Cell Viability Assessment Using Fluorescence Vital Dyes and Confocal Microscopy in Evaluating Freezing and Thawing Protocols Used in Cryopreservation of Allogeneic Venous Grafts.

The authors present their contribution to the improvement of methods suitable for the detection of the freezing and thawing damage of cells of cryopreserved venous grafts used for lower limb revascularization procedures. They studied the post-thaw viability of cells of the wall of cryopreserved venous grafts (CVG) immediately after thawing and after 24 and 48 h culture at +37 °C in two groups of six CVG selected randomly for slow thawing in the refrigerator and rapid thawing in a water bath at +37 °C. The grafts were collected from multi-organ and tissue brain-dead donors, cryopreserved, and stored in a liquid nitrogen vapor phase for five years. The viability was assessed from tissue slices obtained by perpendicular and longitudinal cuts of the thawed graft samples using in situ staining with fluorescence vital dyes. The mean and median immediate post-thaw viability values above 70% were found in using both thawing protocols and both types of cutting. The statistically significant decline in viability after the 48-h culture was observed only when using the slow thawing protocol and perpendicular cutting. The possible explanation might be the "solution effect damage" during slow thawing, which caused a gentle reduction in the graft cellularity. The possible influence of this phenomenon on the immunogenicity of CVG should be the subject of further investigations.

Pharmacologic assessment of bovine ruminal and mesenteric vascular serotonin receptor populations.

Prior work using a contractility bioassay determined that the serotonin (5-HT) receptor subtype 5-HT2A is present in bovine lateral saphenous veins and plays a role in ergot alkaloid (EA)-induced vascular contraction in steers grazing endophyte-infected (Epichloë coenophiala) tall fescue (Lolium arundinaceum). Ergot alkaloids have also been shown to be vasoactive in bovine gut vasculature. To determine what 5-HT receptors are involved in vasoconstriction of gut vasculature, contractility of ruminal and mesenteric arteries and veins collected from cattle was evaluated in the presence of agonists selective for 5-HT1B (CP 93129), 5-HT1D (L-694, 247), 5-HT2A (TCB-2), 5-HT2B (BW 723C86), 5-HT4 (BIMU-8), and 5-HT7 (LP 44) receptors. Segments of ruminal and mesenteric veins and arteries were collected and suspended in a multimyograph containing continuously oxygenated Krebs-Henseleit buffer. Blood vessels were exposed to increasing concentrations of 5-HT agonists every 15 min and contractile response data were normalized as a percentage of the maximum contractile response induced by 120 mM KCl. Analysis of variance was evaluated using mixed models procedure of SAS for effects of agonist concentration for each vessel type. Receptor agonists for 5-HT2B, 5-HT1D, and 5-HT7 did not induce a contractile response for ruminal or mesenteric vasculature (P > 0.05). However, when exposed to agonists for 5-HT2B or 5-HT1D, mesenteric veins relaxed below zero (P < 0.05). Exposure of all 4 blood vessel types to 5-HT2A agonist induced contractile responses (P < 0.05). The findings of this study indicate that 5-HT1D and 5-HT2B are present in mesenteric veins and may play a role in vasorelaxation. Further, 5-HT2A is present in ruminal and mesenteric vasculature, plays a role in vasoconstriction of these vessels, and could be influenced by EA exposure as has been demonstrated in peripheral blood vessels.

Assessment of vasoconstrictive potential of D-lysergic acid using an isolated bovine lateral saphenous vein bioassay.

Vasoconstriction has been associated with several symptoms of fescue toxicosis thought to be alkaloid induced. Lysergic acid, an ergot alkaloid, has been proposed as a toxic component of endophyte-infected tall fescue. The objective of this study was to examine the vasoconstrictive potential of D-lysergic acid using a bovine lateral (cranial branch) saphenous vein bioassay. Before testing lysergic acid, validation of the bovine lateral saphenous vein bioassay for use with a multimyograph apparatus was conducted using a dose-response to norepinephrine to evaluate the effects of limb of origin (right vs. left) and overnight storage on vessel contractile response. Segments (2 to 3 cm) of the cranial branch of the lateral saphenous vein were collected from healthy mixed breed cattle (n = 12 and n = 7 for the lysergic acid and norepinephrine experiments, respectively) at local abattoirs. Tissue was placed in modified Krebs-Henseleit, oxygenated buffer and kept on ice or stored at 2 to 8 degrees C until used. Veins were trimmed of excess fat and connective tissue, sliced into 2- to 3-mm sections, and suspended in a myograph chamber containing 5 mL of oxygenated Krebs-Henseleit buffer (95% O2, 5% CO2; pH = 7.4; 37 degrees C). Tissue was allowed to equilibrate at 1 g of tension for 90 min before initiation of treatment additions. Increasing doses of norepinephrine (1 x 10(-8) to 5 x 10(-4) M) or lysergic acid (1 x 10(-11) to 1 x 10(-4) M) were administered every 15 min after buffer replacement. Data were normalized as a percentage of the contractile response induced by a reference dose of norepinephrine. Veins from both left and right limbs demonstrated contractions in a dose-dependent manner (P < 0.01) but did not differ between limbs. There were no differences in dose-response to norepinephrine between tissue tested the day of dissection and tissue tested 24 h later. Exposure of vein segments to increasing concentrations of lysergic acid did not result in an appreciable contractile response until the addition of 1 x 10(-4) M lysergic acid (15.6 +/- 2.3% of the 1 x 10(-4) M norepinephrine response). These data indicate that only highly elevated concentrations of lysergic acid result in vasoconstriction. Thus, in relation to the symptoms associated with vasoconstriction, lysergic acid may only play a minor role in the manifestation of fescue toxicosis.

5-HT1B-receptors and vascular reactivity in human isolated blood vessels: assessment of the potential craniovascular selectivity of sumatriptan.

AIMS: 5-HT1B-receptor mediated vasoconstriction of cranial arteries is a potential mechanism by which 5-HT1B/1D-receptor agonists such as sumatriptan produce their antimigraine effects. 5-HT1B-receptors exist in other blood vessels which may give rise to unwanted vascular effects. Therefore we examined the distribution of 5-HT1B-receptor immunoreactivity (i.r.) in human blood vessels (including target and nontarget vessels) and confirmed the functionality of this receptor protein, by comparing the vasoconstrictor effects of sumatriptan and 5-HT (the endogenous ligand) in isolated vessels. METHODS: Blood vessels (middle meningeal, pial, temporal and uterine arteries and saphenous veins) were obtained from surgical patients (with consent). Sections of the vessels were prepared for routine immunohistochemical studies using specific 5-HT1B- and 5-HT1D-receptor antibodies. For functional studies, ring segments of the vessels were mounted in organ baths for isometric tension recording. RESULTS: 5-HT1B-receptor i.r. was detected on the smooth muscle layer in middle meningeal, pial and uterine arteries and in saphenous vein and sumatriptan produced contractions in these vessels with potency values (mean pEC50) of 7.00, 7.08, 6.44 and 6.61, respectively, the magnitude of contraction was greatest in the cranial arteries with Emax values of 100.7, 60.3, 23.0 and 35.9%, respectively (expressed as a percentage of the reference agonist 45 mm KCl). 5-HT1B-receptor i.r. was not detected in temporal artery and sumatriptan had no effect in this artery. 5-HT1D-receptor i.r. was not detected in any of the vessels studied. CONCLUSIONS: Sumatriptan can evoke vasoconstriction in antimigraine target vessels and also in nontarget vessels through an action at 5-HT1B-rcceptors. Sumatriptan acts preferentially to cause contraction in human cranial arteries compared with the other blood vessels we examined and this effect is likely to be shared by other drugs of this class.

Assessment of the global two-stage method to EC50 determination.

Preclinical studies of a new drug supply sets of concentration-effect observations. For each experiment, EC50 is estimated through fitting of the Emax model. Naive pooling of these individual estimates, also called the Standard Two-Stage method (STS), is usually performed. A better combination is obtained by the Global Two-Stage method (GTS) which takes into account the variability of the nonlinear regression estimation errors. The performances of STS and GTS are compared on real and simulated data. The results show that GTS performs better than STS in terms of bias or RMSE, especially in case of poorly designed experiments. Degradation of the quality of STS results in simulations appears to be mainly due to some experiments that are usually rejected by experimenters. Such rejections are avoided when using GTS, which hence is particularly suitable for systematic treatment of this kind of data.

Assessment of human long saphenous vein function with minimally invasive harvesting with the Mayo stripper.

BACKGROUND: The use of the Mayo Stripper to harvest the long saphenous vein has been shown to improve morbidity from leg wound incisions. It has not been universally accepted because of a perceived increase in injury to the venous conduit. OBJECTIVE: To compare the function of undistended autologous long saphenous vein harvested by a Mayo stripper with the traditional 'open' technique in the same patient (n = 12) appearance. METHODS: Vascular reactivity was assessed in isolated organ baths. Contractile function was measured in response to increasing concentrations (10(-9)-10(-5) mol) of 5-hydroxytryptamine and noradrenaline. This was calculated as a percentage of the maximum contractile response to 90 mM KCl measured in millinewtons (mN) (control 41.4 +/- 12.1, (n = 11), open technique 35.8 +/- 11.1, (n = 11), Mayo stripper 33.7 +/- 15.9, (n = 11)). The endothelial dependent and independent function was assessed with acetylcholine and sodium nitroprusside, respectively. RESULTS: There was no significant difference in response to both constrictors and dilators between vein taken with the Mayo stripper compared with the traditional open technique (n = 6 for each observation; P > 0.05 by ANOVA). Histological examination by light microscopy of the vessel segments removed with the Mayo stripper was unable to show any significant damage to the vessel wall. Both functional and morphological studies were conducted by 'blinded' observers. One-year follow-up with magnetic resonance angiography (MRA) and stress thallium tomography demonstrated a patency rate with lower and upper estimates of 80 and 94%. CONCLUSIONS: We have shown that harvesting the long saphenous vein with a Mayo stripper does not compromise vascular reactivity of the long saphenous vein or long-term patency.

Adult human saphenous vein endothelial cells: assessment of their reproductive capacity for use in endothelial seeding of vascular prostheses.

Autogenous endothelial seeding (AES) of vascular prostheses (VP) using venous endothelial cells (EC) reduces platelet-VP interactions and improves patency rates in small caliber VP in dogs. To conserve patients' veins for use in coronary or limb bypass surgery, human trials of AES should require proof that adequate numbers of EC with the growth capacity to cover VP can be harvested from acceptably small pieces of peripheral vein. EC were isolated from excess saphenous vein segments remaining after coronary bypass surgery by filling veins with 0.1% CLS II collagenase at 37 degrees C for 15 min and removing EC by flushing the veins with culture medium. EC were cultured on fibronectin-coated dishes in medium 199 with 30% human serum and 300 micrograms/ml of endothelial cell growth factor. These cells grew to form confluent monolayers, and were identified as EC by tests for factor VIII antigen. Veins from 53 patients with a mean age of 55.8 +/- 9.8 (SD) years yielded vein segments with an average area of 1.9 +/- 0.6 cm2, from which an average of 5.3 +/- 2.8 X 10(4) cells were removed per cm2 of vein area. EC in culture underwent 14.3 +/- 1.4 population doublings with an average population doubling time of 1.8 +/- 0.3 days (N = 14 cultures), which allowed an 100-fold increase in cell number to occur in 11 to 12 days. These data suggest that the EC available from small vein segments in adult humans have the growth capacity to cover areas comparable in size to the luminal areas of VP commonly used in arterial surgery.