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Gene expression analysis of vastus medialis cells after tourniquet-induced ischemia during Total Knee Arthroplasty: A randomized clinical trial

ABSTRACT: Gene expression analysis of vastus medialis cells after tourniquet-induced ischemia during Total Knee Arthroplasty: A randomized clinical trial

PROVIDER: PRJNA512618 | ENA |

REPOSITORIES: ENA

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Gene expression analysis of vastus medialis cells after tourniquet-induced ischemia during Total Knee Arthroplasty: A randomized clinical trial

Project description:Purpose The effects of the commonly utilized intraoperative tourniquet on gene expression within the skeletal muscle cells are barely examined. Our group already showed that tourniquet application results in higher proteolytic activity within vastus medialis cells (KSSTA 2016), without influence on the amount and function of mitochondria (EJOT 2016). The purpose of the present study was to examine the gene expression within the skeletal muscle cells after tourniquet-induced ischemia and to identify differential expressed genes (DEGs). Methods As part of a randomized, controlled, monocentric trial (Clinical-Trials.gov NCT02475603) we included 20 patients scheduled to undergo primary total knee arthroplasty (TKA). The patients received a written consent and were randomly assigned to Group A (TKA with tourniquet) (n=10) and Group B (TKA without tourniquet) (n=10). Muscle biopsies of (5×5×5 mm) were obtained from the vastus medialis immediately after performing the surgical approach and exactly 60 min later. After preparation of a muscle homogenate, RNA extraction was performed (RNeasy Plus Universal Mini Kit Qiagen) and RNA integrity (RIN) was determined (Agilent 2100 Bioanalyzer,RNA 6000 Pico Kit). Gene expression profiling was performed methodological validated (GeneChip™ Human Transcriptome Array 2.0;Affymetrix). Statistical analysis include the number of significant DEGs with p-value < 0,05, number of DEGs with relative difference >25% and pathway analysis (SPSS-Version 24; SAS JMP10 Genomics, Version 6). The power analysis considered an effect size of 0,4 and a statistical power > 0,8, sample size n=10 for each group. The protocol was approved by our Institutional Ethics Committee (File reference 2012-334N-MA). Results and conclusion Numerous genes were differencially expressed during tourniquet induced ischemia. Further investigations and statistical analysis are ongoning.

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Project description:Ischaemic preconditioning is a method of protecting tissue against ischaemia-reperfusion injury. It is an innate protective mechanism that increases a tissue's tolerance to prolonged ischaemia when it is first subjected to short burst of ischaemia and reperfusion. It is thought to provide this protection by increasing the tissue's tolerance to ischaemia, therby reducing oxidative stress, inflammation and apoptosis in the preconditioned tissue. We used microarrays to investigate the genomic response induced by ischaemic preconditioning in muscle biopsies taken from the operative leg of total knee arthroplasty patients in order to gain insight into the ischaemic preconditioning mechanism. Patients undergoing primary knee arthroplasty were randomised to control and treatment (ischaemic preconditioning) groups. Patients in the treatment group received a preconditioning stimulus immediately prior to surgery. The ischaemic preconditioning stimulus consisted of three five-minute periods of tourniquet insufflation on the lower operative limb, interrupted by five minute periods of reperfusion. All patients had a tourniquet applied to the lower limb after the administration of spinal anaesthesia, as per normal protocol for knee arthroplasty surgery. Muscle biopsies were taken from the quadriceps muscle of the operative knee at the immediate onset of surgery (T0) and at 1 hour into surgery (T1). Total RNA was extracted from biospies of four control and four treatment patients and hybridised to the Affymetrix Human U133 2.0 chip.

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