Caroline Floreoto Baldo

Diabetes and Vascular Disease: Basic Concepts of Nitric Oxide Physiology, Endothelial Dysfunction, Oxidative Stress and Therapeutic Possibilities

The vascular manifestations associated with diabetes mellitus (DM) result from the dysfunction of several vascular physiology components mainly involving the endothelium, vascular smooth muscle and platelets. It is also known that hyperglycemia-induced oxidative stress plays a role in the development of this dysfunction. This review considers the basic physiology of the endothelium, especially related to the synthesis and function of nitric oxide. We also discuss the pathophysiology of vascular disease associated with DM. This includes the role of hyperglycemia in the induction of oxidative stress and the role of advanced glycation end-products. We also consider therapeutic strategies.

Effects of acid-base imbalance on vascular reactivity

Acid-base homeostasis maintains systemic arterial pH within a narrow range. Whereas the normal range of pH for clinical laboratories is 7.35-7.45, in vivo pH is maintained within a much narrower range. In clinical and experimental settings, blood pH can vary in response to respiratory or renal impairment. This altered pH promotes changes in vascular smooth muscle tone with impact on circulation and blood pressure control. Changes in pH can be divided into those occurring in the extracellular space (pHo) and those occurring within the intracellular space (pHi), although, extracellular and intracellular compartments influence each other. Consistent with the multiple events involved in the changes in tone produced by altered pHo, including type of vascular bed, several factors and mechanisms, in addition to hydrogen ion concentration, have been suggested to be involved. The scientific literature has many reports concerning acid-base balance and endothelium function, but these concepts are not clear about acid-base disorders and their relations with the three known mechanisms of endothelium-dependent vascular reactivity: nitric oxide (NO/cGMP-dependent), prostacyclin (PGI2/cAMP-dependent) and hyperpolarization. During the last decades, many studies have been published and have given rise to confronting data on acid-base disorder and endothelial function. Therefore, the main proposal of this review is to provide a critical analysis of the state of art and incentivate researchers to develop more studies about these issues.

Effects of anesthetic drugs on canine splenic volume determined via computed tomography

OBJECTIVE To evaluate effects of commonly used anesthetics administered as single bolus injections on splenic volume. ANIMALS 10 adult Beagles. PROCEDURES A randomized crossover study was conducted. Computed tomography was performed on dogs to determine baseline splenic volume and changes after IV injection of assigned drug treatments. Dogs were allowed to acclimate for 10 minutes in a plastic crate before acquisition of abdominal CT images. Treatments were administered at 7-day intervals and consisted of IV administration of saline (0.9% NaCl) solution (5 mL), acepromazine maleate (0.03 mg/kg), hydromorphone (0.1 mg/kg), and dexmedetomidine (0.005 mg/kg) to all 10 dogs; thiopental (8 mg/kg) to 5 of the dogs; and propofol (5 mg/kg) to the other 5 dogs. Splenic volume was calculated from the CT images with image processing software. A repeated-measures ANOVA was performed, followed by a Bonferroni post hoc test. RESULTS No significant difference in splenic volume was detected between the acepromazine, propofol, and thiopental treatments, but splenic volume was greater with these drugs than with saline solution, hydromorphone, and dexmedetomidine. Splenic volume was less with hydromorphone, compared with dexmedetomidine, but splenic volume with hydromorphone and dexmedetomidine did not differ significantly from that with saline solution. CONCLUSIONS AND CLINICAL RELEVANCE Administration of acepromazine, thiopental, and propofol resulted in splenomegaly. Dexmedetomidine did not alter splenic volume. Hydromorphone slightly decreased splenic volume. Propofol should not be used when splenomegaly is not desirable, whereas hydromorphone and dexmedetomidine may be used when it is best to avoid splenic enlargement.

Plasma nitrate/nitrite (NOx) is not a useful biomarker to predict inherent cardiopulmonary bypass inflammatory response.

Abstract  Background and Aim: There were strong evidences that nitric oxide has capital importance in the progressive vasodilatation associated with varied circulatory shock forms, including systemic inflammatory response syndrome (SIRS), in patients undergoing cardiac surgeries for cardiopulmonary bypass (CPB). If CPB procedures, per se, are the inciting stimulus for inflammation, plasma nitrate/nitrite (NOx) excretion would be expected to be higher in these patients rather than in patients operated without CPB. In consequence, we hypothesized that increased levels of NOx would be predictive for vasoplegic syndrome. Methods: Thirty patients were assigned to three groups: Group 1—coronary artery bypass graft (CABG) roller pump CPB; Group 2—CABG centrifugal vortex pump CPB; and Group 3—heart valve surgery roller pump CPB. Sampling of venous blood for chemiluminescence plasma NOx dosage was achieved at the following time points: (1) before anesthesia induction; (2) after anesthesia induction; (3) before heparin infusion; (4) after heparin infusion; (5) CPB‐30 minutes; (6) CPB‐60 minutes; (7) before protamine infusion; (8) after protamine infusion; and (9) on return to the recovery area. Results: There were no intergroup differences regarding age and anesthetic regimen, and the number of arteries grafted was not different between the CABG groups. There were no NOx statistic differences, neither among the three groups of patients or among the surgery time. In addition, there was no correlation among NOx, lactate, and hemoglobin. Conclusions: Considering the inflammatory process intrinsic to CPB, this study reinforces the idea that plasma NOx is not useful as a biomarker of inflammatory response onset, which may or may not lead to SIRS and/or vasoplegic syndrome.

Extracellular alkalinization induces endothelium-derived nitric oxide dependent relaxation in rat thoracic aorta

AIM To investigate the mechanism through which the extracellular alkalinization promotes relaxation in rat thoracic aorta. METHODS The relaxation response to NaOH-induced extracellular alkalinization (7.4-8.5) was measured in aortic rings pre-contracted with phenylephrine (Phe, 10(-6) M). The vascular reactivity experiments were performed in endothelium-intact and -denuded rings, in the presence or and absence of indomethacin (10(-5) M), NG-nitro-l-arginine methyl ester (L-NAME, 10(-4) M), N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide/HCl (W-7, 10(-7) M), 2,5-dimethylbenzimidazole (DMB, 2×10(-5) M) and methyl-β-cyclodextrin (10(-2) M). In addition, the effects of NaOH-induced extracellular alkalinization (pH 8.0 and 8.5) on the intracellular nitric oxide (NO) concentration was evaluated in isolated endothelial cells loaded with diaminofluorescein-FM diacetate (DAF-FM DA, 5 μM), in the presence and absence of DMB (2×10(-5) M). RESULTS The extracellular alkalinization failed to induce any change in vascular tone in aortic rings pre-contracted with KCl. In rings pre-contracted with Phe, the extracellular alkalinization caused relaxation in the endothelium-intact rings only, and this relaxation was maintained after cyclooxygenase inhibition; completely abolished by the inhibition of nitric oxide synthase (NOS), Ca(2+)/calmodulin and Na(+)/Ca(2+) exchanger (NCX), and partially blunted by the caveolae disassembly. CONCLUSIONS These results suggest that, in rat thoracic aorta, that extracellular alkalinization with NaOH activates the NCX reverse mode of endothelial cells in rat thoracic aorta, thereby the intracellular Ca(2+) concentration and activating the Ca(2+)/calmodulin-dependent NOS. In turn, NO is released promoting relaxation.

Oxidative stress is not associated with vascular dysfunction in a model of alloxan-induced diabetic rats.

OBJECTIVES To verify if an experimental model of alloxan-diabetic rats promotes oxidative stress, reduces nitric oxide bioavailability and causes vascular dysfunction, and to evaluate the effect of N-acetylcysteine (NAC) on these parameters. METHODS Alloxan-diabetic rats were treated or not with NAC for four weeks. Plasmatic levels of malondialdehyde (MDA) and nitrite/nitrate (NOx), the endothelial and inducible nitric oxide synthase (eNOS and iNOS) immunostaining and the vascular reactivity of aorta were compared among diabetic (D), treated diabetic (TD) and control (C) rats. RESULTS MDA levels increased in D and TD. NOx levels did not differ among groups. Endothelial eNOS immunostaining reduced and adventitial iNOS increased in D and TD. The responsiveness of rings to acetylcholine, sodium nitroprusside, and phenylephrine did not differ among groups. CONCLUSIONS NAC had no effect on the evaluated parameters and this experimental model did not promote vascular dysfunction despite the development of oxidative stress.

Exhaled breath condensate collection for nitrite dosage: a safe and low cost adaptation

PURPOSE Standardization of a simple and low cost technique of exhaled breath condensate (EBC) collection to measure nitrite. METHODS Two devices were mounted in polystyrene boxes filled either with crushed ice/salt crystals or dry ice/crushed ice. Blood samples were stored at -70 degrees C for posterior nitrite dosages by chemiluminescence and the Griess reaction. RESULTS a) The use of crushed ice/dry ice or salt revealed sufficient EBC room air collection, but was not efficient for patients under ventilation support; b) the method using crushed ice/salt collected greater EBC volumes, but the nitrite concentrations were not proportional to the volume collected; c) The EBC nitrite values were higher in the surgical group using both methods; d) In the surgical group the nasal clip use diminished the EBC nitrite concentrations in both methods. CONCLUSIONS The exhaled breath condensate (EBC) methodology collection was efficient on room air breathing. Either cooling methods provided successful EBC collections showing that it is possible to diminish costs, and, amongst the two used methods, the one using crushed ice/salt crystals revealed better efficiency compared to the dry ice method.

Methylene blue improves hemodynamic shock but increases lipoperoxidation in severe acute pancreatitis pig model

PURPOSE Study hemodynamic pattern and lipoperoxidation during methylene blue (MB) treatment on taurocholate - enterokinase induced acute pancreatitis (AP). METHODS Thirty pigs were equally divided in control group; MB group; AP group; MB previous AP group; and MB after 90 min of induced AP group. MB was given iv in a bolus dose (2 mg.kg-1) followed by maintenance dose (2 mg.kg-1.h-1). Hemodynamic parameters were recorded continuously during 180 min by Swan-Ganz catheter. Blood samples were taken every 60 min to determine arterial and venous nitrate, malondialdehyde (MDA) and amylase. Pancreatic tissue was removed for histopathologic study. RESULTS In AP group MBP and CO decreased over time 33% (p<0.05) and 52% (p<0.05), respectively. In MB previous induced-AP group, there was 70 minutes delay (p<0.05) to decrease MBP and CO. In MB group arterial and venous nitrite decreased (p<0.05) over time. MB infusion increased (p>0.05) serum MDA when associated to AP. After induced AP, MB did not reverse MBP and CO decrease. There was no difference in serum amylase and necro-hemorrhagic findings with MB treatment. CONCLUSIONS In this taurocholate-induced AP model MB treatment delayed hemodynamic shock and decreases serum nitrate levels but increases serum MDA levels. No volemic replacement was done and it may have been a mitigated factor to a poor tissue perfusion and impairment microcirculation. Further investigations are needed to elucidate MB treatment role during AP treatment.

Vascular relaxation of canine visceral arteries after ischemia by means of supraceliac aortic cross-clamping followed by reperfusion

BackgroundThe supraceliac aortic cross-clamping can be an option to save patients with hipovolemic shock due to abdominal trauma. However, this maneuver is associated with ischemia/reperfusion (I/R) injury strongly related to oxidative stress and reduction of nitric oxide bioavailability. Moreover, several studies demonstrated impairment in relaxation after I/R, but the time course of I/R necessary to induce vascular dysfunction is still controversial. We investigated whether 60 minutes of ischemia followed by 30 minutes of reperfusion do not change the relaxation of visceral arteries nor the plasma and renal levels of malondialdehyde (MDA) and nitrite plus nitrate (NOx).MethodsMale mongrel dogs (n = 27) were randomly allocated in one of the three groups: sham (no clamping, n = 9), ischemia (supraceliac aortic cross-clamping for 60 minutes, n = 9), and I/R (60 minutes of ischemia followed by reperfusion for 30 minutes, n = 9). Relaxation of visceral arteries (celiac trunk, renal and superior mesenteric arteries) was studied in organ chambers. MDA and NOx concentrations were determined using a commercially available kit and an ozone-based chemiluminescence assay, respectively.ResultsBoth acetylcholine and calcium ionophore caused relaxation in endothelium-intact rings and no statistical differences were observed among the three groups. Sodium nitroprusside promoted relaxation in endothelium-denuded rings, and there were no inter-group statistical differences. Both plasma and renal concentrations of MDA and NOx showed no significant difference among the groups.ConclusionSupraceliac aortic cross-clamping for 60 minutes alone and followed by 30 minutes of reperfusion did not impair relaxation of canine visceral arteries nor evoke biochemical alterations in plasma or renal tissue.

Liver histological, portal flow and plasmatic nitric oxide alterations caused by biliary obstruction and drainage in rats

PURPOSE To evaluate liver alterations caused by biliary obstruction and drainage. METHODS Thirty-nine male Wistar rats were randomly distributed in 4 groups: BO (n=18) bile duct ligation for 20 days, with a periodic evaluation of liver histological alterations, Doppler echography portal flow and measurements of NO and malondialdehyde (MDA); BO/DB (n=13) bile duct occlusion for 20 days followed by biliary drainage by choledochoduodenal anastomosis, 5 days follow-up, same BO group parameters evaluations; group CED (n=4) sham operation and portal flow evaluation trough 20 days; CHB (n=4) sham operation, with hepatic biopsy on 25th day and followed-up trough 25 days, by the same parameters of group BO, with exception of portal flow. Direct bilirubin (DB) and alkaline phosphatase (AP) were evaluated in the group BO, BO/DB and CHB. RESULTS The bile duct ligation led to an increase of DB and AP, development of liver histological alterations, reduction of portal flow and increase of plasmatic NO and of MDA levels. The bile duct clearing resulted in a reduction of DB, AP, NO, MDA histological alterations and increase of portal flow. CONCLUSION The biliary occlusion resulted in cholestasis and portal flow reduction, besides the increase of plasmatic NO and of hepatic MDA levels, and histological liver alterations, with a tendency of normalization after the bile duct clearing.