Morphology and function of preserved microvascular arterial grafts: an experimental study in rats

The aim of this study is to examine the morphology and function and small-caliber, arterial grafts after preservation in the University of Wisconsin solution (UW). Rat carotid arteries were stored in UW (n = 10) or in phosphate-buffered saline (PBS) (n = 10) for 1, 3, 7, and 14 days and were examined with light microscopy (LM) and scanning electron microscopy (SEM). Rat aortic preparations were stored in UW or PBS for 1 hour, 24 hours, 72 hours, 7 days, and 14 days and assessed for functional responses (stimulated contraction and endothelium-dependent relaxation). Segments (5 mm) of rat carotid arteries were stored in UW or PBS for 3 days, 7 days, and 14 days and orthotopically implanted as autografts and allografts. No immunosuppressive or anticoagulant agents were used. After 28 days of implantation, the grafts were assessed for patency and excised for LM and SEM. In UW, the endothelial layer remained intact up to 9 days of storage. In PBS, the endothelial layer showed deterioration after 1 day and was completely lost after 3 days. Functional responses were demonstrated to exist for as long as 7 days storage in UW. In PBS, no responses could be evoked after 24 hours storage. Autografts preserved in UW for 3 days (n = 6), 7 days (n = 6), and 14 days (n = 6) showed patency rates of 83.3%, 66.6%, and 66.6%, respectively, whereas patency rates of allografts were 66.6%, 33.3%, and 33.3%, respectively. Autografts stored in PBS for 3 days (n = 6), 7 days (n = 6), and 14 days (n = 6) showed patency rates of 33.3%, 33.3%, and 50%, respectively, whereas patency rates of allografts were 16.7%, 0%, and 33.3%, respectively. The UW preserved autografts showed normal morphology. All other groups showed vessel wall degeneration which in the allograft groups, were accompanied by lymphocellular infiltration. In conclusion, the endothelial layer and vessel wall of arteries are adequately preserved in UW. Functional responses are retained up to 14 days storage in UW, but, are lost after 24 hours storage in PBS. Autograft implantation studies accordingly show good performance of arterial segments preserved in UW, whereas allografts are subject to degradation as a result of rejectio

Morphology and function of dog arterial grafts preserved in UW-solution

Objectives:To assess the function of arterial grafts after prolonged preservation in the University of Wisconsin solution (UW), in vitro and in vivo.Methods:Carotid arteries were harvested from dogs and stored for 1–21 days at 4°C in UW (n = 10) or in PBS (0.9% NaCl, pH 7.4), (PBS) (n = 10). Slices were examined by lightmicroscopy (LM) and scanning electron microscopy (SEM). For viability testing, specimens were connected to an isometric force transducer (2 × n = 9). Contractile and relaxation responses were examined by adding phenylephrine (200μM) and metacholine (200μM), respectively. For in vivo studies (n = 41), 2.5cm carotid artery segments were implanted orthotopically, as autografts and allografts, after 14 days of storage in UW or in PBS. Autologous veins were used as controls. After 28 days or 56 days, arteriography was performed and the grafts were excised for LM and SEM.Results:The arterial endothelial layer remained intact after up to 14 days of storage in UW. In PBS, the endothelium was lost after 3 days. The functional response after 14 days storage in UW was approximately 50% vs. 0% after 14 days in PBS. In the autografts, total patencies (28 days + 56 days) were 100% (8/8) and 63% (5/8) for UW and PBS stored grafts, respectively. In the allografts, the UW and PBS preserved grafts showed total patencies of 86% (12/14) and 83% (5/6), respectively. Microscopically, the allografts showed fibrotic degeneration.Conclusions:Arteries are well preserved in UW up to 14 days of storage. Arterial autografts preserved in UW showed good patency and better integrity of the vessel wall after implantation, than grafts stored in PBS or allografts (without immunosuppressive therapy)

The influence of oxidative stress on various inotropic responses in isolated reat left atria

The effects of free radicals, generated by electrolysis of a physiological salt solution, on various inotropic responses to drugs in isolated rat left atria were studied. Evidence for the generation of hydroxyl radicals was obtained from an appropriate fluorimetric assay. The amount of free radicals produced by electrolysis of the medium proved current-dependent. Exposure of isolated rat left atria to the medium which had been subjected to electrolysis caused a current-dependent decrease in contractile force. Oxidative stress, as a result of the electrolysis of the medium, caused altered inotropic responses to extra cellular Ca2+ (pD2 control group: 2.62 +/- 0.06 vs. 2.44 +/- 0.07 electrolysis group), sodium withdrawal (rise in contractile force control group: 1.73 +/- 0.19 mN vs. 0.48 +/- 0.21 mN electrolysis group) and lowering of stimulation frequency. The response to isoprenaline was diminished in atria subjected to oxidative stress and led to a rightward shift of the concentration response curves (pD2 control group: 7.56 +/- 0.10 vs. 6.77 +/- 0.11 electrolysis group). In addition, the inotropic responses to forskolin (pD2 control group: 6.17 +/- 0.12 vs. < 4.5 electrolysis group) and dibutyryl cAMP (rise in contractile force caused by 1 x 10(-5) M db-cAMP in control group: 2.15 +/- 0.01 mN vs. 1.21 +/- 0.10 mN electrolysis group) proved blunted as well. Measurement of the adenylyl cyclase activity revealed that free radicals attenuated the basal (by 11.1%) and forskolin stimulated (155.0 +/- 5.1 vs. 48.0 +/- 1.8 pmol cAMP/mg prot./min for control and electrolysis group respectively) activity of the adenylyl cyclase. DMSO, a well known hydroxyl radical scavenger, was able to abolish the free radical-induced decrease in the response to isoprenaline. Surprisingly, addition of alpha-adrenoceptor agonists to atria subjected to electrolysis-generated free radicals led to a rapid decrease in contractile force. DMSO was unable to counteract the negative intropic effect of methoxamine in atria subjected to oxidative stress. This negative inotropic response to alpha-adrenoceptor agonists in atria subjected to electrolysed medium is unlikely to be the direct result of phospholipase C or protein kinase C activation. Angiotensin II (which stimulates PLC, as well) did not reduce contractile force and chelerythrine (a PKC inhibitor) was unable to counteract the negative inotropic effect of the adrenoceptor agonists. In addition, the negative inotropic effect of methoxamine proved insensitive to 10(-6) M phentolamine and 10(-5) M doxazosin, which indicates an alpha-adrenoceptor independent mechanism. From this study we conclude that free radicals alter responses to various inotropic stimuli. These alterations may be the result of injured contractile elements, transporter molecules and molecules involved in signal transduction. Addition of alpha-adrenoceptor agonists after oxidative stress leads to a alpha-adrenoceptor. PLC and PKC independent decrease in contractile forc

Mediation by the same muscarinic receptor subtype of phasic and tonic contractile activities in the rat isolated portal vein.

1. The effects of several agonists on the phasic and tonic contractile responses to muscarinic receptor stimulation have been investigated in the rat portal vein in vitro. 2. Neither chemical denervation with 6-hydroxydopamine nor the presence of the alpha 1-adrenoceptor antagonist, prazosin, influenced the spontaneous or the stimulated myogenic activity of the portal vein. 3. Indomethacin and NG-nitro-L-arginine were used to investigate the influence of vasoactive factors in this preparation. They slightly increased the frequency and the amplitude of the spontaneous myogenic activity of the portal vein, respectively. NG-nitro-L-arginine but not indomethacin enhanced the maximal phasic response to carbachol. Both indomethacin and NG-nitro-L-arginine failed to influence the tonic response to carbachol. 4. Muscarinic agonists increased phasic activity according to the rank order of potency: acetylcholine > muscarine > methacholine > carbachol > aceclidine > bethanechol. These effects were superimposed on a sustained contracture at higher concentrations. Oxotremorine was more potent than arecoline in increasing the mechanical phasic activity, without inducing a sustained contracture. Pilocarpine and McN A343 were weak agonists, producing submaximal effects only on phasic activity. 5. The muscarinic antagonists AF-DX116, 4-diphenylacetoxy-N-methylpiperidine (4-DAMP), P-fluorohexahydrosiladiphenidol (pFHHSiD) and pirenzepine antagonized the phasic and tonic mechanical responses to carbachol. Although the tonic contracture was slightly more sensitive to all antagonists studied, the rank order of potency: 4-DAMP > pFHHSiD > pirenzepine > AF-DX 116 was the same for both types of responses, which is indicative of a M3-receptor subtype.(ABSTRACT TRUNCATED AT 250 WORDS

ATP-sensitive potassium channels in isolated rat aorta during physiologic, hypoxic, and low-glucose conditions

In arterial smooth muscle, adenosine triphosphate (ATP)-sensitive potassium channels are the targets of a variety of synthetic and endogenous vasodilators. In this study, we evaluated the influence of glibenclamide, an ATP-sensitive K(+)-channel blocker, on various vasodilator responses, including those by levcromakalim under hypoxic and low-glucose conditions in isolated rat aortic rings. The concentration-response curves induced by methacholine and sodium nitroprusside (after precontraction with 1 microM phenylephrine) were not affected by glibenclamide. Glibenclamide influenced neither the adenosine- nor the iloprost- (a stable prostacyclin) induced vasodilator effects. Glibenclamide caused a concentration-dependent rightward shift of the concentration-response curves of levcromakalim. The vascular tone induced by phenylephrine was not affected under low-glucose conditions, whereas hypoxia caused a decrease in the phenylephrine-induced contraction when compared with that under normal circumstances. Under all conditions, glibenclamide did not influence the phenylephrine-induced increase in vascular tone. Under low-glucose and hypoxic conditions, the concentration-response curves for levcromakalim showed a significantly less steep slope than under normal conditions, and higher concentrations of glibenclamide were necessary to inhibit the vasodilator response induced by levcromakalim under these experimental conditions adopted to mimic pathologic conditions. In conclusion, methacholine, sodium nitroprusside, adenosine, and iloprost appear not to induce vasodilation in the rat aorta by glibenclamide-sensitive K+ channels, whereas hypoxia and low-glucose levels cause an impaired function of the glibenclamide-sensitive K+ channel

The direct effects of thyroid hormones on rat mesenteric resistance arteries

The direct relaxant effects of thyroid hormones on mesenteric resistance vessels were investigated using an isometric wire myograph. Both the L- and the D-isomers of thyroxine (T4) and triiodothyronine (T3) were studied. In contrast with the long-term effects of thyroid hormones, both T4 enantiomers proved more potent in inducing vascular relaxation than the two T3 enantiomers. The interaction between thyroid hormones and calcium-induced contractions was studied. T4 concentration dependently inhibited the Ca2+ induced contractions, showing noncompetitive interaction. Furthermore, we investigated whether the endothelium was involved in the relaxant effect to L-T4. The T4 induced relaxation proved impaired by prior incubation with the nitric oxide (NO) inhibitor N-omega-nitro-L-arginine methylester HCl (L-NAME, 0.1 microM), indicating that T4 is able to stimulate the production of endothelium-derived NO. L-T4-induced relaxation was enhanced by prior incubation with indomethacin (10 microM), whereas in endothelium-denuded preparations an unaltered response was found. The present results indicate that L-T4-induced relaxation is established by an indirect effect via the endothelium and by a direct effect on vascular smooth muscle cells, possibly by influencing calcium fluxes. Because vascular relaxation is established at supraphysiologic concentrations (approximately 100 times the basal level) of thyroid hormone, it is concluded that the direct effect of thyroid hormone on mesenteric vascular smooth muscle cells are not relevant for the in vivo situatio