DOI: 10.5061/dryad.jsxksn0cj
title: Whole-transcriptome sequencing identifies neuroinflammation, metabolism and blood-brain barrier related processes in the hippocampus of aged mice during perioperative period

description: 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.
description: <strong>Animals</strong>
 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).
 <strong>Surgery and
 Anesthesia</strong> 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.
 <strong>RNA-Seq Library
 Preparation and Sequencing Analysis</strong>
 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).