Multi-photon microscopic evaluation of saphenous vein endothelium and its preservation with a new solution, GALA

doi:10.1016/S0003-4975(02)04705-7

The Annals of Thoracic Surgery

Volume 75, Issue 4, April 2003, Pages 1145-1152

https://doi.org/10.1016/S0003-4975(02)04705-7 Get rights and content

Abstract

Background

Injury to endothelium can compromise the patency of bypass grafts harvested during coronary artery bypass graft (CABG) surgery. Maintaining structural and functional viability of endothelium in grafts may lead to improved long-term patency. The information gained from the application of multi-photon microscopy in transmission and epifluorescence mode was used to assess the structural and functional integrity of human saphenous vein segments stored in multiple preservation solutions, and to design a superior storage solution.

Methods

Multi-photon microscopy was used to image deep within saphenous vein tissue harvested from patients undergoing CABG for analysis of endothelial structure and function. Endothelial cell structural viability, calcium mobilization, and nitric oxide generation were determined using specific fluorescence markers.

Results

Within 60 minutes of harvest and storage in standard preservation solutions, calcium mobilization and nitric oxide generation were markedly diminished with more than 90% of endothelial cells no longer viable in the vein. In contrast, veins could be stored for 24 hours without substantial loss in cell viability in a newly formulated heparinized physiologic buffered salt solution containing glutathione, ascorbic acid, and L-arginine (GALA).

Conclusions

Standard solutions in clinical use today led to a profound decline in saphenous vein endothelial cell viability, whereas the newly designed physiologic salt solution (GALA) maintained endothelial function and structural viability for up to 24 hours. The improvements seen from using GALA as a vessel storage medium may lead to greater long-term vein graft patency following CABG surgery.

Section snippets

Saphenous veins

Saphenous vein segments (inner diameter 0.05 to 0.2 mm, outer diameter 0.2 to 1.0 mm) were obtained before distension or any other manipulation from male patients of age 67.13 ± 9.78 (mean ± SD) years undergoing CABG surgery at the VA Boston Healthcare System, according to an approved Human Studies Subcommittee protocol. The vein segments were placed in the preservation solutions described below at 21°C and processed within 30 minutes. Excess adipose and adventitia were gently excised. Five

Effects of preservation solutions on endothelial cell viability in explanted saphenous vein

Vein segments stored in heparinized lidocaine saline (HLS), autologous heparinized blood (AHB) or tissue culture medium (TCM) exhibited a red fluorescence pattern in the lumenal region within 1 hour of storage indicative of extensive cell membrane damage and compromised viability of endothelial cells (Fig 1, Table 1). In contrast, the endothelial cells were structurally intact (green fluorescence) after 1 hour of storage in Hank’s balanced salt solution (HBSS; Fig 1), but lost cellular

Comment

The preservation of saphenous vein endothelial cells during the course of CABG surgery is essential for the long-term patency of the grafts. Endothelial cells are important mediators in regulating platelet function and in determining the coagulant and fibrinolytic pathways in the vessel wall. Endothelial injury can lead to acute alterations in these essential pathways, leading to thrombosis and stenosis 1, 2, 3, 4, 5. Histochemical analyses have suggested that structural derangements in the

Acknowledgements

We gratefully acknowledge the expert technical assistance of Jin-Hwa Rhee, Thomas McGarry and Sofija Zagarins. We would like to thank Nancy Healey for editorial assistance and Aditi Thatte for her encouragement. This work was supported by National Institutes of Health grants (TM), a Veteran’s Affairs Merit Review Grant (SFK), and the Richard Warren Surgical Research and Educational Fund (HST, SFK).

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To assess the effect of DuraGraft (Somahlution Inc, Jupiter, Fla), an intraoperative graft treatment, on saphenous vein grafts in patients undergoing isolated coronary artery bypass grafting.
Within patients, 2 saphenous vein grafts were randomized to DuraGraft or heparinized saline. Multidetector computed tomography angiography at 1, 3, and 12 months assessed change in wall thickness (primary end point at 3 months), lumen diameter, and maximum narrowing for the whole graft and the proximal 5-cm segment. Safety end points included graft occlusion, death, myocardial infarction, and repeat revascularization.
At 3 months, no significant changes were observed between DuraGraft- and saline-treated grafts (125 each) for wall thickness, lumen diameter, and maximum narrowing. At 12 months, DuraGraft-treated grafts demonstrated smaller mean wall thickness, overall (0.12 ± 0.06 vs 0.20 ± 0.31 mm; P = .02) and in the proximal segment (0.11 ± 0.03 vs 0.21 ± 0.33 mm; P = .01). Changes in wall thickness were greater in the proximal segment of saline-treated grafts (0.09 ± 0.29 vs 0.00 ± 0.03 mm; P = .04). Increase in maximum graft narrowing was larger in the proximal segment in the saline-treated grafts (4.7% ± 12.7% vs 0.2% ± 3.8%; P = .01). Nine DuraGraft and 11 saline grafts had occluded or thrombosed. One myocardial infarction was associated with a saline graft occlusion. No deaths or revascularizations were observed.
DuraGraft demonstrated a favorable effect on wall thickness at 12 months, particularly in the proximal segment. Longer-term follow-up in larger studies is needed to evaluate the effect on clinical outcomes.
Intraoperative Vein Graft Preservation: What Is the Solution?
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Citation Excerpt :
Accordingly, the reduction in NO production that occurs during SVG preparation [40] likely contributes to both thrombosis and intimal hyperplasia. In addition to assessing endothelial structure, as described above, Thatte and colleagues [23] used multiphoton fluorescence microscopy to demonstrate severe attenuation of NO production during storage for 60 minutes in several preservation media, including AHB. In contrast, NO generation actually increased in GALA-stored grafts over an extended 5-hour storage period.
Saphenous vein graft (SVG) disease and subsequent vein graft failure remain a major problem after coronary artery bypass graft operations. In an effort to mitigate loss of endothelial viability, the vein is stored, intraoperatively, in a preservation solution. However, human SVG samples demonstrate endothelial denudation and dysfunction after such storage, the severity of which varies, depending on the medium. The paucity of clinical data evaluating preservation solutions is illustrated by the absence of optimal procedural protocol. This review evaluates the potential efficacy of different storage solutions in preserving vein grafts, in relation to a mechanistic understanding of SVG pathophysiology.
Preservation solution impacts physiologic function and cellular viability of human saphenous vein graft
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GALA, as the most widely recognized of these solutions, was used for physiologic characterization along with more traditional solutions. GALA contains glutathione and l-ascorbic acid, antioxidants, and arginine, a substrate for nitric oxide synthase in endothelial cells, which were added to a buffered electrolyte solution.8 GALA solution, and other transplant harvest solutions have not been directly compared with normal saline using HSV tissue previously.
Recent clinical data suggest intraoperative preservation of human saphenous vein (HSV) in normal saline is associated with vein graft failure. We evaluated the influence of several preservation media on acute physiologic function and cellular viability of HSV conduit.
Unprepared (UP) HSV obtained from coronary artery bypass graft patients was characterized on a muscle bath after 2-hour storage in 6 solutions: Plasma-Lyte A, 0.9% NaCl (normal saline), University of Wisconsin solution, Celsior solution, autologous whole blood, or glutathione-ascorbic acid L-arginine (GALA) solution. Vascular smooth muscle contractility was assessed after exposure to depolarizing KCl and phenylephrine. The relaxation of phenylephrine-precontracted HSV to sodium nitroprusside and carbachol (endothelial-independent and -dependent relaxation, respectively) was also assessed. Cellular viability was determined via the methyl thiazolyl tetrazolium (MTT) assay. Rat aortae were used to assess the effect of pH during graft preservation on endothelial-dependent relaxation.
Preservation of HSV in normal saline and autologous whole blood impaired contractile responses to KCl relative to UP tissues, whereas preservation in University of Wisconsin solution and Celsior solution enhanced contractile responses (P < .05). Relative to UP tissues, responses to phenylephrine were decreased with preservation in normal saline, whereas preservation in University of Wisconsin solution, Celsior solution, and GALA all potentiated these responses (P < .05). Only preservation in normal saline impaired endothelial-independent relaxation (P = .005). Preservation in Plasma-Lyte A (P = .02), normal saline (P = .002), and University of Wisconsin solution (P = .02) impaired endothelial-dependent relaxation. Normal saline preservation decreased MTT viability index relative to UP tissues (0.02 ± 0.002 mg⁻¹0.5 mL⁻¹ vs 0.033 ± 0.005 mg⁻¹0.5 mL⁻¹; P = .03). Endothelial function was impaired by acidic pH in rat aorta.
Preservation of HSV in normal saline causes graft injury leading to impaired physiologic function and decreased viability of the HSV. This harm is mitigated by the use of buffered salt solutions as preservation media.
Further evaluation of Somah: Long-term preservation, temperature effect, and prevention of ischemia-reperfusion injury in rat hearts harvested after cardiocirculatory death
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ATP and CP levels were measured in cardiac tissue after 24-hour storage at 4°C, 10°C, 21°C or 37°C and upon reperfusion using spectrofluorometer and bioluminescent assay kit (Perkin Elmer, Waltham, MASS, USA) as described.13 State-of-the-art multiphoton imaging techniques13–17 using BioRad imaging system (MRC 1024ES) coupled with MaiTai mode-locked titanium:sapphire laser (Spectra-Physics, Mountain View, Calif, USA), were used for deep-imaging cardiac biopsies and semiquantitative fluorescence measurements. Images 512 × 512 pixels were collected in direct detection configuration, at pixel resolution of 0.484 μm.
To identify and evaluate the ideal temperature for long-term storage of hearts from donation after cardiocirculatory death, in the novel organ preservation solution Somah.
DCD hearts from Sprague-Dawley rats were harvested after 30 minutes of euthanasia, preserved in Somah at 4°C, 10°C, 21°C, or 37°C for 24 hours and then reperfused with blood:Somah (3:1) perfusate at 37°C for 30 minutes. Myocardial biopsies were taken during storage and before and after reperfusion to assess the structural and functional viability of tissue using multiphoton imaging, biochemistry, and immunofluorescence.
Myocyte viability, determined by Live-Dead and esterase assays, was similar at 4°C, 10°C, and 21°C (193, 198 and 217 normalized fluorescence counts [NFC]) with a significant decrease at 37°C (131 NFC). Upon reperfusion, esterase activity was enhanced in DCD hearts stored in Somah at 21°C but noticeably decreased at all other temperatures. High-energy adenosine triphosphate/creatine phosphate (ATP/CP) syntheses and the expression of structural/contractile proteins was well preserved at 21°C, both after 24-hour storage and upon reperfusion. In contrast, hearts stored at all other temperatures demonstrated variable degenerative changes, loss of protein expression, and/or deranged ATP/CP synthesis after 24 hours of storage and/or upon reperfusion.
The robust maintenance of structural/functional integrity of cardiac tissue and the preservation of protein expression and cellular energy metabolism in DCD hearts after long-term preservation at subnormothermic temperature suggests that 21°C is ideal for long-term storage of DCD hearts in Somah solution.
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Original article: cardiovascularMulti-photon microscopic evaluation of saphenous vein endothelium and its preservation with a new solution, GALA

Abstract

Background

Methods

Results

Conclusions

Section snippets

Saphenous veins

Effects of preservation solutions on endothelial cell viability in explanted saphenous vein

Comment

Acknowledgements

Eur J Vasc Endovasc Surg

Ann Thorac Surg

J Vasc Surg

Biophys J

Biophys J

FEBS Lett

J Surg Res

Brain Res Protoc

Prog Cardiovasc Dis

J Surg Res

J Biol Chem

Coronary bypass graft surgery improves survival in high risk unstable anginaresults of a VA co-operative study with a eight year followup

Circulation

Original article: cardiovascular
Multi-photon microscopic evaluation of saphenous vein endothelium and its preservation with a new solution, GALA