Aim: Perioperative neurocognitive disorders (PND) occur frequently after surgery and anesthesia, especially in aged patients. Previous studies have shown multiple PND related mechanisms in the hippocampus, however, their relationships remain unclear. Meanwhile, the perioperative neuropathological processes are sophisticated and changeable, single period study could not reveal the accurate mechanisms. Thus, multiperiod whole-transcriptome study is necessary to elucidate the gene expression patterns during perioperative period.

Methods: Aged C57BL/6 mice were subjected to exploratory laparotomy under sevoflurane anesthesia. Whole-transcriptome sequencing (RNA-seq analysis) was performed on the hippocampi from control condition (Con), 30 minutes (Day0), 2 days (Day2) and 7 days (Day7) after surgery. Gene Ontology/Kyoto Encyclopedia of Genes and Genomes analyses, quantitative Real-Time PCR, immunofluorescence and fear conditioning test were also performed to elucidate the pathological processes and modulation networks during the period.

Results: Through RNA-seq analysis, 328, 3597 and 4179 differentially expressed genes (DEGs) were screened out in intraoperative period (Day0 vs Con), early postoperative period (Day2 vs Day0) and late postoperative period (Day7 vs Day2). The involved GO biological processes were divided into 9 categories, and positive-regulated processes were more than negative-regulated ones. Seventy-four transcription factors were highlighted. The potential synaptic and neuroinflammatory pathways were constructed for Neurotransmitter, Synapse and Neuronal alteration categories with 9 DEGs (Htr1a, Rims1, Ezh2, etc.). The metabolic and mitochondrial pathways were constructed for Metabolism, Oxidative stress and Biological rhythm categories with 9 DEGs (Gpld1, Sirt1, Cry2, etc.). The blood-brain barrier and neurotoxicity related pathways were constructed for Blood-brain barrier, Neurotoxicity and Cognitive function categories with 10 DEGs (Mmp2, Itpr1, Nrf1, etc.).

Conclusion: The results revealed gene expression patterns and modulation networks in the aged hippocampus during perioperative period, which provide insights into overall mechanisms and potential therapeutic targets for prevention and treatment of perioperative central nervous system diseases, such as PND, from the genetic level.

Animals

Female C57BL/6 mice, 18-month-old, weighing between 23 and 34 g were used. The mice were housed in cages and maintained on a standard housing condition with food and water ad libitum for 2 weeks. Four study time points were chosen: control condition (Con, preoperative time point), 30 minutes after surgery (Day0, postoperative day 0, the time point between intraoperative period and postoperative period), postoperative day 2 (Day2) and postoperative day 7 (Day7). The perioperative period was divided into intraoperative period (between Con and Day0), early postoperative period (between Day0 and Day2), and late postoperative period (between Day2 and Day7). Mice was randomly assigned to Con, Day0, Day2 and Day7 groups (n=6).

Surgery and Anesthesia

Minimum alveolar concentration of sevoflurane for mice has been reported as 2.4 - 2.7%. In the present study, mice in Day0, Day2 and Day7 groups received 2.5% sevoflurane in 50% oxygen for 30 min through breathing masks, and the control group received 50% oxygen for 30 min. The mice breathed spontaneously, and the sevoflurane concentration was monitored continuously with an anesthetic monitor (Datex, Tewksbury, MA, USA). The surgical procedure (exploratory laparotomy) was performed for the 3 groups. A longitudinal midline incision was made from xiphoid to 0.5 cm proximal pubic symphysis on the skin. The abdominal muscles and peritoneum, then approximately 10 cm of the intestine were exteriorized. The bowel loops remained outside the abdominal cavity for 1 minute and then replaced into the abdominal cavity. The incision was finally sutured layer by layer with 5-0 Vicryl thread. The entire procedure was completed under sevoflurane anesthesia. The rectal temperature was maintained at 37 ± 0.5 °C, and this surgical protocol has been shown not to significantly alter values of blood pressure and blood gas in the preliminary studies. Then the mice were put into a chamber containing 50% oxygen until 10 minutes after the recovery of consciousness. Mice in Day0, Day2 and Day7 groups were sacrificed by decapitation 30 min, 2 days and 7 days after surgery respectively. The brain tissue was removed rapidly, and the hippocampus was dissected out and frozen in liquid nitrogen.

RNA-Seq Library Preparation and Sequencing Analysis

Total RNAs were isolated from the hippocampus using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), then digested with RNase-Free DNase to remove residual DNAs. The Quantity and purity were detected with Nanodrop 2000 (ThermoFisher, Wilmington, DE, USA) and Qubit Fluorometer (Invitrogen, Carlsbad, CA, USA). Library construction was performed according to the Illumina sample preparation for RNA-seq protocol. The mRNA was enriched by magnetic beads with Oligo (dT) after the samples were qualified. When the enrichment was complete, the mRNA was interrupted into short segments with the addition of a fragmentation buffer. Subsequently, double-stranded cDNA was synthesized by reverse transcription using 6-base random primers. The purified double-stranded cDNA was subjected to terminal reparation, singe nucleotide A (Adenine) addition and serial sequencing. The fragment size of double-stranded cDNA was selected by an AMpure XP bead (Beckman coulter, Shanghai, China), and the selected double-stranded cDNA was subjected to PCR enrichment to construct a cDNA library. Constructing and sequencing the RNA-seq library for each sample was conducted (Compass Biotechnology, Beijing, China) based on the protocols of Illumina HiSeqTM2500/MiSeq™ to generate paired-end reads (150 bp in length). The quality of RNA-seq reads from all the brain tissues was checked using FastQC (v0.11.5, Babraham institute, Cambridge, UK).