Adenosine and lidocaine (AL) as a vasodilator in cardiac procedures and a storage solution for vascular banking

Introduction: Cardiovascular disease is one of the most common causes of morbidity and mortality worldwide, and globally has contributed to more than 17 million deaths in a year. Coronary heart disease (CHD) alone is responsible for one in seven deaths in the US, and the mortality rate is expected to rise by 10% per year over the next 20 years. One of the invasive treatments for CHD is coronary artery bypass grafting (CABG), which aims to improve cardiac tissue perfusion by grafting another blood vessel to bypass the narrowed or blocked coronary artery. Currently the artery conduit is the standard choice for this procedure, however, the main concern for the use of artery conduits is that they have a high probability of inducing perioperative vasospasm. Therefore, it is crucial to maintain functionality of the conduit during harvest, pressure testing, storage and implantation. The current strategy to prevent artery vasospasm involves a range of anti-spasmodic agents. Some of the most commonly used vasodilators in the surgical setting include Ca²⁺ antagonists (diltiazem, verapamil), nitrates (nitroglycerin, glyceryl trinitrate), and phosphodiesterase inhibitors (papaverine). However, the results remain unsatisfactory. Accordingly, the search for a vasorelaxation agent to reduce graft spasm remains an ongoing pursuit, which if successful may also be applicable to vascular surgery and neurosurgery. The aim of this thesis is to explore the use of adenosine and lidocaine combination as a potential vasodilator to improve arterial grafting using in vitro models. Methods: In this thesis, vascular reactivity was assessed using two different in vitro methods: 1) Isometric force measurements for the isolated male rat aortic ring studies, and 2) Pressured myography for the isolated guinea pig mesenteric artery studies. Isometric force measurements of vasoreactivity were used as the basis for Chapters 3, 4 and 5; and for Chapter 6 after static cold storage. The pressure myography system was only used in Chapter 5. Chapter 3 investigates the relaxation effect of adenosine as a single drug on rat aorta as well as its possible mechanisms of action. In this chapter, aortic rings were freshly harvested from adult male Sprague Dawley rats and equilibrated in an organ bath containing oxygenated, modified Krebs Henseleit (KH) solution (11 mM glucose, pH 7.4, 37°C). Isolated rings were pre-contracted sub-maximally with 0.3 μM norepinephrine (NE), and the effect of increasing concentrations of adenosine (1 to 1000 μM) was examined. The effect of antagonists on adenosine relaxation, such as Nᴳ-nitro-L-arginine methyl ester (L-NAME), indomethacin, 4-aminopyridine (4-AP), glibenclamide, 5-hydroxydecanoate (5-HD), ouabain, 8-(3-chlorostyryl) caffeine (CSC) and 8-[4-[4-(4-chlorobenzyl)piperazide-1-sulfonyl)phenyl]]-1-propylxanthine (PSB- 0788) were examined in intact and denuded aortic rings. Rings were dilated with 100 μM papaverine after each experiment to confirm viability. In Chapter 4, lidocaine effects and mechanisms of action on rat aorta vasorelaxation were examined. Incremental concentrations of lidocaine (1 to 1000 μM) were administered and tested against 0.3 μM NE pre-contracted rat aorta. The effects of antagonists L-NAME, indomethacin, 4-AP, glibenclamide, 5-HD, ouabain, CSC and PSB-0788 were also examined against lidocaine relaxation. As in Chapter 3, rings were tested for viability after each experiment with maximally dilating 100 μM papaverine. Chapter 5 focused on the effect of the combination of adenosine and lidocaine on rat aortic ring relaxation compared to each drug alone. Rings were pre-contracted submaximally with 0.3 μM norepinephrine, and the effects of increasing AL, A or L (up to 1.0 mM) were examined in intact and denuded rings. In this Chapter 5, the vasorelaxation effect of AL as a combination was further explored in the mesenteric artery of guinea pig. This study used the pressure myograph system to examine mesenteric conduit relaxation and the vascular dilatory response to adenosine, lidocaine and AL during luminal and abluminal administration. This methodology is often used to investigate small vessel function (diameter >60 μM) under near physiological conditions of pressure and flow by measuring vessel diameter and flow in time. Mesenteric artery segments were isolated from guinea pigs and mounted in an arteriograph containing KH solution and pressurized to 60 mmHg. Arteries were preconstricted with 10⁻⁸ M vasopressin and AL, A or L was administered luminally or abluminally. Diameters were measured using video-microscopy. Chapter 6 explores the potential use of AL as an additive in a vessel preservation solution. In this chapter, thoracic aortic vessels were harvested from 300-350 g Sprague Dawley rats and transferred to a container with pre-cooled KH solution. Vessel segments were cleaned and cut in 3-mm length rings and stored at 4°C for six days in one of the following preservation solutions: 1) Krebs Henseleit (KH), 2) modified KH (low Ca²⁺/high Mg²⁺), 3) modified KH + adenosine-lidocaine (KH+AL), or 4) modified KH + AL and melatonin and insulin (KH+ALMI). After 6-day storage, physiological (contraction and relaxation) function of the preserved aortic rings was measured using an isometric force transducer. Contraction was induced by norepinephrine (NE; 0.3 μM) and potassium chloride (KCl; 60 mM). Vessel relaxation in response to acetylcholine (ACh; 10⁻⁶-10⁻³ M) and sodium-nitroprusside (SNP; 10-6- 10-3 M) was tested after preconstriction with 0.3 μM NE. At the end of each experiment, rings were maximally dilated with 100 μM papaverine to confirm viability of the vessels. Results: Adenosine induced a dose-dependent, triphasic relaxation response, and the mechanical removal of the endothelium significantly decreased adenosine relaxation above 10 μM. Interestingly, endothelial removal significantly reduced the responsiveness (defined as % relaxation per μM adenosine) by two-thirds between 10 and 100 μM, but not in the lower (1-10 μM) or higher (>100 μM) ranges. In intact rings, L-NAME, but not indomethacin, significantly reduced relaxation, suggesting a role of nitric oxide (NO) but not prostacyclin in adenosine endothelium-dependent relaxation. Antagonists of voltage-dependent Kᵥ (4-AP), sarcolemmal K(ATP) (glibenclamide) and mitochondrial K(ATP) channels (5-HD) led to significant reductions in adenosine relaxation in both intact and denuded rings, with the Na⁺/K⁺-ATPase antagonist ouabain having little or no effect. Adenosine-induced relaxation appeared to involve the A₂ₐ receptor, but not the A₂(b) subtype. In contrast to adenosine, lidocaine relaxation in intact rings was biphasic between 1 to 10 μM (Phase 1) and 10 to 1000 μM (Phase 2). Mechanical removal of the endothelium resulted in further relaxation, and at lower concentrations ring sensitivity (% relaxation per μM lidocaine) significantly increased 3.5 times compared to intact rings. The relaxing factor(s) responsible for enhancing lidocaine relaxation did not appear to be NO- or prostacyclin-dependent, as L-NAME and indomethacin had little or no effect on intact ring relaxation. In denuded rings, lidocaine relaxation was completely abolished by Kᵥ channel inhibition and significantly reduced by antagonists of the MitoK(ATP) channel, and to a lesser extent the SarcK(ATP) channel. Curiously, A₂ₐ subtype receptor antagonism significantly inhibited lidocaine relaxation above 100 μM, but not the A₂(b) receptor. In combination, adenosine and lidocaine (AL) increased aortic relaxation from 21 to 100% (0.1-1.0 mM) and relaxation was endothelium-independent. Although adenosine alone was also a potent relaxant of aortic rings, unlike AL relaxation, it was partially endothelium-dependent. Further investigation of AL effects on mesenteric artery showed that increasing luminal administration of AL in intact mesenteric artery segments produced a potent endothelium-independent dilation up to 90% (p<0.05). Adenosine dilation was endothelium-independent but not lidocaine, which produced 33% dilation only after endothelial removal. Extra-luminal AL and A led to 76% and 80% dilation in intact segments respectively, whereas L resulted in constriction (10-17%). When exploring the potential use of AL as a preservation solution with 6-day cold storage in Chapter 6, it was found that AL addition in modified KH solution resulted in 100% recovery of NE contractile function in rat aorta, which was superior compared to KH solution alone (89% recovery). However, there was no further recovery in the KCl response over modified KH (76% recovery). A similar result was also shown with ALMI in modified KH, which led to 100% and 86% of contractile function recovery in response to NE and KCl, respectively. Furthermore, AL but not ALMI addition in modified KH significantly improved relaxation function compared to standard KH, with 93% recovery compared to 79% with modified KH alone after six days of storage. Maximal SNP relaxation following 6-day cold storage with either modified KH alone, modified KH with AL or with ALMI recovered 100%. Conclusions: Adenosine is a potent vasodilator of aortic rings. Adenosine relaxation in NEprecontracted rat aortic rings was triphasic and endothelium-dependent above 10 μM, and relaxation involved endothelial nitric oxide (not prostanoids) and a complex interplay between smooth muscle A₂ₐ subtype and voltage-dependent Kᵥ, SarcK(ATP) and MitoK(ATP) channels. In contrast, lidocaine relaxation is not as potent as adenosine relaxation, but it appears to be significantly enhanced by endothelial removal, which did not appear to be NO- or prostacyclin-dependent. The unknown factor(s) responsible for enhanced relaxation was significantly reduced by Kᵥ channel inhibition, MitoK(ATP) channel inhibition, and A₂ₐ subtype inhibition indicating a potential role for crosstalk in lidocaine's vasoreactivity. When combined, AL can dilate aortic rings and mesenteric artery segments by up to 90% regardless of whether the endothelium is intact. This may have potential translational significance of AL to improve conduit protection in cardiac surgery, and other major surgeries where varying degrees of endothelial damage, vasoconstriction or vasospasm are known to occur. In addition, AL has a potential role as an adjuvant in preservation solutions since it improved vascular function after 6-day cold storage. AL addition in modified KH solution significantly improved NE-induced vascular contractility and ACh-induced relaxation compared to standard KH solution. This may indicate that AL improved endothelial preservation during storage, which was not achieved with standard preservation solution

Adenosine relaxation in isolated rat aortic rings and possible roles of smooth muscle Kv channels, KATP channels and A2a receptors.

Background: An area of ongoing controversy is the role adenosine to regulate vascular tone in conduit vessels that regulate compliance, and the role of nitric oxide (NO), potassium channels and receptor subtypes involved. The aim of our study was to investigate adenosine relaxation in rat thoracic aortic rings, and the effect of inhibitors of NO, prostanoids, Kv, KATP channels, and A2a and A2b receptors. Methods: Aortic rings were freshly harvested from adult male Sprague Dawley rats and equilibrated in an organ bath containing oxygenated, modified Krebs-Henseleit solution, 11 mM glucose, pH 7.4, 37 °C. Isolated rings were pre-contracted sub-maximally with 0.3 μM norepinephrine (NE), and the effect of increasing concentrations of adenosine (1 to 1000 μM) were examined. The drugs L-NAME, indomethacin, 4-aminopyridine (4-AP), glibenclamide, 5-hydroxydecanoate, ouabain, 8-(3-chlorostyryl) caffeine and PSB-0788 were examined in intact and denuded rings. Rings were tested for viability after each experiment. RESULTS: Adenosine induced a dose-dependent, triphasic relaxation response, and the mechanical removal of the endothelium significantly deceased adenosine relaxation above 10 μM. Interestingly, endothelial removal significantly decreased the responsiveness (defined as % relaxation per μM adenosine) by two-thirds between 10 and 100 μM, but not in the lower (1-10 μM) or higher (>100 μM) ranges. In intact rings, L-NAME significantly reduced relaxation, but not indomethacin. Antagonists of voltage-dependent Kv (4-AP), sarcolemma KATP (glibenclamide) and mitochondrial KATP channels (5-HD) led to significant reductions in relaxation in both intact and denuded rings, with ouabain having little or no effect. Adenosine-induced relaxation appeared to involve the A2a receptor, but not the A2b subtype. Conclusions:It was concluded that adenosine relaxation in NE-precontracted rat aortic rings was triphasic and endothelium-dependent above 10 μM, and relaxation involved endothelial nitric oxide (not prostanoids) and a complex interplay between smooth muscle A2a subtype and voltage-dependent Kv, SarcKATP and MitoKATP channels. The possible in vivo significance of the regulation of arterial compliance to left ventricular function coupling is discussed

Lidocaine relaxation in isolated rat aortic rings is enhanced by endothelial removal: possible role of Kv, KATP channels and A2a receptor crosstalk

Background: Lidocaine is an approved local anesthetic and Class 1B antiarrhythmic with a number of ancillary properties. Our aim was to investigate lidocaine's vasoreactivity properties in intact versus denuded rat thoracic aortic rings, and the effect of inhibitors of nitric oxide (NO), prostenoids, voltage-dependent Kv and KATP channels, membrane Na+/K+ pump, and A2a and A2b receptors. Methods: Aortic rings were harvested from adult male Sprague Dawley rats and equilibrated in an organ bath containing oxygenated, modified Krebs-Henseleit solution, pH 7.4, 37 °C. The rings were pre-contracted sub-maximally with 0.3 μM norepinephrine (NE), and the effect of increasing lidocaine concentrations was examined. Rings were tested for viability after each experiment with maximally dilating 100 μM papaverine. The drugs 4-aminopyridine (4-AP), glibenclamide, 5-hydroxydecanoate, ouabain, 8-(3-chlorostyryl) caffeine and PSB-0788 were examined. Results: All drugs tested had no significant effect on basal tension. Lidocaine relaxation in intact rings was biphasic between 1 and 10 μM (Phase 1) and 10 and 1000 μM (Phase 2). Mechanical removal of the endothelium resulted in further relaxation, and at lower concentrations ring sensitivity (% relaxation per μM lidocaine) significantly increased 3.5 times compared to intact rings. The relaxing factor(s) responsible for enhancing ring relaxation did not appear to be NO- or prostacyclin-dependent, as L-NAME and indomethacin had little or no effect on intact ring relaxation. In denuded rings, lidocaine relaxation was completely abolished by Kv channel inhibition and significantly reduced by antagonists of the MitoKATP channel, and to a lesser extent the SarcKATP channel. Curiously, A2a subtype receptor antagonism significantly inhibited lidocaine relaxation above 100 μM, but not the A2b receptor

PERBEDAAN KADAR NITRIC OXIDE PADA IBU HAMIL TRIMESTER 1 DENGAN ANEMIA DAN TIDAK ANEMIA

Anemia saat hamil berefek buruk bagi ibu maupun janin, karena dapat mengurangi suplai oksigen pada metabolisme ibu akibat kekurangan kadar hemoglobin untuk mengikat oksigen, dan peran hemoglobin sebagai pengikat nitric oxide dapat menyebabkan vasokontriksi dan mempengaruhi pengiriman oksigen. Tujuannya untuk mengetahui perbedaan kadar nitric oxide pada ibu hamil trimester 1 dengan anemia dan tidak anemia. Desain penelitian cross sectional study dengan masing-masing 35 ibu hamil trimester 1 yang anemia dan tidak anemia dengan teknik consecutive sampling. Hasil penelitian menunjukkan rata-rata kadar nitric oxide pada ibu hamil trimester 1 yang anemia lebih tinggi (128,8μmol/L) dibandingkan yang tidak anemia (89,1μmol/L) nilai p=0,008. Ibu hamil trimester 1 yang anemia kemungkinan 3,692 kali memiliki resiko mengalami peningkatan kadar nitric oxide dibandingkan yang tidak anemia dengan cut off point 92,86μmol/L. Disimpulkan, kadar nitric oxide lebih tinggi pada ibu hamil trimester 1 yang anemia dan memiliki resiko terjadi peningkatan kadar nitric oxide pada ibu hamil trimester 1 dengan anemia sebesar 3,962 kali

The adenosine hypothesis revisited: modulation of coupling between myocardial perfusion and arterial compliance

For over four decades the thoracic aortic ring model has become one of the most widely used methods to study vascular reactivity and electromechanical coupling. A question that is rarely asked, however, is what function does a drug-mediated relaxation (or contraction) in this model serve in the intact system? The physiological significance of adenosine relaxation in rings isolated from large elastic conduit arteries from a wide range of species remains largely unknown. We propose that adenosine relaxation increases aortic compliance in acute stress states and facilitates ventricular-arterial (VA) coupling, and thereby links compliance and coronary artery perfusion to myocardial energy metabolism. In 1963 Berne argued that adenosine acts as a local negative feedback regulator between oxygen supply and demand in the heart during hypoxic/ischemic stress. The adenosine VA coupling hypothesis extends and enhances Berne's “adenosine hypothesis” from a local regulatory scheme in the heart to include conduit arterial function. In multicellular organisms, evolution may have selected adenosine, nitric oxide, and other vascular mediators, to modulate VA coupling for optimal transfer of oxygen (and nutrients) from the lung, heart, large conduit arteries, arterioles and capillaries to respiring mitochondria. Finally, a discussion of the potential clinical significance of adenosine modulation of VA coupling is extended to vascular aging and disease, including hypertension, diabetes, obesity, coronary artery disease and heart failure

COMPARISON OF BLEEDING TIME AND CLOTTING TIME IN WOMEN AGAINST ABO BLOOD TYPE

Kelompok darah ABO dan jenis kelamin yang berbeda dapat memberikan hasil yang berbeda dari waktu perdarahan dan waktu pembekuan. Kebaruan penelitian ini karena meneliti tentang perbandingan bleeding time dan clotting time pada wanita terhadap golongan darah ABO. Penelitian ini bertujuan untuk membandingkan waktu perdarahan dan waktu pembekuan pada wanita berdasarkan golongan darah ABO. Penelitian ini merupakan penelitian deskriptif dengan desain cross sectional dan sampel total 115 subjek. Pengukuran waktu perdarahan menggunakan metode duke, waktu pembekuan menggunakan metode slide dan golongan darah menggunakan metode aglutinasi slide. Penelitian ini menggunakan uji komparatif numerik dengan one way ANOVA, dilanjutkan dengan analisis post hoc untuk membandingkan waktu perdarahan dan waktu pembekuan antara golongan darah ABO. Hasil penelitian ini menunjukkan waktu perdarahan pada golongan darah B yang secara signifikan lebih lama dibandingkan golongan darah lainnya (p=0,001). Sementara itu, waktu pembekuan pada golongan darah B secara signifikan lebih lama dibandingkan golongan darah lainnya (p= 0,001). Kesimpulan bahwa wanita dengan golongan darah B memiliki waktu perdarahan dan waktu pembekuan yang jauh lebih lama daripada golongan darah lainnya.Kata Kunci: Waktu pendarahan; Waktu pembekuan; Golongan darah ABO. AbstractDifferent ABO blood groups and sexes can give different results from bleeding and clotting times. This study's novelty is that it examines the comparison of bleeding time and clotting time in women to the ABO blood type. This study aimed to compare bleeding and clotting time in women based on ABO blood type. This study is a descriptive study with a cross-sectional design and a total sample of 115 subjects. Measure bleeding time using the duke method, clotting time using the slide method, and blood type using the slide agglutination method. This study used a comparative numerical test with a one-way ANOVA, followed by post hoc analysis to compare bleeding and clotting time between ABO blood types. This study showed that the bleeding time in blood type B was significantly longer than in other blood types (p=0.001). Meanwhile, the clotting time in blood type B is considerably longer than in different blood types (p= 0.001). In conclusion, women with blood type B have much longer bleeding and clotting times than other blood types.Keywords: Bleeding time; Freezing time; ABO Blood type.

Perbandingan Perlindungan Minyak Jintan Hitam, Minyak Argan dan Minyak Zaitun terhadap Enzim Hati Tikus Akibat Diet Tinggi Lemak

High-fat diet has become one of the risk factors of liver dysfunction due to accumulation of fat in the liver cells. This disorder might be triggered by increased reactive oxygen species (ROS) activity with high-fat consumption. This study aimed to compare protective effect of natural antioxidants black cumin oil, argan oil and olive oil on liver function in wistar rats fed with high-fat diet. Male wistar rats (n = 24) were divided into four treatment groups. Group 1 (negative control) was not given any oil treatment (0,5 ml/g bw), group 2 was given black cumin oil (0,4 ml/g bw), group 3 was given argan oil (0,5 ml/g bw) and group 4 was given olive oil (0,5 ml/g bw). All rats were fed a high-fat diet of 10 gr/day for 2 months. The analysis of liver function tests was performed before and after treatment. With high-fat diet, the negative controls had SGOT of 93.05 ± 47.91 UI/I and SGPT of 43.10 ± 14.64 UI/l.  Administration of black cumin oil markedly reduced SGOT (62.05 ± 30.67 UI/l) and SGPT levels (28.81 ± 10.60 UI/l) (P0.05). Argan oil can not reduce SGOT levels (97.92 ± 35.07 UI/l) but can reduce SGPT levels (51.67 ± 15.84 UI/l). Olive oil can not reduce SGOT levels (67.38 ± 29.31 UI/l) but can reduce SGPT levels (50.19 ± 9.70 UI/l). It was concluded that administration of black cumin is more effective to reduce SGOT and SGPT levels in rats with high-fat diet compared to argan oil and olive oil treatments

Adenosine and lidocaine (AL) combination dilates intimally damaged rat thoracic aortic rings and guinea pig mesenteric arteries: possible significance to cardiac surgery

New pharmacotherapies are required to improve vessel graft protection and prevent vasoconstriction and spasm in CABG surgery. Previously we have studied adenosine (A) and lidocaine (L) relaxation in rat aortic rings, and reported a possible crosstalk between L relaxation and adenosine A(2a) receptor inhibition. The aim of the present study was to examine the effect of AL combination compared to A and L alone on relaxation in intact and denuded rat aortic rings and in guinea-pig pressurized mesenteric arterial segments. Aortic rings were harvested from Sprague-Dawley rats and equilibrated in an organ bath containing modified Krebs-Henseleit (KH) solution, pH 7.4, 37 degrees C. Rings were pre-contracted sub-maximally with 0.3 mu M norepinephrine, and the effects of increasing AL, A or L (up to 1.0 mM) were examined in intact and denuded rings. Mesenteric artery segments were isolated from guinea-pigs and mounted in an arteriograph containing KH solution and pressurised to 60 mmHg. Arteries were preconstricted with 10(-8) M vasopressin and AL, A, or L was administered luminally or abluminally. Diameters were measured using video-microscopy. We report in intact rat aortic rings, AL increased relaxation from 21 to 100% (0.1-1.0 mM) and relaxation was endothelium-independent. Adenosine alone was also a potent relaxant of aortic rings but, unlike AL relaxation, it was partially endothelium-dependent. In intact mesenteric artery segments, increasing luminal AL produced a potent endothelium-independent dilation (up to 90%). Adenosine dilation was endothelium-independent but not lidocaine, which produced 33% dilation only after endothelial removal. Extra-luminal AL and A led to 76% and 80% dilationin intact segments respectively, whereas L resulted in constriction (10-17%). In conclusion, we show that AL can dilate aortic rings and mesenteric artery segments by up to 90% regardless of whether the endothelium is intact. We discuss the potential translational significance of AL to improve conduit protection in cardiac surgery, and other major surgeries