and cognitive inputs, thus plays an important role in regulating the
   cognitive functions of the brain ([107]43, [108]44). Dysfunction of
   cholinergic neurotransmission has been implicated in neurological
   disorders such as Alzheimer's disease, and attempts have been made to
   use cognitive enhancer such as acetylcholinesterase inhibitors to
   combat dementia ([109]43–[110]45). Additionally, several pathways
   associated with metabolism such as tyrosine metabolism and tryptophan
   metabolism; several other pathways involved in neurogenic diseases,
   such as cocaine addiction and Parkinson's disease, were also affected.
   These results suggest that brain functions are greatly disturbed during
   surgery induced hippocampal inflammation and provide a basis to
   understand the underlying mechanisms of PND.

   Our study also has some limitations. First, in addition to IL-6, other
   cytokines such as TNF-α, MCP-1, and IL-1β, as well as influx of
   inflammatory cells into the brain parenchyma and glial activation, all
   contribute to cognitive deterioration after surgery, thus it would be
   important to fully assay all these changes. Second, we did not test
   permeability of blood brain barrier (BBB) at early stage of PND. BBB
   protects the neural microenvironment with low permeability by tight
   junction and low rates of transcytosis. Previous study showed increased
   BBB permeability and expression changes of tight junction molecules
   after surgery ([111]15), yet transcellular path has not been stressed.
   In fact, accumulating evidences suggested impairment of BBB was a
   stepwise process. In stroke, rates of transcytosis were increased as
   early as 6 h, while profound structural defects of tight junction
   displayed after 2 days ([112]46, [113]47). The future studies should
   assess the kinetics changes of BBB by using tracer molecule and
   TJs-labeled transgenic mice in PND. Besides, RNA-Seq technology also
   has some disadvantages, including an inherent lack of spatial
   information. Applying in situ hybridization (ISH) would allow us to
   further obtain temporal and spatial information about gene expression
   ([114]48).

   In conclusion, we employed RNA-Seq analyses as an unbiased approach to
   screen differentially expressed genes in hippocampus tissues from
   Surgery and Control mice 6 h post-surgery. Two hundred and sixty eight
   genes were screened differentially expressed, including 170
   up-regulated genes and 98 down-regulated genes. Additionally, 15
   dysregulated genes of interest were validated by qRT-PCR. While clear
   associations between these genes and cognitive behavior phenotype in
   PND are not yet elucidated, still, in respect of the dysregulated
   genes, may lead to important advances in our understanding of how early
   immune-related events can influence cognition and memory processes.

Ethics Statement

   This study was carried out in accordance with the National Institutes
   of Health guidelines and regulations. The protocol was approved by the
   Animal Care Committee at Zhejiang University.

Author Contributions

   XX, YY, XT, MC, YZ, and SZ conceived and designed the experiments. XX,
   YY, and XT performed the experiments. XX, YY, XT, MC, YZ, and SZ
   analyzed the data. XX and SZ wrote the paper. All the authors have read
   and approved of the final manuscript.

Conflict of Interest Statement

   The authors declare that the research was conducted in the absence of
   any commercial or financial relationships that could be construed as a
   potential conflict of interest.

Footnotes

   Funding. This work is supported by grants from the National Natural
   Science Foundation of China 81771194, 81471171, and 81271274 (to SZ).

Supplementary Material

   The Supplementary Material for this article can be found online at:
   [115]https://www.frontiersin.org/articles/10.3389/fimmu.2019.00149/full
   #supplementary-material
   Table S1

   Primers used in this study for qRT-PCR.
   [116]Click here for additional data file.^ (18.2KB, DOCX)
   Table S2

   DEGs screened between Surgery and Control groups.
   [117]Click here for additional data file.^ (31.5KB, XLSX)

References