Transcriptome Profile in Hippocampus During Acute Inflammatory Response to Surgery: Toward Early Stage of PND

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: RNA-Seq; cognitive dysfunction; hippocampus; neuroinflammation; surgery.

Figure 1

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.

Figure 2

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.

Figure 3

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.

Figure 4

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.

Figure 5

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.

Figure 6

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.