Impact of Pumps on Internal Thoracic Arteries (IPITA)
- Conditions
- Coronary Disease
- Registration Number
- NCT04168853
- Lead Sponsor
- University Hospital, Angers
- Brief Summary
Cardiopulmonary bypass (CPB) induces a systemic inflammatory response and affects the organ vascular bed. Experimentally, the lack of pulsatility alters myogenic tone of resistance arteries and increases the parietal inflammatory response. The purpose of this study was to compare the vascular reactivity and the inflammatory response of the internal thoracic arteries (ITAs) between patients undergoing coronary artery bypass grafting (CABG) under CPB with a roller pump or with a centrifugal pump.
- Detailed Description
Eighty elective male patients undergoing CABG were selected using one or two internal thoracic arteries under CPB with a roller pump (RP group) or centrifugal pump (CFP group). ITA samples were collected before starting CPB (Time 1) and before the last coronary anastomosis during aortic cross clamping (Time 2). Terminal complement complex activation (SC5b-9) and neutrophil activation (elastase) analysis were performed on arterial blood at the same times.
Recruitment & Eligibility
- Status
- COMPLETED
- Sex
- Male
- Target Recruitment
- 80
- male patients and elective coronary artery bypass grafting using at least one of the two ITAs.
- female patients because their complement activation has been shown to be greater than that in men during surgery under cardiopulmonary bypass ; age < 18 years; CABG requiring additional valve repair or replacement; emergency surgery and insufficient length of the internal thoracic artery
Study & Design
- Study Type
- INTERVENTIONAL
- Study Design
- PARALLEL
- Primary Outcome Measures
Name Time Method Myography 1 day for each patient, 2 fresh segments of ITA (Time 1 and Time 2) stored in PSS were analyzed. On day+1, these segments were mounted on a wire-myograph (DMT, Aarhens, DK) . Two tungsten wires (25 μm diameter) were inserted into the lumen of the arteries and connected to a force transducer and a micrometer, respectively. The arteries were bathed in the PSS solution. Wall tension, equivalent to intra-arterial pressure (90 mmHg), was applied and the blood vessels were allowed to stabilize for thirty minutes. Arterial contractility was assessed with phenylephrine (PE, 10 μmol/L). Acetylcholine-induced (Ach 10 μmol/L) relaxation was then obtained after phenylephrine-induced preconstruction (50% of maximal contraction) in the presence or in the absence of the NO synthesis blocker L-NMMA (3.10-4 mol/L) and in the presence or in the absence of the COX synthesis blocker Indomethacin (10-5 mol/L).
- Secondary Outcome Measures
Name Time Method Quantitative real time transcription-polymerase chain reaction (RT-PCR) analysis 1 day Sections of ITAs were dried and stored at -80°C in RNA later Stabilization Reagent (Qiagen). RNA extraction was performed using the RNeasy® micro kit (Qiagen). 500 ng of RNA extracted from each artery were used to synthesize cDNA using the QuantiTect® Reverse Transcription kit (Qiagen). Quantitative real-time PCR was performed with Sybr® Green PCR Master Mix (Applied Biosystems) using a Light cycler 480 Real-Time PCR System (Roche).
Immunochemistry 1 day sections (10 μm thickness) of arteries were rehydrated by 500 μl of PBS during 10 minutes and fixated with 200 μl of paraformaldehyde (PFA) (pH = 7.4, room temperature) and then were rinsed by PBS. Permeabilization with 200 μl of PBS-BSA (Bovine Serum Albumin - Sigma) 10% - Tween 0.1% during 40 minutes and then saturation with PBS-BSA 10% during 40 minutes were performed. Sections were incubated overnight with 100 μl of anti-CD45 antibody at 1/500th dilution for leukocyte staining or anti-CD80 antibody at 1/200th dilution for lymphocyte staining. Antibodies were labelled with a red fluorochrome (Phycoerythrine). Karyoplasm was stained blue by DAPI solution. On day+1, sections were rinsed with PBS and analyzed with the confocal microscope. Fluorescent images were quantified with the ImageJ (NIH) software.
Superoxide detection and confocal microscopy 1 day : dihydroethidium staining (DHE, Sigma-Aldrich) was used to evaluate the in-situ levels of superoxide anions (O2-). DHE is permeable to cells and is oxidized by superoxide (O2-) to fluorescent products that are trapped by intercalation into the DNA. Sections (10 μm thickness) were incubated with DHE (1 μmol/L) in phosphate-buffered solution (PBS) and DAPI (4',6'-diamidino-2-phénulindole-Molecular probes, Invitrogen) for nuclear cells Fluorescent images of ethidium bromide were obtained using a confocal microscope (Nikon Eclipse TE2000S).
Blood sampling and biochemical analysis 2 hours Serial arterial blood samples for elastase and for SC5b-9, marker of terminal complement complex activation, were collected at Time 1 and Time 2. Specimens were centrifuged (10 minutes, 3,000 rpm, 4°C) immediately to obtain plasma which was stored at - 80°C before analysis.
Enzyme-linked immunosorbent assay techniques were used from 10 μl of plasma to measure terminal complement (SC5b-9; Quidel, San Diego, CA, USA) and 50 μl of plasma for neutrophil elastase (Neutrophil ELA2, Assay pro, St Charles, USA). The limit of sensitivity of each assay undertaken was as follows: SC5b-9 = 16 ng/mL and elastase = 20 ng/mL.